WO2011060015A1

WO2011060015A1 - Methods and compositions for detecting target proteins

Info

Publication number: WO2011060015A1
Application number: PCT/US2010/056149
Authority: WO
Inventors: Judy Young; Laura Deforge; Wai-Lee Wong
Original assignee: Genentech, Inc.; F. Hoffmann-La Roche Ag
Priority date: 2009-11-11
Filing date: 2010-11-10
Publication date: 2011-05-19

Abstract

Disclosed herein are methods and compositions useful for detecting a target protein.

Description

METHODS AND COMPOSITIONS FOR DETECTING TARGET PROTEINS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application Serial No. 61/260,188, entitled Methods and Compositions for Detecting Target Proteins, filed November 11, 2009.

FIELD OF THE INVENTION

[0002] The present invention relates to methods and compositions useful in detecting target proteins.

BACKGROUND OF THE INVENTION

[0003] Numerous techniques are available to detect and measure levels of analytes in test samples. For example, immunoassays are useful in detecting the presence of an antigen using an antibody that specifically binds to the antigen, whereby the antibody forms a complex with the antigen that is detected using various techniques, e.g., radioactivity, fluorescence, etc. A common example of an immunoassay is the ELISA wherein antibodies specific to the target antigen are linked to an insoluble carrier surface. These antibodies are then exposed to a test sample and any antigens present in the sample form a complex with the antibody. The presence of the antibody-antigen complex are typically detected using an enzymatic reaction that produces a signal that can be detected and quantified.

[0004] Although techniques such as immunoassays are available, in some instances, because of the nature of the antigen currently known techniques are not capable of accurately detecting and measuring the antigen. Thus, additional techniques of accurately detecting such antigens in biological samples are desirable.

SUMMARY OF THE INVENTION

[0005] The present invention is based, in part, on the discovery of a novel method of detecting a target protein where presence of the target protein cannot be detected directly. For example, the target protein is bound to one or more therapeutic antibodies that specifically bind to the target protein such that direct detection of the target protein is not possible. In certain circumstances the target protein may associate with one or more of the target proteins or other molecules to create a homo- or hetero-multimer, e.g., a dimer, trimer, tetramer, pentamer, etc. Binding of the therapeutic antibodies to each of the target proteins in the multimer may prevent direct detection of the presence of the target protein because of steric hindrance created by the therapeutic antibodies, especially if the relative size of the target protein is small compared to the size of the bound therapeutic antibody. Additionally, epitopes useful for direct detection of the target protein may be buried within the multimer resulting in inaccessibility of the target protein to direct detection using techniques known in the art. Detection of the target protein may also be difficult if the concentration of the target protein in the sample being analyzed is very low.

[0006] Thus, in one aspect, the invention provides a method of detecting a target protein in a sample, wherein the target protein cannot be detected directly, wherein the method comprises the step of detecting a secondary antibody bound to a primary antibody, wherein the primary antibody is bound to the target protein, and wherein detection of the presence of the secondary antibody in the sample is indicative of the presence of the target protein. In certain embodiments the target protein cannot be detected directly due to steric hindrance by the primary antibody bound to the target protein.

[0007] The target protein may be a multimeric protein, e.g., a protein consisting of multiple monomers, such as but not limited to a dimer, trimer, tetramer, pentamer, etc. The target protein may be a multimeric protein consisting of identical monomers, e.g, a homotrimer, or it may be a multimeric protein consisting of non-identical monomers, e.g., a heterotrimer.

[0008] In some aspects of the methods of the invention, the secondary antibody is an anti-idiotype antibody that specifically binds to the variable region of the primary antibody. In certain embodiments the secondary antibody binds to one or more CDRs of the primary antibody.

[0009] In some embodiments the target protein is LTa and the primary antibody is an anti-LTa antibody. For example the anti-LTa antibody may be 2C8vX. In some embodiments the secondary antibody is an anti-idiotype antibody that specifically binds to the variable region of the anti-LTa antibody. For example, the anti-idiotype antibody specifically binds to CDR-L3 of the anti-LTa antibody, e.g., 2C8vX. [0010] The invention also provides a method of detecting LTa in a sample in the presence of an anti-LTa antibody that specifically binds to LTa, wherein presence of the anti-LTa antibody prevents direct detection of LTa, the method comprising the step of detecting an anti-idiotype antibody that specifically binds to the anti-LTa antibody, wherein the anti-LTa antibody is bound to LTa, and wherein detection of the anti-idiotype antibody is indicative of the presence of LTa. In certain embodiments the anti-idiotype antibody specifically binds to one or more CDRs of the anti-LTa antibody. In some embodiments the anti-idiotype antibody binds to CDR-L3 of the anti-LTa antibody. In one aspect the anti-LTa antibody is 2C8vX and the anti-idiotype antibody binds to CDR- L3 of the anti-LTa antibody.

[0011] In any of the methods of the invention the sample may be obtained from serum, plasma, synovial fluid, tissue culture supernatants, tissue lysates or extracts and lymph or exudates. The sample may be obtained directly from a patient being treated with anti-LTa antibodies or may be obtained from tissue or cell cultures containing or exposed to anti-LTa antibodies.

[0012] Any embodiment described above or any combination thereof applies to any and all methods of the inventions described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 A is a schematic diagram illustrating an assay used for measuring levels of LTa homotrimer in human or cynomolgus monkey serum using two different anti-LTa antibodies for capture and detection. The specificity of this assay for TNF family ligands is shown in Figure IB. The assay is approximately 10 fold less sensitive for the detection of υΓα2β 1 and 100 fold less sensitive for LTai 2 heterotrimers than it is for detecting the homotrimer. The assay does not detect TNFa or LIGHT. RLU = relative light units.

[0014] Figure 2 is a graph showing that the assay depicted in Figure 1 cannot accurately measure the levels of LTa3 at three different biologically relevant

concentrations (4, 20, and 100 pg/mL) in the presence of as little as 6 ng/mL anti-LTa antibody, 2C8vX. RLU = relative light units.

[0015] Figure 3 is a schematic diagram illustrating a modified assay for detecting LTa in the presence of anti-LTa, in which all binding sites on LTa3 in a sample are saturated by adding excess anti-LTa antibody, followed by detection of the anti-LTa antibody using a biotinylated anti-idiotypic (Id) antibody that specifically binds to the anti- LTa antibody.

[0016] Figure 4 are graphs showing that the novel assay depicted in Figure 3 can accurately measure the levels of rhesus monkey LTa in a sample containing up to 50 ug/ml of anti-LTa antibody (Figure 4 A) and up to 30% cynomolgus monkey serum (Figure 4B).

[0017] Figure 5 is a graph showing that the modified assay depicted in Figure 3 can accurately measure the levels of human LTa in a sample containing up to 100 ug/ml of anti-LTa antibody prior to a 1 :4 dilution into the assay, and up to 25% human serum.

[0018] Figure 6 shows results obtained by using the assay depicted in Figure 3 to measure LTa in the serum of cynomolgus monkeys receiving single doses of anti-LTa antibody: group 1 received 0.15 mg/kg intravenously of an afucosylated variant of anti- lymphotoxin alpha; group 2 received 1.5 mg/kg intravenously of afucosylated anti- lymphotoxin alpha; group 3 received 1.5 mg/kg subcutaneously of afucosylated anti- lymphotoxin alpha; group 4 received 15 mg/kg intravenously of afucosylated anti- lymphotoxin alpha; group 5 received 1.5 mg/kg intravenously of wild type anti- lymphotoxin alpha.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)); 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., 1993-8) J. Wiley and Sons; 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); 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); and Cancer: Principles and Practice of Oncology (V. T. DeVita et al., eds., J.B. Lippincott Company, 1993).

[0020] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), provide one skilled in the art with a general guide to many of the terms used in the present application. All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

Definitions

[0021] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0022] The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies {e.g.

bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

[0023] An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, an antibody is purified (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of, for example, a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using, for example, Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0024] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different

immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V_H) followed by a number of constant domains. Each light chain has a variable domain at one end (V_L) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

[0025] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0026] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy- chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta- sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of

Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody- dependent cellular toxicity.

[0027] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0028] Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (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., IgGi, IgG₂, IgG₃, IgG₄, IgAi, and IgA₂ . The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co., 2000). An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

[0029] The terms "full length antibody," "intact antibody" and "whole antibody" are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain an Fc region.

[0030] A "naked antibody" for the purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0031] "Antibody fragments" comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')₂, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. [0032] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')₂ fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0033] "Fv" is the minimum antibody fragment which contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen- binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0034] The Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')₂ antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0035] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0036] The term "diabodies" refers to antibody fragments with two antigen- binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al, Nat. Med. 9: 129- 134 (2003).

[0037] The term "monoclonal antibody" as used herein 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 mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[0038] 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 a variety of techniques, including, for example, the hybridoma method {e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al, Hybridoma, 14 (3): 253- 260 (1995), Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al, in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage-display technologies (see, e.g., Clackson et al, Nature, 352: 624-628 (1991); Marks et al, J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al, J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al, J. Immunol. Methods 284(1-2): 119-132(2004), and technologies for producing human or human- like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al, Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al, Nature 362: 255-258 (1993); Bruggemann et al, Year in

Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;

5,633,425; and 5,661,016; Marks et al, Bio/Technology 10: 779-783 (1992); Lonberg et al, Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al, Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

[0039] The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g.,XJ.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)). Chimeric antibodies include PRIMATIZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.

[0040] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human

immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a HVR of the recipient are replaced by residues from a HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non- human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human

immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human

immunoglobulin. For further details, see, e.g., Jones et al, Nature 321 :522-525 (1986); Riechmann et al, Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593- 596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1 : 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0041] A "human antibody" is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al. , J. Mol. Biol, 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol, 147(l):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin.

Pharmacol, 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,18.1 and 6,150,584 regarding

XENOMOUSE™ technology). See also, for example, Li et al, Proc. Nail. Acad. Sci. USA., 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.

[0042] The term "hypervariable region," "HVR," or "HV," when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248: 1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, e.g., Hamers-Casterman et al, Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0043] A number of HVR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of

Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact

LI L24-L34 L24-L34 L26-L32 L30-L36

L2 L50-L56 L50-L56 L50-L52 L46-L55

L3 L89-L97 L89-L97 L91-L96 L89-L96

HI H31-H35B H26-H35B H26-H32 H30-H35B

(Kabat Numbering)

HI H31-H35 H26-H35 H26-H32 H30-H35

(Chothia Numbering)

H2 H50-H65 H50-H58 H53-H55 H47-H58

H3 H95-H102 H95-H102 H96-H101 H93-H101

[0044] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (HI), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions. [0045] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0046] The term "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat," and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.

[0047] The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "EU index as in Kabat" refers to the residue numbering of the human IgGl EU antibody. Unless stated otherwise herein, references to residue numbers in the variable domain of antibodies means residue numbering by the Kabat numbering system. Unless stated otherwise herein, references to residue numbers in the constant domain of antibodies means residue numbering by the EU numbering system {e.g., see United States Provisional Application No. 60/640,323, Figures for EU numbering).

[0048] An "affinity matured" antibody is one with one or more alterations in one or more HVRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). In one embodiment, an affinity matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies may be produced using certain procedures known in the art. For example, Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random

mutagenesis of HVR and/or framework residues is described by, for example, Barbas et al. Proc Nat. Acad. Sci. USA 91 :3809-3813 (1994); Schier et a/. Gene 169: 147-155 (1995); Yelton et al. J. Immunol. 155: 1994-2004 (1995); Jackson et al, J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

[0049] A "blocking" antibody or an "antagonist" antibody is one which inhibits or reduces biological activity of the antigen it binds. Certain blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0050] An "agonist antibody," as used herein, is an antibody which partially or fully mimics at least one of the functional activities of a polypeptide of interest.

[0051] "Growth inhibitory" antibodies are those that prevent or reduce proliferation of a cell expressing an antigen to which the antibody binds.

[0052] Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.

[0053] The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

[0054] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g. , an antibody variable domain) and can be assessed using various assays. [0055] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a native sequence human IgGl Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof.

[0056] A "variant Fc region" comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). In certain embodiments, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g. from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

[0057] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, an FcR is a native human FcR. In some

embodiments, an FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of those receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (IT AM) in its cytoplasmic domain. Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine- based inhibition motif (ITIM) in its cytoplasmic domain, (see, e.g. , Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al, Immunomethods 4:25-34 (1994); and de Haas et al, J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein.

[0058] The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al, J. Immunol. 117:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)) and regulation of homeostasis of immunoglobulins. Methods of measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al).

[0059] Binding to human FcRn in vivo and serum half life of human FcRn high affinity binding polypeptides can be assayed, e.g. , in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides with a variant Fc region are administered. WO 2000/42072 (Presta) describes antibody variants with improved or diminished binding to FcRs, the entire disclosure of which is expressly incorporated herein by reference. See also, e.g., Shields et al. J. Biol. Chem. 9(2):6591- 6604 (2001). Furthermore, Attorney Docket Number PR4182 describes antibody variants with increased in vivo half life and/or improved binding to FcRn, the entire disclosure of which is expressly incorporated herein by reference.

[0060] An "antigen" is a predetermined antigen to which an antibody can selectively bind. The target antigen may be polypeptide, carbohydrate, nucleic acid, lipid, hapten, or another naturally occurring or synthetic compound. Preferably, the target antigen is a polypeptide. An "acceptor human framework" for the purposes herein is a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework, or from a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or human consensus framework may comprise the same amino acid sequence thereof, or may contain pre-existing amino acid sequence changes. Where pre-existing amino acid changes are present, preferably no more than five, and more preferably four or less, and still more preferably three or less, pre-existing amino acid changes are present. Where pre-existing amino acid changes are present in a VH, preferably those changes are only at three, two, or one of positions 71, 73, and 78; for instance, the histidine residues at those positions may be alanine residues. In one embodiment, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0061] The term "Fc region-comprising antibody" refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody. Accordingly, a composition comprising an antibody having an Fc region according to this invention can comprise an antibody with K447, with all K447 removed, or a mixture of antibodies with and without the K447 residue.

[0062] "Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low- affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative and exemplary

embodiments for measuring binding affinity are described in the following.

[0063] In one embodiment, the "Kd" or "Kd value" according to this invention is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay. Solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal

125

concentration of ( I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To establish conditions for the assay, MICROTITER^® multi-well plates (Thermo Scientific) are coated overnight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ 125 I] -antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab- 12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% TWEEN-20™ in PBS. When the plates have dried, 150 μΐ/well of scintillant (MICROSCINT-20™; Packard) is added, and the plates are counted on a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.

[0064] According to another embodiment, the Kd or Kd value is measured by using surface plasmon resonance assays using a BIACORE^®-2000 or a BIACORE ^®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at ~10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5,

BIACORE, Inc.) are activated with N-ethyl-N'- (3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (-0.2 μΜ) before injection at a flow rate of 5 μΐ/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, serial dilutions of Fab are injected in PBS with 0.05% TWEEN-20™ surfactant (PBST) at 25°C at a flow rate of

approximately 25 μΐ/min. Association rates (k_on) and dissociation rates (k₀ff) are calculated using a simple one-to-one Langmuir binding model (BIACORE ^® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio k₀ff/k_on See, e.g., Chen et al, J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds

106 M^"l s^~l by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25°C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO ™ spectrophotometer (Thermo Spectronic) with a stirred cuvette.

[0065] An "on-rate," "rate of association," "association rate," or "k_on" according to this invention can also be determined as described above using a BIACORE ^®-2000 or a BIACORE ^®-3000 system (BIAcore, Inc., Piscataway, NJ).

[0066] The term "substantially similar" or "substantially the same," as used herein, denotes a sufficiently high degree of similarity between two numeric values (for example, one associated with an antibody of the invention and the other associated with a reference/comparator antibody), such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values is, for example, less than about 50%, less than about 40%, less than about 30%>, less than about 20%>, and/or less than about 10% as a function of the reference/comparator value.

[0067] The phrase "substantially reduced," or "substantially different," as used herein, denotes a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a

reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values is, for example, greater than about 10%>, greater than about 20%>, greater than about 30%, greater than about 40%, and/or greater than about 50% as a function of the value for the reference/comparator molecule.

[0068] "Purified" means that a molecule is present in a sample at a

concentration of at least 95% by weight, or at least 98% by weight of the sample in which it is contained.

[0069] An "isolated" nucleic acid molecule is a nucleic acid molecule that is separated from at least one other nucleic acid molecule with which it is ordinarily associated, for example, in its natural environment. An isolated nucleic acid molecule further includes a nucleic acid molecule contained in cells that ordinarily express the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0070] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA into which additional DNA segments may be ligated. Another type of vector is a phage vector.

Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors," or simply, "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector.

[0071] "Polynucleotide," or "nucleic acid," as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A

polynucleotide may comprise modification(s) made after synthesis, such as conjugation to a label. Other types of modifications include, for example, "caps," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotides(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5' and 3 ' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0- methyl-, 2'-0-allyl-, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S ("thioate"), P(S)S ("dithioate"), (0)NR₂

("amidate"), P(0)R, P(0)OR', CO, or CH2 ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0072] "Oligonucleotide," as used herein, generally refers to short, generally single-stranded, generally synthetic polynucleotides that are generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and

"polynucleotide" are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.

[0073] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0074] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN- 2 computer program.

[0075] "Detection" includes any means of detecting, including direct and indirect detection.

[0076] An "individual," "subject," or "patient" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice and rats. In certain embodiments, a mammal is a human.

[0077] "Inflammatory cytokines associated with rheumatoid arthritis" refer to IL-6, IL-lb, and TNFa, associated with RA pathology, which can be inhibited systemically and or in the joints in an in vivo collagen-induced arthritis assay.

[0078] The word "label" when used herein refers to a compound or

composition which is conjugated or fused directly or indirectly to a reagent such as a nucleic acid probe or an antibody and facilitates detection of the reagent to which it is conjugated or fused. The label may itself be detectable {e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.

[0079] "Lymphotoxin-alpha," "Lymphotoxin-a" or "LTa" is defined herein as a biologically active polypeptide derived from the amino acid sequence shown in FIG. 2A of US Pat. No. 5,824,509. LTa is defined to specifically exclude human TNFa or its natural animal analogues (Pennica et al., Nature 312:20/27 : 724-729 (1984) and Aggarwal et al., J. Biol. Chem. 260: 2345-2354 (1985)). LTa is defined to specifically exclude human υΓβ as defined, for example, in US 5,661,004. [0080] "Lymphotoxin-a3 trimer" or "LT 3" refers to a homotrimer of LTa monomers.

[0081] "Lymphotoxin-αβ" or 'ΤΤαβ" or 'ΤΤαβ complex" refers to a heterotrimer of LTa with LTp. These heterotrimers contain either two subunits of LTa and one subunit of ίΤβ ( Τα2β1), or one subunit of LTa and two of ίΤβ ( Τα1β2).

[0082] A "native sequence" polypeptide comprises a polypeptide having the same amino acid sequence as a polypeptide derived from nature. Thus, a native sequence polypeptide can have the amino acid sequence of naturally occurring polypeptide from any mammal. Such native sequence polypeptide can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence" polypeptide specifically encompasses naturally occurring truncated or secreted forms of the

polypeptide (e.g., an extracellular domain sequence), naturally occurring variant forms (e.g., alternatively spliced forms) and naturally occurring allelic variants of the

polypeptide.

[0083] A polypeptide "variant" means a biologically active polypeptide having at least about 80% amino acid sequence identity with the native sequence polypeptide. Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the polypeptide. Ordinarily, a variant will have at least about 80% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, and even more preferably at least about 95% amino acid sequence identity with the native sequence polypeptide.

[0084] An "amino acid modification" refers to a change in the amino acid sequence of a predetermined amino acid sequence. Exemplary modifications include an amino acid substitution, insertion, and/or deletion. The preferred amino acid modification herein is a substitution.

[0085] An "amino acid modification at" a specified position, e.g. of the Fc region, refers to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent to the specified residue. By insertion "adjacent to" a specified residue is meant insertion within one to two residues thereof. The insertion may be N-terminal or C-terminal to the specified residue.

[0086] An "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence with another different "replacement" amino acid residue. The replacement residue or residues may be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) and selected from the group consisting of: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gin); glutamic acid (Glu); glycine (Gly); histidine (His);

isoleucine (He): leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe);

proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). Preferably, the replacement residue is not cysteine. Substitution with one or more non-naturally occurring amino acid residues is also encompassed by the definition of an amino acid substitution herein. A "non-naturally occurring amino acid residue" refers to a residue, other than those naturally occurring amino acid residues listed above, which is able to covalently bind adjacent amino acid residues(s) in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogues such as those described in Ellman et al., Meth. Enzym. 202:301-336 (1991). For generation of such non-naturally occurring amino acid residues, the procedures of Noren et al, Science 244: 182 (1989) and Ellman et al., supra, can be used. Briefly, these procedures involve chemically activating a suppressor tRNA with a non-naturally occurring amino acid residue followed by in vitro transcription and translation of the RNA.

[0087] The term "conservative" amino acid substitution as used within this invention is meant to refer to amino acid substitutions that substitute functionally equivalent amino acids. Conservative amino acid changes result in silent changes in the amino acid sequence of the resulting polypeptide. For example, one or more amino acids of a similar polarity act as functional equivalents and result in a silent alteration within the amino acid sequence of the polypeptide. In general, substitutions within a group may be considered conservative with respect to structure and function. However, the skilled artisan will recognize that the role of a particular residue is determined by its context within the three-dimensional structure of the molecule in which it occurs. For example, Cys residues may occur in the oxidized (disulfide) form, which is less polar than the reduced (thiol) form. The long aliphatic portion of the Arg side chain may constitute a critical feature of its structural or functional role, and this may be best conserved by substitution of a nonpolar, rather than another basic, residue. Also, it will be recognized that side chains containing aromatic groups (Trp, Tyr, and Phe) can participate in ionic- aromatic or "cation-pi" interactions. In these cases, substitution of one of these side chains with a member of the acidic or uncharged polar group may be conservative with respect to structure and function. Residues such as Pro, Gly, and Cys (disulfide form) can have direct effects on the main-chain conformation, and often may not be substituted without structural distortions.

[0088] An "amino acid insertion" refers to the incorporation of at least one amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, the present application contemplates larger "peptide insertions", e.g., insertion of about three to about five or even up to about ten amino acid residues. The inserted residue(s) may be naturally occurring or non-naturally occurring as disclosed above.

[0089] An "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0090] Amino acids may be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M)

(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q)

(3) acidic: Asp (D), Glu (E)

(4) basic: Lys (K), Arg (R), His(H)

[0091] Alternatively, naturally occurring residues may be divided into groups based on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

[0092] "Regulatory cytokines" are cytokines the abnormal levels of which indicate the presence of an autoimmune disorder in a patient. Such cytokines include, for example, interleukin-1 (IL-1), IL- 2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-14, IL-15, IL- 18, IL-23, IL-24, IL-25, IL-26, BLyS/April, TGF-a, TGF-β, interferon-a (IFN-a), IFN-β, IFN-γ, MIP-1, MIF, MCP-1, M-CSF or G-CSF, a lymphotoxin, LIGHT, 4-1BB ligand, CD27 ligand, CD30 ligand, CD40 ligand, Fas ligand, GITR ligand, OX40 ligand, RANK ligand, THANK, TRAIL, TWEAK and VEG1. This group includes TNF family members, which include but are not limited to, TNF-a, LTs such as LTa, LTp, and LIGHT. For a review of the TNF superfamily, see MacEwan, Br. J. Pharmacology 135: 855-875 (2002). Preferably, the regulatory cytokine is an IL such as IL-lb or IL-6 and/or a TNF family member.

[0093] The term "sample," or "test sample" as used herein, refers to a composition that is obtained or derived from a subject of interest that contains a cellular and/or other molecular entity that is to be characterized and/or identified, for example based on physical, biochemical, chemical and/or physiological characteristics. In one embodiment, the definition encompasses blood and other liquid samples of biological origin and tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom. The source of the tissue sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids; and cells from any time in gestation or development of the subject or plasma.

[0094] In another embodiment, the definition includes biological samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides, or embedding in a semi-solid or solid matrix for sectioning purposes. For the purposes herein a "section" of a tissue sample is meant a single part or piece of a tissue sample, e.g. a thin slice of tissue or cells cut from a tissue sample.

[0095] Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, exudates, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, as well as tissue extracts such as homogenized tissue, tumor tissue, and cellular extracts.

[0096] In one embodiment, the sample is a clinical sample. In another embodiment, the sample is used in a diagnostic assay. In some embodiments, the sample is obtained from a patient suffering from an autoimmune disorder. In some embodiments the autoimmune disorder is, e.g., rheumatoid arthritis (RA), lupus, Wegener's disease, inflammatory bowel disease (IBD), idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis (MS), psoriasis, IgA nephropathy, IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus, Reynaud's syndrome, Sjogren's syndrome, glomerulonephritis, Hashimoto's thyroiditis, Graves' disease, helicobacter-pylori gastritis, and chronic hepatitis C. Tissue biopsy is often used to obtain a representative piece of tissue affected by the autoimmune disorder that the patient suffers from. Alternatively, cells can be obtained indirectly in the form of tissues or fluids that are known or thought to contain the affected cells of interest.

[0097] A "target" or "target protein," as used herein, is a protein of interest, the detection of which is desired.

[0098] A "disorder" is any condition that would benefit from treatment with an antibody or method of the invention, regardless of mechanism, but including inhibiting or blocking the action of LT 3 or LT P and/or by depleting LT -positive cells. This condition includes, but is not limited to, a medical condition or illness mediated by or related to elevated expression or activity, or abnormal activation, of LT 3 and/or LT P by any cell. This includes chronic and acute disorders such as those pathological conditions that predispose the mammal to the disorder in question.

[0099] An "autoimmune disorder" herein is a disease or disorder arising from and directed against an individual's own tissues or organs or a co-segregate or

manifestation thereof or resulting condition therefrom. In many of these autoimmune and inflammatory disorders, a number of clinical and laboratory markers may exist, including, but not limited to, hypergammaglobulinemia, high levels of autoantibodies, antigen- antibody complex deposits in tissues, benefit from corticosteroid or immunosuppressive treatments, and lymphoid cell aggregates in affected tissues. Without being limited to any one theory regarding B-cell mediated autoimmune disorder, it is believed that B cells demonstrate a pathogenic effect in human autoimmune diseases through a multitude of mechanistic pathways, including autoantibody production, immune complex formation, dendritic and T-cell activation, cytokine synthesis, direct chemokine release, and providing a nidus for ectopic neo-lymphogenesis. Each of these pathways may participate to different degrees in the pathology of autoimmune diseases.

[0100] As used herein, an "autoimmune disorder" can be an organ-specific disease (i.e., the immune response is specifically directed against an organ system such as the endocrine system, the hematopoietic system, the skin, the cardiopulmonary system, the gastrointestinal and liver systems, the renal system, the thyroid, the ears, the

neuromuscular system, the central nervous system, etc.) or a systemic disease that can affect multiple organ systems (for example, systemic lupus erythematosus (SLE), rheumatoid arthritis, polymyositis, etc.). Preferred such diseases include autoimmune rheumatologic disorders (such as, for example, rheumatoid arthritis, Sjogren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, anti-phospholipid antibody syndrome, and psoriatic arthritis), autoimmune gastrointestinal and liver disorders (such as, for example, inflammatory bowel diseases (e.g., ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease), vasculitis (such as, for example, ANCA-negative vasculitis and ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's

granulomatosis, and microscopic polyangiitis), autoimmune neurological disorders (such as, for example, multiple sclerosis (MS), opsoclonus myoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathies), renal disorders (such as, for example, glomerulonephritis,

Goodpasture's syndrome, and Berger's disease), autoimmune dermatologic disorders (such as, for example, psoriasis, urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneous lupus erythematosus), hematologic disorders (such as, for example,

thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases (such as, for example, inner ear disease and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplant, and autoimmune endocrine disorders (such as, for example, diabetic-related autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM), Addison's disease, and autoimmune thyroid disease (e.g., Graves' disease and thyroiditis)). More preferred such diseases include, for example, RA, IBD, including Crohn's disease and ulcerative colitis, ANCA-associated vasculitis, lupus, MS, Sjogren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis. Still more preferred are RA, IBD, lupus, and MS, and more preferred RA and IBD, and most preferred RA.

[0101] Specific examples of other autoimmune disorders as defined herein, which in some cases encompass those listed above, include, but are not limited to, arthritis (acute and chronic, rheumatoid arthritis including juvenile-onset rheumatoid arthritis and stages such as rheumatoid synovitis, gout or gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, menopausal arthritis, estrogen-depletion arthritis, and ankylosing spondylitis/rheumatoid spondylitis), autoimmune

lymphoproliferative disease, inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, atopy including atopic diseases such as hay fever and Job's syndrome, dermatitis including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, hives, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, x-linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic

scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spino- optical MS, primary progressive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (for example, Crohn's disease, autoimmune-mediated gastrointestinal diseases, gastrointestinal inflammation, colitis such as ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease), bowel inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, graft- versus-host disease, angioedema such as hereditary angioedema, cranial nerve damage as in meningitis, herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune condition, IgE- mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen's encephalitis and limbic and/or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or acute glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN (RPGN), proliferative nephritis, autoimmune polyglandular endocrine failure, balanitis including balanitis circumscripta plasmacellularis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, eythema multiform, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis, epidermolytic

hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, food allergies, drug allergies, insect allergies, rare allergic disorders such as mastocytosis, allergic reaction, eczema including allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular palmoplantar eczema, asthma such as asthma bronchiale, bronchial asthma, and auto-immune asthma, conditions involving infiltration of T cells and chronic inflammatory responses, immune reactions against foreign antigens such as fetal A-B-0 blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, SLE, such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus, juvenile onset (Type I) diabetes mellitus, including pediatric IDDM, adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy, diabetic colitis, diabetic large-artery disorder, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T- lymphocytes, tuberculosis, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, agranulocytosis, vasculitides (including large-vessel vasculitis such as polymyalgia rheumatica and giant-cell (Takayasu's) arteritis, medium- vessel vasculitis such as Kawasaki's disease and polyarteritis nodosa/periarteritis nodosa,

immuno vasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as fibrinoid necrotizing vasculitis and systemic necrotizing vasculitis, A CA-negative vasculitis, and ANCA-associated vasculitis such as Churg- Strauss syndrome (CSS), Wegener's granulomatosis, and microscopic polyangiitis), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia(s), cytopenias such as pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex- mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, motoneuritis, allergic neuritis, Beliefs disease/syndrome, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid or pemphigus such as pemphigoid bullous, cicatricial (mucous membrane) pemphigoid, skin pemphigoid, pemphigus vulgaris, paraneoplastic pemphigus, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus, epidermolysis bullosa acquisita, ocular inflammation, preferably allergic ocular inflammation such as allergic conjunctivis, linear IgA bullous disease, autoimmune-induced conjunctival inflammation, autoimmune polyendocrinopathies, Reiter's disease or syndrome, thermal injury due to an autoimmune condition, preeclampsia, an immune complex disorder such as immune complex nephritis, antibody-mediated nephritis, neuroinf ammatory disorders, polyneuropathies, chronic neuropathy such as IgM polyneuropathies or IgM-mediated neuropathy, thrombocytopenia (as developed by myocardial infarction patients, for example), including thrombotic thrombocytopenic purpura (TTP), post-transfusion purpura (PTP), heparin-induced thrombocytopenia, and autoimmune or immune-mediated thrombocytopenia including, for example, idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, scleritis such as idiopathic cerato-scleritis, episcleritis, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, Grave's eye disease (ophthalmopathy or thyroid- associated ophthalmopathy), polyglandular syndromes such as autoimmune polyglandular syndromes, for example, type I (or polyglandular endocrinopathy syndromes),

paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as

Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff- person syndrome, encephalomyelitis such as allergic encephalomyelitis or

encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant-cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, pneumonitis such as lymphoid interstitial

pneumonitis (LIP), bronchiolitis obliterans (non-transplant) vs. NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia such as mixed cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsed or relapsing polychondritis, pulmonary alveolar proteinosis, keratitis such as Cogan's

syndrome/nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's disease/syndrome, rosacea autoimmune, zoster-associated pain, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory

demyelinating polyneuropathy, Dressler's syndrome, alopecia areata, alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, e.g., due to anti-spermatozoan antibodies, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, fibrosing mediastinitis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch's cyclitis, Henoch-Schonlein purpura, human

immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis (systemic inflammatory response syndrome (SIRS)), endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, post- vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant-cell polymyalgia, chronic hypersensitivity pneumonitis, conjunctivitis, such as vernal catarrh, keratoconjunctivitis sicca, and epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway/pulmonary disease, silicosis, aphthae, aphthous stomatitis,

arteriosclerotic disorders (cerebral vascular insufficiency) such as arteriosclerotic encephalopathy and arteriosclerotic retinopathy, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia

phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica (sympathetic ophthalmitis), neonatal ophthalmitis, optic neuritis, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired spenic atrophy, non-malignant thymoma, lymphofollicular thymitis, vitiligo, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T- lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex -mediated diseases, antiglomerular basement membrane disease, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, autoimmune polyglandular syndromes, including polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, allergic sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, spondyloarthropathies, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis, autoimmune disorders associated with collagen disease, rheumatism such as chronic arthrorheumatism, lymphadenitis, reduction in blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, ischemic re-perfusion disorder, reperfusion injury of myocardial or other tissues, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine -induced toxicity, narcolepsy, acute serious inflammation, chronic intractable inflammation, pyelitis, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis.

[0102] As used herein, "rheumatoid arthritis" or "RA" refers to a recognized disease state that may be diagnosed according to the 2000 revised American Rheumatoid Association criteria for the classification of RA, or any similar criteria, and includes active, early, and incipient RA, as defined below. Physiological indicators of RA include symmetric joint swelling, which is characteristic though not invariable in rheumatoid arthritis. Fusiform swelling of the proximal interphalangeal (PIP) joints of the hands as well as metacarpophalangeal (MCP), wrists, elbows, knees, ankles, and

metatarsophalangeal (MTP) joints are commonly affected and swelling is easily detected. Pain on passive motion is the most sensitive test for joint inflammation, and inflammation and structural deformity often limit the range of motion for the affected joint. Typical visible changes include ulnar deviation of the fingers at the MCP joints, hyperextension, or hyperflexion of the MCP and PIP joints, flexion contractures of the elbows, and

subluxation of the carpal bones and toes. The subject with RA may be resistant to a disease-modifying anti-rheumatic drug (DMARD), in that the DMARD is not effective or fully effective in treating symptoms. The subject with RA may have experienced an inadequate response to previous or current treatment with TNF inhibitors such as etanercept, infliximab, and/or adalimumab because of toxicity or inadequate efficacy (for example, etanercept for 3 months at 25 mg twice a week or at least 4 infusions of infliximab at 3 mg/kg).

[0103] A patient with "active rheumatoid arthritis" means a patient with active and not latent symptoms of RA. Subjects with "early active rheumatoid arthritis" are those subjects with active RA diagnosed for at least eight weeks but no longer than four years, according to the revised 1987 ACR criteria for the classification of RA. Subjects with "early rheumatoid arthritis" are those subjects with RA diagnosed for at least eight weeks but no longer than four years, according to the revised 1987 ACR criteria for classification of RA. Early RA includes, for example, juvenile-onset RA, juvenile idiopathic arthritis (JIA), or juvenile RA (JRA).

[0104] Patients with "incipient RA" have early polyarthritis that does not fully meet ACR criteria for a diagnosis of RA, but is associated with the presence of RA- specific prognostic biomarkers such as anti-CCP and shared epitope. They include patients with positive anti-CCP antibodies who present with polyarthritis, but do not yet have a diagnosis of RA, and are at high risk for going on to develop bona fide ACR criteria RA (95% probability).

[0105] "Joint damage" is used in the broadest sense and refers to damage or partial or complete destruction to any part of one or more joints, including the connective tissue and cartilage, where damage includes structural and/or functional damage of any cause, and may or may not cause joint pain/arthalgia. It includes, without limitation, joint damage associated with or resulting from inflammatory joint disease as well as noninflammatory joint disease. This damage may be caused by any condition, such as an autoimmune disease, especially arthritis, and most especially RA. Exemplary conditions include acute and chronic arthritis, RA including juvenile-onset RA, juvenile idiopathic arthritis (JIA), or juvenile RA (JRA), and stages such as rheumatoid synovitis, gout or gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, septic arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, menopausal arthritis, estrogen-depletion arthritis, and ankylosing spondylitis/rheumatoid spondylitis), rheumatic autoimmune disease other than RA, and significant systemic involvement secondary to RA (including but not limited to vasculitis, pulmonary fibrosis or Felty's syndrome). For purposes herein, joints are points of contact between elements of a skeleton (of a vertebrate such as an animal) with the parts that surround and support it and include, but are not limited to, for example, hips, joints between the vertebrae of the spine, joints between the spine and pelvis (sacroiliac joints), joints where the tendons and ligaments attach to bones, joints between the ribs and spine, shoulders, knees, feet, elbows, hands, fingers, ankles, and toes, but especially joints in the hands and feet.

[0106] As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishing of any direct or indirect pathological consequences of the disease, prevention of metastasis, decreasing of the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0107] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0108] A "therapeutically effective amount" of a substance/molecule may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance/molecule are outweighed by the therapeutically beneficial effects.

[0109] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.

Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.

[0110] The term "immunosuppressive agent" as used herein for adjunct therapy refers to substances that act to suppress or mask the immune system of the mammal being treated herein. This would include substances that suppress cytokine production, down- regulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see US 4,665,077); nonsteroidal anti-inflammatory drugs (NSAIDs); ganciclovir, tacrolimus, glucocorticoids such as Cortisol or aldosterone, anti-inflammatory agents such as a cyclooxygenase inhibitor, a 5 -lipoxygenase inhibitor, or a leukotriene receptor antagonist; purine antagonists such as azathioprine or mycophenolate mofetil (MMF); trocade (Ro32-355); a peripheral sigma receptor antagonist such as ISR-31747; alkylating agents such as cyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (which masks the MHC antigens, as described in US 4,120,649); anti -idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A; steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs, e.g., prednisone, methylprednisolone, including SOLU-MEDROL^® methylprednisolone sodium succinate, rimexolone, and dexamethasone; dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); anti-malarial agents such as chloroquine and hydroxychloroquine; sulfasalazine; leflunomide; cytokine release inhibitors such as SB-210396 and SB-217969 monoclonal antibodies and a MHC II antagonist such as ZD2315; a PG1 receptor antagonist such as ZD4953; a VLA4 adhesion blocker such as ZD7349; anti-cytokine or anti-cytokine receptor antibodies including anti- interferon-alpha, -beta, or -gamma antibodies, anti- TNF-alpha antibodies (infliximab (REMICADE®) or adalimumab), anti-TNF-alpha immunoadhesin (etanercept), anti-TNF- beta antibodies, interleukin-1 (IL-1) blockers such as recombinant HuIL-lRa and IL-1B inhibitor, anti-interleukin-2 (IL-2) antibodies and anti-IL-2 receptor antibodies; IL-2 fusion toxin; anti-L3T4 antibodies; leflunomide; heterologous anti-lymphocyte globulin; OPC- 14597; NISV (immune response modifier); an essential fatty acid such as gammalinolenic acid or eicosapentaenoic acid; CD-4 blockers and pan-T antibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies; co-stimulatory modifier (e.g., CTLA4-Fc fusion, also known as ABATACEPT™); anti-interleukin-6 (IL-6) receptor antibodies and antagonists; anti- LFA-1 antibodies, including anti-CD 1 la and anti-CD 18 antibodies; soluble peptide containing a LFA-3-binding domain (WO 1990/08187); streptokinase; IL-10; transforming growth factor-beta (TGF-beta); streptodornase; RNA or DNA from the host; FK506; RS- 61443; enlimomab; CDP-855; PNP inhibitor; CH-3298; GW353430; 4162W94, chlorambucil; deoxyspergualin; rapamycin; T-cell receptor (Cohen et al, US 5,114,721); T-cell receptor fragments (Offner et al, Science, 251 : 430-432 (1991); WO 1990/11294; Janeway, Nature, 341 : 482-483 (1989); and WO 91/01133); BAFF antagonists such as BAFF antibodies and BR3 antibodies; zTNF4 antagonists (Mackay and Mackay, Trends Immunol., 23: 113-5 (2002)); biologic agents that interfere with T cell helper signals, such as anti-CD40 receptor or anti-CD40 ligand (CD 154), including blocking antibodies to CD40-CD40 ligand (e.g., Durie et al, Science, 261 : 1328-30 (1993); Mohan et al, J. Immunol, 154: 1470-80 (1995)) and CTLA4-Ig (Finck et al, Science, 265: 1225-7 (1994)); and T-cell receptor antibodies (EP 340,109) such as T10B9. Some preferred immunosuppressive agents herein include cyclophosphamide, chlorambucil, azathioprine, leflunomide, MMF, or methotrexate. [0111] Examples of "disease-modifying anti-rheumatic drugs" or "DMARDs" include chloroquine, hydroxycloroquine, myocrisin, auranofm, sulfasalazine, methotrexate, leflunomide, etanercept, infliximab (plus oral and subcutaneous methotrexate),

azathioprine, D-penicilamine, gold salts (oral), gold salts (intramuscular), minocycline, cyclosporine including cyclosporine A and topical cyclosporine, staphylococcal protein A (Goodyear and Silverman, J. Exp. Med., 197, (9), pi 125-39 (2003)), including salts and derivatives thereof, etc.

[0112] Examples of "non-steroidal anti-inflammatory drugs" or "NSAIDs" include aspirin, acetylsalicylic acid, ibuprofen and ibuprofen retard, fenoprofen, piroxicam, flurbiprofen, naproxen, ketoprofen, naproxen, tenoxicam, benorylate, diclofenac, naproxen, nabumetone, indomethacin, ketoprofen, mefenamic acid, diclofenac, fenbufen, azapropazone, acemetacin, tiaprofenic acid, indomethacin, sulindac, tolmetin,

phenylbutazone, diclofenac and diclofenac retard, cyclooxygenase (COX)-2 inhibitors such as GR 253035, MK966, celecoxib (CELEBREX®; 4-(5-(4-methylphenyl)-3- (trifluoromethyl)-lH-pyrazol-l-yl), benzenesulfonamide and valdecoxib (BEXTRA®), and meloxicam (MOBIC®), including salts and derivatives thereof, etc. Preferably, they are aspirin, naproxen, ibuprofen, indomethacin, or tolmetin. Such NSAIDs are optionally used with an analgesic such as codenine, tramadol, and/or dihydrocodinine or narcotic such as morphine.

[0113] "Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH-buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low-molecular-weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as

polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0114] The term "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal herein is human. [0115] A "medicament" is an active drug to treat the disorder in question or its symptoms or side effects.

[0116] The term "about" as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field.

Methods of the Invention

[0117] The present invention is based, in part, on the discovery of a novel method of detecting a target protein where presence of the target protein cannot be detected directly, e.g., the assay results in high signal to noise ratio. For example, the target protein is bound to one or more antibodies that specifically bind to the target protein such that direct detection of the target protein is difficult. In certain circumstances the target protein may associate with one or more of the target proteins to create a multimer, e.g., a dimer, trimer, tetramer, pentamer, etc. For example the target protein may self associate. Binding of the antibodies to each of the target proteins in the multimer may prevent direct detection of the presence of the target protein because of steric hindrance created by the antibodies, especially if the relative size of the target protein is small compared to the size of the bound antibody. Additionally, epitopes useful for direct detection of the target protein may be buried within the multimer resulting in inaccessibility of the target protein to direct detection using techniques known in the art. Detection of the target protein may also be difficult if the concentration of the target protein in the sample being analyzed is very low.

[0118] Thus, in one aspect, the invention provides a method of detecting a target protein in a sample, wherein the target protein cannot be detected directly, wherein the method comprises the step of detecting a secondary antibody bound to a primary antibody, wherein the primary antibody is bound to the target protein, and wherein detection of the presence of the secondary antibody in the sample is indicative of the presence of the target protein. In certain embodiments the target protein cannot be detected directly due to steric hindrance by the primary antibody bound to the target protein.

[0119] For example, levels of LTa3 in a test sample containing as little as 6 ng/ml anti-LTa antibody 2C8vX cannot be accurately measured using a biotinylated anti- LTa antibody 2C8-biotin. The anti-LTa antibody 2C8vX binds to LTa3 and blocks it from being detected by the anti-LTa detection antibody 2C8-biotin (see also Example 1). In contrast, the presence of LTa3 can be detected and accurately measured using the methods of the present invention. In one embodiment the LTa3 is detected using an antibody that specifically binds to the anti-LTa antibody 2C8vX. The anti-LTa antibody 2C8vX is described in patent application no. PCT/US2007/081154 (published as WO 2008/063776).

[0120] The target protein may be a multimeric protein, e.g., a protein consisting of multiple monomers, such as but not limited to a dimer, trimer, tetramer, pentamer, etc. The target protein may be a multimeric protein consisting of identical monomers, e.g, a homotrimer, or it may be a multimeric protein consisting of non-identical monomers, e.g., a heterotrimer.

[0121] In some aspects of the methods of the invention, the secondary antibody is an anti-idiotype antibody that specifically binds to the variable region of the primary antibody. In certain embodiments the secondary antibody binds to one or more CDRs of the primary antibody.

[0122] In some embodiments the target protein is LTa and the primary antibody is an anti-LTa antibody. For example the anti-LTa antibody may be 2C8vX. In some embodiments the secondary antibody is an anti-idiotype antibody that specifically binds to the variable region of the anti-LTa antibody. For example, the anti-idiotype antibody specifically binds to CDR-L3 of the anti-LTa antibody, e.g., 2C8vX.

[0123] The invention also provides a method of detecting LTa in a sample in the presence of an anti-LTa antibody that specifically binds to LTa, wherein presence of the anti-LTa antibody prevents direct detection of LTa, the method comprising the step of detecting an anti-idiotype antibody that specifically binds to the anti-LTa antibody, wherein the anti-LTa antibody is bound to LTa, and wherein detection of the anti-idiotype antibody is indicative of the presence of LTa. In certain embodiments the anti-idiotype antibody binds specifically binds to one or more CDRs of the anti-LTa antibody. In some embodiments the anti-idiotype antibody binds to CDR-L3 of the anti-LTa antibody. In one aspect the anti-LTa antibody is 2C8vX and the anti-idiotype antibody binds to CDR- L3 of the anti-LTa antibody.

[0124] In any of the methods of the invention the sample may be obtained from serum, plasma, synovial fluid, tissue culture supernatants, tissue lysates or extracts and lymph or exudates. The sample may be obtained directly from a patient being treated with anti-LTa antibodies or may be obtained from tissue or cell cultures containing or exposed to anti-LTa antibodies.

[0125] Any embodiment described above or any combination thereof applies to any and all methods of the inventions described herein. [0126] Although in the foregoing description the invention is illustrated with reference to certain embodiments, it is not so limited. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All references cited throughout the specification, and the references cited therein, are hereby expressly incorporated by reference in their entirety.

EXAMPLES

Example 1 Assay for measurement of LTa3 in biological fluids

[0127] Briefly, Meso Scale Discovery (MSD) MA6000 High Bind 96 well assay plates (cat# LI lXB-1) were spotted with 5 uL of goat anti-TNFp polyclonal antibody (R&D Systems cat# AF-211-NA) at 2 ug/mL in spotting buffer (PBS pH 7.4, 0.05% Tween 20) and incubated for 1 hr at room temperature (RT; about 22-26°C). Plates were blocked with 150 uL/well blocking buffer (PBS + 5%> BSA) for 1-2 hours at room temperature with gentle agitation. Meanwhile, standards (a series of dilutions of recombinant human LTa3), controls (standard material made at three specific

concentrations targeting the high, mid, and low portions of the standard range), and samples were diluted in MSD Human Serum Cytokine Assay Diluent (cat# KOAOOC-4). Standard material (recombinant human LTa3) was diluted to 500 pg/mL and then further diluted serially 1 :2.5 for 8 dilutions (standard range: 500-0.7 pg/mL). Human serum was diluted 1 :2 for the initial dilution, then serially diluted 1 :2 for 8 dilutions. Plates were washed 3x with wash buffer (WB) consisting of PBS pH7.4, and 0.05%> Tween 20.

Twenty-five uL of each dilution of standards, controls, and samples were added to assigned individual wells on the plate according to the plate format. Plates were incubated 2 hr with gentle agitation. A biotinylated anti-LTa antibody, 2C8-biotin, was diluted to 2 ug/mL in Assay Diluent (AD) consisting of PBS pH7.4, 0.05% Tween 20, 0.5% BSA, and 10 ppm Proclin. Plates were washed 3x with WB and 25 uL of diluted 2C8-biotin was added to every well. Plates were incubated for 1 hour at RT with gentle agitation. Plates were washed 3x, and 25 uL Streptavidin-Ruthenium (MSD Cat# RA32-D) diluted to 500 ng/mL in AD was added to every well. Plates were incubated 30 minutes at RT with gentle agitation, washed 3x, then 150 μΐ lx read buffer T (MSD R92TC-3) was added to every well. Plates were read on a Meso Scale Discovery Sector Imager MA6000. A schematic diagram of this assay is depicted in Figure 1 A

[0128] The specificity of this assay is shown in Figure IB. The standard range for the assay is 0.7-500 pg/mL of LTa. The limit of detection of this assay for LTa3 is 0.7 pg/mL. The assay predominantly detects LTa3 homotrimers. It is approximately 10 fold less sensitive for detection of υΓα2β 1 and 100 fold less sensitive for detecting LTai 2. The assay does not detect TNFa or LIGHT.

[0129] The assay described in Figure 1 was tested for its tolerance to the presence of anti-LTa antibody in the sample being analyzed. Three different biologically relevant concentrations of recombinant human LTa (4, 20, and 100 pg/mL) were preincubated with a titration of anti-LT antibody 2C8vX ranging from 3ng/mL to 300 ug/mL. Each mixture was tested in the assay as a sample, to evaluate recovery of LTa3 quantitation.

[0130] The results of this experiment are shown in Figure 2. The data indicate that the assay depicted in Figure 1A cannot accurately measure the levels of LTa3 in the presence of as little as 6 ng/mL anti-LTa antibody 2C8vX. With increasing concentrations of anti-LTa3 antibody 2C8vX present, the ability of the assay to detect LTa3 is inhibited. The anti-LTa antibody 2C8vX binds to LTa3 and blocks it from being detected by the biotinylated anti-LTa detection antibody (2C8-biotin). In addition, a panel of 34 different monoclonal and polyclonal anti-LT a3 antibodies were also unable to accurately measure the levels of LTa3 in the presence of anti-LTa antibody 2C8vX suggesting that the interference of detecting LTa3 is not epitope specific but may be due, at least in part, to steric hindrance caused by the presence of anti-LTa antibody 2C8vX.

Example 2 Modified assay for measuring levels of LTa3 in the presence of anti-LTa antibody

[0131] A schematic diagram for a modified assay for measuring LTa3 in the presence of anti-LTa antibody is shown in Figure 3. Briefly, Nunc 96 well immunoplates (cat# 4-39454) were coated overnight at 4°C with goat anti-TNFp polyclonal antibody (R&D Systems cat# AF-211-NA) at 2 ug/mL in 100 uL of coating buffer (0.05 M carbonate/bicarbonate, pH 9.6). Plates were washed 3x with 250 uL WB, and blocked with 150 uL/well blocking buffer (PBS, 0.5% BSA, 10 ppm Proclin, pH 7.4) for 1-2 hours at room temperature (about 22-26°C) with gentle agitation. Meanwhile, standards, controls, and samples were prepared in High Salt Assay Diluent (HSAD) consisting of PBS pH 7.4, 0.1% Tween 20, 0.5% BSA, lOppp Proclin, 0.2% BGG, 0.25% CHAPS, 5mM EDTA, 350mM NaCl). Fetal bovine serum (Sigma) at 5% was added to the HSAD. Standard material (recombinant rhesus LTa3, or recombinant human LTa3) was diluted to 20 ng/mL (2x), and then further diluted serially 1 :2.5 for 7 dilutions (standard range: 10 ng - 40 pg/mL). Cynomolgus monkey or human serum was diluted 1 :2 for the initial dilution (2x), then serially diluted 1 :2 for 8 dilutions. 2C8vX anti-LTa antibody was diluted to 20 ug/mL (2x). 60 uL of standards, controls, or serum dilutions, was each mixed with 60 uL of 20 ug/mL 2C8vX (total volume in each tube 120 uL), and pre-incubated 1 hour at room temperature (about 22-26°C) with gentle agitation. Plates were then washed three times with WB, and 100 uL of preincubated mixtures were added to individual appropriate wells according to the plate format. Plates were incubated overnight at 4°C with gentle agitation. The next day, plates were washed 6 times with WB and 100 uL of 200 ng/mL biotinylated anti-Id MAb (anti-2C8vX-biotin) diluted in HSAD was added to every well. Plates were incubated for 1 hour at RT (about 22-26°C) with gentle agitation. Plates were washed 6 times with WB and 100 uL SA-HRP polymer (Sigma cat# S-2438) diluted to 500 ng/mL in AD was added to every well. Plates were incubated 30 minutes at RT (about 22-26°C) with gentle agitation, washed again 6 times with WB, then 100 μΐ of Moss TMB

peroxidase (cat # TMBE-1000) was added to every well. Plates were incubated 15-20 minutes at RT (about 22-26°C) to allow color to develop and then 100 μΐ of 1 M H₃PO₄ was added to each well to stop development. Plates were read on a microtiter plate reader at O.D. at 650 nm. A schematic of this assay is shown in Figure 3.

[0132] In Figure 4A, drug tolerance of the assay described above was measured. Each concentration of the recombinant rhesus monkey LTa3 standard curve was combined with each of various 2C8vX concentrations such that the 2C8vX

concentration in assay tubes was 2X that shown in Figure 4 A (0-50ug/mL) in a volume of 60 uL. Then 60uL of 20 ug/mL 2C8vX (2x) was added (total volume in each tube 120 uL), and the mixtures were pre-incubated 1 hour at room temperature (about 22-26°C) with gentle agitation. Plates were then washed and 100 uL of preincubated mixtures were added to individual appropriate wells according to the plate format. The remainder of the assay was carried out as described in paragraph 0123.

[0133] In Figure 4B, cyno serum tolerance of the assay was measured. Each concentration of the recombinant rhesus monkey LTa3 standard curve was combined with each of various serum concentrations such that the serum concentration in assay tubes was 2X that shown in Figure 4B (0-30%) in a volume of 60 uL. Then 60uL of 20 ug/mL 2C8vX (2x) was added (total volume in each tube 120 uL), and the mixtures were pre- incubated 1 hour at room temperature (about 22-26°C) with gentle agitation. Plates were then washed and 100 uL of preincubated mixtures were added to individual appropriate wells according to the plate format. The remainder of the assay was carried out as described in paragraph 0123.

[0134] In Figure 5, both drug and serum tolerance were tested. In this case each dilution of recombinant human LTa3 standard curve was combined with drug or human serum such that the final concentration in the assay tubes was 25ug/mL 2C8vX or 25% serum. This was compared to no drug or serum combined with the standard curve. The remainder of the assay was carried out as described above. Results from these experiments show that the modified assay can accurately measure rhesus monkey LTa3 in the presence of varying amounts of anti-LTa antibody (Figure 4A), varying amounts of cynomolgus monkey serum (Figure 4B).

[0135] The assay can accurately measure human LTa3 in the presence of up to 25 ug/mL anti-LTa antibody and up to 25% human serum (Figure 5).

Example 3 Use of the modified assay to measure endogenous LTa3 levels in animals dosed with anti-LTa antibody 2C8vX

[0136] The assay described in Example 2 was used to measure endogenous LTa3 levels in cynomolgus monkeys dosed with anti-LTa antibody. The levels of anti- LTa antibody in each monkey's serum was also measured. Twenty cynomolgus monkeys (4 animals in each of 5 dosage groups) were given a single dose of wildtype (WT) or afucosylated (AF) 2C8vX. Group 1 received 0.15 mg/kg AF anti-LTa intravenously (IV); group 2 received 1.5 mg/kg AF anti-LTa intravenously, group 3 received 1.5 mg/kg AF anti-LTa subcutaneously, group 4 received 15 mg/kg AF anti-LTa intravenously, group 5 received 1.5 mg/kg WT anti-LTa intravenously. Blood was drawn and serum prepared at the following time points: pre-dose, days 2, 4, 8, 11, 15, 22, 29, 36 and 50. The level of anti-LTa antibody in the serum of each monkey was determined at these time points using the method described below. The level of endogenous LTa in the serum of each monkey was also determined using the modified assay described in Example 2. Measurement of anti-LTa antibody in cyno serum

[0137] Nunc 96 well immunoplates (cat# 4-39454) were coated overnight at 4°C with recombinant human LTa at 0.5 ug/mL in 100 uL of CB. Plates were washed 3x with 250 uL WB and blocked with 200 uL/well blocking buffer (AD + 3% BSA pH 7.4) for 1-2 hours at room temperature (about 22-26°C) with gentle agitation. Meanwhile, standards (a titration curve of various concentrations of anti-LTa antibody 2C8vX), controls (3 concentrations of 2C8vX that quantitate at high mid, and low points on the standard curve), and monkey serum samples were prepared in HSAD. Standard material (anti-LTa antibody 2C8vX) was diluted to 250 ng/mL and then further diluted 1 :2 for 11 dilutions (standard range: 250-0.24 ng/mL final). Cyno serum obtained from each monkey at each time point was diluted into the range of the assay for the initial dilution then serially diluted 1 :3 for 8 dilutions. Plates were then washed 3x with WB and 100 uL of standards, controls, and cyno serum samples were added to appropriate wells according to the plate format. Plates were incubated for 2 hr with gentle agitation. Plates were washed 6x, and 100 uL of 100 ng/mL peroxidase conjugated AffmiPure F(ab')2 fragment goat anti- human IgG, Fey frag specific (Jackson ImmunoResearch cat# 109-036-098) diluted in AD was added to each well. Plates were incubated for 1 hour at RT with gentle agitation. Plates were washed again 6x, then 100 μΐ of TMB (SureBlue Microwell Peroxidase Substrate, 1 -component, KPL Products 52-00-03) was added per well. Plates were incubated 15-20 minutes at RT (about 22-26°C) to allow color to develop and then 100 μΐ of 1 M H₃PO₄ was added to each well to stop development. Plates were read on a microtiter plate reader at O.D. at 450/650 nm.

[0138] Results of these experiments are show in Figure 6. The resulting PK curves show the averages and standard deviations of the 4 monkeys in each group. The results indicate that LTa in the monkey's circulation is stabilized by being bound by antibody, as levels increased up to 12,000 fold (up to 12 ng/mL) following antibody administration. The LTa levels correlated with the dose of antibody the animals received. LTa levels then decreased as the levels of antibody decreased, and fell below the assay detection limit of 200 pg/mL when the antibody levels fell below approximately 200 ng/mL. These assays have also been used to measure anti-LTa and LTa levels in monkeys receiving multiple doses of anti-LTa antibody and similar results were obtained.

Claims

WHAT IS CLAIMED:

1. A method of detecting a target protein in a sample, wherein the target protein cannot be detected directly, the method comprising,

detecting a secondary antibody bound to a primary antibody wherein the primary antibody is bound to the target protein,

wherein detection of the secondary antibody is indicative of the presence of the target protein.

2. The method of claim 1 , wherein the target protein is a multimeric protein.

3. The method of claim 1, wherein the target protein is a homotrimer or heterotrimer.

4. The method of claim 1, wherein the target protein is lymphotoxin alpha (LT ).

5. The method of claim 1, wherein the target protein cannot be detected directly due to steric hindrance by the primary antibody bound to the target.

6. The method of claim 1 , wherein the secondary antibody specifically binds to the variable region of the primary antibody.

7. The method of claim 6, wherein the secondary antibody specifically binds to one or more CDRs of the primary antibody.

8. The method of claim 4, wherein the primary antibody is an anti-LTa antibody.

9. The method of claim 8, wherein the anti-LTa antibody is 2C8vX.

10. The method of claim 8, wherein the secondary antibody is anti-idiotype antibody.

11. The method of claim 1 , wherein the sample is from serum, plasma, lymph, synovial fluid, cerebro-spinal fluid, tissue lysates or extracts, exudates or cell culture supematants.

12. A method of detecting LTa in a sample in the presence of a primary antibody that specifically binds to LTa, wherein presence of the primary antibody prevents direct detection of LTa, the method comprising,

detecting a secondary antibody bound to the primary antibody wherein the primary antibody is bound to LTa,

wherein detection of the secondary antibody is indicative of the presence of LTa.

13. The method of claim 12, wherein the secondary antibody specifically binds to the variable region of the primary antibody.

14. The method of claim 13, wherein the secondary antibody specifically binds to one or more CDRs of the primary antibody.

15. The method of claim 12, wherein the primary antibody is an anti-LTa antibody.

16. The method of claim 15, wherein the anti-LTa antibody is 2C8vX.

17. The method of claim 12, wherein the secondary antibody is an anti-idiotype antibody.

18. The method of claim 12, wherein the sample is from serum, plasma, lymph, synovial fluid, tissue lysates or extracts, exudates or cell culture supernatants.

19. An isolated anti-idiotype antibody that specifically binds to a variable region of an anti-LTa antibody.

20. The antibody of claim 19, wherein the anti-LTa antibody is 2C8vX.

21. The antibody of claim 19, wherein the anti-idiotype antibody binds to one or more CDRs of the anti-LTa antibody.

22. The antibody of claim 21, wherein the anti-idiotype antibody binds to CDR-L3 of the anti-LTa antibody.