WO2017055966A1 - Low viscosity antibody compositions - Google Patents

Low viscosity antibody compositions Download PDF

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Publication number
WO2017055966A1
WO2017055966A1 PCT/IB2016/055612 IB2016055612W WO2017055966A1 WO 2017055966 A1 WO2017055966 A1 WO 2017055966A1 IB 2016055612 W IB2016055612 W IB 2016055612W WO 2017055966 A1 WO2017055966 A1 WO 2017055966A1
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Prior art keywords
amino acid
acid sequence
antibody
seq
sequence shown
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PCT/IB2016/055612
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French (fr)
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Robert Henry WALTERS
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Pfizer Inc.
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Publication of WO2017055966A1 publication Critical patent/WO2017055966A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591Stabilisation, fragmentation
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12Carboxylic acids; Salts or anhydrides thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183Amino acids, e.g. glycine, EDTA or aspartame
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0019Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/14Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52Constant or Fc region; Isotype
    • C07K2317/53Hinge

Definitions

  • the present invention relates to the field of pharmaceutical formulations of antibodies. Specifically, the present invention relates to a low viscosity liquid antibody formulation and its pharmaceutical preparation and use.
  • Antibody preparations intended for therapeutic or prophylactic use require stabilizers to prevent loss of activity or structural integrity of the protein due to the effects of denaturation, oxidation or aggregation over a period of time during storage and transportation prior to use. These problems are exacerbated at the high concentrations of antibody often desired for therapeutic administration.
  • a major aim in the development of antibody formulations is to maintain antibody solubility, stability and potency of its antigen binding. It is particularly desirable to avoid antibody self-association, aggregates, and particulates in solution which would require sterile filtration before use for intravenous or subcutaneous injection and limit route of administration.
  • Antibody aggregates can cause pain and anaphylactoid side effects when the formulation containing them is intravenously injected.
  • self- associated antibodies and aggregates present increased difficulties for subcutaneous (sc) administration.
  • the ease of injection for sc administration is described as extrusion force, the force required to extrude the composition from the syringe.
  • the viscosity of the liquid composition is directly related to the required extrusion force: more viscous compositions require greater extrusion force.
  • a larger diameter needle or a longer injection time may be required to administer the desired dose.
  • high viscosity compositions present a greater risk for pain at the injection site, and thus may negatively impact patient compliance.
  • Self-associated antibodies exhibit high viscosity and result in difficulty in manufacturing. Tangential flow filtration is often used in manufacturing for buffer exchange and protein concentration. High viscosity compositions create additional back pressure and shear stress during this process, which can increase the processing time and destabilize the antibody.
  • One solution to self -association of antibody therapeutics is to formulate the therapeutic in a viscosity lowering composition. Lyophilization or freeze drying is an alternative to the liquid formulation of antibodies. The process has a propensity for inducing denaturation of the antibody and decreasing its antigen-binding activity particularly upon reconstitution.
  • Viscosity lowering excipients, surfactants, pH, and cryoprotectant/tonicity agents such as sugars can contribute to overcoming self-association problems.
  • Formulation of antibody preparations requires careful selection of these factors among others to avoid denaturation of the protein and loss of antigen-binding activity.
  • formulation constituents, such as sugars may further exacerbate the self-association tendency of the antibody and high concentrations of these stabilizing excipients can lead to high viscosities.
  • Some viscosity lowering excipients have been explored including arginine, histidine, lysine, and camphor-10-sulfonic acid.
  • intramuscular, intraperitoneal, intradermal or subcutaneous injection is further desirable that the formulation has minimized risk of anaphylactoid side effects.
  • compositions comprising a. an antibody, wherein the antibody concentration is between about 100 mg/ml to about 400 mg/ml, and b. a viscosity lowering excipient comprising camphorsulfonic acid, sulfosalicylic acid, a salt of camphorsulfonic acid or a salt of sulfosalicylic acid, wherein the viscosity lowering excipient concentration is between about 30 mM to about 200 mM; wherein the pH of said composition is from about 4.0 to about 9.0.
  • FIG. 1 A depicts a graph comparing the viscosity of anti-IL-7R antibody formulation at different pH values.
  • FIG. 1 B depicts a graph comparing the viscosity of anti-IL-7R antibody formulation at different pH values.
  • FIG. 2A depicts a graph comparing the viscosity of anti-IL7R antibody formulation with and without camphorsulfonic acid.
  • FIG. 2B depicts a graph comparing the viscosity of anti-IL7R antibody formulation with and without camphorsulfonic acid.
  • FIG. 3 depicts a graph comparing the viscosity of anti-IL7R antibody formulation with a combination of camphorsulfonic acid and arginine, and arginine HCI.
  • FIG 4 depicts a graph comparing the viscosity of anti-IL7R antibody formulation at varying concentrations of camphorsulfonic acid, camphorsulfonic acid and arginine, and arginine HCI.
  • FIG. 5 depicts a graph comparing the viscosity of anti-glucagon receptor antibody (XX1 ) and anti-PCSK9 receptor antibody (XX2) formulations.
  • FIG. 6 depicts a graph comparing the viscosity of anti-glucagon receptor antibody (XX1 ) and anti-PCSK9 receptor antibody (XX2) with camphorsulfonic acid,
  • FIG. 7A depicts a graph comparing the viscosity of anti-glucagon receptor antibody (XX1 ) and anti-PCSK9 receptor antibody (XX2) with varying levels of camphorsulfonic acid, camphorsulfonic acid and arginine, and arginine HCI.
  • FIG. 7B depicts a graph comparing the viscosity of anti-glucagon receptor antibody (XX1 ) and anti-PCSK9 receptor antibody (XX2) with varying levels of camphorsulfonic acid, camphorsulfonic acid and arginine, and arginine HCI.
  • the present invention may be understood even more readily by reference to the following detailed description of exemplary embodiments of the invention and the examples included therein. Unless stated otherwise, the concentrations listed herein are those concentrations at ambient conditions, [i.e., at 25°C and atmospheric pressure].
  • an “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc. , through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
  • a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.
  • the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv) and domain antibodies), and fusion proteins comprising an antibody portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site.
  • An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2, which denote differences in the constant region, particularly in the hinge and upper CH 2 domain.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Following human lgG1 EU numbering and beginning at the 216 position, the isotypes have the following sequences: lgG1 : EPKSCDKTHTCPPCP lgG2: ERKCCVE— CPPCP lgG4: ESKYGPP— CPSCP
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567.
  • the monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example.
  • variable region of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination.
  • variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) that contain hypervariable regions.
  • FR framework regions
  • CDRs complementarity determining regions
  • the identity of the amino acid residues in a particular antibody that make up a CDR can be determined using methods well known in the art. For example, antibody CDRs may be identified as the
  • hypervariable regions originally defined by Kabat et al (Kabat et al., 1991 , Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C., NIH Publication No. 91 -3242).
  • the positions of the CDRs may also be identified as the structural loop structures described by Chothia and others (Chothia et al., 1989, Nature 342:877-883).
  • CDRs In another approach, referred to herein as the "conformational definition" of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding (Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1 166). Still other CDR boundary definitions may not strictly follow one of the above approaches, but will nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. As used herein, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches.
  • CDRs defined according to any of these approaches.
  • the CDRs (or other residue of the antibody) may be defined in accordance with any of Kabat, Chothia, North, extended, AbM, contact, and/or conformational definitions.
  • a "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.
  • the "hinge region" of an antibody consists of a flexible domain that joins the Fab arms to the Fc region.
  • the antibody of the present invention is selected from the group of monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, ScFv etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion (e.g., a domain antibody), humanized antibodies, human antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • the antibody may be murine, rat, human, or any other origin (including chimeric or humanized antibodies).
  • the antibody can be human but is more preferably humanized.
  • the antibody is isolated, further preferably it is substantially pure. Where the antibody is an antibody fragment this preferably retains the functional characteristics of the original antibody i.e. the ligand binding and/or antagonist or agonist activity.
  • the antibody heavy chain constant region may be from any type of constant region, such as IgG, IgM, IgD, IgA, and IgE; and any isotypes, such as IgGI , lgG2, lgG3, and lgG4.
  • the antibody is an lgG2 antibody.
  • the antibody can comprise the human heavy chain lgG2a constant region.
  • the antibody comprises the human light chain kappa constant region.
  • the antibody comprises a modified constant region, such as a constant region that is immunologically inert, e.g., does not trigger complement mediated lysis, or does not stimulate antibody-dependent cell mediated cytotoxicity (ADCC).
  • the constant region is modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT publication No.
  • the antibody comprises a human heavy chain lgG2a constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering with reference to the wildtype lgG2a sequence), Eur. J. Immunol. (1999) 29:2613-2624.
  • the antibody does not necessarily comprise an identical amino acid sequence of the amino acid sequence described herein.
  • An antibody that has a similar amino acid sequence refers to an antibody analog that satisfies at least one of the following: (a) an amino acid sequence that is at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% identical to the amino acid sequence of any of the antibodies or portions of antibodies described herein; (b) an antibody encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding the antibody of at least about 5 contiguous amino acid residues, at least about 10 contiguous amino acid residues, at least about 15 contiguous amino acid residues, at least about 20 contiguous amino acid residues, at least about
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the two sequences are the same length.
  • One, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et ah, 1990, J. Mol. Biol. 215:403.
  • Gapped BLAST can be utilized as described in Altschul et al, 1997, Nucleic Acids Res. 25:3389-3402.
  • PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id).
  • BLAST Gapped BLAST
  • PSI-Blast programs the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website).
  • Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11 -17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
  • the formulations of the present invention include at least one antibody. In some embodiments, more than one antibody may be present. At least one, at least two, at least three, at least four, at least five, or more, different antibodies may be present. Generally, the two or more different antibodies have complementary activities that do not adversely affect each other. The, or each, antibody can also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the antibodies.
  • the concentration of antibody can range from about 50 mg/ml to about 450 mg/ml. In other embodiments, the concentration of antibody is about 50 mg/ml, about 70 mg/ml, about 90 mg/ml, about 100 mg/ml, about 1 10 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, about 150 mg/ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190 mg/ml, about 200 mg/ml, about 210 mg/ml, about 220 mg/ml, about 230 mg/ml, about 240 mg/ml, about 250 mg/ml, about 260 mg/ml, about 270 mg/ml, about 280 mg/ml, about 290 mg/ml, about 300 mg/ml, about 310 mg/ml, about 320 mg/ml, about 330 mg/ml, about 340 mg/ml, about 350 mg/m/m
  • the concentration of the antibody in the formulation is between about 50 mg/ml and about 400 mg/ml, between about 100 mg/ml and about 400 mg/ml, between about 100 mg/ml and about 350 mg/ml, between about 100 mg/ml and about 300 mg/ml, between about 100 mg/ml and about 250 mg/ml, between about 100 mg/ml and about 200 mg/ml, between about 120 mg/ml and 200 mg/ml, between about 150 mg/ml and about 200 mg/ml, or between about 165 mg/ml and about 215 mg/ml.
  • Isolated when used to describe the various antibodies disclosed herein, means a polypeptide or antibody that has been identified, separated and/or recovered from a component of its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the antibody in the antibody formulation is purified prior to being added to the antibody formulation.
  • At least about 50%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, at least about or 99.9% (w/w) of an impurity is purified from the antibody.
  • CSA camphor-10-sulphonic acid or the compound of formula CSA.
  • CSA is represented by the chemical formula:
  • CSA includes all hydrate/solvate forms including the anhydrous form as well as the free base form, as well as any salt form.
  • CSA includes all enantiomers (e.g. ,
  • (+)camphorsulphonic acid and (-)camphorsulphonic acid any combination of enantiomers (e.g., 50% (+)camphorsulphonic acid and 50% (-)camphorsulphonic acid; 90%-100% (+)camphorsulphonic acid and 10%-0% (-)camphorsulphonic acid, etc.).
  • the term "CSA” includes greater than 99% (+)camphorsulphonic acid and less than 1 % (-)camphorsulphonic acid.
  • the term "CSA” includes an enantomerically pure (+)camphorsulphonic acid.
  • Exemplary pharmaceutically acceptable acid salts may be formed from counterions and include sodium, arginine, lysine, histidine, and include all hydrates, solvates, and enantiomers thereof.
  • the present invention includes various amounts of CSA.
  • the amount of CSA is greater than about 5 mM, greater than about 10 mM, greater than about 15 mM, greater than about 20 mM, greater than about 25 mM, greater than about 30 mM , greater than about 35 mM , greater than about 50 mM , greater than about 75 mM , greater than about 100 mM , greater than about 125 mM , greater than about 150 mM , greater than about 200 mM , greater than about 225 mM , or greater than about 250 mM.
  • the amount of CSA in the formulation is between about 5 mM and about 250 mM , between about 10 mM and about 200 mM, between about 20 mM and about 200 mM , between about 30 mM and about 200 mM, between about 30 mM and about 150 mM, or between about 30 mM and about 100 mM.
  • the concentration of CSA and the counterion can be the same or different.
  • a salt of CSA formed with arginine (CSA-Arg) with a concentration of 50mM has a CSA concentration of 50mM and an arginine concentration of 50mM.
  • more or less arginine (counterion) could be present, whereby not all of the CSA or counterion is in the salt form.
  • Arginine (Arg) is an amino acid that can be represented by the chemical formula:
  • Arginine includes all hydrate/solvate forms including the anhydrous form as well as the free base form, as well as any salt form, including without limitation Arginine
  • Arg-HCI hydrochloride
  • Arginine includes all enantiomers (e.g., L-arginine and D- arginine), and any combination of enantiomers (e.g., 50% L-arginine and 50% D- arginine; 90%-100% L-arginine and 10%-0% D-arginine, etc.).
  • the term "arginine” includes greater than 99% L-arginine and less than 1 % D-arginine.
  • the term "arginine” includes an enantomerically pure L-arginine.
  • the arginine is a pharmaceutical grade arginine.
  • Histidine is an amino acid that can be represented by the chemical formula:
  • Histidine includes all hydrate/so I vate forms including the anhydrous form as well as the free base form, as well as any salt form, including without limitation histidine hydrochloride.
  • Histidine includes all enantiomers (e.g. , L-histidine and D-histidine), and any combination of enantiomers (e.g., 50% L- histidine and 50% D- histidine; 90%- 100%) L- histidine and 10%-0% D- histidine, etc.).
  • the term “histidine” includes greater than 99%) L- histidine and less than 1 % D- histidine.
  • the term "histidine” includes an enantomerically pure L- histidine. In some embodiments, the histidine is a pharmaceutical grade histidine. "Lysine” is an amino acid that can be represented by the chemical formula:
  • “Lysine” includes all hydrate/solvate forms including the anhydrous form as well as the free base form, as well as any salt form, including without limitation lysine hydrochloride
  • “Lysine” includes all enantiomers (e.g., L-lysine and D-lysine), and any combination of enantiomers (e.g., 50% L- lysine and 50% D- lysine; 90%-100% L- lysine and 10%-0% D- lysine, etc.).
  • the term “lysine” includes greater than 99% L- lysine and less than 1 % D- lysine.
  • the term “lysine” includes an enantomerically pure L- lysine.
  • the lysine is a pharmaceutical grade lysine.
  • chelating agent is meant an optional composition component that is a
  • Such chelating agents can reduce or prevent degradation of an antibody that is formulated in comparision to the antibody without the protection of a chelating agent.
  • Exemplary chelating agents include aminopolycarboxylic acids, hydroxyaminocarboxylic acids, N-substituted glycines, 2- (2- amino-2-oxocthyl) aminoethane sulfonic acid (BES), deferoxamine (DEF), citric acid, niacinamide, and desoxycholates and mixtures thereof.
  • the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), diethylenetriamine pentaacetic acid 5 (DTPA), nitrilotriacetic acid (NTA), N-2- acetamido-2-iminodiacetic acid (ADA), bis(aminoethyl)glycolether, ⁇ , ⁇ , ⁇ ', ⁇ '-tetraacetic acid (EGTA), trans-diaminocyclohexane tetraacetic acid (DCTA), glutamic acid, and aspartic acid, N- hydroxyethyliminodiacetic acid (HIMDA), N, N-bis-hydroxyethylglycine (bicine) and N- (trishydroxymethylmethyl) 10 glycine (tricine), glycylglycine, sodium desoxycholate, ethylenediamine; propylenediamine; diethylenetriamine;
  • EDTA ethylenediaminetetraacetic
  • the chelating agent is selected from the group consisting of salts of EDTA including dipotassium EDTA, disodium EDTA, EDTA calcium disodium, sodium EDTA, trisodium EDTA, and potassium EDTA; and a suitable salt of deferoxamine (DEF) is deferoxamine mesylate (DFM), or mixtures thereof.
  • DEF deferoxamine
  • DMF deferoxamine mesylate
  • Chelating agents used in the invention can be present, where possible, as the free acid or free base form or salt form of the compound, also as an anhydrous, solvated or hydrated form of the compound or corresponding salt.
  • the concentration of chelating agent when present, generally ranges from about 0.01 mg/ml to about 50 mg/ml, from about 0.01 mg/ml to about 10.0 mg/ml, from about 0.01 mg/ml to about 5.0 mg/ml, from about 0.01 mg/ml to about 1.0 mg/ml, or from about 0.01 mg/ml to about 0.3 mg/ml.
  • the concentration of chelating agent generally ranges from about 0.01 mM to about 2.0 mM, from about 0.01 mM to about 1.5 mM, from about 0.01 mM to about 0.5 mM, from about 0.01 mM to about 0.4 mM, from about 0.01 mM to about 0.2 mM, from about 0.01 mM to about 0.15 mM, from about 0.01 mM to about 0.1 mM, from about 0.01 mM to about 0.09 mM, from about 0.01 mM to about 0.08 mM, from about 0.01 mM to about 0.07 mM, from about 0.01 mM to about 0.06 mM, from about 0.01 mM to about 0.05 mM, from about 0.01 mM to about 0.04 mM, from about 0.01 mM to about 0.03 mM, from about 0.01 mM to about 0.02 mM or from about 0.005 mM to about 0.01 mM.
  • the concentration of chelating agent can be about 0.01 mg/ml, 0.02 mg/ml, 0.03 mg/ml, about 0.04 mg/ml, about 0.05 mg/ml, about 0.06 mg/ml, about 0.07 mg/ml, about 0.10 mg/ml, about 0.20 mg/ml.
  • cryoprotectant is an optional composition component that is a molecule which, when combined with a protein of interest, significantly prevents or reduces chemical and/or physical instability of the protein upon lyophilization and subsequent storage.
  • cryoprotectants include sugars and their corresponding sugar alcohols; an amino acid such as monosodium glutamate or histidine; a methylamine such as betaine; a lyotropic salt such as magnesium sulfate; a polyol such as trihydric or higher molecular weight sugar alcohols, e.g.
  • cryoprotectants include glycerin and gelatin, and the sugars mellibiose, melezitose, raffinose, mannotriose and stachyose.
  • reducing sugars include glucose, maltose, lactose, maltulose, iso-maltulose and lactulose.
  • non-reducing sugars include non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other straight chain polyalcohols including sucrose, dextrose, mannose and trehalose (including all forms of trehalose such as trehalose monohydrate and trehalsoe dihydrate).
  • Example sugar alcohols are monoglycosides, especially those compounds obtained by reduction of disaccharides such as lactose, maltose, lactulose and maltulose.
  • the glycosidic side group can be either glucosidic or galactosidic. Additional examples of sugar alcohols are glucitol, maltitol, lactitol and iso-maltulose.
  • the concentration of the cryoprotectant, when present in the liquid composition ranges from about 0.1 mg/ml to about 150 mg/ml, from about 0.1 mg/ml to about 100 mg/ml, or from about 1 mg/ml to about 100 mg/ml.
  • the concentration of the cryoprotectant in the liquid composition is about 20 mg/ml, about 25 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 75 mg/ml, about 80 mg/ml, about 85 mg/ml, about 90 mg/ml, about 95 mg/ml, about 100 mg/ml, about 1 10 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, or about 150 mg/ml.
  • the concentration of the salt in the liquid composition ranges from about 1 mg/ml to about 20 mg/ml. Salts that are
  • pharmaceutically acceptable and suitable for this invention include sodium chloride, sodium succinate, sodium sulfate, potassium chloride, magnesium chloride, magnesium sulfate, and calcium chloride.
  • Exemplary salts include sodium chloride and magnesium chloride, magnesium chloride may also improve the antibody stability by protecting the protein from deamidation.
  • the salt in the liquid composition is selected from a range of concentrations of any of about 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8, mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml and 20 mg/ml.
  • injection force is the amount of pressure (in Newtons) required to pass the antibody formulation through a needle.
  • the injection force is correlated with the amount of resistance provided by the antibody formulation when administering the antibody formulation to a subject.
  • the injection force will be dependent on the gauge of the administering needle, as well as temperature.
  • the antibody formulation has an injection force of less than 15 N, 12 N, 10N, or 8 N when passed through a 27 Ga thin wall PFS needle such as defined in the International Organization for Standardization (ISO) document "Stainless steel needle tubing for the manufacture of medical devices" (ISO 9626:1991 ) and manufactured by BD Medical, Pharmaceutical Systems (Franklin Lakes, N.J.).
  • the antibody formulation has an injection force of less than 15 N, 12 N, 10N, or 8 N when passed through a 25 or 26 Gauge needle.
  • an “isotonic” formulation is one which has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to about 350 mOsm.
  • the term “hypotonic” describes a formulation with an osmotic pressure below that of human blood.
  • the term “hypertonic” is used to describe a formulation with an osmotic pressure above that of human blood. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example.
  • the compositions of the present invention are isotonic.
  • IV bag protectant refers to the surfactant added to the intravenous bag prior to dilution of the antibody formulation described herein into the intravenous bag.
  • the IV bag protectant can also be added to the intravenous bag prior to addition of other antibody formulations known to those of skill in the art, e.g. , a lyophilized antibody formulation.
  • Surfactants suitable for use as an IV bag protectant will generally be those suitable for use in IV formulations.
  • the surfactant used in the IV bag protectant is the same buffer used in the antibody formulation. For example, if the antibody formulation comprises polysorbate 80 as a surfactant, then polysorbate 80 would be added to the intravenous bag prior to adding the antibody formulation to the intravenous bag.
  • the surfactant concentration in the IV bag resulting from addition of the IV protectant will be about the same or only a portion of the surfactant concentration in the antibody formulation. Knowing the desired final concentration of surfactant in the IV bag, one can formulate the desired concentration of the surfactant in the IV bag protectant.
  • K off is intended to refer to the off rate constant for dissociation of an antibody from the antibody/antigen complex.
  • Kd is intended to refer to the dissociation constant of an antibody-antigen interaction.
  • One way of determ ining the K d or binding affinity of antibodies to the antigen is by measuring binding affinity of monofunctional Fab fragments of the antibody.
  • an antibody for example, IgG
  • IgG antibody
  • the affinity of a Fab fragment of an antibody can be determ ined by surface plasmon resonance (BIAcorC1 GM000TM surface plasmon resonance (SPR) system , BIAcore, INC, Piscaway NJ).
  • CM5 chips can be activated with N-ethyl-N'-(3-dimethylam inopropyl)-carbodiinide hydrochloride (EDC) and N-hydroxysuccinim ide (NHS) according to the supplier's instructions.
  • EDC N-ethyl-N'-(3-dimethylam inopropyl)-carbodiinide hydrochloride
  • NHS N-hydroxysuccinim ide
  • Human antigen can be diluted into 1 0 mM sodium acetate pH 4.0 and injected over the activated chip at a concentration of 0.005 mg/mL. Using variable flow time across the individual chip channels, two ranges of antigen density can be achieved: 1 00-200 response units (RU) for detailed kinetic studies and 500-600 RU for screening assays. Serial dilutions (0.1 -1 Ox estimated K d ) of purified Fab samples are injected for 1 m in at 100 m icroliters/min and dissociation times of up to 2 hours are allowed.
  • concentrations of the Fab proteins are determined by ELISA and/or SDS-PAGE electrophoresis using a Fab of known concentration (as determ ined by amino acid analysis) as a standard.
  • Kinetic association rates (k on ) and dissociation rates (k 0 ff) are obtained simultaneously by fitting the data to a 1 : 1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6. 99-1 10) using the BIAevaluation program.
  • Equilibrium dissociation constant (Kd) values are calculated as k 0 ff/k on . This protocol is suitable for use in determ ining binding affinity of an antibody to any antibody.
  • the antibody formulations can have different "osmolarity" concentrations.
  • Methods of measuring osmolarity of antibody formulations are known to those in the art, and can include, e.g. , an osmometer (e.g., an Advanced Instrument Inc 2020 freezing point depression osmometer).
  • the formulation has an osmolarity of between 200 and 600 mosm/kg, between 260 and 500 mosm/kg, or between 300 and 450 mosm/kg.
  • the formulation does not comprise an osmolyte.
  • phrases "pharmaceutically acceptable” indicates that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith.
  • a “pharmaceutically acceptable acid” includes inorganic and organic acids which are non toxic at the concentration and manner in which they are formulated.
  • suitable inorganic acids include hydrochloric, perchloric, hydrobromic, hydroiodic, nitric, sulfuric, sulfonic, sulfinic, sulfanilic, phosphoric, carbonic, etc.
  • Suitable organic acids include straight and branched-chain alkyl, aromatic, cyclic, cyloaliphatic, arylaliphatic, heterocyclic, saturated, unsaturated, mono, di- and tri-carboxylic, including for example, form ic, acetic, 2-hydroxyacetic, trifluoroacetic, phenylacetic, trimethylacetic, t-butyl acetic, anthranilic, propanoic, 2-hydroxypropanoic, 2-oxopropanoic, propandioic, cyclopentanepropionic, cyclopentane propionic, 3-phenylpropionic, butanoic, butandioic, benzoic, 3-(4-hydroxybenzoyl)benzoic, 2-acetoxy-benzoic, ascorbic, cinnam ic, lauryl sulfuric, stearic, muconic, mandelic, succinic, embonic, fumaric, malic, maleic, hydroxymale
  • “Pharmaceutically-acceptable bases” include inorganic and organic bases which are non-toxic at the concentration and manner in which they are formulated.
  • suitable bases include those formed from inorganic base forming metals such as lithium, sodium, potassium, magnesium, calcium, ammonium, iron, zinc, copper, manganese, aluminum, N-methylglucamine, morpholine, piperidine and organic nontoxic bases including, primary, secondary and tertiary amine, substituted amines, cyclic amines and basic ion exchange resins, [e.g., N(R').sub.4.sup.+ (where R 1 is independently H or C. sub.1 -4 alkyl, e.g., ammonium, Tris)], for example,
  • ethanolamine 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N- ethylpiperidine, polyamine resins and the like.
  • Additional pharmaceutically acceptable acids and bases useable with the present invention include those which are derived from the amino acids, for example, histidine, glycine, phenylalanine, aspartic acid, glutamic acid, lysine, arginine and asparagine.
  • “Pharmaceutically acceptable” buffers and salts which are an optional composition component, include those derived from both acid and base addition salts of the above indicated acids and bases. According to the present invention, the buffer is used to adjust the pH and it may also enhance the antibody stability.
  • the buffer is selected from the group consisting of acetate, succinate, gluconate, citrate, histidine, arginine, acetic acid, phosphate, phosphoric acid, ascorbate, tartartic acid, maleic acid, glycine, lactate, lactic acid, ascorbic acid, imidazole, bicarbonate and carbonic acid, succinic acid, sodium benzoate, benzoic acid, gluconate, edetate (EDTA), acetate, malate, imidazole, tris, phosphate, and mixtures thereof.
  • the concentration of the buffer can range from about 0.1 millimolar (mM) to about 200 mM. In one embodiment, the concentration of the buffer is from about 0.5 mM to about 200 mM, from about 1 mM to about 100 mM, from about 1 mM to about 65 mM, or from about 1 mM to about 30 mM.
  • the concentration of the buffer is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM.
  • the pH can be in the range of about 4.0 to about 9.0. In another embodiment, the pH is between about 4.5 and about 8.0, between about 5.0 and about 7.5, between about 5.5 and about 7.0, or between about 6.0 and 7.5.
  • the pH is about 4.0, about 4.1 , about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1 , about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 , about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0.
  • viscosity lowering excipient comprising CSA, SSA, CSA salts or SSA salts
  • CSA viscosity lowering excipient
  • SSA CSA salts
  • SSA salts CSA salts
  • a "preservative” is a compound which can be added to the formulations herein to reduce bacterial activity.
  • the addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation. Examples of potential
  • preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride.
  • Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.
  • the composition does not include a preservative.
  • a “stable” formulation is one in which the protein therein essentially retains its physical and chemical stability and integrity upon storage.
  • Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301 , Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991 ) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993).
  • Stability can be measured at a selected temperature for a selected time period. For rapid screening, the formulation may be kept at 40°C for 2 weeks to 1 month, at which time stability is measured.
  • a “stable" formulation may be one wherein less than about 20%, or less than about 15%, or less than about 10%, or less than about 5%, or less than about 2% of the antibody is degraded, denatured, aggregated or unfolded as determined by SEC HPLC when the antibody is stored at 2- 8°C for 6 months, or 12 months, or 18 months, or 24 months.
  • the antibody stability is determined by size exclusion
  • SEC chromatography
  • analytes e.g., macromolecules such as proteins and antibodies
  • SEC can separate antibodies in their natural three-dimensional conformation from antibodies in various states of denaturation, and/or antibodies that have been degraded.
  • the stationary phase is generally composed of inert particles packed into a dense three- dimensional matrix within a glass or steel column.
  • the mobile phase can be pure water, an aqueous buffer, an organic solvent, mixtures of these, or other solvents.
  • the stationary-phase particles have small pores and/or channels which will only allow species below a certain size to enter.
  • SEC is combined with an identification technique to identify or characterize proteins, or fragments thereof.
  • Protein identification and characterization can be accomplished by various techniques, including but not limited chromatographic techniques, e.g., high-performance liquid chromatography (HPLC), immunoassays, electrophoresis, ultra-violet/visible/infrared spectroscopy, raman spectroscopy, surface enhanced raman spectroscopy, mass spectroscopy, gas chromatography, static light scattering (SLS), Fourier Transform Infrared Spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and/or ANS protein binding.
  • HPLC high-performance liquid chromatography
  • immunoassays electrophoresis
  • ultra-violet/visible/infrared spectroscopy raman spectroscopy
  • surface enhanced raman spectroscopy mass spectroscopy
  • gas chromatography gas chromatography
  • SLS static light scattering
  • FTIR Fourier Transform In
  • HPLC high-pressure liquid chromatography
  • Various instruments and apparatuses are known to those of skill in the art to perform HPLC.
  • HPLC involves loading a liquid solvent containing the protein of interest onto a separation column, in which the separation occurs.
  • the HPLC separation column is filled with solid particles (e.g. silica, polymers, or sorbents), and the sample mixture is separated into compounds as it interacts with the column particles.
  • HPLC separation is influenced by the liquid solvent's condition (e.g. pressure, temperature), chemical interactions between the sample mixture and the liquid solvent (e.g. hydrophobicity, protonation, etc.), and chemical interactions between the sample mixture and the solid particles packed inside of the separation column (e.g. ligand affinity, ion exchange, etc.).
  • the SEC and protein identification occurs within the same apparatus, or simultaneously.
  • SEC and HPLC can be combined, often referred to as SE-HPLC.
  • the stability of the antibody in the antibody formulation can be determined.
  • the term “stability” generally is related to maintaining the integrity or to minimizing the degradation, denaturation, aggregation or unfolding of a biologically active agent such as a protein, peptide or another bioactive macromolecule.
  • improved stability generally means that, under conditions known to result in degradation, denaturation, aggregation or unfolding, the protein (e.g., antibody), peptide or another bioactive macromolecule of interest maintains greater stability compared to a control protein, peptide or another bioactive macromolecule.
  • stability refers to an antibody formulation having low to undetectable levels of aggregation.
  • low to undetectable levels of aggregation refers to samples containing no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1 % and no more than 0.5% aggregation by weight of protein as measured by high performance size exclusion chromatography (HPSEC), static light scattering (SLS), Fourier Transform Infrared Spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and 1- anilino-8-naphthalenesulfonic acid (ANS) protein binding techniques.
  • HPSEC high performance size exclusion chromatography
  • SLS static light scattering
  • FTIR Fourier Transform Infrared Spectroscopy
  • CD circular dichroism
  • urea-induced protein unfolding techniques intrinsic tryptophan fluorescence
  • the antibody formulation has low to undetectable levels of fragmentation.
  • low to undetectable levels of fragmentation refers to samples containing equal to or more than 80%, 85%, 90%, 95%, 98% or 99% of the total protein, for example, in a single peak as determined by HPSEC, or in two peaks (e.g., heavy- and light-chains) (or as many peaks as there are subunits) by reduced Capillary Gel Electrophoresis (rCGE), representing the non-degraded antibody or a non-degraded fragment thereof, and containing no other single peaks having more than 5%, more than 4%, more than 3%, more than 2%, more than 1 %, or more than 0.5% of the total protein in each.
  • rCGE reduced Capillary Gel Electrophoresis
  • reduced Capillary Gel Electrophoresis refers to capillary gel electrophoresis under reducing conditions sufficient to reduce disulfide bonds in an antibody.
  • stability of a protein is dependent on other features in addition to the composition of the formulation. For example, stability can be affected by temperature, pressure, humidity, and external forms of radiation. Thus, unless otherwise specified, stability referred to herein is considered to be measured at 2-8. degree. C, one atmosphere pressure, 60% relative humidity, and normal background levels of radiation.
  • various components can be omitted from the antibody formulation, or can be “substantially free” of that component.
  • the term “substantially free” as used herein refers to an antibody formulation, said formulation containing less than 0.01 %, less than 0.001 %, less than 0.0005%, less than 0.0003%, or less than 0.0001 % of the designated component.
  • SSA Sulfosalicyclic acid
  • SSA includes hydrate/solvate forms including the anhydrous form as well as the free base form and any salt form.
  • exemplary pharmaceutically acceptable acid salts may be formed from counter-ions and include sodium, hydrochloride, arginine, lysine, histidine, and include all hydrates, solvates, and enantiomers thereof.
  • the present invention includes various amounts of SSA.
  • the amount of SSA is greater than about 5 mM, greater than about 10 mM, greater than about 15 mM, greater than about 20 mM, greater than about 25 mM, greater than about 30 mM , greater than about 35 mM , greater than about 50 mM , greater than about 75 mM , greater than about 100 mM , greater than about 125 mM , greater than about 150 mM , greater than about 200 mM , greater than about 225 mM , or greater than about 250 mM.
  • the amount of SSA in the formulation is between about 5 mM and about 250 mM , between about 10 mM and about 200 mM, between about 20 mM and about 200 mM , between about 30 mM and about 200 mM, between about 30 mM and about 150 mM, or between about 30 mM and about 100 mM.
  • “Surfactant” is an optional component to the composition which is a pharmaceutically acceptable agent that reduces the tendency for the formation of bubbles in the formulation during preparation and handling of the formulation and preparation for parenteral administration and especially from stress related to shaking and agitation during preparation and also during shipping.
  • exemplary surfactants include
  • the surfactant is selected from the group consisting of polysorbate 20, polysorbate 21 , polysorbate 40, polysorbate 60, polysorbate 61 , polysorbate 65, polysorbate 80, polysorbate 81 , polysorbate 85, and mixtures thereof.
  • the concentration of the surfactant generally ranges from about 0.01 mg/ml to about 5.0 mg/ml, from about 0.01 mg/ml to about 2.0 mg/ml, from about 0.01 mg/ml to about 1.5 mg/ml, from about 0.01 mg/ml to about 1.0 mg/ml, from about 0.01 mg/ml to about 0.5 mg/ml, from about 0.01 mg/ml to about 0.4 mg/ml, from about 0.01 mg/ml to about 0.3 mg/ml, from about 0.01 mg/ml to about 0.2 mg/ml, from about 0.01 mg/ml to about 0.15 mg/ml, from about 0.01 mg/ml to about 0.1 mg/ml, or from about 0.01 mg/ml, to about 0.05 mg/ml.
  • the concentration of the surfactant is about 0.5 mg/ml, about 0.05 mg/ml about 0.06 mg/ml about 0.07 mg/ml about 0.08 mg/ml about 0.09 mg/ml about 0.1 mg/ml about 0.11 mg/ml about 0.12 mg/ml about 0.13 mg/ml about 0.14 mg/ml about 0.15 mg/ml about 0.16 mg/ml about 0.17 mg/ml about 0.18 mg/ml about 0.19 mg/ml, or about 0.2 mg/ml.
  • terapéuticaally effective amount means an amount of a compound of the present invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.
  • treating embrace both preventative, i.e., prophylactic, and palliative treatment.
  • animal refers to humans (male or female), companion animals (e.g., dogs, cats and horses), food-source animals, zoo animals, marine animals, birds and other similar animal species.
  • Edible animals refers to food-source animals such as cows, pigs, sheep and poultry.
  • Viscosity as used herein may be “kinematic viscosity” or “absolute viscosity.”
  • Kininematic viscosity is a measure of the resistive flow of a fluid under the influence of gravity. When two fluids of equal volume are placed in identical capillary viscometers and allowed to flow by gravity, a viscous fluid takes longer than a less viscous fluid to flow through the capillary.
  • Absolute viscosity is the product of kinematic viscosity and fluid density: Absolute
  • Viscosity Kinematic Viscosity x Density
  • the dimension of kinematic viscosity is L 217 where L is a length and T is a time.
  • kinematic viscosity is expressed in centistokes (cSt).
  • the SI unit of kinematic viscosity is mm 2/s , which is 1 cSt.
  • Absolute viscosity is expressed in units of centipoise (cP).
  • the formulation may be in either aqueous or lyophilized form.
  • the formulation may have a viscosity of no greater than about 60 cP.
  • the formulation has a viscosity of no greater than about 50 cP, or no greater than about 40 cP, or no greater than about 30 cP, or no greater than about 20 cP, or no greater than about 40 cP, or no greater than about 15 cP.
  • the composition comprising antibody has a viscosity of between about 1 cP and about 50 cP, between about 1 cP and 40 cP, between about 1 cP and about 30 cP, between about 1 cP and about 20 cP, between about 1 cP and about 15 cP, or between about 1 cP and about 10 cP at 25°C.
  • the formulation has a viscosity of about 50 cP, about 45 cP, about 40 cP, about 35 cP, about 30 cP, about 25 cP, about 20 cP, about 15 cP, or about 10 cP, or about 5 cP.
  • the formulation has a viscosity of between about 10 cP and 50 cP, between about 10 cP and 30 cP, between about 10 cP and 20 cP, or between about 5 cP and 15 cP.
  • the present invention is directed at pharmaceutical compositions that include: a. an antibody, wherein the antibody concentration is between about 100 mg/ml to about 400 mg/ml, and b. a viscosity lowering excipient comprising camphorsulfonic acid, sulfosalicylic acid, or a salt of camphorsulfonic acid or sulfosalicylic acid, wherein the viscosity lowering excipient concentration is between about 30 mM to about 200 mM; wherein the pH of said composition is from about 4.0 to about 9.0.
  • the composition also includes a pharmaceutically acceptable buffer.
  • the pharmaceutically acceptable buffer comprises arginine, histidine, tris, phosphate or lysine, or a salt thereof.
  • the pharmaceutically acceptable buffer comprises histidine, tris, or phosphate, or a salt thereof.
  • the concentration of pharmaceutically acceptable buffer is from about 1.0 to about 200 mM.
  • the composition also includes a surfactant.
  • a surfactant in one embodiment
  • the surfactant is polysorbate 20 or polysorbate 80. In another embodiment, the surfactant is polysorbate 20 or polysorbate 80. In another
  • the concentration of surfactant is from about 0.01 to about 0.3 mg/ml.
  • the composition also includes a chelating agent.
  • the chelating agent is EDTA or disodium EDTA.
  • the concentration of chelating agent is from about 0.01 to about 0.3 mg/ml.
  • the composition also includes a cryoprotectant.
  • the cryoprotectant is sucrose, dextrose, mannose or trehalose.
  • the concentration of the cryoprotectant is from about 1 mg/ml to about 100 mg/ml.
  • the viscosity lowering agent is (+)camphorsulfonic acid or (-)camphorsulfonic acid or a salt thereof. In another embodiment, the viscosity lowering agent concentration is between about 50 mM to about 150 mM. In another
  • the viscosity lowering agent concentration is between about 70 mM to about 1 10 mM.
  • the viscosity lowering agent is a salt of camphorsulfonic acid comprising camphorsulfonic acid and arginine. In another embodiment, the
  • camphorsulfonic acid concentration is between about 50 mM and about 150 mM and the arginine concentration is between about 50 mM and about 150 mM. In another embodiment, the camphor sulfonic acid concentration is between about 70 mM and about 1 10 mM and the arginine concentration is between about 70 mM and about 110 mM.
  • the antibody is a human or humanized monoclonal lgG1 , lgG2 or lgG4 antibody. In another embodiment, the antibody is an anti-IL7R, anti-PCSK9 or anti-glucagon receptor antibody.
  • the antibody includes a heavy chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, 5, or 6; CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7 or 8; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9, and a light chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 1 1 , and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12.
  • the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain variable region amino acid sequence shown in SEQ ID NO: 2, and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain variable region amino acid sequence shown in SEQ ID NO: 3.
  • the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain amino acid sequence shown in SEQ ID NO: 13, and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain amino acid sequence shown in SEQ ID NO: 14.
  • the antibody includes a variable heavy chain sequence comprising the amino acid sequence shown in SEQ ID NO: 2 and a variable light chain sequence comprising the amino acid sequence shown in SEQ ID NO: 3.
  • the antibody includes a heavy chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, 18 or 19; CDR2 comprising the amino acid sequence shown in SEQ ID NO: 20 or 21 ; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 22, and a light chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 23, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 24, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 25.
  • the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain variable region amino acid sequence shown in SEQ ID NO: 15, and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain variable region amino acid sequence shown in SEQ ID NO: 16.
  • the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain amino acid sequence shown in SEQ ID NO: 26 or 27, and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain amino acid sequence shown in SEQ ID NO: 28.
  • the antibody includes a variable heavy chain sequence comprising the amino acid sequence shown in SEQ ID NO: 15 and a variable light chain sequence comprising the amino acid sequence shown in SEQ ID NO: 16.
  • the antibody includes a heavy chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 32, 33, or 34; CDR2 comprising the amino acid sequence shown in SEQ ID NO: 35 or 36; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 37, and a light chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 38, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 39, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 40.
  • the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain variable region amino acid sequence shown in SEQ ID NO: 41 , and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain variable region amino acid sequence shown in SEQ ID NO: 42.
  • the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain amino acid sequence shown in SEQ ID NO: 30, and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain amino acid sequence shown in SEQ ID NO: 31.
  • the antibody includes a variable heavy chain sequence comprising the amino acid sequence shown in SEQ ID NO: 41 and a variable light chain sequence comprising the amino acid sequence shown in SEQ ID NO: 42.
  • the composition is lyophilized. In yet another embodiment, the lyophilized composition is reconstituted and the antibody concentration of the reconstituted composition is between about 250 mg/ml and about 400 mg/ml. In yet another embodiment, the lyophilized composition is reconstituted and the antibody concentration of the reconstituted composition is higher than the antibody concentration before lyophilization. In yet another embodiment, the composition has a viscosity of less than about 50 cP at 25°C. In yet another embodiment, the composition is isotonic.
  • IL-7R refers to any form of IL-7R and variants thereof that retain at least part of the activity of IL-7R. Unless indicated differently, such as by specific reference to human IL-7R, IL-7R includes all mammalian species of native sequence IL-7R, e.g., human, canine, feline, equine, and bovine. One exemplary human IL-7R is found as Uniprot Accession Number P16871 (SEQ ID NO: 1 ).
  • Antagonist IL-7R antibodies encompass antibodies that block, antagonize, suppress or reduce (to any degree including significantly) IL-7R biological activity, including downstream pathways mediated by IL-7R signaling, such interaction with IL-7 and/or elicitation of a cellular response to IL-7.
  • IL-7R antibody (interchangeably termed “IL-7R antagonist antibody,” “antagonist anti-IL-7R antibody” or “anti-IL-7R antagonist antibody”) encompasses all the previously identified terms, titles, and functional states and characteristics whereby the IL-7R itself, an IL-7R biological activity (including but not limited to interaction with IL-7, its ability to mediate any aspect of phosphorylation of STAT5, phosphatidylinositol-3-kinase (PI3K)-Akt pathway activation, p27Kip1 downregulation, Bcl-2 upregulation, Rb hyperphosphorylation, and CXCR4
  • an antagonist IL-7R antibody binds IL-7R and prevents interaction with IL-7.
  • antagonist IL-7R antibodies are provided herein.
  • Anti-IL-7R antagonist antibodies for use in the invention can be identified or characterized using methods known in the art, whereby reduction, amelioration, or neutralization of an IL-7R biological activity is detected and/or measured.
  • C1 GM is used to refer to an antibody comprising the amino acid sequence of the heavy chain and light chain variable regions shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
  • C1 GM The generation and characterization of C1 GM is described in the Examples of
  • C1 GM refers to
  • the antibody is an anti-IL-7R antibody that binds IL-7Ra (such as human IL-7Ra) with a high affinity.
  • high affinity is (a) binding IL- 7R with a K D of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less).
  • antibodies (a) bind IL-7R (such as human IL-7R) with a KD of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less), and/or a k 0 ff of about 4x10 "4 s '
  • the epitope(s) that can be bound by the antibody can be continuous or discontinuous.
  • the antibody binds essentially the same IL-7R epitope as antibody C1 GM.
  • the antibody can be anti-IL-7R antibody comprising a heavy chain variable region comprising: (a) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4
  • GFTFDDSVMH extended or in SEQ ID NO: 5 (DSVMH) (Kabat) or in SEQ ID NO: 6 (GFTFDDS) (Chothia);
  • the antibody can be an anti-IL-7R antibody comprising a light chain variable region comprising:
  • the antibody can be anti-IL-7R antibody comprising three CDRs from a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2.
  • the antibody can be anti-IL-7R antibody comprising three CDRs from a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 3.
  • the anti-IL-7R antibody may comprise a heavy chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 2 and/or a light chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 3, wherein the antibody binds specifically to human IL-7Ra.
  • the anti-IL-7R antibody may comprise a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 2 and/or may comprise a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 3.
  • the anti-IL-7R antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 2 and 3.
  • the anti-IL-7R antibody may comprise a heavy chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 13 and / or a light chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%), 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 14, wherein the antibody binds specifically to human IL-7Ra.
  • the anti-IL-7R antibody may comprise a heavy chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 13 and/or may comprise a light chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 14.
  • the anti-IL-7R antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 13 and 14.
  • the anti-IL-7R antibody may compete for IL-7R binding with an anti-IL-7R antibody as defined herein.
  • the anti-IL-7R antibody may compete for IL-7R binding with an antibody comprising a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 2 and/or a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 3.
  • the anti-IL-7R antibody may be a human and affinity matured antibody, C1 GM, which specifically binds human IL-7Ra.
  • Antibody C1 GM is described in WO2011/104687, the content of which is hereby incorporated by reference in its entirety.
  • the amino acid sequences of the heavy chain and light chain variable regions of C1 GM are shown in SEQ ID NOs: 2 and 3, respectively.
  • the CDR portions of antibody C1 GM (including Chothia and Kabat CDRs) are diagrammatically depicted in Table 1 of
  • Antibody C1 GM is highly potent in blocking IL-7R biological activity.
  • the anti-IL-7R antibody may also comprise a fragment or a region of the antibody C1 GM.
  • the fragment is a light chain of the antibody C1 GM comprising the amino acid sequence as shown in SEQ ID NO: 14 herein.
  • the fragment is a heavy chain of the antibody C1 GM comprising the amino acid sequence as shown in SEQ ID NO: 13 herein.
  • the fragment contains one or more variable regions from a light chain and/or a heavy chain of the antibody C1 GM.
  • the fragment contains one or more CDRs from a light chain and/or a heavy chain of the antibody C1 GM comprising the amino acid sequences as shown in SEQ ID NOS: 14 and 13, respectively, herein.
  • the antibody may comprise any one or more of the following: a) one or more (one, two, three, four, five, or six) CDR(s) derived from antibody C1 GM shown in SEQ ID NOs: 4-12.
  • the CDRs may be Kabat CDRs, Chothia CDRs, or a combination of Kabat and Chothia CDRs (termed “extended” or “combined” CDRs herein).
  • the polypeptides comprise any of the CDR configurations (including combinations, variants, etc.) described herein.
  • the C-terminal lysine of the heavy chain of any of the anti-IL-7R antibodies described herein is deleted.
  • the heavy and/or light chain of the anti-IL-7R antibodies described herein may optionally include a signal sequence.
  • the antibody may be selected from an anti-IL-7R antibody known in the art, such as antibodies described in, for example without limitation, any of the following published PCT applications: WO201 1/104687 (including, for example without limitation, any of the antibodies listed in Table 1 ), WO/201 1/094259 (including, for example without limitation, antibodies H3L4, BPC4401 , BPC4398, BPC1 142, BPC4399, BPC4402, BPC4403, and BPC1 142), WO/2013/056984 (including, for example without limitation, antibodies MD707-1 , MD707-2, MD707-3, MD707-4, MD707-5, MD707-6, MD707-9, MD707-12, and MD707-13), and WO2010/017468 (including, for example without limitation, antibodies 9B7, R34.34, 6A3 and 1A11 ).
  • the antibody may bind to the same epitope as an anti-IL-7R antibody known in the art and/or may compete for binding to
  • Glucagon Receptor refers to any form of glucagon receptor and variants thereof that retain at least part of the activity of the glucagon receptor. Unless indicated differently, such as by specific reference to human glucagon receptor, glucagon receptor includes all mammalian species of native sequence glucagon receptor, e.g., human, canine, feline, equine, and bovine. One exemplary human glucagon receptor is found as Uniprot Accession Number P47871 (SEQ ID NO: 29).
  • an "anti-glucagon receptor antagonist antibody” refers to an antibody that is able to inhibit glucagon receptor biological activity and/or downstream events(s) mediated by glucagon receptor.
  • Anti-glucagon receptor antagonist antibodies encompass antibodies that block, antagonize, suppress or reduce (to any degree including significantly) glucagon receptor biological activity, including downstream events mediated by glucagon receptor, such glucagon binding and downstream signaling, adenylate cyclase activation, increased levels of intracellular cAMP, glycogenolysis stimulation, gluconeogenesis activation, glycogenesis inhibition, glycolysis inhibition, and hepatic glucose production.
  • anti-glucagon receptor antagonist antibody encompasses all the previously identified terms, titles, and functional states and characteristics whereby the glucagon receptor itself, a glucagon receptor biological activity (including but not limited to its ability to bind glucagon, increase intracellular cAMP, stimulate glycogenolysis, activate gluconeogenesis, and promote relase of hepatic glucose), or the consequences of the biological activity, are substantially nullified, decreased, or neutralized in any meaningful degree.
  • an anti-glucagon receptor antagonist antibody binds glucagon receptor and lowers plasma glucose levels. Examples of anti-glucagon receptor antagonist antibodies are provided herein.
  • mAb5 is used to refer to an antibody comprising the amino acid sequence of the heavy chain and light chain variable regions shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively.
  • mAb5 heavy chain variable region is used to refer to an antibody comprising the amino acid sequence of the heavy chain and light chain variable regions shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively.
  • immunoglobulin encoded by (a) a polynucleotide encoding mAb5 light chain variable region that has a deposit number of ATCC No. PTA-120164 and (b) a polynucleotide encoding mAb5 heavy chain variable region that has a deposit number of ATCC No. PTA-120165.
  • the antibody is an anti- glucagon receptor antibody that binds glucagon receptor (such as human glucagon receptor) with a high affinity.
  • high affinity is (a) binding IL-7R with a K D of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less).
  • antibodies bind glucagon receptor (such as human glucagon receptor) with a K D of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less), and/or a k 0 ff Of about 4x10 "4 s '
  • the epitope(s) that can be bound by the antibody can be continuous or discontinuous.
  • the antibody binds essentially the same glucagon receptor epitope as antibody mAb5.
  • the antibody can be anti- glucagon receptor antibody comprising a heavy chain variable region comprising: (a) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17
  • GYTFTDFSVH extended or in SEQ ID NO: 18 (GYTFTDF) (Chothia) or in SEQ ID NO: 19 (DFSVH) (Kabat);
  • the antibody can be an anti-glucagon receptor antibody comprising a light chain variable region comprising:
  • the antibody can be anti-glucagon receptor antibody comprising three CDRs from a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 15.
  • the antibody can be anti-glucagon receptor antibody comprising three CDRs from a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 16.
  • the anti-glucagon receptor antibody may comprise a heavy chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO.
  • a light chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 16, wherein the antibody binds specifically to human glucagon receptor.
  • the anti-glucagon receptor antibody may comprise a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 15 and/or may comprise a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 16.
  • the anti-glucagon receptor antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 15 and 16.
  • the anti-glucagon receptor antibody may comprise a heavy chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 26 and / or a light chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 28, wherein the antibody binds specifically to human glucagon receptor.
  • the amino acid sequence of mAb5 full-length heavy chain (SEQ ID NO: 26) is shown below:
  • amino acid sequence of mAb5 full-length heavy chain without the C-terminal lysine is shown below:
  • amino acid sequence of mAb5 full-length light chain (SEQ ID NO: 28) is shown below:
  • the anti-glucagon receptor antibody may comprise a heavy chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 26 and/or may comprise a light chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 28.
  • the anti-glucagon receptor antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 26 and 28.
  • the anti-glucagon receptor antibody may compete for glucagon receptor binding with an anti-glucagon receptor antibody as defined herein.
  • the anti-glucagon receptor antibody may compete for glucagon receptor binding with an antibody comprising a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 15 and/or a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 16.
  • the anti-glucagon receptor antibody may be a human and affinity matured antibody, mAb5, which specifically binds human glucagon receptor.
  • Antibody mAb5 is described in WO2014/181229, the content of which is hereby incorporated by reference in its entirety for all purposes.
  • the amino acid sequences of the heavy chain and light chain variable regions of mAb5 are shown in SEQ ID NOs: 15 and 16, respectively.
  • the CDR portions of antibody mAb5 (including Chothia and Kabat CDRs) are diagrammatically depicted in Table 1 A of WO2014/181229.
  • Antibody mAb5 is highly potent in blocking glucagon receptor biological activity.
  • the anti-glucagon receptor antibody may also comprise a fragment or a region of the antibody mAb5.
  • the fragment is a light chain of the antibody mAb5 comprising the amino acid sequence as shown in SEQ ID NO: 28 herein.
  • the fragment is a heavy chain of the antibody mAb5 comprising the amino acid sequence as shown in SEQ ID NO: 26 herein.
  • the fragment contains one or more variable regions from a light chain and/or a heavy chain of the antibody mAb5.
  • the fragment contains one or more CDRs from a light chain and/or a heavy chain of the antibody mAb5 comprising the amino acid sequences as shown in SEQ ID NOS: 28 and 26, respectively, herein.
  • the antibody may comprise any one or more of the following: a) one or more (one, two, three, four, five, or six) CDR(s) derived from antibody mAb5 shown in SEQ ID NOs: 17-25.
  • the CDRs may be Kabat CDRs, Chothia CDRs, or a combination of Kabat and Chothia CDRs (termed “extended” or “combined” CDRs herein).
  • the polypeptides comprise any of the CDR configurations (including combinations, variants, etc.) described herein.
  • the C-terminal lysine of the heavy chain of any of the anti-glucagon receptor antibodies described herein is deleted, as in SEQ ID NO. 27.
  • the heavy and/or light chain of the anti-glucagon receptor antibodies described herein may optionally include a signal sequence.
  • the antibody may be selected from an anti-glucagon receptor antibody known in the art, such as antibodies described in, for example without limitation, any of the following published PCT applications: WO2014/181229 (including, for example without limitation, any of the antibodies listed in Tables 1 A and 1 B including, for example without limitation antibodies mAb1 , mAb2, mAb3, mAb4, mAb5, mAb6, H2-A8, H2-A1 1 , H2-C8, H2-E7, H2-F10, H2-F11 , H3-C5, H3-C10, H3-F5, H3- H9, H2-A1 1 -H3-1 , H2-A1 1 -H3-2, H2-A1 1 -H3-3, H2-A1 1 -H3-4, H2-C8-H3-1 , H2-C8-H3- 2, H2-C8-H3-3, H2-C8-H3-4, H2-E7-H3-1 , H2-E7-H3-2,
  • the antibody may bind to the same epitope as an anti-glucagon receptor antibody known in the art and/or may compete for binding to glucagon receptor with such an antibody.
  • PCSK9 refers to any form of PCSK9 and variants thereof that retain at least part of the activity of PCSK9. Unless indicated differently, such as by specific reference to human PCSK9, PCSK9 includes all mammalian species of native sequence PCSK9, e.g., human, canine, feline, equine, and bovine. One exemplary human PCSK9 is found as Uniprot Accession Number Q8NBP7. (SEQ ID NO: 43)
  • PCSK9 antagonist refers to an antibody, peptide, or aptamer that is able to inhibit PCSK9 biological activity and/or downstream pathway(s) mediated by PCSK9 signaling, including PCSK9-mediated down-regulation of the LDLR, and
  • PCSK9-mediated decrease in LDL blood clearance A PCSK9 antagonist antibody encompasses antibodies that block, antagonize, suppress or reduce (to any degree including significantly) PCSK9 biological activity, including downstream pathways mediated by PCSK9 signaling, such as LDLR interaction and/or elicitation of a cellular response to PCSK9.
  • PCSK9 signaling such as LDLR interaction and/or elicitation of a cellular response to PCSK9.
  • PCSK9 antagonist antibody encompasses all the previously identified terms, titles, and functional states and characteristics whereby the PCSK9 itself, a PCSK9 biological activity (including but not limited to its ability to mediate any aspect of interaction with the LDLR, down regulation of LDLR, and decreased blood
  • a PCSK9 antagonist antibody binds PCSK9 and prevents interaction with the LDLR.
  • Examples of PCSK9 antagonist antibodies are provided herein.
  • the term "L1 L3" is used to refer to an antibody comprising the amino acid sequence of the heavy chain and light chain variable regions shown in SEQ ID NO: 41 and SEQ ID NO: 42, respectively.
  • L1 L3 DIQMTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYSASYRYTGVP SRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRTFGQGTKLEIK (SEQ ID NO: 42)
  • SEQ ID NO: 42 The generation and characterization of L1 L3 is described in the Examples of
  • L1 L3 refers to
  • immunoglobulin encoded by (a) a polynucleotide encoding L1 L3 light chain variable region that has a deposit number of ATCC No. PTA-10303 and (b) a polynucleotide encoding L1 L3 heavy chain variable region that has a deposit number of ATCC No. PTA-10302.
  • the antibody is an anti-PCSK9 antibody that binds PCSK9 receptor (such as human PCSK9 receptor) with a high affinity.
  • high affinity is (a) bindingPCSK9 receptor with a K D of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less).
  • antibodies bind PCSK9 receptor (such as human PCSK9 receptor) with a K D of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less), and/or a k off of about 4x10 "4 s '
  • the epitope(s) that can be bound by the antibody can be continuous or discontinuous.
  • the antibody binds essentially the same PCSK9 receptor epitope as antibody L1 L3.
  • the antibody can be anti-PCSK9 receptor antibody comprising a heavy chain variable region comprising:
  • the antibody can be an anti-PCSK9 receptor antibody
  • a light chain variable region comprising: (a) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 38 (RASQGISSALA);
  • the antibody can be anti-PCSK9 antibody comprising three CDRs from a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 41.
  • the antibody can be anti-PCSK9 receptor antibody comprising three CDRs from a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 42.
  • the anti-PCSK9 receptor antibody may comprise a heavy chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 41 and/or a light chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 42, wherein the antibody binds specifically to human PCSK9 receptor.
  • the anti-PCSK9 receptor antibody may comprise a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 41 and/or may comprise a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 42.
  • the anti-PCSK9 receptor antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 41 and 42.
  • the anti-PCSK9 receptor antibody may comprise a heavy chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 30 and / or a light chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 31 , wherein the antibody binds specifically to human PCSK9 receptor.
  • amino acid sequence of L1 L3 full-length heavy chain (SEQ ID NO: 30) is shown below:
  • the anti- PCSK9 receptor antibody may comprise a heavy chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 30 and/or may comprise a light chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 31 .
  • the anti-PCSK9 receptor antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 30 and 31.
  • the anti-PCSK9 receptor antibody may compete for PCSK9 receptor binding with an anti- PCSK9 receptor antibody as defined herein.
  • the anti- PCSK9 receptor antibody may compete for PCSK9 receptor binding with an antibody comprising a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 41 and/or a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 42.
  • the anti-PCSK9 receptor antibody may be a human and affinity matured antibody, L1 L3, which specifically binds human PCSK9 receptor.
  • Antibody L1 L3 is described in WO2010/029513, the content of which is hereby incorporated by reference in its entirety for all purposes.
  • the amino acid sequences of the heavy chain and light chain variable regions of L1 L3 are shown in SEQ ID NOs: 41 and 42, respectively.
  • the CDR portions of antibody L1 L3 (including Chothia and Kabat CDRs) are diagrammatically depicted in Table 7 of WO2010/029513.
  • Antibody L1 L3 is highly potent in blocking PCSK9 receptor biological activity.
  • the anti-PCSK9 receptor antibody may also comprise a fragment or a region of the antibody L1 L3.
  • the fragment is a light chain of the antibody L1 L3 comprising the amino acid sequence as shown in SEQ ID NO: 31 herein.
  • the fragment is a heavy chain of the antibody L1 L3 comprising the amino acid sequence as shown in SEQ ID NO: 30 herein.
  • the fragment contains one or more variable regions from a light chain and/or a heavy chain of the antibody L1 L3.
  • the fragment contains one or more CDRs from a light chain and/or a heavy chain of the antibody L1 L3 comprising the amino acid sequences as shown in SEQ ID NOS: 31 and 30, respectively, herein.
  • the antibody may comprise any one or more of the following: a) one or more (one, two, three, four, five, or six) CDR(s) derived from antibody L1 L3 shown in SEQ ID NOs: 32-40.
  • the CDRs may be Kabat CDRs, Chothia CDRs, or a combination of Kabat and Chothia CDRs (termed “extended” or “combined” CDRs herein).
  • the polypeptides comprise any of the CDR configurations (including combinations, variants, etc.) described herein.
  • the C-terminal lysine of the heavy chain of any of the anti-PCSK9 receptor antibodies described herein is deleted.
  • the heavy and/or light chain of the anti-PCSK9 receptor antibodies described herein may optionally include a signal sequence.
  • the anti-PCSK9 receptor antibody is alirocumab
  • PRALUENTTM evolocumab
  • REPATHATM evolocumab
  • REGN728 LGT209
  • RG7652 evolocumab
  • the anti-PCSK9 antibody is bococizumab, alirocumab
  • the antibody may bind to the same epitope as an anti-PCSK9 receptor antibody known in the art and/or may compete for binding to PCSK9 receptor with such an antibody.
  • camphorsulfonic acid or sulfosalicyclic acid are mixed together and the pH of the mixture is measured and if necessary, adjusted with the use of a buffer or basic component.
  • Other optional components may also be added to the mixture including one or more surfactants, chelating agents, and cryopretectants.
  • the formulation may be utilized in the liquid state, or lyophilized.
  • Many different freeze-dryers are available for this purpose such as Hull50.TM. (Hull, USA) or GT20.TM. (Leybold-Heraeus, Germany) freeze-dryers. Freeze-drying is accomplished by freezing the formulation and subsequently subliming ice from the frozen content at a temperature suitable for primary drying.
  • the product temperature is below the eutectic point or the collapse temperature of the formulation.
  • the shelf temperature for the primary drying will range from about -30 to 25°C (provided the product remains frozen during primary drying) at a suitable pressure, ranging typically from about 50 to 250 mTorr.
  • the formulation, size and type of the container holding the sample (e.g., glass vial) and the volume of liquid will mainly dictate the time required for drying, which can range from a few hours to several days (e.g. 40-60 hrs).
  • a secondary drying stage may also be performed depending upon the desired residual moisture level in the product.
  • the temperature at which the secondary drying is carried out ranges from about 0-4OC, depending primarily on the type and size of container and the type of protein employed.
  • the shelf temperature throughout the entire water removal phase of lyophilization may be from about 15-30C (e.g., about 20°C).
  • the time and pressure required for secondary drying will be that which produces a suitable lyophilized cake, dependent, e.g., on the temperature and other parameters.
  • the secondary drying time is dictated by the desired residual moisture level in the product and typically takes at least about 5 hours (e.g. 10-15 hours).
  • the pressure may be the same as that employed during the primary drying step. Freeze-drying conditions can be varied depending on the formulation and vial size.
  • the formulations described herein may also be prepared as reconstituted lyophilized formulations.
  • the compositions described herein are lyophilized and then reconstituted to produce the reduced-viscosity stable liquid formulations of the invention.
  • a "pre-lyophilized formulation” is produced after preparation of the antibody of interest as described above.
  • the amount of antibody present in the pre- lyophilized formulation is determined taking into account the desired dose volumes and mode(s) of administration.
  • the concentration of an antibody in the pre- lyophilized formulation and in the reconstituted formulation can be as described previously, and may differ such that the reconstituted formulation may have an increased antibody concentration as compared to the pre-lyophilized formulation.
  • a "reconstituted" formulation is one which has been prepared by dissolving a lyophilized formulation in a diluent such that the antibody is distributed throughout the reconstituted formulation.
  • the reconstituted formulation is suitable for administration (e.g. parenteral administration) to a patient to be treated with the antibody of interest and, in certain embodiments of the invention, may be one which is suitable for subcutaneous administration.
  • diluent of interest herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, such as a formulation reconstituted after lyophilization.
  • exemplary diluents include sterile water, bacteriostatic water for injection (BWFI or WFI), a pH buffered solution (e.g. phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.
  • diluents can include aqueous solutions of salts and/or buffers and/or surfactants.
  • Reconstitution generally takes place at a temperature of about 25°C to ensure complete hydration, although other temperatures may be employed as desired.
  • the time required for reconstitution will depend, e.g., on the type of diluent, amount of excipient(s) and protein.
  • the reconstituted formulation has less than 3000 particles per vial which are less than 10 pm per vial and 300 particles per vial which are less than 25 pm per vial for vials containing less than 100 ml. of solution.
  • compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g.
  • injectable and infusible solutions dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with antibodies in general.
  • One mode of administration is parenteral (e.g. intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes).
  • kits comprising antibodies of the invention and pharmaceutical compositions comprising these antibodies.
  • a kit may include, in addition to the antibody or pharmaceutical composition, diagnostic or therapeutic agents.
  • a kit may also include instructions for use in a diagnostic or therapeutic method.
  • the kit includes the antibody or a
  • kits includes the antibody or a pharmaceutical composition thereof and one or more therapeutic agents, such as an additional antineoplastic agent, anti-tumor agent or chemotherapeutic agent.
  • Liposomes containing compounds of the invention are prepared by methods known in the art, such as described in US4485045 and US4544545. Liposomes with enhanced circulation time are disclosed in US5013556. 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. Suitable emulsions may be prepared using commercially available fat emulsions, such as IntralipidTM, LiposynTM, InfonutrolTM, LipofundinTM and LipiphysanTM.
  • the active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water.
  • oil e.g. soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil
  • a phospholipid e.g. egg phospholipids, soybean phospholipids or soybean lecithin
  • Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%.
  • the fat emulsion can comprise fat droplets between 0.1 and ⁇ . ⁇ , particularly 0.1 and 0.5pm, and have a pH in the range of 5.5 to 8.0.
  • the emulsion compositions can be those prepared by mixing a compound of the invention with IntralipidTM or the components thereof (soybean oil, egg phospholipids, glycerol and water).
  • IntralipidTM or the components thereof (soybean oil, egg phospholipids, glycerol and water).
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interracial polymerization, for example,
  • hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • 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. Suitable examples of sustained- release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, 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 (US3773919), copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3- hydroxybutyric acid.
  • polyesters for example, poly(2-hydroxyethyl-methacrylate), or 'poly(vinylalcohol)
  • polylactides US3773919
  • copolymers of L-glutamic acid and 7 ethyl-L-glutamate non
  • compositions to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes.
  • Therapeutic compounds of the invention are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above.
  • the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
  • compositions in preferably sterile pharmaceutically acceptable solvents may be nebulised by use of gases. Nebulised solutions may be breathed directly from the nebulising device or the nebulising device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner. Compositions of the invention may be used in conjunction with established treatments for the relevant indication.
  • the present invention also provides various therapeutic applications for the
  • compositions of the invention can be used for treating various diseases (e.g. cancer, autoimmune diseases, or viral infections) by binding the first protein (e.g. first human antibody variable domain) to an effector antigen and by binding the second protein (e.g. second human antibody variable domain) to a target antigen.
  • the compositions of the invention can be used for redirecting cytotoxicity, delivering thrombolytic agents to clots, for delivering immunotoxins to tumor cells, or for converting enzyme activated prodrugs at a target site (e.g. a tumor).
  • compositions of the invention can be used for increasing specificity of a therapeutic agent and/or modulating synergistic or additive pathways (e.g. metabolic or biochemical pathways).
  • the compositions of the invention can engage receptor/receptor, receptor/ligand, ligand/ligand, cell/cell, ligand/payload, receptor/payload, or single receptor.
  • Dosages and desired drug concentration of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. "The Use of
  • formulations of the present invention are administered to a mammal in need of treatment with the protein, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular,
  • the formulations are administered to the mammal by:
  • an article of manufacture which contains the formulation and preferably provides instructions for its use.
  • the article of manufacture comprises a container. Suitable containers include, for example, bottles, vials (e.g.
  • the container may be formed from a variety of materials such as glass or plastic.
  • the container holds the formulation and the label on, or associated with, the container may indicate directions for reconstitution and/or use.
  • the label may further indicate that the formulation is useful or intended for subcutaneous administration.
  • the container holding the formulation may be a multi-use vial, which allows for repeat administrations (e.g. from 2-6 administrations) of the reconstituted formulation.
  • the article of manufacture may further comprise a second container comprising a suitable diluent (e.g. BWFI).
  • the final protein concentration in the reconstituted formulation will generally be at least 50 mg/ml.
  • the article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • patches that release the medications directly into a subject's skin.
  • patches can contain the compound of the present invention in an optionally buffered, liquid solution, dissolved and/or dispersed in an adhesive, or dispersed in a polymer.
  • the compound may be administered once daily, but may also be administered multiple times. For example, the compound may be administered from once daily to once every six months or longer.
  • the administering may be on a schedule such as three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once every month, once every two months, once every three months and once every six months.
  • the compound may also be administered continuously via a minipump.
  • the compound may be administered at the site of the diseased body part or at a site distant from the site of the diseased body part.
  • the compound may be administered once, at least twice or for at least the period of time until the disease is treated, palliated or cured.
  • the compound generally may be administered for as long as the disease is present.
  • the compound typically would be administered as part of a pharmaceutical composition as described supra.
  • Exemplary, non-limiting dose ranges for administration of the pharmaceutical compositions of the present invention to a subject are from about 0.01 mg/kg to about 200 mg/kg (expressed in terms of milligrams (mg) of the antibody administered per kilogram (kg) of subject weight), from about 0.1 mg/kg to about 100 mg/kg, from about 1.0 mg/kg to about 50 mg/kg, from about 5.0 mg/kg to about 20 mg/kg, or about 15 mg/kg.
  • an average human subject weighs about 70 kg.
  • Ranges intermediate to any of the dosages cited herein, e.g., about 0.02 mg/kg - 199 mg/kg are also intended to be part of this invention. For example, ranges of values using a combination of any of the recited values as upper and/or lower limits are intended to be included.
  • Dosage regimens can also be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response) by administering several divided doses to a subject over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a
  • the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the compound or portion and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an antibody for the treatment of sensitivity in individuals.
  • the liquid compositions of the present invention can be prepared as unit dosage forms.
  • a unit dosage per vial may contain from 1 to 1000 milliliters (mis) of different concentrations of the compound of Formula (A).
  • a unit dosage per vial may contain about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, 20 ml, 30 ml, 40 ml, 50 ml or 100 ml of different concentrations of the compound of Formula (A).
  • these preparations can be adjusted to a desired concentration by adding a sterile diluent to each vial.
  • the liquid compositions of the present invention can also be prepared as unit dosage forms in sterile bags or containers, which are suitable for connection to an intravenous administration line or catheter.
  • Formulation 1 was amenable to achieve concentrations of approximately 50-70 mg/mL C1 GM antibody (in 20 mM histidine, 85 g/L sucrose, 0.05 g/L disodium EDTA dihydrate, 0.2 g/L polysorbate-80, pH 5.8), with suitable stability characteristics.
  • the drug product was formulated as a lyophilized powder for reconstitution with sWFI (Table 1 ). Viscosity was evaluated using an Anton-Paar rheometer in cone- plate configuration, at 25°C. The sample size was approximately 81 pL. The samples were measured with a constant shear rate (898 s "1 ).
  • FIGS. 1 A and 1 B viscosity of formulation at pH 5.8 and pH 5.0 (A) up to approximately 200 mg/mL C1 GM; (B) y-axis scale limited to 100 cP).
  • camphor-10-sulfonic acid also described as camphorsulfonic acid or CSA
  • CSA camphorsulfonic acid
  • Formulation 2 shown in Table 2 below, includes 200 mM CSA. Due to the acidic nature of CSA, nearly an equimolar amount of NaOH was used to neutralize the acid and bring the pH of the formulation to 7.0. Therefore, sodium ions are also present in this formulation at approximately 200 mM.
  • Viscosity was evaluated using an Anton-Paar rheometer in cone-plate configuration, at 25°C. The sample size was approximately 81 ⁇ _. The samples were measured with a constant shear rate (898 s "1 ). Viscosity data are summarized in Table 3 below and FIG. 2A and 2B.
  • Viscosity of formulation 2 containing 200 mM CSA showed significantly reduced viscosity, i.e., approximately 20-40 fold reduction in viscosity, compared to formulation 1 across concentrations tested (Table 3 and FIG. 2A and 2B).
  • viscosity of formulation 2 was 3.2 cP, compared to viscosity of formulation 1 , which was 55.1 cp, a 17.2 fold reduction.
  • viscosity of formulation 2 was 4.4 cP, compared to viscosity of formulation 1 , which was 89.5 cP, a 20.3 fold reduction.
  • viscosity of formulation 2 was 7.5 cP, compared to viscosity of formulation 1 , which was 221.8 cP, a 29.6 fold reduction.
  • viscosity of formulation 2 was 13.1 cP, compared to viscosity of formulation 1 , which was 506.3 cP, a 38.6 fold reduction.
  • viscosity reduction on a fold basis increases as antibody concentration increases.
  • Formulation 2 which contains 200 mM CSA and has pH 7, allows C1 GM protein concentrations of greater than 170 mg/mL with viscosity behavior suitable for use in therapeutic treatment. This was not possible for C1 GM in formulation 1 because of high viscosity.
  • Example 3 Impact of counterion on viscosity reduction This example illustrates how varying the counterion ion for CSA can also impact viscosity.
  • Example 2 NaOH was used to neutralize the acidic nature of CSA in creating formulation 2, and bring the pH of the formulation to 7.0. Therefore, sodium ions are also present in this formulation at approximately 200 mM.
  • other molecules basic in nature may be utilized to neutralize the acidic nature of CSA, effectively forming a salt of CSA.
  • One such species is arginine (Arg).
  • Formulation 3 shown in the table below, includes 200 mM CSA and 200 mM arginine. The basic nature of the arginine largely neutralizes the acidic nature of the CSA, and minimal acid or base needs to be added to adjust the pH to 7.0, avoiding the
  • Formulation 4 utilizes 200 mM arginine-HCI (Arg-HCI).
  • Viscosity was evaluated using an Anton-Paar rheometer in cone-plate configuration, at 25°C. The sample size was approximately 81 ⁇ . The samples were measured with a constant shear rate (898 s "1 ). Viscosity data are summarized in Table 5 below and FIG. 3. Table 5
  • This example illustrates the impact of varying excipient concentration on viscosity in an anti-IL-7R antibody formulation.
  • cryoprotectants, surfactants, and chelating agents may be necessary to include in the formulation, and will contribute to the tonicity of the formulation. Therefore, reduction of CSA and Arg levels may be required to allow these other excipients to be included in the formulation. Even at lower levels of CSA and Arg, formulations of C1 GM that include these excipients have significantly lower viscosity than formulations without these excipients. These results demonstrate that CSA-Arg appears to provide some robust protection against viscosity increases over the range of the ionic strength of the formulation. Example 5. Viscosity Reduction by Camphorsulfonic acid and CSA-Arg formulations of other antibodies
  • mAb5 and L1 L3 were reformulated with the above formulations by adding 200 mM Arginine HCI, 200 mM CSA, or 200 mM CSA-Arg. Viscosity was evaluated using an Anton-Paar rheometer in cone-plate configuration, at 25°C. The sample size was approximately 81 ⁇ _. The samples were measured with a constant shear rate (898 s " ). Viscosity data are summarized in Table 9 below and FIG. 6 (XX 1 is mAb5 antibody and XX2 is L1 L3 antibody).
  • the CSA-Arg combination offers the most effective reduction of viscosity for the mAb5 and L1 L3 antibody formulations.
  • This example illustrates the impact of varying excipient concentration on viscosity on mAb5 and L1 L3 antibody formulations.
  • Excipient concentrations of CSA and Arginine were reduced to either 100 mM or 50 mM from the previous level of 200 mM. Viscosities of these formulations were evaluated at pH 7 using an Anton-Paar rheometer in cone-plate configuration, at 25°C. The sample size was approximately 81 ⁇ _. The samples were measured with a constant shear rate (898 s " ). Results are summarized in FIG. 7A and 7B (XX1 is mAb5 antibody and XX2 is L1 L3 antibody) and Table 10. Lower amounts of excipients led to higher viscosities than observed for the 200 mM excipients.
  • This example illustrates the impact of the choice of hinge region structure on the viscosity of a formulation of monoclonal antibodies.
  • Monoclonal antibodies with identical CDR regions but differing hinge regions were produced and characterized using methods known in the art. Identical CDR regions were incorporated into frameworks of the lgG1 , lgG2, and lgG4 subtypes, and referred to as lgG1 , lgG2, and lgG4, respectively.
  • the lgG4 contained a hinge stabilizing S228P mutation (serine at position 228 of the heavy chain is replaced with proline).
  • Each antibody was formulated at 130 mg/ml_ in 20 mM histidine, 85 g/L sucrose, 0.05 g/L disodium EDTA dihydrate, 0.2 g/L polysorbate-80, pH 5.8.
  • Viscosity of these formulations was evaluated using an Anton-Paar rheometer in cone-plate configuration, at 25°C. The sample size was approximately 81 pL. The samples were measured with a constant shear rate (898 s " ). Viscosity data are summarized in Table 1 1 .

Abstract

The present invention relates generally to the field of pharmaceutical formulations of antibodies. Specifically, the present invention relates to a stable low-viscosity antibody formulation and its pharmaceutical preparation and use.

Description

LOW VISCOSITY ANTIBODY COMPOSITIONS
Reference to Sequence Listing
This application includes an electronically submitted sequence listing in .txt format. The sequence listing contained in this .txt file is part of the specification and is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to the field of pharmaceutical formulations of antibodies. Specifically, the present invention relates to a low viscosity liquid antibody formulation and its pharmaceutical preparation and use.
BACKGROUND
Antibody preparations intended for therapeutic or prophylactic use require stabilizers to prevent loss of activity or structural integrity of the protein due to the effects of denaturation, oxidation or aggregation over a period of time during storage and transportation prior to use. These problems are exacerbated at the high concentrations of antibody often desired for therapeutic administration.
A major aim in the development of antibody formulations is to maintain antibody solubility, stability and potency of its antigen binding. It is particularly desirable to avoid antibody self-association, aggregates, and particulates in solution which would require sterile filtration before use for intravenous or subcutaneous injection and limit route of administration. Antibody aggregates can cause pain and anaphylactoid side effects when the formulation containing them is intravenously injected. Moreover, self- associated antibodies and aggregates present increased difficulties for subcutaneous (sc) administration. The ease of injection for sc administration is described as extrusion force, the force required to extrude the composition from the syringe. The viscosity of the liquid composition is directly related to the required extrusion force: more viscous compositions require greater extrusion force. Alternatively, a larger diameter needle or a longer injection time may be required to administer the desired dose. As a result, high viscosity compositions present a greater risk for pain at the injection site, and thus may negatively impact patient compliance.
Self-associated antibodies exhibit high viscosity and result in difficulty in manufacturing. Tangential flow filtration is often used in manufacturing for buffer exchange and protein concentration. High viscosity compositions create additional back pressure and shear stress during this process, which can increase the processing time and destabilize the antibody. One solution to self -association of antibody therapeutics is to formulate the therapeutic in a viscosity lowering composition. Lyophilization or freeze drying is an alternative to the liquid formulation of antibodies. The process has a propensity for inducing denaturation of the antibody and decreasing its antigen-binding activity particularly upon reconstitution.
Viscosity lowering excipients, surfactants, pH, and cryoprotectant/tonicity agents such as sugars can contribute to overcoming self-association problems. Formulation of antibody preparations requires careful selection of these factors among others to avoid denaturation of the protein and loss of antigen-binding activity. In addition, formulation constituents, such as sugars, may further exacerbate the self-association tendency of the antibody and high concentrations of these stabilizing excipients can lead to high viscosities. Some viscosity lowering excipients have been explored including arginine, histidine, lysine, and camphor-10-sulfonic acid. Zheng Guo et al., "Structure-Activity Relationship for Hydrophobic Salts as Viscosity-Lowering Excipients for Concentrated Solutions of Monoclonal Antibodies", Pharmaceutical Research, vol. 29, no. 1 1 , June 13, 2012, p. 3182-3189. However, a need still exists for stable lowered viscosity antibody compositions which stably support high concentrations of bioactive antibody in solution and is suitable for parenteral administration, including intravenous
intramuscular, intraperitoneal, intradermal or subcutaneous injection. It is further desirable that the formulation has minimized risk of anaphylactoid side effects.
SUMMARY
One aspect of the present invention includes pharmaceutical compositions comprising a. an antibody, wherein the antibody concentration is between about 100 mg/ml to about 400 mg/ml, and b. a viscosity lowering excipient comprising camphorsulfonic acid, sulfosalicylic acid, a salt of camphorsulfonic acid or a salt of sulfosalicylic acid, wherein the viscosity lowering excipient concentration is between about 30 mM to about 200 mM; wherein the pH of said composition is from about 4.0 to about 9.0. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 A depicts a graph comparing the viscosity of anti-IL-7R antibody formulation at different pH values.
FIG. 1 B depicts a graph comparing the viscosity of anti-IL-7R antibody formulation at different pH values.
FIG. 2A depicts a graph comparing the viscosity of anti-IL7R antibody formulation with and without camphorsulfonic acid.
FIG. 2B depicts a graph comparing the viscosity of anti-IL7R antibody formulation with and without camphorsulfonic acid. FIG. 3 depicts a graph comparing the viscosity of anti-IL7R antibody formulation with a combination of camphorsulfonic acid and arginine, and arginine HCI.
FIG 4 depicts a graph comparing the viscosity of anti-IL7R antibody formulation at varying concentrations of camphorsulfonic acid, camphorsulfonic acid and arginine, and arginine HCI. FIG. 5 depicts a graph comparing the viscosity of anti-glucagon receptor antibody (XX1 ) and anti-PCSK9 receptor antibody (XX2) formulations.
FIG. 6 depicts a graph comparing the viscosity of anti-glucagon receptor antibody (XX1 ) and anti-PCSK9 receptor antibody (XX2) with camphorsulfonic acid,
camphorsulfonic acid and arginine, and arginine HCI. FIG. 7A depicts a graph comparing the viscosity of anti-glucagon receptor antibody (XX1 ) and anti-PCSK9 receptor antibody (XX2) with varying levels of camphorsulfonic acid, camphorsulfonic acid and arginine, and arginine HCI.
FIG. 7B depicts a graph comparing the viscosity of anti-glucagon receptor antibody (XX1 ) and anti-PCSK9 receptor antibody (XX2) with varying levels of camphorsulfonic acid, camphorsulfonic acid and arginine, and arginine HCI.
DETAILED DESCRIPTION
The present invention may be understood even more readily by reference to the following detailed description of exemplary embodiments of the invention and the examples included therein. Unless stated otherwise, the concentrations listed herein are those concentrations at ambient conditions, [i.e., at 25°C and atmospheric pressure].
Before the present compositions and methods are disclosed and described, it is to be understood that this invention is not limited to specific synthetic methods of making that may of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. The plural and singular should be treated as interchangeable, other than the indication of number. As used in this specification, the singular forms "a," "an" and "the" specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise.
The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10%.
An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc. , through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv) and domain antibodies), and fusion proteins comprising an antibody portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2, which denote differences in the constant region, particularly in the hinge and upper CH2 domain. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Following human lgG1 EU numbering and beginning at the 216 position, the isotypes have the following sequences: lgG1 : EPKSCDKTHTCPPCP lgG2: ERKCCVE— CPPCP lgG4: ESKYGPP— CPSCP
As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example.
A "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) that contain hypervariable regions. The identity of the amino acid residues in a particular antibody that make up a CDR can be determined using methods well known in the art. For example, antibody CDRs may be identified as the
hypervariable regions originally defined by Kabat et al (Kabat et al., 1991 , Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C., NIH Publication No. 91 -3242). The positions of the CDRs may also be identified as the structural loop structures described by Chothia and others (Chothia et al., 1989, Nature 342:877-883). Other approaches to CDR identification include the "AbM definition," which is a compromise between Kabat and Chothia and is derived the Abysis program (www.abysis.org), or the "contact definition" of CDRs based on observed antigen contacts, set forth in MacCallum et al., 1996, J. Mol. Biol., 262:732- 745. North has identified canonical CDR conformations using a different preferred set of CDR definitions (North et al., 201 1 , J. Mol. Biol, 406: 228-256). In another approach, referred to herein as the "conformational definition" of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding (Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1 166). Still other CDR boundary definitions may not strictly follow one of the above approaches, but will nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. As used herein, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment containing more than one CDR, the CDRs (or other residue of the antibody) may be defined in accordance with any of Kabat, Chothia, North, extended, AbM, contact, and/or conformational definitions. As known in the art a "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.
The "hinge region" of an antibody consists of a flexible domain that joins the Fab arms to the Fc region. The antibody of the present invention is selected from the group of monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, ScFv etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion (e.g., a domain antibody), humanized antibodies, human antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibody may be murine, rat, human, or any other origin (including chimeric or humanized antibodies). In some embodiments, the antibody can be human but is more preferably humanized. Preferably the antibody is isolated, further preferably it is substantially pure. Where the antibody is an antibody fragment this preferably retains the functional characteristics of the original antibody i.e. the ligand binding and/or antagonist or agonist activity.
In one embodiment of the present invention the antibody heavy chain constant region may be from any type of constant region, such as IgG, IgM, IgD, IgA, and IgE; and any isotypes, such as IgGI , lgG2, lgG3, and lgG4. In another embodiment, the antibody is an lgG2 antibody.
According to the present invention, the antibody can comprise the human heavy chain lgG2a constant region. In some embodiments the antibody comprises the human light chain kappa constant region. In some embodiments, the antibody comprises a modified constant region, such as a constant region that is immunologically inert, e.g., does not trigger complement mediated lysis, or does not stimulate antibody-dependent cell mediated cytotoxicity (ADCC). In other embodiments, the constant region is modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT publication No.
WO099/58572; and/or UK Patent Application No. 9809951.8. In still other
embodiments, the antibody comprises a human heavy chain lgG2a constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering with reference to the wildtype lgG2a sequence), Eur. J. Immunol. (1999) 29:2613-2624.
As used herein, the antibody does not necessarily comprise an identical amino acid sequence of the amino acid sequence described herein. An antibody that has a similar amino acid sequence refers to an antibody analog that satisfies at least one of the following: (a) an amino acid sequence that is at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% identical to the amino acid sequence of any of the antibodies or portions of antibodies described herein; (b) an antibody encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding the antibody of at least about 5 contiguous amino acid residues, at least about 10 contiguous amino acid residues, at least about 15 contiguous amino acid residues, at least about 20 contiguous amino acid residues, at least about 25 contiguous amino acid residues, at least about 40 contiguous amino acid residues, at least about 50 contiguous amino acid residues, at least about 60 contiguous amino residues, at least about 70 contiguous amino acid residues, at least about 80 contiguous amino acid residues, at least about 90 contiguous amino acid residues, at least about 100 contiguous amino acid residues, at least about 125 contiguous amino acid residues, or at least about 150 contiguous amino acid residues to the amino acid sequences described herein; or (c) an antibody encoded by a nucleotide sequence that is at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% identical to the nucleotide sequence encoding the any of the antibodies or portions of antibodies described herein.
To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of
positions.times.100%). In one embodiment, the two sequences are the same length.
The determination of percent identity between two sequences can also be
accomplished using a mathematical algorithm. One, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et ah, 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11 -17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
The formulations of the present invention include at least one antibody. In some embodiments, more than one antibody may be present. At least one, at least two, at least three, at least four, at least five, or more, different antibodies may be present. Generally, the two or more different antibodies have complementary activities that do not adversely affect each other. The, or each, antibody can also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the antibodies.
In one embodiment of the present invention the concentration of antibody can range from about 50 mg/ml to about 450 mg/ml. In other embodiments, the concentration of antibody is about 50 mg/ml, about 70 mg/ml, about 90 mg/ml, about 100 mg/ml, about 1 10 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, about 150 mg/ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190 mg/ml, about 200 mg/ml, about 210 mg/ml, about 220 mg/ml, about 230 mg/ml, about 240 mg/ml, about 250 mg/ml, about 260 mg/ml, about 270 mg/ml, about 280 mg/ml, about 290 mg/ml, about 300 mg/ml, about 310 mg/ml, about 320 mg/ml, about 330 mg/ml, about 340 mg/ml, about 350 mg/ml, about 370 mg/ml, about 390 mg/ml, about 400 mg/ml, about 420 mg/ml, or about 450mg/ml. In some embodiments, the concentration of the antibody in the formulation is between about 50 mg/ml and about 400 mg/ml, between about 100 mg/ml and about 400 mg/ml, between about 100 mg/ml and about 350 mg/ml, between about 100 mg/ml and about 300 mg/ml, between about 100 mg/ml and about 250 mg/ml, between about 100 mg/ml and about 200 mg/ml, between about 120 mg/ml and 200 mg/ml, between about 150 mg/ml and about 200 mg/ml, or between about 165 mg/ml and about 215 mg/ml. "Isolated" when used to describe the various antibodies disclosed herein, means a polypeptide or antibody that has been identified, separated and/or recovered from a component of its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody in the antibody formulation is purified prior to being added to the antibody formulation. The terms "isolate," and "purify" refer to separating the antibody from an impurity or other contaminants in the composition which the antibody resides, e.g., a composition comprising host cell proteins. In some embodiments, at least about 50%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, at least about or 99.9% (w/w) of an impurity is purified from the antibody.
Throughout the present disclosure, all expressions of percentage, ratio, and the like are "by weight" unless otherwise indicated. As used herein, "by weight" is synonymous with the term "by mass," and indicates that a ratio or percentage defined herein is done according to weight rather than volume, thickness, or some other measure.
"Camphorsulphonic acid" (CSA) means camphor-10-sulphonic acid or the compound of formula CSA. CSA is represented by the chemical formula:
Figure imgf000012_0001
CSA
"CSA" includes all hydrate/solvate forms including the anhydrous form as well as the free base form, as well as any salt form. "CSA" includes all enantiomers (e.g. ,
(+)camphorsulphonic acid and (-)camphorsulphonic acid), and any combination of enantiomers (e.g., 50% (+)camphorsulphonic acid and 50% (-)camphorsulphonic acid; 90%-100% (+)camphorsulphonic acid and 10%-0% (-)camphorsulphonic acid, etc.). In some embodiments, the term "CSA" includes greater than 99% (+)camphorsulphonic acid and less than 1 % (-)camphorsulphonic acid. In some embodiments, the term "CSA" includes an enantomerically pure (+)camphorsulphonic acid.
Exemplary pharmaceutically acceptable acid salts may be formed from counterions and include sodium, arginine, lysine, histidine, and include all hydrates, solvates, and enantiomers thereof.
The present invention includes various amounts of CSA. In some embodiments, the amount of CSA is greater than about 5 mM, greater than about 10 mM, greater than about 15 mM, greater than about 20 mM, greater than about 25 mM, greater than about 30 mM , greater than about 35 mM , greater than about 50 mM , greater than about 75 mM , greater than about 100 mM , greater than about 125 mM , greater than about 150 mM , greater than about 200 mM , greater than about 225 mM , or greater than about 250 mM. In some embodiments, the amount of CSA in the formulation is between about 5 mM and about 250 mM , between about 10 mM and about 200 mM, between about 20 mM and about 200 mM , between about 30 mM and about 200 mM, between about 30 mM and about 150 mM, or between about 30 mM and about 100 mM.
When present as the CSA salt form, the concentration of CSA and the counterion can be the same or different. For example, a salt of CSA formed with arginine (CSA-Arg) with a concentration of 50mM has a CSA concentration of 50mM and an arginine concentration of 50mM. However, more or less arginine (counterion) could be present, whereby not all of the CSA or counterion is in the salt form.
"Arginine" (Arg) is an amino acid that can be represented by the chemical formula:
Figure imgf000013_0001
Arginine
"Arginine" includes all hydrate/solvate forms including the anhydrous form as well as the free base form, as well as any salt form, including without limitation Arginine
hydrochloride (Arg-HCI). Arginine includes all enantiomers (e.g., L-arginine and D- arginine), and any combination of enantiomers (e.g., 50% L-arginine and 50% D- arginine; 90%-100% L-arginine and 10%-0% D-arginine, etc.). In some embodiments, the term "arginine" includes greater than 99% L-arginine and less than 1 % D-arginine. In some embodiments, the term "arginine" includes an enantomerically pure L-arginine. In some embodiments, the arginine is a pharmaceutical grade arginine.
"Histidine" is an amino acid that can be represented by the chemical formula:
Figure imgf000014_0001
Histidine
"Histidine" includes all hydrate/so I vate forms including the anhydrous form as well as the free base form, as well as any salt form, including without limitation histidine hydrochloride. "Histidine" includes all enantiomers (e.g. , L-histidine and D-histidine), and any combination of enantiomers (e.g., 50% L- histidine and 50% D- histidine; 90%- 100%) L- histidine and 10%-0% D- histidine, etc.). In some embodiments, the term "histidine" includes greater than 99%) L- histidine and less than 1 % D- histidine. In some embodiments, the term "histidine" includes an enantomerically pure L- histidine. In some embodiments, the histidine is a pharmaceutical grade histidine. "Lysine" is an amino acid that can be represented by the chemical formula:
Figure imgf000014_0002
Lysine
"Lysine" includes all hydrate/solvate forms including the anhydrous form as well as the free base form, as well as any salt form, including without limitation lysine hydrochloride "Lysine" includes all enantiomers (e.g., L-lysine and D-lysine), and any combination of enantiomers (e.g., 50% L- lysine and 50% D- lysine; 90%-100% L- lysine and 10%-0% D- lysine, etc.). In some embodiments, the term "lysine" includes greater than 99% L- lysine and less than 1 % D- lysine. In some embodiments, the term "lysine" includes an enantomerically pure L- lysine. In some embodiments, the lysine is a pharmaceutical grade lysine.
By "chelating agent" is meant an optional composition component that is a
pharmaceutically acceptable agent that lowers the formation of reduced oxygen species, reduces acidic species (e.g. , deamidation) formation, reduces antibody aggregation, and/or reduces antibody fragmentation, and/or reduces antibody oxidation in the compositions of the present invention. Such chelating agents can reduce or prevent degradation of an antibody that is formulated in comparision to the antibody without the protection of a chelating agent. Exemplary chelating agents include aminopolycarboxylic acids, hydroxyaminocarboxylic acids, N-substituted glycines, 2- (2- amino-2-oxocthyl) aminoethane sulfonic acid (BES), deferoxamine (DEF), citric acid, niacinamide, and desoxycholates and mixtures thereof. In one embodiment, the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), diethylenetriamine pentaacetic acid 5 (DTPA), nitrilotriacetic acid (NTA), N-2- acetamido-2-iminodiacetic acid (ADA), bis(aminoethyl)glycolether, Ν,Ν, Ν', Ν'-tetraacetic acid (EGTA), trans-diaminocyclohexane tetraacetic acid (DCTA), glutamic acid, and aspartic acid, N- hydroxyethyliminodiacetic acid (HIMDA), N, N-bis-hydroxyethylglycine (bicine) and N- (trishydroxymethylmethyl) 10 glycine (tricine), glycylglycine, sodium desoxycholate, ethylenediamine; propylenediamine; diethylenetriamine;
triethylenetetraamine (trien), ethylenediaminetetraaceto EDTA; disodium EDTA, calcium EDTA oxalic acid, malate, citric acid, citric acid monohydrate, and trisodium citrate- dihydrate, 8-hydroxyquinolate, amino acids, histidine, cysteine, methionine, peptides, polypeptides, and proteins and mixtures thereof. In another embodiment, the chelating agent is selected from the group consisting of salts of EDTA including dipotassium EDTA, disodium EDTA, EDTA calcium disodium, sodium EDTA, trisodium EDTA, and potassium EDTA; and a suitable salt of deferoxamine (DEF) is deferoxamine mesylate (DFM), or mixtures thereof. Chelating agents used in the invention can be present, where possible, as the free acid or free base form or salt form of the compound, also as an anhydrous, solvated or hydrated form of the compound or corresponding salt. The concentration of chelating agent, when present, generally ranges from about 0.01 mg/ml to about 50 mg/ml, from about 0.01 mg/ml to about 10.0 mg/ml, from about 0.01 mg/ml to about 5.0 mg/ml, from about 0.01 mg/ml to about 1.0 mg/ml, or from about 0.01 mg/ml to about 0.3 mg/ml. In another embodiment, the concentration of chelating agent generally ranges from about 0.01 mM to about 2.0 mM, from about 0.01 mM to about 1.5 mM, from about 0.01 mM to about 0.5 mM, from about 0.01 mM to about 0.4 mM, from about 0.01 mM to about 0.2 mM, from about 0.01 mM to about 0.15 mM, from about 0.01 mM to about 0.1 mM, from about 0.01 mM to about 0.09 mM, from about 0.01 mM to about 0.08 mM, from about 0.01 mM to about 0.07 mM, from about 0.01 mM to about 0.06 mM, from about 0.01 mM to about 0.05 mM, from about 0.01 mM to about 0.04 mM, from about 0.01 mM to about 0.03 mM, from about 0.01 mM to about 0.02 mM or from about 0.005 mM to about 0.01 mM. In another embodiment, the concentration of chelating agent can be about 0.01 mg/ml, 0.02 mg/ml, 0.03 mg/ml, about 0.04 mg/ml, about 0.05 mg/ml, about 0.06 mg/ml, about 0.07 mg/ml, about 0.10 mg/ml, about 0.20 mg/ml.
A "cryoprotectant" is an optional composition component that is a molecule which, when combined with a protein of interest, significantly prevents or reduces chemical and/or physical instability of the protein upon lyophilization and subsequent storage. Exemplary cryoprotectants include sugars and their corresponding sugar alcohols; an amino acid such as monosodium glutamate or histidine; a methylamine such as betaine; a lyotropic salt such as magnesium sulfate; a polyol such as trihydric or higher molecular weight sugar alcohols, e.g. glycerin, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; Pluronics.RTM.; and combinations thereof. Additional exemplary cryoprotectants include glycerin and gelatin, and the sugars mellibiose, melezitose, raffinose, mannotriose and stachyose. Examples of reducing sugars include glucose, maltose, lactose, maltulose, iso-maltulose and lactulose. Examples of non-reducing sugars include non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other straight chain polyalcohols including sucrose, dextrose, mannose and trehalose (including all forms of trehalose such as trehalose monohydrate and trehalsoe dihydrate). Example sugar alcohols are monoglycosides, especially those compounds obtained by reduction of disaccharides such as lactose, maltose, lactulose and maltulose. The glycosidic side group can be either glucosidic or galactosidic. Additional examples of sugar alcohols are glucitol, maltitol, lactitol and iso-maltulose. The concentration of the cryoprotectant, when present in the liquid composition ranges from about 0.1 mg/ml to about 150 mg/ml, from about 0.1 mg/ml to about 100 mg/ml, or from about 1 mg/ml to about 100 mg/ml. In one embodiment, the concentration of the cryoprotectant in the liquid composition is about 20 mg/ml, about 25 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 75 mg/ml, about 80 mg/ml, about 85 mg/ml, about 90 mg/ml, about 95 mg/ml, about 100 mg/ml, about 1 10 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, or about 150 mg/ml.
Where the cryoprotectant comprises a salt, the concentration of the salt in the liquid composition ranges from about 1 mg/ml to about 20 mg/ml. Salts that are
pharmaceutically acceptable and suitable for this invention include sodium chloride, sodium succinate, sodium sulfate, potassium chloride, magnesium chloride, magnesium sulfate, and calcium chloride. Exemplary salts include sodium chloride and magnesium chloride, magnesium chloride may also improve the antibody stability by protecting the protein from deamidation. In one embodiment, the salt in the liquid composition is selected from a range of concentrations of any of about 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8, mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml and 20 mg/ml.
The term "injection force" is the amount of pressure (in Newtons) required to pass the antibody formulation through a needle. The injection force is correlated with the amount of resistance provided by the antibody formulation when administering the antibody formulation to a subject. The injection force will be dependent on the gauge of the administering needle, as well as temperature. In some embodiments, the antibody formulation has an injection force of less than 15 N, 12 N, 10N, or 8 N when passed through a 27 Ga thin wall PFS needle such as defined in the International Organization for Standardization (ISO) document "Stainless steel needle tubing for the manufacture of medical devices" (ISO 9626:1991 ) and manufactured by BD Medical, Pharmaceutical Systems (Franklin Lakes, N.J.). In some embodiments, the antibody formulation has an injection force of less than 15 N, 12 N, 10N, or 8 N when passed through a 25 or 26 Gauge needle.
An "isotonic" formulation is one which has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to about 350 mOsm. The term "hypotonic" describes a formulation with an osmotic pressure below that of human blood. Correspondingly, the term "hypertonic" is used to describe a formulation with an osmotic pressure above that of human blood. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example. In one embodiment, the compositions of the present invention are isotonic. The term "IV bag protectant" refers to the surfactant added to the intravenous bag prior to dilution of the antibody formulation described herein into the intravenous bag. The IV bag protectant can also be added to the intravenous bag prior to addition of other antibody formulations known to those of skill in the art, e.g. , a lyophilized antibody formulation. Surfactants suitable for use as an IV bag protectant will generally be those suitable for use in IV formulations. In some embodiments, the surfactant used in the IV bag protectant is the same buffer used in the antibody formulation. For example, if the antibody formulation comprises polysorbate 80 as a surfactant, then polysorbate 80 would be added to the intravenous bag prior to adding the antibody formulation to the intravenous bag. In some embodiments, the surfactant concentration in the IV bag resulting from addition of the IV protectant will be about the same or only a portion of the surfactant concentration in the antibody formulation. Knowing the desired final concentration of surfactant in the IV bag, one can formulate the desired concentration of the surfactant in the IV bag protectant. The term "Koff", as used herein, is intended to refer to the off rate constant for dissociation of an antibody from the antibody/antigen complex. The term "Kd", as used herein, is intended to refer to the dissociation constant of an antibody-antigen interaction. One way of determ ining the Kd or binding affinity of antibodies to the antigen is by measuring binding affinity of monofunctional Fab fragments of the antibody. To obtain monofunctional Fab fragments, an antibody (for example, IgG) can be cleaved with papain or expressed recombinantly. The affinity of a Fab fragment of an antibody can be determ ined by surface plasmon resonance (BIAcorC1 GM000™ surface plasmon resonance (SPR) system , BIAcore, INC, Piscaway NJ). CM5 chips can be activated with N-ethyl-N'-(3-dimethylam inopropyl)-carbodiinide hydrochloride (EDC) and N-hydroxysuccinim ide (NHS) according to the supplier's instructions.
Human antigen can be diluted into 1 0 mM sodium acetate pH 4.0 and injected over the activated chip at a concentration of 0.005 mg/mL. Using variable flow time across the individual chip channels, two ranges of antigen density can be achieved: 1 00-200 response units (RU) for detailed kinetic studies and 500-600 RU for screening assays. Serial dilutions (0.1 -1 Ox estimated Kd) of purified Fab samples are injected for 1 m in at 100 m icroliters/min and dissociation times of up to 2 hours are allowed. The
concentrations of the Fab proteins are determined by ELISA and/or SDS-PAGE electrophoresis using a Fab of known concentration (as determ ined by amino acid analysis) as a standard. Kinetic association rates (kon) and dissociation rates (k0ff) are obtained simultaneously by fitting the data to a 1 : 1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6. 99-1 10) using the BIAevaluation program. Equilibrium dissociation constant (Kd) values are calculated as k0ff/kon. This protocol is suitable for use in determ ining binding affinity of an antibody to any antibody.
The antibody formulations can have different "osmolarity" concentrations. Methods of measuring osmolarity of antibody formulations are known to those in the art, and can include, e.g. , an osmometer (e.g., an Advanced Instrument Inc 2020 freezing point depression osmometer). In some embodiments, the formulation has an osmolarity of between 200 and 600 mosm/kg, between 260 and 500 mosm/kg, or between 300 and 450 mosm/kg. In some embodiments, the formulation does not comprise an osmolyte.
The phrase "pharmaceutically acceptable" indicates that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith.
A "pharmaceutically acceptable acid" includes inorganic and organic acids which are non toxic at the concentration and manner in which they are formulated. For example, suitable inorganic acids include hydrochloric, perchloric, hydrobromic, hydroiodic, nitric, sulfuric, sulfonic, sulfinic, sulfanilic, phosphoric, carbonic, etc. Suitable organic acids include straight and branched-chain alkyl, aromatic, cyclic, cyloaliphatic, arylaliphatic, heterocyclic, saturated, unsaturated, mono, di- and tri-carboxylic, including for example, form ic, acetic, 2-hydroxyacetic, trifluoroacetic, phenylacetic, trimethylacetic, t-butyl acetic, anthranilic, propanoic, 2-hydroxypropanoic, 2-oxopropanoic, propandioic, cyclopentanepropionic, cyclopentane propionic, 3-phenylpropionic, butanoic, butandioic, benzoic, 3-(4-hydroxybenzoyl)benzoic, 2-acetoxy-benzoic, ascorbic, cinnam ic, lauryl sulfuric, stearic, muconic, mandelic, succinic, embonic, fumaric, malic, maleic, hydroxymaleic, malonic, lactic, citric, tartaric, glycolic, glyconic, gluconic, pyruvic, glyoxalic, oxalic, mesylic, succinic, salicylic, phthalic, palmoic, palmeic, thiocyanic, methanesulphonic, ethanesulphonic, 1 ,2-ethanedisulfonic, 2-hydroxyethanesulfonic, benzenesulphonic, 4-chorobenzenesulfonic, napthalene-2-sulphonic, p- toluenesulphonic, camphorsulphonic, 4-methylbicyclo[2.2.2]-oct-2-ene-1 -carboxylic, glucoheptonic, 4, 4' -methylenebis-3-(hydroxy-2-ene-1 -carboxylic acid), hydroxynapthoic.
"Pharmaceutically-acceptable bases" include inorganic and organic bases which are non-toxic at the concentration and manner in which they are formulated. For example, suitable bases include those formed from inorganic base forming metals such as lithium, sodium, potassium, magnesium, calcium, ammonium, iron, zinc, copper, manganese, aluminum, N-methylglucamine, morpholine, piperidine and organic nontoxic bases including, primary, secondary and tertiary amine, substituted amines, cyclic amines and basic ion exchange resins, [e.g., N(R').sub.4.sup.+ (where R1 is independently H or C. sub.1 -4 alkyl, e.g., ammonium, Tris)], for example,
isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine,
ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N- ethylpiperidine, polyamine resins and the like.
Additional pharmaceutically acceptable acids and bases useable with the present invention include those which are derived from the amino acids, for example, histidine, glycine, phenylalanine, aspartic acid, glutamic acid, lysine, arginine and asparagine.
"Pharmaceutically acceptable" buffers and salts, which are an optional composition component, include those derived from both acid and base addition salts of the above indicated acids and bases. According to the present invention, the buffer is used to adjust the pH and it may also enhance the antibody stability. In one embodiment of the present invention, the buffer is selected from the group consisting of acetate, succinate, gluconate, citrate, histidine, arginine, acetic acid, phosphate, phosphoric acid, ascorbate, tartartic acid, maleic acid, glycine, lactate, lactic acid, ascorbic acid, imidazole, bicarbonate and carbonic acid, succinic acid, sodium benzoate, benzoic acid, gluconate, edetate (EDTA), acetate, malate, imidazole, tris, phosphate, and mixtures thereof.
When present, the concentration of the buffer can range from about 0.1 millimolar (mM) to about 200 mM. In one embodiment, the concentration of the buffer is from about 0.5 mM to about 200 mM, from about 1 mM to about 100 mM, from about 1 mM to about 65 mM, or from about 1 mM to about 30 mM. In another embodiment, the concentration of the buffer is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM.
According to one embodiment of the present invention, the pH can be in the range of about 4.0 to about 9.0. In another embodiment, the pH is between about 4.5 and about 8.0, between about 5.0 and about 7.5, between about 5.5 and about 7.0, or between about 6.0 and 7.5. In another embodiment, the pH is about 4.0, about 4.1 , about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1 , about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 , about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0.
Of note, the inclusion of viscosity lowering excipient (comprising CSA, SSA, CSA salts or SSA salts) in the formulation may allow the antibody to be formualted at a pH that is impractical without the viscosity lowering excipient. One of skill in the art would expect that self-association is strongest when the antibody is formulated at the pH of the isoelectric point, where the net charge on the antibody is expected to be zero. In some cases, severe self-association may even lead to phase separation, making it impractical to formulate the molecule at a pH near the isoelectric point. The addition of the viscosity lowering excipient in some cases greatly reduce the self-association, which may allow formulation at pH values which otherwise would be prohibited, and may offer additional benefits with respect to stability of the antibody. Unexpectedly, the self-association behavior may actually be minimized near the isolectric point of the molecule rather than at higher or lower pH values when a viscosity lowering exipient are included in the formulation.
A "preservative" is a compound which can be added to the formulations herein to reduce bacterial activity. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation. Examples of potential
preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. In one embodiment, the composition does not include a preservative.
A "stable" formulation is one in which the protein therein essentially retains its physical and chemical stability and integrity upon storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301 , Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991 ) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected time period. For rapid screening, the formulation may be kept at 40°C for 2 weeks to 1 month, at which time stability is measured. Where the formulation is to be stored at 2-8°C, generally the formulation should be stable at 30°C or 40°C for at least 1 month and/or stable at 2-8°C for at least 2 years. Where the formulation is to be stored at 23-27°C, generally the formulation should be stable for at least 24 months. Thus, a "stable" formulation may be one wherein less than about 20%, or less than about 15%, or less than about 10%, or less than about 5%, or less than about 2% of the antibody is degraded, denatured, aggregated or unfolded as determined by SEC HPLC when the antibody is stored at 2- 8°C for 6 months, or 12 months, or 18 months, or 24 months.
In some embodiments, the antibody stability is determined by size exclusion
chromatography (SEC). SEC separates analytes (e.g., macromolecules such as proteins and antibodies) on the basis of a combination of their hydrodynamic size, diffusion coefficient, and surface properties. Thus, for example, SEC can separate antibodies in their natural three-dimensional conformation from antibodies in various states of denaturation, and/or antibodies that have been degraded. In SEC, the stationary phase is generally composed of inert particles packed into a dense three- dimensional matrix within a glass or steel column. The mobile phase can be pure water, an aqueous buffer, an organic solvent, mixtures of these, or other solvents. The stationary-phase particles have small pores and/or channels which will only allow species below a certain size to enter. Large particles are therefore excluded from these pores and channels, but the smaller particles are removed from the flowing mobile phase. The time particles spend immobilized in the stationary-phase pores depends, in part, on how far into the pores they can penetrate. Their removal from the mobile phase flow causes them to take longer to elute from the column and results in a separation between the particles based on differences in their size. In some embodiments, SEC is combined with an identification technique to identify or characterize proteins, or fragments thereof. Protein identification and characterization can be accomplished by various techniques, including but not limited chromatographic techniques, e.g., high-performance liquid chromatography (HPLC), immunoassays, electrophoresis, ultra-violet/visible/infrared spectroscopy, raman spectroscopy, surface enhanced raman spectroscopy, mass spectroscopy, gas chromatography, static light scattering (SLS), Fourier Transform Infrared Spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and/or ANS protein binding.
In some embodiments, protein identification is achieved by high-pressure liquid chromatography. Various instruments and apparatuses are known to those of skill in the art to perform HPLC. Generally HPLC involves loading a liquid solvent containing the protein of interest onto a separation column, in which the separation occurs. The HPLC separation column is filled with solid particles (e.g. silica, polymers, or sorbents), and the sample mixture is separated into compounds as it interacts with the column particles. HPLC separation is influenced by the liquid solvent's condition (e.g. pressure, temperature), chemical interactions between the sample mixture and the liquid solvent (e.g. hydrophobicity, protonation, etc.), and chemical interactions between the sample mixture and the solid particles packed inside of the separation column (e.g. ligand affinity, ion exchange, etc.).
In some embodiments, the SEC and protein identification occurs within the same apparatus, or simultaneously. For example, SEC and HPLC can be combined, often referred to as SE-HPLC.
By separating the various antibodies and antibody degradation products using known techniques such as those techniques identified herein, the stability of the antibody in the antibody formulation can be determined. As used herein, the term "stability" generally is related to maintaining the integrity or to minimizing the degradation, denaturation, aggregation or unfolding of a biologically active agent such as a protein, peptide or another bioactive macromolecule. As used herein, "improved stability" generally means that, under conditions known to result in degradation, denaturation, aggregation or unfolding, the protein (e.g., antibody), peptide or another bioactive macromolecule of interest maintains greater stability compared to a control protein, peptide or another bioactive macromolecule.
In some embodiments, stability refers to an antibody formulation having low to undetectable levels of aggregation. The phrase "low to undetectable levels of aggregation" as used herein refers to samples containing no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1 % and no more than 0.5% aggregation by weight of protein as measured by high performance size exclusion chromatography (HPSEC), static light scattering (SLS), Fourier Transform Infrared Spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and 1- anilino-8-naphthalenesulfonic acid (ANS) protein binding techniques.
In some embodiments, the antibody formulation has low to undetectable levels of fragmentation. The term "low to undetectable levels of fragmentation" as used herein refers to samples containing equal to or more than 80%, 85%, 90%, 95%, 98% or 99% of the total protein, for example, in a single peak as determined by HPSEC, or in two peaks (e.g., heavy- and light-chains) (or as many peaks as there are subunits) by reduced Capillary Gel Electrophoresis (rCGE), representing the non-degraded antibody or a non-degraded fragment thereof, and containing no other single peaks having more than 5%, more than 4%, more than 3%, more than 2%, more than 1 %, or more than 0.5% of the total protein in each. The term "reduced Capillary Gel Electrophoresis" as used herein refers to capillary gel electrophoresis under reducing conditions sufficient to reduce disulfide bonds in an antibody. One of skill in the art will appreciate that stability of a protein is dependent on other features in addition to the composition of the formulation. For example, stability can be affected by temperature, pressure, humidity, and external forms of radiation. Thus, unless otherwise specified, stability referred to herein is considered to be measured at 2-8. degree. C, one atmosphere pressure, 60% relative humidity, and normal background levels of radiation.
In some embodiments, various components can be omitted from the antibody formulation, or can be "substantially free" of that component. The term "substantially free" as used herein refers to an antibody formulation, said formulation containing less than 0.01 %, less than 0.001 %, less than 0.0005%, less than 0.0003%, or less than 0.0001 % of the designated component.
"Sulfosalicyclic acid" (SSA) means the compound represented by the chemical formula:
Figure imgf000025_0001
SSA
"SSA" includes hydrate/solvate forms including the anhydrous form as well as the free base form and any salt form. Exemplary pharmaceutically acceptable acid salts may be formed from counter-ions and include sodium, hydrochloride, arginine, lysine, histidine, and include all hydrates, solvates, and enantiomers thereof. The present invention includes various amounts of SSA. In some embodiments, the amount of SSA is greater than about 5 mM, greater than about 10 mM, greater than about 15 mM, greater than about 20 mM, greater than about 25 mM, greater than about 30 mM , greater than about 35 mM , greater than about 50 mM , greater than about 75 mM , greater than about 100 mM , greater than about 125 mM , greater than about 150 mM , greater than about 200 mM , greater than about 225 mM , or greater than about 250 mM. In some embodiments, the amount of SSA in the formulation is between about 5 mM and about 250 mM , between about 10 mM and about 200 mM, between about 20 mM and about 200 mM , between about 30 mM and about 200 mM, between about 30 mM and about 150 mM, or between about 30 mM and about 100 mM. "Surfactant" is an optional component to the composition which is a pharmaceutically acceptable agent that reduces the tendency for the formation of bubbles in the formulation during preparation and handling of the formulation and preparation for parenteral administration and especially from stress related to shaking and agitation during preparation and also during shipping. Exemplary surfactants include
polysorbates, poloxamers, tritons, sodium dodecyl sulfate, sodium laurel sulfate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl- sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroamidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyl-taurate, disodium methyl oleyl- taurate, dihydroxypropyl PEG 5 linoleammonium chloride, polyethylene glycol, polypropylene glycol, and mixtures thereof. In further embodiments, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 21 , polysorbate 40, polysorbate 60, polysorbate 61 , polysorbate 65, polysorbate 80, polysorbate 81 , polysorbate 85, and mixtures thereof. When present, the concentration of the surfactant generally ranges from about 0.01 mg/ml to about 5.0 mg/ml, from about 0.01 mg/ml to about 2.0 mg/ml, from about 0.01 mg/ml to about 1.5 mg/ml, from about 0.01 mg/ml to about 1.0 mg/ml, from about 0.01 mg/ml to about 0.5 mg/ml, from about 0.01 mg/ml to about 0.4 mg/ml, from about 0.01 mg/ml to about 0.3 mg/ml, from about 0.01 mg/ml to about 0.2 mg/ml, from about 0.01 mg/ml to about 0.15 mg/ml, from about 0.01 mg/ml to about 0.1 mg/ml, or from about 0.01 mg/ml, to about 0.05 mg/ml. Furthermore, the concentration of the surfactant is about 0.5 mg/ml, about 0.05 mg/ml about 0.06 mg/ml about 0.07 mg/ml about 0.08 mg/ml about 0.09 mg/ml about 0.1 mg/ml about 0.11 mg/ml about 0.12 mg/ml about 0.13 mg/ml about 0.14 mg/ml about 0.15 mg/ml about 0.16 mg/ml about 0.17 mg/ml about 0.18 mg/ml about 0.19 mg/ml, or about 0.2 mg/ml.
The phrase "therapeutically effective amount" means an amount of a compound of the present invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.
The terms "treating", "treat", or "treatment" embrace both preventative, i.e., prophylactic, and palliative treatment.
The term "animal" refers to humans (male or female), companion animals (e.g., dogs, cats and horses), food-source animals, zoo animals, marine animals, birds and other similar animal species. "Edible animals" refers to food-source animals such as cows, pigs, sheep and poultry. "Viscosity" as used herein may be "kinematic viscosity" or "absolute viscosity." "Kinematic viscosity" is a measure of the resistive flow of a fluid under the influence of gravity. When two fluids of equal volume are placed in identical capillary viscometers and allowed to flow by gravity, a viscous fluid takes longer than a less viscous fluid to flow through the capillary. If one fluid takes 200 seconds to complete its flow and another fluid takes 400 seconds, the second fluid is twice as viscous as the first on a kinematic viscosity scale. "Absolute viscosity", sometimes called dynamic or simple viscosity, is the product of kinematic viscosity and fluid density: Absolute
Viscosity=Kinematic Viscosity x Density
The dimension of kinematic viscosity is L 217 where L is a length and T is a time.
Commonly, kinematic viscosity is expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm2/s, which is 1 cSt. Absolute viscosity is expressed in units of centipoise (cP). The SI unit of absolute viscosity is the milliPascal-second (mPa-s), where 1 cP=1 mPa-s.
The formulation may be in either aqueous or lyophilized form. In aqueous form, the formulation may have a viscosity of no greater than about 60 cP. In another embodiment, the formulation has a viscosity of no greater than about 50 cP, or no greater than about 40 cP, or no greater than about 30 cP, or no greater than about 20 cP, or no greater than about 40 cP, or no greater than about 15 cP. In some embodiments the composition comprising antibody has a viscosity of between about 1 cP and about 50 cP, between about 1 cP and 40 cP, between about 1 cP and about 30 cP, between about 1 cP and about 20 cP, between about 1 cP and about 15 cP, or between about 1 cP and about 10 cP at 25°C. In some embodiments, the formulation has a viscosity of about 50 cP, about 45 cP, about 40 cP, about 35 cP, about 30 cP, about 25 cP, about 20 cP, about 15 cP, or about 10 cP, or about 5 cP. In some embodiments, the formulation has a viscosity of between about 10 cP and 50 cP, between about 10 cP and 30 cP, between about 10 cP and 20 cP, or between about 5 cP and 15 cP.
The present invention is directed at pharmaceutical compositions that include: a. an antibody, wherein the antibody concentration is between about 100 mg/ml to about 400 mg/ml, and b. a viscosity lowering excipient comprising camphorsulfonic acid, sulfosalicylic acid, or a salt of camphorsulfonic acid or sulfosalicylic acid, wherein the viscosity lowering excipient concentration is between about 30 mM to about 200 mM; wherein the pH of said composition is from about 4.0 to about 9.0.
In another embodiment, the composition also includes a pharmaceutically acceptable buffer. In one embodiment, the pharmaceutically acceptable buffer comprises arginine, histidine, tris, phosphate or lysine, or a salt thereof. In another embodiment, the pharmaceutically acceptable buffer comprises histidine, tris, or phosphate, or a salt thereof. In another embodiment, the concentration of pharmaceutically acceptable buffer is from about 1.0 to about 200 mM.
In another embodiment, the composition also includes a surfactant. In one
embodiment, the surfactant is polysorbate 20 or polysorbate 80. In another
embodiment, the concentration of surfactant is from about 0.01 to about 0.3 mg/ml.
In another embodiment, the composition also includes a chelating agent. In one embodiment, the chelating agent is EDTA or disodium EDTA. In another embodiment, the concentration of chelating agent is from about 0.01 to about 0.3 mg/ml. In another embodiment, the composition also includes a cryoprotectant. In one embodiment, the cryoprotectant is sucrose, dextrose, mannose or trehalose. In another embodiment, the concentration of the cryoprotectant is from about 1 mg/ml to about 100 mg/ml.
In another embodiment, the viscosity lowering agent is (+)camphorsulfonic acid or (-)camphorsulfonic acid or a salt thereof. In another embodiment, the viscosity lowering agent concentration is between about 50 mM to about 150 mM. In another
embodiment, the viscosity lowering agent concentration is between about 70 mM to about 1 10 mM.
In another embodiment, the viscosity lowering agent is a salt of camphorsulfonic acid comprising camphorsulfonic acid and arginine. In another embodiment, the
camphorsulfonic acid concentration is between about 50 mM and about 150 mM and the arginine concentration is between about 50 mM and about 150 mM. In another embodiment, the camphor sulfonic acid concentration is between about 70 mM and about 1 10 mM and the arginine concentration is between about 70 mM and about 110 mM. In one embodiment, the antibody is a human or humanized monoclonal lgG1 , lgG2 or lgG4 antibody. In another embodiment, the antibody is an anti-IL7R, anti-PCSK9 or anti-glucagon receptor antibody.
In another embodiment, the antibody includes a heavy chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, 5, or 6; CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7 or 8; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9, and a light chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 1 1 , and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12.
In yet another embodiment, the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain variable region amino acid sequence shown in SEQ ID NO: 2, and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain variable region amino acid sequence shown in SEQ ID NO: 3.
In yet another embodiment, the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain amino acid sequence shown in SEQ ID NO: 13, and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain amino acid sequence shown in SEQ ID NO: 14.
In yet another embodiment, the antibody includes a variable heavy chain sequence comprising the amino acid sequence shown in SEQ ID NO: 2 and a variable light chain sequence comprising the amino acid sequence shown in SEQ ID NO: 3. In yet another embodiment, the antibody includes a heavy chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, 18 or 19; CDR2 comprising the amino acid sequence shown in SEQ ID NO: 20 or 21 ; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 22, and a light chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 23, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 24, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 25. In yet another embodiment, the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain variable region amino acid sequence shown in SEQ ID NO: 15, and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain variable region amino acid sequence shown in SEQ ID NO: 16.
In yet another embodiment, the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain amino acid sequence shown in SEQ ID NO: 26 or 27, and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain amino acid sequence shown in SEQ ID NO: 28.
In yet another embodiment, the antibody includes a variable heavy chain sequence comprising the amino acid sequence shown in SEQ ID NO: 15 and a variable light chain sequence comprising the amino acid sequence shown in SEQ ID NO: 16.
In yet another embodiment, the antibody includes a heavy chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 32, 33, or 34; CDR2 comprising the amino acid sequence shown in SEQ ID NO: 35 or 36; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 37, and a light chain comprising one, two or three CDRs selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 38, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 39, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 40.
In yet another embodiment, the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain variable region amino acid sequence shown in SEQ ID NO: 41 , and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain variable region amino acid sequence shown in SEQ ID NO: 42.
In yet another embodiment, the antibody includes an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a heavy chain amino acid sequence shown in SEQ ID NO: 30, and an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95% or 98% identical to a light chain amino acid sequence shown in SEQ ID NO: 31. In yet another embodiment, the antibody includes a variable heavy chain sequence comprising the amino acid sequence shown in SEQ ID NO: 41 and a variable light chain sequence comprising the amino acid sequence shown in SEQ ID NO: 42.
In another embodiment, the composition is lyophilized. In yet another embodiment, the lyophilized composition is reconstituted and the antibody concentration of the reconstituted composition is between about 250 mg/ml and about 400 mg/ml. In yet another embodiment, the lyophilized composition is reconstituted and the antibody concentration of the reconstituted composition is higher than the antibody concentration before lyophilization. In yet another embodiment, the composition has a viscosity of less than about 50 cP at 25°C. In yet another embodiment, the composition is isotonic.
Preparation of the Antibodies
Anti-IL-7R
As used herein, the term "IL-7R" refers to any form of IL-7R and variants thereof that retain at least part of the activity of IL-7R. Unless indicated differently, such as by specific reference to human IL-7R, IL-7R includes all mammalian species of native sequence IL-7R, e.g., human, canine, feline, equine, and bovine. One exemplary human IL-7R is found as Uniprot Accession Number P16871 (SEQ ID NO: 1 ).
MTILGTTFGM VFSLLQVVSG ESGYAQNGDL EDAELDDYSF SCYSQLEVNG SQHSLTCAFE DPDVNTTNLE FEICGALVEV KCLNFRKLQE IYFIETKKFL LIGKSNICVK VGEKSLTCKK IDLTTIVKPE APFDLSVIYR EGANDFVVTF NTSHLQKKYV KVLMHDVAYR QEKDENKWTH VNLSSTKLTL LQRKLQPAAM YEIKVRSIPD HYFKGFWSEW SPSYYFRTPE INNSSGEMDP ILLTISILSF FSVALLVILA CVLWKKRIKP IVWPSLPDHK KTLEHLCKKP RKNLNVSFNP ESFLDCQIHR VDDIQARDEV EGFLQDTFPQ QLEESEKQRL GGDVQSPNCP SEDWITPES FGRDSSLTCL AGNVSACDAP ILSSSRSLDC RESGKNGPHV YQDLLLSLGT TNSTLPPPFS LQSGILTLNP VAQGQPILTS LGSNQEEAYV TMSSFYQNQ (SEQ ID NO: 1 )
Antagonist IL-7R antibodies encompass antibodies that block, antagonize, suppress or reduce (to any degree including significantly) IL-7R biological activity, including downstream pathways mediated by IL-7R signaling, such interaction with IL-7 and/or elicitation of a cellular response to IL-7. For purpose of the present invention, it will be explicitly understood that the term "antagonist IL-7R antibody" (interchangeably termed "IL-7R antagonist antibody," "antagonist anti-IL-7R antibody" or "anti-IL-7R antagonist antibody") encompasses all the previously identified terms, titles, and functional states and characteristics whereby the IL-7R itself, an IL-7R biological activity (including but not limited to interaction with IL-7, its ability to mediate any aspect of phosphorylation of STAT5, phosphatidylinositol-3-kinase (PI3K)-Akt pathway activation, p27Kip1 downregulation, Bcl-2 upregulation, Rb hyperphosphorylation, and CXCR4
upregulation), or the consequences of the biological activity, are substantially nullified, decreased, or neutralized in any meaningful degree. In some embodiments, an antagonist IL-7R antibody binds IL-7R and prevents interaction with IL-7. Examples of antagonist IL-7R antibodies are provided herein. Anti-IL-7R antagonist antibodies for use in the invention can be identified or characterized using methods known in the art, whereby reduction, amelioration, or neutralization of an IL-7R biological activity is detected and/or measured. As used herein, the term "C1 GM" is used to refer to an antibody comprising the amino acid sequence of the heavy chain and light chain variable regions shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
C1 GM heavy chain variable region:
EVQLVESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEVWSLVGWDG FFTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGDYMGNNWGQGT LVTVSS (SEQ ID NO: 2)
C1 GM light chain variable region:
NFMLTQPHSVSESPGKTVTISCTRSSGSIDSSYVQWYQQRPGSSPTTVIYEDDQRPSG VPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDFHHLVFGGGTKLTVL (SEQ ID NO: 3)
The generation and characterization of C1 GM is described in the Examples of
WO201 1/104687, the entire content of which is herein incorporated by reference in its entirety for all purposes. In some embodiments, the term "C1 GM" refers to
immunoglobulin encoded by (a) a polynucleotide encoding C1 GM light chain variable region that has a deposit number of ATCC No. PTA-1 1678, and (b) a polynucleotide encoding C1 GM heavy chain variable region that has a deposit number of ATCC No. PTA-1 1679. In some embodiments, the antibody is an anti-IL-7R antibody that binds IL-7Ra (such as human IL-7Ra) with a high affinity. In some embodiments, high affinity is (a) binding IL- 7R with a KD of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less).
In some embodiments, antibodies (a) bind IL-7R (such as human IL-7R) with a KD of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less), and/or a k0ff of about 4x10"4 s' The epitope(s) that can be bound by the antibody can be continuous or discontinuous. In one embodiment, the antibody binds essentially the same IL-7R epitope as antibody C1 GM.
In some embodiments, the antibody can be anti-IL-7R antibody comprising a heavy chain variable region comprising: (a) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4
(GFTFDDSVMH) (extended) or in SEQ ID NO: 5 (DSVMH) (Kabat) or in SEQ ID NO: 6 (GFTFDDS) (Chothia);
(b) a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7
(LVGWDGFFTYYADSVKG) (Kabat) or in SEQ ID NO: 8 (GWDGFF) (Chothia); and (c) a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9
(QGDYMGNN).
In some embodiments, the antibody can be an anti-IL-7R antibody comprising a light chain variable region comprising:
(a) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10
(TRSSGSIDSSYVQ);
(b) a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 1 1
(EDDQRPS); and
(c) a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12
(QSYDFHHLV). In some embodiments, the antibody can be anti-IL-7R antibody comprising three CDRs from a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2.
EVQLVESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEVWSLVGWDG FFTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGDYMGNNWGQGT LVTVSS (SEQ ID NO: 2)
In some embodiments, the antibody can be anti-IL-7R antibody comprising three CDRs from a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 3. NFMLTQPHSVSESPGKTVTISCTRSSGSIDSSYVQWYQQRPGSSPTTVIYEDDQRPSG VPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDFHHLVFGGGTKLTVL (SEQ ID NO: 3)
In some embodiments, the anti-IL-7R antibody may comprise a heavy chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 2 and/or a light chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 3, wherein the antibody binds specifically to human IL-7Ra.
The anti-IL-7R antibody may comprise a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 2 and/or may comprise a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 3. The anti-IL-7R antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 2 and 3.
The anti-IL-7R antibody may comprise a heavy chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 13 and / or a light chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%), 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 14, wherein the antibody binds specifically to human IL-7Ra.
Heavy chain region sequence
EVQLVESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEVWSLVGWDG FFTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGDYMGNNWGQGT LVTVS S ASTKG P SVF P LAP S S KSTS G GTAALG C LVKDYF P E P VTVS WN S G ALTS G VHT FPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVAPELLGGPSVFLF PPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13)
Light chain region sequence
NFMLTQPHSVSESPGKTVTISCTRSSGSIDSSYVQWYQQRPGSSPTTVIYEDDQRPSG VPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDFHHLVFGGGTKLTVLQPKAAPS VTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKY AASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 14)
The anti-IL-7R antibody may comprise a heavy chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 13 and/or may comprise a light chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 14.
The anti-IL-7R antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 13 and 14.
The anti-IL-7R antibody may compete for IL-7R binding with an anti-IL-7R antibody as defined herein. The anti-IL-7R antibody may compete for IL-7R binding with an antibody comprising a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 2 and/or a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 3. The anti-IL-7R antibody may be a human and affinity matured antibody, C1 GM, which specifically binds human IL-7Ra. Antibody C1 GM is described in WO2011/104687, the content of which is hereby incorporated by reference in its entirety. The amino acid sequences of the heavy chain and light chain variable regions of C1 GM are shown in SEQ ID NOs: 2 and 3, respectively. The CDR portions of antibody C1 GM (including Chothia and Kabat CDRs) are diagrammatically depicted in Table 1 of
WO201 1/104687. Antibody C1 GM is highly potent in blocking IL-7R biological activity.
The anti-IL-7R antibody may also comprise a fragment or a region of the antibody C1 GM. In one embodiment, the fragment is a light chain of the antibody C1 GM comprising the amino acid sequence as shown in SEQ ID NO: 14 herein. In another embodiment, the fragment is a heavy chain of the antibody C1 GM comprising the amino acid sequence as shown in SEQ ID NO: 13 herein. In yet another embodiment, the fragment contains one or more variable regions from a light chain and/or a heavy chain of the antibody C1 GM. In yet another embodiment, the fragment contains one or more CDRs from a light chain and/or a heavy chain of the antibody C1 GM comprising the amino acid sequences as shown in SEQ ID NOS: 14 and 13, respectively, herein.
In some embodiments, the antibody may comprise any one or more of the following: a) one or more (one, two, three, four, five, or six) CDR(s) derived from antibody C1 GM shown in SEQ ID NOs: 4-12. In some embodiments, the CDRs may be Kabat CDRs, Chothia CDRs, or a combination of Kabat and Chothia CDRs (termed "extended" or "combined" CDRs herein). In some embodiments, the polypeptides comprise any of the CDR configurations (including combinations, variants, etc.) described herein.
In some embodiments of the present invention the C-terminal lysine of the heavy chain of any of the anti-IL-7R antibodies described herein is deleted. In various cases the heavy and/or light chain of the anti-IL-7R antibodies described herein may optionally include a signal sequence.
In another embodiment, the antibody may be selected from an anti-IL-7R antibody known in the art, such as antibodies described in, for example without limitation, any of the following published PCT applications: WO201 1/104687 (including, for example without limitation, any of the antibodies listed in Table 1 ), WO/201 1/094259 (including, for example without limitation, antibodies H3L4, BPC4401 , BPC4398, BPC1 142, BPC4399, BPC4402, BPC4403, and BPC1 142), WO/2013/056984 (including, for example without limitation, antibodies MD707-1 , MD707-2, MD707-3, MD707-4, MD707-5, MD707-6, MD707-9, MD707-12, and MD707-13), and WO2010/017468 (including, for example without limitation, antibodies 9B7, R34.34, 6A3 and 1A11 ). The antibody may bind to the same epitope as an anti-IL-7R antibody known in the art and/or may compete for binding to IL-7R with such an antibody.
Anti-Glucagon Receptor
As used herein, the term "Glucagon Receptor" refers to any form of glucagon receptor and variants thereof that retain at least part of the activity of the glucagon receptor. Unless indicated differently, such as by specific reference to human glucagon receptor, glucagon receptor includes all mammalian species of native sequence glucagon receptor, e.g., human, canine, feline, equine, and bovine. One exemplary human glucagon receptor is found as Uniprot Accession Number P47871 (SEQ ID NO: 29).
MPPCQPQRPLLLLLLLLACQPQVPSAQVMDFLFEKWKLYGDQCHHNLSLLPPPTELVC
NRTFDKYSCWPDTPANTTANISCPWYLPWHHKVQHRFVFKRCGPDGQWVRGPRGQ
PWRDASQCQMDGEEIEVQKEVAKMYSSFQVMYTVGYSLSLGALLLALAILGGLSKLHC
TRNAIHANLFASFVLKASSVLVIDGLLRTRYSQKIGDDLSVSTWLSDGAVAGCRVAAVF
MQYGIVANYCWLLVEGLYLHNLLGLATLPERSFFSLYLGIGWGAPMLFVVPWAVVKCL
FENVQCVVTSNDNMGFVWVILRFPVFLAILINFFIFVRIVQLLVAKLRARQMHHTDYKFRL
AKSTLTLIPLLGVHEWFAFVTDEHAQGTLRSAKLFFDLFLSSFQGLLVAVLYCFLNKEV
QSELRRRWHRWRLGKVLWEERNTSNHRASSSPGHGPPSKELQFGRGGGSQDSSAE
TPLAGGLPRLAESPF (SEQ ID NO: 29)
As used herein, an "anti-glucagon receptor antagonist antibody" refers to an antibody that is able to inhibit glucagon receptor biological activity and/or downstream events(s) mediated by glucagon receptor. Anti-glucagon receptor antagonist antibodies encompass antibodies that block, antagonize, suppress or reduce (to any degree including significantly) glucagon receptor biological activity, including downstream events mediated by glucagon receptor, such glucagon binding and downstream signaling, adenylate cyclase activation, increased levels of intracellular cAMP, glycogenolysis stimulation, gluconeogenesis activation, glycogenesis inhibition, glycolysis inhibition, and hepatic glucose production. For purposes of the present invention, it will be explicitly understood that the term "anti-glucagon receptor antagonist antibody" (interchangeably termed "antagonist glucagon receptor antibody", "antagonist anti-glucagon receptor antibody" or "glucagon receptor antagonist antibody") encompasses all the previously identified terms, titles, and functional states and characteristics whereby the glucagon receptor itself, a glucagon receptor biological activity (including but not limited to its ability to bind glucagon, increase intracellular cAMP, stimulate glycogenolysis, activate gluconeogenesis, and promote relase of hepatic glucose), or the consequences of the biological activity, are substantially nullified, decreased, or neutralized in any meaningful degree. In some embodiments, an anti-glucagon receptor antagonist antibody binds glucagon receptor and lowers plasma glucose levels. Examples of anti-glucagon receptor antagonist antibodies are provided herein.
As used herein, the term "mAb5" is used to refer to an antibody comprising the amino acid sequence of the heavy chain and light chain variable regions shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively. mAb5 heavy chain variable region:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDFSVHWVRQAPGQGLEWMGWINTETD ETSYAD D FKG RVTMTRDTSTSTVYM ELSS LRS EDTAVYYCVKS RYWSYGP P DYWGQ GTTVTVSS (SEQ ID NO: 15) mAb5 light chain variable region:
DIQMTQSPSSLSASVGDRVTITCQASQNIRTAWWYQQKPGKAPKLLIYLASNRHSGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCLQHWTYPFTFGGGTKVEIK (SEQ ID NO: 16) The generation and characterization of mAb5 is described in the Examples of
WO2014/181229, the entire content of which is herein incorporated by reference in its entirety for all purposes. In some embodiments, the term "mAb5" refers to
immunoglobulin encoded by (a) a polynucleotide encoding mAb5 light chain variable region that has a deposit number of ATCC No. PTA-120164 and (b) a polynucleotide encoding mAb5 heavy chain variable region that has a deposit number of ATCC No. PTA-120165.
In some embodiments, the antibody is an anti- glucagon receptor antibody that binds glucagon receptor (such as human glucagon receptor) with a high affinity. In some embodiments, high affinity is (a) binding IL-7R with a KD of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less). In some embodiments, antibodies (a) bind glucagon receptor (such as human glucagon receptor) with a KD of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less), and/or a k0ff Of about 4x10"4 s' The epitope(s) that can be bound by the antibody can be continuous or discontinuous. In one embodiment, the antibody binds essentially the same glucagon receptor epitope as antibody mAb5.
In some embodiments, the antibody can be anti- glucagon receptor antibody comprising a heavy chain variable region comprising: (a) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17
(GYTFTDFSVH) (extended) or in SEQ ID NO: 18 (GYTFTDF) (Chothia) or in SEQ ID NO: 19 (DFSVH) (Kabat);
(b) a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 20 (NTETDE) (Chothia) or in SEQ ID NO: 21 (WINTETDETSYADDFKG) (Kabat); and (c) a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 22
(SRYWSYGPPDY).
In some embodiments, the antibody can be an anti-glucagon receptor antibody comprising a light chain variable region comprising:
(a) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 23
(QASQNIRTAVV);
(b) a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 24 (LASNRHS); and
(c) a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 25
(LQHWTYPFT). In some embodiments, the antibody can be anti-glucagon receptor antibody comprising three CDRs from a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 15. QVQLVQSGAEVKKPGASVKVSCKASGYTFTDFSVHWVRQAPGQGLEWMGWINTETD ETSYAD D FKG RVTMTRDTSTSTVYM ELSS LRS EDTAVYYCVKS RYWSYGP P DYWGQ GTTVTVSS (SEQ ID NO: 15)
In some embodiments, the antibody can be anti-glucagon receptor antibody comprising three CDRs from a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 16.
DIQMTQSPSSLSASVGDRVTITCQASQNIRTAWWYQQKPGKAPKLLIYLASNRHSGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCLQHWTYPFTFGGGTKVEIK (SEQ ID NO: 16) In some embodiments, the anti-glucagon receptor antibody may comprise a heavy chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 15 and/or a light chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 16, wherein the antibody binds specifically to human glucagon receptor.
The anti-glucagon receptor antibody may comprise a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 15 and/or may comprise a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 16.
The anti-glucagon receptor antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 15 and 16.
The anti-glucagon receptor antibody may comprise a heavy chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 26 and / or a light chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 28, wherein the antibody binds specifically to human glucagon receptor. The amino acid sequence of mAb5 full-length heavy chain (SEQ ID NO: 26) is shown below:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDFSVHVWRQAPGQGLEWMGWINTETD
ETSYADDFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCVKSRYWSYGPPDYWGQ
GTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPP
CPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHN
AKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPR
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:
26)
The amino acid sequence of mAb5 full-length heavy chain without the C-terminal lysine (SEQ ID NO: 27) is shown below:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDFSVHWVRQAPGQGLEWMGWINTETD ETSYAD D FKG RVTMTRDTSTSTVYM ELSS LRS EDTAVYYCVKS RYWSYGP P DYWGQ GTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPP CPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 88) (SEQ ID NO: 27)
The amino acid sequence of mAb5 full-length light chain (SEQ ID NO: 28) is shown below:
DIQMTQSPSSLSASVGDRVTITCQASQNIRTAWWYQQKPGKAPKLLIYLASNRHSGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCLQHWTYPFTFGGGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 28)
The anti-glucagon receptor antibody may comprise a heavy chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 26 and/or may comprise a light chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 28. The anti-glucagon receptor antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 26 and 28.
The anti-glucagon receptor antibody may compete for glucagon receptor binding with an anti-glucagon receptor antibody as defined herein. The anti-glucagon receptor antibody may compete for glucagon receptor binding with an antibody comprising a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 15 and/or a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 16.
The anti-glucagon receptor antibody may be a human and affinity matured antibody, mAb5, which specifically binds human glucagon receptor. Antibody mAb5 is described in WO2014/181229, the content of which is hereby incorporated by reference in its entirety for all purposes. The amino acid sequences of the heavy chain and light chain variable regions of mAb5 are shown in SEQ ID NOs: 15 and 16, respectively. The CDR portions of antibody mAb5 (including Chothia and Kabat CDRs) are diagrammatically depicted in Table 1 A of WO2014/181229. Antibody mAb5 is highly potent in blocking glucagon receptor biological activity.
The anti-glucagon receptor antibody may also comprise a fragment or a region of the antibody mAb5. In one embodiment, the fragment is a light chain of the antibody mAb5 comprising the amino acid sequence as shown in SEQ ID NO: 28 herein. In another embodiment, the fragment is a heavy chain of the antibody mAb5 comprising the amino acid sequence as shown in SEQ ID NO: 26 herein. In yet another embodiment, the fragment contains one or more variable regions from a light chain and/or a heavy chain of the antibody mAb5. In yet another embodiment, the fragment contains one or more CDRs from a light chain and/or a heavy chain of the antibody mAb5 comprising the amino acid sequences as shown in SEQ ID NOS: 28 and 26, respectively, herein.
In some embodiments, the antibody may comprise any one or more of the following: a) one or more (one, two, three, four, five, or six) CDR(s) derived from antibody mAb5 shown in SEQ ID NOs: 17-25. In some embodiments, the CDRs may be Kabat CDRs, Chothia CDRs, or a combination of Kabat and Chothia CDRs (termed "extended" or "combined" CDRs herein). In some embodiments, the polypeptides comprise any of the CDR configurations (including combinations, variants, etc.) described herein. In some embodiments of the present invention the C-terminal lysine of the heavy chain of any of the anti-glucagon receptor antibodies described herein is deleted, as in SEQ ID NO. 27. In various cases the heavy and/or light chain of the anti-glucagon receptor antibodies described herein may optionally include a signal sequence. In another embodiment, the antibody may be selected from an anti-glucagon receptor antibody known in the art, such as antibodies described in, for example without limitation, any of the following published PCT applications: WO2014/181229 (including, for example without limitation, any of the antibodies listed in Tables 1 A and 1 B including, for example without limitation antibodies mAb1 , mAb2, mAb3, mAb4, mAb5, mAb6, H2-A8, H2-A1 1 , H2-C8, H2-E7, H2-F10, H2-F11 , H3-C5, H3-C10, H3-F5, H3- H9, H2-A1 1 -H3-1 , H2-A1 1 -H3-2, H2-A1 1 -H3-3, H2-A1 1 -H3-4, H2-C8-H3-1 , H2-C8-H3- 2, H2-C8-H3-3, H2-C8-H3-4, H2-E7-H3-1 , H2-E7-H3-2, H2-E7-H3-3, H2-E7-H3-4, FF1 , FF2, FF3, FF4, FF2-H2WT, FF2-H2RG, FF2-H3RY, and FF2-H2WT-H3RY);
WO2012/071372; WO201 1 /030935; WO2013/059531 ; and WO2013/081993. The antibody may bind to the same epitope as an anti-glucagon receptor antibody known in the art and/or may compete for binding to glucagon receptor with such an antibody.
PCSK9
As used herein, the term "PCSK9" refers to any form of PCSK9 and variants thereof that retain at least part of the activity of PCSK9. Unless indicated differently, such as by specific reference to human PCSK9, PCSK9 includes all mammalian species of native sequence PCSK9, e.g., human, canine, feline, equine, and bovine. One exemplary human PCSK9 is found as Uniprot Accession Number Q8NBP7. (SEQ ID NO: 43)
MGTVSSRRSW WPLPLLLLLL LLLGPAGARA QEDEDGDYEE LVLALRSEED GLAEAPEHGT TATFHRCAKD PWRLPGTYW VLKEETHLSQ SERTARRLQA QAARRGYLTK ILHVFHGLLP GFLVKMSGDL LELALKLPHV DYIEEDSSVF AQSIPWNLER ITPPRYRADE YQPPDGGSLV EVYLLDTSIQ SDHREIEGRV MVTDFENVPE EDGTRFHRQA SKCDSHGTHL AGWSGRDAG VAKGASMRSL RVLNCQGKGT VSGTLIGLEF IRKSQLVQPV GPLWLLPLA GGYSRVLNAA CQRLARAGW LVTAAGNFRD DACLYSPASA PEVITVGATN AQDQPVTLGT LGTNFGRCVD LFAPGEDIIG ASSDCSTCFV SQSGTSQAAA HVAGIAAMML SAEPELTLAE LRQRLIHFSA KDVINEAWFP EDQRVLTPNL VAALPPSTHG AGWQLFCRTV WSAHSGPTRM ATAVARCAPD EELLSCSSFS RSGKRRGERM EAQGGKLVCR AHNAFGGEGV YAIARCCLLP QANCSVHTAP PAEASMGTRV HCHQQGHVLT GCSSHWEVED LGTHKPPVLR PRGQPNQCVG HREASIHASC CHAPGLECKV KEHGIPAPQE QVTVACEEGW TLTGCSALPG TSHVLGAYAV DNTCWRSRD VSTTGSTSEG AVTAVAICCR SRHLAQASQE LQ (SEQ ID NO: 43)
As used herein, a "PCSK9 antagonist" refers to an antibody, peptide, or aptamer that is able to inhibit PCSK9 biological activity and/or downstream pathway(s) mediated by PCSK9 signaling, including PCSK9-mediated down-regulation of the LDLR, and
PCSK9-mediated decrease in LDL blood clearance. A PCSK9 antagonist antibody encompasses antibodies that block, antagonize, suppress or reduce (to any degree including significantly) PCSK9 biological activity, including downstream pathways mediated by PCSK9 signaling, such as LDLR interaction and/or elicitation of a cellular response to PCSK9. For purpose of the present invention, it will be explicitly
understood that the term "PCSK9 antagonist antibody" encompasses all the previously identified terms, titles, and functional states and characteristics whereby the PCSK9 itself, a PCSK9 biological activity (including but not limited to its ability to mediate any aspect of interaction with the LDLR, down regulation of LDLR, and decreased blood
LDL clearance), or the consequences of the biological activity, are substantially nullified, decreased, or neutralized in any meaningful degree. In some embodiments, a PCSK9 antagonist antibody binds PCSK9 and prevents interaction with the LDLR. Examples of PCSK9 antagonist antibodies are provided herein. As used herein, the term "L1 L3" is used to refer to an antibody comprising the amino acid sequence of the heavy chain and light chain variable regions shown in SEQ ID NO: 41 and SEQ ID NO: 42, respectively.
L1 L3 heavy chain variable region:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMH WRQAPGQGLEWMGEISPFG GRTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARERPLYASDLWGQGT TVTVSS (SEQ ID NO: 41 )
L1 L3 light chain variable region:
DIQMTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYSASYRYTGVP SRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRTFGQGTKLEIK (SEQ ID NO: 42) The generation and characterization of L1 L3 is described in the Examples of
WO2010/029513, the entire content of which is herein incorporated by reference in its entirety for all purposes. In some embodiments, the term "L1 L3" refers to
immunoglobulin encoded by (a) a polynucleotide encoding L1 L3 light chain variable region that has a deposit number of ATCC No. PTA-10303 and (b) a polynucleotide encoding L1 L3 heavy chain variable region that has a deposit number of ATCC No. PTA-10302.
In some embodiments, the antibody is an anti-PCSK9 antibody that binds PCSK9 receptor (such as human PCSK9 receptor) with a high affinity. In some embodiments, high affinity is (a) bindingPCSK9 receptor with a KD of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less).
In some embodiments, antibodies (a) bind PCSK9 receptor (such as human PCSK9 receptor) with a KD of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30pM, 20pM, 10pM, 5pM or less), and/or a koff of about 4x10"4 s'
The epitope(s) that can be bound by the antibody can be continuous or discontinuous. In one embodiment, the antibody binds essentially the same PCSK9 receptor epitope as antibody L1 L3. In some embodiments, the antibody can be anti-PCSK9 receptor antibody comprising a heavy chain variable region comprising:
(a) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 32
(GYTFTSYYMH) (extended) or in SEQ ID NO: 33 (GYTFTSY) (Chothia) or in SEQ ID NO: 34 (SYYMH) (Kabat); (b) a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 35
(EISPFGGRTNYNEKFKS) (Kabat) or in SEQ ID NO: 36 (SPFGGR) (Chothia); and
(c) a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 37
(ERPLYASDL).
In some embodiments, the antibody can be an anti-PCSK9 receptor antibody
comprising a light chain variable region comprising: (a) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 38 (RASQGISSALA);
(b) a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 39 (SASYRYT); and (c) a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 40
(QQRYSLWRT).
In some embodiments, the antibody can be anti-PCSK9 antibody comprising three CDRs from a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 41. QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMH WRQAPGQGLEWMGEISPFG GRTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARERPLYASDLWGQGT TVTVSS (SEQ ID NO: 41 )
In some embodiments, the antibody can be anti-PCSK9 receptor antibody comprising three CDRs from a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 42.
DIQMTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYSASYRYTGVP SRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRTFGQGTKLEIK (SEQ ID NO: 42)
In some embodiments, the anti-PCSK9 receptor antibody may comprise a heavy chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 41 and/or a light chain variable region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 42, wherein the antibody binds specifically to human PCSK9 receptor.
The anti-PCSK9 receptor antibody may comprise a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 41 and/or may comprise a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 42. The anti-PCSK9 receptor antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 41 and 42.
The anti-PCSK9 receptor antibody may comprise a heavy chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 30 and / or a light chain region comprising an amino acid sequence of any of at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 31 , wherein the antibody binds specifically to human PCSK9 receptor.
The amino acid sequence of L1 L3 full-length heavy chain (SEQ ID NO: 30) is shown below:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEISPFG
GRTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARERPLYASDLWGQGT
TVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSWTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC
PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 30)
The amino acid sequence of L1 L3 full-length light chain (SEQ ID NO: 31 ) is shown below:
DIQMTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYSASYRYTGVP SRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRTFGQGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 31 )
The anti- PCSK9 receptor antibody may comprise a heavy chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 30 and/or may comprise a light chain region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 31 . The anti-PCSK9 receptor antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 30 and 31.
The anti-PCSK9 receptor antibody may compete for PCSK9 receptor binding with an anti- PCSK9 receptor antibody as defined herein. The anti- PCSK9 receptor antibody may compete for PCSK9 receptor binding with an antibody comprising a heavy chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 41 and/or a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 42.
The anti-PCSK9 receptor antibody may be a human and affinity matured antibody, L1 L3, which specifically binds human PCSK9 receptor. Antibody L1 L3 is described in WO2010/029513, the content of which is hereby incorporated by reference in its entirety for all purposes. The amino acid sequences of the heavy chain and light chain variable regions of L1 L3 are shown in SEQ ID NOs: 41 and 42, respectively. The CDR portions of antibody L1 L3 (including Chothia and Kabat CDRs) are diagrammatically depicted in Table 7 of WO2010/029513. Antibody L1 L3 is highly potent in blocking PCSK9 receptor biological activity.
The anti-PCSK9 receptor antibody may also comprise a fragment or a region of the antibody L1 L3. In one embodiment, the fragment is a light chain of the antibody L1 L3 comprising the amino acid sequence as shown in SEQ ID NO: 31 herein. In another embodiment, the fragment is a heavy chain of the antibody L1 L3 comprising the amino acid sequence as shown in SEQ ID NO: 30 herein. In yet another embodiment, the fragment contains one or more variable regions from a light chain and/or a heavy chain of the antibody L1 L3. In yet another embodiment, the fragment contains one or more CDRs from a light chain and/or a heavy chain of the antibody L1 L3 comprising the amino acid sequences as shown in SEQ ID NOS: 31 and 30, respectively, herein.
In some embodiments, the antibody may comprise any one or more of the following: a) one or more (one, two, three, four, five, or six) CDR(s) derived from antibody L1 L3 shown in SEQ ID NOs: 32-40. In some embodiments, the CDRs may be Kabat CDRs, Chothia CDRs, or a combination of Kabat and Chothia CDRs (termed "extended" or "combined" CDRs herein). In some embodiments, the polypeptides comprise any of the CDR configurations (including combinations, variants, etc.) described herein. In some embodiments of the present invention the C-terminal lysine of the heavy chain of any of the anti-PCSK9 receptor antibodies described herein is deleted. In various cases the heavy and/or light chain of the anti-PCSK9 receptor antibodies described herein may optionally include a signal sequence. In some embodiments, the anti-PCSK9 receptor antibody is alirocumab
(PRALUENT™); evolocumab (REPATHA™); REGN728; LGT209; RG7652;
LY3015014; J16, L1 L3 (bococizumab); 31 H4, 1 1 F1 , 12H1 1 , 8A3, 8A1 , or 3C4 (see, e.g., US8,030,457); 300N (see, e.g., US8,062,640); or 1 D05 (see, e.g., US8, 188,234). In some embodiments, the anti-PCSK9 antibody is bococizumab, alirocumab
(PRALUENT™), or evolocumab (REPATHA™). The antibody may bind to the same epitope as an anti-PCSK9 receptor antibody known in the art and/or may compete for binding to PCSK9 receptor with such an antibody.
Preparation of the Formulations
In preparing the compositions of the present invention, the antibody and
camphorsulfonic acid or sulfosalicyclic acid are mixed together and the pH of the mixture is measured and if necessary, adjusted with the use of a buffer or basic component. Other optional components may also be added to the mixture including one or more surfactants, chelating agents, and cryopretectants. Following mixture of the entire composition, the formulation may be utilized in the liquid state, or lyophilized. Many different freeze-dryers are available for this purpose such as Hull50.TM. (Hull, USA) or GT20.TM. (Leybold-Heraeus, Germany) freeze-dryers. Freeze-drying is accomplished by freezing the formulation and subsequently subliming ice from the frozen content at a temperature suitable for primary drying. Under this condition, the product temperature is below the eutectic point or the collapse temperature of the formulation. Typically, the shelf temperature for the primary drying will range from about -30 to 25°C (provided the product remains frozen during primary drying) at a suitable pressure, ranging typically from about 50 to 250 mTorr. The formulation, size and type of the container holding the sample (e.g., glass vial) and the volume of liquid will mainly dictate the time required for drying, which can range from a few hours to several days (e.g. 40-60 hrs). Optionally, a secondary drying stage may also be performed depending upon the desired residual moisture level in the product. The temperature at which the secondary drying is carried out ranges from about 0-4OC, depending primarily on the type and size of container and the type of protein employed. For example, the shelf temperature throughout the entire water removal phase of lyophilization may be from about 15-30C (e.g., about 20°C). The time and pressure required for secondary drying will be that which produces a suitable lyophilized cake, dependent, e.g., on the temperature and other parameters. The secondary drying time is dictated by the desired residual moisture level in the product and typically takes at least about 5 hours (e.g. 10-15 hours). The pressure may be the same as that employed during the primary drying step. Freeze-drying conditions can be varied depending on the formulation and vial size.
The formulations described herein may also be prepared as reconstituted lyophilized formulations. The compositions described herein are lyophilized and then reconstituted to produce the reduced-viscosity stable liquid formulations of the invention. In this particular embodiment, after preparation of the antibody of interest as described above, a "pre-lyophilized formulation" is produced. The amount of antibody present in the pre- lyophilized formulation is determined taking into account the desired dose volumes and mode(s) of administration. For example, the concentration of an antibody in the pre- lyophilized formulation and in the reconstituted formulation can be as described previously, and may differ such that the reconstituted formulation may have an increased antibody concentration as compared to the pre-lyophilized formulation. A "reconstituted" formulation is one which has been prepared by dissolving a lyophilized formulation in a diluent such that the antibody is distributed throughout the reconstituted formulation. The reconstituted formulation is suitable for administration (e.g. parenteral administration) to a patient to be treated with the antibody of interest and, in certain embodiments of the invention, may be one which is suitable for subcutaneous administration.
The "diluent" of interest herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, such as a formulation reconstituted after lyophilization. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI or WFI), a pH buffered solution (e.g. phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution. In an alternative embodiment, diluents can include aqueous solutions of salts and/or buffers and/or surfactants. Reconstitution generally takes place at a temperature of about 25°C to ensure complete hydration, although other temperatures may be employed as desired. The time required for reconstitution will depend, e.g., on the type of diluent, amount of excipient(s) and protein. In one embodiment, the reconstituted formulation has less than 3000 particles per vial which are less than 10 pm per vial and 300 particles per vial which are less than 25 pm per vial for vials containing less than 100 ml. of solution.
Route of Administration
The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g.
injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with antibodies in general. One mode of administration is parenteral (e.g. intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes).
Another aspect of the present invention provides kits comprising antibodies of the invention and pharmaceutical compositions comprising these antibodies. A kit may include, in addition to the antibody or pharmaceutical composition, diagnostic or therapeutic agents. A kit may also include instructions for use in a diagnostic or therapeutic method. In some embodiments, the kit includes the antibody or a
pharmaceutical composition thereof and a diagnostic agent. In other embodiments, the kit includes the antibody or a pharmaceutical composition thereof and one or more therapeutic agents, such as an additional antineoplastic agent, anti-tumor agent or chemotherapeutic agent.
Liposomes containing compounds of the invention are prepared by methods known in the art, such as described in US4485045 and US4544545. Liposomes with enhanced circulation time are disclosed in US5013556. 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. Suitable emulsions may be prepared using commercially available fat emulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%. The fat emulsion can comprise fat droplets between 0.1 and Ι .Ομηη, particularly 0.1 and 0.5pm, and have a pH in the range of 5.5 to 8.0.
The emulsion compositions can be those prepared by mixing a compound of the invention with Intralipid™ or the components thereof (soybean oil, egg phospholipids, glycerol and water). The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interracial polymerization, for example,
hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing (2000).
Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, 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 (US3773919), copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3- hydroxybutyric acid.
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes. Therapeutic compounds of the invention are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulised by use of gases. Nebulised solutions may be breathed directly from the nebulising device or the nebulising device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner. Compositions of the invention may be used in conjunction with established treatments for the relevant indication.
Methods of using proteins of the invention
The present invention also provides various therapeutic applications for the
compositions of the invention. In one aspect, the compositions of the invention can be used for treating various diseases (e.g. cancer, autoimmune diseases, or viral infections) by binding the first protein (e.g. first human antibody variable domain) to an effector antigen and by binding the second protein (e.g. second human antibody variable domain) to a target antigen. For example, the compositions of the invention can be used for redirecting cytotoxicity, delivering thrombolytic agents to clots, for delivering immunotoxins to tumor cells, or for converting enzyme activated prodrugs at a target site (e.g. a tumor).
In another aspect, the compositions of the invention can be used for increasing specificity of a therapeutic agent and/or modulating synergistic or additive pathways (e.g. metabolic or biochemical pathways). For example, the compositions of the invention can engage receptor/receptor, receptor/ligand, ligand/ligand, cell/cell, ligand/payload, receptor/payload, or single receptor. Dosages and desired drug concentration of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. "The Use of
Interspecies Scaling in Toxicokinetics," In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp. 42-46. When in vivo administration of the polypeptides or antibodies described herein are used, normal dosage amounts may vary from about 10 ng/kg up to about 100 mg/kg of mammal body weight or more per day, preferably about 1 mg/kg/day to 10 mg/kg/day, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature; see, for example, U.S. Pat. Nos.
4,657,760; 5,206,344; or 5,225,212. It is within the scope of the invention that different formulations will be effective for different treatments and different disorders, and that administration intended to treat a specific organ or tissue may necessitate delivery in a manner different from that to another organ or tissue. Moreover, dosages may be administered by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs.
However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. The formulations of the present invention, including but not limited to reconstituted formulations, are administered to a mammal in need of treatment with the protein, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular,
intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
In some embodiments, the formulations are administered to the mammal by
subcutaneous (i.e. beneath the skin) administration. For such purposes, the formulation may be injected using a syringe. However, other devices for administration of the formulation are available such as injection devices (e.g. the Inject-ease.TM. and Genject.TM. devices); injector pens (such as the GenPen.TM.); auto-injector devices, needleless devices (e.g. MediJector.TM. and BioJector.TM.); and subcutaneous patch delivery systems. In another embodiment of the invention, an article of manufacture is provided which contains the formulation and preferably provides instructions for its use. The article of manufacture comprises a container. Suitable containers include, for example, bottles, vials (e.g. dual chamber vials), syringes (such as single or dual chamber syringes) and test tubes. The container may be formed from a variety of materials such as glass or plastic. The container holds the formulation and the label on, or associated with, the container may indicate directions for reconstitution and/or use. The label may further indicate that the formulation is useful or intended for subcutaneous administration. The container holding the formulation may be a multi-use vial, which allows for repeat administrations (e.g. from 2-6 administrations) of the reconstituted formulation. The article of manufacture may further comprise a second container comprising a suitable diluent (e.g. BWFI). Upon mixing of the diluent and the lyophilized formulation, the final protein concentration in the reconstituted formulation will generally be at least 50 mg/ml. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
Other methods for administration of the compound of the present invention described herein include dermal patches that release the medications directly into a subject's skin. Such patches can contain the compound of the present invention in an optionally buffered, liquid solution, dissolved and/or dispersed in an adhesive, or dispersed in a polymer.
The compound may be administered once daily, but may also be administered multiple times. For example, the compound may be administered from once daily to once every six months or longer. The administering may be on a schedule such as three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once every month, once every two months, once every three months and once every six months.
The compound may also be administered continuously via a minipump. The compound may be administered at the site of the diseased body part or at a site distant from the site of the diseased body part. The compound may be administered once, at least twice or for at least the period of time until the disease is treated, palliated or cured. The compound generally may be administered for as long as the disease is present. The compound typically would be administered as part of a pharmaceutical composition as described supra.
Exemplary, non-limiting dose ranges for administration of the pharmaceutical compositions of the present invention to a subject are from about 0.01 mg/kg to about 200 mg/kg (expressed in terms of milligrams (mg) of the antibody administered per kilogram (kg) of subject weight), from about 0.1 mg/kg to about 100 mg/kg, from about 1.0 mg/kg to about 50 mg/kg, from about 5.0 mg/kg to about 20 mg/kg, or about 15 mg/kg. For purposes of the present invention, an average human subject weighs about 70 kg. Ranges intermediate to any of the dosages cited herein, e.g., about 0.02 mg/kg - 199 mg/kg, are also intended to be part of this invention. For example, ranges of values using a combination of any of the recited values as upper and/or lower limits are intended to be included.
Dosage regimens can also be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response) by administering several divided doses to a subject over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the compound or portion and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an antibody for the treatment of sensitivity in individuals.
The liquid compositions of the present invention can be prepared as unit dosage forms. For example, a unit dosage per vial may contain from 1 to 1000 milliliters (mis) of different concentrations of the compound of Formula (A). In other embodiments, a unit dosage per vial may contain about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, 20 ml, 30 ml, 40 ml, 50 ml or 100 ml of different concentrations of the compound of Formula (A). If necessary, these preparations can be adjusted to a desired concentration by adding a sterile diluent to each vial. The liquid compositions of the present invention can also be prepared as unit dosage forms in sterile bags or containers, which are suitable for connection to an intravenous administration line or catheter.
Embodiments of the present invention are illustrated by the following Examples. It is to be understood, however, that the embodiments of the invention are not limited to the specific details of these Examples, as other variations thereof will be known, or apparent in light of the instant disclosure, to one of ordinary skill in the art.
EXAMPLES
Unless specified otherwise, starting materials are generally available from commercial sources such as Sigma-Aldrich Corp. (St. Louis, MO), Fisher Chemical (Pittsburgh, PA), Avantor Performance Materials (Center Valley, PA) MP Biomedicals (Santa Ana, CA) Promega Corp. (Madison, Wl), Lancaster Synthesis, Inc. (Windham, NH), Acros Organics (Fairlawn, NJ), Maybridge Chemical Company, Ltd. (Cornwall, England), Tyger Scientific (Princeton, NJ), AstraZeneca Pharmaceuticals (London, England), and Accela ChemBio (San Diego, CA). General Experimental Procedures
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed. , 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of
Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M. P. Calos, eds. , 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds. , 1991 ); Short Protocols in Molecular Biology (Wiley and Sons, 1999);
Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).
Procedures
Example 1 . Anti-IL-7R antibody formulation 1
This example illustrates the viscosity of high concentration anti-IL-7R antibody formulations. Formulation 1 was amenable to achieve concentrations of approximately 50-70 mg/mL C1 GM antibody (in 20 mM histidine, 85 g/L sucrose, 0.05 g/L disodium EDTA dihydrate, 0.2 g/L polysorbate-80, pH 5.8), with suitable stability characteristics.
Studies were conducted to evaluate impact of pH change (below and above isoelectric point, pi). The drug product was formulated as a lyophilized powder for reconstitution with sWFI (Table 1 ). Viscosity was evaluated using an Anton-Paar rheometer in cone- plate configuration, at 25°C. The sample size was approximately 81 pL. The samples were measured with a constant shear rate (898 s"1).
Table 1
Figure imgf000059_0001
High viscosity was observed at both pH 5.0 and 5.8 (FIGS. 1 A and 1 B: viscosity of formulation at pH 5.8 and pH 5.0 (A) up to approximately 200 mg/mL C1 GM; (B) y-axis scale limited to 100 cP).
Example 2. Anti-IL-7R antibody formulations with camphorsulfonic acid.
This example illustrates the impact of camphor-10-sulfonic acid (also described as camphorsulfonic acid or CSA) on viscosity in a new anti-IL-7R antibody formulation, formulation 2.
A study was conducted to assess the viscosity of formulation 2. Formulation 2, shown in Table 2 below, includes 200 mM CSA. Due to the acidic nature of CSA, nearly an equimolar amount of NaOH was used to neutralize the acid and bring the pH of the formulation to 7.0. Therefore, sodium ions are also present in this formulation at approximately 200 mM.
Table 2
Figure imgf000060_0001
Viscosity was evaluated using an Anton-Paar rheometer in cone-plate configuration, at 25°C. The sample size was approximately 81 μΙ_. The samples were measured with a constant shear rate (898 s"1). Viscosity data are summarized in Table 3 below and FIG. 2A and 2B.
Table 3
Figure imgf000061_0001
Viscosity of formulation 2 containing 200 mM CSA showed significantly reduced viscosity, i.e., approximately 20-40 fold reduction in viscosity, compared to formulation 1 across concentrations tested (Table 3 and FIG. 2A and 2B). For example, at about 100 mg/ml antibody, viscosity of formulation 2 was 3.2 cP, compared to viscosity of formulation 1 , which was 55.1 cp, a 17.2 fold reduction. At about 1 16 mg/ml antibody, viscosity of formulation 2 was 4.4 cP, compared to viscosity of formulation 1 , which was 89.5 cP, a 20.3 fold reduction. At about 145 mg/ml antibody, viscosity of formulation 2 was 7.5 cP, compared to viscosity of formulation 1 , which was 221.8 cP, a 29.6 fold reduction. At about 175 mg/ml antibody, viscosity of formulation 2 was 13.1 cP, compared to viscosity of formulation 1 , which was 506.3 cP, a 38.6 fold reduction. Of note, viscosity reduction on a fold basis increases as antibody concentration increases.
These results demonstrate the inclusion of CSA significantly reduces viscosity of an anti-IL-7R antibody formulation. Formulation 2, which contains 200 mM CSA and has pH 7, allows C1 GM protein concentrations of greater than 170 mg/mL with viscosity behavior suitable for use in therapeutic treatment. This was not possible for C1 GM in formulation 1 because of high viscosity.
Example 3. Impact of counterion on viscosity reduction This example illustrates how varying the counterion ion for CSA can also impact viscosity.
As indicated in Example 2, NaOH was used to neutralize the acidic nature of CSA in creating formulation 2, and bring the pH of the formulation to 7.0. Therefore, sodium ions are also present in this formulation at approximately 200 mM. Alternatively, other molecules basic in nature may be utilized to neutralize the acidic nature of CSA, effectively forming a salt of CSA. One such species is arginine (Arg). Formulation 3, shown in the table below, includes 200 mM CSA and 200 mM arginine. The basic nature of the arginine largely neutralizes the acidic nature of the CSA, and minimal acid or base needs to be added to adjust the pH to 7.0, avoiding the
introduction of additional ionic species. For the purpose of comparison, Formulation 4 utilizes 200 mM arginine-HCI (Arg-HCI).
Table 4
Figure imgf000062_0001
Viscosity was evaluated using an Anton-Paar rheometer in cone-plate configuration, at 25°C. The sample size was approximately 81 μί. The samples were measured with a constant shear rate (898 s"1). Viscosity data are summarized in Table 5 below and FIG. 3. Table 5
Figure imgf000063_0001
These results demonstrate that the choice of counter-ion used together with CSA also has an impact on viscosity. With the correct choice of counter-ion, a synergistic effect can occur, resulting in a greater viscosity reduction than is observed for the individual components. The viscosity values for the CSA-Arg combination used in Formulation 3 are lower than what is observed for the CSA based Formulation 2 and far lower than what is observed for the Arg-HCI based Formulation 4. Thus, combinations of CSA with an appropriate counter-ion are particularly advantageous to achieve viscosity reduction. Formulation 3, which contains 200 mM CSA-Arg and has pH 7, allows C1 GM protein concentrations of greater than 190 mg/mL with viscosity behavior suitable for use in therapeutic treatment. This was not possible for C1 GM in Formulation 1 because of high viscosity, and offers further improvements over both Formulations 2 and 4.
Example 4. Impact of excipient concentration on viscosity
This example illustrates the impact of varying excipient concentration on viscosity in an anti-IL-7R antibody formulation.
Excipient concentrations of CSA and Arginine in Formulations 2-4 were reduced to either 100 mM or 50 mM. Viscosities of these formulations were evaluated at pH 7 using an Anton-Paar rheometer in cone-plate configuration, at 25°C. The sample size was approximately 81 μΙ_. The samples were measured with a constant shear rate (898 s"1). Results are summarized in FIG. 4 and Table 6. Table 6
Figure imgf000064_0001
Lower amounts of excipients led to higher viscosities than observed for the 200 mM excipients used previously. However, reduction of excipient levels may be necessary to develop formulations which are roughly isotonic and suitable for parenteral injection. In particular, additional excipients not included in Formulations 2-4, such as
cryoprotectants, surfactants, and chelating agents, may be necessary to include in the formulation, and will contribute to the tonicity of the formulation. Therefore, reduction of CSA and Arg levels may be required to allow these other excipients to be included in the formulation. Even at lower levels of CSA and Arg, formulations of C1 GM that include these excipients have significantly lower viscosity than formulations without these excipients. These results demonstrate that CSA-Arg appears to provide some robust protection against viscosity increases over the range of the ionic strength of the formulation. Example 5. Viscosity Reduction by Camphorsulfonic acid and CSA-Arg formulations of other antibodies
This example illustrates the impact of camphorsulfonic acid (CSA) and CSA-Arg on viscosity new formulations of mAb5 and L1 L3 antibodies. Formulations of mAb5 and L1 L3 antibodies listed in Table 7 exhibited high viscosity at high concentrations, as shown in Figure 5 (XX1 is mAb5 antibody and XX2 is L1 L3 antibody) and Table 8.
Table 7
Figure imgf000065_0001
Table 8
Figure imgf000066_0001
In an effort to lower viscosity of these formulations, mAb5 and L1 L3 were reformulated with the above formulations by adding 200 mM Arginine HCI, 200 mM CSA, or 200 mM CSA-Arg. Viscosity was evaluated using an Anton-Paar rheometer in cone-plate configuration, at 25°C. The sample size was approximately 81 μΙ_. The samples were measured with a constant shear rate (898 s" ). Viscosity data are summarized in Table 9 below and FIG. 6 (XX 1 is mAb5 antibody and XX2 is L1 L3 antibody).
Table 9
Figure imgf000067_0001
The data show that Arg, CSA, and CSA-Arg formulations are all effective in reducing the viscosity of formulations of mAb5 and L1 L3 antibodies. As was observed for
formulations of the C1 GM antibody, the CSA-Arg combination offers the most effective reduction of viscosity for the mAb5 and L1 L3 antibody formulations.
Formulations containing 200 mM CSA-Arg at pH 7.0 allows mAb5 protein
concentrations of greater than 195 mg/mL, and L1 L3 protein concentrations of greater than 185 mg/mL, with viscosity behaviors suitable for use in therapeutic treatment. This was not possible for mAb5 and L1 L3 antibodies without the excipients because of high viscosity. Example 6. Impact of excipient concentration on viscosity for other antibodies.
This example illustrates the impact of varying excipient concentration on viscosity on mAb5 and L1 L3 antibody formulations.
Excipient concentrations of CSA and Arginine were reduced to either 100 mM or 50 mM from the previous level of 200 mM. Viscosities of these formulations were evaluated at pH 7 using an Anton-Paar rheometer in cone-plate configuration, at 25°C. The sample size was approximately 81 μΙ_. The samples were measured with a constant shear rate (898 s" ). Results are summarized in FIG. 7A and 7B (XX1 is mAb5 antibody and XX2 is L1 L3 antibody) and Table 10. Lower amounts of excipients led to higher viscosities than observed for the 200 mM excipients. Even at lower levels of CSA and Arg, formulations of mAb5 and L1 L3 that include these excipients have significantly lower viscosity than formulations without these excipients. These results demonstrate that CSA-arginine appears to provide some robust protection against viscosity increases over the range of the ionic strength of the formulation for a variety of antibodies.
Table 10
Figure imgf000069_0001
Example 7. Impact of hinge region structure on viscosity
This example illustrates the impact of the choice of hinge region structure on the viscosity of a formulation of monoclonal antibodies.
Monoclonal antibodies with identical CDR regions but differing hinge regions were produced and characterized using methods known in the art. Identical CDR regions were incorporated into frameworks of the lgG1 , lgG2, and lgG4 subtypes, and referred to as lgG1 , lgG2, and lgG4, respectively. The lgG4 contained a hinge stabilizing S228P mutation (serine at position 228 of the heavy chain is replaced with proline). Each antibody was formulated at 130 mg/ml_ in 20 mM histidine, 85 g/L sucrose, 0.05 g/L disodium EDTA dihydrate, 0.2 g/L polysorbate-80, pH 5.8. Viscosity of these formulations was evaluated using an Anton-Paar rheometer in cone-plate configuration, at 25°C. The sample size was approximately 81 pL. The samples were measured with a constant shear rate (898 s" ). Viscosity data are summarized in Table 1 1 .
Table 1 1
Figure imgf000070_0001
The data show that the choice of hinge region structure can impact viscosity for an otherwise identical monoclonal antibody. lgG1 exhibits the lowest viscosity, followed by lgG2, and the hinge stabilized lgG4. The data indicate that the flexibility of the hinge region may impact viscosity. These results demonstrate that proper selection or design of the hinge region of an antibody can result in lower viscosity antibody formulations.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application for all purposes.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification including the examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

What is claimed is:
1. A pharmaceutical composition comprising; a. an antibody, wherein the antibody concentration is between about 100 mg/ml to about 400 mg/ml, and b. a viscosity lowering excipient comprising camphorsulfonic acid, sulfosalicylic acid, a salt of camphorsulfonic acid, or a salt of sulfosalicylic acid, wherein the viscosity lowering excipient concentration is between about 30 mM to about 200 mM, wherein the pH of said composition is from about 4.0 to about 9.0.
2. The composition of claim 1 wherein the antibody is a human or humanized monoclonal lgG1 , lgG2 or lgG4 antibody.
3. The composition of claim 1 or 2 wherein the composition further comprises a pharmaceutically acceptable buffer.
4. The composition of claim 3 wherein the pharmaceutically acceptable buffer comprises histidine, tris, phosphate, or a salt thereof.
5. The composition of claim 3 or 4 wherein the concentration of pharmaceutically acceptable buffer is from about 1.0 to about 200 mM.
6. The composition of any of claims 1 -5 wherein the composition further comprises a surfactant.
7. The composition of claim 6 wherein the surfactant is polysorbate 20 or polysorbate 80.
8. The composition of claim 6 or 7 wherein the concentration of surfactant is from about 0.01 to about 0.3 mg/ml.
9. The composition of any of claims 1 -8 wherein the composition further comprises a chelating agent.
The composition of claim 9 wherein the chelating agent is EDTA or disodium
1 1 . The composition of claim 9 or 10 wherein the concentration of chelating agent is from about 0.01 to about 0.3 mg/ml.
12. The composition of any of claims 1 -8 wherein the composition further comprises a cryoprotectant.
13. The composition of claim 12 wherein the cryoprotectant is sucrose, dextrose, mannose or trehalose.
14. The composition according to claim 12 or 13, wherein the concentration of the cryoprotectant is from about 1 mg/ml to about 100 mg/ml.
15. The composition of any of claims 1 -14 wherein the viscosity lowering agent is a salt of camphorsulfonic acid comprising camphorsulfonic acid and arginine .
16. The composition of claim 15 wherein the camphorsulfonic acid concentration is between about 50 mM and about 150 mM and the arginine concentration is between about 50 mM and about 150 mM.
17. The composition of claim 16 wherein the camphor sulfonic acid concentration is between about 70 mM and about 1 10 mM and the arginine concentration is between about 70 mM and about 1 10 mM.
18. The composition according to any one of claims 1 to 17 wherein the antibody comprises a heavy chain comprising at least one CDR selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, 5, or 6; CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7 or 8; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9, and a light chain comprising at least one CDR selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11 , and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12.
19. The composition according to any one of claims 1 to 17 wherein the antibody comprises an amino acid sequence that is at least 85% identical to a heavy chain variable region amino acid sequence shown in SEQ ID NO: 2, and an amino acid sequence that is at least 85% identical to a light chain variable region amino acid sequence shown in SEQ ID NO: 3.
20. The composition according to any one of claims 1 to 17 wherein the antibody comprises an amino acid sequence that is at least 85% identical to a heavy chain amino acid sequence shown in SEQ ID NO: 13, and an amino acid sequence that is at least 85% identical to a light chain amino acid sequence shown in SEQ ID NO: 14.
21 . The composition according to any one of claims 1 to 17 wherein the antibody comprises a variable heavy chain sequence comprising the amino acid sequence shown in SEQ ID NO: 2 and a variable light chain sequence comprising the amino acid sequence shown in SEQ ID NO: 3.
22. The composition according to any one of claims 1 to 17 wherein the antibody comprises a heavy chain comprising at least one CDR selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, 18 or 19; CDR2 comprising the amino acid sequence shown in SEQ ID NO: 20 or 21 ; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 22, and a light chain comprising at least one CDR selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 23, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 24, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 25.
23. The composition according to any one of claims 1 to 17 wherein the antibody comprises an amino acid sequence that is at least 85% identical to a heavy chain variable region amino acid sequence shown in SEQ ID NO: 15, and an amino acid sequence that is at least 85% identical to a light chain variable region amino acid sequence shown in SEQ ID NO: 16.
24. The composition according to any one of claims 1 to 17 wherein the antibody comprises an amino acid sequence that is at least 85% identical to a heavy chain amino acid sequence shown in SEQ ID NO: 26 or 27, and an amino acid sequence that is at least 85% identical to a light chain amino acid sequence shown in SEQ ID NO: 28.
25. The composition according to any one of claims 1 to 17 wherein the antibody comprises a variable heavy chain sequence comprising the amino acid sequence shown in SEQ ID NO: 15 and a variable light chain sequence comprising the amino acid sequence shown in SEQ ID NO: 16.
26. The composition according to any one of claims 1 to 17 wherein the antibody comprises a heavy chain comprising at least one CDR selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 32, 33, or 34; CDR2 comprising the amino acid sequence shown in SEQ ID NO: 35 or 36; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 37, and a light chain comprising at least one CDR selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO: 38, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 39, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 40.
27. The composition according to any one of claims 1 to 17 wherein the antibody comprises an amino acid sequence that is at least 85% identical to a heavy chain variable region amino acid sequence shown in SEQ ID NO: 41 , and an amino acid sequence that is at least 85% identical to a light chain variable region amino acid sequence shown in SEQ ID NO: 42.
28. The composition according to any one of claims 1 to 17 wherein the antibody comprises an amino acid sequence that is at least 85% identical to a heavy chain amino acid sequence shown in SEQ ID NO: 30, and an amino acid sequence that is at least 85% identical to a light chain amino acid sequence shown in SEQ ID NO: 31.
29. The composition according to any one of claims 1 to 17 wherein the antibody comprises a variable heavy chain sequence comprising the amino acid sequence shown in SEQ ID NO: 41 and a variable light chain sequence comprising the amino acid sequence shown in SEQ ID NO: 42.
30. The composition according to any one of claims 1 to 29 wherein the composition is lyophilized.
31 . The composition according to claim 30 wherein the lyophilized composition is reconstituted and the antibody concentration of the reconstituted composition is between about 250 mg/ml and about 400 mg/ml.
32. The composition according to any one of claims 1 to 31 wherein the composition has a viscosity of less than about 50 cP at 25°C.
33. The composition according to any one of claims 1 to 32 wherein the antibody hinge region increases flexibility of the antibody.
34. The composition according to claim 33 wherein the antibody has lowered viscosity.
35. The composition according to any one of claims 1 to 34 wherein the antibody is an lgG4 subtype comprising S228P in the antibody hinge region.
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