WO2017066714A1 - Anti-vsig1 antibodies and drug conjugates - Google Patents

Anti-vsig1 antibodies and drug conjugates Download PDF

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Publication number
WO2017066714A1
WO2017066714A1 PCT/US2016/057231 US2016057231W WO2017066714A1 WO 2017066714 A1 WO2017066714 A1 WO 2017066714A1 US 2016057231 W US2016057231 W US 2016057231W WO 2017066714 A1 WO2017066714 A1 WO 2017066714A1
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sequence
seq
cpa
antibody
vsigl
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PCT/US2016/057231
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French (fr)
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WO2017066714A9 (en
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Andrew W. DRAKE
Andrew POW
Kyle Hansen
Yair BENITA
Gad S. Cojocaru
Shirley Sameah-Greenwald
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Compugen Ltd.
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Publication of WO2017066714A9 publication Critical patent/WO2017066714A9/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6873—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting an immunoglobulin; the antibody being an anti-idiotypic antibody
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57449—Specifically defined cancers of ovaries
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • V-set and immunoglobulin domain containing protein 1 is a recently discovered member of the junctional adhesion molecule (JAM) family, also sometimes referred to as the immunoglobulin superfamily (IgSF).
  • JAM junctional adhesion molecule
  • IgSF immunoglobulin superfamily
  • Members of the IgSF/JAM family have a unique structure containing an N-terminal signal peptide domain, immunoglobulin (Ig)-like domains, and transmembrane and cytoplasmic tail domains. However, very little is known about its physiological functions.
  • Glycoprotein A34 it was first characterized in humans as an X-linked gene and characterized by the presence of two Ig-like domains.
  • VSIGl has been shown to be specifically expressed in the stomach and testis, and overexpressed in some gastric cancers, esophageal carcinomas, and ovarian cancers, but not in lung, breast or colon carcinomas.
  • Scanlon et al Cancer Immun. 6:20 (2010).
  • the stomach there are three altematively spliced isoforms that appear to be involved in differentiation of gastric epithelia; see Oidovsambuu et al, PLOS One 6(10):e25908 (2011).
  • testis it has been associated with spermatogenesis rather than fertilization. See Kim et al, Mol. Cells 30:443-448 (2010).
  • VSIGl has been shown to be preferentially expressed in certain gastric, esophageal and ovarian cancers.
  • decreased expression of VSIGl is associated with poor prognosis in primary gastric cancer; see Chen et al, J. Surgical Oncology 2012 106:286-293.
  • VSIGl expression was completely lost in 126 out of 232 patient samples and remarkably reduced in another 106 patients, and was an independent predictor of overall survival.
  • the present invention is directed to new surprising results associated with VSIGl expression. BRIEF SUMMARY OF THE INVENTION [0006] The present invention provides anti-VSIGl antibodies.
  • the antibodies are antibody drug conjugates that bind V-set and immunoglobulin domain-containing protein 1 (VSIGl) and comprise at least one drug moiety and an anti-VSIGl antibody.
  • the anti-VSIGl antibodies are selected from the group consisting of 1) CPA.4.013, CPA.4.013.VH, CPA.4.013.VL, CPA.4.013.HC, CPA.4.013.LC and CPA.4.013.H1, CPA.4.013.H2, CPA.4.013.H3 and CPA.4.013.H4; 2) CPA.4.016, CPA.4.016.VH, CPA.4.016.VL, CPA.4.016.HC, CPA.4.016.LC, CPA.4.016.H1, CPA.4.016.H2, CPA.4.016.H3 and CPA.4.016.H4; 3) CPA.4.017, CPA.4.017.VH,
  • CPA.4.020.LC CPA.4.020.H1, CPA.4.020.H2, CPA.4.020.H3 and CPA.4.020.H4; 6) CPA.4.023, CPA.4.023.VH, CPA.4.023.VL, CPA.4.023.HC, CPA.4.023.LC, CPA.4.023.H1, CPA.4.023.H2, CPA.4.023.H3 and CPA.4.023.H4; 7) CPA.4.005, CPA.4.005.VH,
  • CPA.4.027.LC CPA.4.027.H1, CPA.4.027.H2, CPA.4.027.H3 and CPA.4.027.H4; 10) CPA.4.028, CPA.4.028.VH, CPA.4.028.VL, CPA.4.028.HC, CPA.4.028.LC, CPA.4.028.H1, CPA.4.028.H2, CPA.4.028.H3 and CPA.4.028.H4; 11) CPA.4.031, CPA.4.031.VH, CPA.4.031.VL, CPA.4.031.HC, CPA.4.031.LC, CPA.4.031.H1, CPA.4.031.H2,
  • CPA.4.012.LC CPA.4.012.H1, CPA.4.012.H2, CPA.4.012.H3 and CPA.4.012.H4; 14) CPA.4.008, CPA.4.008,VH, CPA.4.008.VL, CPA.4.008.HC, CPA.4.008.LC, CPA.4.008.H1, CPA.4.008.H2, CPA.4.008.H3 and CPA.4.008.H4; and 15) CPA.4.011, CPA.4.011.VH, CPA.4.011.VL, CPA.4.011.HC, CPA.4.011.LC, CPA.4.011.H1, CPA.4.011.H2,
  • the drug moiety is attached to the antibody by an ADC linker.
  • the ADC linker is cleavable, and in others, it is self-immolative.
  • the drug moiety is selected from the group consisting of: taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6- thioguanine, cytarabine, 5-fluorouracil decarbazine, mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclothosphamide, busul
  • taxol cyto
  • the anti-VSIGl antibodies have a drug moiety is selected from the group consisting of: the maytansinoid (N2'-deacetyl-N2'-(3-mercapto-l- oxopropyl)maytansine) (DM1), monomethyl auristatin (MMAE), monomethyl auristatin phenylalanine (MMAF), and Duocarmycin.
  • DM1 maytansinoid (N2'-deacetyl-N2'-(3-mercapto-l- oxopropyl)maytansine)
  • MMAE monomethyl auristatin
  • MMAF monomethyl auristatin phenylalanine
  • Duocarmycin Duocarmycin
  • the antibody drug conjugate compound is cytotoxic to cancer cells, and in others it is cytostatic.
  • the anti-VSIGl antibodies of the invention bind to an epitope located within the extracellular domain of VSIG1.
  • the antibody drug conjugate compound of the invetnion competes for binding at an epitope located within the extracellular domain of VSIG1 with any of the enumerated antibodies herein.
  • the antibody component of the drug conjugate compound is a human monoclonal anti-VSIGl antibody.
  • the monoclonal anti-VSIGl antibody comprises a variable heavy and variable light chain pair selected from the group consisting of: CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011.
  • the isolated anti-VSIGl antibodies of the invention comprise:
  • a heavy chain variable domain comprising a vHCDRl having the sequence
  • GFTFSSYS (SEQ ID NO:X)
  • a vhCDR2 having the sequence ISSSSSYI (SEQ ID NO:X)
  • a vhCDR3 having the sequence ARNVLRGAAQYYFDY (SEQ ID NO:X)
  • this antibody comprises a covalently attached drug moiety.
  • the antiVSIGl antibodies comprise:
  • a heavy chain variable domain comprising a vHCDRl having the sequence
  • GFTFSSYG (SEQ ID NO:X)
  • vhCDR2 having the sequence ISYDGSNK (SEQ ID NO:X)
  • vhCDR3 having the sequence VRGINVNFHYYGMDV (SEQ ID NO:X)
  • this antibody comprises a covalently attached drug moiety.
  • the antiVSIGl antibodies comprise:
  • a heavy chain variable domain comprising a vHCDRl having the sequence
  • GFTFSSYA SEQ ID NO:X
  • vhCDR2 having the sequence ISGSGGST
  • vhCDR3 having the sequence AKDSYFDWQIGSNDAFDI
  • this antibody comprises a covalently attached drug moiety.
  • the antiVSIGl antibodies comprise: [0026] a) a heavy chain variable domain comprising a vHCDRl having the sequence
  • GFTFSSYA SEQ ID NO:X
  • vhCDR2 having the sequence ISGSGGST
  • vhCDR3 having the sequence AKAYGS GS YFH YWYFDL
  • QSLLQSNGYNY (SEQ ID NO:X)
  • a vLCDR2 having the sequence LGS (SEQ ID NO:X)
  • a vLCDR3 having the sequence MQALQTPPT (SEQ ID NO:X).
  • this antibody comprises a covalently attached drug moiety.
  • the antiVSIGl antibodies comprise:
  • a heavy chain variable domain comprising a vHCDRl having the sequence
  • GGTFSSYA SEQ ID NO:X
  • vhCDR2 having the sequence IIPIFGTA
  • vhCDR3 having the sequence ATQYS S GWYIWGAFDI
  • this antibody comprises a covalently attached drug moiety.
  • the antiVSIGl antibodies comprise:
  • a heavy chain variable domain comprising a vHCDRl having the sequence
  • GYSFTSYW (SEQ ID NO:X)
  • a vhCDR2 having the sequence IYPGDSDT (SEQ ID NO:X)
  • a vhCDR3 having the sequence ARLGIVDTSWSAFDI (SEQ ID NO:X)
  • this antibody comprises a covalently attached drug moiety.
  • the antiVSIGl antibodies comprise:
  • a heavy chain variable domain comprising a vHCDRl having the sequence
  • GDTFSSYT SEQ ID NO:X
  • vhCDR2 having the sequence FIPPLDIA
  • vhCDR3 having the sequence ATGGATIFFYYFGMDV
  • this antibody comprises a covalently attached drug moiety.
  • the antiVSIGl antibodies comprise:
  • a heavy chain variable domain comprising a vHCDRl having the sequence
  • GYTFTSYA (SEQ ID NO:X)
  • a vhCDR2 having the sequence INAGNGNT (SEQ ID NO:X)
  • a vhCDR3 having the sequence ASSFHGSGSYYNKVVGMWY (SEQ ID NO:X)
  • this antibody comprises a covalently attached drug moiety.
  • the antiVSIGl antibodies comprise:
  • a heavy chain variable domain comprising a vHCDRl having the sequence
  • this antibody comprises a covalently attached drug moiety.
  • the antiVSIGl antibodies comprise:
  • a heavy chain variable domain comprising a vHCDRl having the sequence
  • GFTFSSYG (SEQ ID NO:X)
  • vhCDR2 having the sequence IWYDGSNK (SEQ ID NO:X)
  • vhCDR3 having the sequence ARGGPWGIVVVNQFDY (SEQ ID NO:X)
  • this antibody comprises a covalently attached drug moiety.
  • the antiVSIGl antibodies comprise: [0047] a) a heavy chain variable domain comprising a vHCDRl having the sequence
  • SGSIRSSNW (SEQ ID NO:X)
  • a vhCDR2 having the sequence IYHSGST (SEQ ID NO:X)
  • a vhCDR3 having the sequence AGRNIAGGSFDY (SEQ ID NO:X)
  • SGIDVGPYR SEQ ID NO:X
  • vLCDR2 having the sequence YNSDSDK
  • vLCDR3 having the sequence MIWHNKSRV
  • this antibody comprises a covalently attached drug moiety.
  • the antiVSIGl antibodies comprise:
  • a heavy chain variable domain comprising a vHCDRl having the sequence
  • this antibody comprises a covalently attached drug moiety.
  • the antiVSIGl antibodies comprise:
  • a heavy chain variable domain comprising a vHCDRl having the sequence
  • GFSFSNYV (SEQ ID NO:X)
  • vhCDR2 having the sequence ISYDGSDK
  • vhCDR3 having the sequence ARSLRPAYYDASGV (SEQ ID NO:X)
  • this antibody comprises a covalently attached drug moiety.
  • the antiVSIGl antibodies comprise:
  • a heavy chain variable domain comprising a vHCDRl having the sequence
  • GGTFSSYS (SEQ ID NO:X)
  • vhCDR2 having the sequence IIPLFGTT
  • vhCDR3 having the sequence ARSKDYYGSGMEALLMDV
  • this antibody comprises a covalently attached drug moiety.
  • the antiVSIGl antibodies comprise:
  • a heavy chain variable domain comprising a vHCDRl having the sequence
  • GFTFSSYA SEQ ID NO:X
  • vhCDR2 having the sequence ISGSGGST
  • vhCDR3 having the sequence AKELRGGSYYFTGTDAFDI
  • this antibody comprises a covalently attached drug moiety.
  • Figure 1 shows the amino acid sequence of the extracellular domain of human VSIGl, aligned with mouse VSIGl. The analysis shows 174/212 (82%) identical residues between the two proteins. The consensus sequence is marked in italics.
  • Figure 2A-2B shows the Fc sequences for human IgGl, IgG2, IgG3 and IgG4.
  • Figures 3A to 3H shows the heavy and light chain amino acid sequences for each of the enumerated anti-VSIGl antibodies.
  • Figure 4 shows the variable regions of the heavy and light chains, with frameworks and CDR domains outlined, of each of the enumerated anti-VSIGl antibodies.
  • FIGs 5A and 5B shows IHC analysis as per Example 1.
  • the IHC score is a measure of the amount of staining, with “1" being the lowest amount seen, "2" being medium and "3" being significant staining.
  • the numbers in the columns represents the number of different samples that showed the staining; that is, one normal breast tissue showed staining of "1", and 18 samples of breast cancer tissue showed a staining level of "1".
  • MFI mean fluorescence intensity
  • Table shading highlights weaker and stronger binders (hatched and solid grey, respectively) or binders cross-reactive against human and mouse VSIGl (solid grey).
  • Figure 7 shows sensorgrams of A) VSIGl monomer injected at three concentrations (1.6nM, 4.9nM, 14.8nM) over mAb 156-01. D06 captured to a GLC ProteOn biosensor chip. The higher monomer concentrations appeared to show some nonspecific binding.
  • sensorgrams of antigen injected over the mAbs and the red lines are a global fit of a 1 : 1 kinetic binding model. Binding constants are listed in Table 5. Data were processed and fit using ProteOn Manager Version 3.1.0.6.
  • Figure 8 shows sensorgrams of A) VSIGl monomer injected at four concentrations (1.6nM-44.4nM) over mAb 156-01. C09 captured to a GLC ProteOn biosensor chip. B) VSIGl monomer injected at six concentrations (1.6nM - 400nM) over mAb 156-01. C04 captured to a GLC ProteOn biosensor chip. In both panels the black lines are the
  • sensorgrams of antigen injected over the mAbs and the red lines are a global fit of a 1 : 1 kinetic binding model. Binding constants are listed in Table 5. Data were processed and fit using ProteOn Manager Version 3.1.0.6.
  • Figure 9 shows sensorgrams of A) VSIGl monomer injected at six concentrations (1.6nM-400nM) over mAb 156-01. C03 captured to a GLC ProteOn biosensor chip. Steady- state signal points were taken as an average between the green (109 sec) and red lines (119 sec). B) Steady-state RU signals as a function of [VSIGl] for mAb 156-01. C03 and fit with a simple 1 : 1 equilibrium model. C) VSIGl monomer injected at six concentrations (1.6nM- 400nM) over mAb 156-01. E12 captured to a GLC ProteOn biosensor chip.
  • Steady-state signal points were taken as an average between the green (109 sec) and red lines (119 sec).
  • Figure 10 shows sensorgrams A) VSIGl monomer injected at four concentrations (1.6nM-44.4nM) over mAb 156-01.F04 captured to a GLC ProteOn biosensor chip. B) VSIGl monomer injected at six concentrations (1.6nM - 400nM) over mAb 156-01. B10 captured to a GLC ProteOn biosensor chip. In both panels the black lines are the
  • sensorgrams of antigen injected over the mAbs and the red lines are a global fit of a 1 : 1 kinetic binding model. Binding constants are listed in Table 5. Data were processed using ProteOn Manager Version 3.1.0.6. Data in A were fit using Scrubber ProteOn and the data in B were fit using ProteOn Manager.
  • Figure 11 shows sensorgrams of A) VSIGl monomer injected at six concentrations (1.6nM-400nM) over mAb 156-01.E06 captured to a GLC ProteOn biosensor chip. Steady- state signal points were taken as an average between the green (105 sec) and red lines (113 sec). B) Steady-state RU signals as a function of [VSIGl] for mAb 156-01. E06 and fit with a simple 1 : 1 equilibrium model. C) VSIGl monomer injected at six concentrations (1.6nM
  • Figure 12 shows sensorgrams of A) VSIGl monomer injected at six concentrations (1.6nM-400nM) over mAb 156-01. A03 captured to a GLC ProteOn biosensor chip. Steady- state signal points were taken as an average between the green (105 sec) and red lines (113 sec). B) Steady-state RU signals as a function of [VSIGl] for mAb 156-01. A03 and fit with a simple 1 : 1 equilibrium model. C) VSIGl monomer injected at six concentrations (1.6nM- 400nM) over mAb 156-01. D12 captured to a GLC ProteOn biosensor chip.
  • Steady-state signal points were taken as an average between the green (107 sec) and red lines (113 sec).
  • Figure 13 shows sensorgrams of A) VSIGl monomer injected at six concentrations (1.6nM-400nM) over mAb 156-01. F10 captured to a GLC ProteOn biosensor chip. Steady- state signal points were taken as an average between the green (105 sec) and red lines (113 sec). B) Steady-state RU signals as a function of [VSIGl] for mAb 156-01. F10 and fit with a simple 1 : 1 equilibrium model. C) VSIGl monomer injected at six concentrations (1.6nM
  • Figure 14 shows sensorgrams of A) VSIG1 monomer injected at six concentrations (1.6nM-400nM) over mAb 156-01. D05 captured to a GLC ProteOn biosensor chip. Steady- state signal points were taken as an average between 110 sec and 116 sec. B) Steady-state RU signals as a function of [VSIG1] for mAb 156-01. D05 and fit with a simple 1 : 1 equilibrium model. Binding constants are listed in Table 5. Data were processed and fit using ProteOn Manager Version 3.1.0.6. Because of the apparent low affinity, higher concentrations of VSIG1 should be included for a better estimate of K D .
  • Figure 15 shows an example of a processed and referenced set of sensorgrams over all immobilized mAbs for generating the VSIG1 antigen blocking patterns for mAb CPA.4.023.
  • Each panel represents a different ProteOn chip array spot having an immobilized mAb.
  • the 15 different mAbs immobilized over spots A-0 are also immobilized in identical order over spots P-DD.
  • Black sensorgrams in A-0 are mAb CPA.4.023 injected at a binding site concentration of -400 nM pre-mixed with VSIG1 antigen at a binding site concentration of 23 nM.
  • Black sensorgrams in P-DD are 156-01. C08 injected at ⁇ 400nM without Ag as a control.
  • Figure 16 shows a binary matrix of mAb pair- wise blocking ("0", red box) or sandwiching ("1", green box) for 15 anti-VSIGl mAbs.
  • MAbs listed vertically on the left of the matrix are mAbs covalently immobilized to the ProteOn array.
  • MAbs listed horizontally across the top of the matrix are analytes injected with pre-mixed antigen.
  • the black boxes outline four epitope bins according to the horizontal blocking patterns of the mAbs as immobilized ligands. The bottom two bins differ only in how their component mAbs block or sandwich Ag in the presence of mAb CPA.4.012.
  • Figure 17 shows hierarchical clustering dendrogram of the horizontal binding patterns of each mAb in the binary matrix in Figure 16. There appear to be four bins of mAbs with identical epitope blocking patterns within each group. Clone CPA.4.012 is the only mAb in bin 2. Bins 3 and 4 could be closely related, however, since the mAbs only differ in their antigen blocking with clone CPA.4.012.
  • Figure 18 shows anti-VSIGl antibody binding curves. Binding of anti-VSIGl antibodies to HEK 293 cells transfected with recombinant human (A), cynomolgus (B), or mouse (C) VSIG1.
  • A human
  • B cynomolgus
  • C mouse
  • X-axis nM of antibody.
  • Y-axis median fluoresce intensity.
  • Figure 20 shows in vitro cytotoxic activity of CPA.4.009 on OV-90 and DU145 cells.
  • X-axis nM antibody.
  • Y-axis relative light unit reading from cell titer glo.
  • Figure 21 shows in vitro cytotoxic activity of 15 anti-VISGl ADCs with 4 payloads on OV-90, MKN-45, and HUH-1 Human Cancer Cells.
  • OV-90 and HUH-1 cells were treated with 0.6 ⁇ g/ml anti-VSIGl antibody or hlgGl isotype control.
  • MKN-45 cells were treated with 5 ⁇ g/ml anti-VSIGl antibody or hlgGl isotype control.
  • Antibody alone (gray bars) or media with Protein G preloaded with DM1 (A), MMAE (B), MMAF (C), or Duocarmycin (D) (black bars) was added to each well.
  • "% untreated control” (relative light units of treated samples / relative light units of untreated samples) * 100.
  • Figure 22 shows in vitro cytotoxic activity of 4 anti-VISGl ADCs with 4 payloads on HUH-1 Human Cancer Cells.
  • Cells were treated with anti-VSIGl antibody or hlgGl isotype control.
  • Media only or media with Protein G preloaded with DM1 (A), MMAE (B), MMAF (C), or Duocarmycin (D) was added to each well.
  • Percent untreated control (relative light units of treated samples/ relative light units of untreated samples) * 100.
  • Figure 25A-25F presents a comparison in the level of VSIGl expression on cancerous vs normal tissue in lung tissue (A), pancreatic tissue (B), liver tissue (C), ovarian tissue (D), colorectal tissue (E), uterine tissue (F), head and neck tissue (G), and kidney tissue (H).
  • Figure 26 shows the results of the IHC experiments of Example 1 , including the sample description and IHC scoring for each sample.
  • Figure 27 shows the results of the IHC experiments of Example 1 , including the sample description and IHC scoring for each sample.
  • Figure 28 depicts the closest human germline genes to the VH and VL sequences of the enumerated antibodies of the invention.
  • Figures 29A and 29B depicts the alignment and grouping of the VH amino acid sequences, using IMGT numbering.
  • Figures 30A and 30B depicts the alignment and grouping of the VL amino acid sequences, using IMGT numbering.
  • Figure 31A-31B present MED discovery engine expression results.
  • Figure 31A pancreatic cancer and normal expression of VSIGl .
  • Figures 32A to 32J depict the nucleic acid sequences for the enumerated antibodies of the invention.
  • Figures 33A to 33F depict a number of additional amino acid sequences of the invetnion.
  • Figures 33A - 33C depicts human VSIGl ECD fused to human IgGl Fc (VSIGl H:H, SEQ ID NO:XX which is the natural human Signal Peptide and human ECD (aa22- 232 of VSIG1_HUMAN) fused to Human IgGl Fc mutated at C220S of hinge.
  • Figures 33D to 33F depicts mouse VSIGl ECD fused to mouse IgG2a Fc (VSIGl M:M, SEQ ID NO XX which is (PBG-SPl + Mouse ECD (aal-231 VSIGl _MOUSE) + Mouse IgG2a Fc)), used for anti-VSIGl Fab Antibodies generation by Phage Display.
  • Figure 34 shows immunohistochemistry validation of commercially available anti- human VSIGl antibody (R&D systems, cat # MAB481 8). Antibody used at 5 ⁇ £ ⁇ / ⁇ , ⁇ 6.2 HIER, Biocare Universal HRP Detection, 40X.
  • Figure 35 provides an ovarian cancer tumor microarray.
  • Figure 36 provides a stomach cancer tumor microarray.
  • Figure 37 provides a pancreatic cancer tumor microarray.
  • Figure 38 provides a liver cancer tumor microarray.
  • Figure 39 provides a lung cancer tumor microarray.
  • Figure 40 shows 2D in vitro cytotoxicity of 156-01.E05 directly conjugated to
  • DM1, MMAE, MMAF, and DMSA on OV-90 cells.
  • X-axis nM antibody.
  • Y-axis % of untreated control cells.
  • hIgGl human IgGl isotype control.
  • Figure 41 shows 2D in vitro cytotoxicity of 156-01.E05 directly conjugated to
  • Y-axis % of untreated control cells.
  • hIgGl human IgGl isotype control.
  • Figure 42 shows 2D in vitro cytotoxicity of 156-01.E05 directly conjugated to
  • DM1, MMAE, MMAF, and DMSA on HUH-1 cells.
  • X-axis nM antibody.
  • Y-axis % of untreated control cells.
  • hIgGl human IgGl isotype control.
  • Figure 43 shows 3D in vitro cytotoxicity of 156-01. E05 directly conjugated to
  • DM1, MMAE, MMAF, and DMSA on OV-90 cells.
  • X-axis nM antibody.
  • Y-axis % of untreated control cells.
  • hIgGl human IgGl isotype control.
  • Figure 44 shows 3D in vitro cytotoxicity of 156-01. E05 directly conjugated to
  • Y-axis % of untreated control cells.
  • hIgGl human IgGl isotype control.
  • Figure 45 shows 3D in vitro cytotoxicity of 156-01. E05 directly conjugated to
  • DM1, MMAE, MMAF, and DMSA on HUH-1 cells.
  • X-axis nM antibody.
  • Y-axis % of untreated control cells.
  • hIgGl human IgGl isotype controls.
  • the present invention is directed to antibody compositions against human
  • VSIGl and methods of using the antibodies.
  • increased VSIGl expression has been shown in several cancers, including stomach, ovarian, liver and pancreatic cancers, and thus anti-VSIGl antibodies find use in the treatment of these cancers.
  • the anti-VSIGl antibodies find use in treating non-small cell lung cancer (NSCLC)
  • adenocarcinoma breast carcinoma, hepatocellular carcinoma (HCC), colon cancer, rectal cancer, kidney clear cell carcinoma, kidney papillary cell carcinoma, uterine cancer, stomach cancer, head and neck cancer, pancreatic cancer and ovarian cancer.
  • anti-VSIGl antibodies that include one or more covalently attached drugs, including chemotherapeutic drugs that can be cytotoxic and/or cytostatic, find particular use in the treatment of these particular cancers.
  • ADCs antibody drug conjugates
  • the anti-VSIGl ADC antibody binds to the VSIG1 antigen and then is internalized by the cell, to deliver the drug (sometimes referred to in the art as a "payload") to the inside of the cell, to kill the cell.
  • the linker that attaches the drugs to the antibody (sometimes referred to herein as the "ADC linker", to distinguish it from other linkers used in the invention), can be cleaved, thus releasing the drug for ease of action.
  • the ADC linker is not necessarily immediately cleaved but through the general action of the internal cellular machinery the drug is active.
  • the anti-VSIGl antibody remains on the cell surface.
  • the antibody-drug conjugate compounds (ADC) of the invention may selectively deliver an effective dose of a cytotoxic agent to tumor tissue whereby greater selectivity, i.e. a lower efficacious dose may be achieved.
  • the bioavailability of the ADC, or an intracellular metabolite of the ADC is improved in a mammal when compared to the corresponding drug moiety alone.
  • the bioavailability of the ADC, or an intracellular metabolite of the ADC is improved in a mammal when compared to the corresponding antibody alone (antibody of the ADC, without the drug moiety or linker).
  • the drug moiety of the ADC is not cleaved from the antibody until the antibody-drug conjugate binds to a cell-surface receptor, or enters a cell with a cell-surface receptor specific for the antibody of the antibody-drug conjugate.
  • the drug moiety may be cleaved from the antibody after the antibody-drug conjugate enters the cell.
  • the drug moiety may be intracellularly cleaved in a mammal from the antibody of the compound, or an intracellular metabolite of the compound, by enzymatic action, hydrolysis, oxidation, or other mechanism.
  • V-set and immunoglobulin domain containing protein 1 is a transmembrane protein with an extracellular domain (ECD), the human sequence of which is shown in Figure 1.
  • ECD extracellular domain
  • the antibody used in the antibody-drug conjugate is an antibody that interacts with one or more epitopes on the VSIGl polypeptide.
  • the antibody is specific for the VSIGl extracellular domain.
  • antibody is used generally. Antibodies that find use in the present invention can take on a number of formats as described herein, including traditional antibodies as well as antibody derivatives, fragments and mimetics, described below. In general, the term “antibody” includes any polypeptide that includes at least one antigen binding domain, as more fully described below. Antibodies may be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, as described herein. In some embodiments, antibodies of the invention bind specifically or substantially specifically to VSIGl molecules.
  • monoclonal antibodies and “monoclonal antibody composition”, as used herein, refer to a population of antibody molecules that contain only one species of an antigen-binding site capable of immunoreacting with a particular epitope of an antigen
  • polyclonal antibodies and “polyclonal antibody composition” refer to a population of antibody molecules that contain multiple species of antigen-binding sites capable of interacting with a particular antigen.
  • a monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.
  • Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one "light” (typically having a molecular weight of about 25 kDa) and one "heavy” chain (typically having a molecular weight of about 50-70 kDa).
  • Human light chains are classified as kappa and lambda light chains.
  • the present invention is directed to the IgG class, which has several subclasses, including, but not limited to IgGl, IgG2, IgG3, and IgG4.
  • isotype as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.
  • the anti- VSIG1 antibodies of the invention include those using IgG2, IgG3 and IgG4 sequences, or combinations thereof, as shown in Figure 2.
  • IgG2 IgG3 and IgG4 sequences, or combinations thereof, as shown in Figure 2.
  • IgG2 IgG3 and IgG4 sequences, or combinations thereof, as shown in Figure 2.
  • different IgG isotypes have different effector functions which may or may not be desirable.
  • each chain includes a variable region of about
  • variable domain 100 to 110 or more amino acids primarily responsible for antigen recognition, generally referred to in the art and herein as the "Fv domain” or “Fv region".
  • Fv domain 100 to 110 or more amino acids primarily responsible for antigen recognition
  • CDR complementarity-determining region
  • Variable refers to the fact that certain segments of the variable region differ extensively in sequence among antibodies. Variability within the variable region is not evenly distributed. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions”.
  • Each VH and VL is composed of three hypervariable regions
  • CDRs complementary determining regions
  • the hypervariable region generally encompasses amino acid residues from about amino acid residues 24-34 (LCDR1 ; "L” denotes light chain), 50-56 (LCDR2) and 89- 97 (LCDR3) in the light chain variable region and around about 31-35B (HCDR1 ; "H” denotes heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al, SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or those residues forming a hypervariable loop (e.g.
  • each chain defines a constant region primarily responsible for effector function.
  • Kabat et al. collected numerous primary sequences of the variable regions of heavy chains and light chains. Based on the degree of conservation of the sequences, they classified individual primary sequences into the CDR and the framework and made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5 th edition, NIH publication, No. 91-3242, E. A. Kabat et al, entirely incorporated by reference).
  • immunoglobulin domains in the heavy chain.
  • immunoglobulin (Ig) domain herein is meant a region of an immunoglobulin having a distinct tertiary structure.
  • the heavy chain domains including, the constant heavy (CH) domains and the hinge domains.
  • the IgG isotypes each have three CH regions. Accordingly, "CH” domains in the context of IgG are as follows: “CHI " refers to positions 118-220 according to the EU index as in Kabat. "CH2" refers to positions 237-340 according to the EU index as in Kabat, and “CH3” refers to positions 341-447 according to the EU index as in Kabat.
  • IMTG numbering system or the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately, residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) (e.g, Kabat et al, supra (1991)).
  • EU numbering as in Kabat is generally used for constant domains and/or the Fc domains.
  • the CDRs contribute to the formation of the antigen-binding, or more specifically, epitope binding site of antibodies.
  • Epitope refers to a determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope.
  • the epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen binding peptide; in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide.
  • Epitopes may be either conformational or linear.
  • a conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain.
  • a linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Conformational and nonconformational epitopes may be distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be verified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen, for example "binning.”
  • the present invention provides a number of anti-VSIGl antibodies as outlined herein, and they fall into different "bins", e.g. they are binding to different locations on the VSIG1 molecule.
  • the present invention provides not only the enumerated antibodies but additional antibodies that compete with the enumerated antibodies to specifically bind to the VSIG1 molecule.
  • an antibody of the invention has a minimum functional requirement that it bind to a VSIG1 antigen.
  • antigen fragments and derivatives that retain the ability to bind an antigen and yet have alternative structures, including, but not limited to, (i) the Fab fragment consisting of VL, VH, CL and CHI domains, (ii) the Fd fragment consisting of the VH and CHI domains, (iii) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, 1988, Science 242:423-426, Huston et al, 1988, Proc.
  • scFv single chain Fv molecules
  • domain antibodies or “dAb” (sometimes referred to as an "immunoglobulin single variable domain", including single antibody variable domains from other species such as rodent (for example, as disclosed in WO 00/29004), nurse shark and Camelid V-HH dAbs,
  • SMIPs small molecule immunopharmaceuticals
  • camelbodies nanobodies and IgNAR.
  • an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecules, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides.
  • immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules.
  • Antibody portions such as Fab and F(ab3 ⁇ 4 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.
  • the antibodies of the invention can be modified, or engineered, to alter the amino acid sequences by amino acid substitutions.
  • amino acid substitution or “substitution” herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid.
  • the substitution is to an amino acid that is not naturally occurring at the particular position, either not naturally occurring within the organism or in any organism.
  • substitution E272Y refers to a variant polypeptide, in this case an Fc variant, in which the glutamic acid at position 272 is replaced with tyrosine.
  • a protein which has been engineered to change the nucleic acid coding sequence but not change the starting amino acid is not an "amino acid substitution"; that is, despite the creation of a new gene encoding the same protein, if the protein has the same amino acid at the particular position that it started with, it is not an amino acid substitution.
  • amino acid substitutions can be made to alter the affinity of the CDRs for the VSIG1 protein (including both increasing and decreasing binding), as well as to alter additional functional properties of the antibodies.
  • the antibodies may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • an antibody according to at least some embodiments of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Such embodiments are described further below.
  • the numbering of residues in the Fc region is that of the EU index of Kabat.
  • the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased.
  • This approach is described further in U.S. Pat. No. 5,677,425 by Bodmer et al.
  • the number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
  • the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding.
  • SpA Staphylococcyl protein A
  • amino acid substitutions can be made in the Fc region, in general for altering binding to FcyR receptors.
  • Fc gamma receptor By “Fc gamma receptor”, “FcyR” or “FcgammaR” as used herein is meant any member of the family of proteins that bind the IgG antibody Fc region and is encoded by an FcyR gene.
  • this family includes but is not limited to FcyRI (CD64), including isoforms FcyRIa, FcyRIb, and FcyRIc; FcyRII (CD32), including isoforms FcyRIIa (including allotypes H131 and R131), FcyRIIb (including FcyRIIb-l and FcyRIIb-2), and FcyRIIc; and FcyRIII (CD 16), including isoforms FcyRIIIa (including allotypes V158 and F158) and FcyRIIIb (including allotypes FcyRIIIb-NAl and FcyRIIIb-NA2) (Jefferis et al, 2002, Immunol Lett 82:57-65, entirely incorporated by reference), as well as any undiscovered human FcyRs or FcyR isoforms or allotypes.
  • An FcyR may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys.
  • Mouse FcyRs include but are not limited to FcyRI (CD64), FcyRII (CD32), FcyRIII-l (CD 16), and FcyRIII-2 (CD 16-2), as well as any undiscovered mouse FcyRs or FcyR isoforms or allotypes.
  • Fc substitutions that can be made to alter binding to one or more of the FcyR receptors. Substitutions that result in increased binding as well as decreased binding can be useful. For example, it is known that increased binding to FcyRIIIa generally results in increased ADCC (antibody dependent cell-mediated cytotoxicity; the cell- mediated reaction wherein nonspecific cytotoxic cells that express FcyRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell. Similarly, decreased binding to FcyRIIb (an inhibitory receptor) can be beneficial as well in some circumstances. Amino acid substitutions that find use in the present invention include those listed in U.S. Ser. Nos. 11/124,620 (particularly FIG.
  • the antibodies of the invention are modified to increase its biological half-life.
  • Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to Ward.
  • the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos.
  • the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody.
  • one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the CI component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.
  • one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix
  • the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439.
  • ADCC antibody dependent cellular cytotoxicity
  • the antibody can be modified to abrogate in vivo
  • Fab arm exchange involves the exchange of IgG4 half-molecules (one heavy chain plus one light chain) between other IgG4 antibodies that effectively results in bispecific antibodies which are functionally monovalent. Mutations to the hinge region and constant domains of the heavy chain can abrogate this exchange (see Aalberse, RC, Schuurman J., 2002, Immunology 105:9-19).
  • the glycosylation of an antibody is modified.
  • an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation).
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen or reduce effector function such as ADCC.
  • Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence, for example N297.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures.
  • altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies according to at least some embodiments of the invention to thereby produce an antibody with altered glycosylation.
  • the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (a (1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates.
  • the Ms704, Ms705, and Ms709 FUT8 cell lines are created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22).
  • a cell line with a functionally disrupted FUT8 gene which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the a 1,6 bond-related enzyme.
  • Hanai et al. also describe cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662).
  • PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Led 3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733 -267 '40).
  • glycoprotein-modifying glycosyl transferases e.g., (l,4)-N-acetylglucosaminyltransferase III (GnTIII)
  • GnTIII glycoprotein-modifying glycosyl transferases
  • the fucose residues of the antibody may be cleaved off using a fucosidase enzyme.
  • the fucosidase a-L-fucosidase removes fucosyl residues from antibodies (Tarentino, A. L. et al. (1975) Biochem. 14:5516-23).
  • Another modification of the antibodies herein that is contemplated by the invention is pegylation or the addition of other water soluble moieties, typically polymers, e.g., in order to enhance half-life.
  • An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody.
  • the antibody, or fragment thereof typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
  • PEG polyethylene glycol
  • the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • a reactive PEG molecule or an analogous reactive water-soluble polymer.
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Ci-Cio) alkoxy- or aryloxy -poly ethylene glycol or polyethylene glycol-maleimide.
  • the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies according to at least some embodiments of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al. [00147] In addition to substitutions made to alter binding affinity to FcyRs and/or
  • FcRn and/or increase in vivo serum half life additional antibody modifications can be made, as described in further detail below.
  • affinity maturation is done. Amino acid modifications in the
  • CDRs are sometimes referred to as "affinity maturation".
  • An “affinity matured” antibody is one having one or more alteration(s) in one or more CDRs which results in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). In some cases, although rare, it may be desirable to decrease the affinity of an antibody to its antigen, but this is generally not preferred.
  • one or more amino acid modifications are made in one or more of the CDRs of the VISG1 antibodies of the invention.
  • 1 or 2 or 3- amino acids are substituted in any single CDR, and generally no more than from 1, 2, 3. 4, 5, 6, 7, 8 9 or 10 changes are made within a set of CDRs.
  • any combination of no substitutions, 1, 2 or 3 substitutions in any CDR can be independently and optionally combined with any other substitution.
  • Affinity maturation can be done to increase the binding affinity of the antibody for the VSIG1 antigen by at least about 10% to 50-100-150% or more, or from 1 to 5 fold as compared to the "parent" antibody.
  • Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the VSIG1 antigen.
  • Affinity matured antibodies are produced by known procedures. See, for example, Marks et al, 1992, Biotechnology 10:779-783 that describes affinity maturation by variable heavy chain (VH) and variable light chain (VL) domain shuffling. Random mutagenesis of CDR and/or framework residues is described in: Barbas, et al. 1994, Proc. Nat. Acad.
  • amino acid modifications can be made in one or more of the
  • CDRs of the antibodies of the invention that are "silent", e.g. that do not significantly alter the affinity of the antibody for the antigen. These can be made for a number of reasons, including optimizing expression (as can be done for the nucleic acids encoding the antibodies of the invention).
  • variant CDRs and antibodies included within the definition of the CDRs and antibodies of the invention are variant CDRs and antibodies; that is, the antibodies of the invention can include amino acid modifications in one or more of the CDRs of the enumerated antibodies of the invention.
  • amino acid modifications can also independently and optionally be made in any region outside the CDRs, including framework and constant regions.
  • the present invention provides anti-VISGl antibodies, including ADC compositions.
  • anti-VSIGl antibodies and “VSIGl antibodies” are used interchangeably.
  • the anti-VSIGl antibodies of the invention specifically bind to human VSIGl, and preferably the ECD of human VISG1, as depicted in Figure 1.
  • Specific binding for VSIGl or a VSIGl epitope can be exhibited, for example, by an antibody having a KD of at least about 10 "4 M, at least about 10 "5 M, at least about 10 "6 M, at least about 10 "7 M, at least about 10 "8 M, at least about 10 "9 M, alternatively at least about 10 "10 M, at least about 10 "11 M, at least about 10 "12 M, or greater, where KD refers to a dissociation rate of a particular antibody-antigen interaction.
  • an antibody that specifically binds an antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a control molecule relative to the VSIGl antigen or epitope.
  • specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KA or Ka for a VSIGl antigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the epitope relative to a control, where KA or Ka refers to an association rate of a particular antibody-antigen interaction.
  • Anti-VSIGl antibodies that are Fabs of the invention include those listed in Figure 4, including, but not limited to, CPA.4.013, CPA.4.016, CPA.4.017,
  • CPA.4.013.VH refers to the variable heavy portion of CPA.4.013
  • CPA.4.013.VL is the variable light chain
  • CPA.4.013.HC refers to the entire heavy chain (e.g. variable and constant domain) of this molecule
  • CPA.4.013.LC refers to the entire light light chain (e.g. variable and constant domain) of the same molecule.
  • CPA.4.013. HI refers to a full length antibody comprising the variable heavy and light domains, including the constant domain of Human IgGl (hence, the HI; IgGl, IgG2, IgG3 and IgG4 sequences are shown in Figure 2).
  • CPA.4.013.H2 would be the CPA.4.013 variable domains linked to a Human IgG2.
  • CPA.4.013.H3 would be the CPA.4.013 variable domains linked to a Human IgG3
  • CPA.4.013.H4 would be the CPA.4.013 variable domains linked to a Human IgG4.
  • VSIG1 antibodies include 1) CPA.4.013,
  • CPA.4.009.LC CPA.4.009.H1, CPA.4.009.H2, CPA.4.009.H3 and CPA.4.009.H4; 9) CPA.4.027, CPA.4.027.VH, CPA.4.027.VL, CPA.4.027.HC, CPA.4.027.LC, CPA.4.027.H1, CPA.4.027.H2, CPA.4.027.H3 and CPA.4.027.H4; 10) CPA.4.028, CPA.4.028.VH, CPA.4.028.VL, CPA.4.028.HC, CPA.4.028.LC, CPA.4.028.H1, CPA.4.028.H2,
  • CPA.4.033.LC CPA.4.033.H1, CPA.4.033.H2, CPA.4.033.H3 and CPA.4.033.H4; 13) CPA.4.012, CPA.4.012.VH, CPA.4.012.VL, CPA.4.012.HC, CPA.4.012.LC, CPA.4.012.H1, CPA.4.012.H2, CPA.4.012.H3 and CPA.4.012.H4; 14) CPA.4.008, CPA.4.008,VH, CPA.4.008.VL, CPA.4.008.HC, CPA.4.008.LC, CPA.4.008.H1, CPA.4.008.H2,
  • the human monoclonal antibody has at least one heavy chain CDR and at least one light chain CDR of a human monoclonal anti-VSIGl Fab component selected from the group consisting of: CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011.
  • a human monoclonal anti-VSIGl Fab component selected from the group consisting of: CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011.
  • the human monoclonal antibody has at least two heavy chain CDRs and at least two light chain CDRs of a human monoclonal anti-VSIGl Fab component selected from the group consisting of: CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011..
  • the human monoclonal antibody contains variable regions that are homologous (and preferably identical) to the variable regions of a human monoclonal anti-VSIGl Fab component selected from the group consisting of: CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011.
  • a human monoclonal anti-VSIGl Fab component selected from the group consisting of: CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011.
  • variable regions are both the heavy and light chain variable regions. In some embodiments, the variable region is the heavy chain variable region. In some embodiments, the variable region is the light chain variable region.
  • the human monoclonal antibody has a heavy chain of a human monoclonal anti-VSIGl antibody selected from the group consisting of:
  • the human monoclonal antibody has a light chain of a human monoclonal anti-VSIGl antibody selected from the group consisting of:
  • the human monoclonal antibody competes for binding at an epitope located within the extracellular domain of VSIGl with a Fab domain selected from the group consisting of: CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011 (or the full length antibodies as well).
  • a Fab domain selected from the group consisting of: CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011 (or the full length antibodies as well).
  • the anti-VSIGl antibody that competes for binding at an epitope located within the extracellular domain of VSIGl is selected from the group consisting of CPA.4.008, CPA.4.023, CPA.4.005 and CPA.4.020.
  • the anti-VSIGl antibody that competes for binding at an epitope located within the extracellular domain of VSIGl is selected from the group consisting of CPA.4.013, CPA.4.031, CPA.4.033, CPA.4.009, and CPA.4.016.
  • the anti-VSIGl antibody that competes for binding at an epitope located within the extracellular domain of VSIGl is selected from the group consisting of CPA.4.017, CPA.4.019, CPA.4.027, CPA.4.011, and CPA.4.028.
  • the anti-VSIGl antibody that competes for binding at an epitope located within the extracellular domain of VSIGl is selected from the group consisting of CPA.4.012.
  • an anti-VSIGl antibody according to the invention comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to isolated anti- VSIGl amino acid sequences of preferred anti-VSIGl immune molecules, respectively, wherein the antibodies retain the desired functional properties of the parent anti-VSIGl antibodies.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol.
  • the protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences.
  • Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the percentage identity for comparison between VSIG1 antibodies is at least 75%, at least 80%, at least 90%, with at least about 95, 96, 97, 98 or 99% percent identity being preferred.
  • the percentage identity may be along the whole amino acid sequence, for example the entire heavy or light chain or along a portion of the chains.
  • included within the definition of the anti-VSIGl antibodies of the invention are those that share identity along the entire variable region (for example, where the identity is 95 or 98% identical along the variable regions), or along the entire constant region, or along just the Fc domain.
  • the VH exhibits at least 95% identity or at least 99% identity to a VH of an anti-VSIGl antibody as described herein.
  • the VL exhibits at least 95% identity or at least 99% identity to a VL of an anti-VSIGl antibody as described herein.
  • VSIG1 antibodies include those with CDRs identical to those shown in Figure 4 but whose identity along the variable region can be lower, for example 95 or 98% percent identical.
  • the anti-VSIGl antibodies of the invention bind to several different epitopes on human VSIG1, as shown in the examples. As is known in the art, antibodies binding to different epitopes are said to be in different "bins”. That is, antibodies are said to be in the same "bin” where the binding of one antibody blocks or competes with the binding of a different antibody.
  • the anti-VSIGl antibodies of the invention are recombinant.
  • Recombinant refers broadly with reference to a product, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non- recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • the anti-VSIGl antibody suitable for use in the antibody-drug conjugate compounds of the present invention binds to human VSIGl with a KD of 100 nM or less, 50 nM or less, 10 nM or less, or 1 nM or less (that is, higher binding affinity), or lpM or less, wherein K D is determined by known methods, e.g. surface plasmon resonance (SPR, e.g. Biacore assays), ELISA, KINEXA, and most typically SPR at 25° or 37° C.
  • SPR surface plasmon resonance
  • ELISA e.g. Biacore assays
  • the anti-VSIGl antibody or derivative thereof suitable for use in the antibody-drug conjugate compounds of the present invention include anti- VSIGl antibody or derivative thereof wherein the VH and VL sequences of different anti- VSIGl antibody or derivative thereof can be "mixed and matched" to create other anti-VSIGl antibody or derivative thereof according to at least some embodiments of the invention.
  • VSIGl binding of such "mixed and matched" antibodies can be tested using the binding assays described above, e.g., ELISAs).
  • a VH sequence from a particular VH/VL pairing is replaced with a structurally similar VH sequence.
  • a VL sequence from a particular VH/VL pairing is replaced with a structurally similar VL sequence.
  • the VH and VL sequences of homologous antibodies are particularly amenable for mixing and matching.
  • the antibody comprises CDR amino acid sequences selected from the group consisting of (a) sequences as listed herein; (b) sequences that differ from those CDR amino acid sequences specified in (a) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions except for the Serine residue in heavy chain CDR3 at position 100A (Kabat numbering system); (c) amino acid sequences having 90% or greater, 95% or greater, 98% or greater, or 99% or greater sequence identity to the sequences specified in (a) or (b); (d) a polypeptide having an amino acid sequence encoded by a polynucleotide having a nucleic acid sequence encoding the amino acids as listed herein.
  • Nucleic acid compositions encoding the anti-VSIGl antibodies of the invention are also provided, as well as expression vectors containing the nucleic acids and host cells transformed with the nucleic acid and/or expression vector compositions.
  • nucleic acid compositions that encode the VSIG1 antibodies will depend on the format of the antibody. For traditional, tetrameric antibodies containing two heavy chains and two light chains are encoded by two different nucleic acids, one encoding the heavy chain and one encoding the light chain. These can be put into a single expression vector or two expression vectors, as is known in the art, transformed into host cells, where they are expressed to form the antibodies of the invention. In some embodiments, for example when scFv constructs are used, a single nucleic acid encoding the variable heavy chain-linker-variable light chain is generally used, which can be inserted into an expression vector for transformation into host cells.
  • the nucleic acids can be put into expression vectors that contain the appropriate transcriptional and translational control sequences, including, but not limited to, signal and secretion sequences, regulatory sequences, promoters, origins of replication, selection genes, etc.
  • DNAs encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or gene synthesis) and the DNAs can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences.
  • operatively linked is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector.
  • the antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).
  • the light and heavy chain variable regions of the antibodies described herein can be used to create full- length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segments within the vector and the VL segment is operatively linked to the CL segment within the vector.
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell.
  • the antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
  • the recombinant expression vectors carry regulatory sequences that control the expression of the antibody chain genes in a host cell.
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes.
  • Such regulatory sequences are described, for example, in Goeddel ("Gene Expression Technology", Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)).
  • regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • nonviral regulatory sequences may be used, such as the ubiquitin promoter or ⁇ -globin promoter.
  • regulatory elements composed of sequences from different sources, such as the SR a. promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).
  • the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.).
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin or
  • selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • the expression vectors encoding the heavy and light chains is transfected into a host cell by standard techniques.
  • the various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • Preferred mammalian host cells for expressing the recombinant antibodies include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) o/. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells.
  • Chinese Hamster Ovary CHO cells
  • dhfr- CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220
  • a DHFR selectable marker e.g., as described in R. J. Kaufman and P. A. Sharp (1982) o/. Biol. 159:601-621
  • NSO myeloma cells
  • another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841.
  • the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.
  • Antibodies can be recovered from the culture medium using standard protein purification methods.
  • Antibody coding sequences of interest include those encoded by native sequences, as well as nucleic acids that, by virtue of the degeneracy of the genetic code, are not identical in sequence to the disclosed nucleic acids, and variants thereof.
  • Variant polypeptides can include amino acid substitutions as discussed herein. Techniques for in vitro mutagenesis of cloned genes are known. Also included in the subject invention are polypeptides that have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.
  • the invention further provides nucleic acids which encode an anti-VSIGl antibody according to the invention, or a fragment or conjugate thereof.
  • the nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • a nucleic acid is "isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column
  • a nucleic acid according to at least some embodiments of the invention can be, for example, DNA or RNA and may or may not contain intronic sequences.
  • VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).
  • a flexible linker e.g., encoding the amino acid sequence (Gly4-Ser)3
  • the present invention finds particular use in the antibody drug conjugate
  • ADC antibody-drug conjugates
  • ADCs are used in oncology applications, where the use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents allows for the targeted delivery of the drug moiety to tumors, which can allow higher efficacy, lower toxicity, etc.
  • An overview of this technology is provided in Ducry et al, Bioconjugate Chem, 21 :5-13 (2010), Carter et al., Cancer J. 14(3): 154 (2008) and Senter, Current Opin. Chem. Biol. 13:235-244 (2009), all of which are hereby incorporated by reference in their entirety.
  • the invention provides VISG1 antibodies conjugated to drug moieties.
  • conjugation is done by covalent attachment to the antibody, as further described below, and generally relies on a linker, often a peptide linkage (which, as described below, may be designed to be sensitive to cleavage by proteases at the target site or not).
  • Linkers used for attachment of drug moieties are referred to herein as "ADC linkers", to distinguish them from other linkers herein (although the peptide makeup of the linkers may be the same or different).
  • ADCs are formed using amino acid side chains within the antibody as attachment points for linker-drug components.
  • the number of drug moieties per antibody can change, depending on the conditions of the reaction, and can vary from 1 : 1 to 10: 1 drug: antibody, with from 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1 and 6: 1 finding particular use in some embodiments.
  • the actual number is an average, depending on the drug loading methodologies and specific techniques, as more fully described below.
  • the invention provides VSIG1 antibodies conjugated to drug moieties.
  • the drug of the ADC can be any number of agents, including but not limited to cytotoxic agents such as chemotherapeutic agents, growth inhibitory agents, toxins (for example, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (that is, a radioconjugate) are provided.
  • cytotoxic agents such as chemotherapeutic agents, growth inhibitory agents, toxins (for example, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (that is, a radioconjugate) are provided.
  • toxins for example, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof
  • a radioactive isotope that is, a radioconjugate
  • Drugs for use in the present invention include cytotoxic drugs, particularly those which are used for cancer therapy.
  • cytotoxic drugs include, in general, DNA damaging agents, anti-metabolites, natural products and their analogs.
  • exemplary classes of cytotoxic agents include the enzyme inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNA cleavers, topoisomerase inhibitors, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, the podophyllotoxins, dolastatins, maytansinoids, differentiation inducers, and taxols.
  • enzyme inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNA cleavers, topoisomerase inhibitors, the anth
  • Members of these classes include, for example, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxanes including taxol, taxotere retinoic acid, butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin, esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin, camptothecin, maytansin
  • Toxins may be used as antibody-toxin conjugates and include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as
  • geldanamycin (Mandler et al (2000) J. Nat. Cancer Inst. 92(19): 1573-1581 ; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10: 1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al, (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). Toxins may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.
  • Conjugates of a VSIG1 antibody and one or more small molecule toxins such as a maytansinoids, dolastatins, auristatins, a trichothecene, calicheamicin, and CC1065, and the derivatives of these toxins that have toxin activity, are contemplated.
  • the drug moiety for attachment to the anti-VSIGl antibody is a maytansinoid compound.
  • Maytansine compounds are highly potent microtubule targeting compounds suitable for use as maytansinoid ADC moieties, and can be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al (2002) PNAS 99:7968-7973), or maytansinol and maytansinol analogues prepared synthetically according to known methods.
  • drugs may be modified by the incorporation of a functionally active group such as a thiol or amine group for conjugation to the antibody.
  • Exemplary maytansinoid drug moieties include those having a modified aromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by lithium aluminum hydride reduction of ansamytocin P2); C-20-hydroxy (or C-20-demethyl) +/-C-19- dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and C-20-demethoxy, C-20- acyloxy (--OCOR), +/-dechloro (U.S. Pat. No. 4,294,757) (prepared by acylation using acyl chlorides) and those having modifications at other positions.
  • C-19-dechloro U.S. Pat. No. 4,256,746
  • C-20-hydroxy (or C-20-demethyl) +/-C-19- dechloro U.S. Pat. No
  • Exemplary maytansinoid drug moieties also include those having
  • DM1 Disclosed in US Patent No. 5,208,020, incorporated by reference
  • DM4 Dislosed in US Patent No. 7,276,497, incorporated by reference
  • ADCs containing maytansinoids, methods of making same, and their therapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020; 5,416,064; 6,441,163 and European Patent EP 0 425 235 Bl, the disclosures of which are hereby expressly incorporated by reference.
  • Liu et al, Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described ADCs comprising a maytansinoid designated DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer.
  • the conjugate was found to be highly cytotoxic towards cultured colon cancer cells, and showed antitumor activity in an in vivo tumor growth assay.
  • ADCs Chari et al, Cancer Research 52: 127-131 (1992) describe ADCs in which a maytansinoid was conjugated via a disulfide linker to the murine antibody A7 binding to an antigen on human colon cancer cell lines, or to another murine monoclonal antibody TA.1 that binds the HER-2/neu oncogene.
  • the cytotoxicity of the TA. l-maytansonoid conjugate was tested in vitro on the human breast cancer cell line SK-BR-3, which expresses 3x105 HER-2 surface antigens per cell.
  • the drug conjugate achieved a degree of cytotoxicity similar to the free maytansinoid drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule.
  • the A7-maytansinoid conjugate showed low systemic cytotoxicity in mice.
  • the ADC comprises a VSIG1 antibody conjugated to dolastatins or dolostatin peptidic analogs and derivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).
  • Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob. Agents Chemother.
  • the dolastatin or auristatin drug moiety may be attached to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02/088172).
  • Exemplary auristatin embodiments include the N-terminus linked
  • An exemplary auristatin embodiment is MMAE (shown in Figure 10 wherein the wavy line indicates the covalent attachment to a linker (L) of an antibody drug conjugate; see US Patent No. 6,884,869 expressly incorporated by reference in its entirety).
  • MMAF Another exemplary auristatin embodiment is MMAF, shown in Figure 10 wherein the wavy line indicates the covalent attachment to a linker (L) of an antibody drug conjugate (US 2005/0238649, 5,767,237 and 6,124,431, expressly incorporated by reference in their entirety):
  • peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments.
  • Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schroder and K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry.
  • the auristatin/dolastatin drug moieties may be prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc.
  • the ADC comprises an antibody of the invention conjugated to one or more calicheamicin molecules.
  • Mylotarg is the first commercial ADC drug and utilizes calicheamicin ⁇ as the pay load (see US Patent No. 4,970,198, incorporated by reference in its entirety). Additional calicheamicin derivatives are described in US Patent Nos. 5,264,586, 5,384,412, 5,550,246, 5,739,116, 5,773,001, 5,767,285 and 5,877,296, all expressly incorporated by reference.
  • the calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations.
  • Structural analogues of calicheamicin which may be used include, but are not limited to, ⁇ , ⁇ 2 ⁇ , ⁇ 2 ⁇ , N-acetyl- ⁇ , PSAG and ⁇ 1 (Hinman et al, Cancer Research 53:3336-3342 (1993), Lode et al, Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patents to American Cyanamid).
  • QFA Another anti-tumor drug that the antibody can be conjugated is QFA which is an antifolate.
  • QFA is an antifolate.
  • calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects.
  • duocarmycin is members of a family of antitumor antibiotics utilized in ADCs. These antibiotics appear to work through sequence-selectively alkylating DNA at the N3 of adenine in the minor groove, which initiates a cascade of events that result in apoptosis.
  • duocarmycin herein is meant duocarmycin and analogs.
  • duocarmycin A US Patent
  • WO2009/017394A1 5,703,080, 6,989,452, 7,087,600, 7,129,261, 7,498,302, and 7,507,420, all of which are expressly incorporated by reference, in particular for the structure of the duocarmycin.
  • the antibody conjugates according to at least some embodiments of the invention can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may also include, for example, a protein such as tumor necrosis factor or interferon- ⁇ ; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAP II, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published Oct. 28, 1993.
  • the present invention further contemplates an ADC formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA
  • endonuclease such as a deoxyribonuclease; DNase).
  • the antibody may comprise a highly radioactive atom.
  • radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32, Pb212 and radioactive isotopes of Lu.
  • the radio- or other labels may be incorporated in the conjugate in known ways.
  • the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen.
  • Labels such as Tc99m or 1123, Rel86, Rel88 and Inl l l can be attached via a cysteine residue in the peptide.
  • Yttrium-90 can be attached via a lysine residue.
  • the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate Iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.
  • p is a certain value from Antibody-Drug- Conjugates with other drug loadings
  • separation, purification, and characterization of homogeneous Antibody-Drug-conjugates where p is a certain value from Antibody-Drug- Conjugates with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.
  • p is 2, 3, 4, 5, 6, 7, or 8 or a fraction thereof.
  • a number of different reactions are available for covalent attachment of drugs and/or linkers to binding agents.
  • a commonly used nonspecific methods of covalent attachment is the carbodiimide reaction to link a carboxy (or amino) group of a compound to amino (or carboxy) groups of the antibody.
  • bifunctional agents such as dialdehydes or imidoesters have been used to link the amino group of a compound to amino groups of an antibody molecule.
  • an intermediate which is the precursor of the linker, is reacted with the drug under appropriate conditions.
  • reactive groups are used on the drug and/or the intermediate.
  • the product of the reaction between the drug and the intermediate, or the derivatized drug, is subsequently reacted with an Fc variant antibody of the invention under appropriate conditions.
  • the antibody-drug conjugate compounds comprise a linker unit between the drug unit and the antibody unit.
  • the antibody causes the ADC to bind to the target cancer cells.
  • the ADC is then internalized by the cell and the drug is released into the cell. Because of the targeting, the side effects are lower and give a wider therapeutic window.
  • Hydrophilic linkers e.g., PEG4Mal
  • MDR multiple drug resistance
  • the linker is cleavable under intracellular or extracellular conditions, such that cleavage of the linker releases the drug unit from the antibody in the appropriate environment.
  • solid tumors that secrete certain proteases may serve as the target of the cleavable linker; in other embodiments, it is the intracellular proteases that are utilized.
  • the linker unit is not cleavable and the drug is released, for example, by antibody degradation in lysosomes.
  • the linker is cleavable by a cleaving agent that is present in the intracellular environment (for example, within a lysosome or endosome or caveolea).
  • the linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease.
  • the peptidyl linker is at least two amino acids long or at least three amino acids long or more.
  • linker types that have been used to conjugate a drug moiety to an antibody include, but are not limited to, polyethylene glycol, hydrazones, thioethers, esters, disulfides and peptide-containing linkers.
  • a linker can be chosen that is, for example, susceptible to cleavage by low pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).
  • a cleavable linker is typically susceptible to cleavage under intracellular conditions.
  • the linker can be any suitable linker, such as, for example, a peptidyl, hydrazine, or disulfide linker.
  • the linker prevents the drug moiety from being removed from the cancer cell.
  • the choice of the linker enhances the plasma stability of the antibody-drug conjugate, preventing unwanted release of the drug at non-tumor sites. In some embodiments, the choice of linker reduces the risk of impairing the affinity of the antibody to VSIG1.
  • Cleaving agents can include, without limitation, cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123).
  • a peptidyl linker that is cleavable by the thiol- dependent protease cathepsin-B which is highly expressed in cancerous tissue, can be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Gly linker (SEQ ID NO: 11).
  • Other examples of such linkers are described, e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference in its entirety and for all purposes.
  • the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the val-cit linker).
  • the cleavable linker is pH-sensitive, that is, sensitive to hydrolysis at certain pH values.
  • the pH-sensitive linker hydrolyzable under acidic conditions.
  • an acid-labile linker that is hydrolyzable in the lysosome for example, a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like
  • an acid-labile linker that is hydrolyzable in the lysosome for example, a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like.
  • the hydrolyzable linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via an
  • the linker is cleavable under reducing conditions
  • disulfide linker for example, a disulfide linker
  • disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2- pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2- pyridyl-dithio)toluene)- , SPDB and SMPT.
  • SATA N-succinimidyl-5-acetylthioacetate
  • SPDP N-succinimidyl-3-(2-pyridyldithio)propionate
  • SPDB N-succinimidyl-3-(2-
  • the linker is a malonate linker (Johnson et al, 1995,
  • Anticancer Res. 15: 1387-93 a maleimidobenzoyl linker (Lau et al, 1995, Bioorg-Med- Chem. 3(10): 1299-1304), or a 3'-N-amide analog (Lau et al, 1995, Bioorg-Med-Chem. 3(10): 1305-12).
  • the linker unit is not cleavable and the drug is released by antibody degradation. (See U.S. Publication No. 2005/0238649 incorporated by reference herein in its entirety and for all purposes).
  • the linker is self-immolative.
  • self-immolative spacer refers to a bifunctional chemical moiety that is capable of covalently linking together two spaced chemical moieties into a stable tripartite molecule. It will spontaneously separate from the second chemical moiety if its bond to the first moiety is cleaved.
  • WO 2007059404A2, WO06110476A2, WO05112919A2, WO2010/062171, WO09/017394, WO07/089149, WO 07/018431, WO04/043493 and WO02/083180 which are directed to drug-cleavable substrate conjugates where the drug and cleavable substrate are optionally linked through a self-immolative linker and which are all expressly incorporated by reference.
  • these can also be called "self elimination spacers" or "self elimination connectors", the idea being that upon cleavage by a tumor-associated protease or enzyme, the linker then self-eliminates to release the cytotoxic drug.
  • SpaceLink platform of Syntarga, as described in US Patent No. 7,223,837, hereby incorporated by reference in its entirety, and particularly for the exemplification of these linkers and structures, the associated description therein.
  • linker is not substantially sensitive to the extracellular environment.
  • linker not substantially sensitive to the extracellular environment, in the context of a linker, means that no more than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of the linkers, in a sample of antibody-drug conjugate compound, are cleaved when the antibody-drug conjugate compound presents in an extracellular environment (for example, in plasma).
  • Whether a linker is not substantially sensitive to the extracellular environment can be determined, for example, by incubating with plasma the antibody-drug conjugate compound for a predetermined time period (for example, 2, 4, 8, 16, or 24 hours) and then quantitating the amount of free drug present in the plasma.
  • the linker promotes cellular internalization.
  • the linker promotes cellular internalization when conjugated to the therapeutic agent (that is, in the milieu of the linker-therapeutic agent moiety of the antibody-drug conjugate compound as described herein).
  • the linker promotes cellular internalization when conjugated to both the auristatin compound and the Fc variant antibodies of the invention.
  • the linker has multiple purposes, including increasing drug solubility and pharmacokinetics, as well as increased drug load, such as the "Fleximer” polymer of Mersana Therapeutics; see Yurkovetskiy et al, Cancer Research, doi
  • the drug loading is represented by p, the average number of drug molecules per antibody.
  • Drug loading may range from 1 to 20 drugs (D) per Antibody.
  • the average number of drugs per antibody in preparation of conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC.
  • the quantitative distribution of Antibody- Drug-Conjugates in terms of p may also be determined.
  • Drug loading may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
  • ADCs of the invention include collections of antibodies conjugated with a range of drug moieties, from 1 to 20.
  • the average number of drug moieties per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as mass spectroscopy and, ELISA assay.
  • ADC quantitative distribution of ADC in terms of p may also be determined.
  • separation, purification, and characterization of homogeneous ADC where p is a certain value from ADC with other drug loadings may be achieved by means such as electrophoresis.
  • p may be limited by the number of attachment sites on the antibody.
  • an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached.
  • higher drug loading e.g. p>5
  • the drug loading for an ADC of the invention ranges from 1 to about 8; from about 2 to about 6; from about 3 to about 5; from about 3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8; from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3 to about 3.8; or from about 3.3 to about 3.7.
  • the optimal ratio of drug moieties per antibody may be less than 8, and may be about 2 to about 5. See US 2005-0238649 Al (herein incorporated by reference in its entirety).
  • an antibody may contain, for example, lysine residues that do not react with the drug-linker intermediate or linker reagent. Generally, antibodies do not contain many free and reactive cysteine thiol groups which may be linked to a drug moiety; indeed most cysteine thiol residues in antibodies exist as disulfide bridges.
  • an antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups.
  • DTT dithiothreitol
  • TCEP tricarbonylethylphosphine
  • an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine.
  • the loading (drug/antibody ratio) of an ADC may be controlled in different ways, e.g., by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reductive conditions for cysteine thiol modification, (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of cysteine residues is modified for control of the number and/or position of linker-drug attachements (such as thioMab or thioFab prepared as disclosed herein and in
  • the resulting product is a mixture of ADC compounds with a distribution of one or more drug moieties attached to an antibody.
  • the average number of drugs per antibody may be calculated from the mixture by a dual ELIS A antibody assay, which is specific for antibody and specific for the drug.
  • Individual ADC molecules may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction chromatography.
  • a homogeneous ADC with a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography.
  • cytotoxic or cytostatic activity of an ADC can be measured by: exposing mammalian cells expressing a target protein of the ADC in a cell culture medium; culturing the cells for a period from about 6 hours to about 5 days; and measuring cell viability.
  • Cell-based in vitro assays can be used to measure viability
  • a thymidine incorporation assay may be used.
  • cancer cells expressing a target antigen at a density of 5,000 cells/well of a 96-well plated can be cultured for a 72-hour period and exposed to 0.5 of H-thymidine during the final 8 hours of the 72-hour period.
  • the incorporation of H-thymidine into cells of the culture is measured in the presence and absence of the ADC.
  • necrosis or apoptosis (programmed cell death) can be measured.
  • necrosis is typically accompanied by increased permeability of the plasma membrane; swelling of the cell, and rupture of the plasma membrane.
  • Apoptosis is typically characterized by membrane blebbing, condensation of cytoplasm, and the activation of endogenous endonucleases. Determination of any of these effects on cancer cells indicates that an ADC is useful in the treatment of cancers.
  • Cell viability can be measured by determining in a cell the uptake of a dye such as neutral red, trypan blue, or ALAMARTM blue (see, e.g., Page et al, 1993, Intl. J. Oncology 3:473-476).
  • a dye such as neutral red, trypan blue, or ALAMARTM blue
  • the cells are incubated in media containing the dye, the cells are washed, and the remaining dye, reflecting cellular uptake of the dye, is measured spectrophotometrically.
  • the protein-binding dye sulforhodamine B (SRB) can also be used to measure cytoxicity (Skehan et al, 1990, J. Natl. Cancer Inst. 82: 1107-12).
  • a tetrazolium salt such as MTT
  • MTT a tetrazolium salt
  • Apoptosis can be quantitated by measuring, for example, DNA fragmentation.
  • Apoptosis can also be determined by measuring morphological changes in a cell. For example, as with necrosis, loss of plasma membrane integrity can be determined by measuring uptake of certain dyes (e.g., a fluorescent dye such as, for example, acridine orange or ethidium bromide).
  • a fluorescent dye such as, for example, acridine orange or ethidium bromide.
  • Cells also can be labeled with a DNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide) and the cells observed for chromatin condensation and margination along the inner nuclear membrane.
  • a DNA dye e.g., acridine orange, ethidium bromide, or propidium iodide
  • Other morphological changes that can be measured to determine apoptosis include, e.g., cytoplasmic condensation, increased membrane blebbing, and cellular shrinkage.
  • the presence of apoptotic cells can be measured in both the attached and "floating" compartments of the cultures.
  • both compartments can be collected by removing the supernatant, trypsinizing the attached cells, combining the preparations following a centrifugation wash step (e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., by measuring DNA fragmentation).
  • a centrifugation wash step e.g. 10 minutes at 2000 rpm
  • detecting apoptosis e.g., by measuring DNA fragmentation.
  • a therapeutic composition of the VSIGl antibody of the invention can be evaluated in a suitable animal model.
  • xenogenic cancer models can be used, wherein cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Klein et al, 1997, Nature Medicine 3: 402-408). Efficacy can be measured using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.
  • anti-VSIGl antibodies of the invention are used to treat certain cancers.
  • cancer cells express VSIGl at a sufficient level and said cancer is as described herein, wherein VSIGl expression on any the cancer cells could be either present prior to cancer treatment or induced post treatment.
  • expressing VSIGl at a sufficient level it is meant that such cells express VSIGl protein at a high enough level according to an assay.
  • the assay is IHC (immunohistochemistry), and expression is measured on a scale of 0 to 3 (0-no expression, 1- faint staining, 2-moderate and 3-strong expression), then a sufficient level of VSIGl expression would optionally be at least 1, alternatively be at least 2, alternatively be at least 3.
  • the antibody-drug conjugate compounds as described herein may be used for such an assay.
  • an antibody-drug conjugate according to the present invention is used for the treatment and/or diagnosis of cancer, wherein the cancer, and/or immune cells infiltrating the cancer, and/or stromal cells of the subject express VSIGl, e.g. prior to, or following cancer therapy, and wherein said cancer is e.g., selected from the group consisting of stomach cancer, ovarian cancer, liver cancer and prostate cancer, as shown in the IHC experiments as outlined herein.
  • the anti-VSIGl antibodies provided also find use in the in vitro or in vivo imaging of tumors or autoimmune disease states associated with VSIGl . In some
  • the antibodies described herein are used for both diagnosis and treatment, or for diagnosis alone.
  • anti-VSIGl antibodies are used for both diagnosis and treatment, some embodiments rely on two different anti-VSIGl antibodies to two different epitopes, such that the diagnostic antibody does not compete for binding with the therapeutic antibody, although in some cases the same antibody can be used for both.
  • compositions comprising a diagnostic antibody and a therapeutic antibody, and in some embodiments, the diagnostic antibody is labeled as described herein.
  • the composition of therapeutic and diagnostic antibodies can also be co-administered with other drugs as outlined herein.
  • Particularly useful antibodies for use in diagnosis include, but are not limited to, those encompassing the following Fabs: CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011, and particularly the ".HI" constructs as described herein.
  • a diagnostic antibody is labeled.
  • labeled herein is meant that the antibodies disclosed herein have one or more elements, isotopes, or chemical compounds attached to enable the detection in a screen or diagnostic procedure.
  • labels fall into several classes: a) immune labels, which may be an epitope incorporated as a fusion partner that is recognized by an antibody, b) isotopic labels, which may be radioactive or heavy isotopes, c) small molecule labels, which may include fluorescent and colorimetric dyes, or molecules such as biotin that enable other labeling methods, and d) labels such as particles (including bubbles for ultrasound labeling) or paramagnetic labels that allow body imagining. Labels may be incorporated into the antibodies at any position and may be incorporated in vitro or in vivo during protein expression, as is known in the art.
  • Diagnosis can be done either in vivo, by administration of a diagnostic antibody that allows whole body imaging as described below, or in vitro, on samples removed from a patient.
  • sample in this context includes any number of things, including, but not limited to, bodily fluids (including, but not limited to, blood, urine, serum, lymph, saliva, anal and vaginal secretions, perspiration and semen), as well as tissue samples such as result from biopsies of relevant tissues.
  • in vivo imaging is done, including but not limited to ultrasound, CT scans, X-rays, MRI and PET scans, as well as optical techniques, such as those using optical labels for tumors near the surface of the body.
  • In vivo imaging of diseases associated with VSIG1 may be performed by any suitable technique.
  • 99Tc-labeling or labeling with another .beta. -ray emitting isotope may be used to label anti-VSIGl antibodies.
  • Variations on this technique may include the use of magnetic resonance imaging (MRI) to improve imaging over gamma camera techniques.
  • MRI magnetic resonance imaging
  • the present invention provides an in vivo imaging method wherein an anti-VSIGl antibody is conjugated to a detection-promoting agent, the conjugated antibody is administered to a host, such as by injection into the bloodstream, and the presence and location of the labeled antibody in the host is assayed.
  • the present invention provides a method for screening for the presence of disease-related cells in a human patient or a biological sample taken from a human patient.
  • radioisotopes may be bound to an anti-VSIGl antibody either directly, or indirectly by using an intermediary functional group.
  • intermediary functional groups include chelators, such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid (see for instance U.S. Pat. No. 5,057,313), in such diagnostic assays involving radioisotope-conjugated anti-VSIGl antibodies, the dosage of conjugated anti-VSIGl antibody delivered to the patient typically is maintained at as low a level as possible through the choice of isotope for the best combination of minimum half-life, minimum retention in the body, and minimum quantity of isotope, which will permit detection and accurate measurement.
  • diagnostic methods may be performed using anti-VSIGl antibodies that are conjugated to dyes (such as with the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules and enhancing agents (e.g. paramagnetic ions) for magnetic resonance imaging (MRI) (see, e.g., U.S. Pat. No. 6,331,175, which describes MRI techniques and the preparation of antibodies conjugated to a MRI enhancing agent).
  • dyes such as with the biotin-streptavidin complex
  • contrast agents e.g. paramagnetic ions
  • fluorescent compounds or molecules e.g. paramagnetic ions
  • enhancing agents e.g. paramagnetic ions
  • Such diagnostic/detection agents may be selected from agents for use in magnetic resonance imaging, and fluorescent compounds.
  • a reagent having a long tail to which are attached a multiplicity of chelating groups for binding the ions.
  • a tail may be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which can be bound chelating groups such as, e.g., porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes, and like groups known to be useful for this purpose.
  • Chelates may be coupled to anti-VSIGl antibodies using standard chemistries.
  • a chelate is normally linked to an anti-VSIGl antibody by a group that enables formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and/or internal cross-linking.
  • Examples of potentially useful metal-chelate combinations include 2-benzyl-
  • DTPA and its monomethyl and cyclohexyl analogs used with diagnostic isotopes in the general energy range of 60 to 4,000 keV, such as 125 I, 12 I, 124 I, 62 Cu, 64 Cu, 18 F, m In, 67 Ga, 99 Tc, 94 TC, n C, 1 N, 5 0, and 76 Br, for radio-imaging.
  • Labels include a radionuclide, a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photoactive agent.
  • diagnostic agents are well known and any such known diagnostic agent may be used.
  • Non-limiting examples of diagnostic agents may include a radionuclide such as HOIn, l l lln, 177Lu, 18F, 52Fe, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 86Y, 90Y, 89Zr, 94mTc, 94Tc, 99mTc, 1201, 1231, 1241, 1251, 1311, 154-158Gd, 32P, 11C, 13N, 150, 186Re, 188Re, 51Mn, 52mMn, 55Co, 72As, 75Br, 76Br, 82mRb, 83Sr, or other .gamma.-, .beta.-, or positron-emitters.
  • a radionuclide such as HOIn, l l lln, 177Lu, 18F, 52Fe, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 86Y,
  • Paramagnetic ions of use may include chromium (III), manganese (II), iron
  • Ultrasound contrast agents may comprise liposomes, such as gas filled liposomes. Radiopaque diagnostic agents may be selected from compounds, barium compounds, gallium compounds, and thallium compounds.
  • chelates when complexed with non-radioactive metals, such as manganese, iron, and gadolinium may be useful for MRI diagnostic methods in connection with anti-VSIGl antibodies.
  • Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with a variety of metals and radiometals, most particularly with
  • radionuclides of gallium, yttrium, and copper may be made very stable by tailoring the ring size to the metal of interest.
  • Other ring-type chelates such as macrocyclic poly ethers, which are of interest for stably binding nuclides, such as 223Ra may also be suitable in diagnostic methods.
  • the present invention provides diagnostic anti-VSIGl antibody conjugates, wherein the anti-VSIGl antibody conjugate is conjugated to a contrast agent (such as for magnetic resonance imaging, computed tomography, or ultrasound contrast- enhancing agent) or a radionuclide that may be, for example, a .gamma.-, .beta.-, .alpha.-, Auger electron-, or positron-emitting isotope.
  • a contrast agent such as for magnetic resonance imaging, computed tomography, or ultrasound contrast- enhancing agent
  • a radionuclide that may be, for example, a .gamma.-, .beta.-, .alpha.-, Auger electron-, or positron-emitting isotope.
  • Anti-VSIGl antibodies may also be useful in, for example, detecting expression of an antigen of interest in specific cells, tissues, or serum.
  • the antibody typically will be labeled with a detectable moiety for in vitro assays.
  • suitable labels for use in in vitro testing.
  • Suitable dyes for use in this aspect of the invention include, but are not limited to, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl- coumarins, quantum dots (also referred to as "nanocrystals"; see U.S. Ser. No.
  • Stained tissues may then be assessed for radioactivity counting as an indicator of the amount of VSIGl -associated peptides in the tumor.
  • the images obtained by the use of such techniques may be used to assess biodistribution of VSIGl in a patient, mammal, or tissue, for example in the context of using VSIG1 as a biomarker for the presence of invasive cancer cells.
  • compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method.
  • Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16 th Edition, A. Osal., Ed., 1980).
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, acetate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, hist
  • coloring agents such as coloring agents; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
  • salt-forming counter-ions such as sodium
  • metal complexes e.g. Zn-protein complexes
  • non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
  • the pharmaceutical composition that comprises the antibodies of the invention may be in a water-soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, gly colic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • the formulations to be used for in vivo administration are preferrably sterile. This is readily accomplished by filtration through sterile filtration membranes or other methods.
  • Administration of the pharmaceutical composition comprising antibodies of the present invention may be done in a variety of ways, including, but not limited to subcutaneously and intravenously.
  • Subcutaneous administration may be preferable in some circumstances because the patient may self-administer the pharmaceutical composition.
  • Many protein therapeutics are not sufficiently potent to allow for formulation of a therapeutically effective dose in the maximum acceptable volume for subcutaneous administration. This problem may be addressed in part by the use of protein formulations comprising arginine-HCl, histidine, and polysorbate (see WO 04091658).
  • Fc polypeptides of the present invention may be more amenable to subcutaneous administration due to, for example, increased potency, improved serum half-life, or enhanced solubility.
  • protein therapeutics are often delivered by IV infusion or bolus.
  • the antibodies of the present invention may also be delivered using such methods.
  • administration may venious be by intravenous infusion with 0.9% sodium chloride as an infusion vehicle.
  • any of a number of delivery systems are known in the art and may be used to administer the Fc variants of the present invention. Examples include, but are not limited to, encapsulation in liposomes, microparticles, microspheres (eg. PLA/PGA microspheres), and the like.
  • an implant of a porous, non-porous, or gelatinous material, including membranes or fibers may be used.
  • Sustained release systems may comprise a polymeric material or matrix such as polyesters, hydrogels, poly(vinylalcohol), polylactides, copolymers of L-glutamic acid and ethyl-L-gutamate, ethylene-vinyl acetate, lactic acid-gly colic acid copolymers such as the LUPRON DEPOT.RTM., and poly-D-(-)-3- hydroxyburyric acid.
  • the antibodies disclosed herein may also be formulated as
  • a liposome is a small vesicle comprising various types of lipids, phospholipids and/or surfactant that is useful for delivery of a therapeutic agent to a mammal.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al, 1985, Proc Natl Acad Sci USA, 82:3688; Hwang et al, 1980, Proc Natl Acad Sci USA, 77:4030; U.S. Pat. No. 4,485,045; U.S. Pat. No. 4,544,545; and PCT WO 97/38731.
  • Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE).
  • PEG-PE PEG-derivatized phosphatidylethanolamine
  • Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • a chemotherapeutic agent or other therapeutically active agent is optionally contained within the liposome (Gabizon et al, 1989, J National Cancer Inst 81 : 1484).
  • the antibodies may also be entrapped in microcapsules prepared by methods including but not limited to coacervation techniques, interfacial polymerization (for example using hydroxymethylcellulose or gelatin-mi crocapsules, or poly-(methylmethacylate) microcapsules), colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), and macroemulsions.
  • coacervation techniques for example using hydroxymethylcellulose or gelatin-mi crocapsules, or poly-(methylmethacylate) microcapsules
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • macroemulsions for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared.
  • sustained-release preparations include semipermeable matrices of solid hydrophobic polymer, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid- glycolic acid copolymers such as the LUPRON DEPOT.RTM.
  • the dosing amounts and frequencies of administration are, in a preferred embodiment, selected to be therapeutically or prophylactically effective.
  • adjustments for protein degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
  • the concentration of the antibody in the formulation may vary from about 0.1 to 100 weight %. In a preferred embodiment, the concentration of the Fc variant is in the range of 0.003 to 1.0 molar.
  • a therapeutically effective dose of the Fc variant of the present invention may be administered.
  • therapeutically effective dose herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. Dosages may range from 0.0001 to 100 mg/kg of body weight or greater, for example 0.1, 1, 10, or 50 mg/kg of body weight, with 1 to 10 mg/kg being preferred.
  • the antibodies of the present invention may be administered concomitantly with one or more other therapeutic regimens or agents.
  • the additional therapeutic regimes or agents may be used to improve the efficacy or safety of the anti-VSIGl antibody.
  • the additional therapeutic regimes or agents may be used to treat the same disease or a comorbidity rather than to alter the action of the VSIG1 antibody.
  • a VSIG1 antibody of the present invention may be administered to the patient along with
  • the antibodies of the present invention may be administered in combination with one or more other prophylactic or therapeutic agents, including but not limited to cytotoxic agents,
  • chemotherapeutic agents include cytokines, growth inhibitory agents, anti-hormonal agents, kinase inhibitors, anti-angiogenic agents, cardioprotectants, immunostimulatory agents,
  • immunosuppressive agents agents that promote proliferation of hematological cells, angiogenesis inhibitors, protein tyrosine kinase (PTK) inhibitors, or other therapeutic agents.
  • angiogenesis inhibitors agents that promote proliferation of hematological cells
  • protein tyrosine kinase (PTK) inhibitors or other therapeutic agents.
  • the terms "in combination with” and “co-administration” are not limited to the administration of said prophylactic or therapeutic agents at exactly the same time. Instead, it is meant that the anti-VSIGl antibody and the other agent or agents are administered in a sequence and within a time interval such that they may act together to provide a benefit that is increased versus treatment with only either anti-VSIGl antibody of the present invention or the other agent or agents. It is preferred that the anti-VSIGl antibody and the other agent or agents act additively, and especially preferred that they act synergistically. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The skilled medical practitioner can determine empirically, or by considering the
  • TMA comprised 11 tissue types: breast, colon, lymphoid and prostate (8 tumor and 2 normal samples of each), gastric, ovary, brain, kidney, liver and skin (4 tumor and 2 normal samples of each), and lung (8 non-small cell tumor and 4 small cell tumor samples, and 4 normal lung samples).
  • Antigen retrieval and staining The sections were de-paraffinized; antigen retrieved and rehydrated using pH9.0 Flex + 3-in- 1 antigen retrieval buffers, in PT Link apparatus at 95°C for 20 min with automatic heating and cooling.
  • the negative control sections were incubated with non-immune rabbit IgG antibodies (Alere #X0936032) at ⁇ g/ml or in antibody diluent alone ('no primary' control).
  • Anti Von Willebrand's Factor (vWF) antibody was applied at ⁇ g/ml.
  • the negative control sections were incubated with non-immune rabbit IgG antibodies (Dako, CAT #0936) at 3 and ⁇ g/ml or in DAKO Envision Flex antibody diluent ('no primary' control).
  • the sections were then rinsed twice in FLEX buffer, incubated with anti-mouse/ rabbit Flex + HRP for 20 min, rinsed twice in FLEX buffer and then incubated with diaminobenzidine (DAB) substrate for 10 min.
  • DAB diaminobenzidine
  • Results The sections were analyzed for the intensity of the specific staining and a semi-quantitative scoring system was used.
  • the core in the tissue array with the most intense VSIG1 (Sigma Aldrich pAb Cat# HPA036311) -ir was assigned a score of 3+ and the intensities of the immunoreactivity in the other cores were scored relative to that of the 3+ core.
  • the percentage of VSIGl-ir tumor was estimated and recorded using the following ranges: 0-25%, 25-50%, 50-75% and 75-100%. Where scoring was too low to quantitate - an assigned '+' was used to denote the presence of staining.
  • the intracellular distribution of the immune-stained cells in the tumor was also recorded.
  • Anti VSIG1 pAb was tested in positive and negative control HEK-293 cell lines and in a positive control tissue (stomach fundus).
  • the optimal IHC conditions for detection of the target determined in that calibration study (heat mediated antigen retrieval (AR) at pH 6.1 and anti-VSIGl antibody applied at ⁇ g/ml) were used for the current 'Multitumour' TMA (Asternad, UK) study and the TOP4 (Asternad, UK) study.
  • Multitumor TMA results VSIGl-ir was noted in nuclei, cytoplasm or plasma membranes with some samples exhibiting either both nuclear-cytoplasmic or plasma membrane-cytoplasmic staining. The most intense VSIGl-ir was observed in liver tumor and the least intense VSIG1 -ir being observed in the breast tumor. Weak VSIG1 -ir was also observed in the majority of the ovary tumor samples immunostaining less than 25% of the tumour cells, with exception to one sample - showing strong VSIGl-ir in 75-100% of the tumor cells. Only one of the five breast tumors analyzed was immunostained where ⁇ 25% of the tumor was immunoreactive . The other four samples were negative.
  • the most consistent tissue type was liver having consistently high levels of VSIGl-ir within the tumor cells however the percentage of tumor cells immunostained varied between all four cores from 0-25% and 75%-100%. No other correlation between staining intensity and tumor differentiation was apparent.
  • Stomach tumors were the most inconsistent tissue type - having varying staining patterns including nuclear, plasma membrane, cytoplasmic, to plasma membrane-cytoplasmic.
  • the staining intensity of VSIG1 also varied from weak to strong in generally less than 50% of the tumor cells.
  • Normal tissues also showed moderate to strong VSIGl expression in the liver within the hepatocytes, and stomach within the crypts and surface epithelium, which was expected due to the positive control tissue being stomach fundus. No VSIGl-ir was observed in the normal breast, lung and ovary tissues. Overall, the expression of VSIGl was variable in both intensity and percentage of tumor cells stained both within and between tumor types.
  • TOP 4 TMA results In this study, sections of Asterand's Top4 TMA were examined for the presence of VSIGl-ir using antibody HPA036311. The results focus on the breast and lung tissue only, as in these tissues demonstrated clear over expression over normal tissue. Data from the other tumors represented on the array included in Figure 27. In normal breast and lung tissues very little VSIGl-ir was present. VSIGl-ir was present to some degree in the majority of breast and lung tumors tested, although the intensity of immunoreactivity was generally weak. Grade II adenocarcinomas of the breast included some examples of strong VSIGl-ir, while Grade III breast adenocarcinomas only exhibited weak immunoreactivity.
  • MED is a proprietary software platform for collection of public gene- expression data, normalization, annotation and performance of various queries.
  • Expression data from the most widely used Affymetrix microarrays is downloaded from the Gene Expression Omnibus (GEO - www.ncbi . nlm. nih. gov/ GEO) .
  • Experiments are annotated, first automatically, and then manually, to identify tissue and condition, and chips are grouped according to this annotation, and cross verification of this grouping by comparing the overall expression pattern of the genes of each chip to the overall average expression partem of the genes in this group.
  • Each probeset in each group is assigned an expression value which is the median of the expressions of that probeset in all chips included in the group.
  • the vector of expression of all probesets within a certain group forms the virtual chip of that group, and the collection of all such virtual chips is a virtual panel.
  • the panel (or sub-panels) can be queried to identify probesets with a required behavior (e.g. specific expression in a sub-set of tissues, or differential expression between disease and healthy tissues).
  • These probesets are linked to LEADS contigs and to RefSeqs (http : / / www. ncbi.nlm.nih. go v/RefS eq/) by probe-level mapping, for further analysis.
  • the Affymetrix platforms that are downloaded are HG-U95A and the HG-
  • U133 family ( ⁇ , ⁇ , A2.0 and PLUS 2.0). Three virtual panels were created: U95 and U133 Plus 2.0, based on the corresponding Affymetrix platforms, and U133 which uses the set of common probesets for HG-U133A, HG-U133A2.0 and HG-U133 PLUS 2.0+.
  • results of the MED discovery engine are presented for selected cancer not represented in the two cancer TMAs which were analyzed using an anti VSIGl antibody.
  • the y-axis the (normalized) expression and the x-axis describes the groups in the panel. For each group, the median expression is represented by a bar, and the standard deviation.
  • the MED discovery engine was used to assess the expression of VSIGl transcripts.
  • Expression data for Affymetrix probe sets 234370_at representing the VSIGl gene data is shown in Figure 31.
  • the expression of VSIGl transcripts detectable with the above probe sets was observed in several cancer types mainly pancreatic cancer, and specifically in pancreas intraductal papillary mucinous adenocarcinoma ( Figure 31). Cervical cancer also demonstrated over expression of VSIGl.
  • NGS Next Generation Sequencing
  • TCGA cancer tissue
  • GTEx normal tissue
  • IHC Figure 25A for lung cancer and data not shown for breast cancer
  • the expression in TCGA and GTEx also validated the over expression seen using the MED database in pancreatic cancer (which is the cancer with the highest mRNA expression in TCGA Figure 25B) and based on the
  • TCGA/GTEx analysis liver and ovary cancer also show over expression of VSIGl ( Figure 25 C and D respectively).
  • VSIGl membrane over expression of VSIGl in multiple cancers, such as NSCLC adenocarcinoma, breast carcinoma, HCC, colon and rectal cancer, kidney clear cell carcinoma, kidney papillary cell carcinoma, uterus cancer, head and neck cancer, pancreatic cancer, stomach cancer and ovarian cancer , as well as the limited normal expression indicates that VSIGl could serve as a target for ADC treatment in cancer. This further supported by the cytotoxic effect seen on cell lines once an anti VSIGl antibody is conjugated to a toxin.
  • cancers such as NSCLC adenocarcinoma, breast carcinoma, HCC, colon and rectal cancer, kidney clear cell carcinoma, kidney papillary cell carcinoma, uterus cancer, head and neck cancer, pancreatic cancer, stomach cancer and ovarian cancer , as well as the limited normal expression indicates that VSIGl could serve as a target for ADC treatment in cancer. This further supported by the cytotoxic effect seen on cell lines once an anti VSIGl antibody is conjugated to
  • a human antibody phage display library was panned against a recombinant protein comprising the VSIGl extracellular domain (ECD) fused to a human IgGi Fc region.
  • ECD VSIGl extracellular domain
  • Free biotin was removed by dialyzing samples overnight against phosphate buffered saline (PBS) pH 7.4 using a 3500 MWCO Slide-A-Lyzer cassette (Pierce). The approximate number of biotin moieties per protein was determined using an EZ Biotin Quantitation Kit (Pierce). Dialyzed proteins were stored at -80°C. CGEN-XXXHH, negative control protein comprising an irrelevant ECD fused to the same human Fc, was prepared in the same way (data not shown).
  • VSIGlHH-1 preparation was assessed by SDS-PAGE. Approximately 2 ⁇ g of each protein was diluted to 10 in PBS, mixed with 2.5 5x LDS sample loading dye, heated to 100°C for 5 mins and centrifuged to remove precipitate. Sample Reducing Agent was also added where necessary (1.4 ⁇ ). Proteins were separated by electrophoresis on NuPage 4- 12% tris-glycine gels and stained with SimplyBlue SafeStain. SeeBlue Plus2 molecular weight markers were run alongside samples for size comparison. All protein electrophoresis reagents and equipment were sourced from Life Technologies.
  • Phage panning of human antibody library Panning reactions were carried out in solution using streptavidin-coated magnetic beads to capture the biotinylated antigens. Note that all washing and elution steps were conducted using a magnetic rack to capture the beads (Promega). All incubation steps were conducted at room temperature with gentle mixing on a tube rotator. Four panning sub-campaigns were conducted, each with a different combination of antigens, washes and Fc-binder depletion steps (Table 1).
  • Sub-campaigns A and B applied depletion steps to remove binders against the CGEN-150XX control protein and streptavidin-coated magnetic beads.
  • Sub-campaigns C and D only applied depletion to remove binders against the beads. All campaigns used 100 pmols of the appropriate CGEN-VSIGlHH-1 or C GEN- 150XXHH protein per panning or depletion step (this was the total amount of protein, not the final protein concentration).
  • Table 1 Antigen and washing stringency used for phage panning against CGEN-VSIG1HH- 1. Note that the washing protocol involved a set of washes in PBS-T following by identical washes in PBS. There were either three, six or eight (3X, 6X, 8X) washes in each cycle depending on the sub-campaign and panning round.
  • phage library for panning All phage panning experiments used the XOMA031 human fab antibody phage display library (XOMA Corporation). Sufficient phage for a 50-fold over-representation of the library were blocked by mixing 1 : 1 with 10% skim milk powder in PBS (final skim milk concentration 5%) and incubating for lhr.
  • Biotin-labeled antigens were coupled to the beads for 1 hr at RT with gentle mixing on a tube rotator. Bead suspensions were washed twice with PBS to remove free antigen and re-suspended in 100 blocking buffer prior to phage panning.
  • Phage panning round 1 The blocked and depleted phage library was mixed with biotinylated CGEN-VSIGlHH-1 coupled to streptavidin-coated magnetic beads. This suspension was incubated for lhr at RT with gentle rotation to allow binding of CGEN- VSIG1 specific phage. Non-specific binders were removed by washing according to the protocols in Table 1. Each wash was conducted by re-suspending the beads in 1 mL of wash buffer using five aspirations with a pipette. After washing, bound phage were eluted by incubation with 500 ⁇ , of 100 mM triethylamine (TEA) (EMD Millpore) for 10 - 15 mins at RT. The eluate was neutralized by adding 500 of 1 M Tris-HCl pH 8.0 (Teknova).
  • TAA triethylamine
  • phage titer ⁇ 0 ⁇ . of the initial phage library (input titer) or panning eluate (output titer) was serially diluted (10-fold) in PBS. A 90 aliquot of each phage dilution was mixed with 90 ⁇ . of TGI E. coli cells (Lucigen) grown to an optical density of -0.5 at 600 nm (OD 600nm). Phage were allowed to infect the cells by stationary incubation for 30 mins, then shaking incubation (250 rpm) for 30 mins, all at 37°C.
  • Phage rescue The remaining phage eluate ( ⁇ 1 mL) was mixed with 10 mL of
  • TGI E. coli grown to an OD of 0.5 at 600 nm. Phage were infected into cells as detailed above, infected cells were pelleted by centrifugation at 2500xG and re-suspended in 750 ⁇ . 2YT medium (Teknova). The cell suspension spread on 2YTCG agar plates (Teknova). These were incubated overnight at 37°C and the resulting E. coli lawns were scraped and re- suspended in -20 mL liquid 2YTCG (Teknova).
  • a small aliquot of re-suspended cells was inoculated into 50 mL 2YTCG to achieve an OD 600nm of 0.05, and then grown at 37°C with 250 rpm shaking until the OD reached 0.5.
  • This culture was infected with M13K07 helper phage (New England Biolabs) and incubated overnight at 25°C with shaking to allow phage production.
  • the culture supernatant containing rescued phage particles was cleared by centrifugation at 2500xG and 1 mL was carried over for either a) a subsequent round of panning or b) fab binding screens.
  • Phage panning rounds 2-3 second and third rounds of panning were conducted as per the steps above, except that the rescued phage supernatant from the previous round was used in place of the phage library.
  • Fab expression vectors The XOMA031 library is based on phagemid constructs that also function as fab expression vectors. These vectors contain fab heavy chain and light chain expression cassettes, a lac promoter to drive expression of the antibody genes, and an ampicillin resistance gene. The antibody chains are appended with N-terminal signal peptides to drive their secretion into the periplasmic space. The C-terminal of the heavy chain carries a truncated gene III protein sequence for incorporation into phage particles. The heavy chain also carries hexa-histidine, c-myc and V5 affinity tags. Transformation of these vectors into E. coli and induction with isopropyl ⁇ -D-l-thiogalactopyranoside (IPTG) results in periplasmic expression of soluble fab molecules.
  • IPTG isopropyl ⁇ -D-l-thiogalactopyranoside
  • Fab PPE production Eluted phage pools from panning round 3 were diluted and infected into TGI E. coli cells (Lucigen, Middleton, WI) so that single colonies were generated when spread on a 2YTCG agar plate. This resulted in each colony carrying a single fab clone. Individual clones were inoculated into 1 mL 2YTCG starter cultures in 96-well deepwell blocks (VWR) using a Qpix2 instrument (Molecular Devices). These starter cultures were grown overnight in a Multitron 3mm incubator (Infors) at 37°C with 700 rpm shaking.
  • VWR 96-well deepwell blocks
  • Qpix2 instrument Molecular Devices
  • CGEN-VSIGlHH-1 The target protein was diluted to 1 ⁇ g/mL in PBS and 50 aliquots were coated on the wells of high binding EIA/RIA microplates (Costar) overnight at 4°C. Coated plate wells were rinsed twice with PBS and incubated with 300 blocking buffer (5% skim milk powder in PBS pH 7.4) at room temperature (RT) for 1 hr. Blocking buffer was removed and plates were rinsed twice more with PBS. 50 ⁇ . aliquots of each PPE sample were added to individual plate wells and incubated at RT for 1 hr.
  • Assay signals were read on a Victor II plate reader (Perkin-Elmer) absorbance 450 nm. Each ELISA plate also contained two negative control wells (blanks) where no fab was present. Fab binding was expressed as the fab binding signal divided by the blank signal. Positive hits were identified as those giving a fab/blank ratio equal or greater than five.
  • Flow cytometry binding screen Fab PPE samples were also tested for binding to Expi293 cells (Life Technologies) over-expressing the human or mouse orthologs of CGEN-VSIG1. Parental (i.e. not transfected) Expi293 cells were used as the negative control. All reagent preparation and wash steps were carried out in FACS buffer (PBS (Life Technologies), 1% BSA (Sigma Aldrich), and 0.1% Sodium Azide.
  • CGEN-VSIG1 expressing was mixed with 25 ⁇ . of PPE and incubated for 30 mins at 4°C. After incubation, cells were washed once in 200 ⁇ of FACS buffer and re-suspended in 30 ⁇ of a monoclonal mouse anti c-myc antibody (Roche) diluted 1 : 1000 (10 ⁇ g/ml). This was incubated for 30 mins at 4°C followed by another wash step. Cell were re-suspended in 25 ⁇ of a polyclonal goat anti-mouse- AF 647 antibody (Jackson Immunoresearch) diluted 1 :300 (5 ⁇ g/ml) and incubated for 25 mins at 4°C. After two final washes cells were re-suspended in 50 ⁇ of FACS buffer containing 2% paraformaldehyde.
  • VSIGlHH-1 did not change the SDS-PAGE banding partem compared to the starting material (data not shown).
  • the non-reduced protein showed a major band slightly larger than 100 kDa (expected size based on amino acid sequence is 98 kDa).
  • Phage panning the phage output titer results indicate the number of individual phage particles recovered at the end of each panning round (Table 2).
  • the output titer after round 1 ranged from 2xl0 4 to lxlO 5 . This is at the lower range generally obtained with the XOMA031 library (typical round 1 outputs are between lxlO 4 and lxlO 6 ).
  • Titers for each sub-campaign increased to 1.4xl0 5 - 4.6x10 5 in round 2 and 7.6x10 s - 3x10 9 in round 3.
  • Fab PPE screening A set of 186 fab clones from each sub-campaign (744 in total) were tested by ELISA and FACS for binding to the recombinant CGEN-VSIGlHH-1 protein and Expi293-VSIG1 cell lines, respectively (Table 2).
  • the ELISA hit rate i.e. percentage of clones positive for binding
  • the FACS hit rate for each sub-campaign was slightly lower, ranging from 17% - 25%.
  • Only a sub-set of fabs had binding activity that correlated between FACS and ELISA (40 - 61%, depending on the sub- campaign). This was expected, given that phage panning was conducted against the recombinant CGEN-VSIGlHH-1 protein and some fabs may recognize epitopes that are different from, or do not occur, on the natural cell-surface form of the protein.
  • FACS results are against CGEN-VSIG1HH- 1.
  • FACS results include binding against Expi293-huVSIGl and Expi293-moVSIGl cells.
  • FACS/ELISA correlation refers to the proportion of fabs that bound in both assays (FACS and ELISA) compared to combined number of binders observed in either assay.
  • Step 1 High density surfaces of anti -human fc pAb (Invitrogen HI 0500) were immobilized on each spot of a ProteOn GLC chip using standard amine coupling chemistry. Each of the six channels were activated in the horizontal direction for 6 minutes with EDC- NHS prior to a 5 minute injection in all vertical channels of anti -human fc pAb diluted to 9 ⁇ g/mL in 10 mM NaOAc, pH 5.0. This was followed by a 6 minute block of excess activated carboxyls using 1M ethanolamine pH 8.5 in all vertical and horizontal channels. This immobilization protocol was performed in order to be able to use interspot referencing. Final immobilization levels of pAb ranged from ⁇ 4900-5100 RU. The immobilization of anti- human fc pAb was performed using degassed PBST as the running buffer.
  • Step 2 For each kinetic experiment cycle, six of the anti-VSIGl mAbs listed below were each diluted to -0.25 ⁇ g/mL in running buffer (degassed PBST withlOO ⁇ g/mL BSA) and captured in the vertical direction for 1 minute at a flow rate of 25 ⁇ / ⁇ :
  • Step 3 Human ECD VSIG1 (Lot# 150518-02) monomer was simultaneously injected at six different concentrations which ranged from 1.6 nM - 400 nM over all captured anti-VSIGl mAbs for two minutes followed by 10 minutes of dissociation at a flow rate of 50 ⁇ . Monomer injections were preceded by an identical buffer injection for double- referencing. Capture surfaces were regenerated with a 30-second pulse of 146 mM phosphoric acid in both the horizontal and vertical directions.
  • Step 4 Sensorgram data were zeroed, aligned, and double-referenced (interspot, buffer blanks) using ProteOn Manager Version 3.1.0.6 and Scrubber ProteOn software. Where appropriate, data were fit with a 1 : 1 kinetic model including a term for mass transport. A steady-state approach to estimate K D was used for those data which showed unusually fast on-rates and off-rates. In those cases an identical time point at the end of the injection cycle was selected for each sensorgram where the association phase appeared to have reached equilibrium. A simple 1 : 1 equilibrium binding model was then used to fit the RU signals at the identical time point as a function of monomer concentration to estimate K D . Kinetic and equilibrium data fitting were performed using both ProteOn Manager and Scrubber ProteOn software. The binding constants of each mAb are listed in order of decreasing affinity in Figure 23. All data are shown in the Figures.
  • Step 5 Sensorgram data for mAb CPA.4.016 appeared to be complex when acquired with the ProteOn so an additional experiment was performed using the Biacore 3000 to attempt to acquire data sufficient for a steady-state estimate for K D .
  • mAb CPA.4.016 was covalently coupled to flow cell 1 of a CM5 chip at ⁇ 11,670 RU while flow cell 2 was activated and blocked as a reference surface.
  • Triplicate concentrations of VSIGl monomer that ranged from 2.3 nM - 148 nM were injected for three minutes followed by three minutes of dissociation at a flow rate of 50 ⁇ / ⁇ . The surface was regenerated with a single 20 second pulse of lOmM glycine, pH 2.5.
  • FIG 23 shows mAb CPA.4.009 as having the highest affinity of the 15 mAbs in this study.
  • ADC Antibody Drug Conjugate
  • Higher monomer concentrations are needed for a better estimate of the K D for mAb 156-01. D05 because of its apparent low affinity. Therefore the K D listed here for mAb D05 should only be considered a rough estimate.
  • Step 1 The following anti-VSIGl mAbs were each diluted to ⁇ 10 ⁇ g/mL in lOmM sodium acetate, pH 5.0 and covalently immobilized on two independent spots on a ProteOn GLC biosensor chip using standard amine coupling: CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011. [00366] The activation step occurred in the horizontal flow direction while the immobilization step occurred in the vertical flow direction. MAbs were injected for four minutes after surface activation. The blocking step occurred in both the vertical and horizontal positions so that the horizontal "interspots" could be used as reference surfaces. MAbs were immobilized at a range of -4700RU-5600RU.
  • Step 2 Preliminary experiments involved several cycles of injecting -23 nM VSIGl antigen (hVSIGl ECD-his, Lot #150518-02) over all immobilized mAbs for three minutes at a flow rate of 25 ⁇ / ⁇ followed by regeneration with a 30-second pulse of 10 mM glycine-HCl, pH 2.5. Antigen samples were prepared in degassed PBST running buffer with 100 ⁇ g/mL BSA. These preliminary experiments showed that the VSIGl antigen bound reproducibly to all immobilized mAbs when using glycine at pH 2.5 as the regeneration reagent.
  • Step 3 Earlier SPR kinetics experiments with these mAbs and the identical antigen (6/10/15) showed that most interactions had relatively fast off-rates, therefore a "pre- mix" binning protocol was performed.
  • each mAb listed in Step 1 was pre- mixed with VSIGl antigen and then injected for three minutes over all immobilized mAbs.
  • the molar binding site concentration of each mAb was in excess of the molar antigen binding site concentration.
  • the final binding site concentration of each mAb was ⁇ 400nM and the final binding site concentration of the antigen was ⁇ 23nM.
  • An antigen-only control cycle was performed after every five mAb injection cycles to monitor the activity of the immobilized mAbs throughout the experiment.
  • Step 4 Sensorgram data were processed and referenced using ProteOn Manager version 3.1.0.6.
  • the mAb-only control injections were used as the injection references where significant binding with the mAb-only injections was observed.
  • An antibody pair was classified as having a shared antigen binding epitope (designated as a red "0" in the matrix in Figure 12) if no binding was observed from the injection of mixed mAb and antigen over the immobilized mAb, or if binding was significantly reduced as compared to the antigen-only control injection over the same immobilized mAb.
  • An antibody pair was classified as binding to different antigen epitopes (designated as a green "1" in the matrix in Figure 16) if the injection of mixed mAb and antigen showed binding to the immobilized mAb similar to or greater than the antigen-only control over the same immobilized mAb.
  • Step 5 Hierarchical clustering of the binding patterns in the binary matrix for each immobilized mAb (horizontal patterns in Figure 15) was performed using JMP software version 11.0.0.
  • the blocking patterns of the mAbs as analytes in solution were also clustered as a comparison to the blocking patterns of the immobilized mAbs (data not shown, see Results for discussion).
  • Figure 14 is an example of the processed and referenced sensorgram data generated for one mAb (CPA.4.023) injected with pre-mixed antigen over each immobilized mAb (panels A-O) as well as the mAb-only control injection (panels P-DD).
  • Figure 14 shows the binary matrix of the blocking ("0") or sandwiching ("1") between each mAb pair where the mAbs are listed in identical order both vertically (mAbs on the surface - "ligands”) and horizontally (mAbs in solution - "analytes”).
  • Identical "bins" of blocking patterns for mAbs as ligands are highlighted in Figure 16 with a black box around each group of similar patterns.
  • Figure 17 shows the dendrogram of the horizontal (ligand) blocking patterns in the matrix in Figure 16.
  • Hierarchical clustering of the blocking patterns of the mAbs as analytes was identical to the clustering of the mAbs in solution with the exception of mAb CPA.4.009 which, as an immobilized ligand, appeared to be able to sandwich the antigen with mAb CPA.4.012 injected in solution, while as an analyte mAb CPA.4.009 appeared to block the antigen from binding to immobilized mAb 156- 01.F10.
  • Bins 3 and 4 differ by only how they bind to the antigen in the presence of mAb CPA.4.012. MAbs in Bin 3 appear to be able to sandwich with
  • OV-90, MK 45, HUH-1 or DU145 cells were cultured in complete OV-90 media (1 : 1 mixture of MCDB 105 media containing a final concentration of 1.5 g/L sodium bicarbonate and Media 199 containing a final concentration of 2.2 g/L sodium bicarbonate with a final concentration of 15% fetal bovine serum) or DU145 media (EMEM + 4 nM Glutamine + 10% fetal bovine serum) or MK 45/HUH-1 media (RPMI + 4mM Glutamax + 10% FBS).
  • Cells were harvested in log phase growth and 8,000 OV-90, 5,000 DU145/HUH-1, or 3,000 MKN45 cells in 80 ul per well were seeded in 96 well plates and incubated for 24hrs at 37°C in a humidified water jacketed with 5% C02.
  • Anti- human VSIGl human IgGl antibodies or isotype control were added in single point dilutions, 2-fold, or 3-fold dilution series starting at 5 ⁇ g/ml.
  • Protein G preloaded with cytotoxic payloads DM1 (non-cleavable linker), MMAE (cleavable linker), MMAF (non-cleavable linker), or Duocarmycin (cleavable linker) (Concortis, San Diego CA) were added at 10 nM, 25 nM, 50 nM, and 50 nM respectively, and plates were incubated at 37°C in a humidified water jacketed incubator with 5% C02. Cell killing was measured after 72 hrs using Cell Titer Glo (Promega) reagent according to the manufacturer's instructions. Data was analyzed using Prism software (GraphPad, La Jolla, CA).
  • Antibody Binding to Human, Mouse and Cynomolgus VSIGl ExPi 293 (Life Technologies) or HEK 293 (ATCC) cells were transfected by electroporation with pcDNA3.1 (Life Technologies) vectors containing full length human, mouse, and cynomolgus VSIG1. Cells were cultured in ExPi 293 Expression media or DMEM + 4mM Glutamine + 10% fetal bovine serum and passaged according the manufacturer's instructions. Anti- VSIG1 antibodies at ⁇ in FACS buffer (PBS + 1% BSA + 0.1% sodium azide) were serially diluted 3-fold, added to 50,000 cells, and incubated for 1 hr at 4°C.
  • FACS buffer PBS + 1% BSA + 0.1% sodium azide
  • VSIG1 To determine the specificity and cross-reactivity of antibodies against VSIG1, we screened a panel of antibodies against human, mouse, and cynomolgus VSIG1 recombinant over expressing cell lines. A majority of the antibodies tested bound to cell lines
  • Anti-VSIGl Antibody drug conjugates exhibit potent in vitro cytotoxic activity: To examine the potency of anti-VSIG antibody drug conjugates, we assayed the in vitro efficacy of a panel of antibodies in a piggyback cytotoxicity assay with protein G preloaded payloads DM1, MMAE, MMAF, and Duocarmycin. OV-90 (ovarian carcinoma), MK 45 (stomach carcinoma), and HUH-1 (hepatocellular carcinoma) human cancer cell lines were selected based on high expression of VSIG1 mRNA by microarray and RNAseq. VSIG1 protein surface expression on the human cancer cells was verified by FACS ( Figure 15).
  • DU145 a prostate cancer cell line
  • Anti-VSIGl clone 156-01. E05 demonstrated potent cytotoxic cell killing of OV-90 cells and no detectable killing of VSIG1 negative cell line DU145 ( Figure 20).
  • CPA.4.005, CPA.4.009, and CPA.4.028 exhibited the greatest cell killing activity across all 4 payloads.
  • the weaker activity observed with 156-01. F10 was expected since it has a weaker avidity to antigen than the other 3 clones tested. ( Figure 22).
  • test proteins were diluted to 1 ⁇ g/mL in phosphate buffered saline (PBS) and 50 aliquots were coated on the wells of a Maxisorp ELISA plate (Thermo Fisher Scientific, Waltham, MA) overnight at 4°C, or for lhr at 37°C. Coated plate wells were rinsed twice with PBS and incubated with 300 blocking buffer (5% skim milk powder in PBS pH 7.4) at room temperature (RT) for 1 hr. Blocking buffer was removed and plates were rinsed twice more with PBS. Plate-bound proteins were detected by adding 50 ⁇ ⁇ of a primary antibody and incubating at RT for 1 hr.
  • PBS phosphate buffered saline
  • TMB Sureblue 3,3',5,5'-Tetramethylbenzidine
  • KPL Inc Gaithersburg, MD
  • HRP reaction was stopped by adding 50 ⁇ . 2N H2SO4 (VWR, Radnor, PA) and assay signals were read on a Fluostar (BMG Labtech, Cary, NC) plate reader at absorbance 450 nm.
  • VSIGl antigen A range of proteins required for phage display experiments were biotinylated to facilitate solution-based panning. These included human VSIGl ECD fused to human IgGl Fc (VSIGl H:H, SEQ ID NO 12) and mouse VSIGl ECD fused to mouse IgG2a Fc (VSIGl M:M, SEQ ID NO 13). A negative control human IgGl Fc-fusion protein, i.e. irrelevant ECD fused to the same human IgGl Fc as VSIGl H:H, was biotinylated to use for depletion steps in the panning experiments.
  • Sub-campaigns A and B alternated between the human and mouse ECD versions of VSIG1 in an attempt to enrich binders with human/mouse species cross- reactivity.
  • Sub-campaigns C and D were focused on the human ECD version of the antigen, and used depletion steps against the negative control human IgGl Fc-fusion protein to remove binders against the human IgGl Fc. All campaigns used 100 pmol of the appropriate VSIG1 or negative Fc-fusion control protein per round.
  • phage library for panning All phage panning experiments used the XOMA031 human Fab antibody phage display library (XOMA Corporation, Berkeley, CA). Sufficient phage for a 50-fold over-representation of the library were blocked by mixing 1 : 1 with 10% skim milk powder in PBS (final skim milk concentration 5%) and incubated for lhr.
  • Phage panning round 1 The blocked and depleted phage library was mixed with the VSIG1 beads described above. This suspension was incubated for lhr at RT with gentle rotation to allow binding of VSIG1 specific phage. Non-specific binders were removed using a sequence of washes. The washing conditions used in all displays were: Round 1, three washes with PBS-T and three washes with PBS; round 2 and round 3, six washes with each buffer.
  • the bound phage were eluted by incubation with 500 of 100 mM triethylamine (TEA) (EMD Millpore, Rockland, MA) for 20 mins at RT.
  • TAA triethylamine
  • the eluate was neutralized by adding 500 of 1 M Tris-HCl pH 8.0 (Teknova, Hollister, CA).
  • phage titer 10 ⁇ of the initial phage library (input titer) or panning eluate (output titer) was serially diluted (10-fold) in PBS. A 90 ⁇ . aliquot of each phage dilution was mixed with 90 ⁇ . of TGI E. coli cells grown to an optical density of -0.5 at 600 nm (OD 600nm). Phage were allowed to infect the cells by stationary incubation for 30 mins, then shaking incubation (250 rpm) for 30 mins, all at 37°C.
  • Phage rescue The remaining phage eluate ( ⁇ 1 mL) was mixed with 10 mL of TGI E. coli grown to an OD 600 nm of 0.5. Phage was infected into cells as detailed above, infected cells were pelleted by centrifugation at 2500xG and re-suspended in 750 ⁇ . 2YT medium (Teknova). The cell suspension was divided into three equal aliquots that were spread on 2YTCG agar plates. These plates were incubated overnight at 37°C and the resulting E. coli lawns were scraped and re-suspended in -20 mL liquid 2YTCG (Teknova).
  • This cell suspension was used to make 1 mL glycerol stocks for each panning round.
  • a small aliquot of re-suspended cells was inoculated into 50 mL 2YTCG to achieve an OD 600nm of 0.05, and then grown at 37°C with 250 rpm shaking until the OD reached 0.5.
  • the resulting culture was infected with M13K07 helper phage (New England Biolabs, Ipswich, MA) and incubated overnight at 25 °C with shaking to allow phage packaging.
  • the culture supernatant containing rescued phage particles was cleared by centrifugation at 2500 X G and 1 mL was carried over for either a) a subsequent round of panning or b) Fab binding screens. Phage in the remaining supernatant were concentrated and purified for phage pool ELISAs (see below).
  • Phage panning rounds 2-3 Second and third rounds of panning were conducted as per the steps above, except that the rescued phage supernatant from the previous round was used in place of the phage library.
  • Phage pool enrichment ELISA Phage from each panning round was precipitated from rescue culture supernatants by adding 1/5 volume of PEG-6000/NaCl solution (Teknova). Precipitated phage were harvested by centrifugation at 8000 rpm and re- suspended in 1 mL PBS.
  • Phage aliquots were diluted 1 : 10 in blocking buffer (5% skim milk powder in PBS) and 50 aliquots were added to the wells of ELISA plates coated with VSIGl-ECD-Ig H:H (SEQ ID NO 12), VSIG1 ECD-Ig M:M (SEQ ID NO 13), or the negative control protein, human IgGl isotype control and mouse IgG2 isotype control.
  • Bound phage were detected with a HRP -conjugated anti-M13 phage coat antibody (GE Healthcare, Pittsburgh, PA) diluted 1 :2000 in PBS-T. All other assays steps are conducted as described in the general ELISA protocol.
  • Fab expression vectors The pXHMV-Fab-kappa and pXHMV-Fab-lambda phagemid vectors used in the XOMA031 library also functioned as Fab expression vectors. These vectors contained Fab heavy chain and light chain expression cassettes, a lac promoter (plac) to drive expression of the antibody genes, and an ampicillin resistance gene. The antibody chains were appended with N-terminal signal peptides to drive their secretion into the periplasmic space. The C-terminal of the heavy chain carried a truncated gene III protein sequence for incorporation into phage particles. The heavy chain also carried hexa-histidine, c-myc and V5 affinity tags.
  • Fab PPE production Eluted phage pools from panning round 3 were diluted and infected into TGI E. coli cells (Lucigen, Middleton, WI) so that single colonies were generated when spread on a 2YTCG agar plate. This resulted in each colony carrying a pXHMV-Fab vector encoding a single Fab clone.
  • Fab expression For Fab expression, 20 of 1 mL starter cultures were transferred into a second set of deepwell plates containing 1 mL 2YT with 0.1% glucose and 100 ⁇ g/mL ampicillin. Cultures were grown until the average OD 600nm was 0.5-1.0 and protein expression induced by adding IPTG (Teknova) to a final concentration of 1 mM. Expression cultures were incubated overnight in the Multitron instrument at 25°C with 700 rpm shaking.
  • ELISA binding Assays Each plate of PPE extracts was tested for binding to biotinylated VSIGl-ECD-Ig H:H (SEQ ID NO 12) and the negative Fc-fusion control protein. The ELISA followed the general protocol above, except that the biotinylated antigen was captured on a streptavidin-coated 96-well plate (Pierce) instead of a standard ELISA plate. A 50 ⁇ . aliquot of each PPE was added to plate wells coated with these antigens and the remainder of the ELISA followed the general method. Bound Fab was detected using a HRP-conjugated anti-human Fab'2 antibody (Jackson Immunoresearch) diluted 1 :2000 in PBS with 5% skim milk.
  • HRP-conjugated anti-human Fab'2 antibody Jackson Immunoresearch
  • Protein expression constructs for human anti-VSIGl IgGs were derived by PCR-amplification of variable heavy, lambda and kappa domain genes, which were sub-cloned into pFUSE-CHIg- hGl (human IgGl heavy chain), pFUSE2-CLIg-hK (human kappa light chain) or pFUSE2- CLIg-hL2 (human lambda 2 light chain) vectors, respectively (all expression vectors sourced from Invivogen).
  • Expi293 cells (Life Technologies) were seeded at 6xl0 5 cells/ml in in
  • Expi293TM medium (Life Technologies) and incubated for 72 hrs at 37°C in a humidified atmosphere of 8% CO2 with shaking at 125 rpm. This cell stock was used to seed expression cultures at 2.0 xlO 6 cells/ml in Expi293TM medium. These cultures were incubated as above for 24 hrs with shaking at 135 rpm.
  • Fusion proteins composed of the extracellular domain of the VSIG1 linked to a mouse IgG2 Fc polypeptide are generated by standard recombinant methods and used as antigen for immunization.
  • Fully human monoclonal antibodies to VSIG1 are prepared using mice from the HCo7 strain of the transgenic HuMab MouseTM which expresses human antibody genes.
  • the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al. (1993) EMBO J. 12:811-820 and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of PCT Publication WO 01/09187.
  • this mouse strain carries a human kappa light chain transgene, KCo5, as described in Fishwild et al. (1996) Nature Biotechnology 14:845-851, and a human heavy chain transgene, HCo7, as described in U.S. Pat. Nos. 5,545,806;
  • mice of the HCo7 were generated fully human monoclonal antibodies to VSIG1, mice of the HCo7
  • HuMab Mouse strain can be immunized with purified recombinant VSIG1 fusion protein derived from mammalian cells that are transfected with an expression vector containing the gene encoding the fusion protein.
  • Purified recombinant VSIG1 antigen preparation (5-50 ⁇ g, purified from transfected mammalian cells expressing VSIG1 fusion protein) is used to immunize the HuMab mice intraperitoneally.
  • Transgenic mice are immunized twice with antigen in complete Freund's adjuvant or Ribi adjuvant IP, followed by 3-21 days IP (up to a total of 11 immunizations) with the antigen in incomplete Freund's or Ribi adjuvant.
  • the immune response is monitored by retroorbital bleeds.
  • the plasma is screened by ELISA (as described below), and mice with sufficient titers of anti-VSIGl human immunoglobulin are used for fusions. Mice are boosted intravenously with antigen 3 days before sacrifice and removal of the spleen. Selection of HuMab mice producing anti- VSIG1 Antibodies
  • HuMab mice producing antibodies that bind VSIG1 sera from immunized mice is tested by a modified ELISA as originally described by Fishwild, D. et al. (1996). Briefly, microtiter plates are coated with purified recombinant VSIG1 fusion protein at l-2ug/ml in PBS, 50ul/wells incubated 4° C. overnight then blocked with 200ul/well of 5% BSA in PBS. Dilutions of plasma from VSIG1 -immunized mice are added to each well and incubated for 1-2 hours at ambient temperature.
  • the plates are washed with PBS/Tween and then incubated with a goat-anti-human kappa light chain polyclonal antibody conjugated with alkaline phosphatase for 1 hour at room temperature. After washing, the plates are developed with pNPP substrate and analyzed by spectrophotometer at OD 415-650. Mice that developed the highest titers of anti-VSIGl antibodies are used for fusions. Fusions are performed as described below and hybridoma supernatants are tested for anti-VSIGl activity by ELISA.
  • mice The mouse splenocytes, isolated from the HuMab mice, are fused with PEG to a mouse myeloma cell line based upon standard protocols. The resulting hybridomas are then screened for the production of antigen-specific antibodies. Single cell suspensions of splenic lymphocytes from immunized mice are fused to one-fourth the numbers of P3X63 Ag8.6.53 (ATCC CRL 1580) nonsecreting mouse myeloma cells with 50% PEG (Sigma).
  • Cells are plated at approximately 1X10 "5 /well in flat bottom microtiter plate, followed by about two week incubation in selective medium containing 10% fetal calf serum, supplemented with origen (IGEN) in RPMI, L-glutamine, sodium pyruvate, HEPES, penicillin, streptomycin, gentamycin, lx HAT, and beta-mercaptoethanol. After 1-2 weeks, cells are cultured in medium in which the HAT is replaced with HT. Individual wells are then screened by ELISA (described above) for human anti-VSIGl monoclonal IgG antibodies. Once extensive hybridoma growth occurred, medium is monitored usually after 10-14 days.
  • IGEN origen
  • the antibody secreting hybridomas are replated, screened again and, if still positive for human IgG, anti- VSIGl monoclonal antibodies are subcloned at least twice by limiting dilution.
  • the stable subclones are then cultured in vitro to generate small amounts of antibody in tissue culture medium for further characterization.
  • the hybridoma clones are selected for further analysis. Structural Characterization of Desired anti-VSIGl Human Monoclonal Antibodies
  • the cDNA sequences encoding the heavy and light chain variable regions of the obtained anti-VSIGl monoclonal antibodies are obtained from the resultant hybridomas, respectively, using standard PCR techniques and are sequenced using standard DNA sequencing techniques.
  • nucleotide and amino acid sequences of the heavy chain variable region and of the light chain variable region are identified. These sequences may be compared to known human germline immunoglobulin light and heavy chain sequences and the CDRs of each heavy and light of the obtained anti-VSIGl sequences identified.
  • binding affinity, binding kinetics, binding specificity, and cross- competition of anti-VSIGl antibodies are examined by Biacore analysis. Also, binding specificity is examined by flow cytometry.
  • Anti- VSIG1 antibodies produced according to the invention are characterized for affinities and binding kinetics by Biacore analysis (Biacore AB, Uppsala, Sweden).
  • Purified recombinant human VSIG1 fusion protein is covalently linked to a CM5 chip (carboxy methyl dextran coated chip) via primary amines, using standard amine coupling chemistry and kit provided by Biacore.
  • Binding is measured by flowing the antibodies in HBS EP buffer (provided by Biacore AB) at a concentration of 267 nM at a flow rate of 50 ⁇ 1/ ⁇ .
  • the antigen-association antibodies association kinetics is followed for 3 minutes and the dissociation kinetics is followed for 7 minutes.
  • association and dissociation curves are fit to a 1 : 1 Langmuir binding model using BlAevaluation software (Biacore AB). To minimize the effects of avidity in the estimation of the binding constants, only the initial segment of data corresponding to association and dissociation phases are used for fitting.
  • Biacore is used to determine epitope grouping of anti-VSIGl HuMAbs.
  • Obtained anti-VSIGl antibodies are used to map their epitopes on the VSIGl antigen. These different antibodies are coated on three different surfaces of the same chip to 8000 RUs each. Dilutions of each of the mAbs are made, starting at 10 ⁇ g/mL and is incubated with Fc fused VSIGl (50 nM) for one hour. The incubated complex is injected over all the three surfaces (and a blank surface) at the same time for 1.5 minutes at a flow rate of 20.mu.L/min. Signal from each surface at end of 1.5 minutes, after subtraction of appropriate blanks, has been plotted against concentration of mAb in the complex. Upon analysis of the data, the anti- VSIGl antibodies are categorized into different epitope groups depending on the epitope mapping results. The functional properties thereof are also compared.
  • CHO cell lines that express VSIGl protein at the cell surface are developed and used to determine the specificity of the VSIGl HuMAbs by flow cytometry.
  • CHO cells are transfected with expression plasmids containing full length cDNA encoding a transmembrane forms of VSIGl antigen or a variant thereof.
  • the transfected proteins contained an epitope tag at the N-terminus are used for detection by an antibody specific for the epitope. Binding of an anti-VSIGl MAb is assessed by incubating the transfected cells with each of the r VSIGl Abs at a concentration of 10 ⁇ g/ml.
  • the cells are washed and binding is detected with a FITC -labeled anti-human IgG Ab.
  • a murine anti- epitope tag Ab, followed by labeled anti-murine IgG, is used as the positive control.
  • Nonspecific human and murine Abs are used as negative controls.
  • the obtained data is used to assess the specificity of the HuMAbs for the VSIGl antigen target.
  • Antibody conjugates with MMAF using the mc linker show anti -tumor efficacy against human tumor xenografts in mice. Marked inhibition of tumor growth is observed upon treatment of various ovarian, liver, pancreas, lung, and stomach tumor xenografts with anti-VSIGl -mc-MMAF. In each experiment, one group of animals bearing established tumors is treated with anti-VSIGl -mc-MMAF. Tumor sizes are measured periodically and graphed vs. time after tumor inoculation. Treated animals have significant tumor growth inhibition. This anti -tumor activity is observed at conjugate doses that have no effect on mouse body weight, a measure of drug toxicity.
  • the closest human germline gene was determined for each VH and VL domain sequence using the sequence as input to the IgBLAST program on the NCBI website.
  • IgBLAST takes a VH or VL sequence and compares it to a library of known human germline sequences; the databases used were IMGT human VH genes (F + ORF, 273 germline sequences), IMGT human V L kappa genes (F + ORF, 74 germline sequences) and IMGT human V L lambda genes (F + ORF, 78 germline sequences).
  • IgBLAST returned the top 10 human germline sequences according to score.
  • VH domains are members of four different human VH subgroups: 1, 3, 4, 5.
  • VL both kappa and lambda VL are represented with multiple subgroups for each. No apparent correlation of any VH germline with any VL germline is apparent.
  • Figure 25 shows alignments of VH sequences and are assigned into groups based on sequence similarity. Residue numbering and CDR definitions are according to IMGT (compiled at IMGT® the international ImMunoGeneTics information system®. The closest human IGHV germlines are shown aligned in gray boxes. For most CDR-Hl (27-38) and CDR-H2 (56-65), the sequences are similar or identical to the germline. For CDRH3 (105-117), the sequences of all 15 antibodies are very different.
  • Figure 26 shows alignments of V L -kappa and V L -lambda sequences and are assigned into groups based on sequence similarity. Residue numbering and CDR definitions are according to IMGT (compiled at IMGT® the international ImMunoGeneTics information system®. The closest human IGKV or IGLV germlines are shown aligned in gray boxes. For the
  • V L -kappa sequences most CDR-L1 (27-38), CDR-L2 (56-65), and CDR-L3 (105-117) the sequences are similar or identical to the germline. For the V L -lambda sequences, there is only slightly more variance from the germline.
  • VSIG1, HUH-1, OV-90, MKN45, Pane 05.04, or DU145 cells were removed from their original plate or container using Cell Dissociation Buffer in PBS (Invitrogen). Cells were then harvested in 50 ml conical tubes and centrifuged for 5 min. at lOOOrpms. After 2x wash in PBS, cells were fixed by gently pipetting in 10% Neutral Buffered Formalin (10% NBF) to the inside wall of the conical tube so as not to disturb the cell pellet. Cells were allowed to fix for 24 hrs. After 24 hrs. of fixation, NBF was removed from tubes and replaced with 70% ethanol . Cells pellets were embedded in paraffin within 48 hrs.
  • Anti-human VSIGl MAB4818 showed specific binding to HEK 293 cells recominantly expressing VSIGl as well as endogenous cell lines that were predicted to express VSIGl by RNAseq ( Figure 34).
  • Anti-VSIGl MAB4814 did not bind to HEK parental, HEK empty vector, or VSIGl RNA negative cell line DU145.
  • VSIGl protein was detected on the cell surface of all the cancer tissues surveyed. The highest prevalence was seen in samples from ovarian and stomach cancer ( Figures 35 and 36).
  • OV-90, Li-7, or HUH-1 cells were cultured in complete OV-90 media (1 : 1 mixture of MCDB 105 media containing a final concentration of 1.5 g/L sodium bicarbonate and Media 199 containing a final concentration of 2.2 g/L sodium bicarbonate with a final concentration of 15% f
  • RPMI + 4mM Glutamax + 10% FBS RPMI + 4mM Glutamax + 10% FBS.
  • Cells were harvested in log phase growth and 8,000 OV-90, 5,000 DU145/HUH-1, or 3,000 MKN45 cells in 80 ul per well were seeded in 96 well plates and incubated for 24hrs at 37°C in a humidified water jacketed incubator with 5% CO2.
  • Anti- human VSIG1 human IgGl antibodies or isotype control antibodies directly conjugated to DM1 (non-cleavable linker), MMAE (cleavable linker), MMAF (non-cleavable linker), or Duocarmycin (cleavable linker) (Moradec LLC, San Diego CA) were added in single point dilutions, 2-fold starting at 10 ⁇ g/ml. Plates were incubated at 37°C in a humidified water jacketed incubator with 5% CO2. Cell killing was measured after 72 hrs using Cell Titer Glo (Promega) reagent according to the manufacturer's instructions. Data was analyzed using Prism software (GraphPad, La Jolla, CA).
  • OV-90, Li-7, or HUH-1 cells were cultured in complete OV-90 media (1 : 1 mixture of MCDB 105 media containing a final concentration of 1.5 g/L sodium bicarbonate and Media 199 containing a final concentration of 2.2 g/L sodium bicarbonate with a final concentration of 15% fetal bovine serum) or Li- 7/HUH-l media (RPMI + 4mM Glutamax + 10% FBS). Cells were harvested in log phase growth and 10,000 OV-90, Li-7, or HUH-1 cells were plated in ultra-low adherence plates (Coming).
  • MMAE conjugates are more soluble and may penetrate into the necrotic core of 3D tumor spheroids better than DMSA.
  • HUH-1 cells have high expression of VSIGl protein, only the CPA.4.028-DMSA showed significant cytotoxic cell killing.
  • HUH-1 cells may have a higher resistance to the payloads, or metabolize the payloads differently than the other VSIGl + cancer cell lines tested.

Abstract

The disclosure relates generally to compounds with anti-cancer activity and more specifically to antibodies conjugated with chemotherapeutic drugs or toxins. The disclosure also relates to methods of using antibody-drug conjugate compounds for in vitro, in situ, and in vivo diagnosis or treatment of specific cancers.

Description

ANTI- VSIGl ANTIBODIES AND DRUG CONJUGATES
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority under 35 U.S.C. §119 to USSN 62/242,908, filed October 16, 2015, and to USSN 62/247,414, filed October 28, 2015, all of which are expressly incorporated herein by reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] V-set and immunoglobulin domain containing protein 1 (VSIGl) is a recently discovered member of the junctional adhesion molecule (JAM) family, also sometimes referred to as the immunoglobulin superfamily (IgSF). Members of the IgSF/JAM family have a unique structure containing an N-terminal signal peptide domain, immunoglobulin (Ig)-like domains, and transmembrane and cytoplasmic tail domains. However, very little is known about its physiological functions.
[0003] First identified and named "Glycoprotein A34", it was first characterized in humans as an X-linked gene and characterized by the presence of two Ig-like domains. VSIGl has been shown to be specifically expressed in the stomach and testis, and overexpressed in some gastric cancers, esophageal carcinomas, and ovarian cancers, but not in lung, breast or colon carcinomas. See Scanlon et al, Cancer Immun. 6:20 (2010). In the stomach, there are three altematively spliced isoforms that appear to be involved in differentiation of gastric epithelia; see Oidovsambuu et al, PLOS One 6(10):e25908 (2011). In testis, it has been associated with spermatogenesis rather than fertilization. See Kim et al, Mol. Cells 30:443-448 (2010).
[0004] Additionally, VSIGl has been shown to be preferentially expressed in certain gastric, esophageal and ovarian cancers. For example, decreased expression of VSIGl is associated with poor prognosis in primary gastric cancer; see Chen et al, J. Surgical Oncology 2012 106:286-293. In this study, VSIGl expression was completely lost in 126 out of 232 patient samples and remarkably reduced in another 106 patients, and was an independent predictor of overall survival.
[0005] The present invention is directed to new surprising results associated with VSIGl expression. BRIEF SUMMARY OF THE INVENTION [0006] The present invention provides anti-VSIGl antibodies.
[0007] In some aspects, the antibodies are antibody drug conjugates that bind V-set and immunoglobulin domain-containing protein 1 (VSIGl) and comprise at least one drug moiety and an anti-VSIGl antibody. In these embodiments, the anti-VSIGl antibodies are selected from the group consisting of 1) CPA.4.013, CPA.4.013.VH, CPA.4.013.VL, CPA.4.013.HC, CPA.4.013.LC and CPA.4.013.H1, CPA.4.013.H2, CPA.4.013.H3 and CPA.4.013.H4; 2) CPA.4.016, CPA.4.016.VH, CPA.4.016.VL, CPA.4.016.HC, CPA.4.016.LC, CPA.4.016.H1, CPA.4.016.H2, CPA.4.016.H3 and CPA.4.016.H4; 3) CPA.4.017, CPA.4.017.VH,
CPA.4.017.VL, CPA.4.017.HC, CPA.4.017.LC, CPA.4.017.H1, CPA.4.017.H2,
CPA.4.017.H3 and CPA.4.017.H4; 4) CPA.4.019, CPA.4.019.VH, CPA.4.019.VL,
CPA.4.019.HC, CPA.4.019.LC, CPA.4.019.H1, CPA.4.019.H2, CPA.4.019.H3 and
CPA.4.019.H4; 5) CPA.4.020, CPA.4.020.VH, CPA.4.020.VL, CPA.4.020.HC,
CPA.4.020.LC, CPA.4.020.H1, CPA.4.020.H2, CPA.4.020.H3 and CPA.4.020.H4; 6) CPA.4.023, CPA.4.023.VH, CPA.4.023.VL, CPA.4.023.HC, CPA.4.023.LC, CPA.4.023.H1, CPA.4.023.H2, CPA.4.023.H3 and CPA.4.023.H4; 7) CPA.4.005, CPA.4.005.VH,
CPA.4.005.VL, CPA.4.005.HC, CPA.4.005.LC, CPA.4.005.H1, CPA.4.005.H2,
CPA.4.005.H3 and CPA.4.005.H4; 8) CPA.4.009, CPA.4.009.VH, CPA.4.009.VL,
CPA.4.009.HC, CPA.4.009.LC, CPA.4.009.H1, CPA.4.009.H2, CPA.4.009.H3 and
CPA.4.009.H4; 9) CPA.4.027, CPA.4.027.VH, CPA.4.027.VL, CPA.4.027.HC,
CPA.4.027.LC, CPA.4.027.H1, CPA.4.027.H2, CPA.4.027.H3 and CPA.4.027.H4; 10) CPA.4.028, CPA.4.028.VH, CPA.4.028.VL, CPA.4.028.HC, CPA.4.028.LC, CPA.4.028.H1, CPA.4.028.H2, CPA.4.028.H3 and CPA.4.028.H4; 11) CPA.4.031, CPA.4.031.VH, CPA.4.031.VL, CPA.4.031.HC, CPA.4.031.LC, CPA.4.031.H1, CPA.4.031.H2,
CPA.4.031.H3 and CPA.4.031.H4; 12) CPA.4.033, CPA.4.033.VH, CPA.4.033.VL, CPA.4.033.HC, CPA.4.033.LC, CPA.4.033.H1, CPA.4.033.H2, CPA.4.033.H3 and
CPA.4.033.H4; 13) CPA.4.012, CPA.4.012.VH, CPA.4.012.VL, CPA.4.012.HC,
CPA.4.012.LC, CPA.4.012.H1, CPA.4.012.H2, CPA.4.012.H3 and CPA.4.012.H4; 14) CPA.4.008, CPA.4.008,VH, CPA.4.008.VL, CPA.4.008.HC, CPA.4.008.LC, CPA.4.008.H1, CPA.4.008.H2, CPA.4.008.H3 and CPA.4.008.H4; and 15) CPA.4.011, CPA.4.011.VH, CPA.4.011.VL, CPA.4.011.HC, CPA.4.011.LC, CPA.4.011.H1, CPA.4.011.H2,
CPA.4.011.H3 and CPA.4.011.H4.
[0008] In some aspects, the drug moiety is attached to the antibody by an ADC linker. In some aspects, the ADC linker is cleavable, and in others, it is self-immolative.
[0009] In further aspects, the drug moiety is selected from the group consisting of: taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6- thioguanine, cytarabine, 5-fluorouracil decarbazine, mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), daunorubicin, doxorubicin), dactinomycin, bleomycin, mithramycin, anthramycin (AMC)), vincristine, vinblastine, a duocarmycin, a calicheamicin, a maytansine, an auristatin, iodine 131, indium 111, yttrium 90, and lutetium 177.
[0010] In further aspects, the anti-VSIGl antibodies have a drug moiety is selected from the group consisting of: the maytansinoid (N2'-deacetyl-N2'-(3-mercapto-l- oxopropyl)maytansine) (DM1), monomethyl auristatin (MMAE), monomethyl auristatin phenylalanine (MMAF), and Duocarmycin.
[0011] In some aspects, the antibody drug conjugate compound is cytotoxic to cancer cells, and in others it is cytostatic.
[0012] In some embodiments, the anti-VSIGl antibodies of the invention bind to an epitope located within the extracellular domain of VSIG1.
[0013] In further embodiments, the antibody drug conjugate compound of the invetnion competes for binding at an epitope located within the extracellular domain of VSIG1 with any of the enumerated antibodies herein.
[0014] In additional aspects, the antibody component of the drug conjugate compound is a human monoclonal anti-VSIGl antibody. [0015] In further aspects, the monoclonal anti-VSIGl antibody comprises a variable heavy and variable light chain pair selected from the group consisting of: CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011.
[0016] In some embodiments, the isolated anti-VSIGl antibodies of the invention comprise:
[0017] a) a heavy chain variable domain comprising a vHCDRl having the sequence
GFTFSSYS (SEQ ID NO:X), a vhCDR2 having the sequence ISSSSSYI (SEQ ID NO:X) and a vhCDR3 having the sequence ARNVLRGAAQYYFDY (SEQ ID NO:X); and
[0018] b) a light chain variable domain comprising a vLCDRl having the sequence
QSLLHGTGYNY (SEQ ID NO:X), a vLCDR2 having the sequence LGS (SEQ ID NO:X) and a vLCDR3 having the sequence MQALQTPLT (SEQ ID NO:X). In further
embodiments, this antibody comprises a covalently attached drug moiety.
[0019] In some embodiments, the antiVSIGl antibodies comprise:
[0020] a) a heavy chain variable domain comprising a vHCDRl having the sequence
GFTFSSYG (SEQ ID NO:X), a vhCDR2 having the sequence ISYDGSNK (SEQ ID NO:X) and a vhCDR3 having the sequence VRGINVNFHYYGMDV (SEQ ID NO:X); and
[0021] b) a light chain variable domain comprising a vLCDRl having the sequence
NTNIGADYH (SEQ ID NO:X), a vLCDR2 having the sequence SNN (SEQ ID NO:X) and a vLCDR3 having the sequence QSFDSSLSAWV (SEQ ID NO:X). In further embodiments, this antibody comprises a covalently attached drug moiety.
[0022] In some embodiments, the antiVSIGl antibodies comprise:
[0023] a) a heavy chain variable domain comprising a vHCDRl having the sequence
GFTFSSYA (SEQ ID NO:X), a vhCDR2 having the sequence ISGSGGST (SEQ ID NO:X) and a vhCDR3 having the sequence AKDSYFDWQIGSNDAFDI (SEQ ID NO:X); and
[0024] b) a light chain variable domain comprising a vLCDRl having the sequence
SNNVGYQG (SEQ ID NO:X), a vLCDR2 having the sequence RNN (SEQ ID NO:X) and a vLCDR3 having the sequence SAWDSSLTAWV (SEQ ID NO:X). In further embodiments, this antibody comprises a covalently attached drug moiety.
[0025] In some embodiments, the antiVSIGl antibodies comprise: [0026] a) a heavy chain variable domain comprising a vHCDRl having the sequence
GFTFSSYA (SEQ ID NO:X), a vhCDR2 having the sequence ISGSGGST (SEQ ID NO:X) and a vhCDR3 having the sequence AKAYGS GS YFH YWYFDL (SEQ ID NO:X); and
[0027] b) a light chain variable domain comprising a vLCDRl having the sequence
QSLLQSNGYNY (SEQ ID NO:X), a vLCDR2 having the sequence LGS (SEQ ID NO:X) and a vLCDR3 having the sequence MQALQTPPT (SEQ ID NO:X). In further
embodiments, this antibody comprises a covalently attached drug moiety.
[0028] In some embodiments, the antiVSIGl antibodies comprise:
[0029] a) a heavy chain variable domain comprising a vHCDRl having the sequence
GGTFSSYA (SEQ ID NO:X), a vhCDR2 having the sequence IIPIFGTA (SEQ ID NO:X) and a vhCDR3 having the sequence ATQYS S GWYIWGAFDI (SEQ ID NO:X); and
[0030] b) a light chain variable domain comprising a vLCDRl having the sequence
QSLLHSSGDNF (SEQ ID NO:X), a vLCDR2 having the sequence LAS (SEQ ID NO:X) and a vLCDR3 having the sequence MQTLQTPLT (SEQ ID NO:X). In further embodiments, this antibody comprises a covalently attached drug moiety.
[0031] In some embodiments, the antiVSIGl antibodies comprise:
[0032] a) a heavy chain variable domain comprising a vHCDRl having the sequence
GYSFTSYW (SEQ ID NO:X), a vhCDR2 having the sequence IYPGDSDT (SEQ ID NO:X) and a vhCDR3 having the sequence ARLGIVDTSWSAFDI (SEQ ID NO:X); and
[0033] b) a light chain variable domain comprising a vLCDRl having the sequence
SSNIGSNA (SEQ ID NO:X), a vLCDR2 having the sequence YDD (SEQ ID NO:X) and a vLCDR3 having the sequence AAWDDSLNGVV (SEQ ID NO:X). In further embodiments, this antibody comprises a covalently attached drug moiety.
[0034] In some embodiments, the antiVSIGl antibodies comprise:
[0035] a) a heavy chain variable domain comprising a vHCDRl having the sequence
GDTFSSYT (SEQ ID NO:X), a vhCDR2 having the sequence FIPPLDIA (SEQ ID NO:X) and a vhCDR3 having the sequence ATGGATIFFYYFGMDV (SEQ ID NO:X); and
[0036] b) a light chain variable domain comprising a vLCDRl having the sequence
RSNIGSGS (SEQ ID NO:X), a vLCDR2 having the sequence TNS (SEQ ID NO:X) and a vLCDR.3 having the sequence AAWDDRLNGLV (SEQ ID NO:X). In further embodiments, this antibody comprises a covalently attached drug moiety.
[0037] In some embodiments, the antiVSIGl antibodies comprise:
[0038] a) a heavy chain variable domain comprising a vHCDRl having the sequence
GYTFTSYA (SEQ ID NO:X), a vhCDR2 having the sequence INAGNGNT (SEQ ID NO:X) and a vhCDR3 having the sequence ASSFHGSGSYYNKVVGMWY (SEQ ID NO:X); and
[0039] b) a light chain variable domain comprising a vLCDRl having the sequence
QSLKHNNGYSY (SEQ ID NO:X), a vLCDR2 having the sequence LDS (SEQ ID NO:X) and a vLCDR3 having the sequence MQGLQIPVT (SEQ ID NO:X). In further embodiments, this antibody comprises a covalently attached drug moiety.
[0040] In some embodiments, the antiVSIGl antibodies comprise:
[0041] a) a heavy chain variable domain comprising a vHCDRl having the sequence
GFSISDYW (SEQ ID NO:X), a vhCDR2 having the sequence VSPGGGHLT (SEQ ID NO:X) and a vhCDR3 having the sequence VRGTHLWRGVDY (SEQ ID NO:X); and
[0042] b) a light chain variable domain comprising a vLCDRl having the sequence
QSVSSY (SEQ ID NO:X), a vLCDR2 having the sequence DAS (SEQ ID NO:X) and a vLCDR3 having the sequence QQRSNWPVT (SEQ ID NO:X). In further embodiments, this antibody comprises a covalently attached drug moiety.
[0043] In some embodiments, the antiVSIGl antibodies comprise:
[0044] a) a heavy chain variable domain comprising a vHCDRl having the sequence
GFTFSSYG (SEQ ID NO:X), a vhCDR2 having the sequence IWYDGSNK (SEQ ID NO:X) and a vhCDR3 having the sequence ARGGPWGIVVVNQFDY (SEQ ID NO:X); and
[0045] b) a light chain variable domain comprising a vLCDRl having the sequence
SDINVSSYN (SEQ ID NO:X), a vLCDR2 having the sequence YYSDSDK (SEQ ID NO:X) and a vLCDR3 having the sequence MIWPSTGRWV (SEQ ID NO:X). In further embodiments, this antibody comprises a covalently attached drug moiety.
[0046] In some embodiments, the antiVSIGl antibodies comprise: [0047] a) a heavy chain variable domain comprising a vHCDRl having the sequence
SGSIRSSNW (SEQ ID NO:X), a vhCDR2 having the sequence IYHSGST (SEQ ID NO:X) and a vhCDR3 having the sequence AGRNIAGGSFDY (SEQ ID NO:X); and
[0048] b) a light chain variable domain comprising a vLCDRl having the sequence
SGIDVGPYR (SEQ ID NO:X), a vLCDR2 having the sequence YNSDSDK (SEQ ID NO:X) and a vLCDR3 having the sequence MIWHNKSRV (SEQ ID NO:X). In further
embodiments, this antibody comprises a covalently attached drug moiety.
[0049] In some embodiments, the antiVSIGl antibodies comprise:
[0050] a) a heavy chain variable domain comprising a vHCDRl having the sequence
GGSISTYY (SEQ ID NO:X), a vhCDR2 having the sequence IYFNDIT (SEQ ID NO:X) and a vhCDR3 having the sequence VRGRGGSPALDY (SEQ ID NO:X); and
[0051] b) a light chain variable domain comprising a vLCDRl having the sequence
QSLLHSNGYNY (SEQ ID NO:X), a vLCDR2 having the sequence LGS (SEQ ID NO:X) and a vLCDR3 having the sequence KQALQTIT (SEQ ID NO:X). In further embodiments, this antibody comprises a covalently attached drug moiety.
[0052] In some embodiments, the antiVSIGl antibodies comprise:
[0053] a) a heavy chain variable domain comprising a vHCDRl having the sequence
GFSFSNYV (SEQ ID NO:X), a vhCDR2 having the sequence ISYDGSDK (SEQ ID NO:X) and a vhCDR3 having the sequence ARSLRPAYYDASGV (SEQ ID NO:X); and
[0054] b) a light chain variable domain comprising a vLCDRl having the sequence
RSNIGAGFD (SEQ ID NO:X), a vLCDR2 having the sequence GDT (SEQ ID NO:X) and a vLCDR3 having the sequence QSYDSSLSVFYV (SEQ ID NO:X). In further embodiments, this antibody comprises a covalently attached drug moiety.
[0055] In some embodiments, the antiVSIGl antibodies comprise:
[0056] a) a heavy chain variable domain comprising a vHCDRl having the sequence
GGTFSSYS (SEQ ID NO:X), a vhCDR2 having the sequence IIPLFGTT (SEQ ID NO:X) and a vhCDR3 having the sequence ARSKDYYGSGMEALLMDV (SEQ ID NO:X); and
[0057] b) a light chain variable domain comprising a vLCDRl having the sequence
SGSVSTRNY (SEQ ID NO:X), a vLCDR2 having the sequence NTN (SEQ ID NO:X) and a vLCDR.3 having the sequence VFYMGSGRWV (SEQ ID NO:X). In further embodiments, this antibody comprises a covalently attached drug moiety.
[0058] In some embodiments, the antiVSIGl antibodies comprise:
[0059] a) a heavy chain variable domain comprising a vHCDRl having the sequence
GFTFSSYA (SEQ ID NO:X), a vhCDR2 having the sequence ISGSGGST (SEQ ID NO:X) and a vhCDR3 having the sequence AKELRGGSYYFTGTDAFDI (SEQ ID NO:X); and
[0060] b) a light chain variable domain comprising a vLCDRl having the sequence
QSLLKSNGYNY (SEQ ID NO:X), a vLCDR2 having the sequence LGS (SEQ ID NO:X) and a vLCDR3 having the sequence MQALQTPPT (SEQ ID NO:X). In further
embodiments, this antibody comprises a covalently attached drug moiety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] Figure 1 shows the amino acid sequence of the extracellular domain of human VSIGl, aligned with mouse VSIGl. The analysis shows 174/212 (82%) identical residues between the two proteins. The consensus sequence is marked in italics.
[0062] Figure 2A-2B shows the Fc sequences for human IgGl, IgG2, IgG3 and IgG4.
[0063] Figures 3A to 3H shows the heavy and light chain amino acid sequences for each of the enumerated anti-VSIGl antibodies.
[0064] Figure 4 shows the variable regions of the heavy and light chains, with frameworks and CDR domains outlined, of each of the enumerated anti-VSIGl antibodies.
[0065] Figures 5A and 5B shows IHC analysis as per Example 1. The IHC score is a measure of the amount of staining, with "1" being the lowest amount seen, "2" being medium and "3" being significant staining. The numbers in the columns represents the number of different samples that showed the staining; that is, one normal breast tissue showed staining of "1", and 18 samples of breast cancer tissue showed a staining level of "1".
[0066] Figures 6A and 6B depicts a summary of FACS binding results for anti-VSIGl Fabs selected for conversion to human IgGi. Results are expressed as the mean fluorescence intensity (MFI) of Fab binding against cells overexpressing VSIGl, divided by the MFI of Fab binding to parental control cells (i.e. MFI VSIGl cells/MFI parental cells = MFI ratio). Legend: (-) MFI ratio <5, designated as non-binding; (+) MFI ratio 5 - 20; (++) MFI ratio 20 - 50; (+++) MFI ratio 50 - 200; (++++) MFI ratio >200. Table shading highlights weaker and stronger binders (hatched and solid grey, respectively) or binders cross-reactive against human and mouse VSIGl (solid grey).
[0067] Figure 7 shows sensorgrams of A) VSIGl monomer injected at three concentrations (1.6nM, 4.9nM, 14.8nM) over mAb 156-01. D06 captured to a GLC ProteOn biosensor chip. The higher monomer concentrations appeared to show some nonspecific binding. B) VSIGl monomer injected at six concentrations (1.6nM - 400nM) over mAb 156-01. E05 captured to a GLC ProteOn biosensor chip. In both panels the black lines are the
sensorgrams of antigen injected over the mAbs and the red lines are a global fit of a 1 : 1 kinetic binding model. Binding constants are listed in Table 5. Data were processed and fit using ProteOn Manager Version 3.1.0.6.
[0068] Figure 8 shows sensorgrams of A) VSIGl monomer injected at four concentrations (1.6nM-44.4nM) over mAb 156-01. C09 captured to a GLC ProteOn biosensor chip. B) VSIGl monomer injected at six concentrations (1.6nM - 400nM) over mAb 156-01. C04 captured to a GLC ProteOn biosensor chip. In both panels the black lines are the
sensorgrams of antigen injected over the mAbs and the red lines are a global fit of a 1 : 1 kinetic binding model. Binding constants are listed in Table 5. Data were processed and fit using ProteOn Manager Version 3.1.0.6.
[0069] Figure 9 shows sensorgrams of A) VSIGl monomer injected at six concentrations (1.6nM-400nM) over mAb 156-01. C03 captured to a GLC ProteOn biosensor chip. Steady- state signal points were taken as an average between the green (109 sec) and red lines (119 sec). B) Steady-state RU signals as a function of [VSIGl] for mAb 156-01. C03 and fit with a simple 1 : 1 equilibrium model. C) VSIGl monomer injected at six concentrations (1.6nM- 400nM) over mAb 156-01. E12 captured to a GLC ProteOn biosensor chip. Steady-state signal points were taken as an average between the green (109 sec) and red lines (119 sec). D) Steady-state RU signals as a function of [VSIGl] for mAb 156-01. E12 and fit with a simple 1 : 1 equilibrium model. Binding constants are listed in Table 5. Data were processed and fit using Scrubber ProteOn software.
[0070] Figure 10 shows sensorgrams A) VSIGl monomer injected at four concentrations (1.6nM-44.4nM) over mAb 156-01.F04 captured to a GLC ProteOn biosensor chip. B) VSIGl monomer injected at six concentrations (1.6nM - 400nM) over mAb 156-01. B10 captured to a GLC ProteOn biosensor chip. In both panels the black lines are the
sensorgrams of antigen injected over the mAbs and the red lines are a global fit of a 1 : 1 kinetic binding model. Binding constants are listed in Table 5. Data were processed using ProteOn Manager Version 3.1.0.6. Data in A were fit using Scrubber ProteOn and the data in B were fit using ProteOn Manager.
[0071] Figure 11 shows sensorgrams of A) VSIGl monomer injected at six concentrations (1.6nM-400nM) over mAb 156-01.E06 captured to a GLC ProteOn biosensor chip. Steady- state signal points were taken as an average between the green (105 sec) and red lines (113 sec). B) Steady-state RU signals as a function of [VSIGl] for mAb 156-01. E06 and fit with a simple 1 : 1 equilibrium model. C) VSIGl monomer injected at six concentrations (1.6nM
- 400nM) over mAb 156-01. C08 captured to a GLC ProteOn biosensor chip. The black lines are the sensorgrams of antigen injected over the mAb and the red lines are a global fit of a 1 : 1 kinetic binding model. Binding constants are listed in Table 5. For both mAbs data were processed and fit using ProteOn Scrubber software.
[0072] Figure 12 shows sensorgrams of A) VSIGl monomer injected at six concentrations (1.6nM-400nM) over mAb 156-01. A03 captured to a GLC ProteOn biosensor chip. Steady- state signal points were taken as an average between the green (105 sec) and red lines (113 sec). B) Steady-state RU signals as a function of [VSIGl] for mAb 156-01. A03 and fit with a simple 1 : 1 equilibrium model. C) VSIGl monomer injected at six concentrations (1.6nM- 400nM) over mAb 156-01. D12 captured to a GLC ProteOn biosensor chip. Steady-state signal points were taken as an average between the green (107 sec) and red lines (113 sec). D) Steady-state RU signals as a function of [VSIGl] for mAb 156-01. D12 and fit with a simple 1 : 1 equilibrium model. Binding constants are listed in Table 5. Data were processed and fit using Scrubber ProteOn software.
[0073] Figure 13 shows sensorgrams of A) VSIGl monomer injected at six concentrations (1.6nM-400nM) over mAb 156-01. F10 captured to a GLC ProteOn biosensor chip. Steady- state signal points were taken as an average between the green (105 sec) and red lines (113 sec). B) Steady-state RU signals as a function of [VSIGl] for mAb 156-01. F10 and fit with a simple 1 : 1 equilibrium model. C) VSIGl monomer injected at six concentrations (1.6nM
- 400nM) over mAb 156-01. B08 covalently immobilized to a CM5 Biacore biosensor chip. The black lines are the sensorgrams of antigen injected over the mAb and the red lines are a global fit of a 1 : 1 kinetic binding model. Binding constants are listed in Table 5. Data were processed and fit using ProteOn Scrubber software for both mAbs.
[0074] Figure 14 shows sensorgrams of A) VSIG1 monomer injected at six concentrations (1.6nM-400nM) over mAb 156-01. D05 captured to a GLC ProteOn biosensor chip. Steady- state signal points were taken as an average between 110 sec and 116 sec. B) Steady-state RU signals as a function of [VSIG1] for mAb 156-01. D05 and fit with a simple 1 : 1 equilibrium model. Binding constants are listed in Table 5. Data were processed and fit using ProteOn Manager Version 3.1.0.6. Because of the apparent low affinity, higher concentrations of VSIG1 should be included for a better estimate of KD.
[0075] Figure 15 shows an example of a processed and referenced set of sensorgrams over all immobilized mAbs for generating the VSIG1 antigen blocking patterns for mAb CPA.4.023. Each panel represents a different ProteOn chip array spot having an immobilized mAb. The 15 different mAbs immobilized over spots A-0 are also immobilized in identical order over spots P-DD. Black sensorgrams in A-0 are mAb CPA.4.023 injected at a binding site concentration of -400 nM pre-mixed with VSIG1 antigen at a binding site concentration of 23 nM. Black sensorgrams in P-DD are 156-01. C08 injected at ~400nM without Ag as a control. All blue sensorgrams are VSIG1 injected at 23 nM without mAb. Blocking examples are seen in panels A, I (CPA.4.023 blocking itself), K, and N where the black response is significantly lowered from the blue response. Ag sandwiching with CPA.4.023 is in B,C,D,E,F,G,H,J,L,M,and O (black greater or equal to blue).
[0076] Figure 16 shows a binary matrix of mAb pair- wise blocking ("0", red box) or sandwiching ("1", green box) for 15 anti-VSIGl mAbs. MAbs listed vertically on the left of the matrix are mAbs covalently immobilized to the ProteOn array. MAbs listed horizontally across the top of the matrix are analytes injected with pre-mixed antigen. The black boxes outline four epitope bins according to the horizontal blocking patterns of the mAbs as immobilized ligands. The bottom two bins differ only in how their component mAbs block or sandwich Ag in the presence of mAb CPA.4.012.
[0077] Figure 17 shows hierarchical clustering dendrogram of the horizontal binding patterns of each mAb in the binary matrix in Figure 16. There appear to be four bins of mAbs with identical epitope blocking patterns within each group. Clone CPA.4.012 is the only mAb in bin 2. Bins 3 and 4 could be closely related, however, since the mAbs only differ in their antigen blocking with clone CPA.4.012.
[0078] Figure 18 shows anti-VSIGl antibody binding curves. Binding of anti-VSIGl antibodies to HEK 293 cells transfected with recombinant human (A), cynomolgus (B), or mouse (C) VSIG1. X-axis= nM of antibody. Y-axis= median fluoresce intensity.
[0079] Figure 19 shows expression of VSIG1 on OV-90, MKN-45, HUH-1, and DU145 Human Cancer Cell Lines. 5 μg/ml anti-VSIGl hlgGl or hlgGl Isotype control staining 50,000 cells in 50μ1 FACS buffer. rMFI = ratio MFI of anti-VSIGl MFI vs. hlgGl isotype control MFI.
[0080] Figure 20 shows in vitro cytotoxic activity of CPA.4.009 on OV-90 and DU145 cells. X-axis= nM antibody. Y-axis= relative light unit reading from cell titer glo.
[0081] Figure 21 shows in vitro cytotoxic activity of 15 anti-VISGl ADCs with 4 payloads on OV-90, MKN-45, and HUH-1 Human Cancer Cells. OV-90 and HUH-1 cells were treated with 0.6 μg/ml anti-VSIGl antibody or hlgGl isotype control. MKN-45 cells were treated with 5 μg/ml anti-VSIGl antibody or hlgGl isotype control. Antibody alone (gray bars) or media with Protein G preloaded with DM1 (A), MMAE (B), MMAF (C), or Duocarmycin (D) (black bars) was added to each well. "% untreated control" = (relative light units of treated samples / relative light units of untreated samples) * 100.
[0082] Figure 22 shows in vitro cytotoxic activity of 4 anti-VISGl ADCs with 4 payloads on HUH-1 Human Cancer Cells. Cells were treated with anti-VSIGl antibody or hlgGl isotype control. Media only or media with Protein G preloaded with DM1 (A), MMAE (B), MMAF (C), or Duocarmycin (D) was added to each well. Percent untreated control = (relative light units of treated samples/ relative light units of untreated samples) * 100.
[0083] Figure 23 Kinetic and thermodynamic binding constants of 15 anti-VSIGl human mAbs binding to VSIG1 monomer measured using SPR kinetics and steady-state methods. Results represent N=l .
[0084] Figure 24 shows the IC50 values From in Vitro Cytotoxic Assay with 4 anti-VSIGl ADCs. IC50 values shown are in nM. ND= not determined. [0085] Figure 25A-25F presents a comparison in the level of VSIGl expression on cancerous vs normal tissue in lung tissue (A), pancreatic tissue (B), liver tissue (C), ovarian tissue (D), colorectal tissue (E), uterine tissue (F), head and neck tissue (G), and kidney tissue (H).
[0086] Figure 26 shows the results of the IHC experiments of Example 1 , including the sample description and IHC scoring for each sample.
[0087] Figure 27 shows the results of the IHC experiments of Example 1 , including the sample description and IHC scoring for each sample.
[0088] Figure 28 depicts the closest human germline genes to the VH and VL sequences of the enumerated antibodies of the invention.
[0089] Figures 29A and 29B depicts the alignment and grouping of the VH amino acid sequences, using IMGT numbering.
[0090] Figures 30A and 30B depicts the alignment and grouping of the VL amino acid sequences, using IMGT numbering.
[0091] Figure 31A-31B present MED discovery engine expression results. Figure 31A - pancreatic cancer and normal expression of VSIGl . Figure 3 IB - cervix cancer and normal expression of VSIGl .
[0092] Figures 32A to 32J depict the nucleic acid sequences for the enumerated antibodies of the invention.
[0093] Figures 33A to 33F depict a number of additional amino acid sequences of the invetnion. Figures 33A - 33C depicts human VSIGl ECD fused to human IgGl Fc (VSIGl H:H, SEQ ID NO:XX which is the natural human Signal Peptide and human ECD (aa22- 232 of VSIG1_HUMAN) fused to Human IgGl Fc mutated at C220S of hinge. Figures 33D to 33F depicts mouse VSIGl ECD fused to mouse IgG2a Fc (VSIGl M:M, SEQ ID NO XX which is (PBG-SPl + Mouse ECD (aal-231 VSIGl _MOUSE) + Mouse IgG2a Fc)), used for anti-VSIGl Fab Antibodies generation by Phage Display.
[0094] Figure 34 shows immunohistochemistry validation of commercially available anti- human VSIGl antibody (R&D systems, cat # MAB481 8). Antibody used at 5 μ£< /νύ\,ρ 6.2 HIER, Biocare Universal HRP Detection, 40X.
[0095] Figure 35 provides an ovarian cancer tumor microarray. [0096] Figure 36 provides a stomach cancer tumor microarray.
[0097] Figure 37 provides a pancreatic cancer tumor microarray.
[0098] Figure 38 provides a liver cancer tumor microarray.
[0099] Figure 39 provides a lung cancer tumor microarray.
[00100] Figure 40 shows 2D in vitro cytotoxicity of 156-01.E05 directly conjugated to
DM1, MMAE, MMAF, and DMSA on OV-90 cells. X-axis = nM antibody. Y-axis = % of untreated control cells. hIgGl= human IgGl isotype control.
[00101] Figure 41 shows 2D in vitro cytotoxicity of 156-01.E05 directly conjugated to
DM1, MMAE, MMAF, and DMSA on Li-7 cells. X-axis = nM antibody. Y-axis = % of untreated control cells. hIgGl= human IgGl isotype control.
[00102] Figure 42 shows 2D in vitro cytotoxicity of 156-01.E05 directly conjugated to
DM1, MMAE, MMAF, and DMSA on HUH-1 cells. X-axis = nM antibody. Y-axis = % of untreated control cells. hIgGl= human IgGl isotype control.
[00103] Figure 43 shows 3D in vitro cytotoxicity of 156-01. E05 directly conjugated to
DM1, MMAE, MMAF, and DMSA on OV-90 cells. X-axis = nM antibody. Y-axis = % of untreated control cells. hIgGl= human IgGl isotype control.
[00104] Figure 44 shows 3D in vitro cytotoxicity of 156-01. E05 directly conjugated to
DM1, MMAE, MMAF, and DMSA on Li-7 cells. X-axis = nM antibody. Y-axis = % of untreated control cells. hIgGl= human IgGl isotype control.
[00105] Figure 45 shows 3D in vitro cytotoxicity of 156-01. E05 directly conjugated to
DM1, MMAE, MMAF, and DMSA on HUH-1 cells. X-axis = nM antibody. Y-axis = % of untreated control cells. hIgGl= human IgGl isotype controls.
DETAILED DESCRIPTION OF THE INVENTION
I. Overview
[00106] The present invention is directed to antibody compositions against human
VSIGl and methods of using the antibodies. As outlined herein, increased VSIGl expression has been shown in several cancers, including stomach, ovarian, liver and pancreatic cancers, and thus anti-VSIGl antibodies find use in the treatment of these cancers. In particular, the anti-VSIGl antibodies find use in treating non-small cell lung cancer (NSCLC)
adenocarcinoma, breast carcinoma, hepatocellular carcinoma (HCC), colon cancer, rectal cancer, kidney clear cell carcinoma, kidney papillary cell carcinoma, uterine cancer, stomach cancer, head and neck cancer, pancreatic cancer and ovarian cancer.
[00107] In addition, anti-VSIGl antibodies that include one or more covalently attached drugs, including chemotherapeutic drugs that can be cytotoxic and/or cytostatic, find particular use in the treatment of these particular cancers. These constructs are generally referred to as "antibody drug conjugates", or "ADCs", as described herein.
[00108] In some embodiments, the anti-VSIGl ADC antibody binds to the VSIG1 antigen and then is internalized by the cell, to deliver the drug (sometimes referred to in the art as a "payload") to the inside of the cell, to kill the cell. In some embodiments, as described more fully below, the linker that attaches the drugs to the antibody (sometimes referred to herein as the "ADC linker", to distinguish it from other linkers used in the invention), can be cleaved, thus releasing the drug for ease of action. In other embodiments, similarly described below, the ADC linker is not necessarily immediately cleaved but through the general action of the internal cellular machinery the drug is active. In some
embodiments, the anti-VSIGl antibody remains on the cell surface.
[00109] In some embodiments, the antibody-drug conjugate compounds (ADC) of the invention may selectively deliver an effective dose of a cytotoxic agent to tumor tissue whereby greater selectivity, i.e. a lower efficacious dose may be achieved.
[00110] In some embodiments, the bioavailability of the ADC, or an intracellular metabolite of the ADC, is improved in a mammal when compared to the corresponding drug moiety alone.
[00111] In some embodiments, the bioavailability of the ADC, or an intracellular metabolite of the ADC is improved in a mammal when compared to the corresponding antibody alone (antibody of the ADC, without the drug moiety or linker).
[00112] In some embodiments, the drug moiety of the ADC is not cleaved from the antibody until the antibody-drug conjugate binds to a cell-surface receptor, or enters a cell with a cell-surface receptor specific for the antibody of the antibody-drug conjugate. The drug moiety may be cleaved from the antibody after the antibody-drug conjugate enters the cell. The drug moiety may be intracellularly cleaved in a mammal from the antibody of the compound, or an intracellular metabolite of the compound, by enzymatic action, hydrolysis, oxidation, or other mechanism.
[00113] Antibodies
[00114] Accordingly, the invention provides anti-VSIGl antibodies. V-set and immunoglobulin domain containing protein 1 (VSIGl) is a transmembrane protein with an extracellular domain (ECD), the human sequence of which is shown in Figure 1. In some embodiments, the antibody used in the antibody-drug conjugate is an antibody that interacts with one or more epitopes on the VSIGl polypeptide. In some embodiments, the antibody is specific for the VSIGl extracellular domain.
[00115] As is discussed below, the term "antibody" is used generally. Antibodies that find use in the present invention can take on a number of formats as described herein, including traditional antibodies as well as antibody derivatives, fragments and mimetics, described below. In general, the term "antibody" includes any polypeptide that includes at least one antigen binding domain, as more fully described below. Antibodies may be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, as described herein. In some embodiments, antibodies of the invention bind specifically or substantially specifically to VSIGl molecules. The terms "monoclonal antibodies" and "monoclonal antibody composition", as used herein, refer to a population of antibody molecules that contain only one species of an antigen-binding site capable of immunoreacting with a particular epitope of an antigen, whereas the term "polyclonal antibodies" and "polyclonal antibody composition" refer to a population of antibody molecules that contain multiple species of antigen-binding sites capable of interacting with a particular antigen. A monoclonal antibody composition, typically displays a single binding affinity for a particular antigen with which it immunoreacts.
[00116] Traditional full length antibody structural units typically comprise a tetramer.
Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one "light" (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. The present invention is directed to the IgG class, which has several subclasses, including, but not limited to IgGl, IgG2, IgG3, and IgG4. Thus, "isotype" as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. While the exemplary antibodies herein are based on IgGl heavy constant regions, as shown in Figure 2, the anti- VSIG1 antibodies of the invention include those using IgG2, IgG3 and IgG4 sequences, or combinations thereof, as shown in Figure 2. For example, as more fully described below, different IgG isotypes have different effector functions which may or may not be desirable.
[00117] The amino-terminal portion of each chain includes a variable region of about
100 to 110 or more amino acids primarily responsible for antigen recognition, generally referred to in the art and herein as the "Fv domain" or "Fv region". In the variable region, three loops are gathered for each of the V domains of the heavy chain and light chain to form an antigen-binding site. Each of the loops is referred to as a complementarity-determining region (hereinafter referred to as a "CDR"), in which the variation in the amino acid sequence is most significant. "Variable" refers to the fact that certain segments of the variable region differ extensively in sequence among antibodies. Variability within the variable region is not evenly distributed. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions".
[00118] Each VH and VL is composed of three hypervariable regions
("complementary determining regions," "CDRs") and four FRs, arranged from amino- terminus to carboxy -terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4.
[00119] The hypervariable region generally encompasses amino acid residues from about amino acid residues 24-34 (LCDR1 ; "L" denotes light chain), 50-56 (LCDR2) and 89- 97 (LCDR3) in the light chain variable region and around about 31-35B (HCDR1 ; "H" denotes heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al, SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or those residues forming a hypervariable loop (e.g. residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. Specific CDRs of the invention are described below.
[00120] The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Kabat et al. collected numerous primary sequences of the variable regions of heavy chains and light chains. Based on the degree of conservation of the sequences, they classified individual primary sequences into the CDR and the framework and made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5 th edition, NIH publication, No. 91-3242, E. A. Kabat et al, entirely incorporated by reference).
[00121] In the IgG subclass of immunoglobulins, there are several immunoglobulin domains in the heavy chain. By "immunoglobulin (Ig) domain" herein is meant a region of an immunoglobulin having a distinct tertiary structure. Of interest in the present invention are the heavy chain domains, including, the constant heavy (CH) domains and the hinge domains. In the context of IgG antibodies, the IgG isotypes each have three CH regions. Accordingly, "CH" domains in the context of IgG are as follows: "CHI " refers to positions 118-220 according to the EU index as in Kabat. "CH2" refers to positions 237-340 according to the EU index as in Kabat, and "CH3" refers to positions 341-447 according to the EU index as in Kabat.
[00122] Throughout the present specification, either the IMTG numbering system or the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately, residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) (e.g, Kabat et al, supra (1991)). EU numbering as in Kabat is generally used for constant domains and/or the Fc domains.
[00123] The CDRs contribute to the formation of the antigen-binding, or more specifically, epitope binding site of antibodies. "Epitope" refers to a determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope.
[00124] The epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen binding peptide; in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide.
[00125] Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Conformational and nonconformational epitopes may be distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
[00126] An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be verified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen, for example "binning."
[00127] The present invention provides a number of anti-VSIGl antibodies as outlined herein, and they fall into different "bins", e.g. they are binding to different locations on the VSIG1 molecule. The present invention provides not only the enumerated antibodies but additional antibodies that compete with the enumerated antibodies to specifically bind to the VSIG1 molecule.
[00128] Included within the definition of "antibody" are "antigen-binding portion" of an antibody (also used interchangeably with "antigen-binding fragment", "antibody fragment" and "antibody derivative"). That is, for the purposes of the invention, an antibody of the invention has a minimum functional requirement that it bind to a VSIG1 antigen. As will be appreciated by those in the art, there are a large number of antigen fragments and derivatives that retain the ability to bind an antigen and yet have alternative structures, including, but not limited to, (i) the Fab fragment consisting of VL, VH, CL and CHI domains, (ii) the Fd fragment consisting of the VH and CHI domains, (iii) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, 1988, Science 242:423-426, Huston et al, 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883, entirely incorporated by reference), (iv) "diabodies" or "triabodies", multivalent or multispecific fragments constructed by gene fusion (Tomlinson et. al, 2000, Methods Enzymol. 326:461- 479; WO94/13804; Holliger et al, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448, all entirely incorporated by reference), (v) "domain antibodies" or "dAb" (sometimes referred to as an "immunoglobulin single variable domain", including single antibody variable domains from other species such as rodent (for example, as disclosed in WO 00/29004), nurse shark and Camelid V-HH dAbs, (vi) SMIPs (small molecule immunopharmaceuticals), camelbodies, nanobodies and IgNAR.
[00129] Still further, an antibody or antigen-binding portion thereof (antigen-binding fragment, antibody fragment, antibody portion) may be part of a larger immunoadhesion molecules, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules. Antibody portions, such as Fab and F(ab¾ fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.
[00130] Optional Antibody Engineering
[00131] The antibodies of the invention can be modified, or engineered, to alter the amino acid sequences by amino acid substitutions.
[00132] By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is to an amino acid that is not naturally occurring at the particular position, either not naturally occurring within the organism or in any organism. For example, the substitution E272Y refers to a variant polypeptide, in this case an Fc variant, in which the glutamic acid at position 272 is replaced with tyrosine. For clarity, a protein which has been engineered to change the nucleic acid coding sequence but not change the starting amino acid (for example exchanging CGG (encoding arginine) to CGA (still encoding arginine) to increase host organism expression levels) is not an "amino acid substitution"; that is, despite the creation of a new gene encoding the same protein, if the protein has the same amino acid at the particular position that it started with, it is not an amino acid substitution.
[00133] As discussed herein, amino acid substitutions can be made to alter the affinity of the CDRs for the VSIG1 protein (including both increasing and decreasing binding), as well as to alter additional functional properties of the antibodies. For example, the antibodies may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody according to at least some embodiments of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Such embodiments are described further below. The numbering of residues in the Fc region is that of the EU index of Kabat.
[00134] In one embodiment, the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
[00135] In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Pat. No. 6,165,745 by Ward et al.
[00136] In some embodiments, amino acid substitutions can be made in the Fc region, in general for altering binding to FcyR receptors. By "Fc gamma receptor", "FcyR" or "FcgammaR" as used herein is meant any member of the family of proteins that bind the IgG antibody Fc region and is encoded by an FcyR gene. In humans this family includes but is not limited to FcyRI (CD64), including isoforms FcyRIa, FcyRIb, and FcyRIc; FcyRII (CD32), including isoforms FcyRIIa (including allotypes H131 and R131), FcyRIIb (including FcyRIIb-l and FcyRIIb-2), and FcyRIIc; and FcyRIII (CD 16), including isoforms FcyRIIIa (including allotypes V158 and F158) and FcyRIIIb (including allotypes FcyRIIIb-NAl and FcyRIIIb-NA2) (Jefferis et al, 2002, Immunol Lett 82:57-65, entirely incorporated by reference), as well as any undiscovered human FcyRs or FcyR isoforms or allotypes. An FcyR may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcyRs include but are not limited to FcyRI (CD64), FcyRII (CD32), FcyRIII-l (CD 16), and FcyRIII-2 (CD 16-2), as well as any undiscovered mouse FcyRs or FcyR isoforms or allotypes.
[00137] There are a number of useful Fc substitutions that can be made to alter binding to one or more of the FcyR receptors. Substitutions that result in increased binding as well as decreased binding can be useful. For example, it is known that increased binding to FcyRIIIa generally results in increased ADCC (antibody dependent cell-mediated cytotoxicity; the cell- mediated reaction wherein nonspecific cytotoxic cells that express FcyRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell. Similarly, decreased binding to FcyRIIb (an inhibitory receptor) can be beneficial as well in some circumstances. Amino acid substitutions that find use in the present invention include those listed in U.S. Ser. Nos. 11/124,620 (particularly FIG. 41) and U.S. Patent No. 6,737,056, both of which are expressly incorporated herein by reference in their entirety and specifically for the variants disclosed therein. Particular variants that find use include, but are not limited to, 236A, 239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E,
239D/332E/330Y, 239D, 332E/330L, 299T and 297N.
[00138] In addition, the antibodies of the invention are modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively, to increase the biological half-life, the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos.
5,869,046 and 6,121,022 by Presta et al. Additional mutations to increase serum half life are disclosed in U.S. Patent Nos. 8,883,973, 6,737,056 and 7,371,826, and include 428L, 434A, 434S, and 428L/434S.
[00139] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the CI component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.
[00140] In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. Nos. 6,194,551 by Idusogie et al.
[00141] In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix
complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al.
[00142] In yet another example, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is described further in PCT Publication WO 00/42072 by Presta. Moreover, the binding sites on human IgGl for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R. L. et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 are shown to improve binding to FcyRIII. Additionally, the following combination mutants are shown to improve FcyRIII binding: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A. Furthermore, mutations such as M252Y/S254T/T256E or
M428L/N434S improve binding to FcRn and increase antibody circulation half-life (see Chan CA and Carter PJ (2010) Nature Rev Immunol 10:301-316). [00143] In still another embodiment, the antibody can be modified to abrogate in vivo
Fab arm exchange. Specifically, this process involves the exchange of IgG4 half-molecules (one heavy chain plus one light chain) between other IgG4 antibodies that effectively results in bispecific antibodies which are functionally monovalent. Mutations to the hinge region and constant domains of the heavy chain can abrogate this exchange (see Aalberse, RC, Schuurman J., 2002, Immunology 105:9-19).
[00144] In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen or reduce effector function such as ADCC. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence, for example N297. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
[00145] Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies according to at least some embodiments of the invention to thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (a (1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8 cell lines are created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 by Hanai et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the a 1,6 bond-related enzyme. Hanai et al. also describe cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Led 3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733 -267 '40). PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., (l,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17: 176-180). Alternatively, the fucose residues of the antibody may be cleaved off using a fucosidase enzyme. For example, the fucosidase a-L-fucosidase removes fucosyl residues from antibodies (Tarentino, A. L. et al. (1975) Biochem. 14:5516-23).
[00146] Another modification of the antibodies herein that is contemplated by the invention is pegylation or the addition of other water soluble moieties, typically polymers, e.g., in order to enhance half-life. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Ci-Cio) alkoxy- or aryloxy -poly ethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies according to at least some embodiments of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al. [00147] In addition to substitutions made to alter binding affinity to FcyRs and/or
FcRn and/or increase in vivo serum half life, additional antibody modifications can be made, as described in further detail below.
[00148] In some cases, affinity maturation is done. Amino acid modifications in the
CDRs are sometimes referred to as "affinity maturation". An "affinity matured" antibody is one having one or more alteration(s) in one or more CDRs which results in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). In some cases, although rare, it may be desirable to decrease the affinity of an antibody to its antigen, but this is generally not preferred.
[00149] In some embodiments, one or more amino acid modifications are made in one or more of the CDRs of the VISG1 antibodies of the invention. In general, only 1 or 2 or 3- amino acids are substituted in any single CDR, and generally no more than from 1, 2, 3. 4, 5, 6, 7, 8 9 or 10 changes are made within a set of CDRs. However, it should be appreciated that any combination of no substitutions, 1, 2 or 3 substitutions in any CDR can be independently and optionally combined with any other substitution.
[00150] Affinity maturation can be done to increase the binding affinity of the antibody for the VSIG1 antigen by at least about 10% to 50-100-150% or more, or from 1 to 5 fold as compared to the "parent" antibody. Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the VSIG1 antigen. Affinity matured antibodies are produced by known procedures. See, for example, Marks et al, 1992, Biotechnology 10:779-783 that describes affinity maturation by variable heavy chain (VH) and variable light chain (VL) domain shuffling. Random mutagenesis of CDR and/or framework residues is described in: Barbas, et al. 1994, Proc. Nat. Acad. Sci, USA 91 :3809-3813; Shier et al, 1995, Gene 169: 147-155; Yelton et al, 1995, J. Immunol. 155: 1994-2004; Jackson et al, 1995, J. Immunol. 154(7):3310-9; and Hawkins et al, 1992, J. Mol. Biol. 226:889-896, for example.
[00151] Alternatively, amino acid modifications can be made in one or more of the
CDRs of the antibodies of the invention that are "silent", e.g. that do not significantly alter the affinity of the antibody for the antigen. These can be made for a number of reasons, including optimizing expression (as can be done for the nucleic acids encoding the antibodies of the invention). [00152] Thus, included within the definition of the CDRs and antibodies of the invention are variant CDRs and antibodies; that is, the antibodies of the invention can include amino acid modifications in one or more of the CDRs of the enumerated antibodies of the invention. In addition, as outlined below, amino acid modifications can also independently and optionally be made in any region outside the CDRs, including framework and constant regions.
[00153] VSIGl Antibodies
[00154] The present invention provides anti-VISGl antibodies, including ADC compositions. (For convenience, "anti-VSIGl antibodies" and "VSIGl antibodies" are used interchangeably). The anti-VSIGl antibodies of the invention specifically bind to human VSIGl, and preferably the ECD of human VISG1, as depicted in Figure 1.
[00155] Specific binding for VSIGl or a VSIGl epitope can be exhibited, for example, by an antibody having a KD of at least about 10"4 M, at least about 10"5 M, at least about 10"6 M, at least about 10"7 M, at least about 10"8 M, at least about 10"9 M, alternatively at least about 10"10 M, at least about 10"11 M, at least about 10"12 M, or greater, where KD refers to a dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds an antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a control molecule relative to the VSIGl antigen or epitope.
[00156] To test whether an antibody competes for binding with one of the enumerated antibodies herein, generally BIACORE™ SPR assays are run, as outlined in the Examples in the "binning" section.
[00157] Also, specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KA or Ka for a VSIGl antigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the epitope relative to a control, where KA or Ka refers to an association rate of a particular antibody-antigen interaction.
[00158] Specific anti-VSIGl antibodies that are Fabs of the invention include those listed in Figure 4, including, but not limited to, CPA.4.013, CPA.4.016, CPA.4.017,
CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011. [00159] The antibodies described herein as labeled as follows. The antibodies have reference numbers, for example "CPA.4.013". This represents the combination of the variable heavy and variable light chains, as depicted in Figure 4, for example.
"CPA.4.013.VH" refers to the variable heavy portion of CPA.4.013, while "CPA.4.013.VL" is the variable light chain. "CPA.4.013.HC" refers to the entire heavy chain (e.g. variable and constant domain) of this molecule, and "CPA.4.013.LC" refers to the entire light light chain (e.g. variable and constant domain) of the same molecule. "CPA.4.013. HI" refers to a full length antibody comprising the variable heavy and light domains, including the constant domain of Human IgGl (hence, the HI; IgGl, IgG2, IgG3 and IgG4 sequences are shown in Figure 2). Accordingly, "CPA.4.013.H2" would be the CPA.4.013 variable domains linked to a Human IgG2. "CPA.4.013.H3" would be the CPA.4.013 variable domains linked to a Human IgG3, and "CPA.4.013.H4" would be the CPA.4.013 variable domains linked to a Human IgG4.
[00160] Thus, included in the definition of VSIG1 antibodies are 1) CPA.4.013,
CPA.4.013.VH, CPA.4.013.VL, CPA.4.013.HC, CPA.4.013.LC and CPA.4.013.H1, CPA.4.013.H2, CPA.4.013.H3 and CPA.4.013.H4; 2) CPA.4.016, CPA.4.016.VH,
CPA.4.016.VL, CPA.4.016.HC, CPA.4.016.LC, CPA.4.016.H1, CPA.4.016.H2,
CPA.4.016.H3 and CPA.4.016.H4; 3) CPA.4.017, CPA.4.017.VH, CPA.4.017.VL,
CPA.4.017.HC, CPA.4.017.LC, CPA.4.017.H1, CPA.4.017.H2, CPA.4.017.H3 and
CPA.4.017.H4; 4) CPA.4.019, CPA.4.019.VH, CPA.4.019.VL, CPA.4.019.HC,
CPA.4.019.LC, CPA.4.019.H1, CPA.4.019.H2, CPA.4.019.H3 and CPA.4.019.H4; 5) CPA.4.020, CPA.4.020.VH, CPA.4.020.VL, CPA.4.020.HC, CPA.4.020.LC, CPA.4.020.H1, CPA.4.020.H2, CPA.4.020.H3 and CPA.4.020.H4; 6) CPA.4.023, CPA.4.023.VH,
CPA.4.023.VL, CPA.4.023.HC, CPA.4.023.LC, CPA.4.023.H1, CPA.4.023.H2,
CPA.4.023.H3 and CPA.4.023.H4; 7) CPA.4.005, CPA.4.005.VH, CPA.4.005.VL,
CPA.4.005.HC, CPA.4.005.LC, CPA.4.005.H1, CPA.4.005.H2, CPA.4.005.H3 and
CPA.4.005.H4; 8) CPA.4.009, CPA.4.009.VH, CPA.4.009.VL, CPA.4.009.HC,
CPA.4.009.LC, CPA.4.009.H1, CPA.4.009.H2, CPA.4.009.H3 and CPA.4.009.H4; 9) CPA.4.027, CPA.4.027.VH, CPA.4.027.VL, CPA.4.027.HC, CPA.4.027.LC, CPA.4.027.H1, CPA.4.027.H2, CPA.4.027.H3 and CPA.4.027.H4; 10) CPA.4.028, CPA.4.028.VH, CPA.4.028.VL, CPA.4.028.HC, CPA.4.028.LC, CPA.4.028.H1, CPA.4.028.H2,
CPA.4.028.H3 and CPA.4.028.H4; 11) CPA.4.031, CPA.4.031.VH, CPA.4.031.VL, CPA.4.031.HC, CPA.4.031.LC, CPA.4.031.H1, CPA.4.031.H2, CPA.4.031.H3 and
CPA.4.031.H4; 12) CPA.4.033, CPA.4.033.VH, CPA.4.033.VL, CPA.4.033.HC,
CPA.4.033.LC, CPA.4.033.H1, CPA.4.033.H2, CPA.4.033.H3 and CPA.4.033.H4; 13) CPA.4.012, CPA.4.012.VH, CPA.4.012.VL, CPA.4.012.HC, CPA.4.012.LC, CPA.4.012.H1, CPA.4.012.H2, CPA.4.012.H3 and CPA.4.012.H4; 14) CPA.4.008, CPA.4.008,VH, CPA.4.008.VL, CPA.4.008.HC, CPA.4.008.LC, CPA.4.008.H1, CPA.4.008.H2,
CPA.4.008.H3 and CPA.4.008.H4; and 15) CPA.4.011, CPA.4.011.VH, CPA.4.011.VL, CPA.4.011.HC, CPA.4.011.LC, CPA.4.011.H1, CPA.4.011.H2, CPA.4.011.H3 and
CPA.4.011.H4.
[00161] In one embodiment, the human monoclonal antibody has at least one heavy chain CDR and at least one light chain CDR of a human monoclonal anti-VSIGl Fab component selected from the group consisting of: CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011.
[00162] In one embodiment, the human monoclonal antibody has at least two heavy chain CDRs and at least two light chain CDRs of a human monoclonal anti-VSIGl Fab component selected from the group consisting of: CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011..
[00163] In one embodiment, the human monoclonal antibody contains variable regions that are homologous (and preferably identical) to the variable regions of a human monoclonal anti-VSIGl Fab component selected from the group consisting of: CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011.
[00164] In some embodiments, the variable regions are both the heavy and light chain variable regions. In some embodiments, the variable region is the heavy chain variable region. In some embodiments, the variable region is the light chain variable region.
[00165] In one embodiment, the human monoclonal antibody has a heavy chain of a human monoclonal anti-VSIGl antibody selected from the group consisting of:
CPA.4.013.HC, CPA.4.016.HC, CPA.4.017.HC, CPA.4.019.HC, CPA.4.020.HC, CPA.4.023.HC, CPA.4.005.HC, CPA.4.009.HC, CPA.4.027.HC, CPA.4.028.HC,
CPA.4.031.HC, CPA.4.033.HC, CPA.4.012.HC, CPA.4.008.HC and CPA.4.011.HC.
[00166] In one embodiment, the human monoclonal antibody has a light chain of a human monoclonal anti-VSIGl antibody selected from the group consisting of:
CPA.4.013.LC, CPA.4.016.LC, CPA.4.017.LC, CPA.4.019.LC, CPA.4.020.LC,
CPA.4.023.LC, CPA.4.005.LC, CPA.4.009.LC, CPA.4.027.LC, CPA.4.028.LC,
CPA.4.031.LC, CPA.4.033.LC, CPA.4.012.LC, CPA.4.008.LC and CPA.4.011.LC.
[00167] In one embodiment, the human monoclonal antibody competes for binding at an epitope located within the extracellular domain of VSIGl with a Fab domain selected from the group consisting of: CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011 (or the full length antibodies as well).
[00168] In some embodiments, the anti-VSIGl antibody that competes for binding at an epitope located within the extracellular domain of VSIGl is selected from the group consisting of CPA.4.008, CPA.4.023, CPA.4.005 and CPA.4.020.
[00169] In some embodiments, the anti-VSIGl antibody that competes for binding at an epitope located within the extracellular domain of VSIGl is selected from the group consisting of CPA.4.013, CPA.4.031, CPA.4.033, CPA.4.009, and CPA.4.016.
[00170] In some embodiments, the anti-VSIGl antibody that competes for binding at an epitope located within the extracellular domain of VSIGl is selected from the group consisting of CPA.4.017, CPA.4.019, CPA.4.027, CPA.4.011, and CPA.4.028.
[00171] In some embodiments, the anti-VSIGl antibody that competes for binding at an epitope located within the extracellular domain of VSIGl is selected from the group consisting of CPA.4.012.
[00172] Included in the definition of VSIGl antibodies are antibodies that share homology or identity to the VSIGl antibodies enumerated herein. That is, in certain embodiments, an anti-VSIGl antibody according to the invention comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to isolated anti- VSIGl amino acid sequences of preferred anti-VSIGl immune molecules, respectively, wherein the antibodies retain the desired functional properties of the parent anti-VSIGl antibodies. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions X 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
[00173] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available commercially), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
[00174] Additionally or alternatively, the protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the antibody molecules according to at least some embodiments of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
[00175] In general, the percentage identity for comparison between VSIG1 antibodies is at least 75%, at least 80%, at least 90%, with at least about 95, 96, 97, 98 or 99% percent identity being preferred. The percentage identity may be along the whole amino acid sequence, for example the entire heavy or light chain or along a portion of the chains. For example, included within the definition of the anti-VSIGl antibodies of the invention are those that share identity along the entire variable region (for example, where the identity is 95 or 98% identical along the variable regions), or along the entire constant region, or along just the Fc domain. In some embodiments, the VH exhibits at least 95% identity or at least 99% identity to a VH of an anti-VSIGl antibody as described herein. In some embodiments, the VL exhibits at least 95% identity or at least 99% identity to a VL of an anti-VSIGl antibody as described herein.
[00176] In addition, also included are sequences that may have the identical CDRs but changes in the variable domain (or entire heavy or light chain). For example, VSIG1 antibodies include those with CDRs identical to those shown in Figure 4 but whose identity along the variable region can be lower, for example 95 or 98% percent identical.
[00177] The anti-VSIGl antibodies of the invention bind to several different epitopes on human VSIG1, as shown in the examples. As is known in the art, antibodies binding to different epitopes are said to be in different "bins". That is, antibodies are said to be in the same "bin" where the binding of one antibody blocks or competes with the binding of a different antibody.
[00178] In general, the anti-VSIGl antibodies of the invention are recombinant.
"Recombinant" as used herein, refers broadly with reference to a product, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non- recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
[00179] The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain
embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
[00180] In some embodiments, the anti-VSIGl antibody suitable for use in the antibody-drug conjugate compounds of the present invention binds to human VSIGl with a KD of 100 nM or less, 50 nM or less, 10 nM or less, or 1 nM or less (that is, higher binding affinity), or lpM or less, wherein KD is determined by known methods, e.g. surface plasmon resonance (SPR, e.g. Biacore assays), ELISA, KINEXA, and most typically SPR at 25° or 37° C.
[00181] In some embodiments, the anti-VSIGl antibody or derivative thereof suitable for use in the antibody-drug conjugate compounds of the present invention include anti- VSIGl antibody or derivative thereof wherein the VH and VL sequences of different anti- VSIGl antibody or derivative thereof can be "mixed and matched" to create other anti-VSIGl antibody or derivative thereof according to at least some embodiments of the invention. VSIGl binding of such "mixed and matched" antibodies can be tested using the binding assays described above, e.g., ELISAs). In some embodiments, when VH and VL chains are mixed and matched, a VH sequence from a particular VH/VL pairing is replaced with a structurally similar VH sequence. Likewise, in some embodiments, a VL sequence from a particular VH/VL pairing is replaced with a structurally similar VL sequence. For example, the VH and VL sequences of homologous antibodies are particularly amenable for mixing and matching.
[00182] Optionally, the antibody comprises CDR amino acid sequences selected from the group consisting of (a) sequences as listed herein; (b) sequences that differ from those CDR amino acid sequences specified in (a) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions except for the Serine residue in heavy chain CDR3 at position 100A (Kabat numbering system); (c) amino acid sequences having 90% or greater, 95% or greater, 98% or greater, or 99% or greater sequence identity to the sequences specified in (a) or (b); (d) a polypeptide having an amino acid sequence encoded by a polynucleotide having a nucleic acid sequence encoding the amino acids as listed herein.
[00183] Nucleic Acid Compositions
[00184] Nucleic acid compositions encoding the anti-VSIGl antibodies of the invention are also provided, as well as expression vectors containing the nucleic acids and host cells transformed with the nucleic acid and/or expression vector compositions.
[00185] The nucleic acid compositions that encode the VSIG1 antibodies will depend on the format of the antibody. For traditional, tetrameric antibodies containing two heavy chains and two light chains are encoded by two different nucleic acids, one encoding the heavy chain and one encoding the light chain. These can be put into a single expression vector or two expression vectors, as is known in the art, transformed into host cells, where they are expressed to form the antibodies of the invention. In some embodiments, for example when scFv constructs are used, a single nucleic acid encoding the variable heavy chain-linker-variable light chain is generally used, which can be inserted into an expression vector for transformation into host cells. The nucleic acids can be put into expression vectors that contain the appropriate transcriptional and translational control sequences, including, but not limited to, signal and secretion sequences, regulatory sequences, promoters, origins of replication, selection genes, etc.
[00186] For example, to express the antibodies DNAs encoding partial or full-length light and heavy chains, can be obtained by standard molecular biology techniques (e.g., PCR amplification or gene synthesis) and the DNAs can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term "operatively linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). The light and heavy chain variable regions of the antibodies described herein can be used to create full- length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segments within the vector and the VL segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
[00187] In addition to the antibody chain genes, the recombinant expression vectors according to at least some embodiments of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel ("Gene Expression Technology", Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP) and polyoma. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter or β-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SR a. promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).
[00188] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors according to at least some embodiments of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or
methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
[00189] For expression of the light and heavy chains, the expression vectors encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the antibodies according to at least some embodiments of the invention in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, and most preferably mammalian host cells, is the most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody. Prokaryotic expression of antibody genes has been reported to be ineffective for production of high yields of active antibody (Boss, M. A. and Wood, C. R. (1985)
Immunology Today 6: 12-13).
[00190] Preferred mammalian host cells for expressing the recombinant antibodies according to at least some embodiments of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) o/. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.
Antibodies can be recovered from the culture medium using standard protein purification methods.
[00191] Antibody coding sequences of interest include those encoded by native sequences, as well as nucleic acids that, by virtue of the degeneracy of the genetic code, are not identical in sequence to the disclosed nucleic acids, and variants thereof. Variant polypeptides can include amino acid substitutions as discussed herein. Techniques for in vitro mutagenesis of cloned genes are known. Also included in the subject invention are polypeptides that have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.
[00192] The invention further provides nucleic acids which encode an anti-VSIGl antibody according to the invention, or a fragment or conjugate thereof. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column
chromatography, agarose gel electrophoresis and others well known in the art. See, F.
Ausubel, et al, ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. A nucleic acid according to at least some embodiments of the invention can be, for example, DNA or RNA and may or may not contain intronic sequences.
[00193] To create a scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).
[00194] Antibody Drug Conjugates
[00195] The present invention finds particular use in the antibody drug conjugate
(ADC) arena. In these embodiments, the VSIG1 antibodies of the invention are conjugated with drugs to form antibody-drug conjugates (ADCs), sometimes also referred to herein as "antibody-drug compositions". In general, ADCs are used in oncology applications, where the use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents allows for the targeted delivery of the drug moiety to tumors, which can allow higher efficacy, lower toxicity, etc. An overview of this technology is provided in Ducry et al, Bioconjugate Chem, 21 :5-13 (2010), Carter et al., Cancer J. 14(3): 154 (2008) and Senter, Current Opin. Chem. Biol. 13:235-244 (2009), all of which are hereby incorporated by reference in their entirety.
[00196] Thus the invention provides VISG1 antibodies conjugated to drug moieties.
Generally, conjugation is done by covalent attachment to the antibody, as further described below, and generally relies on a linker, often a peptide linkage (which, as described below, may be designed to be sensitive to cleavage by proteases at the target site or not). Linkers used for attachment of drug moieties are referred to herein as "ADC linkers", to distinguish them from other linkers herein (although the peptide makeup of the linkers may be the same or different).
[00197] Generally, ADCs are formed using amino acid side chains within the antibody as attachment points for linker-drug components. As will be appreciated by those in the art, the number of drug moieties per antibody can change, depending on the conditions of the reaction, and can vary from 1 : 1 to 10: 1 drug: antibody, with from 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1 and 6: 1 finding particular use in some embodiments. As will be appreciated by those in the art, the actual number is an average, depending on the drug loading methodologies and specific techniques, as more fully described below.
[00198] Thus the invention provides VSIG1 antibodies conjugated to drug moieties.
As described below, the drug of the ADC can be any number of agents, including but not limited to cytotoxic agents such as chemotherapeutic agents, growth inhibitory agents, toxins (for example, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (that is, a radioconjugate) are provided. In other embodiments, the invention further provides methods of using the ADCs.
[00199] Drugs for use in the present invention include cytotoxic drugs, particularly those which are used for cancer therapy. Such drugs include, in general, DNA damaging agents, anti-metabolites, natural products and their analogs. Exemplary classes of cytotoxic agents include the enzyme inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNA cleavers, topoisomerase inhibitors, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, the podophyllotoxins, dolastatins, maytansinoids, differentiation inducers, and taxols.
[00200] Members of these classes include, for example, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxanes including taxol, taxotere retinoic acid, butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin, esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin, camptothecin, maytansinoids (including DM1), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), and maytansinoids (DM4) and their analogues.
[00201] Toxins may be used as antibody-toxin conjugates and include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as
geldanamycin (Mandler et al (2000) J. Nat. Cancer Inst. 92(19): 1573-1581 ; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10: 1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al, (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). Toxins may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.
[00202] Conjugates of a VSIG1 antibody and one or more small molecule toxins, such as a maytansinoids, dolastatins, auristatins, a trichothecene, calicheamicin, and CC1065, and the derivatives of these toxins that have toxin activity, are contemplated.
Maytansinoids
[00203] In some embodiments, the drug moiety for attachment to the anti-VSIGl antibody is a maytansinoid compound. Maytansine compounds are highly potent microtubule targeting compounds suitable for use as maytansinoid ADC moieties, and can be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al (2002) PNAS 99:7968-7973), or maytansinol and maytansinol analogues prepared synthetically according to known methods. As described below, drugs may be modified by the incorporation of a functionally active group such as a thiol or amine group for conjugation to the antibody.
[00204] Exemplary maytansinoid drug moieties include those having a modified aromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by lithium aluminum hydride reduction of ansamytocin P2); C-20-hydroxy (or C-20-demethyl) +/-C-19- dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and C-20-demethoxy, C-20- acyloxy (--OCOR), +/-dechloro (U.S. Pat. No. 4,294,757) (prepared by acylation using acyl chlorides) and those having modifications at other positions.
[00205] Exemplary maytansinoid drug moieties also include those having
modifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by the reaction of maytansinol with H2S or P2S5); C-14-alkoxymethyl(demethoxy/CH20R) (U.S. Pat. No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH20H or CH20Ac) (U.S. Pat. No. 4,450,254) (prepared from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by the conversion of maytansinol by Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by the demethylation of maytansinol by Streptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by the titanium trichloride/LAH reduction of maytansinol).
[00206] Of particular use are DM1 (disclosed in US Patent No. 5,208,020, incorporated by reference) and DM4 (disclosed in US Patent No. 7,276,497, incorporated by reference). See also a number of additional maytansinoid derivatives and methods in 5,416,064, WO/01/24763, 7,303,749, 7,601,354, USSN 12/631,508, WO02/098883, 6,441,163, 7,368,565, WO02/16368 and WO04/1033272, all of which are expressly incorporated by reference in their entirety.
[00207] ADCs containing maytansinoids, methods of making same, and their therapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020; 5,416,064; 6,441,163 and European Patent EP 0 425 235 Bl, the disclosures of which are hereby expressly incorporated by reference. Liu et al, Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described ADCs comprising a maytansinoid designated DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxic towards cultured colon cancer cells, and showed antitumor activity in an in vivo tumor growth assay.
[00208] Chari et al, Cancer Research 52: 127-131 (1992) describe ADCs in which a maytansinoid was conjugated via a disulfide linker to the murine antibody A7 binding to an antigen on human colon cancer cell lines, or to another murine monoclonal antibody TA.1 that binds the HER-2/neu oncogene. The cytotoxicity of the TA. l-maytansonoid conjugate was tested in vitro on the human breast cancer cell line SK-BR-3, which expresses 3x105 HER-2 surface antigens per cell. The drug conjugate achieved a degree of cytotoxicity similar to the free maytansinoid drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule. The A7-maytansinoid conjugate showed low systemic cytotoxicity in mice.
[00209] Auristatins and Dolastatins
[00210] In some embodiments, the ADC comprises a VSIG1 antibody conjugated to dolastatins or dolostatin peptidic analogs and derivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588). Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob. Agents Chemother.
42:2961-2965). The dolastatin or auristatin drug moiety may be attached to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02/088172).
[00211] Exemplary auristatin embodiments include the N-terminus linked
monomethylauristatin drug moieties DE and DF, disclosed in "Senter et al, Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, presented Mar. 28, 2004 and described in United States Patent Publication No. 2005/0238648, the disclosure of which is expressly incorporated by reference in its entirety. [00212] An exemplary auristatin embodiment is MMAE (shown in Figure 10 wherein the wavy line indicates the covalent attachment to a linker (L) of an antibody drug conjugate; see US Patent No. 6,884,869 expressly incorporated by reference in its entirety).
[00213] Another exemplary auristatin embodiment is MMAF, shown in Figure 10 wherein the wavy line indicates the covalent attachment to a linker (L) of an antibody drug conjugate (US 2005/0238649, 5,767,237 and 6,124,431, expressly incorporated by reference in their entirety):
[00214] Additional exemplary embodiments comprising MMAE or MMAF and various linker components (described further herein) have the following structures and abbreviations (wherein Ab means antibody and p is 1 to about 8):
[00215] Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schroder and K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry. The auristatin/dolastatin drug moieties may be prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111 :5463-5465; Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R., et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol 21(7):778-784.
[00216] Calicheamicin
[00217] In other embodiments, the ADC comprises an antibody of the invention conjugated to one or more calicheamicin molecules. For example, Mylotarg is the first commercial ADC drug and utilizes calicheamicin γΐ as the pay load (see US Patent No. 4,970,198, incorporated by reference in its entirety). Additional calicheamicin derivatives are described in US Patent Nos. 5,264,586, 5,384,412, 5,550,246, 5,739,116, 5,773,001, 5,767,285 and 5,877,296, all expressly incorporated by reference. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company). Structural analogues of calicheamicin which may be used include, but are not limited to, γΐΐ, α2Ι, α2Ι, N-acetyl- γΐΐ, PSAG and ΘΙ1 (Hinman et al, Cancer Research 53:3336-3342 (1993), Lode et al, Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patents to American Cyanamid). Another anti-tumor drug that the antibody can be conjugated is QFA which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects.
[00218] Duocarmycins
CC-1065 (see 4,169,888, incorporated by reference) and duocarmycins are members of a family of antitumor antibiotics utilized in ADCs. These antibiotics appear to work through sequence-selectively alkylating DNA at the N3 of adenine in the minor groove, which initiates a cascade of events that result in apoptosis. By "duocarmycin" herein is meant duocarmycin and analogs.
[00219] Important members of the duocarmycins include duocarmycin A (US Patent
No. 4,923,990, incorporated by reference, particularly for the duocarmycin A structure) and duocarmycin SA (U.S. Pat. No. 5,101,038, incorporated by reference, particularly for the duocarmycin SA structure), and a large number of analogues as described in US Patent Nos. 7,517,903, 7,691,962, 5,101,038; 5,641,780; 5,187,186; 5,070,092; 5,070,092; 5,641,780; 5,101,038; 5,084,468, 5,475,092, 5,585,499, 5,846,545, WO2007/089149,
WO2009/017394A1, 5,703,080, 6,989,452, 7,087,600, 7,129,261, 7,498,302, and 7,507,420, all of which are expressly incorporated by reference, in particular for the structure of the duocarmycin.
VI. Other Cytotoxic Agents
[00220] Other antitumor agents that can be conjugated to the antibodies of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296). The antibody conjugates according to at least some embodiments of the invention can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may also include, for example, a protein such as tumor necrosis factor or interferon-γ; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.
[00221] Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAP II, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published Oct. 28, 1993.
[00222] The present invention further contemplates an ADC formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA
endonuclease such as a deoxyribonuclease; DNase).
[00223] For selective destruction of the tumor, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32, Pb212 and radioactive isotopes of Lu.
[00224] The radio- or other labels may be incorporated in the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as Tc99m or 1123, Rel86, Rel88 and Inl l l can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate Iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.
[00225] In some instances, separation, purification, and characterization of homogeneous Antibody-Drug-conjugates where p is a certain value from Antibody-Drug- Conjugates with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis. In exemplary embodiments, p is 2, 3, 4, 5, 6, 7, or 8 or a fraction thereof. [00226] A number of different reactions are available for covalent attachment of drugs and/or linkers to binding agents. This is can be accomplished by reaction of the amino acid residues of the binding agent, for example, antibody molecule, including the amine groups of lysine, the free carboxylic acid groups of glutamic and aspartic acid, the sulfhydryl groups of cysteine and the various moieties of the aromatic amino acids. A commonly used nonspecific methods of covalent attachment is the carbodiimide reaction to link a carboxy (or amino) group of a compound to amino (or carboxy) groups of the antibody. Additionally, bifunctional agents such as dialdehydes or imidoesters have been used to link the amino group of a compound to amino groups of an antibody molecule.
[00227] Also available for attachment of drugs to binding agents is the Schiff base reaction. This method involves the periodate oxidation of a drug that contains glycol or hydroxy groups, thus forming an aldehyde which is then reacted with the binding agent. Attachment occurs via formation of a Schiff base with amino groups of the binding agent. Isothiocyanates can also be used as coupling agents for covalently attaching drugs to binding agents. Other techniques are known to the skilled artisan and within the scope of the present invention.
[00228] In some embodiments, an intermediate, which is the precursor of the linker, is reacted with the drug under appropriate conditions. In other embodiments, reactive groups are used on the drug and/or the intermediate. The product of the reaction between the drug and the intermediate, or the derivatized drug, is subsequently reacted with an Fc variant antibody of the invention under appropriate conditions.
[00229] It will be understood that chemical modifications may also be made to the desired compound in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention. For example a functional group e.g. amine, hydroxyl, or sulfhydryl, may be appended to the drug at a position which has minimal or an acceptable effect on the activity or other properties of the drug
ADC Linker Units
[00230] Typically, the antibody-drug conjugate compounds comprise a linker unit between the drug unit and the antibody unit. In some embodiments, the antibody causes the ADC to bind to the target cancer cells. In some embodiments, the ADC is then internalized by the cell and the drug is released into the cell. Because of the targeting, the side effects are lower and give a wider therapeutic window. Hydrophilic linkers (e.g., PEG4Mal) help prevent the drug being pumped out of resistant cancer cells through MDR (multiple drug resistance) transporters. In some embodiments, the linker is cleavable under intracellular or extracellular conditions, such that cleavage of the linker releases the drug unit from the antibody in the appropriate environment. For example, solid tumors that secrete certain proteases may serve as the target of the cleavable linker; in other embodiments, it is the intracellular proteases that are utilized. In yet other embodiments, the linker unit is not cleavable and the drug is released, for example, by antibody degradation in lysosomes.
[00231] In some embodiments, the linker is cleavable by a cleaving agent that is present in the intracellular environment (for example, within a lysosome or endosome or caveolea). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long or more. Examples of linker types that have been used to conjugate a drug moiety to an antibody include, but are not limited to, polyethylene glycol, hydrazones, thioethers, esters, disulfides and peptide-containing linkers. A linker can be chosen that is, for example, susceptible to cleavage by low pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D). A cleavable linker is typically susceptible to cleavage under intracellular conditions. The linker can be any suitable linker, such as, for example, a peptidyl, hydrazine, or disulfide linker.
[00232] In some embodiments, the linker prevents the drug moiety from being removed from the cancer cell.
[00233] In some embodiments, the choice of the linker enhances the plasma stability of the antibody-drug conjugate, preventing unwanted release of the drug at non-tumor sites. In some embodiments, the choice of linker reduces the risk of impairing the affinity of the antibody to VSIG1.
[00234] Cleaving agents can include, without limitation, cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, a peptidyl linker that is cleavable by the thiol- dependent protease cathepsin-B, which is highly expressed in cancerous tissue, can be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Gly linker (SEQ ID NO: 11). Other examples of such linkers are described, e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference in its entirety and for all purposes.
[00235] In some embodiments, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the val-cit linker).
[00236] In other embodiments, the cleavable linker is pH-sensitive, that is, sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker hydrolyzable under acidic conditions. For example, an acid-labile linker that is hydrolyzable in the lysosome (for example, a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) may be used. (See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805;
5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al, 1989, Biol. Chem. 264: 14653-14661.) Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via an
acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).
[00237] In yet other embodiments, the linker is cleavable under reducing conditions
(for example, a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2- pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2- pyridyl-dithio)toluene)- , SPDB and SMPT. (See, e.g., Thorpe et al, 1987, Cancer Res.
47:5924-5931 ; Wawrzynczak et al, In Immunoconjugates: Antibody Conjugates in
Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)
[00238] In other embodiments, the linker is a malonate linker (Johnson et al, 1995,
Anticancer Res. 15: 1387-93), a maleimidobenzoyl linker (Lau et al, 1995, Bioorg-Med- Chem. 3(10): 1299-1304), or a 3'-N-amide analog (Lau et al, 1995, Bioorg-Med-Chem. 3(10): 1305-12).
[00239] In yet other embodiments, the linker unit is not cleavable and the drug is released by antibody degradation. (See U.S. Publication No. 2005/0238649 incorporated by reference herein in its entirety and for all purposes).
[00240] In many embodiments, the linker is self-immolative. As used herein, the term
"self-immolative spacer" refers to a bifunctional chemical moiety that is capable of covalently linking together two spaced chemical moieties into a stable tripartite molecule. It will spontaneously separate from the second chemical moiety if its bond to the first moiety is cleaved. See for example, WO 2007059404A2, WO06110476A2, WO05112919A2, WO2010/062171, WO09/017394, WO07/089149, WO 07/018431, WO04/043493 and WO02/083180, which are directed to drug-cleavable substrate conjugates where the drug and cleavable substrate are optionally linked through a self-immolative linker and which are all expressly incorporated by reference. In some embodiments, these can also be called "self elimination spacers" or "self elimination connectors", the idea being that upon cleavage by a tumor-associated protease or enzyme, the linker then self-eliminates to release the cytotoxic drug. Particularly useful self-immolative or self-elimination spacers are referred to as the SpaceLink platform of Syntarga, as described in US Patent No. 7,223,837, hereby incorporated by reference in its entirety, and particularly for the exemplification of these linkers and structures, the associated description therein.
[00241] Often the linker is not substantially sensitive to the extracellular environment.
As used herein, "not substantially sensitive to the extracellular environment," in the context of a linker, means that no more than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of the linkers, in a sample of antibody-drug conjugate compound, are cleaved when the antibody-drug conjugate compound presents in an extracellular environment (for example, in plasma).
[00242] Whether a linker is not substantially sensitive to the extracellular environment can be determined, for example, by incubating with plasma the antibody-drug conjugate compound for a predetermined time period (for example, 2, 4, 8, 16, or 24 hours) and then quantitating the amount of free drug present in the plasma. [00243] In other, non-mutually exclusive embodiments, the linker promotes cellular internalization. In certain embodiments, the linker promotes cellular internalization when conjugated to the therapeutic agent (that is, in the milieu of the linker-therapeutic agent moiety of the antibody-drug conjugate compound as described herein). In yet other embodiments, the linker promotes cellular internalization when conjugated to both the auristatin compound and the Fc variant antibodies of the invention.
[00244] A variety of exemplary linkers that can be used with the present compositions and methods are described in WO 2004-010957, U.S. Publication No. 2006/0074008, U.S. Publication No. 20050238649, and U.S. Publication No. 2006/0024317 (each of which is incorporated by reference herein in its entirety and for all purposes).
[00245] In some embodiments, the linker has multiple purposes, including increasing drug solubility and pharmacokinetics, as well as increased drug load, such as the "Fleximer" polymer of Mersana Therapeutics; see Yurkovetskiy et al, Cancer Research, doi
10.0.1158/0008-5472.CAN-15-0129.
[00246] Drug Loading
[00247] For compositions comprising a plurality of antibodies, the drug loading is represented by p, the average number of drug molecules per antibody. Drug loading may range from 1 to 20 drugs (D) per Antibody. The average number of drugs per antibody in preparation of conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC. The quantitative distribution of Antibody- Drug-Conjugates in terms of p may also be determined.
[00248] Drug loading ("p") may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20 or more moieties (D) per antibody, although frequently the average number is a fraction or a decimal. Generally, drug loading of from 1 to 4 is frequently useful, and from 1 to 2 is also useful. ADCs of the invention include collections of antibodies conjugated with a range of drug moieties, from 1 to 20. The average number of drug moieties per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as mass spectroscopy and, ELISA assay.
[00249] The quantitative distribution of ADC in terms of p may also be determined. In some instances, separation, purification, and characterization of homogeneous ADC where p is a certain value from ADC with other drug loadings may be achieved by means such as electrophoresis.
[00250] For some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, where the attachment is a cysteine thiol, as in the exemplary embodiments above, an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached. In certain embodiments, higher drug loading, e.g. p>5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates. In certain embodiments, the drug loading for an ADC of the invention ranges from 1 to about 8; from about 2 to about 6; from about 3 to about 5; from about 3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8; from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3 to about 3.8; or from about 3.3 to about 3.7. Indeed, it has been shown that for certain ADCs, the optimal ratio of drug moieties per antibody may be less than 8, and may be about 2 to about 5. See US 2005-0238649 Al (herein incorporated by reference in its entirety).
[00251] In certain embodiments, fewer than the theoretical maximum of drug moieties are conjugated to an antibody during a conjugation reaction. An antibody may contain, for example, lysine residues that do not react with the drug-linker intermediate or linker reagent. Generally, antibodies do not contain many free and reactive cysteine thiol groups which may be linked to a drug moiety; indeed most cysteine thiol residues in antibodies exist as disulfide bridges. In certain embodiments, an antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups. In certain embodiments, an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine.
[00252] The loading (drug/antibody ratio) of an ADC may be controlled in different ways, e.g., by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reductive conditions for cysteine thiol modification, (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of cysteine residues is modified for control of the number and/or position of linker-drug attachements (such as thioMab or thioFab prepared as disclosed herein and in
WO2006/034488 (herein incorporated by reference in its entirety)).
[00253] It is to be understood that where more than one nucleophilic group reacts with a drug-linker intermediate or linker reagent followed by drug moiety reagent, then the resulting product is a mixture of ADC compounds with a distribution of one or more drug moieties attached to an antibody. The average number of drugs per antibody may be calculated from the mixture by a dual ELIS A antibody assay, which is specific for antibody and specific for the drug. Individual ADC molecules may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction chromatography.
[00254] In some embodiments, a homogeneous ADC with a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography.
Methods of Determining Cytotoxic Effect of ADCs
[00255] Methods of determining whether a drug or ADC exerts a cytostatic and/or cytotoxic effect on a cell are known. Generally, the cytotoxic or cytostatic activity of an ADC can be measured by: exposing mammalian cells expressing a target protein of the ADC in a cell culture medium; culturing the cells for a period from about 6 hours to about 5 days; and measuring cell viability. Cell-based in vitro assays can be used to measure viability
(proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of the Antibody Drug conjugate.
[00256] For determining whether an ADC exerts a cytostatic effect, a thymidine incorporation assay may be used. For example, cancer cells expressing a target antigen at a density of 5,000 cells/well of a 96-well plated can be cultured for a 72-hour period and exposed to 0.5 of H-thymidine during the final 8 hours of the 72-hour period. The incorporation of H-thymidine into cells of the culture is measured in the presence and absence of the ADC.
[00257] For determining cytotoxicity, necrosis or apoptosis (programmed cell death) can be measured. Necrosis is typically accompanied by increased permeability of the plasma membrane; swelling of the cell, and rupture of the plasma membrane. Apoptosis is typically characterized by membrane blebbing, condensation of cytoplasm, and the activation of endogenous endonucleases. Determination of any of these effects on cancer cells indicates that an ADC is useful in the treatment of cancers.
[00258] Cell viability can be measured by determining in a cell the uptake of a dye such as neutral red, trypan blue, or ALAMAR™ blue (see, e.g., Page et al, 1993, Intl. J. Oncology 3:473-476). In such an assay, the cells are incubated in media containing the dye, the cells are washed, and the remaining dye, reflecting cellular uptake of the dye, is measured spectrophotometrically. The protein-binding dye sulforhodamine B (SRB) can also be used to measure cytoxicity (Skehan et al, 1990, J. Natl. Cancer Inst. 82: 1107-12).
[00259] Alternatively, a tetrazolium salt, such as MTT, is used in a quantitative colorimetric assay for mammalian cell survival and proliferation by detecting living, but not dead, cells (see, e.g., Mosmann, 1983, J. Immunol. Methods 65:55-63).
[00260] Apoptosis can be quantitated by measuring, for example, DNA fragmentation.
Commercial photometric methods for the quantitative in vitro determination of DNA fragmentation are available. Examples of such assays, including TUNEL (which detects incorporation of labeled nucleotides in fragmented DNA) and ELISA-based assays, are described in Biochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).
[00261] Apoptosis can also be determined by measuring morphological changes in a cell. For example, as with necrosis, loss of plasma membrane integrity can be determined by measuring uptake of certain dyes (e.g., a fluorescent dye such as, for example, acridine orange or ethidium bromide). A method for measuring apoptotic cell number has been described by Duke and Cohen, Current Protocols in Immunology (Coligan et al. eds., 1992, pp. 3.17.1-3.17.16). Cells also can be labeled with a DNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide) and the cells observed for chromatin condensation and margination along the inner nuclear membrane. Other morphological changes that can be measured to determine apoptosis include, e.g., cytoplasmic condensation, increased membrane blebbing, and cellular shrinkage.
[00262] The presence of apoptotic cells can be measured in both the attached and "floating" compartments of the cultures. For example, both compartments can be collected by removing the supernatant, trypsinizing the attached cells, combining the preparations following a centrifugation wash step (e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., by measuring DNA fragmentation). (See, e.g., Piazza et al, 1995, Cancer Research 55:3110-16).
[00263] In vivo, the effect of a therapeutic composition of the VSIGl antibody of the invention can be evaluated in a suitable animal model. For example, xenogenic cancer models can be used, wherein cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Klein et al, 1997, Nature Medicine 3: 402-408). Efficacy can be measured using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.
[00264] Treatment
[00265] The anti-VSIGl antibodies of the invention are used to treat certain cancers.
According to some embodiments, cancer cells express VSIGl at a sufficient level and said cancer is as described herein, wherein VSIGl expression on any the cancer cells could be either present prior to cancer treatment or induced post treatment. By "expressing VSIGl at a sufficient level" it is meant that such cells express VSIGl protein at a high enough level according to an assay. For example, if the assay is IHC (immunohistochemistry), and expression is measured on a scale of 0 to 3 (0-no expression, 1- faint staining, 2-moderate and 3-strong expression), then a sufficient level of VSIGl expression would optionally be at least 1, alternatively be at least 2, alternatively be at least 3. Optionally the antibody-drug conjugate compounds as described herein may be used for such an assay.
[00266] In some embodiments, an antibody-drug conjugate according to the present invention is used for the treatment and/or diagnosis of cancer, wherein the cancer, and/or immune cells infiltrating the cancer, and/or stromal cells of the subject express VSIGl, e.g. prior to, or following cancer therapy, and wherein said cancer is e.g., selected from the group consisting of stomach cancer, ovarian cancer, liver cancer and prostate cancer, as shown in the IHC experiments as outlined herein.
Diagnostic uses of anit-VSIGl antibodies
[00267] The anti-VSIGl antibodies provided also find use in the in vitro or in vivo imaging of tumors or autoimmune disease states associated with VSIGl . In some
embodiments, the antibodies described herein are used for both diagnosis and treatment, or for diagnosis alone. When anti-VSIGl antibodies are used for both diagnosis and treatment, some embodiments rely on two different anti-VSIGl antibodies to two different epitopes, such that the diagnostic antibody does not compete for binding with the therapeutic antibody, although in some cases the same antibody can be used for both. Thus included in the invention are compositions comprising a diagnostic antibody and a therapeutic antibody, and in some embodiments, the diagnostic antibody is labeled as described herein. In addition, the composition of therapeutic and diagnostic antibodies can also be co-administered with other drugs as outlined herein.
[00268] Particularly useful antibodies for use in diagnosis include, but are not limited to, those encompassing the following Fabs: CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011, and particularly the ".HI" constructs as described herein.
[00269] In many embodiments, a diagnostic antibody is labeled. By "labeled" herein is meant that the antibodies disclosed herein have one or more elements, isotopes, or chemical compounds attached to enable the detection in a screen or diagnostic procedure. In general, labels fall into several classes: a) immune labels, which may be an epitope incorporated as a fusion partner that is recognized by an antibody, b) isotopic labels, which may be radioactive or heavy isotopes, c) small molecule labels, which may include fluorescent and colorimetric dyes, or molecules such as biotin that enable other labeling methods, and d) labels such as particles (including bubbles for ultrasound labeling) or paramagnetic labels that allow body imagining. Labels may be incorporated into the antibodies at any position and may be incorporated in vitro or in vivo during protein expression, as is known in the art.
[00270] Diagnosis can be done either in vivo, by administration of a diagnostic antibody that allows whole body imaging as described below, or in vitro, on samples removed from a patient. "Sample" in this context includes any number of things, including, but not limited to, bodily fluids (including, but not limited to, blood, urine, serum, lymph, saliva, anal and vaginal secretions, perspiration and semen), as well as tissue samples such as result from biopsies of relevant tissues. [00271] In some embodiments, in vivo imaging is done, including but not limited to ultrasound, CT scans, X-rays, MRI and PET scans, as well as optical techniques, such as those using optical labels for tumors near the surface of the body.
[00272] In vivo imaging of diseases associated with VSIG1 may be performed by any suitable technique. For example, 99Tc-labeling or labeling with another .beta. -ray emitting isotope may be used to label anti-VSIGl antibodies. Variations on this technique may include the use of magnetic resonance imaging (MRI) to improve imaging over gamma camera techniques.
[00273] In one embodiment, the present invention provides an in vivo imaging method wherein an anti-VSIGl antibody is conjugated to a detection-promoting agent, the conjugated antibody is administered to a host, such as by injection into the bloodstream, and the presence and location of the labeled antibody in the host is assayed. Through this technique and any other diagnostic method provided herein, the present invention provides a method for screening for the presence of disease-related cells in a human patient or a biological sample taken from a human patient.
[00274] For diagnostic imaging, radioisotopes may be bound to an anti-VSIGl antibody either directly, or indirectly by using an intermediary functional group. Useful intermediary functional groups include chelators, such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid (see for instance U.S. Pat. No. 5,057,313), in such diagnostic assays involving radioisotope-conjugated anti-VSIGl antibodies, the dosage of conjugated anti-VSIGl antibody delivered to the patient typically is maintained at as low a level as possible through the choice of isotope for the best combination of minimum half-life, minimum retention in the body, and minimum quantity of isotope, which will permit detection and accurate measurement.
[00275] In addition to radioisotopes and radio-opaque agents, diagnostic methods may be performed using anti-VSIGl antibodies that are conjugated to dyes (such as with the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules and enhancing agents (e.g. paramagnetic ions) for magnetic resonance imaging (MRI) (see, e.g., U.S. Pat. No. 6,331,175, which describes MRI techniques and the preparation of antibodies conjugated to a MRI enhancing agent). Such diagnostic/detection agents may be selected from agents for use in magnetic resonance imaging, and fluorescent compounds. [00276] In order to load an anti-VSIGl antibody with radioactive metals or paramagnetic ions, it may be necessary to react it with a reagent having a long tail to which are attached a multiplicity of chelating groups for binding the ions. Such a tail may be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which can be bound chelating groups such as, e.g., porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes, and like groups known to be useful for this purpose.
[00277] Chelates may be coupled to anti-VSIGl antibodies using standard chemistries. A chelate is normally linked to an anti-VSIGl antibody by a group that enables formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and/or internal cross-linking.
[00278] Examples of potentially useful metal-chelate combinations include 2-benzyl-
DTPA and its monomethyl and cyclohexyl analogs, used with diagnostic isotopes in the general energy range of 60 to 4,000 keV, such as 125I, 12 I, 124I, 62Cu, 64Cu, 18F, mIn, 67Ga, 99Tc, 94TC, nC, 1 N, 50, and 76Br, for radio-imaging.
[00279] Labels include a radionuclide, a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photoactive agent. Such diagnostic agents are well known and any such known diagnostic agent may be used. Non-limiting examples of diagnostic agents may include a radionuclide such as HOIn, l l lln, 177Lu, 18F, 52Fe, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 86Y, 90Y, 89Zr, 94mTc, 94Tc, 99mTc, 1201, 1231, 1241, 1251, 1311, 154-158Gd, 32P, 11C, 13N, 150, 186Re, 188Re, 51Mn, 52mMn, 55Co, 72As, 75Br, 76Br, 82mRb, 83Sr, or other .gamma.-, .beta.-, or positron-emitters.
[00280] Paramagnetic ions of use may include chromium (III), manganese (II), iron
(III), iron (II), cobalt (II), nickel (III), copper (III), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium (III), Metal contrast agents may include lanthanum (III), gold (III), lead (II) or bismuth (III). [00281] Ultrasound contrast agents may comprise liposomes, such as gas filled liposomes. Radiopaque diagnostic agents may be selected from compounds, barium compounds, gallium compounds, and thallium compounds.
[00282] These and similar chelates, when complexed with non-radioactive metals, such as manganese, iron, and gadolinium may be useful for MRI diagnostic methods in connection with anti-VSIGl antibodies. Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with a variety of metals and radiometals, most particularly with
radionuclides of gallium, yttrium, and copper, respectively. Such metal-chelate complexes may be made very stable by tailoring the ring size to the metal of interest. Other ring-type chelates such as macrocyclic poly ethers, which are of interest for stably binding nuclides, such as 223Ra may also be suitable in diagnostic methods.
[00283] Thus, the present invention provides diagnostic anti-VSIGl antibody conjugates, wherein the anti-VSIGl antibody conjugate is conjugated to a contrast agent (such as for magnetic resonance imaging, computed tomography, or ultrasound contrast- enhancing agent) or a radionuclide that may be, for example, a .gamma.-, .beta.-, .alpha.-, Auger electron-, or positron-emitting isotope.
[00284] Anti-VSIGl antibodies may also be useful in, for example, detecting expression of an antigen of interest in specific cells, tissues, or serum. For diagnostic applications, the antibody typically will be labeled with a detectable moiety for in vitro assays. As will be appreciated by those in the art, there are a wide variety of suitable labels for use in in vitro testing. Suitable dyes for use in this aspect of the invention include, but are not limited to, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl- coumarins, quantum dots (also referred to as "nanocrystals"; see U.S. Ser. No. 09/315,584, hereby incorporated by reference), pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue.TM., Texas Red, Cy dyes (Cy3, Cy5, etc.), alexa dyes (including Alexa, phycoerythin, bodipy, and others described in the 6th Edition of the Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference.
[00285] Stained tissues may then be assessed for radioactivity counting as an indicator of the amount of VSIGl -associated peptides in the tumor. The images obtained by the use of such techniques may be used to assess biodistribution of VSIGl in a patient, mammal, or tissue, for example in the context of using VSIG1 as a biomarker for the presence of invasive cancer cells.
[00286] Formulation
[00287] The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, acetate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; sweeteners and other flavoring agents; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; additives;
coloring agents; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[00288] In a preferred embodiment, the pharmaceutical composition that comprises the antibodies of the invention may be in a water-soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, gly colic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. "Pharmaceutically acceptable base addition salts" include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. The formulations to be used for in vivo administration are preferrably sterile. This is readily accomplished by filtration through sterile filtration membranes or other methods.
[00289] Administration of the pharmaceutical composition comprising antibodies of the present invention, preferably in the form of a sterile aqueous solution, may be done in a variety of ways, including, but not limited to subcutaneously and intravenously.
Subcutaneous administration may be preferable in some circumstances because the patient may self-administer the pharmaceutical composition. Many protein therapeutics are not sufficiently potent to allow for formulation of a therapeutically effective dose in the maximum acceptable volume for subcutaneous administration. This problem may be addressed in part by the use of protein formulations comprising arginine-HCl, histidine, and polysorbate (see WO 04091658). Fc polypeptides of the present invention may be more amenable to subcutaneous administration due to, for example, increased potency, improved serum half-life, or enhanced solubility.
[00290] As is known in the art, protein therapeutics are often delivered by IV infusion or bolus. The antibodies of the present invention may also be delivered using such methods. For example, administration may venious be by intravenous infusion with 0.9% sodium chloride as an infusion vehicle. [00291] In addition, any of a number of delivery systems are known in the art and may be used to administer the Fc variants of the present invention. Examples include, but are not limited to, encapsulation in liposomes, microparticles, microspheres (eg. PLA/PGA microspheres), and the like. Alternatively, an implant of a porous, non-porous, or gelatinous material, including membranes or fibers, may be used. Sustained release systems may comprise a polymeric material or matrix such as polyesters, hydrogels, poly(vinylalcohol), polylactides, copolymers of L-glutamic acid and ethyl-L-gutamate, ethylene-vinyl acetate, lactic acid-gly colic acid copolymers such as the LUPRON DEPOT.RTM., and poly-D-(-)-3- hydroxyburyric acid. The antibodies disclosed herein may also be formulated as
immunoliposomes. A liposome is a small vesicle comprising various types of lipids, phospholipids and/or surfactant that is useful for delivery of a therapeutic agent to a mammal. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al, 1985, Proc Natl Acad Sci USA, 82:3688; Hwang et al, 1980, Proc Natl Acad Sci USA, 77:4030; U.S. Pat. No. 4,485,045; U.S. Pat. No. 4,544,545; and PCT WO 97/38731. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. A chemotherapeutic agent or other therapeutically active agent is optionally contained within the liposome (Gabizon et al, 1989, J National Cancer Inst 81 : 1484).
[00292] The antibodies may also be entrapped in microcapsules prepared by methods including but not limited to coacervation techniques, interfacial polymerization (for example using hydroxymethylcellulose or gelatin-mi crocapsules, or poly-(methylmethacylate) microcapsules), colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), and macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980. Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymer, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid- glycolic acid copolymers such as the LUPRON DEPOT.RTM. (which are injectable microspheres composed of lactic acid-gly colic acid copolymer and leuprolide acetate), poly- D-(-)-3 -hydroxy butyric acid, and ProLease.RTM. (commercially available from Alkermes), which is a microsphere-based delivery system composed of the desired bioactive molecule incorporated into a matrix of poly-DL-lactide-co-glycolide (PLG).
[00293] The dosing amounts and frequencies of administration are, in a preferred embodiment, selected to be therapeutically or prophylactically effective. As is known in the art, adjustments for protein degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
[00294] The concentration of the antibody in the formulation may vary from about 0.1 to 100 weight %. In a preferred embodiment, the concentration of the Fc variant is in the range of 0.003 to 1.0 molar. In order to treat a patient, a therapeutically effective dose of the Fc variant of the present invention may be administered. By "therapeutically effective dose" herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. Dosages may range from 0.0001 to 100 mg/kg of body weight or greater, for example 0.1, 1, 10, or 50 mg/kg of body weight, with 1 to 10 mg/kg being preferred.
[00295] Combination- and Co-Therapies
[00296] The antibodies of the present invention may be administered concomitantly with one or more other therapeutic regimens or agents. The additional therapeutic regimes or agents may be used to improve the efficacy or safety of the anti-VSIGl antibody. Also, the additional therapeutic regimes or agents may be used to treat the same disease or a comorbidity rather than to alter the action of the VSIG1 antibody. For example, a VSIG1 antibody of the present invention may be administered to the patient along with
chemotherapy, radiation therapy, or both chemotherapy and radiation therapy. The antibodies of the present invention may be administered in combination with one or more other prophylactic or therapeutic agents, including but not limited to cytotoxic agents,
chemotherapeutic agents, cytokines, growth inhibitory agents, anti-hormonal agents, kinase inhibitors, anti-angiogenic agents, cardioprotectants, immunostimulatory agents,
immunosuppressive agents, agents that promote proliferation of hematological cells, angiogenesis inhibitors, protein tyrosine kinase (PTK) inhibitors, or other therapeutic agents.
[00297] The terms "in combination with" and "co-administration" are not limited to the administration of said prophylactic or therapeutic agents at exactly the same time. Instead, it is meant that the anti-VSIGl antibody and the other agent or agents are administered in a sequence and within a time interval such that they may act together to provide a benefit that is increased versus treatment with only either anti-VSIGl antibody of the present invention or the other agent or agents. It is preferred that the anti-VSIGl antibody and the other agent or agents act additively, and especially preferred that they act synergistically. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The skilled medical practitioner can determine empirically, or by considering the
pharmacokinetics and modes of action of the agents, the appropriate dose or doses of each therapeutic agent, as well as the appropriate timings and methods of administration.
EXAMPLES EXAMPLE 1
[00298] In order to evaluate VSIG1 expression in cancer and normal tissues several
IHC studies were performed using FFPE (Formalin-Fixed, Paraffin-Embedded) samples or TMAs (Tissue MicroArray) by Asterand (Royston, UK).
[00299] Tissue details: 'multi-tumor ' TMA: As described in detail in Figure 26 the
TMA comprised 11 tissue types: breast, colon, lymphoid and prostate (8 tumor and 2 normal samples of each), gastric, ovary, brain, kidney, liver and skin (4 tumor and 2 normal samples of each), and lung (8 non-small cell tumor and 4 small cell tumor samples, and 4 normal lung samples). [00300] Antigen retrieval and staining: The sections were de-paraffinized; antigen retrieved and rehydrated using pH9.0 Flex+ 3-in- 1 antigen retrieval buffers, in PT Link apparatus at 95°C for 20 min with automatic heating and cooling.
[00301] Following antigen retrieval, sections were washed in Flex (TBST) buffer for
2x5 min then loaded into a DAKO Autostainer Plus. The sections were then incubated for 10 min with Flex+ Peroxidase Blocking reagent, rinsed twice in 50mM Tris. HCl, 150mM NaCl, 0.1% Tween-20, pH 7.6 (TBST), followed by a 10 min incubation with Protein Block reagent (DAKO X0909).
[00302] The sections were incubated for 30 min with primary antibody diluted in
DAKO Envision Flex antibody diluent (DAKO Cytomation, Cat # K8006).
[00303] (Sigma, Cat #: HPA036311, lot #: R33236) was used at a concentration of
^g/ml. The negative control sections were incubated with non-immune rabbit IgG antibodies (Alere #X0936032) at ^g/ml or in antibody diluent alone ('no primary' control). Anti Von Willebrand's Factor (vWF) antibody was applied at ^g/ml. The negative control sections were incubated with non-immune rabbit IgG antibodies (Dako, CAT #0936) at 3 and ^g/ml or in DAKO Envision Flex antibody diluent ('no primary' control). Following incubation with primary antibodies, the sections were then rinsed twice in FLEX buffer, incubated with anti-mouse/ rabbit Flex+ HRP for 20 min, rinsed twice in FLEX buffer and then incubated with diaminobenzidine (DAB) substrate for 10 min. The chromogenic reaction was stopped by rinsing the slides with distilled water.
[00304] Following chromagenesis, the sections were counterstained with
haematoxylin, dehydrated in an ascending series of ethanols (90-99-100%), and cleared in three changes of xylene and coverslipped under DePeX.
[00305] Stained sections were analyzed, and suitable digital scanned images captured using Aperio ScanScope AT Turbo system.
[00306] Results: The sections were analyzed for the intensity of the specific staining and a semi-quantitative scoring system was used. The core in the tissue array with the most intense VSIG1 (Sigma Aldrich pAb Cat# HPA036311) -ir was assigned a score of 3+ and the intensities of the immunoreactivity in the other cores were scored relative to that of the 3+ core. The percentage of VSIGl-ir tumor was estimated and recorded using the following ranges: 0-25%, 25-50%, 50-75% and 75-100%. Where scoring was too low to quantitate - an assigned '+' was used to denote the presence of staining. The intracellular distribution of the immune-stained cells in the tumor was also recorded.
[00307] Anti VSIG1 pAb was tested in positive and negative control HEK-293 cell lines and in a positive control tissue (stomach fundus). The optimal IHC conditions for detection of the target determined in that calibration study (heat mediated antigen retrieval (AR) at pH 6.1 and anti-VSIGl antibody applied at ^g/ml) were used for the current 'Multitumour' TMA (Asternad, UK) study and the TOP4 (Asternad, UK) study.
[00308] Multitumor TMA results: VSIGl-ir was noted in nuclei, cytoplasm or plasma membranes with some samples exhibiting either both nuclear-cytoplasmic or plasma membrane-cytoplasmic staining. The most intense VSIGl-ir was observed in liver tumor and the least intense VSIG1 -ir being observed in the breast tumor. Weak VSIG1 -ir was also observed in the majority of the ovary tumor samples immunostaining less than 25% of the tumour cells, with exception to one sample - showing strong VSIGl-ir in 75-100% of the tumor cells. Only one of the five breast tumors analyzed was immunostained where <25% of the tumor was immunoreactive . The other four samples were negative.
[00309] Another interesting observation was noted in the non-small cell lung tumor samples, where in general stronger VSIGl-ir was seen to that of the small cell tumors. All samples of small cell lung carcinoma for which data was obtained were VSIGl-ir, whereas small cell tumors only exhibited weak VSIGl-ir. However there was no difference in the percentage distribution between the tumor types, as both small cell and non-small cell tumors exhibited VSIGl-ir less than 25% of the tumor cells. However the finding between the lung tumors is only indicative due to core loss of the small cell lung tumor samples. The most consistent tissue type was liver having consistently high levels of VSIGl-ir within the tumor cells however the percentage of tumor cells immunostained varied between all four cores from 0-25% and 75%-100%. No other correlation between staining intensity and tumor differentiation was apparent. Stomach tumors were the most inconsistent tissue type - having varying staining patterns including nuclear, plasma membrane, cytoplasmic, to plasma membrane-cytoplasmic. The staining intensity of VSIG1 also varied from weak to strong in generally less than 50% of the tumor cells. [00310] Normal tissues also showed moderate to strong VSIGl expression in the liver within the hepatocytes, and stomach within the crypts and surface epithelium, which was expected due to the positive control tissue being stomach fundus. No VSIGl-ir was observed in the normal breast, lung and ovary tissues. Overall, the expression of VSIGl was variable in both intensity and percentage of tumor cells stained both within and between tumor types.
[00311] In comparison of normal to tumor tissues of the same type, there appears to be an up-regulation of stronger staining seen in the diseased tissues; very weak to no staining was observed in the majority of normal. Figure 26 includes the sample description and IHC scoring for each sample.
[00312] TOP 4 TMA results: In this study, sections of Asterand's Top4 TMA were examined for the presence of VSIGl-ir using antibody HPA036311. The results focus on the breast and lung tissue only, as in these tissues demonstrated clear over expression over normal tissue. Data from the other tumors represented on the array included in Figure 27. In normal breast and lung tissues very little VSIGl-ir was present. VSIGl-ir was present to some degree in the majority of breast and lung tumors tested, although the intensity of immunoreactivity was generally weak. Grade II adenocarcinomas of the breast included some examples of strong VSIGl-ir, while Grade III breast adenocarcinomas only exhibited weak immunoreactivity. Strong membrane-associated VSIGl-ir was observed in a number of breast tumor samples. The most striking example of plasma membrane-associated VSIGl-ir was seen in a Grade II infiltrating ductal carcinoma of the breast from one case, where the entire tumor scored 3+.
[00313] One sample of lung adenocarcinoma was particularly interesting in that the tumor appeared to contain two distinct populations of tumor cells: one with low-level cytoplasmic VSIGl-ir which made up the majority of the tumor mass, and the other with very intense cytoplasmic and membrane-associated immunoreactivity.
Expression Pattern of VSIGl Proteins Using MED Discovery Engine
[00314] MED is a proprietary software platform for collection of public gene- expression data, normalization, annotation and performance of various queries. Expression data from the most widely used Affymetrix microarrays is downloaded from the Gene Expression Omnibus (GEO - www.ncbi . nlm. nih. gov/ GEO) . Data is multiplicatively normalized by setting the 95 percentile to a constant value (normalized expression=1200), and noise is filtered by setting the lower 30% to 0. Experiments are annotated, first automatically, and then manually, to identify tissue and condition, and chips are grouped according to this annotation, and cross verification of this grouping by comparing the overall expression pattern of the genes of each chip to the overall average expression partem of the genes in this group. Each probeset in each group is assigned an expression value which is the median of the expressions of that probeset in all chips included in the group. The vector of expression of all probesets within a certain group forms the virtual chip of that group, and the collection of all such virtual chips is a virtual panel. The panel (or sub-panels) can be queried to identify probesets with a required behavior (e.g. specific expression in a sub-set of tissues, or differential expression between disease and healthy tissues). These probesets are linked to LEADS contigs and to RefSeqs (http : / / www. ncbi.nlm.nih. go v/RefS eq/) by probe-level mapping, for further analysis.
[00315] The Affymetrix platforms that are downloaded are HG-U95A and the HG-
U133 family (Α,Β, A2.0 and PLUS 2.0). Three virtual panels were created: U95 and U133 Plus 2.0, based on the corresponding Affymetrix platforms, and U133 which uses the set of common probesets for HG-U133A, HG-U133A2.0 and HG-U133 PLUS 2.0+.
[00316] The results of the MED discovery engine are presented for selected cancer not represented in the two cancer TMAs which were analyzed using an anti VSIGl antibody. The y-axis the (normalized) expression and the x-axis describes the groups in the panel. For each group, the median expression is represented by a bar, and the standard deviation.
[00317] The MED discovery engine was used to assess the expression of VSIGl transcripts. Expression data for Affymetrix probe sets 234370_at representing the VSIGl gene data is shown in Figure 31. As evident from the graph in Figure 31, the expression of VSIGl transcripts detectable with the above probe sets was observed in several cancer types mainly pancreatic cancer, and specifically in pancreas intraductal papillary mucinous adenocarcinoma (Figure 31). Cervical cancer also demonstrated over expression of VSIGl.
[00318] Furthermore using database analysis of Next Generation Sequencing (NGS) data such as TCGA (cancer tissue), GTEx (normal tissue) validated the result of VSIGl over expression in NSCLC adenocarcinoma and breast cancer seen by IHC (Figure 25A for lung cancer and data not shown for breast cancer). The expression in TCGA and GTEx also validated the over expression seen using the MED database in pancreatic cancer (which is the cancer with the highest mRNA expression in TCGA Figure 25B) and based on the
TCGA/GTEx analysis liver and ovary cancer also show over expression of VSIGl (Figure 25 C and D respectively).
[00319] Integrative analysis of normal expression patterns (GTEx data), adjacent normal tissues (TCGA data) and cancer tissue (TCGA data) also confirmed over expression of VSIGl in cancer tissues compared to normal in colon, rectal (Figure 25E), uterus (Figure 25F), head and neck (Figure 25G), kidney (Figure 25H) and stomach (data not shown) cancers.
[00320] Overall, the above results, which comprise a wide variety of different human tumor samples, and which are derived from different types of tumor tissues indicate that VSIGl protein is expressed in a large proportion of tumor types.
[00321] The membrane over expression of VSIGl in multiple cancers, such as NSCLC adenocarcinoma, breast carcinoma, HCC, colon and rectal cancer, kidney clear cell carcinoma, kidney papillary cell carcinoma, uterus cancer, head and neck cancer, pancreatic cancer, stomach cancer and ovarian cancer , as well as the limited normal expression indicates that VSIGl could serve as a target for ADC treatment in cancer. This further supported by the cytotoxic effect seen on cell lines once an anti VSIGl antibody is conjugated to a toxin.
[00322] The overall results of the two TMAs indicate expression of VSIGl in lung and breast tumor as most consistent, additional cancers that exhibit VSIGl expression include colon, liver, gastric, lymphomas, melanoma, prostate, ovary, astrocytoma and kidney cancer (Figure 25 summarizes the results of the two studies).
EXAMPLE 2
[00323] A human antibody phage display library was panned against a recombinant protein comprising the VSIGl extracellular domain (ECD) fused to a human IgGi Fc region.
[00324] Protocols
[00325] Preparation of biotinylated VSIGl : [00326] A recombinant protein comprising the human VSIG1 ECD (the sequence of which is shown in Figure 1) was fused to human IgGl Fc (see Figure 2; CGEN-VSIGlHH-1 batch #124) and labelled using an EZ-Link Sulfo-NHS-LC biotinylation kit (Pierce). Two aliquots of protein were labelled, one at a 3: 1 biotin: protein ratio and the other at a 6: 1 ratio. Free biotin was removed by dialyzing samples overnight against phosphate buffered saline (PBS) pH 7.4 using a 3500 MWCO Slide-A-Lyzer cassette (Pierce). The approximate number of biotin moieties per protein was determined using an EZ Biotin Quantitation Kit (Pierce). Dialyzed proteins were stored at -80°C. CGEN-XXXHH, negative control protein comprising an irrelevant ECD fused to the same human Fc, was prepared in the same way (data not shown).
[00327] Analysis of biotinylated antigens: The quality of the biotin-labeled CGEN-
VSIGlHH-1 preparation was assessed by SDS-PAGE. Approximately 2 μg of each protein was diluted to 10 in PBS, mixed with 2.5 5x LDS sample loading dye, heated to 100°C for 5 mins and centrifuged to remove precipitate. Sample Reducing Agent was also added where necessary (1.4 μί). Proteins were separated by electrophoresis on NuPage 4- 12% tris-glycine gels and stained with SimplyBlue SafeStain. SeeBlue Plus2 molecular weight markers were run alongside samples for size comparison. All protein electrophoresis reagents and equipment were sourced from Life Technologies.
[00328] Phage panning of human antibody library: Panning reactions were carried out in solution using streptavidin-coated magnetic beads to capture the biotinylated antigens. Note that all washing and elution steps were conducted using a magnetic rack to capture the beads (Promega). All incubation steps were conducted at room temperature with gentle mixing on a tube rotator. Four panning sub-campaigns were conducted, each with a different combination of antigens, washes and Fc-binder depletion steps (Table 1).
[00329] Sub-campaigns A and B applied depletion steps to remove binders against the CGEN-150XX control protein and streptavidin-coated magnetic beads. Sub-campaigns C and D only applied depletion to remove binders against the beads. All campaigns used 100 pmols of the appropriate CGEN-VSIGlHH-1 or C GEN- 150XXHH protein per panning or depletion step (this was the total amount of protein, not the final protein concentration).
Table 1
Sub-campaign | Washing protocol | Depletion with CGEN- 150XXHH?
A 3x each buffer, all rounds yes
B 3x each buffer, round 1 yes
6x each buffer, round 2
8x each buffer, round 3
C Identical to A No
D Identical to B no
Table 1: Antigen and washing stringency used for phage panning against CGEN-VSIG1HH- 1. Note that the washing protocol involved a set of washes in PBS-T following by identical washes in PBS. There were either three, six or eight (3X, 6X, 8X) washes in each cycle depending on the sub-campaign and panning round.
[00330] Preparation of phage library for panning: All phage panning experiments used the XOMA031 human fab antibody phage display library (XOMA Corporation). Sufficient phage for a 50-fold over-representation of the library were blocked by mixing 1 : 1 with 10% skim milk powder in PBS (final skim milk concentration 5%) and incubating for lhr.
[00331] Antigen coupling to streptavidin beads: For each sub-campaign, three 100 aliquots of Dynal streptavidin-coated magnetic beads (Life Technologies) were blocked by suspension in 1 mL of blocking buffer (5% skim milk powder in PBS) and incubated for 30 mins. One blocked bead aliquot was mixed 50 pmols each of CGEN-VSIGlHH-1 biotinylated at the 3: 1 and 6: 1 ratios (100 pmols of antigen in total). The other two aliquots were either mixed with 100 pmols of the 'depletion' antigen CGEN-150XXHH (sub- campaigns A and B), or not coupled to a biotinylated protein (C and D). Biotin-labeled antigens were coupled to the beads for 1 hr at RT with gentle mixing on a tube rotator. Bead suspensions were washed twice with PBS to remove free antigen and re-suspended in 100 blocking buffer prior to phage panning.
[00332] Depletion of human IgGl Fc and streptavidin-coated bead binders from the phage library: It was necessary to remove unwanted binders to streptavidin beads (all sub- campaigns) and the Fc region of CGEN-VSIGlHH-1 (sub-campaigns A and B) prior to phage panning. The blocked phage suspension was mixed with one 100 aliquot of streptavidin beads coupled to CGEN-150XXHH (A and B) or uncoupled beads (C and D) and at incubated for 45 mins with gentle mixing on a tube rotator. [00333] The beads (and presumably unwanted bead and human IgGl Fc-binders) were discarded. This step was repeated once for all sub-campaigns and the 'depleted' phage library supernatants were reserved for panning.
[00334] Phage panning round 1 : The blocked and depleted phage library was mixed with biotinylated CGEN-VSIGlHH-1 coupled to streptavidin-coated magnetic beads. This suspension was incubated for lhr at RT with gentle rotation to allow binding of CGEN- VSIG1 specific phage. Non-specific binders were removed by washing according to the protocols in Table 1. Each wash was conducted by re-suspending the beads in 1 mL of wash buffer using five aspirations with a pipette. After washing, bound phage were eluted by incubation with 500 μΐ, of 100 mM triethylamine (TEA) (EMD Millpore) for 10 - 15 mins at RT. The eluate was neutralized by adding 500 of 1 M Tris-HCl pH 8.0 (Teknova).
[00335] Determination of phage titer: \0 μΐ. of the initial phage library (input titer) or panning eluate (output titer) was serially diluted (10-fold) in PBS. A 90 aliquot of each phage dilution was mixed with 90 μΐ. of TGI E. coli cells (Lucigen) grown to an optical density of -0.5 at 600 nm (OD 600nm). Phage were allowed to infect the cells by stationary incubation for 30 mins, then shaking incubation (250 rpm) for 30 mins, all at 37°C. A 10 aliquot of each infected cell culture was spotted on a 2YT agar plate supplemented with 2% glucose and 100 μg/mL carbenicillin (2YTCG, Teknova). Plates were incubated overnight at 30°C. Colonies growing from each \0 μΐ. spot were counted and used to calculate input and output titers.
[00336] Phage rescue: The remaining phage eluate (~1 mL) was mixed with 10 mL of
TGI E. coli grown to an OD of 0.5 at 600 nm. Phage were infected into cells as detailed above, infected cells were pelleted by centrifugation at 2500xG and re-suspended in 750 μΐ. 2YT medium (Teknova). The cell suspension spread on 2YTCG agar plates (Teknova). These were incubated overnight at 37°C and the resulting E. coli lawns were scraped and re- suspended in -20 mL liquid 2YTCG (Teknova). A small aliquot of re-suspended cells was inoculated into 50 mL 2YTCG to achieve an OD 600nm of 0.05, and then grown at 37°C with 250 rpm shaking until the OD reached 0.5. This culture was infected with M13K07 helper phage (New England Biolabs) and incubated overnight at 25°C with shaking to allow phage production. The culture supernatant containing rescued phage particles was cleared by centrifugation at 2500xG and 1 mL was carried over for either a) a subsequent round of panning or b) fab binding screens.
[00337] Phage panning rounds 2-3: second and third rounds of panning were conducted as per the steps above, except that the rescued phage supernatant from the previous round was used in place of the phage library.
[00338] Binding screens using fabs prepared in periplasmic extracts
[00339] Fab expression vectors: The XOMA031 library is based on phagemid constructs that also function as fab expression vectors. These vectors contain fab heavy chain and light chain expression cassettes, a lac promoter to drive expression of the antibody genes, and an ampicillin resistance gene. The antibody chains are appended with N-terminal signal peptides to drive their secretion into the periplasmic space. The C-terminal of the heavy chain carries a truncated gene III protein sequence for incorporation into phage particles. The heavy chain also carries hexa-histidine, c-myc and V5 affinity tags. Transformation of these vectors into E. coli and induction with isopropyl β-D-l-thiogalactopyranoside (IPTG) results in periplasmic expression of soluble fab molecules.
[00340] Fab PPE production: Eluted phage pools from panning round 3 were diluted and infected into TGI E. coli cells (Lucigen, Middleton, WI) so that single colonies were generated when spread on a 2YTCG agar plate. This resulted in each colony carrying a single fab clone. Individual clones were inoculated into 1 mL 2YTCG starter cultures in 96-well deepwell blocks (VWR) using a Qpix2 instrument (Molecular Devices). These starter cultures were grown overnight in a Multitron 3mm incubator (Infors) at 37°C with 700 rpm shaking. For fab expression, 20 of 1 mL starter cultures were transferred into a second set of deepwell plates containing 1 mL 2YT with 0.1% glucose and 100 μg/mL ampicillin. Cultures were grown until the average OD at 600 nm was 0.5 - 1.0. Protein expression was then induced by adding IPTG (Teknova) to a final concentration of 1 mM. Expression cultures were incubated overnight in the Multitron instrument at 25°C with 700 rpm shaking.
[00341] Fab proteins secreted into the E. coli periplasm were extracted for analysis.
Cells were harvested by centrifugation at 2500xG, the supernatants were discarded and pellets were re-suspended in 75 ice-cold PPB buffer (Teknova). Extracts were incubated for 10 mins at 4°C with 1000 rpm shaking, and then 225 μΐ. ice-cold ddH20 was then added and incubated for a further lhr. The resulting periplasmic extract (PPE) was cleared by centrifugation at 2500xG and transferred to separate plates for ELISA and FACS analysis. All extraction buffers contained EDTA-free Complete Protease Inhibitors (Roche).
[00342] ELISA binding screen: each fab PPE was tested for binding to recombinant
CGEN-VSIGlHH-1. The target protein was diluted to 1 μg/mL in PBS and 50 aliquots were coated on the wells of high binding EIA/RIA microplates (Costar) overnight at 4°C. Coated plate wells were rinsed twice with PBS and incubated with 300 blocking buffer (5% skim milk powder in PBS pH 7.4) at room temperature (RT) for 1 hr. Blocking buffer was removed and plates were rinsed twice more with PBS. 50 μΐ. aliquots of each PPE sample were added to individual plate wells and incubated at RT for 1 hr. Plates were washed three times with PBS-T (PBS 7.4, 0.05% Tween20), then three times with PBS and 50μίΛνβ11 of a F(ab')2 fragment Specific Goat Anti-Human IgG (Jackson Immunoresearch) was added as the secondary detection antibody (1 :2000 dilution in PBS-T). This was incubated at RT for lhr and plates were washed again. ELISA signals due to fab binding were developed by adding 50 μί of Sureblue TMB substrate (KPL Inc) and incubating for 5 - 20 mins. The HRP reaction was stopped by adding 50 μΐ, 2N H2S04 (VWR). Assay signals were read on a Victor II plate reader (Perkin-Elmer) absorbance 450 nm. Each ELISA plate also contained two negative control wells (blanks) where no fab was present. Fab binding was expressed as the fab binding signal divided by the blank signal. Positive hits were identified as those giving a fab/blank ratio equal or greater than five.
[00343] Flow cytometry binding screen: Fab PPE samples were also tested for binding to Expi293 cells (Life Technologies) over-expressing the human or mouse orthologs of CGEN-VSIG1. Parental (i.e. not transfected) Expi293 cells were used as the negative control. All reagent preparation and wash steps were carried out in FACS buffer (PBS (Life Technologies), 1% BSA (Sigma Aldrich), and 0.1% Sodium Azide.
[00344] For each fab sample and cell line, a 25 μΐ. aliquot of cells (either parental or
CGEN-VSIG1 expressing) was mixed with 25 μΐ. of PPE and incubated for 30 mins at 4°C. After incubation, cells were washed once in 200 μΐ of FACS buffer and re-suspended in 30 μΐ of a monoclonal mouse anti c-myc antibody (Roche) diluted 1 : 1000 (10 μg/ml). This was incubated for 30 mins at 4°C followed by another wash step. Cell were re-suspended in 25 μΐ of a polyclonal goat anti-mouse- AF 647 antibody (Jackson Immunoresearch) diluted 1 :300 (5 μg/ml) and incubated for 25 mins at 4°C. After two final washes cells were re-suspended in 50 μΐ of FACS buffer containing 2% paraformaldehyde.
[00345] Samples were analyzed using an Intellicyt HTFC screening system (Intellicyt), recording approximately 5000 events per well in a designated live gate. Data was analyzed using FloJo (De Novo Software, CA, USA) and exported to Excel (Microsoft). The ratio of mean fluorescence intensity (MFI) of CGEN-VSIGl over-expressing cells: MFI signal of parental cells was calculated and exported into Xabtracker (XOMA). Positive hits were identified as those giving an MFI ratio equal or greater than five.
[00346] Results
[00347] Preparation of biotinylated VSIG1 antigen: biotinylation of CGEN-
VSIGlHH-1 did not change the SDS-PAGE banding partem compared to the starting material (data not shown). The non-reduced protein showed a major band slightly larger than 100 kDa (expected size based on amino acid sequence is 98 kDa). However, there was also evidence of higher molecular proteins in each preparation, which may indicate formation of higher-order multimers.
[00348] Under reducing conditions the CGEN-VSIGlHH-1 samples showed a single band of -60 kDa (expected size is 49 kDa). There was no sign of larger molecular weight products, suggesting that the aggregation seen in the non-reduced samples was disulfide mediated. There was no evidence of protein degradation. Note that the CGEN-VSIGlHH-1 protein was estimated at >90% of total protein in all cases, which is acceptable for phage panning.
[00349] Phage panning: the phage output titer results indicate the number of individual phage particles recovered at the end of each panning round (Table 2). The output titer after round 1 ranged from 2xl04 to lxlO5. This is at the lower range generally obtained with the XOMA031 library (typical round 1 outputs are between lxlO4 and lxlO6). Titers for each sub-campaign increased to 1.4xl05 - 4.6x105 in round 2 and 7.6x10s - 3x109 in round 3. Overall, these data suggest progressive enrichment of CGEN-VSIGl binding phage throughout the phage display process, but it is possible that antibody diversity was restricted by the relatively low round 1 output titers. [00350] Table 2: phage panning output titers by sub-campaign (results are total number of phage recovered).
Figure imgf000076_0001
[00351] Fab PPE screening: A set of 186 fab clones from each sub-campaign (744 in total) were tested by ELISA and FACS for binding to the recombinant CGEN-VSIGlHH-1 protein and Expi293-VSIG1 cell lines, respectively (Table 2). The ELISA hit rate (i.e. percentage of clones positive for binding) ranged from 24% - 39%. The FACS hit rate for each sub-campaign was slightly lower, ranging from 17% - 25%. Only a sub-set of fabs had binding activity that correlated between FACS and ELISA (40 - 61%, depending on the sub- campaign). This was expected, given that phage panning was conducted against the recombinant CGEN-VSIGlHH-1 protein and some fabs may recognize epitopes that are different from, or do not occur, on the natural cell-surface form of the protein.
All clones designated as hits by FACS and/or ELISA were sequenced to eliminate redundant fabs (i.e. fabs containing the same VH and VL sequence). Sequence diversity ranged from 27 - 37%. In addition, the fab pools were high quality, with 72 - 78% of VH sequenced containing full open reading frames (Table 2). All unique binders against Expi293-huVSIGl cells were re-tested against the Expi293-muVSIGl . This revealed seven fabs that appears to be cross-reactive against human and mouse CGEN-VSIG1.
Table 3: Summary of screening of fab PPEs. ELISA results are against CGEN-VSIG1HH- 1. FACS results include binding against Expi293-huVSIGl and Expi293-moVSIGl cells. FACS/ELISA correlation refers to the proportion of fabs that bound in both assays (FACS and ELISA) compared to combined number of binders observed in either assay. Sequence diversity refers to the number of different fabs present among the analyzed binders (i.e. 27% = 27 different fab sequences found for every 100 CGEN-VSIG1 binders).
Su b- FACS/ELISA Sequence % of VH with
ELISA hit rate FACS hit rate
ca mpaign correlation diversity fu ll ORF
A 34% 25% 61% 37% 86%
B 24% 19% 51% 35% 87%
C 28% 22% 69% 48% 72%
D 39% 17% 40% 27% 78%
Tota l nu mber of VSIG1 binding fa bs identified 73
Su b-set of fabs binding to Expi293-huVSIG l cel l line 36
Sub-set of fa bs binding to Expi293-moVSIGl cel l line 7 [00352] The total number of unique CGEN-VSIG1 ELISA binders identified was 73, with 36 of those also positive against the human antigen by FACS.
Of the 36 FACS-binding fabs, there were also seven that appeared cross-reactive against mouse CGEN-VSIG1. The binding signals against the mouse antigen were weaker in all cases. However, it is unclear whether this is due to lower binding affinities against the mouse protein, or lower antigen expression levels on the Expi293-moVSIGl cell line. This low rate of mouse cross reactivity (seven potential mouse VSIG1 binders out of 36 fabs) probably results from sequence differences between the human and mouse orthologues, since the ECD is only 82% homologous between the two species (Figure 1).
[00353] These data confirmed that phage panning was successful and the resulting fab pools were a suitable source of VISGl binding fabs. All sub-campaigns used in the panning process yielded acceptable hit rates, good fab sequence diversity and high quality fab pools. A set of 36 fabs with specific binding for cell-based human VISGl were identified, and seven of these also showed binding against mouse VISGl . All FACS-binding fabs were reformatted into full-length human IgGs as below.
EXAMPLE 3
[00354] Affinity Measurements of 15 Anti-VSIGl Human Antibodies Using Surface Plasmon Resonance
Materials and Methods
[00355] Experiments were performed using ProteOn XPR 36 and Biacore 3000 instruments at 22°C.
[00356] Step 1: High density surfaces of anti -human fc pAb (Invitrogen HI 0500) were immobilized on each spot of a ProteOn GLC chip using standard amine coupling chemistry. Each of the six channels were activated in the horizontal direction for 6 minutes with EDC- NHS prior to a 5 minute injection in all vertical channels of anti -human fc pAb diluted to 9 μg/mL in 10 mM NaOAc, pH 5.0. This was followed by a 6 minute block of excess activated carboxyls using 1M ethanolamine pH 8.5 in all vertical and horizontal channels. This immobilization protocol was performed in order to be able to use interspot referencing. Final immobilization levels of pAb ranged from ~ 4900-5100 RU. The immobilization of anti- human fc pAb was performed using degassed PBST as the running buffer.
[00357] Step 2: For each kinetic experiment cycle, six of the anti-VSIGl mAbs listed below were each diluted to -0.25 μg/mL in running buffer (degassed PBST withlOO μg/mL BSA) and captured in the vertical direction for 1 minute at a flow rate of 25 μί/ιηίη:
CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011.
[00358] After the mAb capture phase, the instrument flow was shifted to the horizontal direction and paused for 10 minutes followed by two buffer blank injections to allow for signal stabilization.
[00359] Step 3: Human ECD VSIG1 (Lot# 150518-02) monomer was simultaneously injected at six different concentrations which ranged from 1.6 nM - 400 nM over all captured anti-VSIGl mAbs for two minutes followed by 10 minutes of dissociation at a flow rate of 50 μΐνηϋη. Monomer injections were preceded by an identical buffer injection for double- referencing. Capture surfaces were regenerated with a 30-second pulse of 146 mM phosphoric acid in both the horizontal and vertical directions.
[00360] Step 4: Sensorgram data were zeroed, aligned, and double-referenced (interspot, buffer blanks) using ProteOn Manager Version 3.1.0.6 and Scrubber ProteOn software. Where appropriate, data were fit with a 1 : 1 kinetic model including a term for mass transport. A steady-state approach to estimate KD was used for those data which showed unusually fast on-rates and off-rates. In those cases an identical time point at the end of the injection cycle was selected for each sensorgram where the association phase appeared to have reached equilibrium. A simple 1 : 1 equilibrium binding model was then used to fit the RU signals at the identical time point as a function of monomer concentration to estimate KD. Kinetic and equilibrium data fitting were performed using both ProteOn Manager and Scrubber ProteOn software. The binding constants of each mAb are listed in order of decreasing affinity in Figure 23. All data are shown in the Figures.
[00361] Step 5: Sensorgram data for mAb CPA.4.016 appeared to be complex when acquired with the ProteOn so an additional experiment was performed using the Biacore 3000 to attempt to acquire data sufficient for a steady-state estimate for KD. Here mAb CPA.4.016 was covalently coupled to flow cell 1 of a CM5 chip at ~11,670 RU while flow cell 2 was activated and blocked as a reference surface. Triplicate concentrations of VSIGl monomer that ranged from 2.3 nM - 148 nM were injected for three minutes followed by three minutes of dissociation at a flow rate of 50 μί/ιηίη. The surface was regenerated with a single 20 second pulse of lOmM glycine, pH 2.5. Several blank injections were interspersed for double-referencing. All samples were prepared in the running buffer which was HBSP buffer with 100 μg/mL BSA. A longer injection time was used for this experiment with the idea that the sensorgrams would equilibrate for a steady-state estimate of KD. Instead the resulting data showed reliable kinetics and were fit with a 1 : 1 kinetic model with a term for mass transport. Data were processed and fit using Scrubber ProteOn software. The results are shown in Figure 13.
[00362] Figure 23 shows mAb CPA.4.009 as having the highest affinity of the 15 mAbs in this study. It should be noted however that D06 has a relatively fast off-rate (kj = 1.9 x 10"2/sec) which may make D06 a less than ideal candidate for Antibody Drug Conjugate (ADC) considerations. Higher monomer concentrations are needed for a better estimate of the KD for mAb 156-01. D05 because of its apparent low affinity. Therefore the KD listed here for mAb D05 should only be considered a rough estimate.
EXAMPLE 4
[00363] Surface Plasmon Resonance Study of Epitope Binning 15 Anti- VSIGl
IgG Antibodies Binding to Human VSIGl Monomer
Materials and Methods
[00364] Experiments were performed using a ProteOn XPR 36 instrument at 22°C with all samples kept at 4°C during the experiment.
[00365] Step 1: The following anti-VSIGl mAbs were each diluted to ~10μg/mL in lOmM sodium acetate, pH 5.0 and covalently immobilized on two independent spots on a ProteOn GLC biosensor chip using standard amine coupling: CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.028, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008 and CPA.4.011. [00366] The activation step occurred in the horizontal flow direction while the immobilization step occurred in the vertical flow direction. MAbs were injected for four minutes after surface activation. The blocking step occurred in both the vertical and horizontal positions so that the horizontal "interspots" could be used as reference surfaces. MAbs were immobilized at a range of -4700RU-5600RU.
[00367] Step 2: Preliminary experiments involved several cycles of injecting -23 nM VSIGl antigen (hVSIGl ECD-his, Lot #150518-02) over all immobilized mAbs for three minutes at a flow rate of 25μί/ηϋη followed by regeneration with a 30-second pulse of 10 mM glycine-HCl, pH 2.5. Antigen samples were prepared in degassed PBST running buffer with 100 μg/mL BSA. These preliminary experiments showed that the VSIGl antigen bound reproducibly to all immobilized mAbs when using glycine at pH 2.5 as the regeneration reagent.
[00368] Step 3: Earlier SPR kinetics experiments with these mAbs and the identical antigen (6/10/15) showed that most interactions had relatively fast off-rates, therefore a "pre- mix" binning protocol was performed. In this protocol each mAb listed in Step 1 was pre- mixed with VSIGl antigen and then injected for three minutes over all immobilized mAbs. The molar binding site concentration of each mAb was in excess of the molar antigen binding site concentration. The final binding site concentration of each mAb was ~400nM and the final binding site concentration of the antigen was ~23nM. An antigen-only control cycle was performed after every five mAb injection cycles to monitor the activity of the immobilized mAbs throughout the experiment. Additional controls included each mAb injected alone over the second spots of immobilized mAbs simultaneously with the identical mAb pre-mixed with antigen. All surfaces were regenerated with a 30 second pulse of 10 mM glycine-HCl at pH 2.5, and all cycles were run at a flow rate of 25 μΕ/ιηίη. MAb and antigen samples were prepared in degassed PBST running buffer with 100 μg/mL BSA.
[00369] Step 4: Sensorgram data were processed and referenced using ProteOn Manager version 3.1.0.6. The mAb-only control injections were used as the injection references where significant binding with the mAb-only injections was observed. An antibody pair was classified as having a shared antigen binding epitope (designated as a red "0" in the matrix in Figure 12) if no binding was observed from the injection of mixed mAb and antigen over the immobilized mAb, or if binding was significantly reduced as compared to the antigen-only control injection over the same immobilized mAb. An antibody pair was classified as binding to different antigen epitopes (designated as a green "1" in the matrix in Figure 16) if the injection of mixed mAb and antigen showed binding to the immobilized mAb similar to or greater than the antigen-only control over the same immobilized mAb.
[00370] Step 5: Hierarchical clustering of the binding patterns in the binary matrix for each immobilized mAb (horizontal patterns in Figure 15) was performed using JMP software version 11.0.0. The blocking patterns of the mAbs as analytes in solution (vertical patterns in Figure 15) were also clustered as a comparison to the blocking patterns of the immobilized mAbs (data not shown, see Results for discussion).
[00371] Results. Figure 14 is an example of the processed and referenced sensorgram data generated for one mAb (CPA.4.023) injected with pre-mixed antigen over each immobilized mAb (panels A-O) as well as the mAb-only control injection (panels P-DD). Figure 14 shows the binary matrix of the blocking ("0") or sandwiching ("1") between each mAb pair where the mAbs are listed in identical order both vertically (mAbs on the surface - "ligands") and horizontally (mAbs in solution - "analytes"). Identical "bins" of blocking patterns for mAbs as ligands are highlighted in Figure 16 with a black box around each group of similar patterns. Figure 17 shows the dendrogram of the horizontal (ligand) blocking patterns in the matrix in Figure 16. For the strictest definition of an epitope "bin" where only those mAbs which show identical blocking patterns technically bin together, there are a total of 4 discrete bins listed below. Hierarchical clustering of the blocking patterns of the mAbs as analytes (vertical patterns in Figure 16) was identical to the clustering of the mAbs in solution with the exception of mAb CPA.4.009 which, as an immobilized ligand, appeared to be able to sandwich the antigen with mAb CPA.4.012 injected in solution, while as an analyte mAb CPA.4.009 appeared to block the antigen from binding to immobilized mAb 156- 01.F10. The former case placed mAb CPA.4.009 in Bin 3 below while the latter case would have placed it in Bin 4. Actually, Bins 3 and 4 differ by only how they bind to the antigen in the presence of mAb CPA.4.012. MAbs in Bin 3 appear to be able to sandwich with
CPA.4.012 as ligands while mAbs in Bin 4 appear to block 156-01.F10 as ligands. Therefore Bins 3 and 4 could be closely related even though there is a definitive difference regarding mAb ICPA.4.012, meaning there could only be 3 related communities rather than 4 discrete bins of identical blocking patterns. Clone CPA.4.012 forms its own bin. 156-01.D05 156-01.F10 156-0:
156-01X08 156-01.E12 156-01.C03
156-01.C09 156-Q1.F04 156-01,012
156-G1.C04 156-01.D06 156-01.E06
156-01.BOS 156-01.EQ5
[00372] Summary: 15 anti -VSIGl IgG mAbs were binned using SPR according to their pair-wise blocking patterns with monomeric antigen VSIGl . By the strictest definition of an epitope bin, there are a total of four discrete bins with two bins differing only by how their component mAbs block/sandwich antigen with one particular clone.
EXAMPLE 5
[00373] Binding and In vitro Cytotoxic Activity of Antibodies Against VSIG 1
Materials and Methods
[00374] In vitro Cytotoxicity Assay: To measure in vitro cell killing, OV-90, MK 45, HUH-1 or DU145 cells were cultured in complete OV-90 media (1 : 1 mixture of MCDB 105 media containing a final concentration of 1.5 g/L sodium bicarbonate and Media 199 containing a final concentration of 2.2 g/L sodium bicarbonate with a final concentration of 15% fetal bovine serum) or DU145 media (EMEM + 4 nM Glutamine + 10% fetal bovine serum) or MK 45/HUH-1 media (RPMI + 4mM Glutamax + 10% FBS). Cells were harvested in log phase growth and 8,000 OV-90, 5,000 DU145/HUH-1, or 3,000 MKN45 cells in 80 ul per well were seeded in 96 well plates and incubated for 24hrs at 37°C in a humidified water jacketed with 5% C02. Anti- human VSIGl human IgGl antibodies or isotype control were added in single point dilutions, 2-fold, or 3-fold dilution series starting at 5 μg/ml. Protein G preloaded with cytotoxic payloads DM1 (non-cleavable linker), MMAE (cleavable linker), MMAF (non-cleavable linker), or Duocarmycin (cleavable linker) (Concortis, San Diego CA) were added at 10 nM, 25 nM, 50 nM, and 50 nM respectively, and plates were incubated at 37°C in a humidified water jacketed incubator with 5% C02. Cell killing was measured after 72 hrs using Cell Titer Glo (Promega) reagent according to the manufacturer's instructions. Data was analyzed using Prism software (GraphPad, La Jolla, CA).
[00375] Antibody Binding to Human, Mouse and Cynomolgus VSIGl: ExPi 293 (Life Technologies) or HEK 293 (ATCC) cells were transfected by electroporation with pcDNA3.1 (Life Technologies) vectors containing full length human, mouse, and cynomolgus VSIG1. Cells were cultured in ExPi 293 Expression media or DMEM + 4mM Glutamine + 10% fetal bovine serum and passaged according the manufacturer's instructions. Anti- VSIG1 antibodies at ΪΟμ^νπΙ in FACS buffer (PBS + 1% BSA + 0.1% sodium azide) were serially diluted 3-fold, added to 50,000 cells, and incubated for 1 hr at 4°C. Cells were washed 2x in FACS buffer and incubated for 30 min at 4°C in the dark with 7.5 μg/ml goat-anti-human alexa 647 secondary. Samples were washed 2x in FACS buffer and median fluorescence was measured using a BD Accuri C6 flow cytometer. Data was analyzed with FlowJo (Treestar Inc, Ashland, OR) and Prism software (GraphPad. La Jolla, CA).
[00376] Results: Anti-VSIGl antibodies recognize human, mouse, and cynomolgus
VSIG1: To determine the specificity and cross-reactivity of antibodies against VSIG1, we screened a panel of antibodies against human, mouse, and cynomolgus VSIG1 recombinant over expressing cell lines. A majority of the antibodies tested bound to cell lines
overexpressing human and cynomolgus VSIG1 (Figure 18 A and B). Anti-VSIGl clone CPA.4.008 was found to preferentially bind to recombinant mouse VSIG1 (Figure 18 C).
[00377] Anti-VSIGl Antibody drug conjugates exhibit potent in vitro cytotoxic activity: To examine the potency of anti-VSIG antibody drug conjugates, we assayed the in vitro efficacy of a panel of antibodies in a piggyback cytotoxicity assay with protein G preloaded payloads DM1, MMAE, MMAF, and Duocarmycin. OV-90 (ovarian carcinoma), MK 45 (stomach carcinoma), and HUH-1 (hepatocellular carcinoma) human cancer cell lines were selected based on high expression of VSIG1 mRNA by microarray and RNAseq. VSIG1 protein surface expression on the human cancer cells was verified by FACS (Figure 15). DU145, a prostate cancer cell line, was selected as a negative control because it lacks VSIG1 protein expression (Figure 15). Anti-VSIGl clone 156-01. E05 demonstrated potent cytotoxic cell killing of OV-90 cells and no detectable killing of VSIG1 negative cell line DU145 (Figure 20).
[00378] To determine the cytotoxic potential of a panel of anti-VSIGl ADCs, we tested 15 antibodies coupled to 4 payloads in a piggyback assay with OV-90, MK 45, and HUH-1 cells (Figure 21 A-D). All anti-VSIGl antibodies tested showed some degree of cytotoxic activity against all three cell lines. The greatest cytotoxic effect was observed on HUH-1 cells with protein G-Duocarmycin coupled ADCs. As expected, the cell lines with the highest VSIG1 protein cell surface expression were the most sensitive to anti-VSIGl ADCs. Additionally, anti-VSIGl antibodies with the strongest avidity for antigen demonstrated the greatest degree of cell killing.
[00379] To further investigate anti-VSIGl ADCs, we selected one anti-VSIGl antibody from each epitope bin to test in a piggyback cytotoxic assay on HUH-1 cells.
CPA.4.005, CPA.4.009, and CPA.4.028exhibited the greatest cell killing activity across all 4 payloads. The weaker activity observed with 156-01. F10 was expected since it has a weaker avidity to antigen than the other 3 clones tested. (Figure 22).
[00380] Summary and Conclusions: The results show conclusive evidence of the specificity of anti-VSIGl antibodies to human and cynomolgus VSIG1 protein. Only one antibody assayed, CPA.4.008, showed specific binding to mouse VSIG1 protein. This likely due to the low homology between the human and mouse VSIG1. OV-90, HUH-1, and MK 45 were found to have significant cell surface VSIG1 protein, with HUH-1 exhibiting the highest expression and MK 45 the lowest expression of the three. In cytotoxic piggyback assays, anti-VSIGl ADCs had potent cytotoxic effects on OV-90 and HUH-1 cells. Although some cytotoxic activity was observed with MK 45, it was not as robust as the other two cell lines. This could be a result of the reduced VSIG1 protein on the surface of MK 45 compared to OV-90 and HUH-1. Anti-VSIGl antibodies coupled with the auristatin MMAF had the strongest potency of all the ADCs tested. Interestingly, antibodies coupled to another auristatin derivative, MMAE, had lower potency than those coupled to MMAF. This could be due to differences in internalization or trafficking of these two payloads. Taken together, this data shows that anti-VSIGl antibody drug conjugates bind VSIG1 on the cell surface, are internalized, and release toxic payload leading to cell cycle arrest. These studies lay the groundwork for future in vitro and in vivo studies to evaluate the potential of VSIG1 as a target for antibody drug conjugates.
EXAMPLE 6
[00381] Generation of High Affinity Anti-VSIGl Fab Antibodies By Phage
Display [00382] Using the Materials & Methods described below a panel of different Fab antibodies of different epitopic specificities that bind VSIGl with high specificity and affinity were produced.
[00383] General method for direct binding ELISA: Unless otherwise noted, test proteins were diluted to 1 μg/mL in phosphate buffered saline (PBS) and 50 aliquots were coated on the wells of a Maxisorp ELISA plate (Thermo Fisher Scientific, Waltham, MA) overnight at 4°C, or for lhr at 37°C. Coated plate wells were rinsed twice with PBS and incubated with 300 blocking buffer (5% skim milk powder in PBS pH 7.4) at room temperature (RT) for 1 hr. Blocking buffer was removed and plates were rinsed twice more with PBS. Plate-bound proteins were detected by adding 50 μίΛ βΙΙ of a primary antibody and incubating at RT for 1 hr. Plates were washed three times with PBS-Tween20 (PBS 7.4, 0.05% Tween20), then three times with PBS and 50μίΛνβ11 of a F(ab')2 fragment Specific Goat Anti-Human IgG (Jackson Immunoresearch, West Grove, PA) was added as the secondary detection antibody. This was incubated at RT for lhr and plates were washed again. Note that in some cases a HRP-conjugated primary antibody (or other detection protein) was used directly, with no secondary detection step. ELISA signals were developed in all wells by adding 50 μΐ. of Sureblue 3,3',5,5'-Tetramethylbenzidine (TMB) substrate (KPL Inc, Gaithersburg, MD) and incubating for 5-20 mins. The HRP reaction was stopped by adding 50 μΐ. 2N H2SO4 (VWR, Radnor, PA) and assay signals were read on a Fluostar (BMG Labtech, Cary, NC) plate reader at absorbance 450 nm.
[00384] Preparation ofbiotinylated VSIGl antigen: A range of proteins required for phage display experiments were biotinylated to facilitate solution-based panning. These included human VSIGl ECD fused to human IgGl Fc (VSIGl H:H, SEQ ID NO 12) and mouse VSIGl ECD fused to mouse IgG2a Fc (VSIGl M:M, SEQ ID NO 13). A negative control human IgGl Fc-fusion protein, i.e. irrelevant ECD fused to the same human IgGl Fc as VSIGl H:H, was biotinylated to use for depletion steps in the panning experiments. All proteins were diluted to 1 mg/mL in 1 mL PBS, and then labeled with a Sulfo-NHS-LC- Biotin kit at a 3: 1 biotin: protein ratio, as per manufacturer's instructions (Pierce, Rockford, IL). After conducting the binding reaction, free biotin was removed by dialyzing samples overnight against PBS pH 7.4 using 3500 MWCO Slide-A-Lyzer cassettes (Pierce). Dialyzed proteins were stored at -80°C. [00385] Phage panning of human antibody library: Panning reactions were carried out in solution using streptavidin-coated magnetic beads to capture the biotinylated antigens. All washing and elution steps were conducted using a magnetic rack to capture the beads (Promega, Madison, WI). All incubation steps were conducted at room temperature with gentle mixing on a tube rotator (BioExpress, Kaysville, UT). Four panning sub-campaigns were conducted, each with a different combination of antigens, washes and Fc-binder depletion steps.
[00386] Sub-campaigns A and B alternated between the human and mouse ECD versions of VSIG1 in an attempt to enrich binders with human/mouse species cross- reactivity. Sub-campaigns C and D were focused on the human ECD version of the antigen, and used depletion steps against the negative control human IgGl Fc-fusion protein to remove binders against the human IgGl Fc. All campaigns used 100 pmol of the appropriate VSIG1 or negative Fc-fusion control protein per round.
[00387] Preparation of phage library for panning: All phage panning experiments used the XOMA031 human Fab antibody phage display library (XOMA Corporation, Berkeley, CA). Sufficient phage for a 50-fold over-representation of the library were blocked by mixing 1 : 1 with 10% skim milk powder in PBS (final skim milk concentration 5%) and incubated for lhr.
[00388] Antigen binding to streptavidin beads: For each sub-campaign, three 100 aliquots of Dynal streptavidin-coated magnetic beads (Life Technologies) were blocked by suspension in 1 mL of blocking buffer (5% skim milk powder in PBS) and incubated for 30 mins. One blocked bead aliquot was mixed with an amount of biotinylated VSIG1 antigen dependent on the panning round and reaction conditions. The other two aliquots were either mixed with 100 pmols of the negative control human IgGl Fc-fusion protein (C and D), or not coupled to a biotinylated protein (A and B). Biotin-labeled antigens were coupled to the beads for 1 hr at RT. Bead suspensions for C and D were washed twice with PBS to remove free antigen and re-suspended in 100 blocking buffer. Blocked beads for A and B are washed and re-suspended in the same way.
[00389] Depletion of human IgGl Fc and streptavidin bead binders from the phage library: Unwanted binders to streptavidin beads (all sub-campaigns) and the Fc region of VSIG1 H:H (sub-campaigns C and D) were removed before phage panning. This was accomplished using blocked phage mixed with one 100 aliquot of uncoupled streptavidin beads (A and B) or beads coupled to the Fc-fusion human IgGl control protein (C and D) and incubated for 45 mins. The beads (and presumably unwanted bead and human IgGl Fc- binders) were discarded. This step was repeated with a second 100 of beads (with or without negative control protein, as appropriate) and the 'depleted' phage library supematants are reserved for panning.
[00390] Phage panning round 1: The blocked and depleted phage library was mixed with the VSIG1 beads described above. This suspension was incubated for lhr at RT with gentle rotation to allow binding of VSIG1 specific phage. Non-specific binders were removed using a sequence of washes. The washing conditions used in all displays were: Round 1, three washes with PBS-T and three washes with PBS; round 2 and round 3, six washes with each buffer.
[00391] After washing, the bound phage were eluted by incubation with 500 of 100 mM triethylamine (TEA) (EMD Millpore, Rockland, MA) for 20 mins at RT. The eluate was neutralized by adding 500 of 1 M Tris-HCl pH 8.0 (Teknova, Hollister, CA).
[00392] Determination of phage titer: 10 μί of the initial phage library (input titer) or panning eluate (output titer) was serially diluted (10-fold) in PBS. A 90 μΐ. aliquot of each phage dilution was mixed with 90 μΐ. of TGI E. coli cells grown to an optical density of -0.5 at 600 nm (OD 600nm). Phage were allowed to infect the cells by stationary incubation for 30 mins, then shaking incubation (250 rpm) for 30 mins, all at 37°C. A 10 μί aliquot of each infected cell culture was spotted on a 2YT agar plate supplemented with 2% glucose and 100 μg/mL carbenicillin (2YTCG, Teknova). Plates were incubated overnight at 30°C. Colonies growing from each \0 μΐ. spot were counted and used to calculate input and output titers.
[00393] Phage rescue: The remaining phage eluate (~1 mL) was mixed with 10 mL of TGI E. coli grown to an OD 600 nm of 0.5. Phage was infected into cells as detailed above, infected cells were pelleted by centrifugation at 2500xG and re-suspended in 750 μΐ. 2YT medium (Teknova). The cell suspension was divided into three equal aliquots that were spread on 2YTCG agar plates. These plates were incubated overnight at 37°C and the resulting E. coli lawns were scraped and re-suspended in -20 mL liquid 2YTCG (Teknova). This cell suspension was used to make 1 mL glycerol stocks for each panning round. A small aliquot of re-suspended cells was inoculated into 50 mL 2YTCG to achieve an OD 600nm of 0.05, and then grown at 37°C with 250 rpm shaking until the OD reached 0.5. The resulting culture was infected with M13K07 helper phage (New England Biolabs, Ipswich, MA) and incubated overnight at 25 °C with shaking to allow phage packaging. The culture supernatant containing rescued phage particles was cleared by centrifugation at 2500 X G and 1 mL was carried over for either a) a subsequent round of panning or b) Fab binding screens. Phage in the remaining supernatant were concentrated and purified for phage pool ELISAs (see below).
[00394] Phage panning rounds 2-3: Second and third rounds of panning were conducted as per the steps above, except that the rescued phage supernatant from the previous round was used in place of the phage library.
[00395] Phage pool enrichment ELISA: Phage from each panning round was precipitated from rescue culture supernatants by adding 1/5 volume of PEG-6000/NaCl solution (Teknova). Precipitated phage were harvested by centrifugation at 8000 rpm and re- suspended in 1 mL PBS. Phage aliquots were diluted 1 : 10 in blocking buffer (5% skim milk powder in PBS) and 50 aliquots were added to the wells of ELISA plates coated with VSIGl-ECD-Ig H:H (SEQ ID NO 12), VSIG1 ECD-Ig M:M (SEQ ID NO 13), or the negative control protein, human IgGl isotype control and mouse IgG2 isotype control.
Bound phage were detected with a HRP -conjugated anti-M13 phage coat antibody (GE Healthcare, Pittsburgh, PA) diluted 1 :2000 in PBS-T. All other assays steps are conducted as described in the general ELISA protocol.
[00396] Fab expression vectors: The pXHMV-Fab-kappa and pXHMV-Fab-lambda phagemid vectors used in the XOMA031 library also functioned as Fab expression vectors. These vectors contained Fab heavy chain and light chain expression cassettes, a lac promoter (plac) to drive expression of the antibody genes, and an ampicillin resistance gene. The antibody chains were appended with N-terminal signal peptides to drive their secretion into the periplasmic space. The C-terminal of the heavy chain carried a truncated gene III protein sequence for incorporation into phage particles. The heavy chain also carried hexa-histidine, c-myc and V5 affinity tags. Transformation of these vectors into E. coli and induction with isopropyl β-D-l-thiogalactopyranoside (IPTG) resulted in periplasmic expression of soluble Fab molecules. [00397] Fab PPE production: Eluted phage pools from panning round 3 were diluted and infected into TGI E. coli cells (Lucigen, Middleton, WI) so that single colonies were generated when spread on a 2YTCG agar plate. This resulted in each colony carrying a pXHMV-Fab vector encoding a single Fab clone. Individual clones were inoculated into 1 mL 2YTCG starter cultures in 96-well deepwell blocks (Greiner Bio-One, Frickenhausen, Germany) using a Qpix2 instrument (Molecular Devices, Sunnyvale, CA). These starter cultures were grown overnight in a Multitron 3mm incubator (ATR Biotech, Laurel, MD) at 37°C with 1000 rpm shaking.
[00398] For Fab expression, 20 of 1 mL starter cultures were transferred into a second set of deepwell plates containing 1 mL 2YT with 0.1% glucose and 100 μg/mL ampicillin. Cultures were grown until the average OD 600nm was 0.5-1.0 and protein expression induced by adding IPTG (Teknova) to a final concentration of 1 mM. Expression cultures were incubated overnight in the Multitron instrument at 25°C with 700 rpm shaking.
[00399] Fab proteins secreted into the E. coli periplasm were extracted for analysis.
Cells were harvested by centrifugation at 2500xG, the supernatants were discarded and pellets were re-suspended in 75 ice-cold PPB buffer (Teknova). Extracts were incubated for 10 mins at 4°C with 1000 rpm shaking, and then 225 μΐ. ice-cold ddH20 was then added and incubated for a further lhr. The resulting periplasmic extract (PPE) was cleared by centrifugation at 2500xG and transferred to separate plates or tubes for ELISA and FACS analysis. Note that all extraction buffers contain EDTA-free Complete Protease Inhibitors® (Roche, Basel, Switzerland).
[00400] ELISA binding Assays: Each plate of PPE extracts was tested for binding to biotinylated VSIGl-ECD-Ig H:H (SEQ ID NO 12) and the negative Fc-fusion control protein. The ELISA followed the general protocol above, except that the biotinylated antigen was captured on a streptavidin-coated 96-well plate (Pierce) instead of a standard ELISA plate. A 50 μΐ. aliquot of each PPE was added to plate wells coated with these antigens and the remainder of the ELISA followed the general method. Bound Fab was detected using a HRP-conjugated anti-human Fab'2 antibody (Jackson Immunoresearch) diluted 1 :2000 in PBS with 5% skim milk.
[00401] Reformatting of Fab hits and production as human IgG molecules: Protein expression constructs for human anti-VSIGl IgGs were derived by PCR-amplification of variable heavy, lambda and kappa domain genes, which were sub-cloned into pFUSE-CHIg- hGl (human IgGl heavy chain), pFUSE2-CLIg-hK (human kappa light chain) or pFUSE2- CLIg-hL2 (human lambda 2 light chain) vectors, respectively (all expression vectors sourced from Invivogen).
[00402] Expi293 cells (Life Technologies) were seeded at 6xl05 cells/ml in in
Expi293™ medium (Life Technologies) and incubated for 72 hrs at 37°C in a humidified atmosphere of 8% CO2 with shaking at 125 rpm. This cell stock was used to seed expression cultures at 2.0 xlO6 cells/ml in Expi293™ medium. These cultures were incubated as above for 24 hrs with shaking at 135 rpm.
[00403] For transfection, cells were diluted again to 2.5xl06 cells/ml in Expi293 medium. The protein expression constructs for antibody heavy chain and light chain were mixed at a ratio of 1:2. For every 30 mL of expression culture volume, 30 μg of DNA and 81 of Expifectamine (Life Technologies) was each diluted separately to 1.5 mL with Opti- MEM (Life Technologies) and incubated for five minutes. Diluted DNA and Expifectamine were then mixed and incubated at RT for 20 mins. This was then added to the expression culture in a shaker flask and incubated as described above, with shaking at 125 rpm.
[00404] Approximately 20 hrs post-transfection, 150μί of ExpiFectamine 293 transfection Enhancer 1 and 1.5mL of ExpiFectamine™ 293 Transfection Enhancer 2 was added to each flask. Cultures were incubated for a further five days (six days post- transfection in total) and supematants were harvested by centrifugation. IgGs were purified from the supematants using an AKTA Pure FPLC (GE Healthcare Bio-Sciences) and HiTrap MabS elect Sure® affinity columns (GE Healthcare Bio-Sciences) according to the manufacturer's instructions.
[00405] Antibodies generated according to the methods disclosed in this Example are listed in Table 1.
EXAMPLE 7
[00406] Development of Fully Human Anti-VSIGl Antibodies by Other Methods
Generation of Human Monoclonal Antibodies Against VSIG1 Antigen [00407] Fusion proteins composed of the extracellular domain of the VSIG1 linked to a mouse IgG2 Fc polypeptide are generated by standard recombinant methods and used as antigen for immunization.
Transgenic HuMab Mouse
[00408] Fully human monoclonal antibodies to VSIG1 are prepared using mice from the HCo7 strain of the transgenic HuMab Mouse™ which expresses human antibody genes. In this mouse strain, the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al. (1993) EMBO J. 12:811-820 and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of PCT Publication WO 01/09187. Furthermore, this mouse strain carries a human kappa light chain transgene, KCo5, as described in Fishwild et al. (1996) Nature Biotechnology 14:845-851, and a human heavy chain transgene, HCo7, as described in U.S. Pat. Nos. 5,545,806;
5,625,825; and 5,545,807.
HuMab immunizations
[00409] To generate fully human monoclonal antibodies to VSIG1, mice of the HCo7
HuMab Mouse strain can be immunized with purified recombinant VSIG1 fusion protein derived from mammalian cells that are transfected with an expression vector containing the gene encoding the fusion protein. General immunization schemes for the HuMab Mouse are described in Lonberg, N. et al (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851 and PCT Publication WO 98/24884. The mice are 6-16 weeks of age upon the first infusion of antigen. A purified recombinant VSIG1 antigen preparation (5-50μg, purified from transfected mammalian cells expressing VSIG1 fusion protein) is used to immunize the HuMab mice intraperitoneally.
[00410] Transgenic mice are immunized twice with antigen in complete Freund's adjuvant or Ribi adjuvant IP, followed by 3-21 days IP (up to a total of 11 immunizations) with the antigen in incomplete Freund's or Ribi adjuvant. The immune response is monitored by retroorbital bleeds. The plasma is screened by ELISA (as described below), and mice with sufficient titers of anti-VSIGl human immunoglobulin are used for fusions. Mice are boosted intravenously with antigen 3 days before sacrifice and removal of the spleen. Selection of HuMab mice producing anti- VSIG1 Antibodies
[00411] To select HuMab mice producing antibodies that bind VSIG1 sera from immunized mice is tested by a modified ELISA as originally described by Fishwild, D. et al. (1996). Briefly, microtiter plates are coated with purified recombinant VSIG1 fusion protein at l-2ug/ml in PBS, 50ul/wells incubated 4° C. overnight then blocked with 200ul/well of 5% BSA in PBS. Dilutions of plasma from VSIG1 -immunized mice are added to each well and incubated for 1-2 hours at ambient temperature. The plates are washed with PBS/Tween and then incubated with a goat-anti-human kappa light chain polyclonal antibody conjugated with alkaline phosphatase for 1 hour at room temperature. After washing, the plates are developed with pNPP substrate and analyzed by spectrophotometer at OD 415-650. Mice that developed the highest titers of anti-VSIGl antibodies are used for fusions. Fusions are performed as described below and hybridoma supernatants are tested for anti-VSIGl activity by ELISA.
Generation of Hybridomas Producing Human Monoclonal Antibodies to VSIG1
[00412] The mouse splenocytes, isolated from the HuMab mice, are fused with PEG to a mouse myeloma cell line based upon standard protocols. The resulting hybridomas are then screened for the production of antigen-specific antibodies. Single cell suspensions of splenic lymphocytes from immunized mice are fused to one-fourth the numbers of P3X63 Ag8.6.53 (ATCC CRL 1580) nonsecreting mouse myeloma cells with 50% PEG (Sigma). Cells are plated at approximately 1X10"5 /well in flat bottom microtiter plate, followed by about two week incubation in selective medium containing 10% fetal calf serum, supplemented with origen (IGEN) in RPMI, L-glutamine, sodium pyruvate, HEPES, penicillin, streptomycin, gentamycin, lx HAT, and beta-mercaptoethanol. After 1-2 weeks, cells are cultured in medium in which the HAT is replaced with HT. Individual wells are then screened by ELISA (described above) for human anti-VSIGl monoclonal IgG antibodies. Once extensive hybridoma growth occurred, medium is monitored usually after 10-14 days. The antibody secreting hybridomas are replated, screened again and, if still positive for human IgG, anti- VSIGl monoclonal antibodies are subcloned at least twice by limiting dilution. The stable subclones are then cultured in vitro to generate small amounts of antibody in tissue culture medium for further characterization. The hybridoma clones are selected for further analysis. Structural Characterization of Desired anti-VSIGl Human Monoclonal Antibodies
[00413] The cDNA sequences encoding the heavy and light chain variable regions of the obtained anti-VSIGl monoclonal antibodies are obtained from the resultant hybridomas, respectively, using standard PCR techniques and are sequenced using standard DNA sequencing techniques.
[00414] The nucleotide and amino acid sequences of the heavy chain variable region and of the light chain variable region are identified. These sequences may be compared to known human germline immunoglobulin light and heavy chain sequences and the CDRs of each heavy and light of the obtained anti-VSIGl sequences identified.
Characterization of Binding Specificity and Binding Kinetics of anti-VSIGl Human Monoclonal Antibodies
[00415] The binding affinity, binding kinetics, binding specificity, and cross- competition of anti-VSIGl antibodies are examined by Biacore analysis. Also, binding specificity is examined by flow cytometry.
Binding affinity and kinetics
[00416] Anti- VSIG1 antibodies produced according to the invention are characterized for affinities and binding kinetics by Biacore analysis (Biacore AB, Uppsala, Sweden). Purified recombinant human VSIG1 fusion protein is covalently linked to a CM5 chip (carboxy methyl dextran coated chip) via primary amines, using standard amine coupling chemistry and kit provided by Biacore. Binding is measured by flowing the antibodies in HBS EP buffer (provided by Biacore AB) at a concentration of 267 nM at a flow rate of 50μ1/ιηίη. The antigen-association antibodies association kinetics is followed for 3 minutes and the dissociation kinetics is followed for 7 minutes. The association and dissociation curves are fit to a 1 : 1 Langmuir binding model using BlAevaluation software (Biacore AB). To minimize the effects of avidity in the estimation of the binding constants, only the initial segment of data corresponding to association and dissociation phases are used for fitting.
Epitope mapping of obtained anti-VSIGl antibodies [00417] Biacore is used to determine epitope grouping of anti-VSIGl HuMAbs.
Obtained anti-VSIGl antibodies are used to map their epitopes on the VSIGl antigen. These different antibodies are coated on three different surfaces of the same chip to 8000 RUs each. Dilutions of each of the mAbs are made, starting at 10 μg/mL and is incubated with Fc fused VSIGl (50 nM) for one hour. The incubated complex is injected over all the three surfaces (and a blank surface) at the same time for 1.5 minutes at a flow rate of 20.mu.L/min. Signal from each surface at end of 1.5 minutes, after subtraction of appropriate blanks, has been plotted against concentration of mAb in the complex. Upon analysis of the data, the anti- VSIGl antibodies are categorized into different epitope groups depending on the epitope mapping results. The functional properties thereof are also compared.
[00418] Chinese hamster ovary (CHO) cell lines that express VSIGl protein at the cell surface are developed and used to determine the specificity of the VSIGl HuMAbs by flow cytometry. CHO cells are transfected with expression plasmids containing full length cDNA encoding a transmembrane forms of VSIGl antigen or a variant thereof. The transfected proteins contained an epitope tag at the N-terminus are used for detection by an antibody specific for the epitope. Binding of an anti-VSIGl MAb is assessed by incubating the transfected cells with each of the r VSIGl Abs at a concentration of 10 μg/ml. The cells are washed and binding is detected with a FITC -labeled anti-human IgG Ab. A murine anti- epitope tag Ab, followed by labeled anti-murine IgG, is used as the positive control. Nonspecific human and murine Abs are used as negative controls. The obtained data is used to assess the specificity of the HuMAbs for the VSIGl antigen target.
EXAMPLE 8
[00419] In vivo Efficacy of the Antibody Drug Conjugate Compounds of the Present Invention
[00420] Antibody conjugates with MMAF using the mc linker show anti -tumor efficacy against human tumor xenografts in mice. Marked inhibition of tumor growth is observed upon treatment of various ovarian, liver, pancreas, lung, and stomach tumor xenografts with anti-VSIGl -mc-MMAF. In each experiment, one group of animals bearing established tumors is treated with anti-VSIGl -mc-MMAF. Tumor sizes are measured periodically and graphed vs. time after tumor inoculation. Treated animals have significant tumor growth inhibition. This anti -tumor activity is observed at conjugate doses that have no effect on mouse body weight, a measure of drug toxicity.
[00421] EXAMPLE 9 In vivo Efficacy of the Antibody Drug Conjugate
Compounds of the Present Invention
[00422] In vivo testing of antibody CPA.4.028 yielded no efficacy, both in the form of MMAE and MMAF conjugates. NOD/SCID_mice bearing OV90 tumors of approximately 200mm3 were dosed 3x with lOmg/kg of conjugate over 2 weeks, with no observed decrease in tumor growth relative to mice dosed with control antibodies. IHC analysis of
representative tumors from treatment and control groups revealed a marked loss of VSIG1 expression in vivo, with very low expression of VSIG1 in OV90 tumors relative to that seen in cells grown in vitro, which may account for the observed lack of efficacy.
EXAMPLE 10
[00423] Analysis of the Human Monoclonal Antibodies of the Present Invention
[00424] For all the 15 anti-VSIGl antibodies disclosed herein, the closest human germline gene was determined for each VH and VL domain sequence using the sequence as input to the IgBLAST program on the NCBI website. IgBLAST takes a VH or VL sequence and compares it to a library of known human germline sequences; the databases used were IMGT human VH genes (F+ORF, 273 germline sequences), IMGT human VLkappa genes (F+ORF, 74 germline sequences) and IMGT human VLlambda genes (F+ORF, 78 germline sequences). IgBLAST returned the top 10 human germline sequences according to score.
[00425] The closest human germline genes are listed in Figure 24. The antibodies have been grouped by IGHV gene similarity. VH domains are members of four different human VH subgroups: 1, 3, 4, 5. For VL, both kappa and lambda VL are represented with multiple subgroups for each. No apparent correlation of any VH germline with any VL germline is apparent.
[00426] Figure 25 shows alignments of VH sequences and are assigned into groups based on sequence similarity. Residue numbering and CDR definitions are according to IMGT (compiled at IMGT® the international ImMunoGeneTics information system®. The closest human IGHV germlines are shown aligned in gray boxes. For most CDR-Hl (27-38) and CDR-H2 (56-65), the sequences are similar or identical to the germline. For CDRH3 (105-117), the sequences of all 15 antibodies are very different.
[00427] Figure 26 shows alignments of VL-kappa and VL-lambda sequences and are assigned into groups based on sequence similarity. Residue numbering and CDR definitions are according to IMGT (compiled at IMGT® the international ImMunoGeneTics information system®. The closest human IGKV or IGLV germlines are shown aligned in gray boxes. For the
[00428] VL-kappa sequences, most CDR-L1 (27-38), CDR-L2 (56-65), and CDR-L3 (105-117) the sequences are similar or identical to the germline. For the VL-lambda sequences, there is only slightly more variance from the germline.
EXAMPLE 11
[00429] The expression of VSIG1 protein in human cancer was adressed.
Protocols
Cell Line Pellet Preparation
Cell Line Preparation
[00430] 20 million HEK 293 parental, HEK 293 empty vector, HEK 293 human
VSIG1, HUH-1, OV-90, MKN45, Pane 05.04, or DU145 cells were removed from their original plate or container using Cell Dissociation Buffer in PBS (Invitrogen). Cells were then harvested in 50 ml conical tubes and centrifuged for 5 min. at lOOOrpms. After 2x wash in PBS, cells were fixed by gently pipetting in 10% Neutral Buffered Formalin (10% NBF) to the inside wall of the conical tube so as not to disturb the cell pellet. Cells were allowed to fix for 24 hrs. After 24 hrs. of fixation, NBF was removed from tubes and replaced with 70% ethanol . Cells pellets were embedded in paraffin within 48 hrs.
Immunohistochemistry
[00431] All IHC stains were performed on a Biocare IntelliPath FLX Automated IHC staining platform using 5 μg/ml of primary antibody. Heat-induced epitope retrieval (HIER) was performed off-line in a Biocare decloaking chamber using a citrate-based pH 6.2 buffer (Diva). Anti -human VISIG1 antibody (R&D systems, MAB4818) was stained Two-step Universal detection (Biocare). Images were captured on a Nikon Eclipse E600 microscope with a 40X objective lens and an Olympus DP70 camera using standard bright field settings.
Results
VSIGl Protein is Present on Cell Surface of Solid Tumors
[00432] To validate anti-human VSIGl antibody MAB48418 for
immunohistochemistry, several cell lines were pelleted, fixed, and paraffin embedded to serve as controls. Anti-human VSIGl MAB4818 showed specific binding to HEK 293 cells recominantly expressing VSIGl as well as endogenous cell lines that were predicted to express VSIGl by RNAseq (Figure 34). Anti-VSIGl MAB4814 did not bind to HEK parental, HEK empty vector, or VSIGl RNA negative cell line DU145.
[00433] To examine the prevalence of VSIGl protein in cancer, tissue microarrays from ovarian, stomach, pancreatic, liver, and lung tumors were stained with anti-human VSIGl antibody MAB4814 (Figures 35-39). VSIGl protein was detected on the cell surface of all the cancer tissues surveyed. The highest prevalence was seen in samples from ovarian and stomach cancer (Figures 35 and 36).
EXAMPLE 12
[00434] The cancer therapeutic potential of anti-VSIGl antibodies conjugated to potent cyto-toxins by evaluating in vitro cytotoxic activity was addressed.
Protocols
In vitro Cytotoxicity Assay
[00435] To measure in vitro cell killing, OV-90, Li-7, or HUH-1 cells were cultured in complete OV-90 media (1 : 1 mixture of MCDB 105 media containing a final concentration of 1.5 g/L sodium bicarbonate and Media 199 containing a final concentration of 2.2 g/L sodium bicarbonate with a final concentration of 15% f
(RPMI + 4mM Glutamax + 10% FBS). Cells were harvested in log phase growth and 8,000 OV-90, 5,000 DU145/HUH-1, or 3,000 MKN45 cells in 80 ul per well were seeded in 96 well plates and incubated for 24hrs at 37°C in a humidified water jacketed incubator with 5% CO2. Anti- human VSIG1 human IgGl antibodies or isotype control antibodies directly conjugated to DM1 (non-cleavable linker), MMAE (cleavable linker), MMAF (non-cleavable linker), or Duocarmycin (cleavable linker) (Moradec LLC, San Diego CA) were added in single point dilutions, 2-fold starting at 10 μg/ml. Plates were incubated at 37°C in a humidified water jacketed incubator with 5% CO2. Cell killing was measured after 72 hrs using Cell Titer Glo (Promega) reagent according to the manufacturer's instructions. Data was analyzed using Prism software (GraphPad, La Jolla, CA).
Tumor Spheroid Model In-Vitro Cytotoxicity Assay
[00436] To measure tumor spheroid killing, OV-90, Li-7, or HUH-1 cells were cultured in complete OV-90 media (1 : 1 mixture of MCDB 105 media containing a final concentration of 1.5 g/L sodium bicarbonate and Media 199 containing a final concentration of 2.2 g/L sodium bicarbonate with a final concentration of 15% fetal bovine serum) or Li- 7/HUH-l media (RPMI + 4mM Glutamax + 10% FBS). Cells were harvested in log phase growth and 10,000 OV-90, Li-7, or HUH-1 cells were plated in ultra-low adherence plates (Coming). Anti- human VSIG1 human IgGl antibodies or isotype control antibodies directly conjugated to DM1 (non-cleavable linker), MMAE (cleavable linker), MMAF (non-cleavable linker), or Duocarmycin (cleavable linker) (Moradec LLC, San Diego CA) were added in single point dilutions, 2-fold starting at 10 μg/ml.
[00437] Cells and ADCs were incubated for 72hrs at 37°C in a humidified water jacketed incubator with 5% CO2. Cells were put on a shaker at 100 rpm during the incubation. Plates were incubated at 37°C in a humidified water jacketed incubator with 5% CO2. Cell killing was measured after 72 hrs using 3D Cell Titer Glo (Promega) reagent according to the manufacturer's instructions. Data was analyzed using Prism software (GraphPad, La Jolla, CA).
Results Anti-VSIGl Direct Antibody Drug Conjugate;
[00438] To examine the potency of anti-VSIG antibody drug conjugates, we assayed the in vitro efficacy of anti-VSIGl clone CPA.4.028 conjugated to DM1, MMAE, MMAF, and Duocarmycin (DMSA). OV-90 (ovarian carcinoma), Li-7 (stomach carcinoma), and HUH-1 (hepatocellular carcinoma) human cancer cell lines were selected based on high expression of VSIGl mRNA by microarray and RNAseq. VSIGl protein surface expression on the human cancer cells was verified by FACS. Anti-VSIGl clone CPA.4.028 conjugated to DM1, MMAE, demonstrated potent cytotoxic cell killing of OV-90 and Li-7 cells (Figures 40 and 41). In contrast, HUH-1 cells were only killed by CPA.4.028 conjugated to DMSA (Figure 42).
[00439] To further investigate anti-VSIGl ADCs, we developed a spheroid tumor model using OV-90, Li-7, and HUH-1 cells. Anti-VSIGl clone CPA.4.028 conjugated with cytotoxic payloads showed potent cytotoxicity on OV-90 and Li-7 cells (Figures 43 and 44). Of all the ADCs tested, CPA.4.028 conjugated to MMAE with a cleavable linker had the most potent activity, with IC50's less than 1 nM (Figures 43 and 44).
Table 4. IC50 values From in Vitro Cytotoxic Assay with 4 anti-VSIGl ADCs. IC50 values shown are in nM. ND= not determined.
Figure imgf000099_0001
Conclusion
[00440] Taken together, these data show the in-vitro efficacy of anti-VSIGl antibody drug conjugates. Direct conjugates of anti-VSIGl antibody CPA.4.028 to DM1, MMAE, MMAF, and DMSA all showed activity against OV-90 and Li-7 cancer cell lines in both 2D and 3D tumor spheroid models. Interestingly, CPA.4.028-DMSA showed more potent activity in 2D cytotoxicity models but less in 3D models. In contrast, CPA.4.028-vc-MMAE showed more potent activity in 3D tumor spheroid models but less in 2D models. These diverging results could be due to differences in the penetrance of the DMSA and MMAE conjugates. MMAE conjugates are more soluble and may penetrate into the necrotic core of 3D tumor spheroids better than DMSA. Although HUH-1 cells have high expression of VSIGl protein, only the CPA.4.028-DMSA showed significant cytotoxic cell killing. HUH-1 cells may have a higher resistance to the payloads, or metabolize the payloads differently than the other VSIGl + cancer cell lines tested.

Claims

WHAT IS CLAIMED;
1. An antibody that binds V-set and immunoglobulin domain-containing protein 1 (VSIGl), wherein said antibody is selected from the group consisting of: CPA.4.028, CPA.4.013, CPA.4.016, CPA.4.017, CPA.4.019, CPA.4.020, CPA.4.023, CPA.4.005, CPA.4.009, CPA.4.027, CPA.4.031, CPA.4.033, CPA.4.012, CPA.4.008, and CPA.4.011.
2. The anti-VSIGl antibody of claim 1 where said antibody is an antibody drug conjugate compound that binds V-set and immunoglobulin domain-containing protein 1 (VSIGl), comprising at least one drug moiety and an anti-VSIGl antibody according to claim 1.
3. The anti-VSIGl antibody according to claim 2 wherein said drug moiety is attached to said antibody by an ADC linker.
4. The anti-VSIGl antibody according to claim 2 wherein said ADC linker is cleavable.
5. The anti-VSIGl antibody according to claims 3 or 4 wherein said ADC linker is self- immolative.
6. The anti-VSIGl antibody according to any of claims 2 to 5 wherein said drug moiety is selected from the group consisting of: taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine, mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) ((DDP) cisplatin), daunorubicin, doxorubicin), dactinomycin, bleomycin, mithramycin, anthramycin (AMC)), vincristine, vinblastine, a duocarmycin, a calicheamicin, a dolastatin, a maytansine, an auristatin, iodine 131, indium 111, yttrium 90, and lutetium 177.
7. The anti-VSIGl antibody according to any of claims 2 to 6 wherein said drug moiety is selected from the group consisting of: the maytansinoid (N2'-deacetyl-N2'-(3-mercapto-l- oxopropyl)maytansine) (DM1), monomethyl auristatin (MMAE), monomethyl auristatin phenylalanine (MMAF), and duocarmycin.
8. The anti-VSIGl antibody of any of claims 2 to 7, wherein the antibody-drug conjugate compound is cytostatic to cancer cells.
9. The anti-VSIGl antibody of any of claims 1 to 8, wherein said anti-VSIGl antibody binds to an epitope located within the extracellular domain of VSIGl .
10. The anti -VSIGl antibody of any of claims
competes for binding at an epitope located within the extracellular domain of VSIGl.
11. The anti -VSIGl antibody of any of claims 1 to 10, wherein the anti -VSIGl antibody is a human monoclonal anti-VSIGl antibody.
12. The anti-VSIGl antibody of claim 11, wherein the human monoclonal anti-VSIGl antibody is CPA.4.028.
13. The anti-VSIGl antibody of claim 11, wherein the human monoclonal anti-VSIGl antibody comprises a variable heavy and variable light chain pair selected from the group consisting of: the variable heavy and variable light chain pair from CPA.4.028, the variable heavy and variable light chain pair from CPA.4.013, the variable heavy and variable light chain pair from CPA.4.016, the variable heavy and variable light chain pair from CPA.4.017, the variable heavy and variable light chain pair from CPA.4.019, the variable heavy and variable light chain pair from CPA.4.020, the variable heavy and variable light chain pair from CPA.4.023, the variable heavy and variable light chain pair from CPA.4.005, the variable heavy and variable light chain pair from CPA.4.009, the variable heavy and variable light chain pair from CPA.4.027, the variable heavy and variable light chain pair from CPA.4.031, the variable heavy and variable light chain pair from CPA.4.033, the variable heavy and variable light chain pair from CPA.4.012, the variable heavy and variable light chain pair from CPA.4.008, and the variable heavy and variable light chain pair from
CPA.4.011.
14. An antibody that binds V-set and immunoglobulin domain-containing protein 1 (VSIGl), wherein said antibody comprises a variable heavy chain sequence and a variable light chain sequence selected from the group consisting of:
i) a variable heavy chain sequence and a variable light chain sequence from
CPA.4.028, wherein
a) the variable heavy chain sequence comprises a vHCDRl having the sequence GFTFSSYG (SEQ ID NO:X), a vHCDR2 having the sequence IWYDGSNK (SEQ ID NO:X), and a vHCDR3 having the sequence
ARGGPWGIVVVNQFDY (SEQ ID NO:X); and
b) the variable light chain sequence comprises a vLCDRl having the sequence SDINVSSYN (SEQ ID NO:X), a vLCDR2 having the sequence YYSDSDK (SEQ ID NO:X), and a vLCDR3 having the sequence MIWPSTGRWV (SEQ ID NO:X); and ii) a variable heavy chain sequence and
4.013, wherein:
a) the variable heavy chain sequence comprises a vHCDRl having the sequence GFTFSSYS (SEQ ID NO:X), a vHCDR2 having the sequence ISSSSSYI (SEQ ID NO:X), and a vHCDR3 having the sequence
ARNVLRGAAQYYFDY (SEQ ID NO:X); and
b) the variable light chain sequence comprises a vLCDRl having the sequence QSLLHGTGYNY (SEQ ID NO:X), a vLCDR2 having the sequence LGS (SEQ ID NO:X), and a vLCDR3 having the sequence MQALQTPLT (SEQ ID NO:X); and
iii) a variable heavy chain sequence and a variable light chain sequence from
CPA.4.016, wherein:
a) the variable heavy chain sequence comprises a vHCDRl having the sequence GFTFSSYG (SEQ ID NO:X), a vHCDR2 having the sequence ISYDGSNK (SEQ ID NO:X), and a vHCDR3 having the sequence
VRGINVNFHYYGMDV (SEQ ID NO:X); and
b) the variable light chain sequence comprises a vLCDRl having the sequence NTNIGADYH (SEQ ID NO:X), a vLCDR2 having the sequence SNN (SEQ ID NO:X), and a vLCDR3 having the sequence QSFDSSLSAWV (SEQ ID NO:X); and
iv) a variable heavy chain sequence and a variable light chain sequence from
CPA.4.017, wherein:
a) the variable heavy chain sequence comprises a vHCDRl having the sequence GFTFSSYA (SEQ ID NO:X), a vHCDR2 having the sequence ISGSGGST (SEQ ID NO:X), and a vHCDR3 having the sequence
AKDSYFDWQIGSNDAFDI (SEQ ID NO:X); and
b) the variable light chain sequence comprises a vLCDRl having the sequence SNNVGYQG (SEQ ID NO:X), a vLCDR2 having the sequence RNN (SEQ ID NO:X), and a vLCDR3 having the sequence
SAWDSSLTAWV (SEQ ID NO:X); and
v) a variable heavy chain sequence and a variable light chain sequence from
CP A.4.019, wherein:
a) the variable heavy chain sequence comprises a vHCDRl having the sequence GFTFSSYA (SEQ ID NO:X), a vHCDR2 having the sequence ISGSGGST (SEQ ID NO:X), an
AKAYGSGSYFHYWYFDL (SEQ ID NO:X); and
b) the variable light chain sequence comprises a vLCDRl having the sequence QSLLQSNGYNY (SEQ ID NO:X), a vLCDR2 having the sequence LGS (SEQ ID NO:X), and a vLCDR3 having the sequence MQALQTPPT (SEQ ID NO:X); and
vi) a variable heavy chain sequence and a variable light chain sequence from
CPA.4.020, wherein:
a) the variable heavy chain sequence comprises a vHCDRl having the sequence GGTFSSYA (SEQ ID NO:X), a vHCDR2 having the sequence IIPIFGTA (SEQ ID NO:X), and a vHCDR3 having the sequence
ATQYSSGWYIWGAFDI (SEQ ID NO:X); and
b) the variable light chain sequence comprises a vLCDRl having the sequence QSLLHSSGDNF (SEQ ID NO:X), a vLCDR2 having the sequence LAS (SEQ ID NO:X), and a vLCDR3 having the sequence MQTLQTPLT (SEQ ID NO:X); and
vii) a variable heavy chain sequence and a variable light chain sequence from
CPA.4.023, wherein:
a) the variable heavy chain sequence comprises a vHCDRl having the sequence GYSFTSYW (SEQ ID NO:X), a vHCDR2 having the sequence IYPGDSDT (SEQ ID NO:X), and a vHCDR3 having the sequence
ARLGIVDTSWSAFDI (SEQ ID NO:X); and
b) the variable light chain sequence comprises a vLCDRl having the sequence SSNIGSNA (SEQ ID NO:X), a vLCDR2 having the sequence YDD (SEQ ID NO:X), and a vLCDR3 having the sequence AAWDDSLNGVV (SEQ ID NO:X); and
viii) a variable heavy chain sequence and a variable light chain sequence from
CPA.4.005, wherein:
a) the variable heavy chain sequence comprises a vHCDRl having the sequence GDTFSSYT (SEQ ID NO:X), a vHCDR2 having the sequence FIPPLDIA (SEQ ID NO:X), and a vHCDR3 having the sequence
ATGGATIFFYYFGMDV (SEQ ID NO:X); and
b) the variable light chain sequence comprises a vLCDRl having the sequence RSNIGSGS (SEQ ID NO:X), a vLCDR2 having the sequence TNS (SEQ ID NO:X), and a vLCDR3
(SEQ ID NO:X); and
) a variable heavy chain sequence and a variable light chain sequence from
CPA.4.009, wherein:
a) the variable heavy chain sequence comprises a vHCDRl having the sequence GYTFTSYA (SEQ ID NO:X), a vHCDR2 having the sequence INAGNGNT (SEQ ID NO:X), and a vHCDR3 having the sequence
ASS FHGS GS YYNKV VGMWY (SEQ ID NO:X); and
b) the variable light chain sequence comprises a vLCDRl having the sequence QSLKHNNGYSY (SEQ ID NO:X), a vLCDR2 having the sequence LDS (SEQ ID NO:X), and a vLCDR3 having the sequence MQGLQIPVT (SEQ ID NO:X); and
a variable heavy chain sequence and a variable light chain sequence from
CPA.4.027, wherein:
a) the variable heavy chain sequence comprises a vHCDRl having the sequence GFSISDYW (SEQ ID NO:X), a vHCDR2 having the sequence VSPGGGHLT (SEQ ID NO:X), and a vHCDR3 having the sequence VRGTHLWRGVDY (SEQ ID NO:X); and
b) the variable light chain sequence comprises a vLCDRl having the sequence QSVSSY (SEQ ID NO:X), a vLCDR2 having the sequence DAS (SEQ ID NO:X), and a vLCDR3 having the sequence QQRSNWPVT (SEQ ID NO:X); and
) a variable heavy chain sequence and a variable light chain sequence from
CPA.4.031, wherein
a) the variable heavy chain sequence comprises a vHCDRl having the sequence SGSIRSSNW (SEQ ID NO:X), a vHCDR2 having the sequence IYHSGST (SEQ ID NO:X), and a vHCDR3 having the sequence
AGRNIAGGSFDY (SEQ ID NO:X); and
b) the variable light chain sequence comprises a vLCDRl having the sequence SGIDVGPYR (SEQ ID NO:X), a vLCDR2 having the sequence YNSDSDK (SEQ ID NO:X), and a vLCDR3 having the sequence
MIWHNKSRV (SEQ ID NO:X); and
') a variable heavy chain sequence and a variable light chain sequence from
CPA.4.033, wherein: a) the variable heavy chain s
sequence GGSISTYY (SEQ ID NO:X), a vHCDR2 having the sequence IYFNDIT (SEQ ID NO:X), and a vHCDR3 having the sequence
VRGRGGSPALDY (SEQ ID NO:X); and
b) the variable light chain sequence comprises a vLCDRl having the sequence QSLLHSNGYNY (SEQ ID NO:X), a vLCDR2 having the sequence LGS (SEQ ID NO:X), and a vLCDR3 having the sequence KQALQTIT (SEQ ID NO:X); and
xiii) a variable heavy chain sequence and a variable light chain sequence from
CPA.4.012, wherein:
a) the variable heavy chain sequence comprises a vHCDRl having the sequence GFSFSNYV (SEQ ID NO:X), a vHCDR2 having the sequence ISYDGSDK (SEQ ID NO:X), and a vHCDR3 having the sequence
ARSLRPAYYDASGV (SEQ ID NO:X); and
b) the variable light chain sequence comprises a vLCDRl having the sequence RSNIGAGFD (SEQ ID NO:X), a vLCDR2 having the sequence GDT (SEQ ID NO:X), and a vLCDR3 having the sequence
QSYDSSLSVFYV (SEQ ID NO:X); and
xiv) a variable heavy chain sequence and a variable light chain sequence from
CPA.4.008, wherein:
a) the variable heavy chain sequence comprises a vHCDRl having the sequence GGTFSSYS (SEQ ID NO:X), a vHCDR2 having the sequence IIPLFGTT (SEQ ID NO:X), and a vHCDR3 having the sequence
ARSKDYYGSGMEALLMDV (SEQ ID NO:X); and
b) the variable light chain sequence comprises a vLCDRl having the sequence SGSVSTRNY (SEQ ID NO:X), a vLCDR2 having the sequence NTN (SEQ ID NO:X), and a vLCDR3 having the sequence VFYMGSGRWV (SEQ ID NO:X); and
xv) a variable heavy chain sequence and a variable light chain sequence from
CPA.4.011, wherein:
a) the variable heavy chain sequence comprises a vHCDRl having the sequence GFTFSSYA (SEQ ID NO:X), a vHCDR2 having the sequence ISGSGGST (SEQ ID NO:X), and a vHCDR3 having the sequence
AKELRGGSYYFTGTDAFDI (SEQ ID NO:X); and b) the variable light chain sei
sequence QSLLKSNGYNY (SEQ ID NO:X), a vLCDR2 having the sequence LGS (SEQ ID NO:X), and a vLCDR3 having the sequence MQALQTPPT (SEQ ID NO:X).
15. The anti-VSIGl antibody according to claim 14 wherein said variable heavy chain is CPA.4.028.VH.
16. The anti-VSIGl antibody according to claim 14 wherein said variable light chain is CPA.4.028.VL.
17. The anti-VSIGl antibody according to any of the preceding claims further comprising at least one covalently attached drug moiety.
18. A nucleic acid composition comprising:
a) a first nucleic acid encoding a variable heavy chain, said variable heavy chain comprising a vHCDRl , a vHCDR2, and a vHCDR3 according to any of the preceding claims; and
b) a second nucleic acid encoding a variable light chain, said variable light chain comprising a vLCDRl, a vLCDR2, and a vLCDR3 according to any of the preceding claims.
19. An expression vector composition comprising:
a) a first expression vector comprising the first nucleic acid of claim 18; and b) a second expression vector comprising the second nucleic acid of claim 18.
20. An expression vector comprising the first nucleic acid of claim 18 and the second nucleic acid of claim 18.
21. A host cell comprising a nucleic acid composition consisting of said nucleic acids of claim 18.
22. A host cell comprising an expression vector composition selected from the group consisting of the expression vector compositions of claims 19 and 20.
23. A method of making a composition comprising an anti-VSIGl antibody according to any of claims 1 to 17 comprising culturing a host cell according to either claim 21 or 22 under conditions whereby said antibody is expressed, and recovering said antibody.
24. A method of making a composition according to any of claims 1 to 17 comprising: a) culturing a host cell according to either claim 21 or 22 under conditions whereby said antibody is expressed;
b) recovering said antibody; and c) contacting said antibody with an actn
wherein at least one drug is covalently attached to said antibody.
25. An isolated anti-VSIGl ADC antibody comprising:
a) a variable heavy chain comprising a vHCDRl having the sequence GFTFSSYG (SEQ ID NO:X), a vHCDR2 having the sequence IWYDGSNK (SEQ ID NO:X), and a vHCDR.3 having the sequence ARGGPWGIVVVNQFDY (SEQ ID NO:X); and b) a variable light chain comprising a vLCDRl having the sequence SDINVSSYN (SEQ ID NO:X), a vLCDR2 having the sequence YYSDSDK (SEQ ID NO:X), and a vLCDR3 having the sequence MIWPSTGRWV (SEQ ID NO:X).
wherein said antibody further comprises a chemotherapeutic drug covalently attached via an ADC linker.
26. The anti-VSIGl antibody according to claim 25 wherein said drug is selected from the group consisting of duocarmycin, maytansinoid, auristatin, dolastatin, and calicheamicin.
27. The anti-VSIGl antibody according to claim 26 wherein said duocarmycin is a duocarmycin analog.
28. The anti-VSIGl antibody according to claim 26 wherein said maytansinoid is a maytansinoid analog.
29. The anti-VSIGl antibody according to claim 26 wherein said auristatin is an auristatin analog.
30. The anti-VSIGl antibody according to claim 29 wherein said auristatin analog is selected from the group consisting of monomethyl auristatin (MMAE) and monomethyl auristatin phenylalanine (MMAF).
31. The anti-VSIGl antibody according to claim 26 wherein said dolastatin is a dolastatin analog.
32. The anti-VSIGl antibody according to claim 26 wherein said calicheamicin is a calicheamicin analog.
33. The anti-VSIGl antibody according to any of claims 25 to 32 wherein said ADC linker is susceptible to cleavage by an enzyme.
34. The anti-VSIGl antibody according to claim 33 wherein said enzyme is a protease.
35. The anti-VSIGl antibody according to any of claims 25 to 34 wherein said ADC linker is self-immolative.
36. The anti-VSIGl antibody according to any of claims 25 to 35 wherein said ADC linker is stable under physiological conditions.
37. The anti-VSIGl antibody according to any
heavy chain is CPA.4.028.VH and said variable light chain is CPA.4.028.VL.
38. A method of treating liver cancer comprising administering an anti-VSIGl ADC antibody according to any of claims 25 to 37 to a patient with liver cancer.
39. A method of treating stomach cancer comprising administering an anti-VSIGl ADC antibody according to any of claims 25 to 37 to a patient with stomach cancer.
40. A method of treating ovarian cancer comprising administering an anti-VSIGl ADC antibody according to any of claims 25 to 37 to a patient with ovarian cancer.
41. A method of treating pancreatic cancer comprising administering an anti-VSIGl ADC antibody according to any of claims 25 to 37 to a patient with pancreatic cancer.
42. A nucleic acid composition comprising:
a) a first nucleic acid encoding a variable heavy chain from CPA.4.028; and b) a second nucleic acid encoding a variable light chain from CPA.4.028.
43. An expression vector composition comprising:
a) a first expression vector comprising the first nucleic acid of claim 42; and b) a second expression vector comprising the second nucleic acid of claim 42.
44. An expression vector comprising the first nucleic acid of claim 42 and the second nucleic acid of claim 42.
45. A host cell comprising a nucleic acid composition consisting of said nucleic acids of claim 42.
46. A host cell comprising an expression vector composition selected from the group consisting of the expression vector compositions of claims 43 and 44.
47. A method of making a composition according to any of claims 25 to 37 comprising culturing a host cell according to either claim 45 or 46 under conditions whereby said antibody is expressed, and recovering said antibody.
48. A method of making a composition according to any of claims 25 to 37 comprising: a) culturing a host cell according to either claim 45 or 46 under conditions whereby said antibody is expressed;
b) recovering said antibody; and
c) contacting said antibody with an activated chemotherapeutic agent under conditions wherein at least one drug is covalently attached to said antibody.
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