CA2581017C - Compositions monovalent for cd40l binding and methods of use - Google Patents

Abstract

The invention relates to antibody polypeptides that monovalently bind CD40L. Antibody polypeptides that are monovalent for binding of CD40L can inhibit CD40L activity while avoiding potential undesirable effects that can occur with antibodies capable of divalent or multivalent binding of CD40L. In one aspect, a monovalent anti-CD40L antibody polypeptide consists of or comprises a single immunoglobulin variable domain that specifically binds and antagonizes the activity of CD40L, preferably without substantially agonizing CD40 and/or CD40L activity. In another aspect, the monovalent anti-CD40L antibody polypeptide is a human antibody polypeptide. The invention further encompasses methods of antagonizing CD40/CD40L interactions in an individual and methods of treating diseases or disorders involving CD40/CD40L interactions, the methods involving administering a monovalent anti-CD40L antibody polypeptide to the individual.

Description

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OF USE
BACKGROUND OF THE INVENTION
CD40 is a 50 lcD cell surface glycoprotein molecule expressed on the surface of mature and immature B cells, macrophages, follicular dendritic cells, thymic epithelium, normal basal epithelium, and some tumor-derived cell lines. The molecule is a member of the TNF receptor family, and has important signaling functions leading to a variety of downstream effects in various cell types.
Early studies showed that cross-linking of CD40 on the B cell surface with an antibody resulted in B cell proliferation and activation. Antibody cross linking of CD40 in the presence of IL-4 induces proliferation and class switching in vitro, B cell aggregation via LFA-1 (Gordon et al., 1988, J. Immunol. 140: 1425), and serine/threonine and tyrosine phosphorylation of a number of intracellular substrates (Gordon et al., 1988, supra; Uckun et al., 1991, J. Biol. Chem. 266:17478). Anti-CD40 monoclonal antibodies also prime B cells to proliferate in response to agents such as PMA
(Gordon et al., 1987, Eur. J. Immunol. 17: 1535) and anti-CD20 antibody (Clark &
Ledbetter, 1986, Proc. Natl. Acad. Sci. U.S.A. 83: 4494).
The receptor homology of CD40 and the antibody cross-linking studies showing a central role for CD40 in B cell activation prompted the search for a natural ligand. A mutant of the Jurkat T cell line was found to constitutively activate human B cells to secrete immunoglobulin (Yellin et al., 1991, J. Immunol. 147: 3389-3395).
A monoclonal antibody, termed 5c8, was raised which specifically reacted with the mutant line, but not with the parental Jurkat cell line. The 5c8 antibody immunoprecipitated a 30 lcD (more accurately, 29.3 IcD, 261 amino acids) cell surface polypeptide and was found to specifically inhibit the B cell helper function of the mutant cell line. (Lederman et al., 1992, J. Exp. Med., 175: 1091-1101;
Lederman et al., 1992, J. Immunol. 149: 3817-3826; Lederman et al., 1993, Curr. Opin.
Immunol.
5: 439-444;). The 30 IcD polypeptide ligand of the 5c8 antibody was termed T-BAM, for T-B-cell Activating Molecule. A second line of studies used molecular cloning SUBSTITUTE SHEET (RULE 26) techniques to identify polypeptides that specifically bind the CD40 molecule.
cDNA
clones for a specific ligand of CD40 were identified in a CD40 binding assay and alternately termed CD40 Ligand (CD4OL), gp39, CD154, or TRAP (Graf et al., 1992, Eur. J. Immunol. 22: 3191-3194; Armitage et al., 1992, Nature 357: 80-82; and Aruffo et al., 1993, Cell 72: 291-300). Subsequently, the CD4OL clone was found to have the same structure as T-BAM (Covey et al., 1994, Mol. Immunol. 31: 471-484).
Human CD4OL protein shows 82.8% and 77.4% identity at the nucleic acid and amino acid levels, respectively, to a similar protein isolated from murine EL4 thymoma cells. Both of these proteins are ligands for CD40 cell surface antigen expressed on resting B cells. CD4OL has also been described as IMD3, a protein involved in hyper-IgM immunodeficiency syndrome.
The human gene encoding CD4OL maps to chromosome Xq26.3-q27. The gene contains five exons. Deletions, point mutations and frameshift mutations clustering within a limited region of the CD4OL extracellular domain have been found to be the basis of a rare X-linked immunodeficiency syndrome (Hyper-IgM
immunodeficiency syndrome, HIGM1) characterized by recurrent bacterial infections, very low or absent IgG, IgA and IgE, and noimal to increased IgM and IgD serum levels. Causally-related mutations have been found to consist of clustered deletions arising by splice-donor mutations with exon skipping, splice-acceptor mutations with utilization of a cryptic splice site, and deletion/insertion events with the creation of a new splice site.
CD4OL is expressed on activated, but not resting CD4+ T cells, and was found to play a particularly important role in the humoral immune response, being linked to B cell proliferation, antibody and cytokine production, and cell viability. In vivo, deletion or mutation of CD4OL leads to severe immunodeficiency, both in mice and in humans, characterized by hypogammaglobulinemia and T cell deficits in cell-mediated immunity (Chess, C., 2001, in Therapeutic Immunology, 2nd edition, Austen, K.F., Burakoff, S., Rosen, F. and Strom, T., eds., Blackwell Sciences, pp.
441-456). Human CD4+ T cells infected by HIV1, which causes severe dysfunction of cellular immunity, but paradoxically results in intense polyclonal activation of B

2 cells, do not express CD4OL. Gene and cell surface expression of the CD4OL by activated T cells has been shown to be depressed in a subgroup of patients with common variable immunodeficiency (CVI). Thus, inefficient signaling via CD40 may be responsible, at least in part, for the failure of B cell differentiation in these patients.
The functional consequences of CD4OL binding to CD40 include, for example, a) rescuing B cells from apoptosis induced by Fas or cross-linking of IgM, b) induction of the co-stimulator molecules CD80 (B7-1) and CD86 (B7-2) which interact with CD28 and CD152 (CTLA-4) on the surface of activated T cells; c) =
increased expression of other cell surface activation molecules including CD23, CD54, CD95 and lymphotoxin-a; and d) inducing immunoglobulin class switching (see Chess, supra, and references 25, 44, and 47-60 cited therein). CD4OL
binding to CD40 also augments the antigen-presenting functions of dendritic cells, inducing maintenance of high levels of 1VIEIC class II antigens and upregulation of accessory molecules including CD58 (LFA-3). CD4OL induces cytokine production and tumoricidal activity in peripheral blood monocytes. CD4OL also co-stimulates the proliferation of activated T cells, and the co-stimulation is accompanied by the production of IFN-y, TNF-a and IL2. The expression of CD4OL on murine T-helper cells and CD4+ T cells is inhibited by IFNI, and is inhibited on T-helper-type 2 cells by TGF-P.
CD4OL upregulates the expression of CD54 by cultured Hodgkin and Reed-Sternberg cells. The increased CD54 surface expression is accompanied by increased shedding of surface-bound CD54.
CD4OL has also been suggested to be important in the induction of tolerance ¨
CD80 and CD86, which are upregulated by CD4OL, interact with CD28 to provide essential co-stimulation of T cells, in concert with T cell receptor activation, that results in full activation of T cells. In the absence of CD80 and CD86-triggered activation of CD28, anergy or tolerance occurs as a consequence of antigen triggering (Linsley & Ledbetter, 1993, Ann. Rev. Immunol. 11 191-212; Jenkins et al., 1993,

3 Curr Opin. Immunol. 5: 361-367; and Boussiotis et al., 1996, Immunol. Rev.
153: 5-26).
The CD4OL/CD40 pathway has been implicated in the in vivo priming of CD8+ cytotoxic T lymphocytes (CTSs) by CD4+ T cells. As noted, CD4OL
expressed on the surface of activated CD4+ T cells interacts with CD40 expressed on dendritic cells, inducing the dendritic cells to express more MEIC, and signaling through CD40 can replace the requirement for CD4+ T-helper cells in priming CD8+
CTL responses. Blockade of CD4OL inhibits CTL priming, emphasizing the vital role of CD4OL/CD40 interactions in CTL priming by helper T cells (Ridge et al., 1998, Nature 393: 474-478; Schoenberger et al., 1998, Nature 393: 480-483; Bennett et al., 1998, Nature 393: 478-480).
CD4OL can also mediate functional interactions of CD4+ T cells with other cells that express CD40, such as fibroblasts, synovial cells and endothelial cells (Yellin et al., 1995, J. Leuko. Biol. 58: 209-216; Yellin et al., 1995, J.
Exp. Med. 182:
1857-1864). CD4OL induces the expression of CD54 (ICAM-1) and CD106 (VCAM-1) by fibroblasts, as well as increasing fibroblast IL-6, collagenase and collagen production and inducing fibroblast proliferation. Thus, CD4OL/CD40 interactions may be involved in the induction of fibrosis associated with autoinimunity and immune responses.
CD4OL interaction with CD40 induces endothelial cells to express CD62E (E-selectin), ICAM-1 and VCAM-1. The upregulation of these adhesion molecules may be involved in the binding of inflammatory cells to vascular endothelium and the subsequent migration of the inflammatory cells to sites of inflammation. CD4OL

blockade retards the migration of leukocytes through endothelial cell barriers. In animal models of autoimmunity, antibodies to CD4OL interfere with the accumulation of inflammatory cells at the site of inflammation.
= CD40/CD4OL interactions have been implicated in diseases having an immune or autoimmune connection. Animal models of immune-related disease in which the CD4OL/CD40 pathway has been demonstrated to play a role in the

4 pathology include, for example, murine models of systemic lupus erythematosis (Lupus or SLE; see, e.g., Kalled et al., 1998, J. Immunol. 160: 2158-2165), arthritis (collagen-induced arthritis, see, e.g., Durie et al., 1993, Science 261: 1328-1330), multiple sclerosis (experimental autoimmune encephalomyelitis, EAE; see, e.g., Howard et al., 1999, J. Clin . Invest. 103: 281-290), autoimmune thyroiditis (experimental autoimmune thyroiditis, EAT; see, e.g., Caryanniotis et al., 1997, Immunology 90: 421-426), colitis (hapten-induced colitis; see, e.g., Stuber et al., 1996, J. Exp. Med. 183: 693-698), atherosclerosis and coronary artery disease (see, e.g., Mach et al., 1998, Nature 394: 200-203), and allog,raft rejection (see, e.g., Parker et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 92: 9560-9564; Kirk et al., 1997, Proc.
Natl. Acad. Sci. U.S.A. 94: 8789-8794; Larsen et al., 1996, Nature 381: 434-438 and Blazar et al., 1997, J. Immunol. 158: 29-39).
CD4OL antibody trials for treatment of human immune-related diseases include studies in patients with Lupus (see, e.g., Huang et al., 2002, Arthritis Rheum.
46: 1554-1562). A phase I trial demonstrated that anti-CD4OL humanized monoclonal antibody (IDEC-131) is safe and well tolerated by patients with Lupus (Davis et al., 2001, J. Rheumatol. 28: 95-101). A phase II study with the IDEC-antibody showed improvement in clinical symptoms, but efficacy of the drug over placebo controls was not demonstrated (Kalunian et al., 2002, Arthritis Rheum.
46:
3251-3258). In a phase II study with BG9588 anti-CD4OL antibody, clinical efficacy was demonstrated, but the study was terminated due to the occurrence of thromboembolic events (Boumpas et al., 2003, Arthritis Rheum. 48: 719-727).
U.S. Patent Nos. 5,474,771 (Lederman et al.) and 5,876,950 (Siadak et al.) disclose murine monoclonal antibodies specific for different epitopes of human gp39.
W095/06666 (Noelle & Foy) discloses murine anti-gp39 antibodies.
U.S. Patent No. 6,328,964 (Noelle & Claassen) discloses methods for the treatment of multiple sclerosis using gp39-specific antibodies.
U.S. Patent No. 5,747,037 (Noelle et al.), and EP0721469B1 (Ledbetter et al.) and its U.S. counterpart U.S. 5,869,049 disclose anti-human monoclonal (mouse) .5 antibodies specific for gp39. U.S. Patent No. 5,876,718 (Noelle et al.) discloses methods of inducing T cell non-responsiveness to transplanted tissues and of treating graft-versus-host disease with anti-gp39 monoclonal (mouse) antibodies.
EP0742721B1 (Noelle et al.) discloses methods of inhibiting a 'humoral immune response to a thymus-dependent ,antigen that use anti-gp39 monoclonal (mouse) antibodies. U.S. Patent No. 6,375,950 describes methods for inducing T cell unresponsiveness to donor tissue or organs in a transplant recipient through use of anti-gp39 monoclonal (murine) antibodies.
EP1005372B1 (De Boer et al.) describes methods for the selective killing of autoreactive CD40L+ T cells using anti-CD4OL monoclonal (mouse) antibody-toxin fusion proteins.
U.S. Patent No. 6,340,459 (Yellin et al.) describes the use of murine anti gp39 monoclonal antibody 5c8 for the treatment or prevention of reperfusion injury.
EP0831906B1 (Claassen et al.) describes methods for the treatment of T cell-mediated tissue destruction in autoimmune diseases such as multiple sclerosis using anti-gp39 monoclonal (mouse) antibodies. Antibodies used in therapeutic approaches in the prior art have been divalent antibodies of murine origin.
A number of smaller antigen binding fragments of naturally occurring antibodies have been identified following protease digestion. These include, for example, the "Fab fragment" (VL-CL-CH1-VH), "Fab' fragment" (a Fab with the heavy chain hinge region) and "F(ab'), fragment" (a dimer of Fab' fragments joined by the heavy chain hinge region). Recombinant methods have been used to generate even smaller antigen-binding fragments, referred to as "single chain Fv"
(variable fragment) or "scFv," consisting of VL and VH joined by a synthetic peptide linker.
While the antigen binding unit of a natura11y-o6curring antibody (e.g., in humans and most other mammals) is generally known to be comprised of a pair of V
regions (VL/VH), camelid species express a large proportion of fully functional, highly specific antibodies that are devoid of light chain sequences. The camelid heavy chain antibodies are found as homodimers of a single heavy chain, dimerized via their constant regions. The variable domains of these camelid heavy chain antibodies are referred to as VH14 domains and retain the ability, when isolated as fragments of the VH chain, to bind antigen with high specificity ((Hamers-Casterman et al., 1993, Nature 363: 446-448; Gahroudi et al., 1997, FEBS Lett. 414: 521-526). Antigen binding single VH domains have also been identified from, for example, a library of murine VH genes amplified from genomic DNA from the spleens of immunized mice and expressed in E. coli (Ward et al., 1989, Nature 341: 544-546). Ward et al.
named the isolated single VH domains "dAbs," for "domain antibodies." The term "dAb"

will refer herein to an antibody single variable domain (VH or VL) polypeptide that specifically binds antigen. A "dAb" binds antigen independently of other V
domains;
however, as the term is used herein, a "dAb" can be present in a homo- or heteromultimer with other VH or VL domains where the other domains are not required for antigen binding by the dAb, i.e., where the dAb binds antigen independently of the additional VH or VL domains.
Antibody single variable domains, for example, VHH, are the smallest antigen-binding antibody unit known. For use in therapy, human antibodies are preferred, primarily because they are not as likely to provoke an immune response when administered to a patient. As noted above, isolated non-camelid VH domains tend to be relatively insoluble and are often poorly expressed. Comparisons of camelid VHH
with the VH domains of human antibodies reveals several key differences in the framework regions of the camelid VBH domain corresponding to the VH/VL
interface of the human VH domains. Mutation of these residues of human VH3 to more closely resemble the Vat.' sequence (specifically Gly 44¨*Glu, Leu 45--->Arg and Trp 47¨Gly) has been performed to produce "camelized" human VH domains that retain antigen binding activity (Davies & Riechmann, 1994, FEBS Lett. 339: 285-290) yet have improved expression and solubility. (Variable domain amino acid numbering used herein is consistent with the Kabat numbering convention (Kabat et al., 1991, Sequences of Immunological Interest, 5th ed. U.S. Dept. Health & Human Services, Washington, D.C.)) WO 03/035694 (Muyldermans) reports that the Trp 103¨ Arg mutation improves the solubility of non-camelid VH domains. Davies & Riechmann (1995, Biotechnology N.Y. 13: 475-479) also report production of a phage-displayed repertoire of camelized human VH domains and selection of clones that bind hapten with affinities in the range of 100-400 nM, but clones selected for binding to protein antigen had weaker affinities.
While many antibodies and their derivatives are useful for diagnosis and therapy, the ideal pharmacokinetics of antibodies are often not achieved for a particular application. In order to provide improvement in the pharmacokinetics of antibody molecules, the present invention provides single domain variable region polypeptides that are linked to polymers which provide increased stability and half-life. The attachment of polymer molecules (e.g., polyethylene glycol; PEG) to proteins is well established and has been shown to modulate the phamiacokinetic properties of the modified proteins. For example, PEG modification of proteins has been shown to alter the in vivo circulating half-life, antigenicity, solubility, and resistance to proteolysis of the protein (Abuchowski et al., J. Biol. Chem.
1977, 252:3578; Nucci et al., Adv. Drug Delivery .Reviews 1991, 6:133; Francis et al., Pharmaceutical Biotechnology Vol. 3 (Borchardt, R. T. ed.); and Stability of Protein Pharmaceuticals: in vivo Pathways of Degradation and Strategies for Protein Stabilization 1991 pp235-263, Plenum, NY).
Both site-specific and random PEGylation of protein molecules is known in the art (See, for example, Zalipsky and Lee, Polv(ethvlene glycol) Chemistry:
Biotechnical and Biomedical Applications 1992, pp 347-370, Plenum, NY; Goodson and Katre, 1990, Bio/Techno/op-,y, 8:343; Hershfield et al., 1991, PNAS
88:7185).
More specifically, random PEGylation of antibody molecules has been described at lysine residues and thiolated derivatives (Ling and Mattiasson, 1983, Immunol.

Methods 59: 327; Wilkinson et al., 1987, Immunol. Letters, 15: 17; Kitamura et al., 1991, Cancer Res. 51:4310; Delgado et al., 1996 Br. J. Cancer, 73: 175; Pedley et al., 1994, Br. J. Cancer, 70:1126).
SUMMARY OF THE INVENTION
The invention relates to antibody polypeptides that monovalently bind CD4OL. Because of the clear importance of CD4OL in the production of antibodies, the CD40/CD4OL interaction and pathways present important targets for the development of therapeutic approaches for the treatment of diseases and disorders that involve inappropriate or excessive antibody responses, such as autoimmune diseases. Antibody polypeptides that are monovalent for binding of CD4OL can inhibit CD4OL activity, including binding and activation of CD40 on the B cell surface and downstream effects, while avoiding potential undesirable effects that can occur with antibodies capable of divalent or multivalent binding of CD4OL.
Monovalent anti-CD4OL antibody polypeptides can also be applied to any of a number of uses for which standard divalent antibodies are also used, e.g., in vivo imaging and diagnosis.
In one aspect, the antibody polypeptide consists of or comprises a single immunoglobulin variable domain that specifically binds and antagonizes the activity of CD4OL, preferably without substantially agonizing CD40 and/or CD4OL
activity.
In another aspect, because human antibodies will avoid the generation of an immune response to the antibodies when administered to human subjects for the treatment or prevention of disease, the antibody polypeptide is a human antibody polypeptide that monovalently binds CD4OL, preferably without substantially agonizing CD40 and/or CD4OL activity.
In summary then, in one embodiment, the invention provides an antibody polypeptide, preferably a human antibody polypeptide, that is monovalent for binding to CD4OL (gp39).
In one embodiment, the human antibody polypeptide dissociates from human CD4OL with a Kd in the range of 50 nM to 20 pM, inclusive, as measured by surface plasmon resonance. For example, the Kd for human CD4OL can be 25 nM to 20 pM, 10 nM to 20 pM, 5 nm to 20 pM, 1 nM to 20 pM, 0.5 nM to 20 pM, 0.1 nM to 20 pM, 0.1 nM to 50 nM, 75 pM to 20 pM or even 50 pM to 20 pM.
Unless otherwise stated, all ranges described herein are inclusive of the specific endpoints.

In another embodiment, the antibody polypeptide inhibits the binding of CD4OL to CD40.
In another embodiment, the binding of the antibody polypeptide to CD4OL
does not substantially agonize CD40 and/or CD4OL activity.
In another embodiment, the human antibody polypeptide inhibits the binding of CD40 to CD4OL, and does not substantially agonize signaling by CD40.
In another embodiment, the binding of the antibody polypeptide to CD4OL
does not substantially induce JNK phosphorylation in Jurkat T-cells.
In another embodiment, the binding of the antibody polypeptide to CD4OL
does not substantially induce IFN-y secretion by Jurkat T-cells co-stimulated with anti-CD3 antibody.
In another embodiment, the presence of the antibody polypeptide in a standard platelet aggregation assay does not result in aggregation of more than 25%
over the aggregation observed in a negative control assay performed without the addition of antibody.
In another embodiment, the human antibody polypeptide comprises a single immunoglobulin variable domain that binds CD4OL. In a preferred embodiment, the single immunoglobulin variable domain is a VH or a VL domain.
In another embodiment, the antibody polypeptide is selected from the group consisting of a dAb, a FAb, an scFv, an Fv, or a disulfide-bonded Fv.
In another embodiment, the human antibody polypeptide is PEG-linked. In one embodiment, the PEG is covalently linked to the human antibody polypeptide. In one preferred embodiment, the PEG-linked human antibody polypeptide has a hydrodynamic size of at least 24 k.D. In another preferred embodiment, the PEG
is linked to the antibody polypeptide at a cysteine or lysine residue. In another preferred embodiment, the total PEG size is from 20 to 60 kl), inclusive. In another preferred embodiment, the PEG-linked human antibody polypeptide has a hydrodynamic size of at least 200 kl).
In one embodiment, the antibody polypeptide has an increased in vivo half-life relative to the same antibody polypeptide composition lacking polyethylene glycol.
In another embodiment, the t-half life of the antibody polypeptide composition is increased by 10% or more. In another embodiment, the t-half life of the antibody polypeptide composition is increased by 50% or more. In another embodiment, the ta-half life of the antibody polypeptide composition is increased by 2X or more. In another embodiment, the t-half life of the antibody polypeptide composition is increased by 5X or more, e.g., 10X, 15X, 20X, 25X, 30X, 40X, or more. In another embodiment, the t-half life of the antibody polypeptide composition is increased by 50X or more.
In another embodiment, the PEG-linked antibody polypeptide has a ta half-life of 0.25 to 6 hours, inclusive. In another embodiment, the tcx half-life is in the range of 30 minutes to 12 hours, inclusive. In another embodiment, the ta-half life of the antibody polypeptide composition is in the range of 1 to 6 hours.
In another embodiment, the t13-half life of the antibody polypeptide composition is increased by 10% or more. In another embodiment, the ti3-half life of the antibody polypeptide composition is increased by 50% or more. In another embodiment, the 13-half life of the antibody polypeptide composition is increased by 2X or more. In another embodiment, the t13-half life of the antibody polypeptide composition is increased by 5X or more, e.g., 10X, 15X, 20X, 25X, 30X, 40X, .or more. In another embodiment, the 43-half life of the antibody polypeptide composition is increased by 50X or more.
In another embodiment, the antibody polypeptide composition has a ti3 half-life of 1 to 170 hours, inclusive. In another embodiment, the 43-half life is in the range of 12 to 48 hours, inclusive. In another embodiment, the ti3-half life is in the range of 12 to 26 hours, inclusive.
In addition, or alternatively to the above criteria, the present invention provides a dAb containing composition comprising a ligand according to the invention having an AUC value (area under the curve) in the range of 1 mg.min/ml or more. In one embodiment, the lower end of the range is 5, 10, 15, 20, 30, 100, 200 or 300 mg.min/ml. In addition, or alternatively, a ligand or composition according to the invention has an AUC in the range of up to 600 mg.min/ml. In one embodiment, the upper end of the range is 500, 400, 300, 200, 150, 100, 75 or 50 mg.min/ml.
Advantageously a ligand according to the invention will have an AUC in the range selected from the group consisting of the following: 15 to 150 mg.min/ml, 15 to 100 mg.min/ml, 15 to 75 mg.min/ml, and 15 to 50 mg.min/ml.
In another embodiment, the antibody polypeptides described herein can be linked to human serum albumin (HSA), which also has the effect of increasing the in vivo half life of the molecule. The human serum albumin coding sequences can be obtained by PCR using primers derived from the cDNA sequence available at GenBank Accession No. NM000477. Such coding sequences can be fused to the coding sequence for a monovalent anti-CD4OL antibody polypeptide as described herein, and the fusion can be expressed by one of skill in the art.
In another embodiment, the ta-half life of the HSA-linked human antibody polypeptide composition is increased by 10% or more.
In another embodiment, the ta-half life of the HSA-linked human antibody polypeptide composition is in the range of 0.25 hours to 6 hours.
In another embodiment, the ti3.-half life of the HSA-linked human antibody polypeptide composition is increased by 10% or more.
In another embodiment, the ti3-half life of the HSA-linked human antibody polypeptide composition is in the range of 12 to 48 hours.

In another embodiment; the human antibody polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 7-82 and 246-360.
In another embodiment, the human antibody polypeptide inhibits binding of CD4OL to CD40 with an IC50 in the range of 20 p1\4 to 1.5 1\4, inclusive;
IC50 for inhibition of CD4OL binding to CD40 in any embodiment described herein is preferably measured as described herein in Example 6. The IC50 can preferably be in the range of 20 p1\4 to 1 u1\4, 20 p1\4 to 900 nM, 20 p1\4 to 800 nM, 20 pM to 700 nM, 20 p1\4 to 600 nM, 20 pM to 500 nM, 20 pIVI to 400 nI\4, 20 pM to 300 n1\4, 20 pM to 200 n1\4, 20 pM to 100 nM, or 20 ply' to 50 nI\4. Further acceptable or preferred ranges include, for example, 50 pM to 1 uM, 100 pM to 500 nM, 125 pM to 250 n_1\1, 150 p1\4 to 200 nM, 150 pM to 100 nly1 and 200 pM to 50 nM.
In another embodiment, the antibody polypeptide is fused to a second antibody polypeptide which binds a ligand other than CD4OL. In a preferred embodiment, the antibody polypeptide which binds a ligand other than CD4OL
binds a ligand selected from the group consisting of HSA, TNFa, IL-1, 11 -2, IL-4;
IL-6, TT -8, IL-12, 1L-18, IFN-y, CD2, CD4, CD8, CTLA4, LFA1, LFA3, VLA4, CD80 (B7-1), CD28, CD86 (B7-2), and CTLA-4.
In another embodiment, the human antibody polypeptide is free of an Fc domain. The limits of an Fc domain are set out in Kabat et al. (1991, Sequences of Immunological Interest, 5th ed. U.S Dept. Health & Human Services, Washington, D.C..). In the alternative, an Fc domain consists of the CFI2-CH3 regions, optionally including a hinge region linked to the 42H2.
In a preferred embodiment, the human antibody polypeptide does not mediate platelet aggregation in a standard platelet aggregation assay.
2.5 The invention further encompasses a human antibody polypeptide which has an amino acid sequence at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs 7-82 and 246-360, which antibody polypeptide specifically and monovalently binds CD4OL.

The invention further encompasses an antigen-binding polypeptide, the polypeptide comprising a single immunoglobulin variable domain which specifically and monovalently binds CD4OL. Recited differently, the invention further encompasses a polypeptide comprising a moiety which specifically binds CD4OL, which moiety consists of a single immunoglobulin variable domain.
In one embodiment, the polypeptide consists of a human single immunoglobulin variable domain.
In another embodiment, the polypeptide has a Kd for human CD4OL in the range of 50 nM to 20 pM, inclusive, as determined by surface plasmon resonance.
For example, the Kd for human CD4OL can be 25 nM to 20 pM, 10 nM to 20 pM, 5 nm to 20 pM, 1 nM to 20 pM, 0.5 nM to 20 pM, 0.1 nM to 20 pM, 75 pM to 20 pM
or even 50 pM to 20 pM.
In another embodiment, the polypeptide inhibits the binding of CD4OL to CD40.
In another embodiment, the polypeptide inhibits the binding of CD40 to CD4OL and has an IC50 in the range of 20 pM to 1.5 p.M, inclusive. For example, the IC50 can be in the range of 20 pM to 1 1.1M, 20 pM to 900 nM, 20 pM to 800 nM, pM to 700 nM, 20 pM to 600 nM, 20 pM to 500 nM, 20 pM to 400 nM, 20 pM to 300 nM, 20 pM to 200 nM, 20 pM to 100 nM, or 20 pM to 50 nM. Further acceptable or preferred ranges include, for example, 50 pM to 1 M, 100 pM to 500 nM, 125 pM
to 250 nM, 150 pM to 200 nM, 150 pM to 100 nM and 200 pM to 50 nM.
In another embodiment, the binding of the polypeptide to CD4OL does not substantially agonize CD40 and/or CD4OL activity.
In another embodiment, the binding of the polypeptide to CD4OL does not substantially induce JNK phosphorylation in Jurkat T-cells.

In another embodiment, the binding of the polypeptide to CD4OL does not substantially induce IFN-y secretion by Jurkat T-cells co-stimulated with anti-antibody.
In another embodiment, the presence of the antibody polypeptide in a standard platelet aggregation assay does not result in aggregation more than 25% over the aggregation observed in a negative control assay lacking antibody polypeptide.
In another embodiment, the single immunoglobulin variable domain is a human single immunoglobulin variable domain.
In another embodiment, the single immunoglobulin variable domain is a VH or a VL domain.
In one embodiment, the p. olypeptide is PEG-linked. In one embodiment, the PEG is covalently linked. In one preferred embodiment, the PEG-linked antigen-binding polypeptide has a hydrodynamic size of at least 24 ka In another preferred embodiment, the PEG is linked to the antigen-binding polypeptide at a cysteine or lysine residue. In another preferred embodiment, the total PEG size is from 20 to 60 kl), inclusive. In another preferred embodiment, the PEG-linked antigen-binding polypeptide has a hydrodynamic size of at least 200 k.D.
In another embodiment, the PEG-linked polypeptide has an increased in vivo half-life relative to the same polypeptide composition lacking linked polyethylene glycol. In another embodiment, the ta-half life of the polypeptide composition is increased by 10% or more. In another embodiment, the ta-half life of the polypeptide composition is increased by 50% or more. In another embodiment, the ta-half life of the polypeptide composition is increased by 2X or more. In another embodiment, the ta-half life of the polypeptide composition is increased by 5X or more, e.g., 10X, 15X, 20X, 25X, 30X, 40X, or more. In another embodiment, the ta-half life of the polypeptide composition is increased by 50X or more.

In another embodiment, the PEG-linked antibody polypeptide has a ta half-life of 0.25 to 6 hours, inclusive. In another embodiment, the ta half-life is in the range of 30 minutes to 12 hours, inclusive. In another embodiment, the ta-half life of the polypeptide composition is in the range of 1 to 6 hours.
In another embodiment, the tP-half life of the polypeptide composition is increased by 10% or more. In another embodiment, the tf3-half life of the polypeptide composition is increased by 50% or more. In another embodiment, the tP-half life of the polypeptide composition is increased by 2X or more. In another embodiment, the tP-half life of the polypeptide composition is increased by 5X or more, e.g., 10X, 15X, 20X, 25X, 30X, 40X, .or more. In another embodiment, the tP-half life of the polypeptide composition is increased by 50X or more.
In another embodiment, the antibody polypeptide composition has a tf3 half-life of 1 to 170 hours, inclusive. In another embodiment, the tP-half life is in the range of 12 to 48 hours, inclusive. In another embodiment, the tP-half life is in the range of 12 to 26 hours, inclusive.
In another embodiment, the composition has an AUC value (area under the curve) in the range of 1 mg.min/ml or more. In one embodiment, the lower end of the range is 5, 10, 15, 20, 30, 100, 200 or 300 mg.miniml. In addition, or alternatively, a ligand or composition according to the invention has an AUC in the range of up to 600 mg.min/ml. In one embodiment, the upper end of the range is 500, 400, 300, 200, 150, 100, 75 or 50 mg.min/ml. Advantageously a ligand according to the invention will have an AUC in the range selected from the group consisting of the following: 15 to 150 mg.min/ml, 15 to 100 mg.min/ml, 15 to 75 mg.min/ml, and to 50 mg.min/ml.
In another embodiment, the antibody polypeptide is linked to human serum albumin (HSA). In another embodiment, the antibody polypeptide has an increased in vivo half-life relative to the same polypeptide composition lacking linked HSA. In another embodiment, the antibody polypeptide has a ta-half life that is increased by 10% or more relative to a molecule lacking linked HSA. In another embodiment, the t-half life of the polypeptide composition is in the range of 0.25 minutes to 6 hours.
In another embodiment, the tP-half life of the polypeptide composition is increased by 10% or more. In another embodiment, the tj3-half life is in the range of 12 to hours.
In another embodiment, the antigen-binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 7-82 and 246-360.
In another embodiment, the antigen-binding polypeptide is free of an Fc domain.
In another aspect, the invention encompasses an immunoglobulin variable domain polypeptide which has an amino acid sequence at least 85% identical to a _ sequence selected from the group consisting of SEQ ID NOs 7-82 and 246-360, which polypeptide specifically and monovalently binds CD4OL.
In one embodiment, the immunoglobulin variable domain polypeptide antagonizes the binding of CD4OL to CD40.
In another embodiment, the immunoglobulin variable domain polypeptide inhibits the binding of CD40 to CD4OL and has an IC50 in the range of 20 pM to 1.5 1\4, inclusive. For example, the IC50 can be in the range of 20 pM to 1 1.1,M, 20 pM to 900 nM, 20 pM to 800 nM, 20 pM to 700 n1\4, 20 pM to 600 nM, 20 pM to 500 nM, pM to 400 nM, 20 pM to 300 nM, 20 pM to 200 nM, 20 pM to 100 nM, or 20 pM
20 to 50 nM. Further acceptable or preferred ranges include, for example, 50 pM to 1 11M, 100 pM to 500 nM, 125 pM to 250 nM, 150 pM to 200 nM, 150 pM to 100 nM
and 200 pM to 50 nM.
In another embodiment, the immunoglobulin variable domain polypeptide inhibits the interaction of CD40 with CD4OL, but does not substantially agonize intracellular signaling by CD40. In a preferred embodiment, the binding of the polypeptide to CD4OL does not substantially induce INK phosphorylation in Jurkat T-cells. In another preferred embodiment, the binding of the polypeptide to does not substantially induce IFN-y secretion by Jurkat T-cells co-stimulated with anti-CD3 antibody. In another preferred embodiment, the binding of the antibody polypeptide to CD4OL does not substantially induce platelet aggregation in a platelet aggregation assay.
In another embodiment, the antigen-binding polypeptide further comprises a second antibody polypeptide which binds a ligand other than CD4OL. In a preferred embodiment, the second antibody polypeptide binds a ligand selected from the group consisting of I-I-SA, TNFa., IL-1, IL-2, IL-4, 1L-6, IL-8, 1L-12, 1L-18, IFN-y, CD2, CD4, CD8, CTLA4, LFA I, LFA3 and VLA4.
In one embodiment, the invention relates to an antibody polypeptide comprising an immunoglobulin variable domain which specifically and monovalently binds CD4OL (e.g., an anti-CD4OL dAb, FAb, an scFv, an Fv, or a disulfide-bonded Fv), and which comprises one or more framework regions comprising an amino acid sequence that is the same as the amino acid sequence of a corresponding framework region encoded by a human geiniline antibody gene segment, or the amino acid sequence of one or more of said framework regions collectively comprises up to

5 amino acid differences relative to the amino acid sequence of said corresponding framework region encoded by a human germline antibody gene segment.
In one embodiment, the amino acid sequences of FW1, FW2, FW3 and FW4 of the anti-CD4OL variable domain or dAb are the same as the amino acid sequences of corresponding framework regions encoded by a human germline antibody gene segment, or the amino acid sequences of FW1, FW2, FW3 and FW4 collectively contain up to 10 amino acid differences relative to the amino acid sequences of corresponding framework regions encoded by said human germline antibody gene segment. In a further embodiment, the amino acid sequences of FW1, FW2 and FW3 of the anti-CD4OL variable domain or dAb are the same as the amino acid sequences of corresponding framework regions encoded by human germline antibody gene segments.

In a further embodiment of the foregoing, the human germline antibody gene segment can be selected from the group consisting of DP47, DP45, DP48 and DPK9.
The invention further encompasses a method of antagonizing the binding of CD40 to CD4OL in an individual, the method comprising administering a monovalent anti-CD4OL antibody polypeptide as described herein to the individual, wherein the polypeptide antagonizes the binding of CD40 to CD4OL in the individual.
The invention further encompasses a method of antagonizing an activity of CD40 or CD4OL in an individual, the method comprising administering a monovalent anti-CD4OL antibody polypeptide as described herein to the individual, wherein the polypeptide antagonizes an activity of CD40 or CD4OL or both.
The invention further encompasses a composition comprising an extended release formulation comprising a monovalent anti-CD4OL antibody polypeptide, preferably, but not limited to, a polypeptide comprising a single immunoglobulin variable domain that binds CD4OL. In one embodiment, the single immunoglobulin variable domain is a non-human mammalian single immunoglobulin variable domain.
In another embodiment, the single immunoglobulin variable domain is a human single immunoglobulin variable domain.
The invention further encompasses a method of treating or preventing a disease or disorder mediated by CD4OL in an individual in need of such treatment, the method comprising administering to the individual a therapeutically effective amount of a composition comprising a monovalent anti-CD4OL antibody polypeptide, preferably a composition comprising a single human immunoglobulin variable domain that binds CD4OL. In one embodiment, the disease or disorder is an autoimmune disease or disorder.
The invention further encompasses a method of treating or preventing a symptom of systemic lupus erythematosus (SLE) in an individual, the method comprising administering a monovalent anti-CD4OL antibody polypeptide to said individual in an amount effective to treat or prevent a symptom of SLE. The invention further encompasses a method of reducing or alleviating a symptom of a disease such as systemic lupus erythematosis, multiple sclerosis, rheumatoid arthritis, allog,raft rejection, xenograft rejection, and Diabetes, including insulin-dependent Type I Diabetes.
The invention further encompasses an antibody polypeptide that is monovalent for binding to CD4OL, wherein the antibody polypeptide comprises a universal framework.
In one embodiment, the universal framework comprises a VH framework selected from the group consisting of DP47, DP45 and DP38, and/or the VL
framework is DPK9.
In another embodiment, the antibody polypeptide comprises a generic ligand binding site. In another embodiment, the generic ligand binding site binds a generic ligand selected from the group consisting of protein A, protein L and protein G.
In another embodiment, the antibody polypeptide comprises a variable domain having one or more framework regions comprising an amino acid sequence that is the same as the amino acid sequence of a corresponding framework region encoded by a human germline antibody gene segment, or the amino acid sequences of one or more of the framework regions collectively comprises up to 5 amino acid differences relative to the amino acid sequence of the corresponding framework region encoded by a human germline antibody gene segment.
In another embodiment, the antibody polypeptide comprises a variable domain, wherein the amino acid sequences of FW1, FW2, FW3 and FW4 are the same as the amino acid sequences of corresponding framework regions encoded by a human gemiline antibody gene segment, or the antibody sequences of FW1, FW2, FW3 and FW4 collectively contain up to 10 amino acid differences relative to the amino acid sequences of corresponding framework regions encoded by the human germline antibody gene segment.

In another embodiment, the antibody polypeptide comprises an antibody variable domain comprising FW1, FW2 and FW3 regions, and the amino acid sequence of said PAIL FW2 and FW3 are the same as the amino acid sequences of corresponding framework regions encoded by human germline antibody gene segments. In another embodiment, the human germline antibody gene segment is selected from the group consisting of DP47, DP45, DP48 and DPK9.
The invention includes an antibody single variable domain polypeptide that binds to CD4OL, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of DOM8-24, or differs from the amino acid sequence of DOM8-24 at no more than 25 amino acid positions and has a sequence that is at least 80% homologous to the sequence of DOM8-24. In one embodiment, the antibody single variable domain polypeptide differs faun the amino acid sequence of 24 at 25 or fewer amino acid positions, 20 or fewer amino acid positions, 15 or fewer amino acid positions, 10 or fewer amino acid positions, 5 or fewer amino acid positions, 2 or fewer amino acid positions, or as few as one amino acid position. In a further embodiment, the antibody single variable domain polypeptide is at least 80%
homologous to the sequence of DOM8-24, for example, at least 85% homologous, at least 90% homologous, at least 95% homologous, and up to and including 96%, 97%, 98%, or 99% homologous.
The invention includes an antibody single variable domain polypeptide that binds to CD4OL, wherein the polypeptide has an amino acid sequence that is identical to the amino acid sequence of DOM8-24, or differs from the amino acid sequence of DOM8-24 at no more than 25 amino acid positions and has a CDR1 sequence that is at least 50% homologous to the CDR1 sequence of DOM8-24, or has a CDR2 sequence that is at least 50% homologous to the CDR2 sequence of DOM8-24, or has a CDR3 sequence that is at least 50% homologous to the CDR3 sequence of DOM8-24.
The invention also includes an antibody single variable domain polypeptide that binds CD4OL, wherein the dAb has an amino acid sequence that is identical to the amino acid sequence of DOM8-24, or differs from the amino acid sequence of DOM8-24 at no more than 25 amino acid positions and has a CDR1 sequence that is at least 50% homologous to the CDR1 sequence of DOM8-24 and has a CDR2 sequence that is at least 50% homologous to the CDR2 sequence of DOM8-24 .
The invention also includes an antibody single variable domain polypeptide that binds CD4OL, wherein the dAb has an amino acid sequence that is identical to the amino acid sequence of DOM8-24, or differs from the amino acid sequence of DOM8-24 at no more than 25 amino acid positions and has a CDR2 sequence that is at least 50% homologous to the CDR2 sequence of DOM8-24 and has a CDR3 sequence that is at least 50% homologous to the CDR3 sequence of DOM8-24 .
The invention also includes an antibody single variable domain polypeptide that binds CD4OL, wherein the dAb has an amino acid sequence that is identical to the amino acid sequence of DOM8-24, or differs from the amino acid sequence of DOM8-24 at no more than 25 amino acid positions and has a CDR1 sequence that is at least 50% homologous to the CDR1 sequence of DOM8-24 and has a CDR3 sequence that is at least 50% homologous to the CDR3 sequence of DOM8-24 .
The invention also includes an antibody single variable domain polypeptide that binds CD4OL, wherein the dAb has an amino acid sequence that is identical to the amino acid sequence of DOM8-24, or differs from the amino acid sequence of DOM8-24 at no more than 25 amino acid positions and has a CDR1 sequence that is at least 50% homologous to the CDR1 sequence of DOM8-24 and has a CDR2 sequence that is at least 50% homologous to the CDR2 sequence of DOM8-24 and has a CDR3 sequence that is at least 50% homologous to the CDR3 sequence of DOM8-24.
In one embodiment, the antibody single variable domain polypeptide that binds to CD4OL, if not identical in sequence to that of DOM8-24, differs folla the amino acid sequence of DOM8-24 at 25 or fewer amino acid positions, 20 or fewer amino acid positions, 15 or fewer amino acid positions, 10 or fewer amino acid positions, 5 or fewer amino acid positions, 2 or fewer amino acid positions, or as few as one amino acid position.
The invention also includes a CD4OL antagonist having a CDR1 sequence that is at least 50% homologous to the CDR1 sequence of DOM8-24.
The invention also includes a CD4OL antagonist having a CDR2 sequence that is at least 50% homologous to the CDR2 sequence of DOM8-24.
The invention also includes a CD4OL antagonist having a CDR3 sequence that is at least 50% homologous to the CDR3 sequence of DOM8-24.
The invention also includes a CD4OL antagonist having a CDR1 sequence that is at least 50% homologous to the CDR1 sequence of DOM8-24 and a CDR2 sequence that is at least 50% homologous to the CDR2 sequence of DOM8-24.
The invention also includes a CD4OL antagonist having a CDR2 sequence that is at least 50% homologous to the CDR2 sequence of DOM8-24 and a CDR3 sequence that is at least 50% homologous to the CDR3 sequence of DOM8-24.
The invention also includes a CD4OL antagonist having a CDR1 sequence that is at least 50% homologous to the CDR1 sequence of DOM8-24 and a CDR3 sequence that is at least 50% homologous to the CDR3 sequence of DOM8-24.
The invention also includes a CD4OL antagonist having a CDR1 sequence that is at least 50% homologous to the CDR1 sequence of DOM8-24 and a CDR2 sequence that is at least 50% homologous to the CDR2 sequence of DOM8-24 and a CDR3 sequence that is at least 50% homologous to the CDR3 sequence of DOM8-24.
In one embodiment the CD4OL antagonist inhibits the binding of CD40 to CD4OL, and/or inhibits an activity of CD40 and/or CD4OL, and/or results in no more than 25% platelet aggregation in a platelet aggregation assay. In one embodiment, the antagonist results in platelet aggregation of 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, and as little as zero platelet aggregation.

The invention- also includes a dual specific ligand comprising a first immunoglobulin single variable domain having a binding specificity to a first antigen and a second single variable domain having a binding activity to a second antigen, wherein the first antigen is CD4OL and binding of the second single variable domain to the second antigen acts to increase the half-life of the ligand in vivo. In one embodiment, the dual specific ligand is a four chain IgG immunoglobulin.
In one embodiment, the four chain IgG comprises two dual specific ligands, said dual specific ligands being different in their variable domains.
The invention also includes a dual specific ligand comprising an anti-human CD4OL dAb and an anti-SA dAb.
In one embodiment, the dAbs are Camelid VHH domains.
In one embodiment of the dual specific ligand, either (i) the first and second immunoglobulin variable domains are heavy chain variable domains; or (ii) the first and the second immunoglobulin variable domains are light chain variable domains.
In one embodiment, the ligand is provided as an IgG immunoglobulin comprising four heavy chain single variable domains or four light chain single variable domains. The heavy chain can comprise Camelid VHH domains.
In a further embodiment of the dual specific ligand, the first and second domains bind independently, such that the dual specific ligand may simultaneously bind both the first and second antigens.
In one embodiment of the dual specific ligand, the first single variable domain has a dissociation constant (Kd) of 1x10-8 M or less for human CD4OL, and a Koff rate constant of 1x10-3 s-1 or less, as determined by surface plasmon resonance.
In one embodiment of the dual specific ligand, the second single variable domain is specific for serum albumin (SA) and has a dissociation constant (Kd) of 1nM to 500um for SA, as determined by surface plasmon resonance.

In a further embodiment, the second domain binds SA in a standard ligand binding assay with an IC50 of lnIVI to 500p.M. The second single variable domain may be specific for SA, and comprise the amino acid sequence of MSA-16 or a sequence that is at least 80% homologous thereto. Alternatively, the second single variable domain may be specific for SA, and comprise the amino acid sequence of MSA-26 or a sequence that is at last 80% homologous thereto.
In one embodiment of the dual specific ligand, the anti-CD4OL variable domain or dAb comprises a universal framework. The anti-CD4OL variable domain or dAb may also comprise a VH framework selected from the group consisting of DP47, DP45 and DP38; or a VL framework which is DPK9. In a further embodiment, the dual specific ligand or dAb can comprise a binding site for a generic ligand.
In one embodiment, the generic ligand binding site is selected from the group consisting of protein A, protein L and protein G binding site.
In one embodiment of the dual specific ligand, the anti-CD4OL variable domain or dAb comprises one or more framework regions comprising an amino acid sequence that is the same as the amino acid sequence of a corresponding framework region encoded by a human germline antibody gene segment, or the amino acid sequence of one or more of said framework regions collectively comprises up to amino acid differences relative to the amino acid sequence of said corresponding framework region encoded by a human germline antibody gene segment.
In one embodiment, the amino acid sequences of FW1, FW2, FW3 and FW4 of the anti-CD4OL variable domain or dAb are the same as the amino acid sequences of corresponding framework regions encoded by a human germline antibody gene segment, or the amino acid sequences of FW1, FW2, FW3 and FW4 collectively contain up to 10 amino acid differences relative to the amino acid sequences of corresponding framework regions encoded by said human germline antibody gene segment.

In one embodiment, the amino acid sequences of said FWI, F2 and FW3 of the anti-CD4OL variable domain or dAb are the same as the amino acid sequences of corresponding framework regions encoded by human germline antibody gene segments. The human germline antibody gene segments are preferably selected from the group consisting of DP47, DP45, DP48 and DPK9.
The invention also includes a method for producing a dual specific ligand as described herein, comprising a first immunoglobulin single variable domain having a binding specificity for CD4OL and a second single immunoglobulin single variable domain having a binding specificity for a protein which increases the half-life of the ligand in vivo, the method comprising the steps of:selecting a first variable domain by its ability to bind CD4OL; selecting a second variable domain by its ability to bind to said protein; combining the variable domains; and selecting the ligand by its ability to bind to CD4OL and said protein.
In one embodiment, the first variable domain is selected for binding to CD4OL
in absence of a complementary variable domain.
The invention also includes nucleic acid encoding a dual specific ligand described herein. The nucleic acid may comprise the nucleic acid sequence of MSA-16 or a sequence that is at least 80% homologous thereto, or alternatively may comprise, the nucleic acid sequence of MSA-26 or a sequence that is at least 70%
homologous thereto. The nucleic acid may be incorporated into a vector, which may be incorporated into a host cell.
The invention also includes a pharmaceutical composition comprising a dual specific ligand as described herein and a pharmaceutically acceptable excipient, carrier or diluent.
The invention also includes a dAb monomer specific for CD4OL, which monomer has a dissociation constant (Kd) of Ix10-8 M or less for human CD4OL, and a Koff rate constant of lx10-3 si or less, as determined by surface plasmon resonance.

In one embodiment, the dAb monomer specific for CD4OL has a dissociation constant (Kd) of lx10-7 M or less, as determined by surface plasmon resonance.
In one embodiment, the dAb monomer has binding specificity to CD4OL with a dissociation constant (Kd) of lx10-8 M or less, as determined by surface plasmon resonance.
In one embodiment, the dAb monomer has binding specificity to CD4OL with a dissociation constant (Kd) of 50nM to 20pM, as determined by surface plasmon resonance.
In one embodiment, the monomer inhibits binding of CD40 to CD4OL with an 1050 of 50nM or less.
In a further embodiment, the dAb monomer has binding specificity to CD4OL
with a K.ff rate constant of lx10-3 s-1 or less, lx10-4 s-1 or less, 1x10-5 s-1 or less, or lx10-6 s-1 or less,as determined by surface plasmon resonance.
In one embodiment, the dAb monomer neutralizes CD4OL in a standard assay with an ND50 of 50nM or less.
In invention also includes a dual specific ligand comprising first and second heavy chain single variable domains, or first and second light chain single variable domains, wherein the first variable domain is an anti-CD4OL dAb monomer.
In one embodiment, the second variable domain has binding specificity for an antigen other than CD4OL.
In a further embodiment, the second variable domain has binding specificity for an antigen selected from the group consisting of EPO receptor, ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor, ENa-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic, fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-bl, insulin, IFN-g, IGF-I, IGF-II, IL-la, II-lb, IL-2, 11-3, IL-4, IL-5, IL-6, IL-7, 1L-8 (72 a.a.), 1L-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin a, Inhibin b, JP-10, keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian inhibitory substance, monocyte colony inhibitory factor, monocyte attractant protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MIG, MIP-1 a, MIP- lb, MIP-3a, MIP-3b, MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor, b-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDFla, DFG1b SCF, SCGF, stem cell factor, (SCF), TARC, TGF-a, TGF-b, TGF-b2; TGF-b3, tumour necrosis factor (TNF), TNF-a, TNF-b, TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor-3, GCP-2, GRO/MGSA, GRO-b, GRO-g, HCC1, 1-309, HER 1, HER 2, HER 3, HER 4, TACE
recognition site, TNF BP-1, TNF BP-II, and an antigen disclosed in Annex 2 or 3.
The invention also includes an antibody polypeptide that antagonizes or inhibits the binding of DOM8-24 to CD4OL, or an antibody polypeptide that binds to the same epitope of CD4OL bound by DOM8-24.
The invention also includes a dual specific ligand comprising a = first immunoglobulin single variable domain having a binding specificity to a first antigen and a second single variable domain having a binding activity to a second antigen, wherein the first antigen is CD4OL and the second single variable domain is an Antigen Presenting Cell surface antigen or a T cell surface antigen. The Antigen Presenting Cell surface antigen can be selected from one of the group consisting of dendritic cell surface antigens, activated macrophage surface antigens, activated B
cell surface antigens, co-stimulatory signal pathway surface antigens, and MHC.
In one embodiment, the MHC is class II, and the class II can be alpha or beta.
The Antigen Presenting Cell surface antigen may be selected from the group consisting of CD28, Inducible costimulatory molecule (ICOS), CD27, CD30, 0X40, CD45, CD69, CD3, CD70, Inducible costimulatory molecule ligand (ICOSL), OX4OL, CD80, CD86, HVEM (Herpes Virus Entry Mediator), and LIGHT, but is preferably one of CD28, Inducible costimulatory molecule (ICOS), CD27, CD30, 0X40, CD45, CD69, or CD3.

The surface antigen is preferably a B7 gene surface antigen such as B7-2 or B7-1.
Specifically, the invention includes:
- use of an antibody polypeptide comprising an antibody single variable domain polypeptide in the preparation of a medicament for treating or preventing a symptom of autoimmune disease, wherein said single variable domain polypeptide is monovalent for binding to CD4OL and antagonizes an activity of CD40 or CD4OL or both, and wherein said antibody polypeptide inhibits the binding of an antibody single variable domain comprising the amino acid sequence of SEQ ID NO: 26 to CD4OL;
- use of an antibody polypeptide comprising an antibody single variable domain polypeptide for treating or preventing a symptom of autoimmune disease, wherein said single variable domain polypeptide is monovalent for binding to CD4OL and antagonizes an activity of CD40 or CD4OL or both, and wherein said antibody polypeptide inhibits the binding of an antibody single variable domain comprising the amino acid sequence of SEQ ID NO: 26 to CD4OL2; and - an antibody polypeptide comprising or consisting of an antibody single variable domain which specifically and monovalently binds CD4OL, wherein said polypeptide inhibits the binding of CD4OL to CD40, wherein binding of said antibody polypeptide to CD4OL does not agonize CD40 or CD4OL activity, and wherein said antibody polypeptide inhibits the binding of an antibody single variable domain comprising the amino acid sequence of SEQ ID NO: 26 to CD4OL.

Definiti As used herein, the term "human" when applied to au antibody polypeptide or to au inimunoglobulin variable domain means that the polypeptide has a sequence derived from a human immunoglobulin. A sequence is "derived from" a human immunoglobulin coding sequence when the sequence is either: a) isolated from a human individual or from cells or a cell line from a human individual; b) isolated from a library of cloned human antibody (2.ene sequences (or a library of human antibody V domain sequences); or c) when a cloned human antibody gene sequence (or a cloned human V region sequence (including, e.g., a gemiline V gene sev,ment)) was used to generate one or more diversified sequences that were then selected for binding to a desired target antigen. The term "human" as applied herein to an antibody polypeptide or to an immunoglobulin variable domain does not encompass an immunoglobulin from another species, e.g., mouse, camel, etc., that has been "humanized" through grafting of human constant region sequences onto an antibody polypeptide (i.e., replacing non-human constant regions with human constant regions) or through grafting of human -V region framework sequences onto an immunoglobulin variable domain from a non-human mammal (i.e., replacing non-human framework regions of a V domain with human framework regions).
At a minimum, a human variable domain has at least 85% amino acid similarity (including, for example, 87%, 90%, 93%, 95%, 97%, 99% or higher similarity) to a naturally-occurring human inununoglobulin variable domain =
sequence.
As used herein, the term "domain" refers to a folded protein structure which retains its tertiary structure independently of the rest of the protein.
Generally, domains are responsible for discrete functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.
29a By "single immunoglobulin variable domain" is meant a folded polypeptide domain which comprises a sequence characteristic of immunoglobulin variable domains and which specifically binds an antigen (e.g., dissociation constant of 500 nM or less). A "single immunoglobulin variable domain" therefore includes complete antibody variable domains as well as modified variable domains, for example in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain a dissociation constant of 500 nM or less (e.g., 450 nM or less, 400 nM or less, 350 n1V1 or less, 300 nM or less, 250 nM or less, 200 nIVI
or less, 150 nM or less, 100 nM or less) and the target antigen specificity of the full-length domain. Where necessary or in case of any doubt, the numbering convention and boundaries set forth by Kabat et al. (1991, supra) are applicable to immunoglobulin variable and constant domains referred to herein.
An antibody single variable domain polypeptide, as used herein refers to a mammalian single immunoglobulin variable domain polypeptide, preferably human, but also includes rodent (for example, as disclosed in W000/29004) or camelid VBH dAbs. Camelid dAbs are antibody single variable domain polypeptides which are derived from species including camel, llama, alpaca, dromedary, and guanaco, and comprise heavy chain antibodies naturally devoid of light chain: VHEI. VBH molecules are about 10x smaller than IgG molecules, and as single polypeptides, they are very stable, resisting extreme pH and temperature conditions. Moreover, camelid antibody single variable domain polypeptides are resistant to the action of proteases. Camelid antibodies are described in, for example, U.S. Pat. Nos. 5,759,808; 5,800,988; 5,840,526; 5,874,541;

6,005,079; and 6,015,695, the contents of each of which are incorporated herein in their entirety. Camelid VI-TH antibody single variable domain polypeptides useful according to the invention include a class of camelid antibody single variable domain polypeptides having human-like sequences, wherein the class is characterized in that SO the VHB domains carry an amino acid from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine, or glutamine at position 45, such as for example L45, and further comprise a tryptophan at position 103 according to the Kabat numbering-.
Humanized camelid Vi H polypeptides are taught, for example in W004/041862.
It will be understood by one of skill in the art that naturally occurring camelid antibody single variable domain polypeptides may be modified according to the teachings of W004/041862 (e.g., amino acid substitutions at positions 45 and 103) to generate humanized camelid VFHI
polypeptides. Also included in the present invention are antibody single variable domain polypeptides which are nurse shark VIE-1. Nurse shark dAbs are antibody single variable domain polypeptides derived from the nurse shark, that comprise-heavy chain antibodies naturally devoid of light chain: VHE. Nurse Shark VHH
dAbs are described, for example, in Greenberg et al. (Nature 374 pp168-173 1995) and -U.S.
20050043519.
The phrase "single immunoglobulin variable domain polypeptide"
encompasses not only an isolated single immunoglobulin variable domain polypeptide, but also larger polypeptides that comprise a monomer of a single immunoglobulin variable domain polypeptide sequence. A "domain antibody" or "dAb" is equivalent to a "single immunoalobulin variable domain polypeptide"
as the terin is used herein. With regard to a single immunoglobulin variable domain polypeptide, the binding to antigen, e.g., CD4OL, is mediated by the single immunoglobulin V domain without a requirement for a complementary -V domain.
According to the invention, the terms "antibody single variable domain polypeptide", "antibody single variable domain", "single antibody variable domain", and "single imniunoglobulin variable domain" are understood to be equivalent.
As used herein, the phrase "sequence characteristic of immunoglobulin variable domains" refers to an amino acid sequence that is homologous, over 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or even 50 or more contiguous amino acids, to a sequence comprised by an inununoglobulin variable domain sequence.

Sequences similar or homologous (e.g., at least about 70% sequence identity) to the sequences disclosed herein are also part of the invention. In some embodiments, the sequence identity at the amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, the sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively, substantial identity exists when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., very high stringency hybridization conditions), to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
As used herein, the terms "homology" or "similarity" refer to the degree with which two nucleotide or amino acid sequences structurally resemble each other.
As used herein, sequence "similarity" is a measure of the degree to which amino acid sequences share similar amino acid residues at corresponding positions in an alignment of the sequences. Amino acids are similar to each other where their side chains are similar. Specifically, "similarity" encompasses amino acids that are conservative substitutes for each other. A "conservative" substitution is any substitution that has a positive score in the blosum62 substitution matrix (Hentikoff and Hentikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919). By the statement "sequence A is n% similar to sequence B" is meant that n% of the positions of an optimal global alignment between sequences A and B consists of identical amino acids or conservative substitutions. Optimal global alignments can be performed using the following parameters in the Needleman-Wunsch alignment algorithm:
For polypeptides:
Substitution matrix: blosum62.
Gap scoring function: -A -B*LG, where A=11 (the gap penalty), B=1 (the gap length penalty) and LG is the length of the gap.
For nucleotide sequences:
Substitution matrix: 10 for matches, 0 for mismatches.
Gap scoring function: -A -B*LG where A=50 (the gap penalty), B=3 (the gap length penalty) and LG is the length of the gap.

Typical conservative substitutions are among Met, Val, Leu and Ile; among Ser and Thr; among the residues Asp, Glu and Asn; among the residues Gln, Lys and Arg; or aromatic residues Phe and Tyr.
As used herein, two sequences are "homologous" or "similar" to each other where they have at least 70%, 80%, or 85% sequence similarity to each other, including, e.g., 90%, 95%, 97%, 99% or even 100% sequence similarity, when aligned using either the Needleman-Wunsch algorithm or the "BLAST 2 sequences"

algorithm described by Tatusova & Madden, 1999, FEMS Microbiol Lett. 174:247-250. Where amino acid sequences are aligned using the "BLAST 2 sequences algorithm," the Blosum 62 matrix is the default matrix.
As used herein, the terms "inhibit," "inhibits" and "inhibited" refer to a decrease in a given measurable activity (e.g., binding activity) by at least 10% relative to a reference. Where inhibition is desired, such inhibition is preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, up to and including 100%, i.e., complete inhibition or absence of the given activity. One way that inhibition of CD4OL binding to CD40 is measured is as described in Example 6 herein. As used herein, the term "substantially inhibits" refers to a decrease in a given measurable activity (e.g., the binding of CD4OL to CD40) by at least 50% relative to a reference.
For example, "substantially inhibits" refers to a decrease in a given measurable activity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and up to and including 100% relative to a reference. As used herein, "inhibits the binding", with reference to the binding of an antibody polypeptide binding to CD4OL, or binding to CD4OL, refers to a decrease in binding by at least 10% relative to a reference. "Inhibits the binding" preferably refers to a decrease in binding of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, up to and including 100%.
As used herein, the terms "activate," "activates" and "activated" refer to an increase in a given measurable activity by at least 5% relative to a reference, for example, at least 10%, 25%, 50%, 75% or even 100%.

As used herein, the term "antagonist" refers to an agent that inhibits at least one activity mediated by CD4OL, inhibits the binding of CD40 to CD4OL, and/or results in no more than 25% platelet activation and/or aggregation in a platelet aggregation assay or platelet activation assay as described herein, and preferably results in 25% or less platelet activation and/or aggregation, 20% or less, 15% or less, 10% or less, 5% or less, and as little as zero platelet activation and/or aggregation.
An activity is "antagonized" if the activity (i.e., CD4OL mediated activity, binding of CD40 or CD4OL, or platelet activation and/or aggregation) is reduced by at least 10%, and preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97% or even 100% (i.e., no activity) in the presence, relative to the absence of an antagonist.
An antagonist as the term is used herein preferably comprises a single immunoglobulin variable domain that binds monovalently to CD4OL.
As used herein, the term "agonist" refers to an agent that activates at least one activity mediated by CD4OL, either alone or when combined with another co-stimulus, relative to a reference. An activity is "agonized" if the activity is increased by at least 10%, e.g., 50%, in the presence, relative to the absence of an agonist.
As used herein, the term "epitope" refers to a unit of structure conventionally bound by an immunoglobulin VH/VL pair. Epitopes define the minimum binding site for an antibody, and thus represent the target of specificity of an antibody.
In the case of a single immunoglobulin variable domain, an epitope represents the unit of structure bound by a single variable domain in isolation. That is, the binding site is provided by one, single immunoglobulin variable domain.
As used herein, the term "extended release" or the equivalent terms "controlled release" or "slow release" refer to drug formulations that release active drug, such as a polypeptide drug, over a period of time following administration to an individual. Extended release of polypeptide drugs, which can occur over a range of desired times, e.g., minutes, hours, days, weeks or longer, depending upon the drug formulation, is in contrast to standard formulations in which substantially the entire dosage unit is available for immediate absorption or immediate distribution via the bloodstream. Preferred extended release formulations result in a level of circulating drug from a single administration that is sustained, for example, for 8 hours or more, 12 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 60 hours or more, 72 hours or more 84 hours or more, 96 hours or more, or even, for example, for 1 week or 2 weeks or more, for example, 1 month or more.
As used herein, a "CD4OL activity" is an activity involving or resulting from the binding of CD4OL to CD40, and includes, but is not limited to binding to (assayed, for example, according to the method described in Example 6), activation of Jun-N-terminal Kinase (JNK), the induction of T cells to produce and secrete cytokines including, for example, IL-10, IFN-y and TNF-a, and the mediation of platelet activation and/or aggregation. Assays for these activities are provided herein below.
As used herein, the term "does not substantially agonize" means that a given agent, e.g., an anti-CD4OL antibody polypeptide, does not activate one or more of the CD4OL activities including Jun-N-terminal kinase activation (phosphorylation) in Jurkat T cells and induction of IFN-y production or secretion in anti-CD3-stimulated Jurkat T cells, as the term "activate" is defined herein. As used herein, "does not substantially agonize" means that the agent does not activate more than 20% of the activity which is activated by CD40 binding to CD4OL, preferably, the agent does not activate more than 10%, 8%, 5%, 3%, or more than 2% or less, including zero activation, of the activity which is activated by CD40 binding to CD4OL.
As used herein, the term "antibody polypeptide" refers to a polypeptide which either is an antibody or is a part of an antibody, modified or unmodified, which =
retains the ability to specifically bind antigen. Thus, the term antibody polypeptide includes an antigen-binding heavy chain, light chain, heavy chain-light chain dimer, Fab fragment, F(ab1)2 fragment, dAb, or an Fv fragment, including a single chain Fv (scFv). The phrase "antibody polypeptide" is intended to encompass recombinant fusion polypeptides that comprise an antibody polypeptide sequence that retains the ability to specifically bind antigen in the context of the fusion.

As used herein, the term "monovalent" means that a given antibody polypeptide or single immunoglobulin variable domain polypeptide can bind only a single molecule of its target. Naturally-occurring antibodies are generally divalent, in that they have two functional antigen-binding arms, each comprising a VI-I and a VL
domain. Where steric hindrance is not an issue, a divalent antibody can bind two separate molecules of the same antigen. In contrast, a "monovalent" antibody has the capacity to bind only one such antigen molecule. As the term is used herein, a "monovalent" antibody can also comprise more than one antigen binding site, e.g., two antigen binding sites, but the binding sites must be for different antigens, such that the antibody can only bind one molecule of CD4OL at a time. The antigen-binding domain of a monovalent antibody can comprise a VH and a "Vi, domain, but preferably comprises only a single imrnunoglobulin variable dotnain, i.e., a VH or a VL domain, that has the capacity to bind CD4OL without the need for a corresponding VL or VH domain, respectively. A monovalent antibody lacks the capacity to cross link molecules of a single antigen.
As used herein, the term "standard platelet aggregation assay" means the assay described in the section herein below, entitled "Platelet Aggregation Assay."
As used herein, the terms "VH domain" and "VL domain" refer to immunoglobulin variable regions as defined by Kabat et al.(supra).
'70 .As used herein, "linked" refers to the attachment of a polymer moiety, such as PEG to an amino acid residue of an antibody polypeptide. Attachment of a PEG
polymer to an amino acid residue of an antibody polypeptide, e.g., an anti-dAb, is referred to as "PEGylation" and may be achieved using several PEG
attachment moieties including, but not limited to N-hydroxylsuccinimide (NHS) active ester, succinimidyl propionate (SPA), maleimide (MAL), vinyl sulfone (VS), or thiol. A PEG polymer, or other polymer, can be linked to an antibody polypeptide at either a predetermined position, or may be randomly linked to the an antibody polypeptide molecule. It is preferred, however, that the PEG polymer be linked to an .36 antibody polypeptide at a predetermined position. A PEG polymer may be linked to any residue in the an antibody polypeptide, however, it is preferable that the polymer is linked to either a lysine or cysteine, which is either naturally occurring in the antibody polypeptide, or which has been engineered into the antibody polypeptide, for example, by mutagenesis of a naturally occurring residue in the antibody polypeptide to either a cysteine or lysine. PEG-linkage can also be mediated through a peptide linker attached to an antibody polypeptide. That is, the PEG moiety can be attached to a peptide linker fused to an antibody polypeptide, where the linker provides the site, e.g., a free cysteine or lysine, for PEG attachment. As used herein, "linked" can also refer to the association of two or more antibody polypeptides, e.g., dAb monomers, to form a dimer, trimer, tetramer, or other multimer. Antibody polypeptide monomers can be linked to form a multimer by several methods known in the art, including, but not limited to, expression of the antibody polypeptide monomers as a fusion protein, linkage of two or more monomers via a Peptide linker between monomers, or by chemically joining monomers after translation, either to each other directly, or through a linker by disulfide bonds, or by linkage to a di-, tri-or multivalent linking moiety (e.g., a multi-arm PEG). While dAb multimers are specifically contemplated herein, e.g., in the context of dual- or multi-specific antibody polypeptide constructs, it is emphasized that for any given antibody polypeptide construct, the construct should only be able to bind one molecule of CD4OL, i.e., the constructs can have only one CD4OL-binding element, and cannot cross link CD4OL.
As used herein, "polymer" refers to a macromolecule made up of repeating monomeric units, and can refer to a synthetic or naturally occurring polymer such as an optionally substituted straight or branched chain polyalkylene, polyalkenylene, or polyoxyalkylene polymer or a branched or unbranched polysaccharide. A
"polymer"
as used herein, specifically refers to an optionally substituted or branched chain poly(ethylene glycol), poly(propylene glycol), or poly(vinyl alcohol) and derivatives thereof.

As used herein, "PEG" or "PEG polymer" refers to polyethylene glycol, and more specifically can refer to a derivitized form of PEG, including, but not limited to N-hydroxylsuccinimide (NHS) active esters of PEG such as succinimidyl propionate, benzotriazole active esters, PEG derivatized with maleimide, vinyl sulfones, or thiol groups. Particular PEG formulations can include PEG-0-CH2CH2CH2-0O2-NHS;
PEG-O-CH,-NHS; PEG-O-CH2CH2-0O2-NHS; PEG-S-CH7CH2-CO-NHS; PEG-02CNH-CH(R)-0O2-NHS; PEG-NHCO-CH2CH2-CO-NHS; and PEG-0-CH2-007-NHS; where R is (CH))4)NHCO2(mPEG). PEG polymers useful in the invention may be linear molecules, or may be branched wherein multiple PEG moieties are present in a single polymer. Some particularly preferred PEG conformations that are useful in the invention include, but are not limited to the following:

II
mPEG-0-C-NH
I
0 [CNA 0 /

./
Chi ...- Ii 7 µ= II
mPEG-N . mirsG-0-C-Nli \C-NH-C-Ii7CH.2-NH-C-CHCi47---N I ;
ii \\_.-----j \-/-- ' 0 # #
mPEG-MAL mPEG2-MAL
'c\
CH2CONH(CH2CH20)2¨ CH2CH2N I PiG ivt.S. MG
1 \-----0 /o\ .
mPEG -CONHCH , HO- PEG \ Pit-0.-Oirl ;
h-0.4 ./cf------CH2CONH(CH2CH20)2¨ CH2CH2N
\------ multi-arm PEG
e mPEG-(MAL)2 c\1 r--1 O cH2coNH(cH2cH2o)2¨a-i2cH2N

\-----I
mPEG- 0 - C-NH

CH2CH2CH2CH2CHCONHCH .
I
1 , mPEG- 0 - liC- NH
0 CH2CONH(CH2q120)2-CH2CH2N
O
mPEG2-(MAL)2 .

nine¨D¨C¨NH

11 10',124 0 0 1 illi a 1.----- 0 i"---il mPEG-0-0.120-12¨C-0¨N trtPEO-0¨c¨N/CH\H r¨o¨si i \-----. .....
o fi -if mPEG2-NHS
mPEG-SPA

As used herein, a "sulfhydryl-selective reagent" is a reagent which is useful for the attachment of a PEG polymer to a thiol-containing amino acid. Thiol groups on the amino acid residue cysteine are particularly useful for interaction with a sulfhydryl-selective reagent. Sulfhydryl-selective reagents which are useful for such attachment include, but are not limited to maleirnide, vinyl sulfone, and thiol. The use of sulfhydryl-selective reagents for coupling to cysteine residues is known in the art and may be adapted as needed according to the present invention (See Eg., Zalipsky, 1995, Bioconjug. Chem. 6:150; Greenwald et al., 2000, Crit. Rev.
Ther.
Drug Carrier Sjist. 17:101; Herman et al., 1994, Macromol. Chem. Phys.
195:203).
The attachment of PEG or another agent, e.g., HSA, to an antibody polypeptide or to a single immunoglobulin variable domain polypeptide as described herein will preferably not impair the ability of the polypeptide to specifically bind CD4OL. That is, the PEG-linked antibody polypeptide or single immunoglobulin variable domain polypeptide will retain its binding activity relative to a non-PEG-linked counterpart. As used herein, "retains activity" refers to a level of activity of a PEG-linked antibody polypeptide which is at least 10% of the level of activity of a non-PEG-linked antibody polypeptide, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80% and up to 90%, preferably up to 95%, 98%, and up to 100% of the activity of a non-PEG-linked antibody polypeptide comprising the same antigen-binding domain or domains. More specifically, the activity of a PEG-linked antibody polypeptide compared to a non-PEG linked antibody variable domain should be determined on an antibody polypeptide molar basis; that is equivalent numbers of moles of each of the PEG-linked and non-PEG-linked antibody polypeptides should be used in each trial. In determining whether a particular PEG-linked antibody polypeptide "retains activity", it is preferred that the activity of a PEG-linked antibody polypeptide be compared with the activity of the same antibody polypeptide in the absence of PEG.
As used herein, the term "in vivo half-life" refers to the time taken for the serum concentration of a ligand (e.g., a single immunoglobulin variable domain) to reduce by 50%, in vivo, for example due to degradation of the ligand and/or clearance or sequestration of the ligand by natural mechanisms. The anti CD4OL antibody polypeptides or single immunoglobulin variable domain polypeptides described herein can be stabilized in vivo and their half-life increased by binding to molecules, such as PEG, which resist degradation and/or clearance or sequestration. The half-life of an antibody polypeptide is increased if its functional activity persists, in vivo, for a longer period than a similar antibody polypeptide which is not linked to a PEG

polymer. Typically, the half life of a PEGylated antibody polypeptide is increased by 10%, 20%, 30%, 40%, 50% or more relative to a non-PEGylated antibody polypeptide. Increases in the range of 2x, 3x, 4x, 5x, 10x, 20x, 30x, 40x, 50x or more of the half life are possible. Alternatively, or in addition, increases in the range of up to 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x of the half life are possible.
According to the invention, a PEG-linked antibody single variable domain has a half-life of between 0.25 and 170 hours, preferably between 1 and 100 hours, more preferably between 30 and 100 hours, and still more preferably between 50 and hours, and up to 170, 180, 190, and 200 hours or more.
As used herein, "resistant to degradation" or "resists degradation" with respect to a PEG or other polymer-linked antibody polypeptide monomer or multimer means that the PEG- or other polymer-linked antibody polypeptide monomer or multimer is degraded by no more than 10% when exposed to pepsin at pH 2.0 for 30 minutes and preferably not degraded at all.
As used herein, "hydrodynamic size" refers to the apparent size of a molecule (e.g., a protein molecule) based on the diffusion of the molecule through an aqueous solution. The diffusion, or motion of a protein through solution can be processed to derive an apparent size of the protein, where the size is given by the "Stokes radius"
- or "hydrodynamic radius" of the protein particle. The "hydrodynamic size" of a protein depends on both mass and shape (conformation), such that two proteins having the same molecular mass may have differing hydrodynamic sizes based on the overall conformation of the protein. Hydrodynamic size is measured, for example, by size exclusion chromatography. The hydrodynamic size of a PEG-linked antibody polypeptide, e.g., a single immunoglobulin variable domain (including antibody variable domain multimers as described herein), can be in the range of 24 kDa to 500 kDa; 30 to 500 lc-Da; 40 to 500 kDa; 50 to 500 kDa; 100 to 500 kDa; 150 to 500 kDa;
200 to 500 kDa; 250 to 500 kDa; 300 to 500 kDa; 350 to 500 kDa; 400 to 500 kDa and 450 to 500 kDa. Preferably the hydrodynamic size of a PEGylated antibody polypeptide of the invention is 30 to 40 kDa; 70 to 80 kDa or 200 to 300 kDa.
Where a single immunoglobulin variable domain polypeptide is desired for use in imaging applications, the polypeptide should have a hydrodynamic size of between 50 and 100 kDa. Alternatively, where a single immunoglobulin variable domain polypeptide is desired for therapeutic applications, the polypeptide preparation should have a hydrodynamic size of greater than 200 kDa.
As used herein, the term "IC50" refers to the concentration of an inhibitor necessary to inhibit a given activity by 50%. IC50 is determined by assaying a given activity, e.g., binding of CD4OL to CD40, in the presence of varying amounts of the inhibitor (e.g., monovalent anti-CD4OL antibody polypeptide), and plotting the inhibitor concentration versus the activity being targeted. Binding of CD4OL
to CD40 is measured herein by the method described in Example 6. Alternatively, SPR
can be used.
As used herein, the term "fused to an antibody polypeptide" means that a polypeptide is fused to a given antibody through use of recombinant DNA
techniques.
Thus, an antibody "fused to" another polypeptide, e.g., to another antibody of different binding specificity, does not exist in nature and is generated through recombinant means. The term "fused to an antibody polypeptide" also encompasses the linkage of a polypeptide to a given antibody polypeptide through, for example, disulfide or other chemical linkages, where the fused polypeptide is not naturally found fused to the antibody polypeptide. Recombinant and chemical methods of fusing a polypeptide to another polypeptide, e.g., to an antibody, are well known in the art.
As used herein, the term "Fc domain" refers to the constant region antibody sequences comprising CH2 and CH3 constant domains as delimited according to Kabat et al., supra. The Fc portion of the heavy chain polypeptide has the ability to self-associate, a function which facilitates the formation of divalent antibodies. The term "lacks an Fc domain" means that a given antibody polypeptide lacks at least the portion of an immunoglobulin Fc domain (as such domains are defined according to Kabat et al., supra) sufficient to mediate the dimerization of Fc-containing antibody polypeptides. Dimerization of Fc-containing antibody polypeptides is measured, for example, by chromatographic methods or by surface plasmon resonance. An antibody polypeptide lacking an Fc domain avoids Fc-platelet interactions and therefore avoids induction of platelet aggregation.
As used herein "treat", "reduce", "prevent", or "alleviate" as it relates to a symptom of disease refer to a decrease of the a symptom by at least 10% based on a a clinically measurable parameter, or by at least one point on a clinically-accepted scale of disease or symptom severity. As used herein, the term "symptom of systemic lupus erythematosus" refers to any of the clinically relevant symptoms of SLE
known to those of skill in the art. Non-limiting examples include the accumulation of IgG
autoantibodies (e.g., against nuclear antigens such as chromatin, snRNPs (especially U1, Sm, Ro/SSA and La/SSB), phospholipids and cell surface molecules), hemolytic anemia, thrombocytopenia, leukopenia, glomerulonephritis, vasculitis, arthritis, and serositis). A reduction in such a symptom is a reduction by at least 10% in a clinically measurable parameter, or by at least one point on a clinically-accepted scale of disease severity.
As used herein, the phrase "specifically binds" refers to the binding of an antigen by an immunoglobulin variable domain with a dissociation constant (Ka) of 1 uM or lower as measured by surface plasmon resonance analysis using, for example, a BIAcoreTM surface plasmon resonance system and BIAcOreTM kinetic evaluation software (e.g., version 2.1). The affinity or Ka for a specific binding interaction is preferably about 500 nM or lower, more preferably about 300 nM or lower.
As used herein, a "generic ligand" is a ligand that binds a substantial proportion of functional members in a given repertoire, e.g., in a phage display library. Thus, the same generic ligand can bind many members of the repertoire regardless of their target lig.-and specificities. In general, the presence of a functional generic ligand binding site indicates that the repertoire member is expressed and folded correctly. Thus, binding of the generic ligand to its binding site provides a method for preselecting functional polypeptides from a repertoire of polypeptides.
Generic ligands include, for example, Protein A, Protein G and Protein L.
As used herein, the term "universal framework" refers to a single antibody framework sequence corresponding to the regions of an antibody conserved in sequence as defined by Kabat (supra) or corresponding to the human germline immunoglobulin repertoire or structure as defined by Chothia and Lesk, (1987) J.
Mol. Biol. 196:910-917. The invention provides for the use of a single framework, or a set of such frameworks, which has been found to permit the derivation of virtually any binding specificity though variation in the hypervariable regions alone.
BR1FF DESCRIPTION OF T}-[E FIGURES
Figure 1 shows gel analysis of the quality of biotin-labeled CD4OL used in the screening procedures described herein. (a) 1 lig of non-biotinylated-CD4OL
(Lane 1) and 0.3 ug of biotin-CD4OL (Lane 2) were analysed on SDS-PAGE and detected by Simply Blue Safe-Stain.TM (b) 0.1 lig of biotin-CD4OL (Lane 1) and 0.02 pg of biotin-CD4OL (Lane 2) were detected by Western-blot probing with 1:5000 Streptavidin-HRP.
Figure 2 shows a graphical representation of a dose response receptor binding assay (RBA) readout, analysing the inhibition of CD4OL binding to CD4O-Fc by dAbs DOM-10, -20, -27, -30, -31, -62, -77, titrated from 1 p.M down to 10 pM.
dAbs DOM-20, -30, and -31 are the most potent with IC50 values of approximately 8 nM.
Figure 3 shows a graphical representation of a dose response receptor binding assay readout, analysing the inhibition of CD4OL binding to CD4O-Fc by dAbs DOM-4 and DOM-5, titrated from 1 111\4 down to 500 pM. The IC50 values for dAbs DOM-and DOM-4 are approximately 3 nM and 100 nM respectively.
Figure 4 shows a graphical representation of a dose response receptor binding assay readout, analysing the inhibition of CD4OL binding to CD4O-Fc by dAb DOM-5 24, titrated from 100 nM down to 0.5 pM. The data were curve-fitted using GraphPad Prism software.
Figure 5 shows the sequence of the VH framework based on germline sequence DP47 ¨ JH4b (SEQ ID NO: 1, amino acid sequence; SEQ NO: 2, nucleotide sequence ¨ both sense and antisense strands are shown ¨ SEQ ID NO:
2 is the top strand (sense) and SEQ ID NO: 476 is the lower strand (anti-sense)).
HCDRs 1-3 are indicated by underlining.
Figure 6 shows the sequence of the V, framework based on gennline sequence DPK9 - JK1 (SEQ ID NO: 3, amino acid sequence; SEQ 1D NO: 4, nucleotide sequence ¨ both sense and antisense strands are shown ¨ SEQ ID NO:
4 is the top strand (sense) and SEQ 1D NO: 477 is the lower strand (anti-sense)).
LCDRs 1-3 are indicated by underlining.
Figure 7 shows a schematic representation of the CD4OL binding assay used herein, e.g., in Example 6.
Figure 8 shows various GAS1 secretion signal peptide coding sequences.
GAS wt: The natural occurring sequence in yeast. GAS E.Coli: The nucleotide sequence according to optimal E. Coli codon usage (Wada et al. 1992 NAR 20 p 2111). GAS leader AT: AT rich nucleotide sequence. All nucleotide sequences encode the same amino acid sequence. Yellow (light grey in greyscale) indicates nucleotides that are similar for all sequences. Blue (dark grey in greyscale) indicates nucleotides that are similar to the wt sequence. White (white in greyscale) indicates nucleotides that are different from the wt sequence.

Figure 9 shows the results of a receptor binding assay which demonstrates the affinity of PEGylated DOM8-24cys with either 30K PEG MAL or 40K PEG2-1vIAL.
Figure 10 shows the results of an assay to assess the simultaneous binding of a dual specific dimer to HSA and CD4OL (shaded bars). Binding to control BSA
antigen is also shown (solid bars).
Figure 11 shows the results of an assay to assess the simultaneous binding of a dual specific Fab to HSA and CD4OL (shaded bars). Binding to control skimmed mild powder antigen is also shown (solid bars).
45a Figure 12 shows the results of FACS analysis of the inhibitory effect of monomeric DOM-24 (grey dotted line). Control stimulated cells ae shown as the solid black line and a control dAb is shown as the grey solid line.
Figure 13 shows the results of FACS analysis of the inhibitory effect of the Vk dAb DOM-116 (dotted line). Control stimulated cells are shown as the solid black line and a control dAb is shown as the grey solid line.
DETAILED DESCRIPTION
The invention provides antibody polypeptides that are monovalent for binding to CD4OL. Monovalency for CD4OL binding removes the possibility for cross-linking that occurs with prior art antibodies, and which plays a role in undesirable side effects observed with anti-CD4OL monoclonal antibodies. Further, while not wishing to be limited to any specific mechanism or theory, because antibody polypeptides monovalent for CD4OL cannot cross link CD4OL, the possibility is eliminated that cross-linked CD4OL may in turn cross-link cell surface CD40 and result in agonism of CD40 signaling activity. Thus, in a preferred aspect, the anti-CD4OL antibodies disclosed herein not only inhibit or antagonize the binding of CD4OL to CD40, they do not substantially agonize CD40 and/or CD4OL activity.
In one aspect, the antibodies monovalent for CD4OL binding are human antibody polypeptides. Human antibody polypeptides can be administered to human patients while largely avoiding the anti-antibody immune response often provoked by the administration of antibodies from other species, e.g., mouse. While murine antibodies can be "humanized" by grafting human constant domains onto the murine antigen-binding domains, human antibodies as disclosed herein are produced without the need for laborious and time-consuming genetic manipulation of a murine antibody sequence.
Monovalent antibody polypeptides:
The heavy and light polypeptide chains of antibodies comprise variable (V) regions that directly participate in antigen interactions, and constant (C) regions that provide structural support and function in non-antigen-specific interactions with immune effectors. The antigen binding domain of a conventional antibody is comprised of two separate domains: a heavy chain variable domain (VH) and a light chain variable domain (VL: which can be either VK or Vx). The antigen binding site itself is formed by six polypeptide loops: three from the VH domain (H1, H2 and H3) and three from the VL domain (L1, L2 and L3). In vivo, a diverse primary repertoire of V genes that encode the VH and VL domains is produced by the combinatorial rearrangement of gene segments. C regions include the light chain C regions (referred to as CL regions) and the heavy chain C regions (referred to as CH1, CH2 and CH3 regions). A naturally-occurring antibody generally comprises two antigen binding domains and is therefore divalent.
A number of smaller antigen binding fragments of naturally occurring antibodies have been identified following protease digestion. These include, for example, the "Fab fragment" (VL-CL-CH1-VH), "Fab' fragment" (a Fab with the heavy chain hinge region), and "F(ab')2 fragment" (a dimer of Fab' fragments joined by the heavy chain hinge region). Recombinant methods have been used to generate such fragments and to generate even smaller antigen-binding fragments, e.g., those referred to as "single chain Fv" (variable fragment) or "scFv," consisting of VL and VH joined by a synthetic peptide linker (VL-linker-VH) Fab fragments, Fab' fragments and scFv fragments are monovalent for antigen binding, as they each comprise only one antigen binding domain comprising one VH/VL dimer. Even smaller monovalent antibody fragments are the "domain antibodies," or "dAbs,"
which comprise only a single immunoglobulin variable domain, e.g., VH or VL, that alone specifically binds antigen, i.e., without the need for a complementary VL or VH
domain, respectively.
The term "dAb" will refer herein to a single immunoglobulin variable domain (VH or VL) polypeptide that specifically binds antigen. A. "dAb" binds antigen independently of other V domains; however, a "dAb" can be present in a homo-or heteromultimer with other VH or VL domains where the other domains are not required for antigen binding by the dAb, i.e., where the dAb binds antigen independently of the additional .VH or VL domains. The preparation of single immunoglobulin variable domains is described and exemplified herein below.
Monovalent antibody polypeptides can be generated in several different ways.
For example, the nucleic acid sequence encoding heavy and light chains of an antibody known to bind CD4OL can be manipulated to generate a number of different antibody polypeptides that are monovalent for CD4OL binding. Thus, given the sequences encoding the heavy and light chain polypeptides that constitute an antibody and standard molecular cloning methodologies, one can generate monovalent antigen-binding polypeptide constructs such as Fab fragments, scFv, dAbs, or even bispecific antibodies (i.e., antibodies that comprise two different antigen-binding moieties and can therefore bind two separate antigens, preferably simultaneously) that are monovalent for CD4OL.
Thus, one means of generating monovalent antibody polypeptides specific for CD4OL is to amplify and express the VH and VL regions of the heavy chain and light chain gene sequences isolated, for example, from a hybridoma (e.g., a mouse hybridoma) that expresses anti-CD4OL monoclonal antibody. The boundaries of VH

and VL domains are set out by Kabat et al. (1991, supra). The information regarding the boundaries of the VH and VL domains of heavy and light chain genes is used to design PCR primers that amplify the V domain from a heavy or light chain coding sequence encoding an antibody known to bind CD4OL. The amplified V domains are inserted into a suitable expression vector, e.g., pHEN-1 (Hoogenboom et al.,.
1991, Nucleic Acids Res. 19: 4133-4137) and expressed, e.g., as a fusion of the VH
and VL
in an scFv or other suitable monovalent format. The resulting polypeptide is then screened for high affinity monovalent binding to CD4OL. For all aspects of the present invention, screening for binding is performed as known in the art or as described herein below.
Alternatively, library screening methods can be used to identify monovalent CD4OL-specific binding proteins. Phage display technology (see, e.g., Smith, 1985, Science 228: 1315; Scott & Smith, 1990, Science 249: 386; McCafferty et al., 1990, 48 =

Nature 348: 552) provides an approach for the selection of antibody polypeptides which bind a desired target from among large, diverse repertoires of antibody polypeptides. These phage-antibody libraries can be grouped into two categories:
natural libraries which use rearranged V genes harvested from human B cells (Marks et al., 1991, J. Mol. Biol., 222: 581; Vaughan et al., 1996, Nature Biotech., 14: 309) or synthetic libraries whereby germline V gene segments or other antibody polypeptide coding sequences are 'rearranged' in vitro (Hoogenboorn & Winter, 1992, Mol. Biol., 227: 381; Nissirn et al., 1994, EMBO J., 13: 692; Griffiths et al., 1994, EMBO J., 13: 3245; De Kruif et al., 1995, J. Mot. Biol., 248: 97) or where synthetic CDRs are incorporated into a single rearranged V gene (Barbas et al., 1992.
Proc.
Natl. Acad. Sci. USA, 89: 4457). Methods involving genetic display packages (e.g., phage display, polysome display) are well-suited for the selection of monovalent CD4OL-specific antibody constructs because they generally express only monovalent fragments, rather than whole, divalent antibodies, on the display packages.
Methods for the preparation of phage display libraries displaying various antibody fragments are described in the preceding references. Such methods are also described, for example, in U.S. Patent No. 6,696,245.
The methods described in the '245 patent generally involve the randomization of selected regions of immunoglobulin gene coding regions, in particular VH and VL
coding regions, while leaving other regions non-randomized (see below). The "245 patent also describes the generation of scFv constructs comprising individually randomized VH and VL domains.
The VH gene is produced by the recombination of three gene segments, VH, D
and JH. In humans, there are approximately 51 functional VH segments (Cook and Tomlinson (1995) Immunol Today 16: 237), 25 functional D segments (Corbett et al.
(1997) J. Mol. Biol. 268: 69) and 6 functional JH segments (Ravetch et al.
(1981) Cell 27: 583), depending on the haplotype. The VII segment encodes the region of the polypeptide chain which forms the first and second antigen binding loops of the VH
domain (H1 and H2), while the VH, D and JH segments combine to form the third antigen binding loop of the VH domain (H3).

The VL gene is produced by the recombination of only two gene segments, VL
and JL. In humans, there are approximately 40 functional Vic segments (Schable and Zachau (1993) Biol. Chem. Hoppe-Seyler 374: 1001), 31 functional Vx, segments (Williams et al. (1996) J. Mol. Biol. 264: 220; Kawasaki et al. (1997) Genome Res. 7:
250), 5 functional Jx. segments (Hieter et al. (1982) J. Biol. Chem. 257:
1516) and 4 functional Jx, segments (Vasicek and Leder (1990) J. Exp. Med. 172: 609), depending on the haplotype. The VL segment encodes the region of the polypeptide chain which forms the first and second antigen binding loops of the VL domain (L1 and L2), while the VL and JL segments combine to form the third antigen binding loop of the VL
domain (L3). Antibodies selected from this primary repertoire are believed to be sufficiently diverse to bind almost all antigens with at least moderate affinity. High affinity antibodies are produced in vivo by "affinity maturation" of the rearranged genes, in which point mutations are generated and selected by the immune system on the basis of improved binding.
Analysis of the structures and sequences of antibodies has shown that five of the six antigen binding loops (111, H2, LI, L2, L3) possess a limited number of main-chain conformations or canonical structures (Chothia and Lesk (1987) J. Mol.
Biol.
196: 901; Chothia et al. (1989) Nature 342: 877). The main-chain conformations are determined by (i) the length of the antigen binding loop, and (ii) particular residues, or types of residue, at certain key positions in the antigen binding loop and the antibody framework. Analysis of the loop lengths and key residues has enabled the prediction of the main-chain conformations of H1, H2, L1, L2 and L3 encoded by the majority of human antibody sequences (Chothia et al. (1992) J. Mol. Biol. 227:
799;
Tomlinson et al. (1995) EMBO J. 14: 4628; Williams et al. (1996) J. Mol. Biol.
264:
220). Although the H3 region is much more diverse in terms of sequence, length and structure (due to the use of D segments), it also forms a limited number of main-chain conformations for short loop lengths which depend on the length and the presence of particular residues, or types of residue, at key positions in the loop and the antibody framework (Martin et al. (1996) J. Mol. Biol. 263: 800; Shirai et al. (1996) FEBS
Letters 399: 1.

While, in one approach, diversity can be added to synthetic repertoires at any site in the CDRs of the various antigen-binding loops, this approach results in a greater proportion of V domains that do not properly fold and therefore contribute to a lower proportion of molecules with the potential to bind antigen. An understanding of the residues contributing to the main chain conformation of the antigen-binding loops permits the identification of specific residues to diversify in a synthetic repertoire of VH or VL domains. That is, diversity is best introduced in residues that are not essential to maintaining the main chain conformation. As an example, for the diversification of loop L2, the conventional approach would be to diversify all the residues in the corresponding CDR (CDR2) as defined by Kabat et al. (1991, supra), some seven residues. However, for L2, it is known that positions 50 and 53 are diverse in naturally occurring antibodies and are observed to make contact with the antigen. The preferred approach would be to diversify only those two residues in this loop. This represents a significant improvement in terms of the functional diversity required to create a range of antigen binding specificities.
Immunoglobulin polypeptide libraries can advantageously be designed to be based on predetermined variable doniain main chain conformation. Such libraries may be constructed as described in International Patent Publication WO
99/20749, the contents of which are incorporated herein by reference. Thus, in one aspeat, an antibody polypeptide comprises the amino acid sequence of a given human germline V region gene segment, e.g., VH germline gene segrnent DP-47, or V, germline gene segment DPK9. Such variable regionpolypeptides can be used for the production of scFvs or. Fabs, e.g., an scFv or Fab comprising (i) an antibody heavy chain variable domain (VH), or antigen binding fragment thereof, which comprises the amino acid sequence of germline VH segment DP-47 and (ii) an antibody light chain variable domain (VL), or antigen binding fragment thereof, which comprises the amino acid sequence of germline V, segment DPK9. Diversification of sequences within the context of the selected heavy and light chain germline gene segments,.e.g., DP-47, DPK 9, DP45, DP38, etc. can generate a repertoire of diverse immunoglobulin coding sequences. One approach to diversification is described below in the context of generating a library of diversified dAb or scFv sequences. These variable region polypeptides can also be expressed as dAbs and screened for high affinity binding to CD4OL. The repertoire can be cloned into or generated in a vector suitable for phage display, e.g., a lambda or filamentous bacteriophage display vector and is then screened for binding to a given target antigen, e.g., CD4OL.
Preparation of Human Single Immunoglobulin Variable Domain Polvpeptides:
A single immunoglobulin variable domain is a folded polypeptide domain which comprises sequences characteristic of immunoglobulin variable domains and which specifically binds an antigen (e.g., dissociation constant of 500 nM or less), and which binds antigen as a single variable domain; that is, there is one binding site provided by a single immunoglobulin variable domain without any complementary variable domain. A single immunoglobulin variable domain therefore includes complete antibody variable domains as well as modified variable domains, for example in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain a dissociation constant of 500 nIVI or less (e.g., 450 nM or less, 400 nM or less, 350 nM or less, 300 nM or less, 250 nM or less, 200 nM
or less, 150 nM or less, 100 nM or less) and the target antigen specificity of the full-length domain. Preferably an antibody single variable domain useful in the invention is selected from the group of VH and VL, including Vkappa and Viambda. The single immunoglobulin variable domains of use herein are preferably "human" as that term is defined herein.
Preparation of Single Immunoglobulin Variable Domains:
Single immunoglobulin variable domains are prepared in a number of ways.
For each of these approaches, well-known methods of preparing (e.g., amplifying, =
mutating, etc.) and manipulating nucleic acid sequences are applicable.
One means of preparing single immunoglobulin variable domains is to amplify and express the VH or VL region of a heavy chain or light chain gene for a cloned antibody known to bind the desired antigen. That is, the VH or VL
domain of a known anti-CD4OL antibody coding region can be amplified and expressed as a single domain (or as a fusion of a single domain) and evaluated for binding to CD4OL.
The boundaries of VH and VL domains are set out by Kabat et al. (1991, supra). The infoimation regarding the boundaries of the VH and VL domains of heavy and light chain genes is used to design PCR primers that amplify the V domain from a cloned heavy or light chain coding sequence encoding an antibody known to bind CD4OL.

The amplified V domain is inserted into a suitable expression vector, e.g., pHEN-1 (Hoogenboom et al., 1991, Nucleic Acids Res. 19: 4133-4137) and expressed, either alone or as a fusion with another polypeptide sequence.
In a preferred approach, a repertoire of VH or VL domains, preferably human VH or VL domains, is screened by, for example, phage display, panning against the desired antigen. Methods for the construction of bacteriophage display libraries and lambda phage expression libraries are well known in the art, and taught, for example, by: McCafferty et al., 1990, Nature 348: 552; Kang et al., 1991, Proc. Natl.
Acad.
Sci. U.S.A., 88: 4363; Clackson et al., 1991, Nature 352: 624; Lowman et al., 1991, Biochemistry 30: 10832; Burton et al., 1991, Proc. Natl. Acad. Sci U.S.A. 88:
10134;
Hoogenboom et al., 1991, Nucleic Acids Res. 19: 4133; Chang et al.,1991, J.
Immunol. 147: 3610; Breitling et al., 1991, Gene 104: 147; Marks et al., 1991, J. Mol.
Biol. 222: 581; Barbas et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89: 4457;
Hawkins and Winter (1992) J. Immunol., 22: 867; Marks et al. (1992) J. Biol. Chem., 267:
16007; and Lerner et al. (1992) Science, 258: 1313. Fab phage display libraries are taught, for example, by U.S. 5,922,545. scFv phage libraries are taught, for example, by Huston et al., 1988, Proc. Natl. Acad. Sci U.S.A. 85: 5879-5883; Chaudhary et al., 1990, Proc. Natl. Acad. Sci U.S.A. 87: 1066-1070; McCafferty et al., 1990, supra;
Clackson et al., 1991, supra; Marks et al., 1991, supra; Chiswell et al., 1992, Trends Biotech. 10: 80; and Marks et al., 1992, supra. Various embodiments of scFv libraries displayed on bacteriophage coat proteins have been described.
Refinements of phage display approaches are also known, for example as described in W096/06213 and W092/01047 (Medical Research Council et al.) and W097/08320 (Morphosys, supra).

The repertoire of VH or VL domains can be a naturally-occurring repertoire of immunoglobulin sequences or a synthetic repertoire. A naturally-occurring repertoire is one prepared, for example, from immunoglobulin-expressing cells harvested from one or more individuals. Such repertoires can be "nave," i.e., prepared, for example, from human fetal or newborn immunoglobulin-expressing cells, or rearranged, i.e., prepared from, for example, adult human B cells. Natural repertoires are described, for example, by Marks et al., 1991, J. Mol. Biol. 222: 581 and Vaughan et al., 1996, Nature Biotech. 14: 309. If desired, clones identified from a natural repertoire, or any repertoire, for that matter, that bind the target antigen are then subjected to mutagenesis and further screening in order to produce and select variants with improved binding characteristics.
Synthetic repertoires of single immunoglobulin variable domains are prepared by artificially introducing diversity into a cloned V domain. Synthetic repertoires are described, for example, by Hoogenboom & Winter, 1992, J. Mol. Biol. 227: 381;
Barbas et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89: 4457; Nissim et al., 1994, EMBO J. 13: 692; Griffiths et al., 1994, EMBO J. 13: 3245; DeKriuf et al., 1995, J.
Mol. Biol. 248: 97; and WO 99/20749.
In one aspect, synthetic variable domain repertoires are prepared in VH or VK
backgrounds, based on artificially diversified gennline VII or Vic sequences.
For example, the VH domain repertoire can be based on cloned germline VH gene segments V3-23/DP47 (Tomlinson et al., 1992, J. Mol. Biol. 227: 7768-) and JH4b.
The Vic domain repertoire can be based, for example, on germline Vic gene segments 02/012/DPK9 (Cox et al., 1994, Eur. J. Immunol. 24: 827) and JO.. Diversity is introduced into these or other gene segments by, for example, PCR mutagenesis.
Diversity can be randomly introduced, for example, by error prone PCR
(Hawkins, et al., 1992, J. Mol. Biol. 226: 889) or chemical mutagenesis. As discussed above, however it is preferred that the introduction of diversity is targeted to particular residues. It is further preferred that the desired residues are targeted by introduction of the codon NNK using mutagenic primers (using the IUPAC nomenclature, where N = G, A, T or C, and K = G or T), which encodes all amino acids and the TAG
stop codon. Other codons which achieve similar ends are also of use, including the NNN
codon (which leads to the production of the additional stop codons TGA and TAA), DVT codon ((A/G/T) (A/G/C)T ), DVC codon ((A/G/T)(A/G/C)C), and DVY codon ((A/G/T)(A/G/C)(C/T). The DVT codon encodes 22% serine and 11% tyrosine, =
asgpargine, glycine, alanine, aspartate, threonine and cysteine, which most closely mimics the distribution of amino acid residues for the antigen binding sites of natural human antibodies. Repertoires are made using PCR primers having the selected degenerate codon or codons at each site to be diversified. PCR mutagenesis is well known in the art.
In one aspect, diversity is introduced into the sequence of human germline VH
gene segments V3-23/DP47 (Tomlinson et al., 1992, J. Mol. Biol. 227: 7768) and .11-14b using the NNK codon at sites H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, H95, H97 and H98, corresponding to diversity in CDRs 1, 2 and 3, with the numbering as used in U.S. 6,696,245.
In another aspect, diversity is also introduced into the sequence of human germline VH gene segments V3-23/DP47 and JH4b, for example, using the NNK
codon at sites H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, H95, H97, H98, H99, H100, H100a and H100b, corresponding to diversity in CDRs 1, 2 and 3,with the numbering as used in U.S. 6,696,245.
In another aspect, diversity is introduced into the sequence of human germline gene segments 02/012/DPK9 and J,(1, for example, using the NNK codon at sites L30, L31, L32, L34, L50, L53, L91, L92, L93, L94 and L96, corresponding to diversity in CDRs 1, 2 and 3,with the numbering as used in U.S. 6,696,245.
Diversified repertoires are cloned into phage display vectors as known in the art and as described, for example, in WO 99/20749. In general, the nucleic acid molecules and vector constructs required for the performance of the present invention are available in the art and are constructed and manipulated as set forth in standard laboratory manuals, such as Sambrook et al. (1989). Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, USA.

The manipulation of nucleic acids in the present invention is typically carried out in recombinant vectors. As used herein, "vector" refers to a discrete element that is used to introduce heterologous DNA into cells for the expression and/or replication thereof. Methods by which to select or construct and, subsequently, use such vectors are well known to one of skill in the art. Numerous vectors are publicly available, including bacterial plasmids, bacteriophage, artificial chromosomes and episomal vectors. Such vectors may be used for simple cloning and mutagenesis;
alternatively, as is typical of vectors in which repertoire (or pre-repertoire) members of the invention are carried, a gene expression vector is employed. A vector of use according to the invention is selected to accommodate a polypeptide coding sequence of a desired size, typically from 0.25 kilobase (kb) to 40 kb in length. A
suitable host cell is transformed with the vector after in vitro cloning manipulations. Each vector contains various functional components, which generally include a cloning (or "polylinker") site, an origin of replication and at least one selectable marker gene. If a given vector is an expression vector, it additionally possesses one or more of the following:
enhancer element, promoter, transcription teimination and signal sequences, each positioned in the vicinity of the cloning site, such that they are operatively linked to the gene encoding a polypeptide repertoire member according to the invention.
Both cloning and expression vectors generally contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells.
Typically in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 micron plasmid origin is suitable for yeast, and various viral origins (e.g. SV 40, adenovirus) are useful for cloning vectors in mammalian cells. Generally, the origin of replication is not needed for mammalian expression vectors unless these are used in mammalian cells able to replicate high levels of DNA, such as cos cells.

Advantageously, a cloning or expression vector also contains a selection gene also referred to as selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium.
Host cells not transformed with the vector containing the selection gene will therefore not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available in the growth media.
Because the replication of vectors according to the present invention is most conveniently performed in E. coli, an E. coli-selectable marker, for example, the f3-lactamase gene that confers resistance to the antibiotic ampicillin, is of use. These can be obtained from E. coli plasmids, such as pBR322 or a pUC plasmid such as pUC18 or pUC19.
Expression vectors usually contain a promoter that is recognized by the host organism and is operably linked to the coding sequence of interest. Such a promoter may be inducible or constitutive. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
Promoters suitable for use with prokaryotic hosts include, for example, the 13-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Promoters for use in bacterial systems will also generally contain a Shine-Dalgamo sequence operably =
linked to the coding sequence.
In libraries or repertoires as described herein, the preferred vectors are expression vectors that enable the expression of a nucleotide sequence corresponding to a polypeptide library member. Thus, selection is perfoimed by separate propagation and expression of a single clone expressing the polypeptide library member or by use of any selection display system. As described above, a preferred selection display system uses bacteriophage display. Thus, phage or phagemid vectors can be used. Preferred vectors are phagemid vectors, which have an E.
coli origin of replication (for double stranded replication) and also a phage origin of replication (for production of single-stranded DNA). The manipulation and expression of such vectors is well known in the art (Hoogenboom and Winter (1992) supra; Nissim et al. (1994) supra). Briefly, the vector contains a f3-lactamase or other selectable marker gene to confer selectivity on the phagemid, and a lac promoter upstream of a expression cassette that consists (N to C terminal) of a pelB
leader sequence (which directs the expressed polypeptide to the periplasmic space), a multiple cloning site (for cloning the nucleotide version of the library member), optionally, one or more peptide tags (for detection), optionally, one or more TAG stop codons and the phage protein pIII. In one embodiment, the vector encodes, rather than the pelB leader sequence, a eukaryotic GAS1 leader sequence which serves to direct the secretion of the fusion polypeptide to the periplasmic space in E.
coli or to the medium in eukaryotic cell systems. Using various suppressor and non-suppressor strains of E. coli and with the addition of glucose, iso-propyl thio-13-D-galactoside (IPTG) or a helper phage, such as VCS M13, the vector is able to replicate as a plasmid with no expression, produce large quantities of the polypeptide library member only, or produce phage, some of which contain at least one copy of the polypeptide-pIII fusion on their surface.
An example of a preferred vector is the pliEN1 phagemid vector (Hoogenboom et al., 1991, Nucl. Acids Res. 19: 4133-4137; sequence is available, e.g., as SEQ ID NO: 7 in WO 03/031611), in which the production of pIII fusion protein is under the control of the LacZ promoter, which is inhibited in the presence of glucose and induced with IPTG. When grown in suppressor strains of E. coli, e.g., TG1, the gene III fusion protein is produced and packaged into phage, while growth in non-suppressor strains, e.g., B1B2151, permits the secretion of soluble fusion protein into the bacterial periplasm and into the culture medium. Because the expression of gene 111 prevents later infection with helper phage, the bacteria harboring the phagemid vectors are propagated in the presence of glucose before infection with VCSM13 helper phage for phage rescue.
Construction of vectors according to the invention employs conventional ligation techniques. Isolated vectors or DNA fragments are cleaved, tailored, and re-ligated in the form desired to generate the required vector. If desired, sequence analysis to confirm that the correct sequences are present in the constructed vector is perfouned using standard methods. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing expression and function are known to those skilled in the art. The presence of a gene sequence in a sample is detected, or its amplification and/or expression quantified by conventional methods, such as Southern or Northern analysis, Western blotting, dot blotting of DNA, RNA or protein, in situ hybridization, immunocytochemistry or sequence analysis of nucleic acid or protein molecules. Those skilled in the art will readily envisage how these methods may be modified, if desired.
Screening Single Immunoglobulin Variable Domains for Antig.en Binding:
Following expression of a repertoire of single immunoglobulin variable domains on the surface of phage, selection is performed by contacting the phage repertoire with immobilized target antigen (e.g., CD4OL and/or an epitope bound by DOM8-24), washing to remove unbound phage, and propagation of the bound phage, the whole process frequently referred to as "panning." This process is applicable to the screening of single immunoglobulin variable domains as well as other antibody fragments that can be expressed on a display library, e.g., scFv, Fab, etc.
Alternatively, phage are pre-selected for the expression of properly folded member variants by panning against an immobilized generic ligand (e.g., protein A or protein L) that is only bound by folded members. This has the advantage of reducing the proportion of non-functional members, thereby increasing the proportion of members likely to bind a target antigen. Pre-selection with generic ligands is taught in WO

99/20749. The screening of phage antibody libraries is generally described, for example, by Harrison et al., 1996, Meth. Enzymol. 267: 83-109.
Screening is commonly performed using purified antigen immobilized on a solid support, for example, plastic tubes or wells, or on a chromatography matrix, for example SepharoseTM (Pharmacia). Screening or selection can also be performed on complex antigens, such as the surface of cells (Marks et al., 1993, BioTechnology 11:
1145; de Kruif et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 92: 3938). Another alternative involves selection by binding biotinylated antigen in solution, followed by capture on streptavidin-coated beads.
In a preferred aspect, panning is performed by immobilizing antigen (generic or specific) on tubes or wells in a plate, e.g., Nunc IVIAXISORPTM immunotube 8 well strips. Wells are coated with 150 ul of antigen (100 fi,g/m1 in PBS) and incubated overnight. The wells are then washed 3 times with PBS and blocked with 400 ill PBS-2% skim milk (2%MPBS) at 37 C for 2 hr. The wells are rinsed 3 times with PBS and phage are added in 2%MPBS. The mixture is incubated at room temperature for 90 minutes and the liquid, containing unbound phage, is removed.
Wells are rinsed 10 times with PBS-0.1% tvveen 20, and then 10 times vvith PBS
to remove detergent. Bound phage are eluted by adding 200 1.1,1 of freshly prepared 100 mM triethylamine, mixing well and incubating for 10 min at room temperature.
Eluted phage are transferred to a tube containing 100 111 of 1M Tris-HC1, pH

7.4 and vortexed to neutralize the triethylamine. Exponentially-growing E. coli host cells (e.g., TG1) are infected with, for example, 150 ml of the eluted phage by incubating for 30 min at 37 C. Infected cells are spun down, resuspended in fresh medium and plated in top agarose. Phage plaques are eluted or picked into fresh cultures of host cells to propagate for analysis or for further rounds of selection. One or more rounds of plaque purification are performed if necessary to ensure pure populations of selected phage. Other screening approaches are described by Harrison et al., 1996, supra.

Following identification of phage expressing a single irnmunoglobulin variable domain that binds a desired target, if a phagernid vector such as pHEN1 has been used, the variable domain fusion proteins are easily produced in soluble foini by infecting non-suppressor strains of bacteria, e.g., HB2151 that permit the secretion of soluble gene III fusion protein. If a GAS1 secretion signal peptide is encoded by the vector, the fusion polypepticie can be secreted by eukaryotic (e.g., yeast or mammalian) or prokaryotic (e.g., E. col!) cells. Alternatively, the V domain sequence can be sub-cloned into an appropriate expression vector to produce soluble protein according to methods known in the art.
Purification and Concentration of Single Immunoglobulin Variable Domains:
Single immunoglobulin variable domain polypeptides or other monovalent antibody polypeptides secreted into the periplasmic space or into the medium of bacteria are harvested and purified according to known methods (Harrison et al., 1996, supra). Skerra & Pluckthun (1988, Science 240: 1038) and Breitling et al.
(1991, Gene 104: 147) describe the harvest of antibody polypeptides from the periplasm, and Better et al. (1988, Science 240: 1041) describes harvest from the culture supernatant. For some antibody polypeptides, purification can also be achieved by binding to generic ligands, such as protein A or Protein L.
Alternatively, the variable domains can be expressed with a peptide tag, e.g., the Myc, HA or His tags, which facilitates purification by affinity chromatography.
If necessary, monovalent anti-CD4OL antibody polypeptides are concentrated by any of several methods well known in the art, including, for example, ultrafiltration, diafiltration and tangential flow filtration. The process of ultrafiltration uses semi-permeable membranes and pressure to separate molecular species on the basis of size and shape. The pressure is provided by gas pressure or by centrifugation. Commercial ultrafiltration products are widely available, e.g., from Millipore (Bedford, MA; examples include the CentriconTM and MicroconTM
concentrators) and Vivascience (Hannover, Germany; examples include the VivaspinTM concentrators). By selection of a molecular weight cutoff smaller than the target polypeptide (usually 1/3 to 1/6 the molecular weight of the target polypeptide, although differences of as little as 10 kD can be used successfully), the polypeptide is retained when solvent and smaller solutes pass through the membrane.
Thus, a molecular weight cutoff of about 5 kD is useful for concentration of anti-CD4OL single immunoglobulin variable domain polypeptides described herein.
Diafiltration, which uses ultrafiltration membranes with a "washing" process, is used where it is desired to remove or exchange the salt or buffer in a polypeptide preparation. The polypeptide is concentrated by the passage of solvent and small solutes through the membrane, and remaining salts or buffer are removed by dilution of the retained polypeptide with a new buffer or salt solution or water, as desired, accompanied by continued ultrafiltration. In continuous diafiltration, new buffer is added at the same rate that filtrate passes through the membrane. A
diafiltration volume is the volume of polypeptide solution prior to the start of diafiltration ¨ using continuous diafiltration, greater than 99.5% of a fully permeable solute can be removed by washing through six diafiltration volumes with the new buffer.
Alternatively, the process can be performed in a discontinuous manner, wherein the sample is repeatedly diluted and then filtered back to its original volume to remove or exchange salt or buffer and ultimately concentrate the polypeptide. Equipment for diafiltration and detailed methodologies for its use are available, for example, from Pall Life Sciences (Ann Arbor, MI) and Sartorius AG/Vivascience (Hannover, Germany).
Tangential flow filtration (TFF), also known as "cross-flow filtration," also uses ultrafiltration membrane. Fluid containing the target polypeptide is pumped tangentially along the surface of the membrane. The pressure causes a portion of the fluid to pass through the membrane while the target polypeptide is retained above the filter. In contrast to standard ultrafiltration, however, the retained molecules do not accumulate on the surface of the membrane, but are carried along by the tangential flow. The solution that does not pass through the filter (containing the target polypeptide) can be repeatedly circulated across the membrane to achieve the desired degree of concentration. Equipment for TFF and detailed methodologies for its use =
are available, for example, from Millipore (e.g., the ProFlux M12TM Benchtop TFF

system and the pelljconTM systems), Pall Life Sciences (e.g., the 1/IinirnTM
Tangential Flow Filtration system).
Protein concentration is measured in a number of ways that are well known in the art. These include, for example, amino acid analysis, absorbance at 280 run, the "Bradford" and "Lowry" methods, and SDS-PAGE. The most accurate method is total hydrolysis followed by amino acid analysis by 1-IPLC, concentration is then determined then comparison with the known sequence of the single immunoglobulin variable domain polypeptide. While this method is the most accurate, it is expensive and time-consuming. Protein determination by measurement of LTV absorbance at 280 nm faster and much less expensive, yet relatively accurate and is preferred as a compromise over amino acid analysis. Absorbance at 280 nm was used to deteitnine protein concentrations reported in the Examples described herein.
"Bradford" and "Lowry" protein assays (Bradford, 1976, Anal. Biochem. 72:
248-254; Lowry et al.,1951, J. Biol. Chem. 193: 265-275) compare sample protein concentration to a standard curve most often based on bovine serum albumin (BSA).
These methods are less accurate, tending to undersetitnate the concentration of single immunoglobulin variable domains. Their accuracy could be improved, however, by using a Vll or V, single domain polypeptide as a standard.
An additional protein assn:. method is the bicinchoninic acid assay described in U.S. Patent No. 4,839,295 and marketed by Pierce. Biotechnology (Rockford, IL) as the "BCA Protein Assay" (e.g., Pierce Catalog No. 23227).
The SDS-PAGE method uses gel electrophoresis and Coomassie Blue staining in comparison to known concentration standards, e.g., known amounts of a single immunoglobulin variable domain polypeptide. Quantitation can be done by eye or by densitometry.
Single human immunoglobulin variable domain antigen-binding polypeptides described herein retain solubility at high concentration (e.g., at least 4.8 mg (-400 M) in aqueous solution (e.g., PBS), and preferably at least 5 mg/ml (-417 M), mg/ml (-833 pM), 20 mg/ml (-1.7 mM), 25 mg/ml (-2.1 mM), 30 mg/ml (-2.5 mM), 35 mg/ml (-2.9 mM), 40 mg/ml (-3.3 mM), 45 mg/ml (-3.75 mM), 50 mg/ml (-4.2 mM), 55 mg/ml (-4.6 mM), 60 mg/ml (-5.0 mM), 65 mg/ml (-5.4 mM), 70 mg/ml (-5.8 mM), 75 mg/ml (-6.3 mM), 100 mg/ml (-8.33 mM), 150 mg/ml (-12.5 mM), 200 mg/ml (-16.7 mM), 240 mg/ml (-20 mM) or higher). One structural feature that promotes high solubility is the relatively small size of the single immunoglobulin variable domain polypeptides. A full length conventional four chain antibody, e.g., IgG is about 150 kD in size. In contrast, single immunoglobulin variable domains, which all have a general structure comprising 4 framework (FW) regions and 3 CDRs, have a size of approximately 12 kD, or less than 1/10 the size of a conventional antibody. Similarly, single immunoglobulin variable domains are approximately 4 the size of an scFv molecule (-26 10), and approximately 1/5 the size of a Fab molecule (-60 kD). It is preferred that the size of a single immunoglobulin variable domain-containing structure disclosed herein is 100 kD
or less, including structures of, for example, about 90 kD or less, 80 kD or less, 70 10 or less, 60 kD or less, 50 kD or less, 40 kD or less, 30 kD or less, 20 kD or less, down to and including about 12 kD, or a single immunoglobulin variable domain in isolation.
The solubility of a polypeptide is primarily determined by the interactions of the amino acid side chains with the surrounding solvent. Hydrophobic side chains tend to be localized internally as a polypeptide folds, away from the solvent-interacting surfaces of the polypeptide. Conversely, hydrophilic residues tend to be localized at the solvent-interacting surfaces of a polypeptide. Generally, polypeptides having a primary sequence that permits the molecule to fold to expose more hydrophilic residues to the aqueous environment are more soluble than one that folds to expose fewer hydrophilic residues to the surface. Thus, the arrangement and number of hydrophobic and hydrophilic residues is an important determinant of solubility. Other parameters that determine polypeptide solubility include solvent pH, temperature, and ionic strength. In a common practice, the solubility of polypeptides can be maintained or enhanced by the addition of glycerol (e.g., ¨10% v/v) to the solution.

As discussed above, specific amino acid residues have been identified in conserved residues of human VH domains that vary in the VH domains of camelid species, which are generally more soluble than human VH domains. These include, for example, Gly 44 (Glu in camelids), Leu 45 (Arg in camelids) and Trp 47 (Gly in camelids). Amino acid residue 103 of VH is also implicated in solubility, with mutation from Trp to Arg tending to confer increased VH solubility.
In preferred aspects of the invention, single immunoglobulin variable domain polypeptides are based on the DP47 germline VH gene segment or the DPK9 germline V, gene segment. Thus, these germline gene segments are capable, particularly when diversified at selected structural locations described herein, of producing specific binding single immunoglobulin variable domain polypeptides that are highly soluble.
In particular, the four framework regions, which are preferably not diversified, can contribute to the high solubility of the resulting proteins.
It is expected that a single human immunoglobulin variable domain that is highly homologous to one having a known high solubility will also tend to be highly soluble. Thus, as one means of prediction or recognition that a given single immunoglobulin variable domain would have the high solubility recited herein, one can compare the sequence of a single immunoglobulin variable domain polypeptide to one or more single immunoglobulin variable domain polypeptides having known solubility. Thus, when a single immunoglobulin variable domain polypeptide is identified that has high binding affinity but unknown solubility, comparison of its amino acid sequence with that of one or more (preferably more) human single immunoglobulin variable domain polypeptides known to have high solubility (e.g., a dAb sequence disclosed herein) can permit prediction of its solubility. While it is not an absolute predictor, where there is a high degree of similarity to a known highly soluble sequence, e.g., 90-95% or greater similarity, and particularly where there is a high degree of similarity with respect to hydrophilic amino acid residues, or residues likely to be exposed at the solvent interface, it is more likely that a newly identified binding polypeptide will have solubility similar to that of the known highly soluble sequence.

Molecular modeling software can also be used to predict the solubility of a polypeptide sequence relative to that of a polypeptide of known solubility.
For example, the substitution or addition of a hydrophobic residue at the solvent-exposed surface, relative to a molecule of known solubility that has a less hydrophobic or even hydrophilic residue exposed in that position is expected to decrease the relative solubility of the polypeptide. Similarly, the substitution or addition of a more hydrophilic residue at such a location is expected to increase the relative solubility.
That is, a change in the net number of hydrophilic or hydrophobic residues located at the surface of the molecule (or the overall hydrophobic or hydrophilic nature of the surface-exposed residues) relative to a single immunoglobulin variable domain polypeptide structure with known solubility can predict the relative solubility of a single immunoglobulin variable domain polypeptide.
Alternatively, or in conjunction with such prediction, one can determine limits of a single immunoglobulin variable domain polypeptide's solubility by simply concentrating the polypeptide.
Affinitv Detei ___ mination:
Isolated single immunoglobulin variable domain- and antibody polypeptide-containing polypeptides as described herein preferably have affinities (dissociation constant, Kd, = Koff/Kõ,,) of at least 500 nM or less, and preferably at least 400 nM-50 pM, 300 nM-50 pM, 200 nM ¨ 50 pM, and more preferably at least 100 nM ¨ 50 pM, 75 nM¨ 50 pM, 50 ¨ 50 pM, 25 nM ¨ 50 p1\4, 10 nM ¨ 50 pM, 5 ¨ 50 pM, 1 nI\4 ¨ 50 p1\4, 950 pM ¨ 50 pM, 900 pM ¨ 50 pM, 850 pM ¨ 50 pM, 800 pM ¨ 50 pM, 750 pM ¨ 50 pM, 700 pM ¨ 50 pM, 650 pM ¨50 pIVI, 600 p1\4 ¨ 50 p1\4, 550 pM ¨

04, 500 pIVI ¨ 50 0\4, 450 pIVI ¨ 50 pM, 400 p1\4 ¨ 50 pM, 350 p1\4 ¨ 50 pM, 300 pM
¨ 50 pM, 250 pI\4 ¨ 50 pM, 200 04¨ 50 pM, 150 pM ¨ 50 pM, 100 pM ¨ 50 pIVI, 90 pM ¨ 50 pl\4., 80 pM ¨ 50 pM, 70 pM ¨ 50 pM, 60 pM ¨ 50 pM, or even as low as p1\4.
The antigen-binding affinity of an antibody. polypeptide, e.g., a single immunoglobulin variable domain polypeptide or other monovalent antibody polypeptide, can be conveniently measured by SPR using the BIAcorermsystem (Pharmacia Biosensor, Piscataway, N.J.). In this method, antigen is coupled to the BIAcore chip at known concentrations, and variable domain polypeptides are introduced. Specific binding between the variable domain polypeptide and the immobilized antigen results in increased protein concentration on the chip matrix and a change in the SPR signal. Changes in SPR signal are recorded as resonance units (RU) and displayed with respect to time along the Y axis of a sensorgram.
Baseline signal is taken with solvent alone (e.g., PBS) passing over the chip. The net difference between baseline signal and signal after completion of antibody polypeptide injection represents the binding value of a given sample. To determine the off rate (Koff), on rate (Kon) and dissociation rate (Kd) constants, BIAcore kinetic evaluation software (e.g., version 2.1) is used.
Thus, SPR can be used to monitor antagonism of CD4OL binding to CD40 by a monovalent anti-CD4OL antibody preparation by measuring the displacement or inhibition of binding of CD4OL to CD40 caused the monovalent antibody preparation.
SPR can also be used to monitor the dimerization, or preferably, the lack of dimerization, occurring via Fc region in antibody preparations as described herein.
High affinity is dependent upon the complementarity between a surface of the antigen and the CDRs of the antibody or antibody fragment. Cornplementarity is determined by the type and strength of the molecular interactions possible between portions of the target and the CDR, for example, the potential ionic interactions, van der Waals attractions, hydrogen bonding or other interactions that can occur.

tends to contribute more to antigen binding interactions than CDRs 1 and 2, probably due to its generally larger size, which provides more opportunity for favorable surface interactions. (See, e.g., Padlan et al., 1994, Mol. Immunol. 31: 169-217;
Chothia &
Lesk, 1987, J. Mol. Biol. 196: 904-917; and Chothia et al., 1985, J. Mol.
Biol. 186:
651-663.) High affinity indicates antibody polypeptide/antigen pairings that have a high degree of complementarity, which is directly related to the structures of the variable domain and the target.

In one aspect, a monovalent anti-CD4OL antibody polypeptide, e.g., a single immunoglobulin variable domain polypeptide, is linked to another antibody polypeptide to form a heterodimer in which each individual antibody polypeptide is capable of binding a different cognate antigen. Fusing antibody polypeptides, such as single immunoglobulin variable domains, as heterodimers, wherein each monomer binds a different target antigen, can produce a dual-specific ligand capable, for example, of bridging the respective target antigens. Such dual specific ligands may be used to target cytokines and other molecules which cooperate synergistically in therapeutic situations in the body of an organism. Thus, there is provided a method for synergising the activity of two or more cytokines, comprising administering a dual specific antibody heterodimer capable of binding to the two or more cytokines.
Non-limiting examples of second targets for anti-CD4OL dual specific antibody polypeptides include the following: TNF-a; IL-1; IL-2; IL-4; IL-6; IL-

8; IL-12; IL-18; IFNI; CD2; CD4; CD8; CTLA4; LFA1 ; LFA3, VLA4, CD80, B7-1, CD28, CD86, B7-2, and CTLA-4. In particular, second targets useful according to the invention include CD80, B7-1, CD28, CD86, B7-2, and CTLA-4. These targets are thought to be involved in a co-stimulatory pathway critical for T-cell activation (termed, co-stimulatory signal pathway antigens). This pathway includes activation of the molecule CD28 on the surface of T cells. This molecule can receive a costimulatory signal delivered by a ligand on B cells or other APCs. Ligands for CD28 include members of the B7 family of B lymphocyte activation antigens, such as B7-1 and/or B7-2 (Freedman, A. S. et al. (1987) J. Immunol. 137, 3260-3267;
Freeman, G. J. et al, (1989) J. Immunol. 143, 2714-2722; Freeman, G. J. et al.
(1991) J. Exp. Med. 174, 625-631; Freeman, G. J. et al. (1993) Science 262, 909-911;
Azuma, M. et al. (1993) Nature 366, 76-79; Freeman, G. J. et al. (1993) J.
Exp. Med.
178, 2185-2192). B7-1 and B7-2 are also ligands for another molecule, CTLA4, present on the surface of activated T cells. Accordingly, the present invention contemplates that members of the CD28 signalling pathway may be useful second targets for the dual-specific format anti-CD4OL antibody polypeptides.
Homologous sequences:

The invention encompasses anti-CD4OL antibody polypeptides, e.g., CD4OL-binding single immunoglobulin variable domain clones, and clones with substantial sequence similarity or homology to them that also bind target antigen with high affinity. As used herein, "substantial" sequence similarity or homology is at least 85% similarity or homology.
Calculations of "homology" or "sequence identity" between two sequences (the terms are equivalent and used interchangeably herein) are performed as follows.
The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity").
The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
As used herein, sequence "similarity" is a measure of the degree to which amino acid sequences share similar amino acid residues at corresponding positions in an alignment of the sequences. Amino acids are similar to each other where their side chains are similar. Specifically, "similarity" encompasses amino acids that are conservative substitutes for each other. A "conservative" substitution is any substitution that has a positive score in the blosum62 substitution matrix (Hentikoff and Hentikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919). By the statement "sequence A is n% similar to sequence B" is meant that n% of the positions of an optimal global alignment between sequences A and B consists of identical amino acids or conservative substitutions. Optimal global alignments can be performed using the following parameters in the Needleman-Wunsch alignment algorithm:
For polypeptides:
Substitution matrix: blosum62.
Gap scoring function: -A -B*LG, where A=11 (the gap penalty), B=1 (the gap length penalty) and LG is the length of the gap.
For nucleotide sequences:
Substitution matrix: 10 for matches, 0 for mismatches.
Gap scoring function: -A -B*LG where A=50 (the gap penalty), B=3 (the gap length penalty) and LG is the length of the gap.
Typical conservative substitutions are among Met, Val, Leu and lle; among Ser and Thr; among the residues Asp, Glu and Asn; among the residues Gln, Lys and Arg; or aromatic residues Phe and Tyr. In calculating the degree (most often as a percentage) of similarity between two polypeptide sequences, one considers the number of positions at which identity or similarity is observed between corresponding amino acid residues in the two polypeptide sequences in relation to the entire lengths of the two molecules being compared.
Alternatively, the BLAST (Basic Local Alignment Search Tool) algorithm is employed for sequence alignment, with parameters set to default values. The BLAST
algorithm "BLAST 2 Sequences" is available at the world wide web site ("www") of the National Center for Biotechnology Information (".ncbi"), of the National Library of Medicine (".nlm") of the National Institutes of Health ("nih") of the U.S.
government (".gov"), in the "/blast/" directory, sub-directories "b12seq/b12.html."
This algorithm aligns two sequences for comparison and is described by Tatusova &
Madden, 1999, FEMS Microbiol Lett. 174:247-250.
An additional measure of homology or similarity is the ability to hybridize under highly stringent hybridization conditions. Thus, a first sequence encoding a single immunoglobulin variable domain polypeptide is substantially similar to a second coding sequence if the first sequence hybridizes to the second sequence (or its complement) under highly stringent hybridization conditions (such as those described by Sambrook et al., Molecular Cloning, Laboratory Manuel, Cold Spring, Harbor Laboratory press, New York). "Highly stringent hybridization conditions" refer to hybridization in 6X SSC at about 45 C, followed by one or more washes in 0.2X
SSC, 0.1% SDS at 65 C. "Very highly stringent hybridization conditions" refer to hybridization in 0.5M sodium phosphate, 7% SDS at 65 C, followed by one or more washes at 0.2X SSC, I% SDS at 65 C.
Assays for CD4OL Activities:
It is preferred that a monovalent anti-CD4OL antibody polypeptides as described herein bind to CD4OL yet do not substantially agonize CD40 signaling.
Activation of the CD4OL/CD40 pathway manifests a number of different outcomes that can be measured in order to assess the effect of a given monovalent anti-antibody polypeptide on the activity of the pathway. However, for the assessment of the antagonist or agonist function of monovalent anti-CD4OL antibody polypeptides described herein, at least one of the following CD4OL assays can be used:
1) Activation of Jun-N-Terminal Kinase (JNK):
Stimulation of T-lymphocytes via CD4OL induces strong activation of JNK.
The ability of a monovalent anti-CD4OL antibody polypeptide to activate this signaling pathway is measured as follows. Human leukemic Jurkat cells are stimulated with a positive control agonistic anti-CD4OL antibody (2 ug/m1 monoclonal anti-human or anti-mouse gp39/CD4OL antibody (Pharmingen, San Diego, CA, USA) or isotype matched hamster or mouse immunoglobulins (Dianova, Hamburg, Germany)), monovalent anti-CD4OL antibody polypeptide, or a negative control irrelevant antibody as described by Brenner et al., 1997, FEBS Lett.
417: 301-306, which is incorporated herein by reference. The cells are lysed and the extract assayed for phosphorylated INK via colorimetric assay= (e.g., TiterzymeTm colorimetric (EIA) phospho-JNK1/2 immunoassay kit, by Assay Designs Inc., Catalog # 900-106). An increase in phospho-JNK (e.g., by 5% or more) for anti-CD4OL-stimulated cells over non-stimulated cells indicates agonism of CD4OL
activity by the antibody polypeptide.
2. Induction of Cytokine Secretion:
Co-stimulation of T cells with anti-CD3 Ab and CD4OL has been shown to upregulate the production of IL-10, IFN-y and TNF-a by those cells. The ability of a monovalent anti-CD4OL antibody polypeptide to activate this sig-naling pathway is measured as follows. Human leukemic Jurkat T cells (or freshly isolated CD4+ T

cells) are plated into 96 well plates containing immobilized anti-CD3 antibody. The cells are then cultured for 72 hours in the presence of a positive control agonistic anti-CD4OL antibody, CD4OL, monovalent anti-CD4OL antibody polypeptide, or a negative control irrelevant antibody, as described by Blair et al., 2000, J.
Exp. Med.
191: 651-660. IFN-y (or IL-10 of TNF-a) is then quantitated in the supernatant by sandwich ELISA using an IFN-g standard to generate a standard curve from which all other unknowns can be calculated. An increase in IFN-g (e.g., by 5% or more) for anti-CD4OL-stimulated cells over non-stimulated cells indicates agonism by the antibody polypeptide.
3. Platelet Aggregation Assay:
Divalent anti-CD4OL antibodies tend to cause platelet aggregation, which is likely associated with the thromboembolic events observed in clinical trials of divalent anti-CD4OL antibodies in the prior art. Monovalent anti-CD4OL
antibody polypeptides as described herein preferably do not substantially mediate or agonize CD4OL-mediated platelet aggregation. With regard tothis aspect, the "standard platelet aggregation assay" is as follows:
Platelets are prepared at 2.5x105/m1 and left stirring in a 500-Ca lumi-aggregometer (or its equivalent, e.g., a Platelet Aggregation Profiler (BioData, Horsham, PA)). Platelets are partially activated by the addition of a dilution series of 0.1-10 M ADP (the 10 M ADP induces aggregation, and is used as a positive control - lower concentrations activate platelets but do not induce aggregation).
CD4OL mediated platelet aggregation is stimulated by addition of either anti-monoclonal antibodies (i.e., divalent monoclonal antibodies, available from, e.g., Pharmingen, San Diego, CA, USA) or soluble CD40/Fc fusion protein (available from R&D Systems). The reaction is allowed to proceed for between 3 and 5 minutes. Stimulation of platelet aggregation above the mininimal aggregation/activation achieved with ADP alone is plotted against stimulating anti-CD4OL or CD40/Fc concentration. The percentage of platelet aggregation is measured by the change in light transmittance following addition of antibody polypeptide being tested or positive control peptide. A value greater than that observed for negative control lacking antibody and amounting to 25% or more of the positive control value (divalent anti-CD4OL or CD40/Fc fusion) is considered to be indicative of induction of platelet aggregation.
Other methods to assess platelet aggregation and/or activation, including events which precede aggregation, or which are downstream from platelet aggregation, include assays which determine various indicators of platelet activation, and are known in the art. For example, platelet activation (and, thus, activity) can be determined by assaying for CD62P expression in platelets (e.g., using anti-CD26P antibodies and flow cytometry), assaying for monocyte-platelet-conjugate formation, assaying for platelet closure time under high shear conditions (e.g., using a PFA-100, Dade Behring, Newark, DE), and assaying for platelet dense granule release. Methods for performing such assays are known in the art and can be found, for example, in Langer et al., 2005 Thromb. Haemost. 93: 1137-46.
PEGylation of monovalent anti-CD4OL antibody polypeptides The present invention provides PEGylated monovalent anti-CD4OL antibody polypeptides which have increased half-life and preferably also resistance to degradation without a loss in activity (e.g., binding affinity) relative to non-PEGylated antibody polypeptides.

Monovalent anti-CD4OL antibody polypeptides according to this aspect can be coupled, using methods known in the art to polymer molecules (preferably PEG) useful for achieving the increased half-life and degradation resistance properties encompassed by the present invention. Polymer moieties which can be utilized in the invention can be synthetic or naturally occurring and include, but are not limited to straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymers, or a branched or unbranched polysaccharide such as a homo- or heteropolysaccharide. Preferred examples of synthetic polymers which may be used in the invention include straight or branched chain poly(ethylene glycol) (PEG), poly(propylene glycol), or poly(vinyl alcohol) and derivatives or substituted forms thereof. Particularly preferred substituted polymers useful in the invention include substituted PEG, including methoxy(polyethylene glycol). Naturally occurring polymer moieties which may be used according to the invention in addition to or in place of PEG include lactose, amylose, dex-tran, or glycogen, as well as derivatives thereof which would be recognized by one of skill in the art. Derivatized forms of polymer molecules of the invention include, for example, derivatives which have additional moieties or reactive groups present therein to permit interaction with amino acid residues of the dAb polypeptides described herein. Such derivatives include N-hydroxylsuccinimide (NHS) active esters, succinimidyl propionate polymers, and sulfhydryl-selective reactive agents such as maleimide, vinyl sulfone, and thiol.
Particularly preferred derivatized polymers include, but are not limited to PEG
polymers having the formulae: PEG-0-CH2CH2CH2-0O2-NHS; PEG-0-CH2-NHS;
PEG-0-CH2CH2-0O2-NHS; PEG- S-CH, CH2-C O-NHS ; PEG-02 CNH-CH(R)-0O2-NHS; PEG-NHCO-CH2CH1-CO-NHS; and PEG-0-CH2-0O2-NHS; where R is (CH2)4)NHCO2(mPEG). PEG polymers useful in the invention may be linear molecules, or may be branched wherein multiple PEG moieties are present in a single polymer. Some particularly preferred PEG derivatives which are useful in the invention include, but are not limited to the following:

roPEO ¨0 ¨t ¨NW
I
0 (C1-1.2)A
mpEG ¨ N 1 .NH¨CHICHI¨NH ¨,t ¨012012¨N I ;
.//
o mPEG-MAL mPEG2-MAL
"..........
CH2CONH(CH2CH20)2-CH2CH2N l ri ri r I \,-#
KG KG KG
I / clo mPEG -CONHCH 0 r, CH2CONH(CH2CH20)2- CH2CH2N I
/i mPEG-(MAL)2 multi-arm PEG
:

li rtilin -0 -C - NH
0 . 1 li,õ...L ICH44 0 0 1 Ilt_ il / - I /CH / ----) mPEO¨O ¨CI-I2CH2 ¨C ¨ 0 ¨N , TOW ¨0 ¨ =¨NI.1 \ C¨O¨N I
fi e mPEG2-NI-IS
mPEG-SPA
;

mPEG2-(MAL)2 /
I I ai,coNH(cH2cH20)2¨ cH2cH2N
mPEG- C-NH

mPEG- C- NH
0 CH2CONH(CH2CH20)2-CH2CH2N

; and The reactive group (e.g., MAL, NHS, SPA, VS, or Thiol) may be attached directly to the PEG polymer or may be attached to PEG via a linker molecule.
The size of polymers useful in the invention can be in the range of between 500 Da to 60 kDa, for example, between 1000 Da and 60 kDa, 10 kDa and 60 kDa, kDa and 60 kDa, 30 kDa and 60 kDa, 40 kDa and 60 kDa, and up to between 50 kDa and 60 kDa. The polymers used in the invention, particularly PEG, can be straight chain polymers or can possess a branched conformation. Depending on the combination of molecular weight and conformation, the polymer molecules useful in the invention, when attached to a monovalent anti-CD4OL antibody polypeptide, will yield a molecule having an average hydrodynamic size of between 24 and 500 kDa.
The hydrodynamic size of a polymer molecule used herein refers to the apparent size of a molecule (e.g., a protein molecule) based on the diffusion of the molecule through an aqueous solution. The diffusion, or motion of a protein through solution can be processed to derive an apparent size of the protein, where the size is given by the Stokes radius or hydrodynamic radius of the protein particle. The "hydrodynamic size" of a protein depends on both mass and shape (conformation), such that two proteins having the same molecular mass may have differing hydrodynamic sizes based on the overall conformation of the protein. The hydrodynamic size of a PEG-linked monovalent anti-CD4OL antibody polypeptide, e.g., an anti-CD4OL single immunoglobulin variable domain as described herein, can be in the range of 24 kDa to 500 kDa; 30 to 500 kDa; 40 to 500 kDa; 50 to 500 kDa; 100 to 500 kDa; 150 to 500 kDa; 200 to 500 kDa; 250 to 500 kDa; 300 to 500 kDa; 350 to 500 kDa; 400 to 500 kDa and 450 to 500 kDa. Preferably the hydrodynamic size of a PEGylated antibody polypeptide as described herein is 30 to 40 kDa; 70 to 80 kDa or 200 to 300 kDa. The size of a polymer molecule attached to a monovalent anti-CD4OL
antibody polypeptide may thus be varied depending upon the desired application. For example, where the PEGylated antibody polypeptide is intended to leave the circulation and enter into peripheral tissues, it is desirable to keep the size of the attached polymer low to facilitate extravazation from the blood stream. Alternatively, where it is desired to have the PEGylated antibody polypeptide remain in the circulation for a longer period of time, a higher molecular weight polymer can be used (e.g., a 30 to 60 kDa polymer).
The polymer (PEG) molecules useful in the invention can be attached to antibody polypeptides using methods that are well known in the art. The first step in the attachment of PEG or other polymer moieties to an antibody polypeptide is the substitution of the hydroxyl end-groups of the PEG polymer by electrophile-containing functional groups. Particularly, PEG polymers are attached to either cysteine or lysine residues present in the antibody polypeptide. The cysteine and lysine residues can be naturally occurring, or can be engineered into the antibody polypeptide molecule. For example, cysteine residues can be recombinantly engineered at the C-terminus of antibody polypeptides, or residues at specific solvent accessible locations in the antibody polypeptide can be substituted with cysteine or lysine. In a preferred embodiment, a PEG moiety is attached to a cysteine residue which is present in the hinge region at the C-terrainus of an antibody polypeptide.
In a further preferred embodiment a PEG moiety or other polymer is attached to a cysteine or lysine residue which is either naturally occurring at or engineered into the N-terminus of antibody single variable domain polypeptide of the invention. In a still further embodiment, a PEG moiety or other polymer is attached to an antibody single variable domain according to the invention at a cysteine or lysine residue (either naturally occurring or engineered) which is at least 2 residues away from (e.g., internal to) the C- and/or N-terminus of the antibody single variable domain polyp epti de.
In one embodiment, the PEG polymer(s) is attached to one or more cysteine or lysine residues present in a framework region (FWs) and one or more heterologous CDRs of a single immunoglobulin variable domain. CDRs and framework regions (e.g., CDR1-CDR3 and FW1-FW4) are those regions of an immunoglobulin variable domain as defined in the Kabat database of Sequences of Proteins of Immunological Interest (Kabat et al., 1991, supra). In a preferred embodiment, a PEG polymer is linked to a cystine or lysine residue in the VH framework segment DP47, or the Vk framework segment DPK9. Cysteine and/or lysine residues of DP47 which may be linked to PEG according to the invention include the cysteine at positions 22, or 96 and the lysine at positions 43, 65, 76, or 98 of SEQ ID NO: 1 (Figure 5).
Cysteine and/or lysine residues of DPK9 which may be linked to PEG according to the invention include the cysteine residues at positions 23, or 88 and the lysine residues at positions 39, 42, 45, 103, or 107 of SEQ ID NO: 3 (Figure 6). In addition, specific cysteine or lysine residues may be linked to PEG in the VH canonical framework region DP38, or DP45.
In addition, specific solvent accessible sites in the antibody molecule which are not naturally occurring cysteine or lysine residues may be mutated to a cysteine or lysine for attachment of a PEG polymer. Solvent accessible residues in any given antibody, e.g., a dAb, can be determined using methods known in the art such as analysis of the crystal structure of the antibody polypeptide. For example, using the solved crystal structure of the VH dAb HEL4 (SEQ ID NO: 3; a dAb that binds hen egg lysozyme), the residues Gln-13, Pro-14, Gly-15, Pro-41, Gly-42, Lys-43, Asp-62, Lys-65, Arg-87, Ala-88, G1u-89, Gln-112, Leu-115, Thr-117, Ser-119, and Ser-have been identified as being solvent accessible, and according to the present ' invention would be attractive candidates for mutation to cysteine or lysine residues for the attachment of a PEG polymer. In addition, using the solved crystal structure of the Vk dummy dAb (SEQ ID NO: 4), the residues Val-15, Pro-40, Gly-41, Ser-56, G1)7-57, Ser-60, Pro-80, Glu-81, Gln-100, Lys-107, and Arg-108 have been identified as being solvent accessible, and according to the present invention would be attractive candidates for mutation to cysteine or lysine residues for the attachment of a PEG
polymer. In one embodiment of the invention, a PEG polymer is linked to multiple solvent accessible cysteine or lysine residues, or to solvent accessible residues which have been mutated to a cysteine or lysine residue. Alternatively, only one solvent accessible residue is linked to PEG, either where the particular antibody polypeptide only possesses one solvent accessible cysteine or lysine (or residue modified to a cysteine or lysine) or where a particular solvent accessible residue is selected from among several such residues for PEGylation.
Primary amino acid sequence of HEL4 (SEQ ID NO: 5).

PGKGLEWVSS
51 IYGP S GS T).7Y AD SVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCASAL

Primary amino acid sequence of Vk dummy (SEQ ID NO: 6).

GKAPKLLIYA

SYSTPNTFGQ
101 GTKVEIKR_ Several PEG attachment schemes which are useful in the invention are TM
provided by the company Nelctar (SanCarlos, CA). For example, where attachment of PEG or other polymer to a lysine residue is desired, active esters of PEG
polymers which have been derivatized with N-hydroxylsuccinimide, such as succinimidyl propionate may be used. Where attachment to a cysteine residue is intended, PEG
polymers which have been derivatized with sulfhydryl-selective reagents such as maleimide, vinyl sulfone, or thiols may be used. Other examples of specific embodiments of PEG derivatives which may be used according to the invention to generate PEGylated antibodies can be found in the Nektar Catalog (available on the world wide web at nektar.com). In addition, several derivitized forms of PEG
may be used according to the invention to facilitate attachment of the PEG polymer to an antibody polypeptide. PEG derivatives useful in the invention include, but are not limited to PEG-succinimidyl succinate, urethane linked PEG, PEG
phenylcarbonate, PEG succinimidyl carbonate, PEG-carboxymethyl azide, dimethylmaleic anhydride PEG, PEG dithiocarbonate derivatives, PEG-tresylates (2,2,2-trifluoroethanesolfonates), mPEG imidoesters, and other as described in Zalipsky and Lee, (1992) ("Use of functionalized poly(ethylene glycol)s for modification of peptides" in Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications, J. Milton Harris, Ed., Plenum Press, NY).
In one embodiment, the invention provides an anti-CD4OL antibody single variable domain composition comprising an antibody single variable domain and PEG
polymer wherein the ratio of PEG polymer to antibody single variable domain is a molar ratio of at least 0.25:1. In a further embodiment, the molar ratio of PEG
polymer to antibody single variable domain is 0.33:1 or greater. In a still further embodiment the molar ratio of PEG polymer to antibody single variable domain is 0.5:1 or greater.
Dual-specific Ligands The invention also provides dual-specific ligands comprising immunoglobulin single variable domains which each have different specificities; that is, the first and the second epitopes bound by the dual-specific ligand are preferably different. As used herein a "dual-specific ligand" refers to a ligand comprising a first immunoglobulin single variable domain and a second immunoglobulin single variable domain as herein defined, wherein the variable regions are capable of binding to two different antigens or two epitopes on the same antigen which are not normally bound by a monospecific immunoglobulin. For example, the two epitopes may be on the same hapten, but are not the same epitope or sufficiently adjacent to be bound by a monospecific ligand. The dual specific ligands according to the invention are =

composed of variable domains which have different specificities, and do not contain mutually complementary variable domain pairs which have the same specificity.
Dual-specific ligands may be, or be part of, polypeptides, proteins or nucleic acids, which may be naturally occurring or synthetic. In this respect, the ligand of the invention may bind an epiotpe or antigen and act as an antagonist or agonist (eg, EPO
receptor agonist). The epitope binding domains of the ligand in one embodiment have the same epitope specificity, and may for example simultaneously bind their epitope when multiple copies of the epitope are present on the same antigen. In another embodiment, these epitopes are provided on different antigens such that the ligand can bind the epitopes and bridge the antigens. One skilled in the art will appreciate that the choice of epitopes and antigens is large and varied. They may be for instance human or animal proteins, cytokines, cytokine receptors, enzymes co-factors for enzymes or DNA binding proteins. Suitable cytokines and growth factors include but are not limited to: ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF
receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic, fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-i31, insulin, IFN-y, IGF-I, IGF-II, IL-la, IL-1P, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin a, Inhibin p, IP-10, keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian inhibitory substance, monocyte colony inhibitory factor, monocyte attractant protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.), MIG, MIP-la, MIP-1p, MIP-3a, MIP-3p, MdP-4, myeloid progenitor inhibitor factor-1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor, P-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDFla, SDF1P, SCF, SCGF, stem cell factor (SCF), TARC, TGF-a, TGF-P, TGF-P2, TGF-P3, tumour necrosis factor (TNF), TNF-a, TNF-P, TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-P, GRO-y, HCC1, 1-309, HER 1, HER 2, HER 3, HER 4, TACE recognition site, TNF
BP-I and TNF BP-II, as well as any target disclosed in Annex 2 or Annex 3 hereto, whether in combination as set forth in the Annexes, in a different combination or individually. Cytokine receptors include receptors for the foregoing cytokines, e.g.
IL-1 R1; IL-6R; IL-10R; IL-18R, as well as receptors for cytokines set forth in Annex 2 or Annex 3 and also receptors disclosed in Annex 2 and 3. It will be appreciated that this list is by no means exhaustive. Where the multispecific ligand binds to two epitopes (on the same or different antigens), the antigen(s) may be selected from this list.
In one embodiment of the second configuration of the invention, the variable domains are derived from an antibody directed against the first and/or second antigen or epitope. In a preferred embodiment the variable domains are derived from a repertoire of single variable antibody domains. In one example, the repertoire is a repertoire that is not created in an animal or a synthetic repertoire. In another example, the single variable domains are not isolated (at least in part) by animal immunisation. Thus, the single domains can be isolated from a neve library.
In another aspect, the invention provides a multi-specific ligand comprising a first epitope binding domain having a first epitope binding specificity and a non-complementary second epitope binding domain having a second epitope binding specificity. The first and second binding specificities may be the same or different.
In a further aspect, the present invention provides a closed conformation multi-specific ligand comprising a first epitope binding domain having a first epitope binding specificity and a non-complementary second epitope binding domain having a second epitope binding specificity wherein the first and second binding specificities are capable of competing for epitope binding such that the closed conformation multi-specific ligand cannot bind both epitopes simultaneously.
In a still further aspect, the invention provides open conformation ligands comprising non-complementary binding domains, wherein the domains are specific for a different epitope on the same target. Such ligands bind to targets with increased avidity. Similarly, the invention provides multivalent ligands comprising non-complementary binding domains specific for the same epitope and directed to targets which comprise multiple copies of said epitope.

In a similar aspect, ligands according to the invention can be configured to bind individual epitopes with low affinity, such that binding to individual epitopes is not therapeutically significant; but the increased avidity resulting from binding to two epitopes provides a therapeutic benefit. In a particular example, epitopes may be targeted which are present individually on normal cell types, but present together only on abnormal or diseased cells, such as tumour cells. In such a situation, only the abnormal or diseased cells are effectively targeted by the bispecific ligands according to the invention.
Ligand specific for multiple copies of the same epitope, or adjacent epitopes, on the same target (known as chelating dAbs) may also be trimeric or polymeric (tertrameric or more) ligands comprising three, four or more non-complementary binding domains. For example, ligands may be constructed comprising three or four VH domains or VI, domains.
Moreover, ligands are provided which bind to multisubunit targets, wherein each binding domain is specific for a subunit of said target. The ligand may be dimeric, trimeric or polymeric.
The invention also includes a dual specific ligand comprising a first immunoglobulin single variable domain having a binding specificity to a first antigen and a second single variable domain having a binding activity to a second antigen, wherein the first antigen is CD4OL and the second single variable domain is an Antigen Presenting Cell surface antigen or a T cell surface antigen. The Antigen Presenting Cell (APC) surface antigen can be selected from one of the group consisting of dendritic cell surface antigens, activated macrophage surface antigens, activated B cell surface antigens, co-stimulatory signal pathway surface antigens, and MHC, such as .1VEIC II alpha or beta.
The (APC) surface antigen may be selected from the group consisting of CD28, Inducible costimulatory molecule (ICOS), CD27, CD30, 0X40, CD45, CD69, CD3, CD70, Inducible costimulatory molecule ligand (ICOSL), OX4OL, CD80, CD86, HVEM (Herpes Virus Entry Mediator), and LIGHT, but is preferably one of CD28, Inducible costimulatory molecule (ICOS), CD27, CD30, 0X40, CD45, CD69, or CD3.
The surface antigen is preferably a B7 gene surface antigen such as B7-2 or B7-1.
Dendritic cell surface antigens are known in the art and can include but are not limited to ICAM-1, ICAM-2, LFA-1, LFA-3, DEC205, MI-1C class I, 1\11-1C class II, B7-1, and B7-2. Activated macrophage surface antigens include, but are not limited to, TNF receptor, CD40, WEE-{C class I and II, and B7 molecules. Activated B-cell surface antigens are known in the art (e.g., including but not limited to CD20 and CD86)and further described above (see, for example, Janeway et al., 1999, Immunobiolog,v, Garland Publishing NY, NY).
Preferably, the multi-specific ligands according to the above aspects of the invention are obtainable by the method comprising the steps of:
a) selecting a first epitope binding domain by its ability to bind to a first epitope, b) selecting a second epitope binding domain by its ability to bind to a second epitope, c) combining the epitope binding domains; and d) selecting the closed conformation multispecific ligand. by its ability to bind to said first second epitope and said second epitope..
Advantageously the first epitope binding domain and the second epitope binding domains are non-complementary immunoglobulin variable domains, as herein defined. That is either vH-vii or vcvL variable domains.
Chelating dAbs in particular may be prepared according to a preferred aspect of the invention, namely the use of anchor dAbs, in which a library of dimeric, trimeric or multimeric dAbs is constructed using a vector which comprises a constant dAb upstream or downstream of a linker sequence, with a repertoire of second, third and further dAbs being inserted on the other side of the linker. In alternative methodologies, the use of linkers may be avoided, for example by the use of non-covalent bonding or natural affinity between binding domains such as VH and V.

The invention accordingly provides a method for preparing a multimeric ligand comprising the steps of:
(a) providing a vector comprising a nucleic acid sequence encoding a single binding domain specific for a first epitope on a target;
(b) providing a vector encoding a repertoire comprising second binding domains specific for a second epitope on said target, which epitope can be the same or different to the first epitope, said second epitope being adjacent to said first epitope;
and (c) expressing said first and second binding domains; and (d) isolating those combinations of first and second binding domains which combine together to produce a target-binding dimer.
The first and second epitopes are adjacent such that a multimeric ligand is capable of binding to both epitopes simultaneously. This provides the ligand with the advantages of increased avidity if binding. Where the epitopes are the same, the increased avidity is obtained by the presence of multiple copies of the epitope on the target, allowing at least two copies to be simultaneously bound in order to obtain the increased avidity effect.
In an alternative embodiment of the above aspect of the second configuration of the invention, at least one epitope binding domain comprises a non-immunoglobulin 'protein scaffold' or 'protein skeleton' as herein defined.
Suitable non-immunoglobulin protein scaffolds include but are not limited to any of those selected from the group consisting of: SpA, fibronectin, GroEL and other chaperones, lipocallin, CCTLA4 and affibodies, as set forth above.

According to the above aspect of the second configuration of the invention, advantageously, the epitope binding domains are attached to a 'protein skeleton'.
Advantageously, a protein skeleton according to the invention is an immunoglobulin skeleton.
According to the present invention, the teriii 'immunoglobulin skeleton' refers to a protein which comprises at least one immunoglobulin fold and which acts as a nucleus for one or more epitope binding domains, as defined herein.
Preferred immunoglobulin skeletons as herein defined includes any one or more of those selected from the following: an immunoglobulin molecule comprising at least (i) the CL (kappa or lambda subclass) domain of an antibody; or (ii) the CH1 domain of an antibody heavy chain; an immunoglobulin molecule comprising the CHI and CH2 domains of an antibody heavy chain; an immunoglobulin molecule comprising the CH1, CH2 and CH3 domains of an antibody heavy chain; or any of the subset (ii) in conjunction with the CL (kappa or lambda subclass) domain of an antibody. A hinge region domain may also be included. Such combinations of domains may, for example, mimic natural antibodies, such as IgG or IgM, or fragments thereof, such as Fv, scFv, Fab or F(ab'), molecules. Those skilled in the art will be aware that this list is not intended to be exhaustive.
Linking of the skeleton to the epitope binding domains, as herein defined may be achieved at the polypeptide level, that is after expression of the nucleic acid encoding the skeleton and/or the epitope binding domains. Altematively, the linking step may be performed at the nucleic acid level. Methods of linking a protein skeleton according to the present invention, to the one or more epitope binding domains include the use of protein chemistry and/or molecular biology techniques which will be familiar to those skilled in the art and are described herein.
Advantageously, the dual- or multispecific ligand may comprise a first domain capable of binding a target molecule, and a second domain capable of binding a molecule or group which extends the half-life of the ligand. For example, the molecule or group may be a bulky agent, such as HSA or a cell matrix protein.
As used herein, the phrase "molecule or group which extends the half-life of a ligand"
refers to a molecule or chemical group which, when bound by a dual-specific ligand as described herein increases the in vivo half-life of such dual specific ligand when administered to an animal, relative to a ligand that does not bind that molecule or -- group. Examples of molecules or groups that extend the half-life of a ligand are described hereinbelow. In a preferred embodiment, the closed conformation multispecific ligand may be capable of binding the target molecule only on displacement of the half-life enhancing molecule or group. Thus, for example, a closed conformation multispecific ligand is maintained in circulation in the -- bloodstream of a subject by a bulky molecule such as HSA. When a target molecule is encountered, competition between the binding domains of the closed conformation multispecific ligand results in displacement of the HSA and binding of the target.
Molecules which increase half-life are discussed in further detail above.
Ligands according to any aspect of the present invention, as well as dAb -- monomers useful in constructing such ligands, may advantageously dissociate from their cognate target(s) with a Kd of 300nM to 5pM (ie, 3 x 10-7 to 5 x 10-12M), preferably 50nM to2OpM, or 5nM to 200pM or 1nM to 100pM, 1 x 10-7 M or less, 1 x 10-8 M or less, 1 x 10-9 M or less, 1 x 10-10 M or less, 1 x 10-11 M or less;
and/or a Koff rate constant of 5 x 10-1 to 1 x 10 S-1, preferably 1 x 10-2 to 1 x 10-6 S-1, or 5 x 10-3 to -- 1 x 10-5 S-1, or 5 x 10-1 S-1 or less, or 1 x 10-2 S-1 or less, or 1 x 10-3 S-1 or less, or 1 x 10-4 S-1 or less, or 1 x 10-5 S-1 or less, or 1 x 10-6 S-1 or less as determined by surface plasmon resonance. The Kd rate constand is defined as Koff/K0.
Furthermore, the invention provides a dAb monomer(or dual specific ligand comprising such a dAb) that binds to serum albumin (SA) with a Kd of 1nM to -- 500g114 (ie, x 10-9 to 5 x 10-4), preferably 100nM to 10 M. Preferably, for a dual specific ligand comprising a first anti-SA dAb and a second dAb to another target, the affinity (eg Kd and/or Koff as measured by surface plasmon resonance, eg using BiaCore) of the second dAb for its target is from 1 to 100000 times (preferably 100 to 100000, more preferably 1000 to 100000, or 10000 to 100000 times) the affinity of -- the first dAb for SA. For example, the first dAb binds SA with an affinity of approximately 1 OpM, while the second dAb binds its target with an affinity of 100pM. Preferably, the serum albumin is human serum albumin (HSA).
In one embodiment, the first dAb (or a dAb monomer) binds SA (eg, HSA) with a Kd of approximately 50, preferably 70, and more preferably 100, 150 or nM.
The invention moreover provides dimers, trimers and polymers of the aforementioned dAb monomers, in accordance with the foregoing aspect of the present invention.
Ligands according to the invention, including dAb monomers, dimers and trimers, can be linked to an antibody Fc region, comprising one or both of CH2 and CH3 domains, and optionally a hinge region. For example, vectors encoding ligands linked as a single nucleotide sequence to an Fc region may be used to prepare such polypeptides. Alternatively, ligands according to the invention may be free of an Fc domain.
In a further aspect, the present invention provides one or more nucleic acid molecules encoding at least a dual- or multispecific ligand as herein defined.
In one embodiment, the ligand is a closed conformation ligand. In another embodiment, it is an open conformation ligand. The multispecific ligand may be encoded on a single nucleic acid molecule; alternatively, each epitope binding domain may be encoded by a separate nucleic acid molecule. Where the ligand is encoded by a single nucleic acid molecule, the domains may be expressed as a fusion polypeptide, or may be separately expressed and subsequently linked together, for example using chemical linking agents. Ligands expressed from separate nucleic acids will be linked together by appropriate means.
The nucleic acid may further encode a signal sequence for export of the polypeptides from a host cell upon expression and may be fused with a surface component of a filamentous bacteriophage particle (or other component of a selection display system) upon expression. Leader sequences, which may be used in bacterial expression and/or phage or phagemid display, include pelB, stII, ompA, phoA, bla and pelA.
In a further aspect of the second configuration of the invention the present invention provides a vector comprising nucleic acid according to the present invention.
In a yet further aspect, the present invention provides a host cell transfected with a vector according to the present invention.
Expression from such a vector may be configured to produce, for example on the surface of a bacteriophage particle, epitope binding domains for selection. This allows selection of displayed domains and thus selection of `multispecific ligands' using the method of the present invention.
Combining single variable domains Domains useful in the invention, once selected using methods exemplified above, may be combined by a variety of methods known in the art, including covalent and non-covalent methods.
Preferred methods include the use of polypeptide linkers, as described, for example, in connection with scPv molecules (Bird et al., (1988) Science 242:423-426). Discussion of suitable linkers is provided in Bird et al. Science 242, 423-426;
Hudson et al, Journal Immunol Methods 231 (1999) 177-189; Hudson et al, Proc Nat Acad Sci USA 85, 5879-5883. Linkers are preferably flexible, allowing the two single domains to interact. One linker example is a (G1y4 Ser)n linker, where n=1 to 8, eg, 2, 3, 4, 5 or 7. The linkers used in diabodies, which are less flexible, may also be employed (Holliger et al., (1993) PNAS (USA) 90:6444-6448).
In one embodiment, the linker employed is not an immunoglobulin hinge region.
Variable domains may be combined using methods other than linkers. For example, the use of disulphide bridges, provided through naturally-occurring or engineered cysteine residues, may be exploited to stabilise H- Ar Ar Ar L-L or VH-VL
dimers (Reiter et al., (1994) Protein Eng. 7:697-704) or by remodelling the interface between the variable domains to improve the "fit" and thus the stability of interaction (Ridgeway et al., (1996) Protein Eng. 7:617-621; Zhu et al., (1997) Protein Science 6:781-788).
Other techniques for joining or stabilizing variable domains of immunoglobulins, and in particular antibody VH domains, may be employed as appropriate.
In accordance with the present invention, dual specific ligands can be in "closed" conformations in solution. A "closed" configuration is that in which the two domains (for example VH and VL) are present in associated form, such as that of an associated VH-VL pair which forms an antibody binding site. For example, scFv may be in a closed conformation, depending on the arrangement of the linker used to link the VH and VL domains. If this is sufficiently flexible to allow the domains to associate, or rigidly holds them in the associated position, it is likely that the domains will adopt a closed conformation.
Similarly, VH domain pairs and VL domain pairs may exist in a closed conformation. Generally, this will be a function of close association of the domains, such as by a rigid linker, in the ligand molecule. Ligands in a closed conformation will be unable to bind both the molecule which increases the half-life of the ligand and a second target molecule. Thus, the ligand will typically only bind the second target molecule on dissociation from the molecule which increases the half-life of the ligand.
Moreover, the construction of VH/VH, VLATL, or VH/VL dimers without linkers provides for competition between the domains.
Ligands according to the invention may moreover be in an open conformation.
In such a conformation, the ligands will be able to simultaneously bind both the molecule which increases the half-life of the ligand and the second target molecule.

Typically, variable domains in an open configuration are (in the case of VH-VL
pairs) held far enough apart for the domains not to interact and form an antibody binding site and not to compete for binding to their respective epitopes. In the case of VH/VH
or VL/VL dimers, the domains are not forced together by rigid linkers.
Naturally, such domain pairings will not compete for antigen binding or form an antibody binding site.
Fab fragments and whole antibodies will exist primarily in the closed confoimation, although it will be appreciated that open and closed dual specific ligands are likely to exist in a variety of equilibria under different circumstances.
Binding of the ligand to a target is likely to shift the balance of the equilibrium towards the open configuration. Thus, certain ligands according to the invention can exist in two confoimations in solution, one of which (the open form) can bind two antigens or epitopes independently, whilst the alternative conformation (the closed form) can only bind one antigen or epitope; antigens or epitopes thus compete for binding to the ligand in this conformation.
Although the open form of the dual specific ligand may thus exist in equilibrium with the closed foini in solution, it is envisaged that the equilibrium will favor the closed form; moreover, the open form can be sequestered by target binding into a closed conformation. Preferably, therefore, certain dual specific ligands of the invention are present in an equilibrium between two (open and closed) conformations.
Dual specific ligands according to the invention may be modified in order to favor an open or closed conformation. For example, stabilisation of VH-Vi, interactions with disulphide bonds stabilises =the closed conformation.
Moreover, linkers used to join the domains, including VH domain and VL domain pairs, may be constructed such that the open from is favoured; for example, the linkers may sterically hinder the association of the domains, such as by incorporation of large amino acid residues in opportune locations, or the designing of a suitable rigid structure which will keep the domains physically spaced apart.

Characterisation of the dual-specific lizand.
The binding of the dual-specific ligand to its specific antigens or epitopes (e.g., CD4OL and/or an epitope bound by DOM8-24) can be tested by methods which will be familiar to those skilled in the art and include ELISA. In a preferred embodiment of the invention binding is tested using monoclonal phage ELISA.
Phage ELISA may be performed according to any suitable procedure: an exemplary protocol is set forth below.
Populations of phage produced at each round of selection can be screened for binding by ELISA to the selected antigen or epitope, to identify "polyclonal"
phage antibodies. Phage from single infected bacterial colonies from these populations can then be screened by ELISA to identify "monoclonal" phage antibodies. It is also desirable to screen soluble antibody fragments for binding to antigen or epitope, and this can also be undertaken by ELISA using reagents, for example, against a C-or N-terminal tag (see for example Winter et al. (1994) Ann. Rev. Immunology 12, and references cited therein.
The diversity of the selected phage monoclonal antibodies may also be assessed by gel electrophoresis of PCR products (Marks et al. 1991, supra;
Nissim et al. 1994 supra), probing (Tomlinson et al., 1992) J. Mol. Biol. 227, 776) or by sequencing of the vector DNA.
Structure of 'Dual-specific ligands'.
As described above, an antibody is herein defined as an antibody (for example IgG, IgM, IgA, IgA, IgE) or fragment (Fab, Fv, disulphide linked Fv, scFv, diabody) which comprises at least one heavy and a light chain variable domain, at least two heavy chain variable domains or at least two light chain variable domains. It may be at least partly derived from any species naturally producing an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria).

In a preferred embodiment of the invention the dual-specific ligand comprises at least one single heavy chain variable domain of an antibody and one single light chain variable domain of an antibody, or two single heavy or light chain variable domains. For example, the ligand may comprise a VH/VL pair, a pair of VH
domains or a pair of VL domains.
The first and the second variable domains of such a ligand may be on the same polypeptide chain. Alternatively they may be on separate polypeptide chains.
In the case that they are on the same polypeptide chain they may be linked by a linker, which is preferentially a peptide sequence, as described above.
The first and second variable domains may be covalently or non-covalently associated. In the case that they are covalently associated, the covalent bonds may be disulphide bonds.
In the case that the variable domains are selected from V-gene repertoires selected for instance using phage display technology as herein described, then these variable domains comprise a universal framework region, such that is they may be recognised by a specific generic ligand as herein defined. The use of universal frameworks, generic ligands and the like is described in W099/20749.
Where V-gene repertoires are used variation in polypeptide sequence is preferably located within the structural loops of the variable domains. The polypeptide sequences of either variable domain may be altered by DNA
shuffling or by mutation in order to enhance the interaction of each variable domain with its complementary pair. DNA shuffling is known in the art and taught, for example, by Stemmer, 1994, Nature 370: 389-391 and U.S. Patent No. 6,297,053.
Other methods of mutagenesis are well known to those of skill in the P rt.
In one embodiment of the invention the 'dual-specific ligand' is a single chain Fv fragment. In an alternative embodiment of the invention, the 'dual-specific ligand' consists of a Fab format.

In a further aspect, the present invention provides nucleic acid encoding at least a 'dual-specific ligand' as herein defined.
One skilled in the art will appreciate that, depending on the aspect of the invention, both antigens or epitopes may bind simultaneously to the same antibody molecule. Alternatively, they may compete for binding to the same antibody molecule. For example, where both epitopes are bound simultaneously, both variable domains of a dual specific ligand are able to independently bind their target epitopes.
Where the domains compete, the one variable domain is capable of binding its target, but not at the same time as the other variable domain binds its cognate target; or the first variable domain is capable of binding its target, but not at the same time as the second variable domain binds its cognate target.
The variable regions may be derived from antibodies directed against target antigens or epitopes. Alternatively they may be derived from a repertoire of single antibody domains such as those expressed on the surface of filamentous bacteriophage. Selection may be performed as described below.
In general, the nucleic acid molecules and vector constructs required for the performance of the present invention may be constructed and manipulated as set forth in standard laboratory manuals, such as Sambrook et al. (1989) Molecular Cloning: A
Laboratoiy Manual, Cold Spring Harbor, USA.
The manipulation of nucleic acids useful in the present invention is typically carried out in recombinant vectors.
Thus in a further aspect, the present invention provides a vector comprising nucleic acid encoding at least a 'dual-specific ligand' as herein defined.
As used herein, vector refers to a discrete element that is used to introduce heterologous DNA into cells for the expression and/or replication. thereof.
Methods by which to select or construct and, subsequently, use such vectors are well known to one of ordinary skill in the art. Numerous vectors are publicly available, including bacterial plasmids, bacteriophage, artificial chromosomes and episomal vectors. Such vectors may be used for simple cloning and mutagenesis; alternatively gene expression vector is employed. A vector of use according to the invention may be selected to accommodate a polypeptide coding sequence of a desired size, typically from 0.25 kilobase (kb) to 40 kb or more in length A suitable host cell is transformed with the vector after in vitro cloning manipulations. Each vector contains various functional components, which generally include a cloning (or "polylinker") site, an origin of replication and at least one selectable marker gene. If given vector is an expression vector, it additionally possesses one or more of the following:
enhancer element, promoter, transcription termination and signal sequences, each positioned in the vicinity of the cloning site, such that they are operatively linked to the gene encoding a ligand according to the invention.
Both cloning and expression vectors generally contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells.
Typically in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 micron plasmid origin is suitable for yeast, and various viral origins (e.g. SV 40, adenovirus) are useful for cloning vectors in mammalian cells. Generally, the origin of replication is not needed for mammalian expression vectors unless these are used in mammalian cells able to replicate high levels of DNA, such as cos cells.
Advantageously, a cloning or expression vector may contain a selection gene also referred to as selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium.
Host cells not transformed with the vector containing the selection gene will therefore not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available in the growth media.

Since the replication of vectors encoding a ligand according to the present invention is most conveniently performed in E. coli, an E. coli-selectable marker, for example, the f3-lactamase gene that confers resistance to the antibiotic ampicillin, is of use. These can be obtained from E. coif plasmids, such as pBR322 or a p-UC
plasmid such as p-UC18 or pUC19.
Expression vectors usually contain a promoter that is recognised by the host organism and is operably linked to the coding sequence of interest. Such a promoter may be inducible or constitutive. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
Promoters suitable for use with prokaryotic hosts include, for example, the [3-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Promoters for use in bacterial systems will also generally contain a Shine-Delgamo sequence operably linked to the coding sequence.
The preferred vectors are expression vectors that enables the expression of a nucleotide sequence corresponding to a polypeptide library member. Thus, selection with the first and/or second antigen or epitope can be performed by separate propagation and expression of a single clone expressing the polypeptide library member or by use of any selection display system. As described above, the preferred = selection display system is bacteriophage display. Thus, phage or phagemid vectors may be used, eg pIT1 or pIT2. Leader sequences useful in the inventiOn include pelB, stII, ompA, phoA, bla and pelA. One example are phagemid vectors which have an E. coli. origin of replication (for double stranded replication) and also a phage origin of replication (for production of single-stranded DNA). The manipulation and expression of such vectors is well known in the art (Hoogenboom and Winter (1992) supra; Nissim et al. (1994) supra). Briefly, the vector contains a P-lactamase gene to confer selectivity on the phagemid and a lac promoter upstream of a expression cassette that consists (N to C terminal) of a pelB leader sequence (which directs the expressed polypeptide to the periplasmic space), a multiple cloning site (for cloning the nucleotide version of the library member), optionally, one or more peptide tag (for detection), optionally, one or more TAG stop codon and the phage protein pIII.
Thus, using various suppressor and non-suppressor strains of E. coli and with the addition of glucose, iso-propyl thio-P-D-galactoside (IPTG) or a helper phage, such as VCS
M13, the vector is able to replicate as a plasmid with no expression, produce large quantities of the polypeptide library member only or produce phage, some of which contain at least one copy of the polypeptide-pIII fusion on their surface.
Construction of vectors encoding ligands according to the invention employs conventional ligation techniques. Isolated vectors or DNA fragments are cleaved, tailored, and religated in the form desired to generate the required vector.
If desired, analysis to confirm that the correct sequences are present in the constructed vector can be performed in a known fashion. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing expression and function are known to those skilled in the art. The presence of a gene sequence in a sample is detected, or its amplification and/or expression quantified by conventional methods, such as Southern or Northern analysis, Western blotting, dot blotting of DNA, RNA or protein, in situ hybridisation, immunocytochemistry or sequence analysis of nucleic acid or protein molecules. Those skilled in the art will readily envisage how these methods may be modified, if desired.
Structure ofli ands According to one aspect of the invention, two or more non-complementary epitope binding domains are linked so that they are in a closed conformation as herein defined. Advantageously, they may be further attached to a skeleton which may, as a alternative, or on addition to a linker described herein, facilitate the formation and/or maintenance of the closed conformation of the epitope binding sites with respect to one another. Alternatively, the monomeric anti-CD4OL antibody single variable domain polypeptides of the invention may be constructed using scaffold or skeleton frameworks as discussed herein.
(I) Skeletons Skeletons may be based on immunoglobulin molecules or may be non-immunoglobulin in origin as set forth above. Preferred immunoglobulin skeletons as herein defined includes any one or more of those selected from the following:
an immunoglobulin molecule comprising at least (i) the CL (kappa or lambda subclass) domain of an antibody; or (ii) the CH1 domain of an antibody heavy chain; an immunoglobulin molecule comprising the CH1 and CH2 domains of an antibody heavy chain; an immunoglobulin molecule comprising the CHI, CH2 and CH3 domains of an antibody heavy chain; or any of the subset (ii) in conjunction with the CL (kappa or lambda subclass) domain of an antibody. A hinge region domain may also be included.. Such combinations of domains may, for example, mimic natural antibodies, such as IgG or IgM, or fragments thereof, such as Fv, scFv, Fab or F(ab')2 molecules. Those skilled in the art will be aware that this list is not intended to be exhaustive.
(II) Protein scaffolds Each epitope binding domain comprises a protein scaffold and one or more CDRs which are involved in the specific interaction of the domain with one or more epitopes. Advantageously, an epitope binding domain according to the present invention comprises three CDRs. Suitable protein scaffolds, in addition to those based on immunoglobulin domains, may also be based on protein scaffolds or skeletons other than immunoglobulin domains. For example natural bacterial receptors such as SpA have been used as scaffolds for the grafting of CDRs to generate ligands which bind specifically to one or more epitopes. Details of this procedure are described in US 5,831,012. Other suitable scaffolds include those based on fibronectin and affibodies (Affibody, Bromma, Sweeden). Details of suitable procedures are described in WO 98/58965. Other suitable= scaffolds include lipocallin and CTLA4, as described in van den Beuken et al., J. Mol. Biol. (2001) 310, 591-601, and scaffolds such as those described in W00069907 (Medical Research Council), which are based for example on the ring structure of bacterial GroEL or other chaperone polypeptides. Other non-immunoglobulin based scaffolds which may be used according to the invention include those based on the LDL receptor class A, EGF domain monomers and mutimers, and scaffolds available from Biorexis (King of Prussia, PA) or Avidia (Mountain View, CA). Other non-immunoglobulin scaffolds which may be used are described, for example, in W005/040229, W004/044011, and Scaffolds for use in Constructing Ligands i. Selection of the main-chain conformation The members of the immunoglobulin superfamily all share a similar fold for their polypeptide chain. For example, although antibodies are highly diverse in terms of their primary sequence, comparison of sequences and crystallographic structures has revealed that, contrary to expectation, five of the six antigen binding loops of antibodies (H1, H2, L1, L2, L3) adopt a limited number of main-chain conformations, or canonical structures (Chothia and Lesk (1987) J. Mol. Biol., 196: 901;
Chothia et al. (1989) Nature, 342: 877). Analysis of loop lengths and key residues has therefore enabled prediction of the main-chain confoiniations of H1, H2, L1, L2 and L3 found in the majority of human antibodies (Chothia et al. (1992) J. Mol. Biol., 227:
799;
Tomlinson et al. (1995) EMBO j., 14: 4628; Williams et al. (1996) J. Mol.
Biol., 264:
220). Although the H3 region is much more diverse in terms of sequence, length and structure (due to the use of D segments), it also forms a limited number of main-chain conformations for short loop lengths which depend on the length and the presence of particular residues, or types of residue, at key positions in the loop and the antibody framework (Martin et al. (1996) J. Mol. Biol., 263: 800; Shirai et al. (1996) FEBS
Letters, 399: 1).
The ligands of the present invention are advantageously selected and/or assembled from libraries of domains, such as libraries of VH domains and/or libraries of VL domains. Moreover, the ligands of the invention may themselves be provided in the form of libraries. In one aspect of the present invention, libraries of ligands and/or domains are designed in which certain loop lengths and key residues have been chosen to ensure that the main-chain conformation of the members is known.
Advantageously, these are real conformations of immunoglobulin superfamily molecules found in nature, to minimise the chances that they are non-functional, as discussed above. Germline V gene segments serve as one suitable basic framework for constructing antibody or T-cell receptor libraries; other sequences are also of use.
Variations may occur at a low frequency, such that a small number of functional members may possess an altered main-chain conformation, which does not affect its function.
Canonical structure theory is also of use to assess the number of different main-chain conformations encoded by ligands, to predict the main-chain conformation based on ligand sequences and to chose residues for diversification which do not affect the canonical structure. It is known that, in the human Vx domain, the L1 loop can adopt one of four canonical structures, the L2 loop has a single canonical structure and that 90% of human Vx domains adopt one of four or five canonical structures for the L3 loop (Tomlinson et al. (1995) supra); thus, in the Vi;
domain alone, different canonical structures can combine to create a range of different main-chain conformations. Given that the V), domain encodes a different range of canonical structures for the L1, L2 and L3 loops and that Vic and \72, domains can pair with any VH domain which can encode several canonical structures for the H1 and H2 loops, the number of canonical structure combinations observed for these five loops is very large. This implies that the generation of diversity in the main-chain conformation may be essential for the production of a wide range of binding specificities. However, by constructing an antibody library based on a single known main-chain conformation it has been found, contrary to expectation, that diversity in the main-chain conformation is not required to generate sufficient diversity to target substantially all antigens. Even more surprisingly, the single main-chain confounation need not be a consensus structure - a single naturally occurring conformation can be used as the basis for an entire library. Thus, in a preferred aspect, the ligands of the invention possess a single known main-chain conformation.
The single main-chain conformation that is chosen is preferably commonplace among molecules of the immunoglobulin superfarnily type in = question. A
conformation is commonplace when a significant number of naturally occurring molecules are observed to adopt it. Accordingly, in a preferred aspect of the invention, the natural occurrence of the different main-chain conformations for each binding loop of an immunoglobulin domain are considered separately and then a naturally occurring variable domain is chosen which possesses the desired combination of main-chain conformations for the different loops. If none is available, the nearest equivalent may be chosen. It is preferable that the desired combination of main-chain conformations for the different loops is created by selecting gennline gene segments which encode the desired main-chain conformations. It is more preferable, that the selected germline gene segments are frequently expressed in nature, and most preferable that they are the most frequently expressed of all natural gatmline gene segments.
In designing ligands or libraries thereof the incidence of the different main-chain conformations for each of the antigen binding loops may be considered separately. For H1, H2, L1, L2 and L3, a given conformation that is adopted by between 20% and 100% of the antigen binding loops of naturally occurring molecules is chosen. Typically, its observed incidence is above 35% (i.e. between 35%
and 100%) and, ideally, above 50% or even above 65%. Since the vast majority of H3 loops do not have canonical structures, it is preferable to select a main-chain conformation which is commonplace among those loops which do display canonical = structures. For each of the loops, the conformation which is observed most often in the natural repertoire is therefore selected. In human antibodies, the most popular canonical structures (CS) for each loop are as follows: H1 - CS 1 (79% of the expressed repertoire), H2 - CS 3 (46%), L1 - CS 2 of V.K. (39%), L2 - CS 1 (100%), L3 - CS 1 of VK (36%) (calculation assumes a ic:2L, ratio of 70:30, Hood et al. (1967) Cold Spring Harbor Symp. Ouant. Biol., 48: 133). For H3 loops that have canonical structures, a CDR3 length (Kabat et al. (1991) Sequences of proteins of immunological interest, U.S. Department of Health and Human Services) of seven residues with a salt-bridge from residue 94 to residue 101 appears to be the most common. There are at least 16 human antibody sequences in the EMBL data library with the required H3 length and key residues to foim this conformation and at least two crystallographic structures in the protein data bank which can be used as a basis for antibody modelling (2cgr and ltet). The most frequently expressed germline gene segments that this combination of canonical structures are the VH segment 3-23 (DP-47), the JH segment TH4b, the V, segment 02/012 (DPK9) and the Jõ segment Jõ1.
VH segments DP45 and DP38 are also suitable. These segments can therefore be used in combination as a basis to construct a library with the desired single main-chain conformation.
Alternatively, instead of choosing the single main-chain conformation based on the natural occurrence of the different main-chain conformations for each of the binding loops in isolation, the natural occurrence of combinations of main-chain conformations is used as the basis for choosing the single main-chain confoimation.
In the case of antibodies, for example, the natural occurrence of canonical structure combinations for any two, three, four, five or for all six of the antigen binding loops can be determined. Here, it is preferable that the chosen conformation is commonplace in naturally occurring antibodies and most preferable that it observed most frequently in the natural repertoire. Thus, in human antibodies, for example, when natural combinations of the five antigen binding loops, H1, H2, Ll, L2 and L3, are considered, the most frequent combination of canonical structures is determined and then combined with the most popular conformation for the H3 loop, as a basis for choosing the single main-chain conformation.
ii. Diversification of the canonical sequence Having selected several known main-chain conformations or, preferably a single known main-chain conformation, ligands according to the invention or libraries for use in the invention can be constructed by varying the binding site of the molecule in order to generate a repertoire with structural and/or functional diversity.
This means that variants are generated such that they possess sufficient diversity in their structure and/or in their function so that they are capable of providing a range of activities.
The desired diversity is typically generated by varying the selected molecule at one or more positions. The positions to be changed can be chosen at random or are preferably selected. The variation can then be achieved either by randomisation, during which the resident amino acid is replaced by any amino acid or analogue thereof, natural or synthetic, producing a very large number of variants or by replacing the resident amino acid with one or more of a defined subset of amino acids, producing a more limited number of variants.
Various methods have been reported for introducing such diversity. Error-prone PCR (Hawkins et al. (1992) 1 Mol. Biol., 226: 889), chemical mutagenesis (Deng et al. (1994) J. Biol. Chem., 269: 9533) or bacterial mutator strains (Low et al.
(1996) J Mol. Biol., 260: 359) can be used to introduce random mutations into the genes that encode the molecule. Methods for mutating selected positions are also well known in the art and include the use of mismatched oligonucleotides or degenerate oligonucleotides, with or without the use of PCR. For example, several synthetic antibody libraries have been created by targeting mutations to the antigen binding loops. The H3 region of a human tetanus toxoid-binding Fab has been randomised to create a range of new binding specificities (Barbas et al. (1992) Proc. Natl.
Acad. Sci.
USA, 89: 4457). Random or semi-random H3 and L3 regions have been appended to germline V gene segments to produce large libraries with unmutated framework regions (Hoogenboom & Winter (1992)1 Mol. Biol., 227: 381; Barbas et al.
(1992) PTOC. Natl. Acad. Sci. USA, 89: 4457; Nissim et al. (1994) EMBO J., 13: 692;
Griffiths et al. (1994) EMBO 1, 13: 3245; De Kruif et al. (1995) J. Mol.
Biol., 248:
97). Such diversification has been extended to include some or all of the other antigen binding loops (Crameri et al. (1996) Nature Med., 2: 100; Riechmann et al.
(1995) Bio/Technology, 13: 475; Morphosys, W097/08320, supra).

Since loop randomisation has the potential to create approximately more than 1015 structures for H3 alone and a similarly large number of variants for the other five loops, it is not feasible using current transformation technology or even by using cell free systems to produce a library representing all possible combinations. For example, in one of the largest libraries constructed to date, 6 x 1010 different antibodies, which is only a fraction of the potential diversity for a library of this design, were generated (Griffiths et al. (1994) supra).
In a preferred embodiment, only those residues which are directly involved in creating or modifying the desired function of the molecule are diversified.
For many molecules, the fill-lotion will be to bind a target and therefore diversity should be concentrated in the target binding site, while avoiding changing residues which are crucial to the overall packing of the molecule or to maintaining the chosen main-chain conformation.
Diversification of the canonical sequence as it applies to antibody domains In the case of the ligands of the invention, the binding site for the target is most often the antigen binding site. Thus, in a highly preferred aspect, the invention provides libraries of or for the assembly of antibody ligands in which only those residues in the antigen binding site are varied. These residues are extremely diverse in the human antibody repertoire and are known to make contacts in high-resolution antibody/antigen complexes. For example, in L2 it is known that positions 50 and 53 are diverse in naturally occurring antibodies and are observed to make contact with the antigen. In contrast, the conventional approach would have been to diversify all the residues in the corresponding Complementarity Determining Region (CDR1) as defined by Kabat et al. (1991, supra), some seven residues compared to the two = 25 diversified in the libi-ary for use according to the invention.
This represents a significant improvement in terms of the functional diversity required to create a range of antigen binding specificities.
In nature, antibody diversity is the result of two processes: somatic recombination of germline V, D and J gene segments to create a naive primary 104=

repertoire (so called germline and junctional diversity) and somatic hypennutation of the resulting rearranged V genes. Analysis of human antibody sequences has shown that diversity in the primary repertoire is focused at the centre of the antigen binding site whereas somatic hypermutation spreads diversity to regions at the periphery of the antigen binding site that are highly conserved in the primary repertoire (see Tomlinson et al. (1996)1 MoL Biol., 256: 813). This complementarity has probably evolved as an efficient strategy for searching sequence space and, although apparently unique to antibodies, it can easily be applied to other polypeptide repertoires. The residues which are varied are a subset of those that form the binding site for the target. Different (including overlapping) subsets of residues in the target binding site are diversified at different stages during selection, if desired.
In the case of an antibody repertoire, an initial 'naive' repertoire is created where some, but not all, of the residues in the antigen binding site are diversified. As used herein in this context, the term "naive" refers to antibody molecules that have no pre-determined target. These molecules resemble those which are encoded by the immunoglobulin genes of an individual who has not undergone immune diversification, as is the case with fetal and newborn individuals, whose immune systems have not yet been challenged by a wide variety of antigenic stimuli.
This repertoire is then selected against a range of antigens or epitopes. If required, further diversity can then be introduced outside the region diversified in the initial repertoire.
This matured repertoire can be selected for modified function, specificity or affinity.
The invention provides two different naive repertoires of binding domains for the construction of ligands, or a naïve library of ligands, in which some or all of the residues in the antigen binding site are varied. The "primary" library mimics the natural primary repertoire, with diversity restricted to residues at the centre of the antigen binding site that are diverse in the germline V gene segments (germline diversity) or diversified during the recombination process (junctional diversity).
Those residues which are diversified include, but are not limited to, H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97, H98, L50, L53, L91, L92, L93, L94 and L96.
In the "somatic" library, diversity is restricted to residues that are diversified during the recombination process (junctional diversity) or are highly somatically mutated).
Those residues which are diversified include, but are not limited to: H31, H33, H35, H95, H96, H97, H98, L30, L31, L32, L34 and L96. All the residues listed above as suitable for diversification in these libraries are known to make contacts in one or more antibody-antigen complexes. Since in both libraries, not all of the residues in the antigen binding site are varied, additional diversity is incorporated during selection by varying the remaining residues, if it is desired to do so. It shall be apparent to one skilled in the art that any subset of any of these residues (or additional residues which comprise the antigen binding site) can be used for the initial and/or subsequent , diversification of the antigen binding site.
In the construction of libraries for use in the invention, diversification of chosen positions is typically achieved at the nucleic acid level, by altering the coding sequence which specifies the sequence of the polypeptide such that a number of possible amino acids (all 20 or a subset thereof) can be incorporated at that position.
Using the IUPAC nomenclature, the most versatile codon is NNK, which encodes all amino acids as well as the TAG stop codon. The NNK codon is preferably used in order to introduce the required diversity. Other codons which achieve the same ends are also of use, including the NN codon, which leads to the production of the additional stop codons TGA and TAA.
A feature of side-chain diversity in the antigen binding site of human antibodies is a pronounced bias which favours certain amino acid residues. If the amino acid composition of the ten most diverse positions in each of the VII, Vx; and regions are summed, more than 76% of the side-chain diversity comes from only seven different residues, these being, serine (24%), tyrosine (14%), asparagine (11%), glycine (9%), alanine (7%), aspartate (6%) ' and threonine (6%). This bias towards hydrophilic residues and small residues which can provide main-chain flexibility probably reflects the evolution of surfaces which are predisposed to binding a wide range of antigens or epitopes and may help to explain the required promiscuity of antibodies in the primary repertoire.

Since it is preferable to mimic this distribution of amino acids, the distribution of amino acids at the positions to be varied preferably mimics that seen in the antigen binding site of antibodies. Such bias in the substitution of amino acids that permits selection of certain polypeptides (not just, antibody polypeptides) against a range of target antigens is easily applied to any polypeptide repertoire. There are various methods for biasing the amino acid distribution at the position to be varied (including the use of tri-nucleotide mutagenesis, see W097/08320), of which the preferred method, due to ease of synthesis, is the use of conventional degenerate codons. By comparing the amino acid profile encoded by all combinations of degenerate codons (with single, double, triple and quadruple degeneracy in equal ratios at each position) with the natural amino acid use it is possible to calculate the most representative codon. The codons (AGT)(AGC)T, (AGT)(AGC)C and (AGT)(AGC)(CT) - that is, DVT, DVC and DVY, respectively using IUPAC nomenclature - are those closest to the desired amino acid profile: they encode 22% serine and 11% tyrosine, asparagine, glycine, alanine, aspartate, threonine and cysteine. Preferably, therefore, libraries are constructed using either the DVT, DVC or DVY codon at each of the diversified positions.
Increased Half-life In vivo, the PEGylated monovalent anti-CD4OL antibodies as described herein confer a distinct advantage over non-PEGylated antibody polypeptides, in that the PEGylated antibody molecules will have a greatly prolonged in vivo half life.
Without being bound to one particular theory, it is believed that the increased half-life of the molecules described herein is conferred by the increased hydrodynamic size of the antibody polypeptide resulting from the attachment of PEG polymer(s). More specifically, it is believed that two parameters play an important role in determining the serum half-life of PEGylated antibody polypeptides. The first criterion is the nature and size of the PEG attachment, i.e., if the polymer used is simply a linear chain or a branched/forked chain, wherein the branched/forked chain gives rise to a longer half-life. The second is the location of the PEG moiety or moieties on the antibody polypeptide in the final format and how many "free" unmodified PEG
arms the molecule has. The resulting hydrodynamic size of the PEGylated antibody polypeptide, as estimated; for example, by size exclusion chromatography, reflects the serum half-life of the molecule. Accordingly, the larger the hydrodynamic size of the PEGylated molecule, the greater the serum half life.
Increased half-life is useful in vivo applications of immunoglobulins, especially antibodies and most especially antibody fragments of small size.
Such fragments (Fvs, Fabs, sat's, dAbs) suffer from rapid clearance from the body;
thus, while they are able to reach most parts of the body rapidly, and are quick to produce and easier to handle, their in vivo applications have been limited by their only brief persistence in vivo.
In one aspect, a monovalent anti-CD4OL antibody polypeptide as described herein is stabilized in vivo by fusion with a moiety, such as PEG, that increases the hydrodynamic size of the antibody polypeptide. Methods for pharmacolcinetic analysis and determination of half-life will be familiar to those skilled in the art.
Details may be found in Kenneth et al: Chemical Stability of Pharmaceuticals:
A
Handbook for Pharmacists and in Peters et al, Pharmacokinetc analysis: A
Practical Approach (1996). Reference is also made to "Pharmacokinetics", M Gibaldi & D
Perron, published by Marcel Dekker, 211d Rev. ex edition (1982), which describes pharmacokinetic parameters such as t alpha and t beta half lives and area under the curve (AIJC).
Typically, the half life of a PEGylated antibody polypeptide as described herein is increased by 10%, 20%, 30%, 40%, 50% or more relative to a non-PEGylated dAb (wherein the antibody polypeptide of the PEGylated antibody polypeptide and non-PEGylated antibody polypeptide are the same). Increases in the range of 2x, 3x, 4x, 5x, 7x, 10x, 20x, 30x, 40x, and up to 50x or more of the half life are possible. Alternatively, or in addition, increases in the range of up to 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x of the half life are possible.
Half lives (t1/2, alpha and t'/2 beta) and ALJC can be determined from a curve of serum concentration of ligand against time. The WinNonlinrmanalysis package (available from Pharsight Corp., Mountain View, CA 94040, USA) can be used, for example, to model the curve. In a first phase (the alpha phase) the ligand is undergoing mainly distribution in the patient, with some elimination. A second phase (beta phase) is the terminal phase when the ligand has been distributed and the serum concentration is decreasing as the ligand is cleared from the patient. The to half life is the half life of the first phas and the tf3 half life is the half life of the second phase.
"Half-life" as used herein, unless otherwise noted, refers to the overall half-life of an antibody single variable domain of the invention determined by non-compartment modeling (as contrasted with biphasic modeling, for example). Beta half-life is a measurement of the time it takes for the amount of dAb monomer or multimer to be cleared from the mammal to which it is administered. Thus, advantageously, the present invention provides a dAb-containing composition, e.g., a dAb-effector group composition, having a t half-life in the range of 0.25 hours to 6 hours or more. In one embodiment, the lower end of the range is 30 minutes, 45 minutes, 1 hour, 1.3 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours. In addition or alternatively, a dAb containing composition will have a ta half-life in the range of up to and including 12 hours. In one embodiment, the upper end of the range is 11, 10, 9, 8, 7, 6, or 5 hours. An example of a suitable range is 1.3 to 6 hours, 2 to 5 hours or 3 to 4 hours.
Advantageously, the present invention provides a dAb containing composition comprising a ligand according to the invention having a tf3 half-life in the range of 1-170 hours or more. In one embodiment, the lower end of the range is 2.5 hours, hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours. In addition, or alternatively, a dAb containing composition, e.g. a dAb-effector group composition has a t13 half-life in the range of up to and including 21 days. In one embodiment, the upper end of the range is 12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days, or 20 days. Advantageously a dAb containing composition according to the invention will have a ti3 half-life in the range 2-100 hours, hours, and 10-40 hours. In a further embodiment, it will be in the range 12-48 hours.
In a further embodiment still, it will be in the range 12-26 hours. The present invention provides a dAb containing composition comprising a ligand according to the invention having a half-life in the range of 1-170 hours or more. In one embodiment, the lower end of the range is 1.3 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours. In addition, or alternatively, a dAb containing composition, e.g. a dAb-effector group composition has a half-life in the range of up to and including 21 days. In one embodiment, the upper end of the range is 12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days, or 20 days.
In addition, or alternatively to the above criteria, the present invention provides a dAb containing composition comprising a ligand according to the invention having an AUC value (area under the curve) in the range of 1 mg.min/ml or more. In one embodiment, the lower end of the range is 5, 10, 15, 20, 30, 100, 200 or 300 mg.min/ml. In addition, or alternatively, a ligand or composition according to the invention has an AUC in the range of up to 600 mg.min/ml. In one embodiment, the upper end of the range is 500, 400, 300, 200, 150, 100, 75 or 50 mg.min/ml.
Advantageously a ligand according to the invention will have an AUC in the range selected from the group consisting of the following: 15 to 150 mg.min/ml, 15 to 100 mg.min/ml, 15 to 75 mg.min/ml, and 15 to 50 mg.min/ml.
The ligands according to the invention, including, mono-, dual- and multi-specific, in one configuration thereof, are capable of binding to one or more molecules which can increase the half-life of the ligand il7 ViVO . Typically, such molecules are polypeptides which occur naturally in vivo and which resist degradation or removal by endogenous mechanisms which remove unwanted material from the organism. For example, the molecule which increases the half-life of the organism may be selected from the following:

Proteins from the extracellular matrix; for example collagen, laminins, integrins and fibronectin. Collagens are the major proteins of the extracellular matrix.
About 15 types of collagen molecules are currently known, found in different parts of the body, eg type I collagen (accounting for 90% of body collagen) found in bone, skin, tendon, ligaments, cornea, internal organs or type II collagen found in cartilage, invertebral disc, notochord, vitreous humour of the eye.
Proteins found in blood, including:
Plasma proteins such as fibrin, a-2 macroglobulin, serum albumin, fibrinogen A, fibrinogen B, serum amyloid protein A, heptaglobin, profilin, ubiquitin, uteroglobulin and f3-2-microglobulin;
Enzymes and inhibitors such as plasminogen, lysozyme, cystatin C, alpha-1-antitrypsin and pancreatic trypsin inhibitor. Plasminogen is the inactive precursor of the trypsin-like serine protease plasmin. It is normally found circulating through the blood stream. When plasminogen becomes activated and is converted to plasmin, it unfolds a potent enzymatic domain that dissolves the fibrinogen fibers that entgangle the blood cells in a blood clot. This is called fibrinolysis.
Immune system proteins, such as IgE, IgG, IgM.
Transport proteins such as retinol binding protein, a-1 microglobulin.
Defensins such as beta-defensin 1, Neutrophil defensins 1,2 and 3.
Proteins found at the blood brain barrier or in neural tissues, such as melanocortin receptor, myelin, ascorbate transporter.
Transferrin receptor specific ligand-neuropharmaceutical agent fusion proteins (see US5977307); brain capillary endothelial cell receptor, transferrin, transferrin receptor, insulin, insulin-like growth factor 1 (IGF 1) receptor, insulin-like growth factor 2 (IGF 2) receptor, insulin receptor.

Proteins localised to the kidney, such as polycystin, type IV collagen, organic anion transporter Kl, Heymann's antigen.
Proteins localised to the liver, for example alcohol dehydrogenase, G250.
Blood coagulation factor X
al antitrypsin HNF
Proteins localised to the lung, such as secretory component (binds IgA).
Proteins localised to the Heart, for example HSP 27. This is assOciated with dilated card i omyop athy.
Proteins localised to the skin, for example keratin.
Bone specific proteins, such as bone morphogenic proteins (BIVIPs), which are a subset of the transforming growth factor f3 superfamily that demonstrate osteogenic activity. Examples include BMP-2, -4, -5, -6, -7 (also referred to as osteogenic protein (OP-1) and -8 (0P-2).
Tumour specific proteins, including human trophoblast antigen, herceptin receptor, oestrogen receptor, cathepsins eg cathepsin B (found in liver and spleen).
Disease-specific proteins, such as antigens expressed only on activated T-cells:
including LAG-3 (lymphocyte activation gene), osteoprotegerin ligand (OPGL) see Nature 402, 304-309; 1999, 0X40 (a member of the TNF receptor family, expressed on activated T cells and the only costimulatory T cell molecule known to be specifically up-regulated in human T cell leukaemia virus type-I (HTLV-I)-producing cells.) See J Inununol. 2000 Jul I ; 1 65 (1): 263-70; Metalloproteases (associated with arthritis/cancers), including CG6512 Drosophila, human paraplegin, human FtsH, human AFG3L2, murine ftsH; angiogenic growth factors, including acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), Vascular endothelial growth factor / vascular permeability factor (VEGF/VPF), transforming growth factor-a (TGF a), tumor necrosis factor-alpha (TNF-0, angiogenin, interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-derived endothelial growth factor (PD-ECGF), placental growth factor (P1GF), midkine platelet-derived growth factor-BB
(PDGF), fi-actalkine.
Stress proteins (heat shock proteins) HSPs are normally found intracellularly. When they are found extracellularly, it is an indicator that a cell has died and spilled out its contents. This unprogrammed cell death (necrosis) only occurs when as a result of trauma, disease or injury and therefore in vivo, extracellular HSPs trigger a response from the immune system that will fight infection and disease. A dual specific which binds to extracellular HSP can be localised to a disease site.
Proteins involved in Fc transport Brambell receptor (also known as FcRB):
This Fc receptor has two functions, both of which are potentially useful for delivery The functions are (1) The transport of IgG from mother to child across the placenta (2) the protection of IgG from degradation thereby prolonging its serum half life of IgG. It is thought that the receptor recycles IgG from endosome.
See Holliger et al, Nat Biotechnol 1997 Jul;15(7):632-6.
Other proteins involved in Fc transport include the neonatal Fc receptor (FcRn) described in Gastinel et al., 1992, PNAS 89:638; and Roopenian et al., 2003 J.
Immunol. 170:3528.

Ligands according to the invention may designed to be specific for the above targets without requiring any increase in or increasing half life in vivo. For example, ligands according to the invention can be specific for targets selected from the foregoing which are tissue-specific, thereby enabling tissue-specific targeting of the dual specific ligand, or a dAb monomer that binds a tissue-specific therapeutically relevant target, irrespective of any increase in half-life, although this may result.
Moreover, where the ligand or dAb monomer targets kidney or liver, this may redirect the ligand or dAb monomer to an alternative clearance pathway in vivo (for example, the ligand may be directed away from liver clearance to kidney clearance).
Polypeptides useful for increasing half-life include, but are not limited to those shown in Annex I.
Increased Protease Stability A further advantage of the present invention is that the PEGylated dAbs and dAb multimers described herein possess increased stability to the action of proteases.
Depending on the assay conditions, dAbs are generally intrinsically stable to the action of proteases. In the presence of pepsin, however, many dAbs are totally degraded at pH 2 because the protein is unfolded under the acid conditions, thus making the protein more accessible to the protease enzyme. The present invention provides PEGylated dAb molecules, including dAb multimers, wherein it is believed that the PEG polymer provides protection of the polypeptide backbone due the physical coverage of the backbone by the PEG polymer, thereby preventing the protease from gaining access to the polypeptide backbone and cleaving it. In a preferred embodiment a PEGylated dAb having a higher hydrodynamic size (e.g., to 500 kDa) is generated according to the invention, because the larger hydrodynamic size will confirm a greater level of protection from protease degradation than a PEGylated dAb having a lower hydrodynamic size. In one embodiment, a PEG- or other polymer-linked antibody single variable domain monomer or multimer is degraded by no more than 10% when exposed to one or more of pepsin, trypsin, elastase, chymotrypsin, or carboxypeptidase, wherein if the protease is pepsin then exposure is carried out at pH 2.0 for 30 minutes, and if the protease is one or more of trypsin, elastase, chymotrypsin, or carboxypeptidase, then exposure is carried out at pH 8.0 for 30 minutes. In a preferred embodiment, a PEG- or other polymer-linked dAb monomer or multimer is degraded by no more than 10% when exposed to pepsin at pH 2.0 for 30 minutes, preferably no more than 5%, and preferably not degraded at all. In a further preferred embodiment, a PEG- or other polymer-linked dAb multimer (e.g., hetero- or homodimer, trimer, tetramer, octamer, etc.) of the invention is degraded by less than 5%, and is preferably not degraded at all in the presence of pepsin at pH 2.0 for 30 minutes. In a preferred embodiment, a PEG- or other polymer-linked dAb monomer or multimer is degraded by no more than 10% when exposed to trypsin, elastase, chymotrypsin, or carboxypeptidase at pH 8.0 for minutes, preferably no more than 5%, and preferably not degraded at all. In a further preferred embodiment, a PEG- or other polymer-linked dAb multimer (e.g., hetero- or homodimer, trimer, tetramer, octamer, etc.) of the invention is degraded by less than 5%, and is preferably not degraded at all in the presence of trypsin, elastase, chymotrypsin, or carboxypeptidase at pH 8.0 for 30 minutes.
The relative ratios of protease:antibody single variable domain polypeptide may be altered according to the invention to achieve the desired level of degradation as described above. For example the ratio or protease to antibody single variable domain may be from about 1:30, to about 10:40, to about 20:50, to about 30:50, about 40:50, about 50:50, about 50:40, about 50:30, about 50:20, about 50:10, about 50:1, about 40:1, and about 30:1.
Accordingly, the present invention provides a method for decreasing the degradation of an antibody single variable domain comprising linking an antibody single variable domain monomer or multimer to a PEG polymer according to any of the embodiments described herein. According to this aspect of the invention, the antibody single variable domain is degraded by no more than 10% in the presence of pepsin at pH2.0 for 30 minutes. In particular, a PEG-linked dAb multimer is degraded by no more than 5%, and preferably not degraded at all in the presence of pepsin at pH 2.0 for 30 minutes. In an alternate embodiment, the antibody single variable domain is degraded by no more than 10% when exposed to trypsin, elastase, chymotrypsin, or carboxypeptidase at pH 8.0 for 30 minutes, preferably no more than 5%, and preferably not degraded at all.
Degradation of PEG-linked dAb monomers and multimers according to the invention may be measured using methods which are well known to those of skill in the art. For example, following incubation of a PEG-linked dAb with pepsin at pH
2.0 for 30 minutes, or with trypsin, elastase, chymotrypsin, or carboxypeptidase at pH
8.0 for 30 minutes, the dAb samples may be analyzed by gel filtration, wherein degradation of the dAb monomer or multimer is evidenced by a gel band of a smaller molecular weight than an un-degraded (i.e., control dAb not treated with pepsin, trypsin, chymotrypsin, elastase, or carboxypeptidase) dAb. Molecular weight of the dAb bands on the gel may be determined by comparing the migration of the band with the migration of a molecular weight ladder (see Figure 5). Other methods of measuring protein degradation are known in the art and may be adapted to evaluate the PEG-linked dAb monomers and multimers of the present invention.
Pharmaceutical Compositions. Dosage and Administration The antibody polypeptides of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject.
Typically, the pharmaceutical composition comprises a monovalent anti-CD4OL antibody polypeptide and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The term "pharmaceutically acceptable carrier" excludes tissue culture medium comprising bovine or horse serum.
Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable substances include minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody polypeptide.

The compositions as described herein may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscurar).
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
=25 The antibody polypeptides described herein can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is intravenous injection or infusion. The polypeptide can also be administered by intramuscular or subcutaneous injection.

As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Single immunoglobulin variable domains and other relatively small monovalent antibody polypeptides are well suited for formulation as extended release preparations due, in part, to their small size ¨ the number of moles per dose can be significantly higher than the dosage of, for example, full sized antibodies. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Additional methods applicable to the controlled or extended release ofpolypeptide agents such as the monovalent antibody polypeptides disclosed herein are described, for example, in U.S. Patent Nos. 6,306,406 and 6,346,274, as well as, for example, in -U.S.
Patent Application Nos. US20020182254 and US20020051808.
In certain embodiments, a monovalent anti-CD4OL antibody polypeptide can be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a = hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the individual's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.

Additional active compounds can also be incorporated into the compositions.
In certain embodiments, a monovalent anti-CD4OL antibody polypeptide is coformulated with and/or coadministered with one or more additional therapeutic agents. For example, a monovalent anti-CD4OL antibody polypeptide can be coforrnulated and/or coadministered with one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules), or, for example, one or more cytokines. Such combination therapies may utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.
The pharmaceutical compositions of the invention can include a "therapeutically effective amount" or a "prophylactically effective amount" of a monovalent anti-CD4OL antibody polypeptide. A "therapeutically effective amount"
refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the antibody polypeptide can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the monovalent anti-CD4OL
antibody polypeptide to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. A
"prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
Typically, because a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is advantageous to formulate parenteral compositions in dosage unit forin for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
A non-limiting range for a therapeutically or prophylactically effective amount of a monovalent anti-CD4OL antibody polypeptide is 0.1-20 mg/kg, more preferably 1-10 mg/kg. It is to be noted that dosage values can vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the administering clinician.
The efficacy of treatment with a monovalent anti-CD4OL antibody polypeptide as described herein is judged by the skilled clinician on the basis of improvement in one or more symptoms or indicators of the disease state or disorder being treated. An improvement of at least 10% (increase or decrease, depending upon the indicator being measured) in one or more clinical indicators is considered "effective treatment," although greater improvements are preferred, such as 20%, 30%, 40%, 50%, 75%, 90%, or even 100%, or, depending upon the indicator being measured, more than 100% (e.g., two-fold, three-fold, ten-fold, etc., up to and including attainment of a disease-free state. Indicators can be physical measurements, e.g., enzyme, cytokine, growth factor or metabolite levels, rate of cell growth or cell death, or the presence or amount of abnormal cells. One can also measure, for example, differences in the amount of time between flare-ups of symptoms of the disease or disorder (e.g., for remitting/relapsing diseases, such as multiple sclerosis).
Alternatively, non-physical measurements, such as a reported reduction in pain or discomfort or other indicator of disease status can be relied upon to gauge the effectiveness of treatment. Where non-physical measurements are made, various clinically acceptable scales or indices can be used, for example, the Crohn's Disease Activity Index, or CDAI (Best et al., 1976, Gastroenterology 70: 439), which combines both physical indicators, such as hematocrit and the number of liquid or very soft stools, among others, with patient-reported factors such as the severity of abdominal pain or cramping and general well-being, to assign a disease score.
As the term is used herein, "prophylaxis" performed using a composition as described herein is "effective" if the onset or severity of one or more symptoms is delayed or reduced by at least 10%, or abolished, relative to such symptoms in a similar individual (human or animal model) not treated with the composition.
Whereas the monovalent anti-CD4OL antibody polypeptides described herein must bind human CD4OL, where one is to evaluate its effect in an animal model system, the polypeptide must cross-react with one or more antigens in the animal model system, preferably at high affinity. One of skill in the art can readily deteimine if this condition is satisfied for a given animal model system and a given monovalent anti-CD4OL antibody polypeptide. If this condition is satisfied, the efficacy of the monovalent anti-CD4OL antibody polypeptide can be examined by administering it to an animal model under conditions which mimic a disease state and monitoring one or more indicators of that disease state for at least a 10% improvement.
Animal Models:
Monovalent anti-CD4OL antibody polypeptides as described herein are useful for the treatment of autoimmune disorders in which CD40/CD4OL signaling is inappropriately active. There are several animal models in which the therapeutic efficacy of a given monovalent anti-CD4OL antibody polypeptide can be assessed, as discussed below.
Systemic Lupus Erythematosis (SLE):
Anti-CD4OL antibody treatment prevents the development of lupus-like nephritis in NZB/NZW and SNF1 SLE mice. Treatment of SNF1 mice with anti-CD4OL antibody reverses established nephritis and preserves kidney function.
See, e.g., Mohan et al., 1995, J. Immunol. 154: 1470-1480; Early et al., 1996, J.
Immunol.
157: 3159-3164; Kalled et al., 1998, J. Immunol. 160: 2158-2165 ,and Chess, 2001, "Blockade of the CD4OL/CD40 Pathway," in Therapeutic Immunology 2nd Edition, Austen, Burakof, Rosen and Strom, Eds., Blackwell Sciences (Pubs.), pp 441-456.
Multiple Sclerosis:
Specific blockade of CD4OL at the time of immunization markedly suppresses the incidence, mortality, day of onset, and clinical scores of experimental autoimmune encephalomyelitis (EAE) in B 1 OP1L and (PLJ x SJL)F1 mice induced by either myelin basic protein or PLP myelin antigens. See, for example, Gerritse, 1996, Proc.
Natl. Acad. Sci. U.S.A. 93: 2494; Grewal et al., 1996, Science 273: 186; Laman et al., 1998, Mult. Scler. 4: 14; and Chess, 2001, supra.
Rheumatoid Arthritis:
Anti-CD4OL blocks the development of joint inflammation, serum antibody titers to collagen, the infiltration of inflammatory cells into the synovial tissue, ant the erosion of cartilage and bone in collagen-induced arthritis. See, e.g., Durie et al., 1993, Science 261: 132; and Chess, 2001, supra.
Insulin-dependent Type I Diabetes Models:
The non-obese diabetic (NOD) mouse spontaneously develops T cell dependent autoimmune diabetes. Anti-CD4OL monoclonal antibody treatment of 3 to 4 week old NOD females (the age at which insulitis typically begins) completely prevented the insulitis and diabetes. Cytokine analysis revealed a dramatic decrease in IFN-g and IL-2 release without a concomitatnt increase in IL-4 production by T
cells from anti-CD4OL-treated mice. See, e.g., Balasa et al., 1997, J.
Immunol. 159:
1420; and Chess, 2001, supra.
Inhibition of Allograft and Xenograft Transplant Rejection:
Anti-CD4OL prevents the development of renal rejection of fully allogeneic grafts in mice. Moreover, the survival of renal allografts transplanted into nephrectomized rehsus monkeys is typically prolonged by anti-CD4OL therapy alone.
Similarly, anti CD4OL therapy has prevented graft rejection of skin, islet cells and cardiac transplants as well as GVHD in rodents. See, e.g., Kirk et al., 1997, Proc.
Natl. Acad. Sci. U.S.A. 94: 8789-8794; Parker et al., 1995, Proc. Natl. Acad.
Sci.
U.S.A. 92: 9560; Larsen et al., 1996, Transplantation 61: 4; and Chess, 2001, supra.
Uses of Monovalent Anti-CD4OL Antibody Polvpeptides Anti-CD4OL antibody polypeptides as described herein are useful for the treatment or prevention of diseases or disorders in which inappropriate activation of a CD4OL/CD40-mediated pathway is involved. In particular, autoimmune diseases frequently involve inappropriate regulation or activity of CD4OL/CD40 pathways.
Administration of an anti-CD4OL antibody polypeptide as described herein to an individual suffering from such a disease, can reduce one or more symptoms of the disease. Non-limiting examples of diseases for which the antibody polypeptides described herein can be therapeutically useful include Systemic Lupus Erythematosus (SLE), Idiotypic Thrombocytopenic Purpura (ITP), transplant rejection, Crohn's Disease, Inflammatory Bowel Disease (IBD), colitis, asthma/allergy, atherosclerosis, Myasthenia Gravis, immune response to recombinant drug products, e.g., factor VII
in hemophilia, Multiple Sclerosis, Psoriasis, Rheumatoid Arthritis, AnIcylosing Spondylitis, Coronary Heart Disease, and Diabetes, including Type 1 and/or Type 2 Diabetes.
The anti-CD4OL antibody polypeptides described herein are additionally useful in the way that generally any antibody preparation is useful, e.g., for in vivo imaging or diagnostic uses, in vitro diagnostic uses, etc. For these and other uses it may be desirable to. label the anti-CD4OL antibody polypeptides, e.g., with a fluorescent, colorimetric, enzymatic or radioactive label. Methods of labeling antibody polypeptides are well known in the art.
EXAMPLES
Example 1. Biotinvlation of Recombinant CD4OL

Recombinant human soluble CD4OL (PeproTech) was biotinylated and used during phage selections. Reagents, equipment and sources from which they are available are provided in Table 1.
Biotinylation of CD4OL was performed by incubating CD4OL (0.5mg/m1) with EZLinkTM Sulfo-NHS-LC-Biotin [Sulfosuccinimidyl-6-(biotinamido)hexanoate]
(Pierce) at a molar ratio of 5:1 on ice for 2 hours according to the product instructions. The biotinylation reaction mixture was then dialysed against 3 exchanges of PBS (1000x sample volume) in a Slide-A-Lyzer Dialysis Cassette at 4 C to remove the unincorporated biotinylation reagent.
The biotinylated-CD4OL was tested by receptor binding assay for binding to CD40/Fc to confirm its biological activity. Quality of biotin-CD4OL was also TM
monitored by analysing on a NuPaGE 4-12% Bis-Tris gel and detected by Simply Blue Safe-Stain (Invitrogen) (Figure la), and western-blotting by probing with Streptavidin-HIRP (Figure lb). The biotinylated-CD4OL was further analysed by mass spectrometry with the majority of CD4OL subunits containing 1 or 2 biotin moieties (data not shown).
Table 1.
Equipment/Reagent Suggested or required supplier Recombinant human PeproTech, Cat No:
soluble CD40 310-02 ligand/TRAP
Reconstituted in 5 mM
Sodium phosphate, pH5.0 to concentration of 0.5 mg/ml EZ_LinkTM Sulfo-NHS- Pierce, Cat No: 21335 LC-Biotin Slide-A-Lyzer Pierce, Cat No: 66110 Dialysis Cassette Recombinant human R&D Systems, Cat No:
CD40/Fc chimera 1493-CD
NuPAGE 4-12 k Bis- Invitrogen life Tris gel technologies Ltd Cat. No NP0322 Streptavidin-HRP Amersham Biosciences Cat No: 1231V
'InvitrogenTM Simply Invitrogen Cat Blue Safe-stain No:LC6065 Example 2. Phage Selections using Biotinvlated Antigen The Domain Antibody (dAb) libraries are based on a single human framework for the VB (DP47 and JH4b) and for the VK (DPK9 and J-K1) with side chain diversity incorporated at positions in the antigen binding site that make contact with antigen in known molecular structures and mirror residues diversified in the human antibody repertoire. The antibodies are displayed as fusion proteins covalently linked to the N ¨ terminus of the Fd-phage protein pIII, using the phage vector pD01\44 (Fd-Tet) with encodes the Fd phage genome with dAb expression under the control of the gene-III promoter. The dAb cassette consists of (5' to 3'): eukaryotic leader sequence, dAb, myc tag, gIII. The vector contains both the ..A1.13 and colEl origins of replication and is selectable using tetracycline. The VF1 and V, libraries each have a calculated size of over 1x101 molecules. Reagents, equipment and sources from which they are available are provided in Table 2.
Approximately lx1011 phage from the each of the Domantis dAb libraries were incubated in a final volume of 1 ml PBS containing 2% Marvell-1'4 at room temperature for 1 11. Biotinylated antigen was added to the blocked phage such that the phage antigen mixture had a final concentration of 2% MarvelTM in PBS. The antigen concentration used for the first round of selection was 60 n.M; the antigen concentration was decreased to 6 nM for round 2, and to 0.6 n1\4 for round 3.
The antigen/phage mix was incubated for 1 h at room temperature with rotation at ¨40 rpm.
TM
For each selection, 100 ul of streptavidin-coated paramagnetic beads (Dynal Biotech) were prepared by washing once in 1 ml of PBS containing 0.1% Tween-20 followed by a second wash in 1 ml of PBS. The beads were then blocked in 1 ml of PBS containing 2% MarvelTM in a 2 ml eppendorf tube at room temperature on a rotating wheel for 1 h.
The tube containing the blocked streptavidin-coated magnetic beads was placed into a magnetic holder, enabling capture of the magnetic beads. The supernatant was removed and the beads resuspended in the antigen/phage mix.
This mixture was rotated for 10 min to allow for bead capture of phage/antigen complexes.
The beads were captured using a magnetic holder and repeatedly washed 19 times using 1 ml of PBS containing 0.1% Tween-m20, followed by a final wash of 1 ml PBS. The eppendorf tubes were changed following washing steps 3, 9, 15 and 19 to minimise background phage carryover.
The washed beads were then recaptured and all washing solution removed.
The phage were eluted through resuspension in 500 p.1 of trypsin solution (50 Ill of 10 mg/ml trypsin stock solution added to 450 p.1 PBS, freshly diluted) and rotated for 10 min at room temperature. The eluted phage were recovered by capturing the beads using the magnetic holder and the liquid containing the eluted phage recovered. The eluted phage were used to infect E. coli TG1 to prepare phage for a further round of selection.
The eluted phage (250 p.1) were mixed with 1.75 nil of log phase E. coli TG1 (0D600 between 0.3 and 0.6) and infection allowed to occur for 30 min at 37 C
without shaking. The infected E. coli TG1 culture was centrifuged at 11,600 g in a micro centrifuge for 1 min at room temperature. The pelleted bacteria were resuspended in 100 ul of 2xTY and plated on regular 9 cm diameter plates containing TYE supplemented with 15 ug/m1 tetracycline. Plates were grown at 37 C
overnight.
After overnight growth, 2 ml of 2xTY containing 15% glycerol was added to the culture plates and cells loosened with .a spreader, ensuring the cells were thoroughly mixed. Two millilitres of the culture were recovered by pipetting into a cryo-vial, from which 50 p.1 was used to inoculate 50 ml of 2xTY supplemented with 15 ug/m1 tetracycline. The remaining cells in the cryo-vial were stored at -80 C.
The 50 nil culture was grown at 37 C for 16 to 24 hours with shaking at 250 rpm.
Following overnight growth, the culture was centrifuged at 3,300 g for 15 min to pellet the bacteria. The phage were then precipitated from the supernatant through the addition of 10 ml of PEG/NaC1 to 40 ml of clarified supernatant. The phage/PEG
solution was mixed and incubated on ice for at least 1 h. To pellet the phage, the solution was centrifuged at 3,300 g for 30 min at 4 C. The supernatant was decanted and any remaining supernatant removed by aspiration.
The resulting phage pellet was resuspended in 2 ml PBS and centrifuged at 11,600 g for 10 min in a micro centrifuge to remove any remaining bacterial debris.
The supernatant was filtered through a 0.45 tan filter (Sartorius, Minisart).
The resuspended phage solution was used for the next round of selection.

Table 2 Equipment/Reagent Suggested or Instrument setting, required supplier reagent preparation Dynabeads M-280 Dynal Biotech UK Resuspend Streptavidin (Prod. 11 Bassendale Road, thoroughly through No.: 112.05) Croft Business Park, repeated pipetting.
Bromborough, Wirral UK
Tween 20 Sigma Chemical 0.1% in PBS.
Company Ltd.
99.5% dried skim MarvelTM (premier 2% in PBS (prepare milk powder brands) from fresh and do not supermarkets. store).
Trypsin (T-8642) Type Sigma Chemical made up in 50 mM
XIII from Bovine Company Ltd. Tris-HCI
pH7.4; 1 mM
Pancreas. CaCl2 and stored at -Fancy Road 20 C.
Dorset BH17 7NH The trypsin stock U K solution should be .
stored aliquotted at -Tel +44 1202 733114 20 C to avoid a utoproteolysis.
Fax +44 1202 PEG/NaCI Sigma Chemical 20%
Polyethylene Company Ltd. glycol 8000 [formally known as 6000], 2.5 M NaCI pre-chilled to 40C.
Dynal MPC-S Dynal Biotech UK =
magnetic particle 11 Bassendale Road, concentrator (Prod. Croft Business Park, Bromborough, Wirral No.: 120.20) CH62 3QL
UK
2xTY 16 g Tryptone, 10 g Yeast Extract and 5 g NaCI in 1 litre.
Autoclave (1210C, 15 min) and store at RT

Example 3: Cloning Enriched Phage Selection Outputs into the Soluble dAb Expression Vector pDOM5 Following the second and third rounds of selection, E. coli cells infected with the enriched dAb displaying fd-phage populations were obtained. An aliquot of these cells was used to prepare phage DNA and the enriched V-genes excised by digestion using the restriction endonucleases, Sall and Notl. The purified V-genes were ligated into the corresponding sites of pDOM5 (expression vector derived from pUC119 with LacZ
promoter, eukaryotic leader, dAb cloning site, myc tag), and the ligated DNA
used to electro-transform E. coli HI32151 cells which were grown overnight on agar plates containing the antibiotic carbenicillin. The resulting colonies were induced to express dAb protein either as 200 I microcultures or 50 ml cultures. The resulting dAb was analysed for inhibitory activity using the CD4OL receptor binding assay.
Following selection of phage, pD01\44 DNA was purified from the cell pellet obtained from a 50 ml overnight E. coli culture using the QIAfilteTMr Plasmid Midi DNA purification kit from Qiagen, following the manufacturer's instructions.
The dAb genes were excised from the pD01\44 vector by mixing: 10 I of 10x Sall buffer;
1 1 of 100x BSA; 20 ug of purified DNA fragment; 2.5 1 of Sall enzyme (10 U/
I);
2.5 .1 of NotI enzyme (10 U/ I); the digestion mix was made up to a final volume of 100 1 using sterile water. The digestion mix was incubated for 5 hours at 37 C.
The digested DNA samples were electrophoresed on a 1.5% agarose gel and the band corresponding to the dAb V-genes (-324 bp to 372 bp) was excised from the gel. The dAb gene DNA was purified from the gel slice using the QIAquic el k m Gel Extraction kit from Qiagen, following the manufacturer's instructions.
The expression vector pDOM5 was digested with Sall and Notl as follows: 10 1 of 10x SalI buffer; 1 I of 100x BSA; 20 jig of plasmid pDOM5; 1.5 1 of Sall enzyme (10 U411); 1.5 I of NotI enzyme (10 U/ I); the digestion mix was made up to a final volume of 100 1 using sterile water. The digestion mix was incubated for 2 hours at 37 C. The digested vector fragment was purified using the QIAquick PCR
Purification Kit.

The digested pDOM5 and digested dAb genes were ligated by mixing: 2 pl of 10x T4 DNA ligase buffer; 400 ng of digested pDOM5 vector; 100 ng of digested dAb genes;
1 1.1.1 of T4 DNA ligase (400 U/p1); the ligation mix was made up to 20 pJ
with sterile water. The ligation mixes were incubated for 2 hours at 25 C.
Two microlitres of the ligation mix was transferred to the bottom of a pre-chilled (on ice) 0.2 cm electroporation cuvette to which 100 p.1 of electrocompetent E.
coli HB2151 cells were added. The DNA / cell mixture was incubated on ice for min, then electroporated at 2.5 kV (25 pF, 200 S2). One millilitre of 2xTY was immediately added to the cuvette and the cells gently resuspended. The resuspended cells were transferred to a 14 ml disposable culture tube and incubated for 1 hour at 37 C with shaking at 250 rpm. Dilutions of the cells from 10-0 to 10-3 were plated on regular 9 cm diameter plates containing TYE supplemented with 5% glucose and p,g/m1 carbenicillin. The cells are incubated overnight at 37 C in an inverted position.
Reagents, equipment and sources from which they are available are provided in Table 3.
=

Table 3 Equipment/Reagent Suggested or required Instrument supplier setting, reagent preparation QIAfilterTM Plasmid Midi Qiagen Ltd Supplied as kit DNA purification kit Cat. No.: 12143 Sall restriction New England Biolabs endonuclease + 10x Sall buffer Cat. No.: R01385 NotI restriction New England Biolabs endonuclease + 10x NotI buffer + 100x Cat. No.: R0189S
BSA
QIAquick Gel Qiagen Ltd Supplied as kit Extraction kit Cat. No.: 28706 Expression plasmid pDOM5 T4 DNA ligase + 10x New England Biolabs The T4 DNA
T4 DNA ligase buffer ligase buffer Cat. No.: M0202L should be stored aliquotted at -2000.
Repeated freeze-thawing should be avoided to minimise the hydrolysis of ATP in the buffer.

Example 4. Microwell Expression of Soluble dAbs Following cloning of the selected phage dAb outputs into pDOM5, individual bacterial colonies were inoculated as microwell cultures and induced using IPTG to express dAb protein which was analysed for inhibitory activity using the CD4OL
receptor binding assay. Reagents, equipment and sources from which they are available are provided in Table 4.
Individual bacterial colonies were carefully picked to ensure that contamination from neighbouring colonies was avoided. The picked colonies were used to inoculate 96 well cell culture plates containing 100 ul per well of 2xTY
supplemented with 5% glucose and 50 ug/m1 carbenicillin. The lids were placed on the cell culture plates which were incubated overnight in a HiGrOrmorbital shakei (GeneMachines, 935 Washington St, San Carlos, CA 94070, USA) under a humidified atmosphere at 37 C with shaking at 450 rpm (4 min shaking orbital diameter), with gas (30% 02 + 70% NO pulsed for 10 seconds every 5 minutes at a flow rate of 5 SLPM (standard litres per minute). [These plates are referred to as Master Plates].
Following overnight growth, a 96 well transfer device was used to transfer between 1-5 ul of the bacterial culture into a fresh 96 well culture plate containing 100 1.11 per well of 2xTY supplemented with 0.1% glucose and 50 .i.g/m1 carbenicillin.
The freshly inoculated plates were incubated at 37 C for 3 to 4 h (shaking at 450 rpm, gas (30% 02 + 70% NO pulsed for 10 seconds every 5 minutes at a flow rate of 5 SLPM) until the culture 0D600 reached approximately 1Ø The cultures were then induced by the addition of 100 ul per well of 2xTY containing 50 pg/m1 carbenicillin and 2 mM IPTG (final IPTG concentration of 1 mM) and incubated overnight at 30 C with shaking at 450 rpm, with gas (30% 02 + 70% 1\12) pulsed for 10 seconds every 5 minutes at a flow rate of 5 SLPM. [These plates are referred to a Induction Plates].

Glycerol stocks of the original Master Plates were made by the addition of 100 ul per well of 2xTY containing 50% sterile glycerol. These plates were stored at -80 C.
Following overnight incubation of the Induction Plates, the bacterial cells were pelleted by centrifugation at 1,800 g for 10 min at 4 C. The supernatant (containing expressed dAb) was then analysed to determine if dAbs were capable of inhibiting binding of CD4OL to CD4O-Fc fusion in a receptor binding assay.
Table 4 Equipment/Reagent Suggested or Instrument setting, required supplier reagent preparation 96 Well Cell Culture Corning Incorporated, Cluster with round Costar.
bottom and lid, Non- Number:3799 pyrogenic, Polystyrene 2xTY 16 g Tryptone, 10 g Yeast Extract and 5 g NaC1 in 1 litre.
Autoclave (1210C, 15min) and store at RT

Example 5. Expression of dAb in E. coli at 50 ml To generate greater quantities of dAb protein for analysis, 50 ml cultures were used for induction. A single colony of the desired dAb (for example DOM-24) grown on TYE plates was inoculated into 10 ml 2xTY supplemented with 5% glucose and 50 ug/m1 carbenicillin in a 30 ml universal tube and grown overnight at 37 C
with shaking at 250 rpm. Five hundred microlitres of the overnight culture was added into 50 ml of 2xTY supplemented with 0.1% glucose and 50 ug/m1 carbenicillin and grown /at 37 C with shaking at 250 rpm. The 0D600 of the culture was monitored regularly in comparison with sterile 2xTY and at an 0D600 of 0.9 the culture was induced by the addition of 1 M IPTG to a final concentration of 1 mM. The inoculated culture was incubated at 30 C with shaking at 250 rpm overnight.
The next day, the culture was centrifuged at 6000 g for 15 min at 4 C and the clarified supernatant mixed with 100 pi of protein-A streamline or protein-L agarose (pre-washed with 5 mM MgSO4) overnight at 4 C. The supernatant/bead mixture was then centrifuged at 180 g at 4 C for 2 minutes. The supernatant was decanted and the retained beads washed with 10 ml of PBS containing 0.5M NaCl. The bead solution was transferred into a 96 well Whatman filter plate and the beads washed once with 400 1 of PBS containing 0.5M NaC1, then once with 400 IA of PBS, followed by centrifugation for 2 minutes at 180 g after each washing step. dAb protein was eluted using 70 [11 of 0.1 M glycine (pH 2.0) and the solution neutralised by the addition of 40 ul of 1 M Tris-HC1 (pH 8.0). The purified dAb concentration was determinate by Reagents, equipment and sources from which they are available are provided in Table 5.
Table 5 Equipment/Reagent Suggested or Instrument setting, required supplier reagent preparation TYE 15 g Bacto-Agar, 8 g NaCI, 10 g Tryptone, g Yeast Extract in 1 litre water. Autoclave (1210C, 15 min) and store at RT
2xTY 16 g Tryptone, 10 g Yeast Extract and 5 g NaCI in 1 litre.
Autoclave (1210C, min) and store at RT
1 M IPTG stock made up in MQ
water is sterile filtered through 0.22 pM filter and stored in aliquots at -Carbenicillin 50 mg/ml stock made in water, 0.2 Jim filter sterilised and stored in aliquots at -200C
40% glucose solution 0.2 m filter sterilise, store at RT
5 mM MgSO4 prepare fresh from 1 M stock solution, filter r sote .2ril iisme and store at RT
0.5M NaCl/PBS Autoclave clave filter sterilise and store at RT
Protein A agarose Sigma P3476 store 40C
Protein L agarose = Sigma P3351 store 40C

=
Streamline rProtein A Amersham store 40C
Biosciences, cat no. 17-1281-02 (300 ml) 1 M Tris-HCI, pH 8.0 0.2 p.m filter sterilise or autoclave and store at RT
0.2 M Glycine, pH2.0 0.2 p.m filter sterilise and store at 40C

Example 6: CD4OL Receptor Bind in Assay The CD4OL assay was used to measure the binding of CD4OL to CD40 and the ability of binding entities (eg, monvalent antibody fragments such a dAbs) to block this interaction, as described below and shown schematically in Figure 7. (The =
soluble proteins from R&D Systems are CD40/Fc homodimer and CD4OL =
hornotrimer).
A 96 well Nunc Maxismp assay plate was coated overnight at 4 C with 100 [41 per well of recombinant human CD40/Fc (R&D Systems) at 0.5 ng/ml in carbonate buffer. The plate was washed 3 times with 300 ul of 0.05% Tween/PBS and 3 times with 300 ul of PBS using a Tecan plate washer. The wells were blocked using 200 u.1 of PBS containing 2% (w/v) BSA and incubated for a minimum of 1 h at room temperature. The wells were washed as above, then 50 ul of purified dAb protein (or unpurified supernatant containing clAb from a micro-culture expression) was added to each well. To each well 50 ul of CD4OL, at 6 ng/ml in diluent (for a final concentration of 3 ng/ml), was also added and the plate incubated for 1 hr at room temperature.
The plate was washed as described previously and 100 ul biotinylated anti-CD4OL antibody, 0.5 ug/m1 in diluent, was added and incubated for 1 hr at room temperature. The plate was washed as described above, then 100 HRP conjugated anti-biotin antibody (1:5000 dilution in diluent) added to each well and the plate incubated for 1 hr at room temperature. The plate was washed again as described above using a Tecan plate washer and the assay developed using 100 ill of SureBluerm 1-Component TMB MicroWelrPeroxidase solution (the plate was left at room temperature for up to 20 min). The reaction was stopped by the addition of 100 ill 1 M hydrochloric acid. The OD45onm of the plate was assayed within 30 minutes of acid addition. The 01)450nm is 'proportional to the atnount of bound streptavidin-HRP
conjugate, therefore the greater the degree of dAb inhibition the lower the OD450.1 of the resulting signal. Reagents, equipment and sources from which they are available are provided in Table 6.

Controls The following controls were in = 0 ng/ml CD4OL (diluent only) = 3 ng/ml CD4OL
= 3 ng/ml CD4OL with 1 tig/m1 anti-CD4OL antibody Table 6 Equipment/Reagent Suggest or required Reagent preparation supplier (specify) _____________ T ________________________________________________________ F96 MaxisorpM 96 well Nunc, Cat No: 439454 immunoplate D.211 sodium Pierce, Cat No: 28382 Dissolve 1 sachet in carbonate bicarbonate 500 ml deionised water buffer pH9.4 and keep solution at 4 C
Recombinant human R&D Systems, Cat No: Stock 50 pg/ml at-80 C
CD40/Fc chimera 1493-CD
Phosphate buffered Sigma, Cat No: P4417 10x solution100 tablets/L
saline (PBS) water.
Wash buffer 0.05% Tween-20/PBS
Diluent 0.1% BSA, 0.050/0 Tween-20 in PBS
Block 2% BSA in PBS
Recombinant human R&D Systems, Cat No: Stock 50 pg/m1 at-80 C

Neutralising anti- Calbiochem, Cat No: Stock 1 mg/ml at 4 C
CD4OL antibody 217595 Biotinylated anti-R&D Systems, Cat No: Stock 50 pg/m1 at -80 C
CD4OL antibody BAF617 Anti-biotin-HRP Stratech, Cat No:
Stock 800 pg/m1 at -80C, conjugate 200-032-096 diluted 1:5000 in antibody diluent. Keep for 1 week only.
SureBlue TMB 1- KPL, Cat No: 52-00-00 at 4 C .
component microwell peroxidase substrate Example 7: Results Receptor binding data for.the most potent inhibitors is summarised in Figures 2, 3, and 4, and in Table 7, below. Table 8, below, provides DNA and translated amino acid sequence of unique dAbs identified in the receptor binding assay as inhibiting CD4OL binding to CD40.
Figure 2 shows a dose response receptor binding assay(RBA) readout, analysing the inhibition of CD4OL binding to CD4O-Fc by dAbs DOM-10, -20, -27, -30, -31, -62, -77, titrated from 1 OA down to 10 p1\4. dAbs D01\4-20, -30, and -31 are the most potent, with IC50 values of approximately 8 nM.
Figure 3 shows a dose response receptor binding assay readout, analysing the inhibition of CD4OL binding to CD4O-Fc by dAbs DOM-4 and DOM-5, titrated from 1 p.M down to 500 pM. The IC50 values for dAbs D01\4-5 and DOM-4 are approximately 3 n114 and 100 nM respectively.
Figure 4 shows a dose response receptor binding assay readout, analysing the inhibition of CD4OL binding to CD4O-Fc by dAb DOM-24, titrated from 100 nM
TM
down to 0.5 p1\4. The data were curve-fitted using GraphPad Prism software.
Table 7 Clone Name dAb Type IC50 (nM) DOM-8 VK 900.

DOM-24 VH 0.417 approx.

Table 8: Summary of dAbs exhibitina a ranae of CD4OL inhibitory 1050 values as determined using the CD4OL / CD4O-Fc receptor inhibition assay.
The DNA and translated amino acid sequence of unique dAbs identified in the receptor binding assay as inhibiting CD4OL binding to CD40 are detailed below:
DOM-2 SEQ ID NO: 7 EVOL LES GGG LVQP GGS L R L SCAA SGF TRSD
Y t M =
GAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC TCCTGTGCAG
CCTCCGGATT CACCTTTTCT
GATTATGAGA
CTCCACGTCGACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAAAGA
CTAATACTCT
=MWVRQA PGKG LEWVST ITSD GIS TYY ADSV
K G R

ATGGTATTTC TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACTACACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTGA TAATGAAGCC TACCATAAAG
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
=F TI FRDN SKN TLY LQMN SLRAED TAVY YCA
K S G

TGCCGAGGAT ACCGCGGTAT ATTACTGTGC
GAAAAGTGGG
CAAGTGGTAG AAGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTA
TGGCGCCATA TAATGACACG
CTTTTCACCC
REED YWG QGT LVTV SS
301 . AGGTTTTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO:
94) TCCAAAAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 95) DOM-4 SEQ ID NO: 8 EVOLLES GGG LVQP GGS LRL SCAA SGF TFDN
Y E M

TCCTGTGCAG CCTCCGGATT CACCTTTGAT
AATTATGAGA
CTCCACGTCG ACPACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGPAACTA
TTAATACTCT
=TWVRQA PGKG LEW VSS ITSD GTS TYYADSV
K G R =

ATGGTACTTC GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACTGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTAGA TAATGCTCAC TACCATGAAG
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SKN TLY LQMN SLRAED TAVY YCA

TGCCGAGGAT ACCGCGGTAT ATTACTGTGC
GPPACCTAAT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTA
TGGCGCCATA TAATGACACG
CTTTGGATTA
PPFDYWG QGT LVTV SS
301 CCGCCGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO:
96) GGCGGCPAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 97) DOM-5 SEQ ID NO: 9 EVOLLES GGG LVQP GGS LRL SCAA SGF TFDG
Y E M =

CCTCCGGATT CACCTTTGAT
GGGTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACTA
CCCATACTCT
=AWVRQA PGKG LEW VSS ITSD GTS TYYADSV
K .G R =

ATGGTACGAG TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTAGA TAATGCTCAC TACCATGCTC
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC

=
.FTI SR 01 SKN TLY LOME SLR AED TAVY YCA
K I G

TGCCGAGGAC ACCGCGGTAN ATTACTGTGC
GAAACCGGGG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
IGGCGCCATA TAATGACALG
CTTTGGCCCC
LRFDYWG QGT LVTV SS
301 CTGCGTTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 98) GACGCAAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 99) DOM-7 SEQ ID NO: 10 EVQLLES GGG LVQP GGS LRL SCAA SGF TFNL
Y E M =

TCCTGTGCAM CCTCCGGATT CACCTTTAAT
TTGTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAATTA
AACATACTCT
=TWVRQA PGKG LEWVSS ITSD GVS TYY ADSV
K G R =

ANGGTGTTTC TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACTGAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTAGA TAATGATCAC TACCACAAAG
ATGTATGANG CGTCTGAGGC
ACTTCCCGGC
.FTI SRDN SKN TLY LQMN SLR AED TAVY YCA
KAG

TGCCGAGGAC ACCGCGGTAN ANTACTGTGC
MAzAGCTGGG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTCGACCC
/IFDYWG QGT LVTV SS
301 GTGATTTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC )SEQ ID NO: 100) CACTAAAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 101) =
=
DOM-8 SEQ ID NO: 11 S L E =

ANCACTTGCC GGGCAAGTCA GTTTATTGAT
ACGTCGTTAG
CTGTAGGTCT ACTGGGTCAG AGGTAGGAGG GACAGACGTA GACATCCTCT GGCACAGTGG TAGTGAACGG
CCCGTTCAGT CAAATAACTA
TGCAGCAATC
=WYQ QKP GKAP KLL IYD GSHL QSG VPS RFSG
S G S =

TGCAAAGTGG GGTCCCATCA CGTTTCAGTG
GCAGTGGATC
TCACCATAGT CGTCTTTGGT CCCTTTCGGG GATTCGAGGA CTAGATACTA CCCAGGGTAA ACGTTTCACC
CCAGGGTAGT GCAAAGTCAC
CGTCACCTAG
=GTD FTLT ISS LQP EDLA TYY CQQ YWVL PLT
FGQ

CTGTCAACAG TATTGGGTTC TTCCTCTGAC
GTTCGGCCAA
ACCCTGTCTAAAGTGAGAGT GGTAGTCGTC AGACGTTGGA CTTCTAAATC GATGCATGAI GACAGTTGTC
ATAACCCAAG AAGGAGACTG
CAAGCCGGTT
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG (SEQ ID NO: 102) CCCTGGTTCC ACCTTTAGTT TGCC (SEQ ID NO: 103) DOM-10 SEQ ID NO: 12 EVQLLES GGGLVQP GGS LRL SCAA SGF TFIA
Y D M =

TCCTGTGCAG CCTCCGGATT CACCTTTATT
GCTTATGATA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AATCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAATAA
CGAATACTAT
=SWVRQA PGKG LEW VSW IDEW GLQ TYY ADSV
K G R =

GGGGTCTGCA GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACTCAACCCA GGCGGTCCGA GGTCCCTTCC CAGACCTCAC CCAGAGTACC TAACTACTCA CCCCAGACGT
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
= SRDN SKE TLY LQMN SLR AED TAVY YCA
K K T

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
cl..AAAAGACG

CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTTTCTGC
PEEP DYW GQG TLVT VSS
301 CCTGAGGAGT TTGACTACTG GGGTCAGGGA ACCCTGGTCA CCGTCTCGAG C (SEQ ID NO:
104) GGACTCCTCA AACTGATGAC CCCAGTCCCT TGGGACCAGT GGCAGAGCTC G (SEQ ID NO: 105) DOM-11 SEQ ID NO: 13 EVQL LES GGG LVQP GGS LRL SCAA SGF TEGD
Y E M

TCCTGTGCAG CCTCCGGATT CACCTTTGGT
GATTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACCA
CTAATACTCT
=SWVRQA PGEGLEW VSG IDGE GSD TYYADSV
E G R

TGAGTTGGGT CCGCCAGGCT CCAGGGGG GTCTAGAGTG GGTCTCAGGG ATTGATGGTG AGGGTTCTGA
TACATACTAC GCAGACTCCG
TGAI,GGGCCG
ACTCAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTCCC TAACTACCAC TCCCAAGACT
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
.FTI SRDN SEN TLY LQMN SLR AED TAVY YCA
KPG

TGCCGAGGAT ACCGCGGTAT ATTACTGTGC
GAAACCGGGG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTA
TGGCGCCATA TAATGACACG
CTTTGGCCCC
RSFD TWG QGT LVTV SS
301 AGGAGTTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO:
106) TCCTCAAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 107) DOM-12 SEQ ID NO: 14 EVQLLES GGG LVQP GGS LRL SCAA SGF TFRL
Y E M

TCCTGTGCAG CCTCCGGATT CACCTTTAGG
TTGTATGAGA
CTCCACGTCGACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAATCC
AACATACTCT

'AWVRQA PGKG LEW VSG IDIL GER TYY ADSV
K G =

TGGGTTCGAG GACATACTAC GCAGACTCCG, TGAAGGGCCG
ACCGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTcAC CCAGAGTCCC TAACTATAAA ACCCAAGCTC
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDM SKN TLY LQMN SLRAED TAVY YCA
KDL

TGCCGAGGAT ACCGCGGTAT ATTACTGTGC
GAAAGATCTG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACANA GACGTTTACT TGTCGGACGC ACGGCTCCTA
TGGCGCCATA TAATGACACG
CTTTCTAGAC
SWQGFDYWGQ GTLVTVSS
301 TCGTGGCAGG GTTTTGACTA CTGGGGTCAG GGAACCCTGG TCACCGTCTC GAGC (SEQ ID NO:
108) AGCACCGTCC CAAAACTGAT GACCCCAGTC CCTTGGGACC AGTGGCAGAG CTCG (SEQ ID NO: 109) DOM-13 SEQ ID NO: 15 EVQLLES GGG LVQP GGS LRL SCAASGF TFSY
Y S M =

TCCTGTGCAG CCTCCGGATT CACCTTTTCT
TATTATTCGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC

ATAATAAGCT
=YWVRQA PGKG LEWVSS ISPF GWG TYYADSV
KGE =

TTGGTTGGGG TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACATAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTAGC TAAAGCGGAA AACCAACCCC
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
'FTI SRDN SKD TLY LQMY SLRAED TAVY YCA
KYG
201 GTTCACCATC TCCCGCGACA ATTCCAAGGA CACGCTGTAT CTGCAAATGA ACAGcCTGCG
TGCCGAGGAC ACCGCGGTAT AfTACTGTGC
GAAATATGGG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCCT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTATACCC
=
ETSG PIS ENF DYWG QGT LVT VSS' GTCTCGAGC )SEQ ID NO: 110) CTCTGCTCAC CAGGCTAAAG ACTCTTAAAA CTGATGACCC CAGTCCCTTG GGACCAGTGG CAGAGCTCG
(SEC) ID NO: 111) DOM-14 SEQ ID NO: 16 EVQLLES GGG LVQP GGS SCAA SGF TEWS
Y D M

TCCTGTGCAG CCTCCGGATT CACCTTTTGG
TCTTATGATA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAAACC
AGAATACTAT
=TWVRQA PGKG LEWVSS INAS GDD TYYADSV
= G R

CGGGTGATGA TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACTGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTAGA TAATACCGAA GCCCACTACT
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SKr TLY LOMN SLR ;LED TAVY YCA
K W D
201 GTTCACCATC TCCCGCGACk ATTCCAAGAA CACGCTGTAT CTGCAAATGA ACAGCCTGCG
TGCCGAGGAT ACCGCGGTAT ATTACTGTGC
GARATGGGAT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACARA GACGTTTACT TGTCGGACGC ACGGCTCCTA
TGGCGCCATA TAATGACACG
CTTTACCCTA
RDFDYWG QGT LVTV SS
301 CGGGATTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO:
112) GCCCTAAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 113) DOM-15 SEQ ID NO: 17 EVO'LLES GG'G LVQP GGS LRL SCAA SGP TFEE
Y V M=

TCCTGTGCAG CCTCCGGATT CACCTTTGAG
GAGTATGTTA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACTC
CTCATACAAT
=SWVRQA PGKG LEWVST ISPI GLT TYYADSV
K G R

TTGGTCTGAC TACATACTAC GCAGACTCCG
TGAAGGGCCG

ACAGCACCCA GGCGGTCCGA GGTCCCTTCC CAGACCTCAC CCAGAGTTGA TAAAGAGGAT AACCAGACTG
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRD14 SEN TLY LQMN SLR AED TAVY YCA
E F P
201 GTTCACCATC TCCCGCGACA ATTCC.AAGAIL CACGCTGTAT CTGCAAATGA ACAGCCTGCG
TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GGAATTTCCT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CCTTAAAGGA
LIIL PDF DYW GQGT LVT VSS
301 TTGATTATTC TTCCTGATTT TGACTACTGG GGTCAGGGA.A. CCCTGGTCAC CGTCTCGAGC
(SEQ ID NO: 114) AACTAATAAG AAGGACTAAA ACTGATGACC CCAGTCCCTT GGGACCAGTG GCAGAGCTCG (SEQ ID NO:
115) DOM-16 SEQ ID NO: 18 EVOL LES GGG LVQP GGS LRL SCAA SGF'TFME
Y A M =

TCCTGTGCAG CCTCCGGATT CACCTTTATG
GAGTATGCGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG GACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGG_LAATAC
CTCATACGCT
=IWVRQA PGKG LEN VSI ISPL GLS TYY ADSV
K G R =

TTGGTTTGTC TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACTAAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTAA TAAAGAGGCG AACCAAACAG
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SEN TLY LQMN SLRAED TAVY YCA
KYQ
201 GTTCACCATC TCCCGCGACA ATTCCAAGAA CACGCTGTAT CfGCAAATGA ACAGCCTGCG
TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAATATCAG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG

OSSD SQY T'NF DYWG QGT LVT VSS

GTCTCGAGC (SEQ ID NO: 116) CTAAGCAGAC TATCAGTCAT ATGCTTAAAA CTGATGACCC CAGTCCCTTG GGACCAGTGG CAGAGCTCG
(SEQ ID NO: 117) DOM-17 SEQ ID NO: 19 EVQLLES GGG LVQP GGS LRL SCAA SGF TFED
Y G M =

TCCTGTGCAG CCTCCGGATT CACCTTTGAG
GATTATGGGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACTC
CTAATACCCT
=GWARQA PGKG LEWVSS IGPL GLW TYY ADSA
= G R

TGGGTCTTTG GACATACTAC GCAGACTCCG
CGAAGGGCCG
ACCCCACCCG GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTCA TAACCAGGAG ACCCAGAAAC
CTGTATGATG CGTCTGAGGC
GCTTCCCGGC
.FTI SRDN SRN TLY LQMN SLR AED TAVY YCA

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAATCTCCG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACARA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTAGAGGC
LEGL ITN FDY WGQG TLVTVSS

(SEQ ID NO: 118) GAACTCCCAA ACTAATGCTT AAAACTGATG ACCCCAGTCC CTTGGGACCA GTGGCAGAGC TCG (SEQ ID
NO: 119) DOM-18 SEQ ID NO: 20 EVQL LES GGG LVQP GGS LRL SCAA SGF TFPE
Y D M

TCCTGTGCAG CCTCCGGATT CACCTTTCCT
GAGTATGATA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAAGGA
CTCATACTAT
=TWVRQA PGKG LEWVSY ISSD GYS TYYADSV
K G R =

ATGGTTATTC TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACTGCACCCA GGCGGTCCGA GGTCCCTTCC CAGACCTCAC CCAGAGTATA TAATCAAGAC TACCAATAAG
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC

=FTI SRDN SKN TLY LQMN SLRAED TAVY YCA
K P H

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACCGCAT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGCGTA
GSPREFD YWG QGTLVTV SS
301 GGGAGTCCGC GGGAGTTTGA CTACTGGGGT CAGGGAACCC TGGTCACCGT CTCGAGC (SEQ ID
NO: 120) CCCTCAGGCG CCCTCAAACT GATGACCCCA GTCCCTTGGG ACCAGTGGCA GAGCTCG (SEQ ID NO:
121) DOM-19 SEQ ID NO: 21 EVQLLES GGG LVQP GGS LRL SCAA SGF PFPQ
YQGG =

TCCTGTGCAG CCTCCGGATT CCCCTTTCCG
CAGTATCAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA. GGGCAAAGGC
GTCATAGTCT
=AWVRQA PGKG LEW VSM ITSD GLD TYY ADSV
K G R =

ATGGTCTTGA TACATATTAC GCAGACTCCG
TGAAGGGCCG
ACCGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTAC TAATGAAGAC TACCAGAACT
ATGTATAATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SKN TLY LQMN SLRAED T.A.VY YCA
K P E

TGCCGAGGAT ACCGCGGTAT ATTACTGTGC
GAAACCTGAG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTA
TGGCGCCATA TAATGACACG
CTTTGGACTC
PLFD YWG QGT LVTV SS
301 CCTCTTTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC .(SEQ ID NO: 122) GGAGAAAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 123) =
DOM-20 SEQ ID NO: 22 EVQL LES GGG LVQP GGS LRL SCAA SGF TFSG
Y Q M =

GAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC TCCTGTGCAG
CCTCCGGATT CACCTTTTCG

GGTTATCAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAAAGC
CCAATAGTCT
=AWV AQA PGKG LEW VSG ISSE GLT TYY ADSV
K G R =

AGGGTCTTAC TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTCCA TAATCAAGCC TCCCAGAATG
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
.FTI SRDN SKN TLY LOMN SLRAED TAVY YCA
K L G

TGCCGAGGAC ACCGCGGTAT ANTACTGTGC
GAAATTGGGG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTAACCCC
RRFD YWG QGT LVTV SS
301 CGTAGGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEC, ID NO: 124) GCATCCAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 125) DOM-21 SEQ ID NO: 23 EVQL LES GGG LVQP GGS LRL SCAA SGF TFAN
Y E M =

TCCTGTGCAG CCTCCGGATT CACCTTTGCG
AATTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGC.AGAG AGGACACGTC
GGAGGCCTAA GTGGAAACGC
TTAATACTCT
=GWARQA PGKG LEW VSV ISSN GYS TYY ADSA
K G R =

GGGGTTATTC TACATACTAC GCAGACTCCG
CGAAGGGCCG
ACCCCACCCG GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTCAA TAAAGACTCA CCCCAATAAG
ATGTATGATG CGTCTGAGGC
GCTTCCCGGC
=FTI SRDN SKN TLY LQMN SLRAED TAVY YC
KLV

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACTTGTG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGAACAC

GGTQ YEF DYW GQGT LVT VSS
301 GGTGGGACTC AGTATGAGTT TGACTACTGG GGTCAGGGAA CCCTGGTCAC CGTCTCGAGC (SEQ
ID NO: 126) CCACCCTGAG TCATACTCAA ACTGATGACC CCAGTCCCTT GGGACCAGTG GCAGAGCTCG (SEQ ID NO:
127) DOM-22 SEQ ID NO: 24 EVQLLES GGG LVQP GGS LRL SCAA SGF TFPN
Y E M =

TCCTGTGCAG CCTCCGGATT CACCTTTCAT
AATTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAAGTA
TTAATACTCT
=SWVRQA PGKG LEWVSS ISSG GSS TYY ADSV
K G R =

GTGGTTCTTC TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACAGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTCA TAAAGAAGCC CACCAAGRAG
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SKN TLY LQMN SLRAED TAVY YCA
K P G

TGCCGAGGAG ACCGCGGTAT ATTACTGTGC
GAAACCGGGG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGCCCC
/KFD YWG QGT LVTV SS
301 GTTAAGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 128) CPATTCAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 129) DOM-23 SEQ ID NO: 25 EVQLLES GGG LVQP GGS LRL SCAA SGF TFGL
Y E M

TCCTGTGCAG CCTGCGGATT CACCTTTGGG
CTGTATGAGA
CTCCACGTCG ACAACCTCAG GCCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACCC
GACATACTCT
=TWVRQA PGKG LEW VSS ITGD GIS TYY ADSV
K G R

ATGGTATTTC GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACTGCACCcA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTCA TAATGCCCAC TACCATAAAG
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SRN TLY LQMN SLRAED TAVY YC
K AG

TGCCGAGGAc ACCGCGGTAT ATTACTGTGC
GLAAGCTGGG
CAAGTGGTAG AGGGCGCTGT TAAGGTCCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTCGACCC
REFDYWG QGT LVTV SS
301 AGGAAGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 130) TCCTTCAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 131) DOM-24 SEQ ID NO: 26 EVQL LES GGG LVQP GGS LRL SCAA SGF TFSN
Y Q M

TCCTGTGCAG CCTCCGGATT CACCTTTAGT
AATTATCAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAATCA
TTAATAGTCT
=AWVRQA PGEG LEWVSS ITSE GGS TYY ADSV
K G R

AGGGTGGTTC GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTCA TAATGATCAC TCCCACCAAG
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC

=FTI SRDN SKN TLY LOMN SLR AED TAVY YCA
K P G

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACCGGGT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGTGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGCCCA
KNFD YWG QGT LVTV SS
301 AAGAATTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 132) 1 0 TTCTTAAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 133) DOM-25 SEQ ID NO: 27 EVQLLES GGG LVQP GGS LRL SCAA SGF TFDN
YEN =

TCCTGTGCAG CCTCCGGATT CACCTTTGAT
AATTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACTA
TTAATACTCT
=TWVRQA PGKG LEWVST ITSQ GTS TYY ADSV
R G R

AGGGTACTAG TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACTGCACCCA GGCGGTCCGA. GGTCCCTTCC CAGATCTCAC CCAGAGTTGA TAATGCAGCG TCCCATGATC
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SRN TLY LQMN SLR AED TAVY YCA
R P D

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACCTGAT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGACTA
. 35 RSFD YWG QGT LVTV SS
301 CGTTCTTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 134) GCAAGAAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 135) DOM-26 SEQ ID NO: 28 EVQL LES'GGG LVQP GGS LRL SCAR SGF TFRS
Y E M

GAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC TCCTGTGCAG
CCTCCGGATT CACCTTTCGT
AETTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAk GTGGAAAGCA
TCAATACTCT
=TWV RQA PGKG LEW VSS ITSD GGT TYY ADSV
K G R

ATGGTGGTAC TACATACTAC GCAGACTCCG
TGAAGGGCCG

TACCACCATG ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SKN TLY LQMN SLRAED TAVY YCA
K P D

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
PAAACCTGAT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGACTA
KTFD YWG QGT LVTV SS
301 AAGACGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 136) TTCTGCAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 137) DOM-27 SEQ ID NO: 29 EVQL LES GGG LVQP GGS LRL SCAA SGF TFNL
Y E M

TCCTGTGCAG CCTCCGGATT CACCTTTAAT
TTGTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGMAATTA
AACATACTCT
=TWVRQAPGKG LEWVSS ITSD GVS TYYADSV
K G R

ATGGTGTTTC TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACTGAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTAGA TAATGATCAC TACCACAAAG
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
.FTI SRDN SKN TLY LQMN SLRAED TAVY YCA
= P D

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACCGGAT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGCCTA

SPFDYWG QGT LVTV SS
301 TCTCCGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 138) AGAGGCAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 139) DOM-28 SEQ ID NO: 30 EVQLLES GGG LVQP GGS LRL SCAA SGF TFGH
Y D M =

TCCTGTGCAG CCTCCGGATT CACCTTTGGG
CATTATGATA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACCC
GTAATACTAT
=AWVRQA PGKG LEWVST ISDN GNG TYYADSV
K G R

ANGGTAATGG GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTGA TAATCACTAT TACCATTACC
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
.FTI SRDN SKN TLY LOMN SLRAED TAVY YCA
K P G

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACCGGGG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGCCCC
RDFD YWG QGT LVTV SS
301 CGTGATTTTG AfTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 140) GCACTAAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 141) DOM-29 SEQ ID NO: 31 EVQLLES GGG LVQP GGSLRL SCAA SGF TFGR
Y Q M=

TCCTGTGCAG CCTCCGGATT CACCTTTGGT
CGTTATCAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG aACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACCA
GCAATAGTCT
=AWVRQA PGKG LEW VSS ISSD GGG TYY ADSV
K G R

ATGGTGGGGG GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTAGA TAAAGAAGAC TACCACCCCC
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
.FTI SRDN SEP TLY LQMN SLR AED TAVY YCA
= P G
201 GTTCACCATC TCCCGCGACA P_TTCCP_iNAA CACGCTGTAT CTGCAAATGA ACAGCCTGCG
TGCCGAGGAT ACCGCGGTAT ATTACTGTGC
cA7,ACCTGGG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTA
TGGCGCCATA TAATGACACG
CTTTGGACCC
RAFDYWG QGT LVTV SS
301 CGGGCGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 142) GCCCGCAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 143) DOM-30 SEQ ID NO: 32 EVQLLES GGG LVQP GGS LRL SCAA SGF TEAR
Y Q M =

TCCTGTGCAG CCTCCGGATT CACCTTTGCG
AGGTATCAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACGC
TCCATAGTCT
=AWVRQA PGKG LEWVST ISDD GDS TYY ADSV
K G R =

ATGGTGATTC GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGAACCCA GGCGGTCCGA GGTCCCTTCC CAGACCTCAC CCAGAGTTGA TAAAGACTAC TACCACTAAG
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC

.FTI SRDN SKN TLY LQMN SLRAED TAVY YCA
K L D

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACTGGAT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGACCTA
KLFDYWG QGT LVTV SS
301 AAGTTGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 144) TTCAACAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 145) DOM-31 SEQ ID NO: 33 EVQLLES GGG LVQP GGS LRL SCAA SGF TFEE
YQN

TCCTGTGCAG CCTCCGGATT CACCTTTGAG
GAGTATCAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGPAAACTC
CTCATAGTCT
=AWVRQA PGKG LEW VST ISDD GSS TYY ADSV
K G R

ATGGTTCTTC GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTGC TAAAGCCTAC TACCAAGAAG
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
"FTI SRDN SKN TLY LQMN SLR AED TAVY YCA
K P D

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
PI,AACCTGAT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGACTA
LYFD YWG QGT LVTV SS
301 CTTTATTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 146) GAAATAAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 147) DOM-32 SEQ ID NO: 34 EVQLIES GGG LVQP GGS LRL SCAA SGF TFEV
Y Q M

TCCTGTGCAG CCTCCGGATT CACCTTTGAG
GTGTATCAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACTC
CACATAGTCT
=GWV RQA PGKG LEW VSF IVPG GDL TYY ADSV
K G R =

GGGGTGATTT GACATACTAC GCAGACTCCG
=
TGAAGGGCCG
ACCCAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTPAA TAACACGGAC CCCCACTAAA
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SKN TLY LOMN SLRAED TAVY YCA
E T W

TGCCGAGGAC ACCGCGGTAg ATTACTGTGC
GGAAACGTGG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CCTTTGCACC
PEFD YWG QGT LVTV SS
301 CCGGAGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 148) GGCCTCAXAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 149) DOM-33 SEQ ID NO: 35 DIQMTQS PSS LSAS VGDRVT ITCR ASQ TIGE
S L H =
GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGAGA CCGTGTCACC ATCACTTGCC
GGGCAAGTCA GACGATTGGG

GAGAGTTTAC
CTGTAGGTCT ACTGGGTCAG AGGTAGGAGG GACAGACGTA GACATCCTCT GGCACAGTGG TAGTGAACGG
CCCGTTCAGT CTGCTAACCC
CTCTCAAATG
=WYQ QKP GKAP ALL IYFASLL QSG VPS RFSG
S G S =
101 ATTGGTACCA GCAPAAACCA GGps=AAGCCC CTAGGCTCCT GATCTATTTT GCTTCCCTGT
TGCAAAGTGG GGTCCCATCG CGTTTCAGTG
GCAGTGGATC
TAACCATGGT CGTCTTTGGT CCCTTTCGGG GATCCGAGGA CTAGATAAAA CGAAGGGACA ACGTTTCACC
CCAGGGTAGC GCAAAGTCAC
CGTCACCTAG
=GTD FTLT ISS LQP EDFA TYY CQQ HHML PST
FGQ

CTGTCAACAG CATCATATGC TTCCTTCTAC
GTTCGGCCAA
ACCCTGTCTAIAATGAGAGT GGTAGTCGTC AGACGTTGGA CTTCTAAAAC GATGCATGAT GACAGTTGTC
GTAGTATACG AAGGAAGATG
CAAGCCGGTT

GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG (SEQ ID NO: 150) CCCTGGTTCC ACCTTTAGTT TGCC (SEQ ID NO: 151) DOM-34 SEQ ID NO: 36 DIQM TQS PS.S LSAS VGD RVT ITCR ASQ WIGD
S L S =
i GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGAGA CCGTGTCACC
ATCACTTGCC GGGCAAGTCA GTGGATTGGT
GATAGTTTAT
CTGTAGGTCT ACTGGGTCAG AGGTAGGAGG GACAGACGTA GAGATCCTCT GGCACAGTGG TAGTGAACGG
CCCGTTCAGT CACCTAACCA
CTA_TCAAATA
=WYQ QRP GRAP KLL IYFASYL QSG VPT RFSG
S G S-TGCAAAGTGG GGTCCCAACA CGTTTCAGTG
GCAGTGGATC
GAACCATGGT CGTCTTTGGT CCCTTTCGGG GATTCGAGGA CTAGATAzAA CGAAGGATAA ACGTTTCACC
CCAGGGTTGT GCAAAGTCAC
CGTCACCTAG
=GTD FTLT ISS LQP EDFA TYY CQQ YFEN PVT
F G
201 TGGGACAGAT TTCACTCTCA. CCATCAGCAG TCTGCAACCT GAAGATTTTG CTACGTACTA
CTGTCAACAG TATTTTGAGA ATCCTGTTAC
GTTCGGCCAA
ACCCTGTCTA AAGTGAGAGT GGTAGTCGTC AGACGTTGGA CTTCTAAAAC GATGCATGAT GACAGTTGTC
ATAAAACTCT TAGGACAATG
CAAGCCGGTT
GTKV GIKR
301 GGGACCAAGG TGGGAATCAA ACGG (SEQ ID NO: 152) CCCTGGTTCC ACCCTTAGTT TGCC (SEQ ID NO: 153) DOM-35 SEQ ID NO: 37 DIQNTQS PSS LSAS VGDRVT ITCRASQ FIGD
S L S =
GACATCCAAA TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGAGA CCGTGTCACC ATCACTTGCC
GGGCAAGTCA GTTTATTGGT
GATTCTTTAT
CTGTAGGTTT ACTGGGTCAG AGGTAGGAGG GACAGACGTA GACATCCTCT GGCACAGTGG TAGTGAACGG
CCCGTTCAGT CAAATAACCA
CTAAGAAATA
=WYQQKP GRAP ELL IYF SSIL QSG VPS RFSG
S G S =

TGCAAAGTGG GGTCCCATCA CGTTTCAGTG
GCAGTGGATC
GAACCATGGT CGTCTTTGGT CCCTTTCGGG GATTCGAGGA CTAGATAAAA AGAAGGTAAA ACGTTTCACC
CCAGGGTAGT GCAAAGTCAC
CGTCACCTAG
=GTD FTLT ISS LQP EDFA TYY CQQ YMDI PIT

CTGTCAACAG TATATGGATA TTCCTATTAC
GTTCGGCCAA
ACCCTGTCTA AAGTGAGAGT GGTAGTCGTC AGACGTTGGA CTTCTAAAAC GATGCATGAT GACAGTTGTC
ATATACCTAT AAGGATAATG
CAAGCCGGTT
GTEVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG (SEQ ID NO: 154) CCCTGGTTCC ACCTTTAGTT TGCC (SEQ ID NO: 155) DOM-3 6 SEQ ID NO: 38 DIQMTQS PSS LSAS VGDRVT ITCR ASQ DIDH
N L E =

ATCACTTGCC GGGCAAGTCA GGATATTGAT
CATAATTTAG
CTGTAGGTCT ACTGGGTCAG AGGTAGGAGG GACAGACGTA GACATCCTCT GGCACAGTGG TAGTGAACGG
CCCGTTCAGT CCTATAACTA
GTATTAAATC
=WYQQRP GRAF ELL IYD SSML QSG VPS RFSG
S G S =

TGCAAAGTGG GGTCCCATCA CGTTTCAGTG
GCAGTGGATC
TCACCATAGT CGTCTTTGGT CCCTTTCGGG GATTCGAGGA CTAGATACTA TCAAGGTACA ACGTTTCACC
CCAGGGTAGT GCAAAGTCAC
CGTCACCTAG
.GTDFTLT ISS LQP EDFATYY CQQ YHSI PVT
F G

CTGTCAACAG TATCATTCTA TTCCTGTTAL
GTTCGGCCAA
ACCCTGTCTA AAGTGAGAGT GGTAGTCGTC AGACGTTGGA CTTCTAAAAC GATGCATGAT GACAGTTGTC
ATAGTAAGAN AAGGACAATG
CAAGCCGGTT
GTEVEIER
301 GGGACCAAGG TGGAKATCAA ACGG (SEQ ID NO: 155) CCCTGGTTCC ACCTTTAGTT TGCC (SEQ ID NO: 157) DOM-37 SEQ ID NO: 39 N L E =

ATCACTTGCC GGGCAAGTCA GCAGATTGAG
ACGAATTTAG
CTGTAGGTCTACTGGGTCAG AGGTAGGAGG GACAGACGTA GACANCCTCT GGCACAGTGG TAGTGAACGG
CCCGTTCAGT CGTCTAACTC
TGCTTAAATC
=WYQQKP GEAP ELI IND GSWL QPG VPS RFSG
S G S =

TGCAAAGTGG GGTCCCATCA CGTTTCAGTG
GCAGTGGATC
TCACCATAGT CGTCTTTGGT CCCTTTCGGG GATTCGAGGA CTAGATACTA CCAAGGACCA ACGTTTCACC
CCAGGGTAGT GCAAAGTCAC
CGTCACCTAG
=GTD FTLT ISE LQP EDFA TYY CQQ YHSL PAT
FGQ

CTGTCAACAG TATCATAGTT TGCCTGCTAC
GTTCGGCCAA
ACCCTGTCTA AAGTGAGAGT GGTAGTCGTC AGACGTTGGA CTTCTAAAAC GATGCATGAT GACAGTTGTC
ATAGTATCAA ACGGACGATG
CAAGCCGGTT
GTEVEIER
301 GGGACCAAGG TGGAAATCAA ACGG (SEC) ID NO: 158) CCCTGGTTCC ACCTTTAGTT TGCC (SEQ ID NO: 159) DOM-38 SEQ ID NO: 40 D IQMTQS PSS LEAS VGDRVT ITCRASQ DIGN
NLE =

ATCACTTGCC GGGCAAGTCA GGATATTGGT
AATAATTTAG
=

CTGTAGGTCT ACTGGGTCAG AGGTAGGAGG GACAGACGTA GACATCCTCT GGCACAGTGG TAGTGAACGG
CCCGTTCAGT CCTATAACCA
TTATTAAATC
=WYQQEP GKAP ALL IYH GSWL (1)SG VPS RFSG
S G S

TGCAAAGTGG GGTCCCATCG CGTTTCAGTG
GCAGTGGATC
TCACCANGGT CGTCTTTGGT CCCTTTCGGG GATCCGAGGA CTAGATAGTA CCCAGGACCA ACGTTTCACC
CCAGGGTAGC GCAAAGTCAC
CGTCACCTAG
.GTDFTLT ISS LOP EDFA TYY CQO YDFN PTT
F G Q

CTGTCAACAG TATGATTTTA ATCCTACTAC
GTTCGGCCAA
ACCCTGTCTA AAGTGAGAAT GGTAGTCGTC AGACGTTGGA CTTCTAAAAC GATGCATGAT GACAGTTGTC
ATACTAAAAT TAGGATGATG
CAAGCCGGTT
GTEVEIER
301 GGGACCAAGG TGGAAATCAA ACGG (SEO ID NO: 160) CCCTGGTTCC ACCTTTAGTT TGCC (SEQ ID NO: 161) DOM-39 SEQ ID NO: 41 DIQMTQS PSS LSAS VGD CVT ITCE ASQ NIDG
L L W =

ATCAETTGCC GGGCAAGTCA GAATATTGAT
GGTCTGTTAT
CTGTAGGTCT ACTGGGTCAG AGGTAGGAGG GACAGACGTA GACATCCTCT GACACAGTGG TAGTGAACGG
CCCGTTCAGT CTTATAACTA
CCAGACAATA
=WYQQEFGEAP ELL IYA GSGL QSG VPS RFSG
= G S =

TGCAAAGTGG GGTCCCATCA CGTTTaAGTG
GCAGTGGATC
CCACCATAGT CGTCTTTGGT CCCTTTCGGG GATTCGAGGA CTAGATAGGC CCCAGGCCCA ACGTTTCACC
CCAGGGTAGT GCAAAGTCAC
CGTCACCTAG
=GTDFTLT ISS LQP EDFA TYY COQ EAFE PFT
PGQ

CTGTCAACAG AAGGCTTTTG AGCCTTTTAC
GTTCGGCCAA
ACCCTGTCTA AAGTGAGAGT GGTAGTCGTC AGACGTTGGA CTTCTAAAAC GATGCATGAT GACAGTTGTC
TTCCGAAAAC TCGGAAAATG
CAAGCCGGTT
GTKVEIRR

301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO: 162) CCCTGGTTCC ACCTTTAGTT TGCC (SEQ ID NO: 163) DOM-40 SEQ ID NO: 42 EVQL LES GGG LVQF GGS LRL SCAA SGF TFKA
Y D M

TCCTGTGCAG CCTCCGGATT CACCTTTAAG
GCGTATGATA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAATTC
CGCATACTAT
=G WV RQA PGKG LEWVSQ IGRD GSF TYY ADSV
G R =

ATGGTTCTTT TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCCAACCCA. GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTGTC TAACCCTCCC TACCAAGAAA_ ATGTA.TGATG CGTCTGAGGC
ACTTCCCGGC
.FTI SRDN SKR TLY LOMN SLR AED TAVY YCA
KPK

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAhCCTCGT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGAGCA
RYAI FTF DRG (2GTL VTV Ss 301 CGGTATGCTA TTTTTACTTT TGATCGGGGT CAGGGAACCC TGGTCACCGT CTCGAGC (SEQ ID
NO: 164) GCCATACGAT AAAAATGAAAACTAGCCCCA GTCCCTTGGG ACCAGTGGCA GAGCTCG (SEQ ID NO: 165) DOM-41 SEQ ID NO: 43 EVQLLES GGG LVQP GGS LRL SCAA SGF TFFE
Y E M =
GAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC TCCTGTGCAG
CCTCCGGATT CACCTTTTTT
GAGTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAAAAA
CTCATACTCT
=TWV RQA PGKG LEWVSS IAND GST TYY ADSV
K G R

ATGGTTCGAC TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACTGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTAGA TAACGCTTAC TACCAAGCTG
ANGTATGATG CGTCTGAGGC
ACTTCCCGGC
,FTI SRDN SKN TLY LQMN SLR AED TAVY YCA
K P D

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACCTGAT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGACTA
APED YWG QGT LVTV SS
301 CGGCAGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 166) GCCGTCAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 167) =
DOM-42 SEQ ID NO: 44 EVQLLES GGG LVQPGGS LRL SCAR SGF TFGP
Y E M
GAGGTGCAGC TOTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC TCCTGTGCAG
CCTCCGGATT CACCTTTGGT

CCGTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACCA
GGCATACTCT
=TWV RQA PGKG LEN VSS IVGD GLD TYY ADSV
K G R =

ATGGTCTGGA TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACTGAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTAGC TAACAACCAC TACCAGACCT
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC

=PTI SRDN SKN TIY LONE SLRAED TAVY ECA
KPD

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACCGGAT
CAAGTGGTAG AGGGGGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGAGACG
CTTTGGCCTA
RVFD YWG QGT LVTV SS
301 CGGGTTTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO:
168) GCCCAA.AAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 169) DOM-43 SEQ ID NO: 45 EVQLIES GGG IVQP GGS LRL SCAA SGF TEAS
Y E N

TCCTGTGCAG CCTCCGGATT CACCTTTGCT
TCTTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACGA
AGAATACTCT
=AWVRQA PGKG LEW VSS IGSD GGP TYY ADSV
K G R

ATGGTGGGCC GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTAGC TAACCATCAC TACCACCCGG
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
.FTI SRDN SKN TLY LQMN SLRAED SAVY YCA
KPD

TGCCGAGGAC TCCGCGGTAT ATTACTGTGC
GAAACCTGAT
cAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
AGGCGCCATA TAATGACACG
CTTTGGACTA
RAFDYWG QGT LVTV SS
301 AGGGCTTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO:
170) TCCCGAAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 171) DOM-44 SEQ ID NO: 46 EVQLLES GGG LVQP GGS LRL SCAA SGF TFTS
Y E M =

TCCTGTGCAG CCTCCGGATT CACCTTTACG
TCTTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAATGC
AGAATACTCT
=GWVRQA PGKG LEW VSS IEPT GIT TYY ADSV
= G R =

CTGGTATTAC GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCCCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTAGA TAACTCGGAT GACCATAATG
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SKN TLY LQMN SLRAED TAVY YCA
KPR

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACCTCAT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGAGTA
FTEL GFD YWG QGTL VTV SS
301 TTTACTGAGC TTGGTTTTGA CTACTGGGGT CAGGGAACCC TGGTCACCGT CTCGAGC (SEQ ID
NO: 172) AAATGACTCG AACCAAAACT GATGACCCCA GTCCCTTGGG ACCAGTGGCA GAGCTCG (SEQ ID NO:
173) DOM-45 SEQ ID NO: 47 EVQLLES GGG LVQP GGS LRL SCAA SGF TFGN
Y A M

TCCTGTGCAG CCTCCGGATT CACCTTTGGT
AATTATGCGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTG(ZAAACCA
TTANTACGCT
=AWVRQA PGKG LEW VSK IGAQ GLH TYY AGSV
K G R =

AGGGTCTTCA TACATACTAC GCAGGCTCCG
TGAAGGGCCG
ACCGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTTC TAACCCCGCG TCCCAGAAGT
ATGTATGATG CGTCCGAGGC
ACTTCCCGGC
=FTI SRDN SEN TLY LQMN SLR AED TAVY YCA
KQT

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACAGACG

CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGTCTGC
TMDY ERE' DYW GOGT LVT VS5 301 ACGATGGATT ATGAGRGGTT TGACTACTGG GGTCAGGGAA CCCTGGTCAC CGTCTCGAGC (SEQ
ID NO: 174) TGCTACCTAA TACTCTCCAA. ACTGATGACC CCAGTCCCTT GGGACCAGTG GCAGAGCTCG (SEQ ID NO:
175) DOM-46 SEQ ID NO: 48 EVQLLES GGG LVQP GGS LRL SCAA SGF TFEL
Y A M =

TCCTGTGCAG CCTCCGGATT CACCTTTGAG
TTGTATGCTA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACTC
AACATACGAT
=AWVRQA FGKG LEW VSG IGAV GET TYY ADSV
K G R =

TGGGTGAGAC TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTCCA TAACCACGAC ACCCACTCTG
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SKN TLY LOMN SLR AED TAVY YCA
K EA

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAAGAGGCT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTCTCCGA
NNLS DNL VFD YWGQ GTL VTV SS

TCGAGC (SEC) ID NO, 176) TTATTAGAAA GACTATTAGA ACACAAACTG ATGACCCCAG TCCCTTGGGA CCAGTGGCAG AGCTCG (SEQ
ID NO: 177) DOM-47 SEQ ID NO: 49 DIQMTOS PSS LSASVGDRVT ITCR ASQ WIGD
S L S =
GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGAGA CCGTGTCACC ATCACTTGCC
GGGCAAGTCA GTGGATTGGG

GATTCGTTAA
CTGTAGGTCT ACTGGGTCAG AGGTAGGAGG GACAGACGTA GACATCCTCT GGCACAGTGG TAGTGAACGG
CCCGTTCAGT CACCTAACCC
CTAAGCAATT
=WYQQKP GKAP KLL IYF GSYL QSG VPS RFSG
S G S =

TGrAAAGTGG GGTCCCATCA CGTTTCAGTG
GCAGTGGATC
CAACCATGGT CGTCTTTGGT CCCTTTCGGG GATTCGAGGA CTAGATAAAA CCAAGGATAA ACGTTTCACC
CCAGGGTAGT GCAAAGTCAC
CGTCACCTAG

.GTD FTLT ISS IQP EDFATYY CQQ YLHT PST
FGO

CTGTCAACAG TATTTGCATA CTCCTTCGAG
GTTCGGCCAA
ACCCTGTCTA AAGTGAGAGT GGTAGTCGTC AGACGTTGGA CTTCTAAAAC GATGCATGAT GACAGTTGTC
ATAAACGTAT GAGGAAGCTG
CAAGCCGGTT
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG (SEQ ID NO: 178) CCCTGGTTCC ACCTTTAGTT TGCC (SEQ ID NO: 179) DOM-48 SEQ ID NO: 50 DIQMTQS PSS LSAS VGDRVT ITCR ASQ WIGD
SLS =
1 GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGAGA CCGTGTC.A.CC
ATCACTTGCC GGGCAAGTCA GTGGATTGGG
GATTCGTTAA
CTGTAGGTCT ACTGGGTCAG AGGTAGGAGG GACAGACGTA GACATCCTCT GGCACAGTGG TAGTGAACGG
CCCGTTCAGT CACCTAACCC
CTAAGCAATT
=WYQ QKP GKAP (<LL IYF GSYL QNG VPS RFSG
S G =

TGCAAAP_TGG GGTCCCATCA CGTTTCAGTG
GCAGTGGATC
CAACCATGGT CGTCTTTGGT CCCTTTCGGG GATTCGAGGA CTAGATALAA CCAAGGATAA ACGTTTTACC
CCAGGGTAGT GCAAAGTCAC
CGTCACCTAG
.GTD FTLT ISS LQP EDFA TYY CQQ YMIT PTT
FGQ

CTGTCAACAG TATATGATTA CTCCTACTAC
GTTCGGCCAA
ACCCTGTCTA AAGTGAGAGT GGTAGTCGTC AGACGTTGGA CTTCTAW,AC GATGCATGAT GACAGTTGTC
ATATACTAAT GAGGATGATG
CAAGCCGGTT
GTKVEIER
301 GGGACCAAGG TGGAAATCAA ACGG (SEQ ID NO: 180) CCCTGGTTCC ACCTTTAGTT TGCC (SEQ ID NO: 161) DOM-49 SEQ ID NO: 51 DVQMTQS PSS LSAs VGDRVT ITCR ASQ WIGD
S L S =

ATCACTTGCC GGGCAAGTCA GTGGATTGGG
GATTCGTTAA
CTGCAGGTCT ACTGGGTCAG AGGTAGGAGG GACAGACGTA GACATCCTCT GGCACAGTGG TAGTGAACGG
CCCGTTCAGT CACCTAACCC
CTAAGCAATT
=NYQQKP GKAP ELL IYF GSYL QSG VPS RFSG
S G S =

TGCAAAGTGG GGTCCCATCA CGTTTCAGTG
GCAGTGGATC
CAACCATGGT CGTCTTTGGT CCCTTTCGGG GATTCGAGGA CTAGATAAAA CCAAGGATAA ACGTTTCACC
CCAGGGTAGT GCAAAGTCAC
CGTCACCTAG
=GTD FTLT ISS LQP EDFATYY CQQ YMSA PST
FGQ

CTGTCAACAG TATATGAGTG CTCCTTCTAC
GTTCGGCCAA
ACCCTGTCTA AAGTGAGAGT GGTAGTCGTC AGACGTTGGA CTTCTAAAAC GATGCATGAT GACAGTTGTC
ATATACTCAC GAGGAAGATG
CAAGCCGGTT
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG (SEQ ID NO: 182) CCCTGGTTCC ACCTTTAGTT TGCC ISEQ ID NO: 183) DOM-50 SEQ ID NO: 52 DIQNTQS PSS LSAS VGDRVT ITCR ASQ WIGD
S L S =
i GACATCCAGATGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGAGA CCGTGTCAcc ATCACTTGCC GGGCAAGTCA GTGGATTGGG
GATTCGTTAA
CTGTAGGTCT ACTGGGTCAG AGGTAGGAGG GACAGACGTA GACATCCTCT GGCACAGTGG TAGTGAACGG
CCCGTTCAGT CACCTAACCC
CTAAGCAATT
=WYQQKP GKAP KLL IYF GSYL QSG VPS RFSG
S G S =

TGCAAAGTGG GGTCCCATCA CGTTTCAGTG
GCAGTGGATC
CAACCATGGT CGTCTTTGGT CCCTTTCGGG GATTCGAGGA CTAGATAAAA CCAAGGATAA ACGTTTCACC
CCAGGGTAGT GCAAAGTCAC
CGTCACCTAG
=G TD PTLT ISS LQP EDSA TYY COQ YQYV PST
FGQ

CTGTCAACAG TATCAGTATG TTCCTTCTAC
GTTCGGCCAA

ACCCTGTCTA AAGTGAGAGT GGTAGTCGTC AGACGTTGGA CTTCTAAGAC GATGCATGAT GACAGTTGTC
ANAGTCATAC AAGGAAGANG
CAAGCCGGTT

301 GGGACCAAGG TGGAAATCAA AEAG (SEQ ID NO: 184) CCCTGGTTCC ACCTTTAGTT TGTC (SEG ID NO: 185) DOM-51 SEQ ID NO: 53 DIQNTQS PSS LSAS VGD RVT ITCR ASQ PIVD
E L. D =
GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGAGA CCGTGTCACC ATCACTTGCC
GGGCAAGTCA GCCTATTGTT
GATGAGTTAG
CTGTAGGTCT ACTGGGTCAG AGGTAGGAGG GACAGACGTA GACATCCTCT GGCACAGTGG TAGTGAACGG
CCCGTTCAGT CGGATAACAA.
CTACTCAATC
=WYQQKP GKAP KLL IYA ASIL QSG VPS RFSG
S G S =

TGCAAAGTGG GGTCCCATCA CGTTTCAGTG
GCAGTGGATC
TAACCATGGT CGTCTTTGGT CCCTTTCGGG GATTCGAGGA CTAGATACGA CGCAGGTAAA ACGTTTCACC
CCAGGGTAGT GCAAAGTCAC
CGTCACCTAG
=GTDFTLT ISS LQP EDFA TYY CHQ WSTY PTT
FGQ

CTGTCANCAG TGGTCTACTT ATCCTACGAZ
GTTCGGCCAA
ACCCTGTCTA AAGTGAGAGT GGTAGTCGTC AGACGTTGGA CTTCTAAAAC GATGCATGAT GACAGTAGTC
ACCAGATGAA TAGGATGCTG
CAAGCCGGTT
GTKVEIKR
301 GGGACCAAGG TGGAAATTAA ACGG (SEQ ID NO: 186) CCCTGGTTCC ACCTTTAATT TGCC (SEQ ID NO: 187) DOM-52 SEQ ID NO: 54 DIQMTQS PSS LSAS VGD RVT ITCRASQ DIGS
A L R

ATCACTTGCC GGGCAAGTCA GGATATTGGG
TCTGCGTTAA
CTGTAGGTCT ACTGGGTCAG AGGTAGGAGG GACAGACGTA GACATCCACT GGCACAGTGG TAGTGAACGG
CCCGTTCAGT CCTATAACCC
AGACGCAATT
=WYQQKP GKAP KLL IYL GSDL QSG VPS RFSG
S G S

TGCAAAGTGG GGTCCCATCA CGTTTCAGTG
GCAGTGGATC
CCACCATAGT CGTCTTTGGT CCCTTTCGGG GATTCGAGGA CTAGATAAAC CCAAGGCTAA ACGTTTCACC
CCAGGGTAGT GCAAAGTCAC
CGTCACCTAG

=GTDFTLT ISS LQP EDFA TYY CQQ TQYF PTT
FGQ

CTGTC_AACAG ACGCAGTATT TTCCTACGAC
GTTCGGCCAA
ACCCTGTCTA AAGTGAGAGT GGTAGTCGTC AGACGTTGGA CTTCTAAAAC GATGCATGAT GACAGTTGTC
TGCGTCATAA AAGGATGCTG
CAAGCCGGTT
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG (SEQ ID NO: 188) CCCTGGTTCC ACCTTTAGTT TGCC (SEQ ID NO: 189) DOM-53 SEQ ID NO: 55 DIQMTQS PSS LSAS VGD RVT ITCR ASQAIYG
G L R =
GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGAGA CCGTGTCACC ATCACTTGCC
GGGCAAGTCA GGCGATTTAT

GGGGGGTTAL
CTGTAGGTCT ACTGGGTCAG AGGTAGGAGG GACAGACGTA GACATCCTCT GGCACAGTGG TAGTGAACGG
CCCGTTCAGT CCGCTAAATA
CCCCCCAATG
=WYQQKP GKAP ELL IYG ESML QSG VPS RFSG
S G S =

TGCAAAGTGG GGTCCCATCA CGTTTCAGTG
GCAGTGGATC
CCACCATGGT CGTCTTTGGT CCCTTTCGGG GATTCGAGGA CTAGATACCC CTCAGGTACA ACGTTTCACC
CCAGGGTAGT GCAAAGTCAC
CGTCACCTAG
=GTD FTLT ISS LHP EDFA TYY CQQ VYHK PFT
FGQ

CTGTCAACAG GTTTATCATA AGCCTTTTAC
GTTCGGCCAA
ACCCTGTCTA AAGTGAGAGT GGTAGTCGTC AGACGTAGGA CTTCTAAAAC GATGCATGAT GACAGTTGTC
CAAATAGTAT TCGGAAAATG
CAAGCCGGTT
GTKVEIKR
301 GGGACCAAGG TGC04ATCAA ACGG (SEQ ID NO: 190) CCCTGGTTCC ACCTTTAGTT TGCC (SEQ ID NO: 191) DOM-54 SEQ ID NO: 56 EVQLLES GGG LVOP GGS LRL SCAA SGE TETA
Y R M =

TCCTGTGCAG CCTCCGGATT CACCTTTACG
GCGTATAGGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAATGC
CGCATATCCT
=AWVRQA PGKG LEWVSW ISPS GSG TYY ADSV
K G R =

CTGGTTCGGG GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTACC TAAAGCGGAA GACCAAGCCC
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
=PTI SRDN SKI( TLY LQMN SLR AED TAVY YCA
= T L

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAAACTTTG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTTGAAAC
TDSP SGE YEE DYWG QGT LVT VSS

GTCTCGAGC (SEQ ID NO: 192) TGCCTAAGCG GCAGCCCCGT AATACTCAAA CTGATGACCC CAGTCCCTTG GGACCAGTGG CAGAGCTCG
(SEQ ID NO: 193) DOM-55 SEQ ID NO: 57 EVQLLES GGG LVQP GGS LRL SCAA SGF TEAR
Y E M =

TCCTGTGCAG CCTCCGGATT CACCTTTGCG
CGGTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACGC
GCCATACTCT
=GWVRQA PGKG LEWVSR ITAQ GLG TYY ADSV
= G R =

AGGGTCTTGG GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCCCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTGCC TAATGACGAG TCCCAGAACC
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC

KYL

ACAGCCTGCG TGCCGAGGAT ACCGCGGTAT ATTACTGTGC
GAAATATCTT

CAAGTGGTAG AGGGCGCTGT TGAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTA
TGGCGCCATA TAATGACACG
CTTTATAGAA.
TDFS SGH DEE' DYNG OGT LVT VSS

GTCTCGAGC (5E4 ID NO: 194) TGACTAAAAT CATCACCCGT AGTCCTCAAA CTGATGACCC CAGTCCCTTG GGACCAGTGG CAGAGCTCG
(SE4 ID NO: 195) DOM-56 SEQ ID NO: 58 EVQL LES GGG LVQP GGS LRL SCAA SGF TFND
Y T M

TCCTGTGCAG CCTCCGGATT CACCTTTAAT
GATTATACTA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAATTA
CTAATATGAT
=GWVRQA PGRGLEWVSW /HGT GGQ TYYADSV
M G R

CTGGTGGTCP. GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCCAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTACC TAAGTACCCT GACCACCAGT
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
.FTI SRDN SKN TLY LQMN SLR AED TAVY YCA
KAL

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAAGCTTTG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG

ADRS GGV VEF DYWG QGT LVT VSS

GTCTCGAGC (SEQ ID NO: 196) CGACTATCCT CACCCCCCCA ACAACTCAAA CTGATGACCC CAGTCCCTTG GGACCAGTGG CAGAGCTCG
(SEQ ID NO: /97) DOM-57 SEQ ID NO: 59 EVQLLES GGGLVQP GGS LRL SCAA SGF TFSE
Y D M

TCCTGTGCAG CCTCCGGATT CACCTTTTCT
GAGTATGATA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAAAGA
CTCATACTAT
=YWVRQA PGKG LEW VSW IDTD GGD TYY ADSV
K G R =

ATTGATACTG ATGGTGGGGA TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACATAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTACC TAACTATGAC TACCACCCCT
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC

=FTI SRDN SEN TLY LQMN SLR AED TAVY YCA
K P G

TGCCGAGGAC ACCGCGGTAN ATTACTGTGC
(1AAkCCTGGT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGACCA
LEFD YWG QGT LVTV SS
301 CTGAAGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 198) GACTTrAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG )SEQ ID NO: 199) DOM-58 SEQ ID NO: 60 EVQLLES GGG LVQP GGS LRL SCAA SGF TFEV
YTM =

TCCTGTGCAG CCTCCGGATT CACCTTTGAG
GTTTAIACTA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGPAAACTC
CAAATATGAT
=AWVRQA PGKG LEW VST IDES GRD TYY ADSV
K G R =

CTGGTCGTGA TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGCACCCA GGCGGTCCGA GGTCCCTTCC CAGACCTCAC CCAGAGTTGC TAACTACTCA GACCAGCACT
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SKN TLY LQMN SLR AED TAVY YCA
K P G
201 GTTCACCATC TCCCGCGACA. ATTCCAAGAA CACGCTGTAT CTGCAAATGA ACAGCCTGCG
TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
PAAACCTGGT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGACCA
VWFD YWG QGT LVTV SS
301 GTTTGGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 200) CAAACCAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 201) DOM-59 SEQ ID NO: 61 EVQLLES GGG LVQPGGS LRL SCAA SGF TFLD
Y A N

TCCTGTGCAG CCTCCGGATT CACCTTTCTG
GATTATGCGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTG=AAGAC
CTAATACGCT
=GWVRQA PGKG LEW VST ISPM GMG TYY ADSV
K G R =

TGGGTATGGG TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCCAACCCA GGCGGTCCGA GGTCCCTTCC CAGACCTCAC CCAGAGTTGA TAAAGAGGCT ACCCATACCC
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SKN TLY LQMN SLRAED TAVY YCA
K S S

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAATCGAGT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTAGCTCA
AISFTSD ISN FDYW GQG TLVTVSS

ACCGTCTCGA. GC (SEQ ID NO: 202) CGATAAAGCA AATGAAGACT ATAAAGATTA AAACTGATGA CCCCAGTCCC TTGGGACCAG TGGCAGAGCT
CG (SEQ ID NO: 203) DOM-61 SEC2 ID NO: 62 EVQL LES GGG LVQP GGS LRL SCAA SGF TFAA
Y A M =
1 GAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTAaAGC CTGGGGGGTC CCTGCGTCTC
TCCTGTGCAG CCTCCGGATT CACCTTTGCT
GCTTATGCTA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACGA
CGAATACGAT
=TWVRQA PGKG LEW VSY ISPN GTA TYY ADSV
K G R

ATGGTACGGC GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACTGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTATA TAATCAGGCT TACCATGCCG
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
.FTI SRDN SKN TLY LQMN SLR AED TAVY YCA
EYV

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GGAATATGTG

CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG

GMRW NSF DYW GOGT LVT VSS
301 GGGATGCGTT GGAATTCTTT TGACTACTGG GGTCAGGGAA CCCTGGTCAC CGTCTCGAGC (SEQ
ID NO: 204) CCCTACGCAA CCTTAAGAAA ACTGATGACC CCAGTCCCTT GGGACCAGTG GCAGAGCTCG (SEQ ID 140:
205) DOM-62 SEQ ID NO: 63 EVQL LES GGG LVQPGGS LRL SCA A SGF TESS
Y E M

TCCTGTGCAG CCTCCGGATT CACCTTTTCG
AGTTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGMAAAAGC
TCAATACTCT
=AWVRQA PGKG LEWVSS ITSL GTS TYY ADSV
R G R

TTGGTACTTC TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTAGA TAATGCTCAG AACCATGAAG
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
.FTI SRDN SRN TLY LQMN SLR AED TAVY YCA
K P G

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACCGGGT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGCCCA
REED YWG QGT LVTV SS
301 AGGAAGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 206) TCCTTCP_zAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 207) DOM-65 SEQ ID NO: 64 EVQLLES GGG LVQP GGS LRL SCAA SGF TFNE
Y E M

TCCTGTGCAG CCTCCGGATT CACCTTTAAT
GAGTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAATTA
CTCATACTCT
=TWVRQA PGKG LEWVST ITSE GSG TYY ADSV
K G R

AGGGTAGTGG GACATACTAC GCAGACTCCG
TAAAGGGCCG
ACTGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTGC TAATGATCAC TCCCATCACC
CTGTATGATG CGTCTGAGGC
ATTTCCCGGC

.FTI SA PP SRN TLY LOMN SLR AED TAVY YCA
E P N

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACCTAAT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGATTA
GEFDYWG QGT LVTV SS
301 GGTAAGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 208) 1 0 CCATTCAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 209) DOM-66 SEQ ID NO: 65 EVOLLES GGG LVQP GGS LRL SCAA SGF TFSD
YEM

TCCTGTGCAG CCTCCGGATT CACCTTTTCT
GATTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAAAGA
CTAATACTCT
=LWVRQA PGEG LEWVST ITSE GHS TYY ADSV
K G R

AGGGTCATTC TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACAACACCCA GGCGGTCCGA GGTCCCTTCC CAGACCTCAC CCAGAGTTGA TAATGATCAC TCCCAGTAAG
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
.FTI SRDN SRN TLY LOMN SLR AED TAVY YCA
EPG

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACCTGGG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGACCC
TSFDYWG QGT LVTV SS
301 ACTTCGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 210) TGAAGCAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 211) DOM-67 SEQ ID NO: 66 EVOLLES GGG LVQP GGS LRL SCAA SGF TFSD
Y E M =
1 , GAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC
TCCTGTGCAG CCTCCGGATT CACCTTTAGT
GATTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAATCA
CTAATACTCT
=SWVRQA PGKG LEW VST 1DSD GSF TYY ADSV
K G R =

ATGGTAGTTT TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACTCAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTGC TAACTAAGAC TACCATCAAA
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SKN TLY LQMN SLR AED TAVY YCA
= P G

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACCGGGT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGCCCA
VKFDYWG QGT LVTV SS
301 GTGAAGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 212) CACTTCAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 213) DOM-68 SEQ ID NO: 67 EVQLLES GGG LVQP GGS LRL SCAA SGF TFKD
Y E M =

TCCTGTGCAG CCTCCGGATT CACCTTTAAG
GATTATGAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAATTC
CTAATACTCT
35 =TWVRQA PGKG LEWVSS ISST GQS TYY ADSV
K G R =

CTGGTCAGTC TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACTGAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTAGA TAAAGAAGAT GACCAGTCAG
ATGTATGATG CGTCTGAGGC

=FTI SRDN SKN TLY LQMN SLRAED TAVY YCA
K P G

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
45 nAAkCCGGGT
=

CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGCCCA
NEEDING OGT LVTV SS
301 AATAAGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (5E0 ID NO: 214) TTATTCAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 215) DOM-69 SEQID NO: 68 EVQLLES GGG LVQP GGS LRL SCAA SGF TFLD
Y G M =

TCCTGTGCAG CCTCCGGATT CACCTTTCTT
GATTATGGTA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAAGAA
CTAATACCAT
=AWVRQA PGKG LEW VSA ISPL GLS TYY ADSV
= S R =

TTGGTCTTAG TACATACTAC GCAGACTCCG
TGAAGAGCCG
ACCGAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTCGA TAAAGCGGAG AACCAGAATC
ATGTATGATG CGTCTGAGGC
ACTTCTCGGC
=FTI SRDN SKN TLY LQMN SLR AED TAVY YCA
K E V
201 GTTCACCATC TCCCGCGACA ATTCCAAGAA CACGCTGTAT CTGChAATGAACAGCCTGCG
TGCCGAGGAT ACCGCGGTAT ATTACTGTGC
nAAAGAGGTG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTA
TGGCGCCATA TAATGACACG
CTTTCTCCAC
RVGR GVH PPK FDYW GQG TLV TVSS

ACCGTCTCGA GC (SEQ ID NO: 216) TCCCACCCAT CCCCACAAGT AGGAGGCTTC AAACTGATGA CCCCAGTCCC TTGGGACCAG TGGCAGAGCT
CG (SEQ ID NO: 217) DOM-70 SEQ ID NO: 69 EVQLLES GGG LVQP GGS LRL SCAA SGF TFEN
Y A M

TCCTGTGCAG CCTCCGGATT CACCTTTGAG
AATTATGCTA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACTC
TTAATACGAT
=SWVRQA PGKG LEW VST IAPL GVP TYY ADSV
K G R =

TGGGTGTTCC GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACAGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTGC TAACGAGGCG ACCCACAAGG
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC

SRDN SEN TLY LQMN SLRAED TAVY YCA
K X E

TGCCGAGGAT ACCGCGGTAT ATTACTGTGC
GAAAAAGAAG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTA
TGGCGCCATA TAATGACACG
CTTTTTCTTC
VGAW LOS RSF DYWG QGT LVT VSS

GTCTCGAGC (SEQ ID NO: 218) CAACCCCGCA CCGACGTCAG CGCCTCAAAA CTGATGACCC CAGTCCCTTG GGACCAGTGG CAGAGCTCG
(SEQ ID NO: 219) DOM-71 SEQ ID NO: 70 EVQLLES GGG LVQP GGS LRL SCAA SGF TFEG
Y P M
GAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC TCCTGTGCAG
CCTCCGGATT CACCTTTGAG
GGTTANCCTA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCaAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACTC
CCAATAGGAT
=SWVRQA PGKG LEWVST ISPL GPD TYY ADSV
K G R

TGGGTCCTGA TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACAGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTGA TAATCAGGAA ACCCAGGACT
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
SRDN SEN TLY LQMN SLRAED TAVY YCA
K L L
201 GTTCACCATC TCCCGCGACk ATTCCAAGAA CACGCTGTAT CTGCAAATGA ACAGCCTGCG
TGCCGAGGAT ACCGCGGTAT ATTACTGTGC
GAAACTGTTG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTA
TGGCGCCATA TAATGACACG
CTTTGACAAC
MGEY LNS RTF DYWG QGT LVT VSS

GTCTCGAGC (SEQ ID NO: 220) TACCCCCTCA TAAACTTAAG ATCCTGCAAA CTGATGACCC CAGTCCCTTG GGACCAGTGG CAGAGCTCG
(SEQ ID NO: 221) DOM-72 SEQ ID NO: 71 EVQI LES GGG IVQP GGS LRL SCAA SGF TFEA
= P M

TCCTGTGCAG CCTCCGGATT CACCTTTGAG
GCGTATCCTA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACTC
CGCATAGGAT
=SWVRQA PGEG LEW VSS ISPL GLW TYYADSV
K G R =

TTGGTTTGTG GACATA.CTAC GCAGACTCCG
TGAAGGGCCG
ACAGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTCA TAAAGGGGAG AACCAAACAC
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
.FTI SRDN SKN TLY IQMN SIRAED TAVY YCA
K L S

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACTTAGT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGAATCA
AGAE THVYRL FDYW GQG TLVTVSS

ACCGTCTCGA GC (5E0 ID NO: 222) CGACCCCGCC TCTGAGTACAAATAGCCGAA AAACTGATGA CCCCAGTCCC TTGGGACCAG TGGCAGAGCT CG
(5E0 ID NO: 223) DOM-73 SEQ ID NO: 72 EVQLLES GGG LVQP GGS LRL SCAA SGF TFSK
Y D M

CCTCCGGATT CACCTTTTCT

AAGTATGATA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGC.AGAG AGGACACGTC
GGAGGCCTAA GTGGAAAAGA
TTCATACTAT
=SWVRQAPGKG LEWVST SLED GLT TYYADS
K G R = ' ATGGTCTGAC TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACAGAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTGA TAAGACCTCC TACCAGACTG
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SKN TLY LQMN SLR AED TAVY YCA
KPG
201 GTTCACCATC TCCCGCGACA ATTCC.AAGAA. CACGCTGTAT CTGCAAATGA
ACAGCCTGCG TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACCGGGG

CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGCCCC
RLED YWG QGT LVTV SS
301 CGTTTGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 224) GCAAACAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 225) DOM-74 SEQ ID NO: 73 EVQLLES GGG LVQP GGS LRL SCAA SGF TFSD
Y P M

TCCTGTGCAG CCTCCGGATT CACCTTTTCG
GATTATCCTA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAAAGC
CTAATAGGAT
=TWVRQA PGKG LEW VST ILSP GTE TYY ADSV
K G R

CGGGTACGGA GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACTGCACCCA GGCGGTCCGA GGTCCCTTCC CAGACCTCAC CCAGAGTTGA TAAGACAGAG GCCCATGCCT
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
.FTI SRDN SKN TLY LQMN SLRAED TAVY YCA
KAE
201 GTTCACCATC TCCCGCGACh ATTCCAAGAA CACGCTGTAT CTGrAAATGA ACAGCCTGCG
TGCCGAGGAC ACCGCGGTAT ATTACTGTGC

GAAAGCTGAG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTCGACTC
KDFD YWG QGT LVTV SS
301 AAGGATTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 226) TTCCTAAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 227) DOM-75 SEQ ID NO: 74 AvaI

Y P M =

TCCTGTGCAG CCTCGGGATT CACCTTTTTG
CAGTATCCGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGCCCTAA GTGGAAAAAC
GTCATAGGCT
=GWV RQA PGKG LEW VST ISPV GLT TYY ADSV
KGR

TTGGTTTGAC TACATACTAC GCAGACTCCG
TGAAGGGCCG

ACCCAACCCA GGCGGTCCGA GGTccCTTCC CAGATCTCAC CCAGAGTTGA TAAAGAGGAC AACCAAACTG
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SEN TLY LQMN SLRAED TAVY YCA
K L F

TGCCGAGGAT ACCGCGGTAT ATTACTGTGC
GAAATTGTTT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTA
TGGCGCCATA TPATGACACG
CTTTAACAAA
EGSR IQR DVG FDYW GQG TLV TVSS

ACCGTCTCGA GC (SEQ ID NO: 228) CTCCCCAGCT CCTAAGTCGC ACTACACCCA AAACTGATGA CCCCAGTCCC TTGGGACCAG TGGCAGAGCT
CG (SEQ ID NO: 229) DOM-77 SEQ ID NO: 75 EVQLLES GGG LVQP GGS LRL SCAA SGF TFEE
Y G M =

TCCTGTGCAG CCTCCGGATT CACCTTTGAG
GAGTATGGTA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACTC
CTCATACCAT
=AWVRQA PGEG LEWVST ISPL GIS TYY ADSV
K G R =

TGGGTATTTC GACATACTAC GCAGACTCCG
TGAT=GGGCCG
ACCGCACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTGA TAAAGAGGCG ACCCATAAAG
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SKU TLY LQMN SLRAED TAVY YCA
KNA

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACATGCT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGTACGA
TSQE SLR SED YWGQ GTLVTV SS

TCGAGC (SEQ ID NO: 230) TGCAGAGTCC TCAGAAACGC CA(ZAAAkCTG ATGACCCCAG TCCCTTGGGA CCAGTGGCAG AGCTCG (SEQ
ID NO: 231) DOM-78 SEQ ID NO: 76 EVQLLES GGG LVQP GGS LRL SCAA SGF TFER
Y Q M =

TCCTGTGCAG CCTCCGGATT CACCTTTGAG
AGGTATCAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTc GGAGGCCTAA GTGGAAACTC
TCCATAGTCT
=AWVRQAPGKG LEW VST ISSD GGG TYY ADSV
K G R =

ATGGTGGGGG GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGCACCCA GGCGGTCCGA GGCCCCTTCC CAGATCTCAC CCAGAGTTGC TAATCAAGAC TACCACCCCC
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SR DR SKN TLY LOMN SLRAED TAVY YCA
K P G
201 GTTCACCATC TCCCGCGACA ATTCCAAGAA CACGCTGTAT CTGrzoaATGA ACAGCCTGCG
TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACCTGGT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGACCA
HRFDYWG QGT LVTV SS
301 CATCGGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 232) GTAGCCAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 233) DOM-80 SEQ ID NO: 77 EVQLLES GGG LVQP GGS LRL SCAA SGF TFGR
Y Q M =

TCCTGTGCAG CCTCCGGATT CACCTTTGGT
CGTTATCAGA
CTCCACGTCGACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACCA
GCAATAGTCT
=AWVRQAPGKG LEW VSS ISSD GGG TYY ADSV
K G R =

ATGGTGGGGG GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTAGA TAAAGAAGAC TACCACCCCC
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
19.3 .FTI SRDN SKN TLY LQMN SAR AED TAVY YCA
K P S

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACCGTCT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCAMA TAATGACACG
CTTTGGCAGA
RRFDYWG QGT LVTV SS
302 CGTCGGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (5E4 ID NO:
234) 1 0 GCAGCCAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 235) DOM-81 SEQ ID NO: 78 EVQL LtS GGG LVQP GGF IRL SCAA SGF TFEL
Y P M =

TCCTGTGC.AG CCTCCGGATT CACCTTTGAG
TTGTATCCGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCA.TGTCG GACCCCCCAA GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACTC
AACATAGGCT
=AWVRQA PGKG LEW VSS ISPV GPL TYY ADSV
K G R =

TTGGTTTTCT GACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGCACCCA GGCGGTCCGA GGTCCCTTCC CAGACCTCAC CCAGAGTAGC TAAAGAGGCC AACCAAP.AGA
CTGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SKN TLY LQMN SLR AED TAVY YCA
K G H

TGCCGAGGAT ACCGCGGTAT ATTACTGTGC
GAAAGGGCAT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTA
TGGCGCCATA TAATGACACG
CTTTCCCGTA
EGSYTPR SAF DYWG QGT LVT VSS

GTCTCGAGC (SEQ ID NO: 236) CTCCCCAGCA TATGAGGCGC CAGCCGAAAA CTGATGACCC CAGTCCCTTG GGACCAGTGG CAGAGCTCG
(SEQ ID NO: 237) DOM-82 SEQ ID NO: 79 EVQLLES GGG LVQP GGS LRL SCAA SGF TFVA
Y P M =

TCCTGTGCAG CCTCCGGATT CACCTTTGTG
GCGTATCCTA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACAC
CGCATAGGAT
=AWVRQA PGKG LEW VST IAPL GGN TYY ADSV
K G R =

TGGGTGGTAA TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGCACCCA GGCGGTCCGA GGTCCCTTCC CAGACCTCAC CCAGAGTTGA TAACGCGGAG ACCCACCATT
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
=

=FTI SRDN SKN TLY LORIN SLR AED TAVY NCR
KRP

TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACGGCCG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGCCGGC
EGLQ IDS QNF DYWG QGT LVT VSS

GTCTCGAGC (SEQ ID NO: 238) CAGAGCTCG (SEQ ID NO: 239 DOM-83 SEQ ID NO: 80 EVQL LES GGG LVQP GGS LRL SCAA SGF TEAL
YQOS =

TCCTGTGCAG CCTCCGGATT CACCTTTGCG
TTGTATCAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAACGC
AACANAGTCT
= ASIVRQA. PGKG LEW VSS IDSS GSD TYYADSV
K G R =

CTGGTAGTGA TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGAACCCA GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTAGC TAACTARGAA GACCATCACT
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
=FTI SRDN SKN TLY LQMN SLRAED TAVY YCA
KPE
201 GTTCACCATC TCCCGCGACA ATTCCAAGAA. CACGCTGTAT CTGCAAANGA ACAGCCTGCG
TGCCGAGGAC ACCGCGGTAT ATTACTGTGC
GAAACCTGAG
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTGGACTC
RDFD YWG QGT LVTV SS
301 CGTGATTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 240) GCACTAAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 241) DOM-84 SEQ ID NO: 81 EVQLLES GGG LVQP GGS LRL SCAA SGF TFRQ
Y Q E =

TCCTGTGCAG CCTCCGGATT CACCTTTAGG
CAGTACCAGA
CTCCACGTCG ACAACCTCAG ACCCCCTCCG AACCATGTCG GACCCCCCAG GGACGCAGAG AGGACACGTC
GGAGGCCTAA GTGGAAATCC
GTCATGGTCT
=AWARQA PGKG LEW VST IASD GVS TYY ADSV
K G R

ATGGTGTTTC TACATACTAC GCAGACTCCG
TGAAGGGCCG
ACCGAACCCG GGCGGTCCGA GGTCCCTTCC CAGATCTCAC CCAGAGTTGC TAACGCAGCC TACCACAAAG
ATGTATGATG CGTCTGAGGC
ACTTCCCGGC
F T I SRDN SRN TLY LQMN SLR AED TAVY YCA
KVG

TGCCGAGGAC ACCGCGGTM ANTACTGTGC
GAAAGTTGGT
CAAGTGGTAG AGGGCGCTGT TAAGGTTCTT GTGCGACATA GACGTTTACT TGTCGGACGC ACGGCTCCTG
TGGCGCCATA TAATGACACG
CTTTCAACCA
RDFD YWG QGT LVTV SS
301 CGTGATTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC (SEQ ID NO: 242) GCACTAAAAC TGATGACCCC AGTCCCTTGG GACCAGTGGC AGAGCTCG (SEQ ID NO: 243) DOM-116 SEQ ID NO: 82 DIQMTQS PSS LSAS VGDRVT ITCRASQ PIGP
D L L

ATCACTTGCC GGGCAAGTCA GCCTATTGGT
CCTGATTTAC
CTGTAGGTCT ACTGGGTCAG AGGTAGGAGG GACAGACGTA GACATCCTCT GGCACAGTGG TAGTGAACGG
CCCGTTCAGT CGGATAACCA
GGACTAAATG
=WYQQKP GKAP KLL IYOTSIL QSG VPS RFSG
S G S =

TGCAAAGTGG GGTCCCATCA CGTTTCAGTG
GCAGTGGATC
ACACCATGGT CGTCTTTGGT CCCTTTCGGG GATTCGAGGA CTAGATAGTC TGCAGGTAAA ACGTTTCACC
CCAGGGTAGT GCAAAGTCAC
CGTCACCTAG
.GTD FTLT ISS LQP EDFA TYY CQQ YWAF PVT
FGQ

CTGTCAACAG TATTGGGCTT TTCCTGTGAC
GTTCGGCCAA

ACCCTGTCTA AAGTGAGAGT GGTAGTCGTC AGACGTTGGA CTTCTAAAAC GATGCATGAT GACAGTTGTC
ATAACCCGAA AAGGACACTG
CAAGCCGGTT
GTRVETER
301 GGGACCAAGG TGGAAATCAA ACGG (SW ID NO: 244) CCCTGGTTCC ACCTTTAGTT TGCC ISEQ ID NO: 245)--SUBSTITUTE SHEET (RULE 26) DOM-85 - SEQ ID NO . : 246 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FE QYDM=

CCTCCGGATT
CACCTTTGAG CAGTATGATA
=RWVRQA P0 KG LEW VSW IDEA GHE TYY AD
SVKGR=

CGGGTCATGA GACATACTAT
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SRN TLY LQMN SLRAED TAVYY
CA KGM

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAAGGGATG
DGFDYWG QGT LVTV SS
301 GATGGGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC - SEQ ID NO.: 361 DOM-86 - SEQ ID NO.: 247 DIQMTQS PSS LSAS VGD RVT ITCRASQD
IG DALF=

GGGCAAGTCA
GGATATTGGG GATGCTTTAT
=WYQ QKP GKAP KLL IYY SSML QSG VPS RF
SG GGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCGGTGGATC
=GTD FTLT ISS LQP EDFA TYY CQQ RHSTP
AT FGQ

CTGTCAACAG CGGCATAGTA
CTCCTGCTAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 362 DOM-87 - SEQ ID NO.: 248 SUBSTITUTE SHEET (RULE 26) DIQMTQS PSS LSASVGDRVT ITCRASQD
ID ESIM=

GGGCAAGTCA
GGATATTGAT GAGTCTTTAA
=WYQ QKP GKAP ALL IYGVSYL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=G TD FTLT ISS LQP EDFA TYY CQQ RWKAP
FT FGQ

CTGTCAACAG CGGTGGAAGG
CTCCTTTTAC GTTCGGCCAA
GTKV EIK R- SEQ ID NO.: 363 DOM-88 - SEQ ID NO.: 249 DI QM TQS PSS LSAS VGD RVT ITCRASQE
IV EDLY=

GGGCAAGTCA
GGAGATTGTG GAGGATTTAT
=WYQ QKP GKAAKLL IYGASWL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=G TD FT LT ISS LQPEDFA TYY CQQ TRRRP
YT FGQ

CTGTCAACAG ACGCGTAGGC
GTCCTTATAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 364 DOM-89 - SEQ ID NO.: 250 DI QM TQS PAS LSAS VGD RVT ITCRASQD
ID PMLR=

SUBSTITUTE SHEET (RULE 26) GGGCAAGTCA
GGATATTGAT CCTATGTTAA
=WYQ QKP GKAP KLL IYAGSIL QSG VPS RF
SGSGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTD FTLT ISS LQP EDFA TYY CQQ TLVTP
YT FGQ

CTGTCAACAG ACGCTGGTGA
CTCCTTATAC GTTCGGCCAA
GTKV EIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 365 DO-9O - SEQ ID NO.: 251 DIQM TQS PSS LSAS VGD RVT ITCR ASQS
IS DALF=

GGGCAAGTCA
GTCGATTTCG GATGCGTTAT
=WYQ QKP GKAP RLL IYY GSVL QSG VPS RE
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTD FTLT ISS LQP EDFA TYY CQQ RFQEP
VT FGQ

CTGTCAACAG CGTTTTCAGG
AGCCTGTGAC GTTCGGCCAA
GTKV EIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 366 DO!-91 - SEQ ID NO.: 252 DIQMTQS PSS LSAS VGDRVT ITCRASQQ
IS DELN=

SUBSTITUTE SHEET (RULE 26) ATCACTTGCC GGGCAAGTCA
GCAGATTAGT GATGAGTTAA
=WYQ QKP GKAP KLL IYAVSIL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTD FTLT ISS LQP EDFATYY CQQ WLSFP
ST FGQ

CTGTCAACAG TGGTTGAGTT
TTCCTTCGAC GTTTGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG ===== SEQ ID NO.: '367 =
DOM-92 - SEQ ID NO.: 253 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FV DYPM=
I GAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC TCCTGTGCAG
CCTCCGGATT
CACCTTTGTT GATTATCCGA
=GWVRQA PGKG LEW VST ISTG GFS TYY AD
SV KGR=
101 TGGGTTGGGT CCGCCAGGCT CCAGGaAAGG GTCTAGAGTG GGTCTCAACG ATTTCTACGG
GGGGTTTTTC GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA WAR

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAAGCGCGG
YYYL SQI KNF DYWG QGT LVT VSS

GTCTCGAGC SEQ ID
NO.: 368 DOM-93 - SEQ ID NO.: 254 EVQL LES GGG LVQP GGS LRL S CAA SGF T
FD IYGM' SUBSTITUTE SHEET (RULE 26) CCTCCGGATT
CACCTTTGAT ATTTATGGGA
=TWV RQA PGKG IEW VSS ISPL GLV TYY AD
PV KGR=

TTGGTCTTGT MACATACTAC
GCAGACCCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KLK

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAACTGAAG
EHGD VPF DYW GQGT LVT VSS

ID NO.:

DO-94 - SEQ ID NO.: 255 EVQILES GGG LVQP GGS LRL SCAA SGFT
FE LYPM=

CCTCCGGATT
CACCTTTGAG CTTTATCCGA
=SWVRQA PG KG LEW VST ISPT GLI TYY AD
SV KGR=

CGGGTTTGTT GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KFK

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAATTTAAG
RSGK TDD TNF DYWG QGT LVT VSS

GTCTCGAGC SEQ ID
NO.: 370 DOM-95 - SEQ ID NO.: 256 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FR EYDM.

SUBSTITUTE SHEET (RULE 26) CCTCCGGATT
CACCTTTCGG GAGTATGATA
=LWV RQA PGKG LEW VST IVGD GNG TYY AD
SV KGR=

ATGGTAATGG TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KQD

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAACAGGAT
RQFD YWG QGT LVTV SS
301 CGTCAGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC SEQ ID NO.:

DO-96 - SEQ ID NO.: 257 EVQPLES GGG LVQP GGS LRL SCAASGFT
FT DYKM=

CCTCCGGATT
CACCTTTACT GATTATAAGA
=LWVRQA PGKG LEW VSS ISPS GRW TYY AD
SV KGR=

GTGGTCGTTG GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLR AED TAVYY
CA KSL
201 GTTCACCATC TCCCGCGAaA ATTCCAAGAA CACGCTGTAT CTGCAAATGA ACAGCCTGCG
TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAAAGTCTT
FEGS FDY WGQ GTLV TVSS

NO.: 372 DO-97 - SEQ ID NO.: 258 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FE EYGM=

SUBSTITUTE SHEET (RULE 26) CCTCCGGATT
CACCTTTGAG GAGTATGGTA
=SWVRQA PGKG LEW VST ISPI GVT TYY AD
SV KGR=

TTGGTGTTAC TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KNA

TGCCGAGGAT ACCGCGGTAT
ATTACTGTGC GAAAAATGCT
YDRK SNF DYW GQGT LVT VSS

ID NO.:

DOM-98 - SEQ ID NO.: 259 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FD RYVM=

CCTCCGGATT
CACCTTTGAT CGGTATGTGA
=VWV RQA PGKD LEWVSG ITPS GRR TYY AD
SV KGR=

GTGGTAGGAG GACATACTAC
GCAGACTCCG TGAAGGGCCG
.FTI SRDN SKD TLY LQMN SLRAED TAVYY
CA KVL

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAAGTGTTG
GRHF DPL LPS FDYW GQG TLV TVSS

ACCGTCTCGA GC - SEQ
ID NO.: 374 DOM-99 - SEQ ID NO.: 260 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FE DYAM.

SUBSTITUTE SHEET (RULE 26) CCTCCGGATT
CACCTTTGAG GATTATGCTA
=SWVRQA PGKG LEW VST ITPG GFW TYY AD
SV KGR=

GTGGTTTTTG GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SRN TLY LQMN SLR AED TAVYY
CA KTS

TGCCGAGGAT ACCGCGGTAT
ATTACTGTGC GAAAACGTCT
SGEL QLV EDF DYWG QGT LVT VSS

GTCTCGAGC SEQ ID
NO.: 375 DOM-100 - SEQ ID NO.: 261 DIQMTQS PSS LSAS VGD RVT ITCR ASQN
IK HSLR.

GAGCAAGTCA
GAATATTAAG CATTCGTTAC
=WYQ QKP GKAP ALL IYHASQL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
.GTDFTLT ISS LQP EDFA TYY CQQ VRHRP
YT FGQ

CTGTCAACAG GTTAGGCATC
GTCCTTATAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 376 DOM-101 - SEQ ID NO.: 262 DIQMTQS PSS ',SAS VGD RVT ITCR ASQA
IG HRLR=

SUBSTITUTE SHEET (RULE 26) GGGCAAGTCA
GGCTATTGGG CATCGGTTAC
=WYQQKP GKAP XII IYH RSKL QSG VPS RF
SGSGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTDFTLT ISS LQP EDFA TYY CQQ VALFP
YT FGQ

CTGTCAACAG GTTGCTTTGT
TTCCCTATAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 377 DOM-102 - SEQ ID NO.: 263 DIQMTQS PSS ',SAS VGDRVT ITCR ASQH
IG HHIR.

GGGCAAGTCA
GCATATTGGT CATCATTTAA
=WYQQKP GKAP KLL IYH RS HL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTDFTIT ISS LQP EDSA TYY CQQ WDRPP
YT FGQ

CTGTCAACAG TGGGATAGGC
CGCCTTATAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 378 DOM-103 - SEQ ID NO.: 264 DIQMTQS PSS ISAS VGDRVT ITCR ASQA
IG HRLR.

SUBSTITUTE SHEET (RULE 26) ATCACTTGCC GGGCAAGTCA
GGCTATTGGG CATCGGTTAC
=WYQ QKP GKAP KLL IYH RSKLQSGVPS RF
SGSGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=G TD FTLT ISS LQP EDFA TYY CQQ VRAVP
YT FGQ

CTGTCAACAG GTGCGGGCTG
TGCCTTATAC GTTTGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATTAA ACGG - SEQ ID NO.: 379 DO11-104 - SEQ ID NO.: 265 DIQMTQS PSS LSAS VGD RVT ITCRASQA
IG HRLR=

GGGCAAGTCA
GGCTATTGGG CATCGGTTAC
=WYQQKP GKAP KLL IYH RSKL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTDFTLT ISS LQP EDFATYY CQQVRFSP
YT FGQ

CTGTCAACAG GTTCGTTTTT
CTCCTTATAC GTTCGGCCAA
GTKV EIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 380 DO-i.05 - SEQ ID NO.: 266 DIQMTQS PSS LSAS VGDRVT ITCR ASQA
IG HRLR=

SUBSTITUTE SHEET (RULE 26) ATCACTTGCC GGGCAAGTCA
GGCTATTGGG CATCGGTTAC
=WyQQKP GKAP KLL IYH RSKL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTDFTLT ISS LQP EDFA TYY CQQ SYARP
VT FGQ

CTGTCAACAG TCTTATGCTA
GGCCTGTGAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 381 DOM-106 - SEQ ID NO.: 267 DIQMTQS PSS LSAS VGDRVT ITCRASQS
IN HRLY=

ATCACTTGCC GGGCAAGTCA
AAGTATTAAT CATAGGTTAT
.WYQQKP GKAP KLL IYH RSRL QSG VPS RF
SG SGS=
101 AfTGGTACCA GCAGAAACCA GGGAAAGCCC CTAAGCTCCT GATCTATCAT CGGTCCAGGT
TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
.GTDFTLT ISS LQP EDFATYY CQQ YK.VRP
NT FGQ

CTGTCAACAG TATAAGGTTA
GGCCTAATAC GTTCGGCCAA
=
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 382 DOM-107 - SEQ ID NO.: 268 D IQMTQS PSS LSAS VGDRVT ITCR ASQA
IG HRLR=

SUBSTITUTE SHEET (RULE 26) ATCACTTGCC GGGCAAGTCA
_ -GGCTATTGGG rT.TrGGTTAC__ =WYQQKP GKAP KLL IYH RSKL QSG VPS RP
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTD FTLT ISS LQP EDFA TYY CQQ TYSSP
HT FGQ

CTGTCAACAG ACTTATTCGT
CTCCTCATAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG - SEQ ID NO.: 383 DOM-108 - SEQ ID NO.: 269 IG HRLR=

GGGCAAGTCA
GGCTATTGGG CATCGGTTAC
=WYQQKP GKAP KLL IYH RSKL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTDFTLT ISS LQP EDFA TYY CQQ RAVRP
FT FGQ

CTGTCAACAG AGGGCGGTGA
GGCCTTTTAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAAG TGGAAATCAA ACGG SEQ ID NO.: 384 DOM-109 - SEQ ID NO.: 270 DIQMTQS PSS LSAS VGD RVT ITCR ASQA
IG HRLR.

SUBSTITUTE SHEET (RULE 26) ATCACTTGCC GGGCAAGTCA
GGCTATTGGG CATCGGTTAC
=WYQQKP GKAP KLL IYH RSKL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTD FTLT ISS LQP EDFA TYY CQQ TYYRP
LT FGQ

CTGTCAACAG ACTTATTATC
GTCCTCTTAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG - SEQ ID NO.:

DOM-110 - SEQ ID NO.: 271 DIQMTQS PAS LSAS VGD RVT ITCR ASQD
ID PMLR=

ATCACTTGCC GGGCAAGTCA
GGATATTGAT CCTATGTTAA
=WYQQKP GKAP KLL IYAGSIL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTDFTLT ISS LQP EDFA TYY CQQ TSIRP
YT FGQ

CTGTCAACAG ACTAGTATTA
GGCCTTATAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 386 DOM8 -111 - SEQ ID NO.: 272 EVQLLES GGG LVQP GGS LRL SCAA SGFT
FE RYPM.

SUBSTITUTE SHEET (RULE 26) CCTCCGGATT
CACCTTTGAG CGTTATCCTA
=TWVRQA PGKG LEW VST IHGS GSATYY AD
SV KGR=

CTGGTAGTGC TACAMACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SR 0R SKN TLY LQMN SLRAED TAVYY
CA KGP

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAAGGGCCG
YTSR HNS LGH FDYW GQG TLVTVSS

ACCGTCTCGA GC - sinQ
ID NO.: 387 DOM-112 - SEQ ID NO.: 273 EVQILES GGG LVQP GGS LRL SCAA SGFT
FM DYPM=

CCTCCGGATT
CACCTTTATG GATTATCCTA
=GWVRQA PGKG LEW VSS IGPV GMS TYY AD
SV KGR-TTGGTATGAG TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KYG

TGCCGAGGAT ACCGCGGTAT
ATTACTGTGC GAAATATGGG
GTSG RHN TKF DYWG QGT LVT VSS

GTCTCGAGC SEQ ID
NO.: 388 DOM-113 - SEQ ID NO.: 274 EVQLLES GGG LVQP GGS LRL SCAR SGFT
FT EYPM.

SUBSTITUTE SHEET (RULE 26) CCTCCGGATT
CACCTTTACT GAGTATCCTA
=swvRQA PGKG LEW VSV ISPL GFT TYY AD
SV KGR=

TTGGTTTTAC GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KWT

TGCCGAGGAT ACCGCGGTAT
ATTACTGTGC GAAATGGACT
GGSG ILN SSFDYWG QGT LVTVSS

GTCTCGAGC SEQ ID
NO.: 389 DOM-114 - SEQ ID NO.: 275 EVQLLES GGG LV:QP GGS LRL SCAASGFR
VS NYDL=

CCTCCGGATT
TAGGGTTAGC AATTACGATT
=TWV RQA PGKG LEW VST ISAT NGS TYY AD
SVKGR=

CAAACGGTAG CACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA AVT

TGCCGAGGAC ACCGCGGTAT
ATTATTGCGC GGCAGTGACG
WWLL RHN DNL GFWG QGT LVT VSS

GTCTCGAGC SEQ ID
NO.: 390 DOM-115 - SEQ ID NO.: 276 EVQLLES GGG LVQP GGS LRL SCAA SGFS
IS YKNM=

SUBSTITUTE SHEET (RULE 26) CCTCCGGATT
TAGCATTAGC TATAAGAATA
=AWV RQA PGKG LEW VSA IKAA NGS TYY AD
SV KGR=

CAAACGGTAG CACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLR AED TAVYY
CA TGS

TGCCGAGGAC ACCGCGGTAT
ATTATTGCGC GACAGGGAGT
QKKR TYT FDF WGQG TLV TVSS

SEQ ID NO.:

DOM-120 - SEQ ID NO.: 277 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FR SYTM=

CCTCCGGATT
CACCTTTAGG TCTTATACGA
=GWVRQA PGKG LEW VSS INPM GYQ TYY AD
SVKGR=

TGGGTTATCA GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KHG

TGCCGAGGAT ACCGCGGTAT
ATTACTGTGC GAAACATGGG
/GKG TKP HNF DYWG QGT LVT VSS

GTCTCGAGC SEQ ID
NO.: 392 DOM-121 - SEQ ID NO.: 278 EVQLLES GGG LVQP GGS LRL SCAA SGFT
FE LYRM==

SUBSTITUTE SHEET (RULE 26) TCCTGTGCAG CCTCCGGATT
CACCTTTGAG CTGTATAGGA
=SWVRQA PGKG LEW VSE ISGS GFP TYYAD
SV KGR=

GTGGTTTTCC TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KSL

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAAAGTCTG
HD KT QHH QEF DYWG QGT LVT VSS

GTCTCGAGC SEQ ID
NO.: 393 DOM-123 - SEQ ID NO.: 279 EVQLLES GGG LVQP GGS LRL SCAA SGFT
FI EYPM.

CCTCCGGATT
CACCTTTATT GAGTATCCTA
=RWVRQA PGKG LEW VSL ISPS GVF TYY AD
SV KGR=

CTGGTGTGTT TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KGD

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAAGGGGAT
ESST FDY WGQ GTLV TVSS
301 GAGTCTAGTA CTTTTGACTA CTGGGGTCAG GGAACCCTGG TCACCGTCTC GAGC SEQ ID NO.:

DOM-124 - SEQ ID NO.: 280 EV QL L ES GGG LVQP GGS LRL S C AA SGF T
FK RYDM.

SUBSTITUTE SHEET (RULE 26) CCTCCGGATT
CACCTTTAAG CGGTATGATA
= 1DWVRQA PGKG LEW VST IGSS GYP TYY AD
sV KGR=

CGGGTTATCC GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA ERM

ATTACTGTGC GGAAAGGATG
PGYFPGFARQ FDYW GQG TLV TVSS

ACCGTCTCGA GC ¨ SEQ
ID NO.: 395 DOM-125 - SEQ ID NO.: 281 EVQLLES GGG LVQP GGS LRL SCAA SGFT
FW RYAN.

CCTCCGGATT
:10 CACCTTTTGG CGGTATGCTA
=GWV,RQA PG KG LEW VST INDE GRE TYY AD
SV KGR=

AGGGTCGGGA GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KKR

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAAAAGCGG
/SSSVNA PYE FDYW GQG TLV TVSS

ACCGTCTCGA GC ¨ SEQ
ID NO.: 396 DOM-126 - SEQ ID NO.: 282 EVQLLES GGG LVQP GGS LRL SCAA SGFT
FA NYSM.

SUBSTITUTE SHEET (RULE 26) CCTCCGGATT
CACCTTTGCG AATTATAGTA
=SWVRQA PGKG LEW VSS IDRL GTH TYY AD
SV KGR=

TTGGTACGCA TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KVL

TGCCGAGGAT ACCGCGGTAT
ATTACTGTGC GAAAGTGCTG
ADLI AGHAEF DYWG QGT LVT VSS

GTCTCGAGC - SEQ ID
NO.: 397 DO-127 - SEQ ID NO.: 283 EVQLLES GGG LVQP GGS LRL SCAASGFT
FP SYDM=
I GAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC TCCTGTGCAG
CCTCCGGATT
CACCTTTCCG TCGTATGATA
=AWVRQAPGKG LEW VSG ISRS GSM TYY AD
SV KGR=

CTGGTTCTAT GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KGV

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAAGGTGTT
DAHVYYM EPF FDYW GQG TLV TVSS

ACCGTCTCGA GC -- SEQ
ID NO.: 398 DO-128 - SEQ ID NO.: 284 EVQLLES GGG LVQP GGS LEL SCAA SGFT
FE RYQM.

SUBSTITUTE SHEET (RULE 26) CCTCCGGATT
CACCTTTGAG AGGTATCAGA
=AWVRQA PC-KG LEW VST Issn GGG TYY AD
SV KGR=

ATGGTGGGGG GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKE TLY IQMN SLRAED TAVYY
CA KPG

TGCCGAGGAC ACCGCGGTAT
ANTACTGTGC GAAACCGGGT
TV FD YWG QGT LVTV SS
301 ACTGTTTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC SEQ ID NO.:

DOM-129 - SEQ ID NO.: 285 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FP KYEM=

CCTCCGGATT
CACATTTCCG AAGTATGAGA
=AWV RQAPGKG LEW VSS IDGD GKS TYY AD
SV KGR=

ATGGTAAGTC TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SRN TLY LQMN SLRAED TAVYY
CA KPD

TGCCGAGGAT ACCGCGGTAT
ATTACTGTGC GAAACCGGAT
QFFD YWG QGT LVTV SS
301 CAGTTTTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC SEQ ID NO.:

DOM-130 - SEQ ID NO.: 286 EVQLLES GGG LVQP GGS LRL SCTA SGFT
FA GYQM.

SUBSTITUTE SHEET (RULE 26) TCCTGTACAG CCTCCGGATT
CACCTTTGCG GGTTATCAGA
=SWVRQA PG KG LEW VSS ITNE GVS TYY AD
SV KGR=

AGGGTGTTTC TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KPG

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAACCGGGG
KY FD YWG QGT LVTV SS
301 AAGTATTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC SEQ ID NO.:

DOM-131 - SEQ ID NO.: 287 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FG EYEM=

CCTCCGGATT
CACCTTTGGG GAGTATGAGA
=VWVRQA PG KG LEW VSS ITSD GLS TYYAD
SV KGR=

ATTACGTCGGATGGTCTGAG TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA EPG

TGCTGAGGAC ACCGCGGTAT
ATTACTGTGC GAAACCGGGT
IRFD YWG QGT LVTV SS
301 ATTCGTTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC SEQ ID NO.:

DOM-132 - SEQ ID NO.: 288 EVQLLES GGG LVQP GGS LRL SCAA SGFT
FA DYDM=

SUBSTITUTE SHEET (RULE 26) CCTCCGGATT
CACCTTTGCT GATTATGATA
=AWV RQA. PGKG LEW VSG IVDD GLM TYY AD
SVKGR=

GCAGACTCCG TGAAGGGCCG
=FTI SRDN SRN TLY LQMN SLR AED TAVYY
CA KPD

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAACCGGAT
/AFD YWG QGT LVTV SN
301 GTTGCTTTTG ACTACTGGGG TCAGGGGACC CTGGTCACCG TCTCGAAC SEQ ID NO.:

DOM-133 - SEQ ID NO.: 289 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FI GYAM.

CCTCCGGATT
CACCTTTATT GGTTATGCTA
=AWVRQA PGKG LEW VSS IGPL GAT TYY AD
SV KGR=

TGGGTGCGAC TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KLP

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAATTGCCT
AGTS SHS VDF DYWG QGT LVT VSS

GTCTCGAGC SEQ ID
NO.: 404 DOM-134 SEQ ID NO.: 290 EVQL LES GGG LVQP GGS LRL S CAA SG F T
F A DYEM=

SUBSTITUTE SHEET (RULE 26) CCTCCGGATT
CACCTTTGCG GATTATGAGA
=TWVRQA PG KG LEW VSS ITSD GVS TYY AD
SV KGR=

ATGGTGTTTC TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KPS

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAACCGTCG
VQFD YWG QGT LVTV SS
301 GTTCAGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC SEQ ID NO.:

DOM-135 - SEQ ID NO.: 291 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FR RYVN=

CCTCCGGATT
CACCTTTCGT AGGTATGTTA
=GWVRQA PGKG LEW VSW IEAD GRT TYY AD
SV KGR=

ATGGTCGTAC GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLR AED TAVYY
CA KGL

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAAGGGCTT
TDQHVIE FDY WGQG TLV TVSS

SEQ ID NO.:

DOM-136 - SEQ ID NO.: 292 EVQL LES GGG LVQP GGS LRL SCAA SGF T
F D GYR1.1=

SUBSTITUTE SHEET (RULE 26) 1 GAGGTGcmc TGTTGGAGTc TGGGGGAGGC TTGGTAcAGC CTGGGGGGTC CcTGCGTCTC TCCTGTGcAG
CCTCCGGATT
CACCTTTGAT GGTTATcGTA
-GWV RQA PGKG LEW VSS IAPD GNY TYY AD
SVKGR=
101 TGGGGTGGGT CCGCCAGGCT CcAGGGAAGG GTCTAGAGTG GGTcTCATcG ATTGCTCCGG
ATGGTAATTA TACATACTAc GtAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLy LQMN SLR AED TAVYY
CA KFW
201 GTTCACCATC TCCCGcGACA ATTCcAAGAA cACGCTGTAT CTGcAAATGA ACAGCCTGcG
TGCCGAGGAc ACCGCGGTAT
ATTACTGTGC GAAATTTTGG
GMQF DyW GQG TLVT Vss 301 GGGATGCAGT TTGACTAcTG GGGTCAGGGA ACCCTGGTCA cCGTCTCGAG c ¨ SEQ ID
NO.: 407 , DOM-137 ¨ SEQ ID NO.: 293 EVQLLES GGG LVQP GGS LRL SCAA sGFT
FA sYpM-1 GAGGTGcAGc TGTTGGAGTc TGGGGGAGGC TTGGTACAGC cTGGGGGGTC CCTGCGTCTc TccTGTGcAG
CCTCCGGATT
CACCTTTGCT TCGTATCcGA
=GWVRQA PGKG LEW VSS TGRI GFT TyY AD
SV KGR=
101 TGGGTTGGGT CCGCCAGGCT CCAGGGAAGG GTcTAGAGTG GGTcTCAAGT ATTGGTCCTA
TTGGTTTTAc TACATAcTAC
GCAGACTCcG TGAAGGGCcG
=FTI SRDN sKN TLy LQMN sLR AED TAVYY
CA EMK
201 GTTCACCATC TCCCGcGACA ATTCCAAGAA CACGcTGTAT CTGCAARTGA ACAGcCTGCG
TGccGAGGAC ACCGCGGTAT
ATTAcTGTGC GGAAATGAAG
spYK PQF DYW GQGT LVT VsS
301 TCGCCTTATAAGcCGcAGTT TGACTACTGG GGTCAGGGAA cccTGGTcAc CGTcTcGAGC SEQ ID
NO.:

DOM-138 ¨ SEQ ID NO.: 294 EVQL LES GGG EVQP GGS LRL S CAA SGF T
FL Ay WM=

SUBSTITUTE SHEET (RULE 26) CCTCCGGATT
CACCTTTTTG GCTTATTGGA
=VWVRQA PGKG LEW VSS ISPS GTH TYY AD
SV KGR=

CGGGTACGCA TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLR VED TAVYY
CA KYT

TGTCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAATAIACT
EPGL GSF DYW GQGT LVTVSS

ID NO.:

DO!-1.39 - SEQ ID NO.: 295 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FS NYEM=

CCTCCGGATT
CACCTTTTCG AATTATGAGA
=GWVRQA PG KG LEW VSV ISEV GSL TYY AD
SV KGR=

TGGGTTCTCT GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KPH

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAACCTCAT
DSSI GFD YWG QGTL VTV SS

NO.:

DOM-141 SEQ ID NO.: 296 DIQMTQS PSS LSAS VGD RVT ITCR ASQW
IG DTLT=
= 223 SUBSTITUTE SHEET (RULE 26) GGGCAAGTCA
GTGGATTGGG GATACGTTAA
=SYQ QKL GKAP KLL IYG GSEL QSG VPP RF
SG SGS=

TGCAAAGTGG GGTCCCACCA
CGTTTCAGTG GCAGTGGATC
=GTD FTLT ISS LOP TD FA TYY CQQ CISSP
CT FGQ

CTGTCAACAG TGTANTAGTA
GTCCTTGTAC GTTCGGCCAA

301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 411 DOM-142 SEQ ID NO.: 297 DI QM TQS PSS LSAS VGD RVT ITCRASQF
IG DSLS=

GGGCAAGTCA
GTTTATTGGT GATTCTTTAT
=wyQ QKP GKAP KLL IYF SSIL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTD FT LT ISS LQP EDFA TYY CQQ YHTSP
TT FGR

CTGTCAACAG TATCATACTT
CGCCTACTAC GTTCGGCCGA
GTKV K.IKR
301 GGGACCAAGG TGAAAATCAA ACGG SEQ ID NO.: 412 DOM-143 - SEQ ID NO.: 298 IE TNLE=

SUBSTITUTE SHEET (RULE 26) GGGCAAGTCA
GACTATTGAG ACTAATTTAG
=WYQ QKP GKAP KLL IYD SSQL QSG VPS RP
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTD FTLT ISS LQP EDLA TYY CQQ YHGY= P
TT FGQ

CTGTCAACAG TATCATGGGT
ATCCTACGAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 413 DOM-144 - SEQ ID NO.: 299 DIQM TQS PSS LSAS VGD RVT ITCR ASQM
ID QDLE=

GGGCAAGTCA
GATGATTGAT CAGGATTTAG
=WYQ QKP GKAP KLL IYN ASWL QSG VPS RF
SG SGS=

CGTTTCAGCG GCAGTGGATC
=GTD FTLT ISS LQP EDFA TYY CQQ YHGYP
IT FGQ

CTGTCAACAG TATCATGGTT
ATCCTATTAC GTTCGGCCAA
GTKV EIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 414 DOM-145 - SEQ ID NO.: 300 DIQMTQS PSS LSAS VGD RVT ITCR ASQT
IY TSLS=

SUBSTITUTE SHEET (RULE 26) 1 GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGAGA ccGTGTaAcc ATCACTTGCC
GGGCAAGTCA
GACGATTTAT ACTTCGTTAA
=WYQ QKP GKAP KIL THY GSVL QSG VPS RF
SGSGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=G TD FTLT ISS IQP EDSA TYY CQQ VHQAP
TT FGQ

CTGTCAACAG GTTCATCAGG
CTCCTACGAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG - SEQ ID NO.: 415 DOM-146 - SEQ ID NO.: 301 D IRMTQS PSS ISAS VGDRVT ITCRASQW
IG DS LA' ATCACTTGCC GGGCAAGTCA
GTGGATTGGG GATTCTTTAG
=WYQQKP GKAP KLL IYG ISEL QSG VPS RF
SGSGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTD FTIT ISS LQP EDSA TYY CQL SSSMP
HT FGQ

CTGTCAACTG TCTAGTAGTA
TGCCTCATAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 416 DOM-147 - SEQ ID NO.: 302 D IQMTQS PSS LSAS VGD RVT ITCR ASQE
IE TNIE.

SUBSTITUTE SHEET (RULE 26) ATCACTTGCC GGGCAAGTCA
GGAGATTGAG ACGAATTTAG
=WYQ QKP GKAP KLL IYD SSHL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTD FTLT ISS LQP EDFA TYY CQQ YHQNP
PT FGQ

CTGTCAACAG TATCATCAGA
ATCCTCCGAC GTTCGGCCAA
GTKVEIKR
301 GGAACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 417 DOM-149 - SEQ ID NO.: 303 DIQM TQS PSS ',SAS VGD RVT ITCR ASQW
IG RQLV.

GGGCAAGTCA
GTGGATTGGG AGGCAGTTAG
=WYQ QKP GKAP KLL IYG ATEL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTTAGTG GCAGTGGATC
=GTD FT LT ISS LQP EDFA TYY CQQ QSKGP
LT FGH

CTGTCAACAG CAGTCGAAGG
GTCCTCTTAC GTTCGGCCAT
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 418 DOM-150 - SEQ ID NO.: 304 DI QM TQS PSS ISAS VGD RVT ITCR ASQG
IG TDIN=

SUBSTITUTE SHEET (RULE 26) ATCACTTGCC GGGCAAGTCA
GGGGATTGGT ACTGATTTAA
=WYQQKP GKAP KLL IYM GSYL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTDFTLT ISS LQP EDFATYY CQQ lYsFp IT FGQ

CTGTCAACAG ATTTATTCTT
TTCCTATTAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 419 DOM-154 - SEQ ID NO.: 305 DIQMTQS PSS LSAS VGD RVT.ITCR ASQD
IE EMLH=

ATCACTTGCC GGGCAAGTCA
GGATATTGAG GAGATGTTAC
=WYQQKP GKAP KLL IYF GSLL QSG VPS RP
SG SRS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTAGATC
=GTDFTLT ISS LQP EDFATYY CQQ HHTRP
YT FGQ

CTGTCAACAG CATCATACTC
GTCCTTATAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 420 DOM-155 - SEQ ID NO.: 306 D IQMTQS PSS LSAS VGD RVT ITCR ASQD
IG MDLE=
=
=

SUBSTITUTE SHEET (RULE 26) ATCACTTGCC GGGCAAGTCA
GGATATTGGG ATGGATTTAG
=WYQ QIP GKVP KIL IYDASYLQSG VPS RF
SGSGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=G TD FTLT ISS LOP EDFA TYY CQQ YRKLP
AT FGQ

CTGTCAACAG TATCATAAGC
TTCCTGCGAC GTTTGGCCAA
GTKVEI'KR
301 GGGACCAAGG TGGAAATCAA ACGG " SEQ ID NO.: 421 DOM-156 - SEQ ID NO.: 307 DIQMTQS PSS LSAS VGD RVT ITCR ASQD
IM DNLE=

ATCACTTGCC GGGCAAGTCA
GGATATTANG GATAATTTAG
=WYQQKP GKAP KLL IYAASWL QSG VPS RF
SGSGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTD FTLT ISS LQP EDFA TYY CQQ YHKLP
VT FGQ

TGCCTGTGAC GTTCGGCCAA
GTKV EIKR
301 GGGACCAAGG TGGAAATCAA AeGG SEQ ID NO.: 422 DOM-157 - SEQ ID NO.: 308 DIQMTQS PSS LSAS VGD RVT ITCR ASQN
IG EDLE=

SUBSTITUTE SHEET (RULE 26) GAGCAAGTCA
GAATATTGGG GAGGATTTAG
=WYQQKP GNAp KLL IYS ASHL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTDFTLT ISS LQP EDFATYY CQQ YSSYP
VT FGQ

CTGTCAACAG TATTCTAGTT
ATCCTGTTAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 423 DOM-158 - SEQ ID NO.: 309 DIQMTQS PSS LSAS VGDRVT ITCRASQP
ID EDLE=

ATCACTTGCC GGGCAAGTCA
GCCGATTGAT GAGGATTTAG
=WYQ QKP GNAP KLL IYSASYL QSG VPS RF
SGSGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGC,ATC
=GTDFTLT ISR LQP EDFATYY CQQ YHLLP
AT FGQ

CTGTCAACAG TATCATCTTC
TGCCTGCTAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 424 DOM-159 - SEQ ID NO.: 310 D IQMIQS PSS LSAS VGDRVT ITCRASQD
IN EDLE=

SUBSTITUTE SHEET (RULE 26) ATCACTTGCC GGGCAAGTCA
GGATANTAAT GAGGATTTAG
=WYQQKP GKAP KLL IYNASMLQSG VPS RF
SG SGS=

TGCAAAGCGG GGTCCCANCA
CGTTTCAGTG GCAGTGGATC
=G TD FTLT ISS LOP KDFA TYY CQQ YHTNP
TT FGQ

CTGTCAACAG TATCPaACTA
ATCCTACTAC GTTCGGCCAA
GTKV EIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 425 DOM-160 - SEQ ID NO.: 311 DIQMTQS PSS LSAS VGD RVT ITCR ASQD
IE ADLE=

ATCACTTGCC GGGCAAGTCA
GGATATTGAG GCGGATTTAG
=WYQ QKP GKAP KLL IYH SSEL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GAAGTGGATC
=GTDFTLT ISS LQP EDFA TYY CQQ YHMSP
VT FGQ

CTGTCAACAG TATCATATGT
CGCCTGTGAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: ,426 DOM-161 - SEQ ID NO.: 312 ID SDLE=

SUBSTITUTE SHEET (RULE 26) ATCACTTGCC GGGCAAGTCA
GGATATTGAT AGTGATTTAG
-WYQ QKP GKAP MLL IYS SEDL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
=
CGTTTCAGTG GCAGTGGATC
=G TD FTLT ISS IQP EDFA TYY CQQ YHSLP
/T FGQ

CTGTCAACAG TATCATAGTC
TGCCTGTTAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 427 DOM-162 - SEQ ID NO.: 313 DIQMTQS PSS LSAS VGD RVT ITCR ASQD
IS DDLE=

ATCACTTGCC GGGCAAGTCA
GGATATTTCG GATGATTTAG
=WYQ QKP GKAP KLL IYN SSFL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GAD FT LT ISS LQP EDFATYY CQQ YHSLP
VT FGQ

CTGTCAACAG TATCATAGTT
TGCCTGTTAC GTTCGGCCAA
GTKV EIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 428 =
DOM-163 - SEQ ID NO.: 314 DIQMTQS PSS LSAS VGD RVT ITCR ASQD
IE GNLE=
=

SUBSTITUTE SHEET (RULE 26) ATCACTTGCC GGGCAAGTCA
GGATATTGAG GGTAATTTAG
=WYQ QKP GKAP KLL IYD SSQL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGGTC
=GTD FTLT ISS LQP EDFA TYY CQQ YHHLP
TT FGQ

CTGTCAACAG TATCATCATC
TTCCTACGAC GTTCGGCCAA
GTKV EIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ 21) NO. : 429 DOM-164 - SEQ ID NO.: 315 DI QM TQS PSS LSAS VGD RVT ITCRASQS
ID TDLE=
J. GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGAGA CCGTGTCACC
ATCACTTGCC GGGCAAGTCA
GAGTATTGAT ACGGATTTAG
=WYQ QKP GKAP KLL IYD GSWL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTD_FTLT ISS LQP EDFA TYY CQQ YRWIP
VT FGQ

CTGTCAACAG TATCGGTGGA
TTCCTGTTAC GTTCGGCCAA
GTRV EIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 430 DOM-165 - SEQ ID NO.: 316 DI QM TQS PSS LSAS VGD RVT ITCRASQS
IS TDLE.

SUBSTITUTE SHEET (RULE 26) ATCACTTGCC GGGCAAGTCA
GAGTATTAGT ACTGAITTAG
=WyQ QKL GKAP KLL IYDASLL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTD FTLT ISS LQP EDFA TYY CQQ YSSLP
/T FGQ

CTGTCAACAG TATTCGAGTC
TGCCTGTTAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 431 DO1-166 - SEQ ID NO.: 317 IT TSLE=

ATCAETTGCC GGGCAAGTCA
GCCTATTACG ACGTCTTTAG
=WYQ QKP GKAP KLL IYD ASML QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCANCA
CGTTTCAGTG GCAGTGGANC
=G TD FTLT ISS LQP EDFA TYY CQQ YWVTP
VT FGQ

CTGTCAACAG TATTGGGTTA
CGCCTGTTAC GTTCGGCCAA
GTKV EIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 432 DO-167 - SEQ ID NO.: 318 D IQMTQS PSS LSAS VGD RVT ITCRASQN
IH TNLE=
= 234 SUBSTITUTE SHEET (RULE 26) ATCACCTGCC GGGCAAGTCA
GAATATTCAT ACGAATTTAG
=WYQ QKP GKAP KLL IYD GSML QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
'G TD FT LT ISS LQP EDFA TYY CQQ YSANP
/T FGQ

CTGTCAACAG TATTCGGCTA
ATCCTGTTAC GTTCGGCCAA
GTKVGIKR
301 GGGACCAAGG TGGGAATCAA ACGG SEQ ID NO.: 433 DOM-168 - SEQ ID NO.: 319 DIQMTQS PSS LSAS VGD RVT ITCRASQW
IN TDLE=

GGGCAAGTCA
GTGGATTCAT ACGGATTTAG
=WYQ QKP GKAP KLL IYD GSML QSG VPS RP
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=G TD FT LT ISS LQP EDFA TYY CQQ YSVSP
VT FGQ

CTGTCAACAG TATAGTGTGT
CGCCTGTTAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 434 DOM-169 - SEQ ID NO.: 320 DIQMTQS PSS LSAS VGD RVT ITCR ASQS
ID NNLE=
=

SUBSTITUTE SHEET (RULE 26) ATCACTTGCC GGGCAAGTCA
GAGTATTGAT AATAATTTAG
'WYQ QKPGEAP KLL IYDGSLL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
-GTD FTLT ISS LQP EDFATYY CQQ YHLHP
/T FGQ

CTGTCAACAG TATCATCTTC
ATCCTGTTAC GTTCGGCCAA
GTKV EIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 435 DOM-170 - SEQ ID NO.: 321 DI QM TQS PSS L SAS VGD RV T I T CR AS Q D
ID TNLE=

GGGCAAGTCA
GGATATTGAT ACGAATTTAG
=WYQ QKP GEAP KLL IYD RSTL QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTDFTLT ISS LQP EDFA TYY CQQ YDSYP
VT FGQ

CTGTCAACAG TATGATTCTT
ATCCTGTGAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 436 DOM-171 - SEQ ID NO.: 322 DIQMTQS PSS ISAS VGD RVT ITCR ASQS
IE SNLE.

SUBSTITUTE SHEET (RULE 26) GGGCAAGTCA
GTCTATTGAG TCTAATTTAG
=WYQ QKP GKAP KII IYN ASELQSG VPS RF
SG SGS.

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTD FTIT ISS LAP EDFA TYY CQQ YDQWP
TT FGQ

CTGTCAACAG TATGATCAGT
GGCCTACGAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 437 DO-172 - SEQ ID NO.: 323 DIQMTQS PSS ISAS VGD RVT ITCR ASQA
IG NTIR=

GGGCAAGTCA
GGCTATTGGT AATACTTTAC
=WYQ QKPGKAP KLL IYL SSRLQSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTD FT LT ISS LQP EDFA TYY CQQ LKKPP
YT FGQ

CTGTCAACAG CTGAAGAAGC
CTCCTTATAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 438 DO-173 - SEQ ID NO.: 324 DIQMTQS PSS ISAS VGD RVT ITCRASQK
IK NRIA.

SUBSTITUTE SHEET (RULE 26) a GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGAGA CCGTGTCACC
ATCACTTGCC GGGCARGTCA
GAAGATTAAG AATCGGTTAG
=WYQ QKP GKAPKLL IYE VSHL QSGVPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTD FTLT IGS LQP EDFA TYY CQQ RRQSP
YT EGQ

CTGTCAACAG AGGAGGCAGT
CGCCTTATAC GTTCGGCCAA
GTKV EIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 439 DO¨L74 ¨ SEQ ID NO.: 325 DIQM TQS PSS LSASVGD RVT ITCRASED
IG EELF=

GGGCAAGTGA
GGATATTGGG GAGGAGTTAT
=WYQ QKP GKAP KLL IYS As TL QSE VPS RF
SG SGS=

TGCAAAGTGA GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTD FT LT ISS LQH EDFA TYY CQQ VYEWP
YT FGQ

CTGTCAACAG GTTTATGAGT
GGCCTTATAC GTTCGGCCAA
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG SEQ ID NO.: 440 DO-175 ¨ SEQ ID NO.: 326 DIQMTQS PSS LSAS VGD RVT ITCRASQP
IS GGLR=

GGGCAAGTCA
GCCTATTTCT GGGGGTTTAA

SUBSTITUTE SHEET (RULE 26) =WYQ QKP GKAP KLL IYS TSML QSG VPS RF
SG SGS=

TGCAAAGTGG GGTCCCATCA
CGTTTCAGTG GCAGTGGATC
=GTID FTLT ISS LOP EDFA TYY CQQ LYSAP
YT FGQ

CTGTCAACAG CTTTATTCTG
CTCCTTATAC GTTCGGCCAA
=
GTKVEIKR
301 GGGACCAAGG TGGAAATCAA ACGG - SEQ ID NO.: 441 DOM-176 - SEQ ID NO.: 327 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FD AYEM=

CCTCCGGATT
CACCTTTGAT GCGTATGAGA
=GWVRQAPGKG LEWVSI IDWD GNS TYY AD
SV KGR=

ATGGTAATTC TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KPG

TGCCGAGGAT ACCGCGGTAT
ATTACTGTGC GAAACCTGGG
DNVG IFD YWG QGTL VTV SS

NO.:

DOM-177 - SEQ ID NO.: 328 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FS NYYM=

CCTCCGGATT
CACCTTTAGT AATTATTATA

SUBSTITUTE SHEET (RULE 26) =VWV RQA PGKG LEW VSAIDEW GFA TYY AD
SVKGR=

GGGGTTTTGC GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLR AED TAVYY
CA KHW

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAACATTGG
EFTS DTS RFD YWGQ GTL VTV SS

TCGAGC - SEQ ID
NO.: 443 DOM-178 - SEQ ID NO.: 329 EVQL LES GGG LVQP GGS LRI SCAA SGFT
FE DEDM=

CCTCCGGATT
CACCTTTGAG GATTTTGATA
=AWV RQA PGKG LEW VSS INDQ GSL TYY AD
SVKGR=
101 TGGCTTGGGT CCGCCAGGCT CCAGGGAAGG GTCTAGAGTG GGTCTCAfCT ATTAATGATC
AGGGTTCTCT GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KPD

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAACCGGAT
QFFD YWG QGT LVTV SS
301 CAGTTTTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC SEQ ID NO.:

DOM-179 - SEQ ID NO.: 330 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FS AYDM=

CCTCCGGATT
CACCTTTAGT GCTTATGATA

SUBSTITUTE SHEET (RULE 26) =MWVRQA PGKG LEW VSR ISPQ GQR TYY AD
SVICGR=

AGGGTCAGCG GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SR DM SEM TLY LQMN SLR AED TAVYy CA KR

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAAATTCGT
GQSR IPM RFD YWGQ GTLV
301 GGGCAGTCGC GGATTCCTAT GAGGTTTGAC TACTGGGGTC AGGGAACCCT GGTC - SEQ ID NO.:

DOM-180 - SEQ ID NO.: 331 EVQLLES GGG LVQP GGS LRL SCAA SGFT
FT DYEM.

CCTCCGGATT
CACCTTMACG GATTATGAGA
=GWVRQA PGKG LEW VST ITSL GES TYY AD
SV KGR=

TGGGTGAGAG TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA I< PG
201 GTTCACCATC TCCCGCGACA AfTCCAAGAA CACGCTGTAT CTGCAAATGA ACAGCCTGCG
TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAACCTGGT
RIFD YWG QGT LVTV SS
301 CGTATTTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC SEQ ID NO.:

DOM-181 - SEQ ID NO.: 332 EVQLLES GGG LVQP GGS LRL SCAA SGFT
FA FYPM.

CCTCCGGATT
CACCTTTGCT TTTTATCCTA

SUBSTITUTE SHEET (RULE 26) =MWV RQA PGKG LEW VSW IDAT GTR TYY AD
SV KGR=

CGGGTACGAG GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SRN TLY LQMN SLRAED TAVYY
CA EGN

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GGAAGGTAAT
YGSS YTM GVF DYWG QGT LVT VSS

GTCTCGAGC SEQ ID
NO.: 447 DOM-182 - SEQ ID NO.: 333 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FD EYPM=

CCTCCGGATT
CACCTTTGAT GAGTATCCGA
=YWV RQA PGKG LEW VSS IGPS GPN TYY AD
SV KGR=

CTGGTCCGAA TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLR AED TAVYY
CA KSP

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAATCTCCG
YFDV IPS YFDYWGQ GTLVTV SS

SEQ ID
NO.: 448 DOM-183 - SEQ ID NO.: 334 EVQL LES GGG LVQP GGS LRL S CAA SGF T
FA DY GM =

CCTCCGGATT
CACCTTTGCG GATTACGGTA

SUBSTITUTE SHEET (RULE 26) =GWV RQA PGKG LEWVSS IQSS GLR TYY AD
SV KGR=

CGGGTTTGCG GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SK14 TLY LQMN SLRAED TAVYY
CA KRA
201 GTTCACCATC TCCCGCGACA AfTCCAAGAA CACGCTGTAT CTGCAAATGA ACAGCCTGCG
TGCCGAGGAT ACCGCGGTAT
ATTACTGTGC GAAACGGGCT
NSRR GFD YWG QGTL VTV SS

NO.:

DOM-184 - SEQ ID NO.: 335 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FS DYEM=

CCTCCGGATT
CACCTTTTCT GATTATGAGA
=MWVRQA PGKG LEWVSS ITSH GGS TYY AD
SVKGR=

AfGGTGGGTC TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLR AED TAVYY
CAKPD

TGCCGAGGAT ACCGCGGTAT
ATTACTGTGC GAAACCTGAT
KDFD YWG QGT LVTV SS
301 AAGGATTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC SEQ ID NO.:

DOM-185 - SEQ ID NO.: 336 EVQLLES GGG LVQP GGS LRL SCAASGFT
FA HYPM-CCTCCGGATT
CACCTTTGCG CATTATCCGA

SUBSTITUTE SHEET (RULE 26) =SWVRQA PGKG LEW VSS IGRL GNR TYY AD
SV KGR=

TGGGTAATCG TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLR AED TAVYY
CA KRA

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAACGTGCT
TPVP IKG LFD YWGQ GTLVTV SS

SEQ ID
NO.: 451 DO¨i.86 ¨ SEQ ID NO.: 337 EVQL LES GGG LVQP GGS LRL SCAR SGLT
FG RYEM=

CCTCCGGACT
CACCTTTGGG AGGTATGAGA
=AWVRQA PGKG LEWVSS IDSD GWV TYY AD
s V KG R=

ATGGTTGGGT GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA QPD

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GCAACCGGAT
SLED YWG QGT LVTV SS
301 TCGTTGTTTG ACTACTGGGG TCAGGGAACC CTGGTCACCG TCTCGAGC SEQ ID NO.:

DOM-187 ¨ SEQ ID NO.: 338 EVQLLES GGG LVQP GGS LRL SCAA SGFT
FS SYSM=

CCTCCGGATT
CACCTTTTCTAGTTATTCTA

SUBSTITUTE SHEET (RULE 26) =VWV RQA PG KG LEN VSG INRG GTR TYY AD
SV KGR=

TGGTGTGGGT CCGCCAGGCT CCAGGGAAGG GTCTAGAGTG GGTCTCAGGT ATTAATCGGG GTGGTACTCG
TACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKR TLY LQMN SLRAED TAVYY
CA KGW

GTTCACCATC TCCCGCGACA ATTCCAAGAA CACGCTGTAT CTGCAAATGA ACAGCCTGCG TGCCGAGGAC
ACCGCGGTAT
ATTACTGTGC GAAAGGTTGG
ARGF DYW GQG TLVTVSS
301 AGGAGGGGGT TTGACTACTG GGGTCAGGGA ACCCTGGTCA CCGTCTCGAG C SEQ ID NO.:

DOM-188 - SEQ ID NO.: 339 EVQL LES GGG LVQP GGS LRL SCAA SGFT
FT RYRM=

CCTCCGGATT
CACCTTTACG CGTTATAGGA
=SWVAQA PGKG LEWVSG ISRD GYR TYY AD
SV KGR=

ATGGTTATCG GACATACTAC
GCAGACTCCG TGAAGGGCCG
=FTI SRDN SKN TLY LQMN SLRAED TAVYY
CA KGM

TGCCGAGGAC ACCGCGGTAT
ATTACTGTGC GAAAGGTATG
TASF DYW GQG TLVT VSS

NO.: 454 DO-189 - SEQ ID NO.: 340 EVQLLES GGG LVQP GGS LRL SCAA SGFT
FQ MYPM.

CCTCCGGATT
CACCTTTCAG ATGTATCCGA

SUBSTITUTE SHEET (RULE 26) DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.

NOTE. Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.

NOTE For additional volumes please contact the Canadian Patent Office.

Claims (55)

CLAIMS:

1. Use of an antibody polypeptide comprising an antibody single variable domain polypeptide in the preparation of a medicament for treating or preventing a symptom of autoimmune disease, wherein said single variable domain polypeptide is monovalent for binding to CD40L and antagonizes an activity of CD40 or CD40L or both, and wherein said antibody polypeptide inhibits the binding of an antibody single variable domain comprising the amino acid sequence of SEQ ID NO: 26 to CD40L.

2. Use of an antibody polypeptide comprising an antibody single variable domain polypeptide for treating or preventing a symptom of autoimmune disease, wherein said single variable domain polypeptide is monovalent for binding to CD40L and antagonizes an activity of CD40 or CD40L or both, and wherein said antibody polypeptide inhibits the binding of an antibody single variable domain comprising the amino acid sequence of SEQ ID
NO: 26 to CD40L.

3. The use according to claim 1 or 2 wherein said autoimmune disease is a disease selected from the group consisting of systemic lupus erythematosis, multiple sclerosis, rheumatoid arthritis, diabetes, allograft rejection and xenograft transplant rejection.

4. The use according to any one of claims 1 to 3 wherein said antibody polypeptide inhibits the binding of CD40L to CD40.

5. The use according to any one of claims 1 to 4 wherein the antibody polypeptide consists of the single variable domain polypeptide.

6. The use according to any one of claims 1 to 5 wherein said single variable domain polypeptide is a human antibody single variable domain polypeptide.

7. The use according to any one of claims 1 to 6 wherein said single variable domain polypeptide is a VH or VL domain.

8. The use according to any one of claims 1 to 7 wherein said antibody polypeptide inhibits binding of CD40L to CD40 with an IC50 in the range of 20 pM
to 100 nM.

9. The use according to any one of claims 1 to 8 wherein said single variable domain polypeptide comprises the amino acid sequence of CDR1, CDR2 and CDR3 of SEQ ID NO: 26.

10. The use according to any one of claims 1 to 9 wherein said antibody polypeptide has an amino acid sequence that is at least 80% homologous to SEQ
ID NO: 26.

11. The use according to any one of claims 1 to 10 wherein said antibody polypeptide has an amino acid sequence that is identical to SEQ ID NO: 26.

12. The use according to any one of claims 1 to 11 wherein binding of said antibody polypeptide to CD40L does not agonize CD40 or CD40L activity.

13. The use according to any one of claims 1 to 12 wherein the presence of said antibody polypeptide in a standard platelet aggregation assay does not result in aggregation of more than 25% over the aggregation observed in a negative control assay.

14. The use according to any one of claims 1 to 13 wherein the single variable domain polypeptide is present as a homo- or heteromultimer with additional V H
or V L
domains, wherein the single variable domain polypeptide binds CD40L
independently of the additional V H or V L domains.

15. An antibody polypeptide comprising or consisting of an antibody single variable domain which specifically and monovalently binds CD40L, wherein said polypeptide inhibits the binding of CD40L to CD40, wherein binding of said antibody polypeptide to CD40L does not agonize CD40 or CD40L activity, and wherein said antibody polypeptide inhibits the binding of an antibody single variable domain comprising the amino acid sequence of SEQ ID NO: 26 to CD40L.

16. An antibody polypeptide consisting of an antibody single variable domain which specifically and monovalently binds CD40L, wherein said polypeptide inhibits the binding of CD40L to CD40, wherein binding of said antibody polypeptide to CD40L does not agonize CD40 or CD40L activity, and wherein said antibody polypeptide inhibits the binding of an antibody single variable domain comprising the amino acid sequence of SEQ ID NO: 26 to CD40L.

17. The antibody polypeptide of claim 15 or 16 which has an amino acid sequence at least 85% identical to a sequence selected from the group consisting of SEQ
ID NOs 7-82 and SEQ ID NOs 246-360.

18. The antibody polypeptide of claim 15 or 16 wherein binding of said antibody polypeptide to CD40L does not induce JNK phosphorylation in Jurkat T-cells.

19. The antibody polypeptide of claim 15 or 16 wherein binding of said antibody polypeptide to CD40L does not induce IFN-.gamma. secretion by Jurkat T-cells co-stimulated with anti-CD3 antibody.

20. The antibody polypeptide of claim 15 or 16 wherein the presence of said antibody polypeptide in a standard platelet aggregation assay does not result in aggregation of more than 25% over the aggregation observed in a negative control assay.

21. The antibody polypeptide of claim 15 or 16 which is PEG-linked.

22. The PEG-linked antibody polypeptide of claim 21 which has a hydrodynamic size of at least 24 kD.

23. The PEG-linked antibody polypeptide of claim 21 wherein said PEG is linked to said antibody at a cysteine or lysine residue.

24. The PEG-linked antibody polypeptide of claim 21 wherein the total PEG
size is from 20 to 60 kD, inclusive.

25. The PEG-linked antibody polypeptide of claim 21 which has a hydrodynamic size of at least 200 kD.

26. The PEG-linked antibody polypeptide of claim 22 which has an increased in vivo half-life relative to the same polypeptide composition lacking linked polyethylene glycol.

27. The PEG-linked antibody polypeptide of claim 21 wherein the t.alpha.-half life of the polypeptide composition is increased by 10% or more.

28. The PEG-linked antibody polypeptide of claim 26 wherein the t.alpha.-half life of the polypeptide composition is in the range of 0.25 minutes to 12 hours.

29. The PEG-linked antibody polypeptide of claim 26 wherein the t.beta.-half life of the polypeptide composition is increased by 10% or more.

30. The PEG-linked antibody polypeptide of claim 26 wherein the t.beta.-half life is in the range of 12 to 48 hours.

31. The antibody polypeptide of claim 15 which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 7-82 and 246-360.

32. The antibody polypeptide of claim 15 which is free of an Fc domain.

33. The antibody polypeptide of claim 15 which inhibits binding of CD40L to CD40 with an IC50 in the range of 20 pM to 100 nM, inclusive.

34. The antibody polypeptide of claim 15 which inhibits the binding of CD40 to CD40L with an IC50 in the range of 20 pM to 100 nM, inclusive.

35. The antibody polypeptide according to claim 15, wherein the antibody polypeptide comprises one or more framework regions comprising an amino acid sequence that is the same as the amino acid sequence of a corresponding framework region encoded by a human germline antibody gene segment, or the amino acid sequence of one or more of said framework regions collectively comprises up to 5 amino acid differences relative to the amino acid sequence of said corresponding framework region encoded by a human germline antibody gene segment.

36. The antibody polypeptide according to claim 15, wherein the amino acid sequences of FW1, FW2, FW3 and FW4 of the antibody polypeptide are the same as the amino acid sequences of corresponding framework regions encoded by a human germline antibody gene segment, or the amino acid sequences of FW1, FW2, FW3 and FW4 collectively contain up to 10 amino acid differences relative to the amino acid sequences of corresponding framework regions encoded by said human germline antibody gene segment.

37. The antibody polypeptide according to claim 36, wherein the amino acid sequences of said FW1, FW2 and FW3 of the antibody polypeptide are the same as the amino acid sequences of corresponding framework regions encoded by human germline antibody gene segments.

38. The antibody polypeptide according to claim 36, wherein said human germline antibody gene segment is selected from the group consisting of DP47, DP45, DP48 and DPK9.

39. The antibody polypeptide of claim 15 which comprises a single immunoglobulin variable domain which specifically and monovalently binds CD40L, and which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 7-82 and SEQ ID NOs 246-360.

40. The antibody polypeptide of claim 39 wherein the single variable domain polypeptide that binds to CD40L comprises the sequence of SEQ ID NO: 26.

41. The antibody polypeptide of claim 15 wherein the single variable domain polypeptide that binds to CD40L has an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 26 at no more than 25 amino acid positions and has a sequence that is at least 80% homologous to the sequence of SEQ ID NO: 26.

42. The antibody polypeptide of claim 39 comprising the sequence of CDR1, CDR2, and CDR3 of an antibody single variable domain polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos. 7-82 and 246-360.

43. A composition comprising the antibody polypeptide of any one of claims 15 to 42 and a second antibody polypeptide which binds a ligand other than CD40L, wherein said antibody polypeptide which binds a ligand other than CD40L binds a ligand selected from the group consisting of USA, TNF.alpha., IL-1, IL-2, IL-4, IL-6, IL-8, IL-12, IL-18, IFN-.gamma., CD2, CD4, CD8, LFA1, LFA3, VLA4, CD80, B7-1, CD28, CD86, B7-2, CTLA-4, CD28, Inducible costimulatory molecule (ICOS), CD27, CD30, OX40, CD45, CD69, CD3, CD70, Inducible costimulatory molecule ligand (ICOSL), OX40L, HVEM (Herpes Virus Entry Mediator), LIGHT.

44. An extended release pharmaceutical formulation comprising the antibody polypeptide of claim 15 together with a biodegradable, biocompatible polymer or an agent that delays adsorption.

45. The extended release pharmaceutical formulation of claim 44 comprising the antibody polypeptide of claim 15.

46. The extended release formulation of claim 44 wherein said antibody polypeptide consists of a single antibody variable domain that binds CD40L.

47. A dual specific ligand comprising an antibody polypeptide of claim 15 and a second single variable domain having a binding activity to a second antigen, wherein the second single variable domain is an Antigen Presenting Cell surface antigen or a T cell surface antigen.

48. A ligand according to claim 47 wherein the Antigen Presenting Cell surface antigen is selected from the group consisting of, activated macrophage surface antigens, activated B cell surface antigens, co-stimulatory signal pathway surface antigens, and MHC.

49. A ligand according to claim 48 wherein the MHC is class II.

50. A ligand according to claim 49 wherein the MHC class II is alpha.

51. A ligand according to claim 49 wherein the MHC class II is beta.

52. A ligand according to claim 48 wherein the Antigen Presenting Cell surface antigen is selected from the group consisting of CD28, Inducible costimulatory molecule (ICOS), CD27, CD30, OX40, CD45, CD69, CD3, CD70, Inducible costimulatory molecule ligand (ICOSL), OX4OL, CD80, CD86, HVEM (Herpes Virus Entry Mediator), and LIGHT.

53. A ligand according to claim 48 wherein the Antigen Presenting Cell surface antigen is selected from the group consisting of CD28, Inducible costimulatory molecule (ICOS), CD27, CD30, OX40, CD45, CD69, and CD3.

54. A ligand according to claim 48 wherein the activated B cell surface antigen is a B7 gene surface antigen.

55. A ligand according to claim 54 wherein the B7 gene surface antigen is B7-2, or B7-1.