MOLECULES THAT BIND TO CD137 AND PSMA

Abstract
The disclosure relates to agents that simultaneously bind CD137 and PSMA and which comprise a single domain antibody specific to CD137 and a moiety that binds PSMA. The disclosure also relates to therapeutic and diagnostic applications of such agents, for example in the treatment or detection of a cancer.
Description
INTRODUCTION

Cancer remains one of the leading causes of death in the world. Recent studies have shown an estimated 12.7 million cancer cases worldwide. This number is expected to increase to 21 million by 2030 (Vinay and Kwon 2014).


CD137 (4-1BB, TNFRS9) is a type 1 transmembrane glycoprotein belonging to the TNF receptor superfamily. It was originally cloned by Kwon et al (1989) from the cDNA of activated murine T cells. It has subsequently been shown to have a broad immune cell expression pattern found on T cells, B cells, NK and NK T cells, dendritic cells (DC), macrophages, neutrophils and eosinophils. Expression has also been reported on non-haematopoetic cells, for example epithelial, endothelial and smooth muscle cells and on tumour cell lines. CD137 expression is mainly activation induced, although low level constitutive expression has been demonstrated on some cell types including Tregs and DC's.


The 255 amino acid human CD137 protein (Genbank accession NP_001552) consists of a 17 amino acid signal peptide sequence, an extracellular region containing four cysteine rich domains, a 27 amino acid transmembrane region and a short 42 amino acid intracellular domain. It exists as both a monomer and dimer on the cell surface. The main ligand for CD137 is CD137 ligand (CD137L, 4-1BB-L, TNFS9), although interactions with galectin-9 which facilitates receptor aggregation (Madireddi et al 2014) and matrix proteins such as fibronectin (Chalupny et al, 1992) have also been reported. CD137 ligand is predominantly expressed on activated antigen presenting cells such as dendritic cells, B-cells and macrophages.


Interaction of the trimeric CD137 ligand with CD137 results in clustering of the receptor and recruitment of signalling molecules such as the TRAF family of proteins leading to kinase modulation and activation of the NfKB pathway. Thus, clustering of CD137 is crucial for initiation and regulation of downstream signalling.


Studies using agonist anti CD137 monoclonal antibodies in vitro and in vivo have shown that upon activation CD137 is rapidly internalised into an endosomal compartment termed the ‘signalosome’ from which it keeps signalling (reviewed in Sanchez-Paulete et al 2016).


Co-stimulatory TNFR family members such as CD137, CD27, OX40 (CD134), HVEM, CD30, and GITR are involved in sustaining the T cell responses after initial T-cell activation. In CD4+ and CD8+ T cells, CD137 acts as a costimulatory receptor that modulates T-cell receptor (TCR) mediated signalling. Ligation of CD137 together with TCR activation promotes proliferation, cytokine production, and inhibits apoptosis through induction of anti-apoptotic B-cell lymphoma-extra large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2) pathways. Cross-linking of CD137 on NK cells has been shown to stimulate IFN-gamma secretion and proliferation. Dendritic cell responses to CD137 stimulation include enhanced maturation and antigen presentation and secretion of cytokines IL-6, IL12—and IL-27 and enzymes such as indoleamine-2,3-dioxygenase (IDO) which can modulate T-cell function. CD137 can also upregulate intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) on tumor vascular endothelium, thus inducing effector cell migration and retention of the activated T-cells in the tumor microenvironment.


Cross linking of CD137 by anti CD137 antibodies has been shown to have potent anti-tumour effects in vivo in a number of models including sarcoma, mastocytoma, glioma, lymphoma, myeloma, and hepatocellular carcinoma. CD8+ cell depletion studies have demonstrated that this effect primarily involves cytolytic T cell expansion and infiltration resulting in tumour cell lysis. However, contributions of other types of cells such as DCs, NK-cells or CD4+ T-cells have been reported in some tumour models. Furthermore, anti CD137 therapy has been shown to trigger an immunologic memory response and to inhibit autoimmune reactions (reviewed in Vinay et al 2012).


It has been shown that existing agonistic therapies result in systemic CD137 effects leading to unwanted side effects. Activation of CD137 signalling has been associated with severe toxicity in murine models. Clinical trials of a fully human IgG4 anti CD137 agonistic antibody (Urelumab®, BMS-663513) reported neutropenia, elevated liver enzymes and at high doses severe hepatic toxicity resulting in trial termination. This severe toxicity has not been observed for a fully human IgG2 (PF-05082566) that is also in clinical trials both as a monotherapy and in combination therapy approaches.


Agonistic antibodies targeting co-stimulatory TNFRs have been shown to require engagement of FcγRs (Bulliard et al). Thus, non-targeted clustering via FcγRs may influence the mechanism by which agonistic antibodies act on these targets.


In light of the toxicity profile observed with existing therapies, there is a need for alternative cancer therapies based on the use of alternative CD137 binding molecules that have reduced toxicity. In particular, there is a clinical need for targeted CD137 agonists that effectively engage CD137 on the surface of cells and have reduced toxicity, including liver toxicity.


Prostate cancer is the most commonly diagnosed non-skin-related malignancy in males in developed countries. It is estimated that one in six males will be diagnosed with prostate cancer.


Current treatments for prostate cancer include surgery, radiation, and adjuvant hormonal therapy. Although these therapies are relatively effective in the early stages of disease, the majority of patients initially diagnosed with localized prostate cancer ultimately relapse. Whilst chemotherapy is one of the most widely used approaches in combating advanced prostate cancer, its therapeutic efficacy is usually insufficient due to lack of specificity and associated toxicity. Lack of targeted delivery to prostate cancer cells is one of the primary obstacles in achieving feasible therapeutic effect. Consequently, there remains a critical need for strategies to increase the selectivity of anti-prostate cancer agents (Barve et al).


The diagnosis of prostate cancer has greatly improved following the use of serum-based markers such as the prostate specific antigen (PSA). In addition, prostate tumour-associated antigens offer targets for tumour imaging, diagnosis, and targeted therapies. The prostate specific membrane antigen (PSMA), a prostate tumour associated marker, is such a target.


PSMA is a 750-residue type II transmembrane glycoprotein highly restricted to prostate secretory epithelial cell membranes. It is highly expressed in prostate cancer cells and in nonprostatic solid tumor neovasculature and other solid tumors and expressed at lower levels in other tissues, including healthy prostate, kidney, liver, small intestine, and brain. PSMA expression increases with prostate disease progression and metastasis and its expression level has thus been correlated with tumour aggressiveness. Various immunohistological studies have demonstrated increased PSMA levels in virtually all cases of prostatic carcinoma compared to those levels in benign prostate epithelial cells. Intense PSMA staining is found in all stages of the disease, including prostatic intraepithelial neoplasia, late stage androgen-independent prostate cancer and secondary prostate tumours localized to lymph nodes, bone, soft tissue, and lungs. PSMA is thus widely used as a biomarker for prostate cancer cells.


PSMA has a 3-part structure: a 19-amino-acid internal portion, a 24-amino-acid transmembrane portion, and a 707-amino-acid external portion. It forms a noncovalent homodimer that possesses glutamate carboxypeptidase activity based on its ability to process the neuropeptide N-acetylaspartylglutamate and glutamate-conjugated folate derivatives. PSMA is rapidly and efficiently internalized by an endocytic pathway and rapidly recycles back to the membrane.


The invention addresses the need for alternative antibody-based treatments for use in the treatment of a cancer.


SUMMARY

The invention relates to novel binding molecules with specificity for both CD137 and PSMA. The inventors have identified single variable heavy chain domain antibodies that bind to CD137 and inhibit binding of CD137L to CD137. They do not cause CD137 signalling when bound to CD137 in monospecific format, that is without being linked to another moiety that binds a second target. However, when linked to a moiety that binds a tumor specific antigen, the single variable heavy chain domain antibodies elicit CD137 signalling. Thus, whilst the single variable heavy chain domain antibodies that bind to CD137 do not induce clustering of the receptor and do not have agonistic activity when bound to CD137 without a binding partner that targets a second antigen, the dual engagement of CD137 and a tumor specific antigen in a bispecific molecule leads to CD137 agonism. The single variable heavy chain domain antibodies that bind to CD137 can therefore be used as a subunit in a multispecific binding molecule that simultaneously engages CD137 and PSMA.


Bi- and multispecific molecules described herein bind to CD137 and PSMA and simultaneously engage both targets. This dual engagement results in CD137 activation, thus restricting the site of action to the tumor microenvironment and potentially minimising undesirable effects of existing CD137 therapies.


In one aspect, there is provided an isolated binding molecule comprising or consisting of a) a single variable heavy chain domain antibody that binds to CD137 and

    • b) a moiety that binds to PSMA.


In one embodiment, the single variable heavy chain domain antibody that binds to CD137 comprises a CDR1 comprising SEQ ID NO. 1 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 2 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 3 or a sequence with at least 40% homology thereto or a CDR1 comprising SEQ ID NO. 425 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 426 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 427 or a sequence with at least 40% homology thereto.


In one embodiment, the single variable heavy chain domain antibody that binds to CD137 comprises human framework regions.


In one embodiment, the single variable heavy chain domain antibody that binds to CD137 comprises a full length sequence as listed in table 2 or 3 or a sequence with at least 75% homology thereto. In one embodiment, the single variable heavy chain domain antibody that binds to CD137 comprises or consists of one of the following sequences: SEQ ID NO. 4, 312, 428, 852, 856, 860, 864, 868, 872, 876 or 880 or a sequence with at least 50% homology thereto.


In one embodiment, the moiety that binds to PSMA is selected from an antibody, an antibody fragment, an antibody mimetic, a protein that mimics the natural ligand of CD137 or other polypeptide.


In one embodiment, said antibody fragment is selected from a Fab, F(ab′)2, Fv, a single chain Fv fragment (scFv), a single domain antibody or fragment thereof.


In one embodiment, said single domain antibody is a single VH domain antibody.


In one embodiment, said single VH domain antibody comprises a CDR1 comprising SEQ ID NO. 812 or a sequence with at least 75% homology thereto, a CDR2 comprising SEQ ID NO. 813 or a sequence with at least 75% homology thereto and a CDR3 comprising SEQ ID NO. 814 or a sequence with at least 75% homology thereto or wherein said VH single domain antibody comprises a CDR1 comprising SEQ ID NO. 837 or a sequence with at least 75% homology thereto, a CDR2 comprising SEQ ID NO. 838 or a sequence with at least 75% homology thereto and a CDR3 comprising SEQ ID NO. 839 or a sequence with at least 75% homology thereto.


In one embodiment, the single variable heavy chain domain antibody that binds to PSMA comprises a full length sequence as listed in table 6 or 7 or a sequence with at least 50% homology thereto.


In one embodiment, the single variable heavy chain domain antibody moiety that binds to PSMA comprises SEQ ID NO. 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825 or 840 or a sequence with at least 50% homology thereto.


In one embodiment, a single domain antibody 1.1 or 2.1 having one of SEQ ID No. 4, 312, 852, 856, 860, 864, 868, 872, 876, 880 or 428 or a variant t (e.g. a molecules with 1 to 10 or 1 to 20 amino acid substitutions) hereof is linked to a single domain antibody 3.1, 3.8 or 4.1 having SEQ ID No. 815, 822 or 840 respectively.


In one embodiment, the isolated binding molecule is capable of binding CD137 with an affinity with a Kd of about of at least about 10-6 M, alternatively at least about 10-7 M, alternatively at least about 10-8 M, alternatively at least about 10-9 M, alternatively at least about 10-10 M, alternatively at least about 10-11 M, alternatively at least about 10-12 M, or greater affinity.


In one embodiment, the isolated binding molecule is capable of binding PSMA with an affinity with a Kd of about of at least about 10-6 M, alternatively at least about 10-7 M, alternatively at least about 10-8 M, alternatively at least about 10-9 M, alternatively at least about 10-10 M, alternatively at least about 10-11 M, alternatively at least about 10-12 M, or greater affinity.


In one embodiment, the single variable heavy chain domain antibody that binds to CD137 is linked to the moiety that binds PSMA by a peptide linker.


In one embodiment, said linker is selected from a (G4S)n linker wherein n is 1 to 10.


An isolated binding molecule of the invention may comprise a single variable heavy chain domain antibody that binds to CD137 linked to a moiety that binds PSMA. In one embodiment, the isolated binding molecule is conjugated to a toxin, enzyme, radioisotope, half-life extending moiety, label, therapeutic molecule or other chemical moiety.


In one embodiment, said half-life extending moiety is selected from the group consisting of an albumin binding moiety, a transferrin binding moiety, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of human serum albumin, and an albumin binding peptide or single domain antibody that binds to human serum albumin.


Generally, the single variable heavy chain domain antibody which binds to human CD137 does not cause CD137 signalling when bound to CD137 as a monospecific entity.


In one embodiment, the single variable heavy chain domain antibody which binds to human CD137 is obtained or obtainable from a transgenic rodent that expresses a transgene comprising human V, D and J regions.


In one embodiment, said rodent, e.g. a mouse, does not produce functional endogenous light and heavy chains.


Also provided is a pharmaceutical composition comprising a binding molecule as described herein, e.g. comprising a single variable heavy chain domain antibody as described herein and a pharmaceutical carrier.


Also provided is a binding molecule or a pharmaceutical composition as described herein for use in the treatment of disease such as cancer, prostate cancer or a PSMA positive tumor.


Also provided is a method for treating cancer comprising administering a therapeutically effective amount of a binding molecule or a pharmaceutical composition as described herein.


In one embodiment, said cancer is prostate cancer lung cancer, glioblastoma, renal, bladder, testicular, neuroendocrine, colon, and breast cancer.


Also provided is a nucleic acid molecule comprising a nucleic acid sequence encoding the binding molecule as described herein.


Also provided is a vector comprising a nucleic acid molecule as described herein.


Also provided is a host cell comprising a nucleic acid molecule or a vector as described herein.


In one embodiment, the host cell is a bacterial, yeast, viral, plant or mammalian cell.


Also provided is a method for producing a binding molecule described herein comprising expressing a nucleic acid encoding said binding molecule in a host cell and isolating the binding molecule from the host cell.


Also provided is a method for promoting CD8+ T cell expansion, inducing activation of cytotoxic T lymphocytes (CTL) and/or cytokine release comprising administering a binding molecule or a pharmaceutical composition as described herein.


Also provided is an in vivo or in vitro method for reducing human PSMA activity comprising contacting human PSMA with a binding molecule as described above.


Also provided is a kit comprising a binding molecule as described herein or a pharmaceutical composition as described herein.


Also provided is a use of a binding molecule or a pharmaceutical composition as described above for activating, e.g. simultaneously activating, downstream signalling pathways of CD137 and PSMA.


Also provided is a method for activating, e.g. simultaneously activating, downstream signalling pathways of CD137 and PSMA comprising administering a binding molecule or a pharmaceutical composition as described herein.


Also provided is a method for dual engagement of CD137 and PSMA comprising administering a binding molecule or a pharmaceutical composition as described herein.


Also provided is a use of a binding molecule or a pharmaceutical composition as described herein for inducing a local T cell response in the vicinity of a PSMA positive tumor cell or tissue.


Also provided is a method for inducing a local T cell response in the vicinity of a PSMA positive tumor cell or tissue comprising administering a binding molecule or a pharmaceutical composition as described herein.





FIGURES

The invention is further described in the following non-limiting figures.



FIG. 1: Dual Binding Cell based ELISA.


CHO human PSMA expressing cells were seeded onto plates and monovalent VH or bispecific molecules added. CD137huFc was subsequently added and binding detected using anti human Fc-HRP. Only bispecific molecule showed increased binding signal confirming dual target binding.



FIG. 2: Activation of CD137 signalling in the Jurkat NF-kB Luciferase Reporter Assay.


(A) CHO PSMA cells, (B) DU145 PSMA cells or (C) DU145 parental cells (bispecific testing)/media only (antibody testing) were cultured with Jurkat human CD137 NF-kB-luciferase reporter cells. Relative luminescence signal (RLU) was measured as a readout of the luciferase reporter gene activity resulting from CD137 mediated activation of the NF-kB signalling pathway. Monovalent Humabody® VH 1.1 and 2.1 were unable to stimulate a response. Bispecific Humabody® VH stimulated CD137 signalling in a PSMA dependent and concentration dependent manner. The level of response was PSMA expression level dependent with higher maximal response observed in the presence of higher expression of PSMA. Anti CD137 antibody responses were PSMA expression independent. Bispecific molecules in the presence of PSMA expressing cells were able to effectively stimulate CD137 signalling.



FIG. 3. Enhancement of cytokine production in T-cells.


Human CD8+ T cells were co-cultured with PSMA expressing cells or non-expressing parental cells in the presence of plate bound anti CD3 antibody. Humabody® VH 1.1 monovalent and bispecific molecules, Humabody® VH 2.1 monovalent and bispecific molecules, anti CD137 comparator antibody and anti PSMA antibody. Supernatants were harvested after 48 hours and levels of IL-2 determined. (A) Enhancement of IL-2 responses in the presence of PSMA expressing and non-PSMA expressing cells (B) IL-2 responses for 3 different T-cell donors in the presence of PSMA expressing cells. (C and D) Concentration dependence of IL-2 response. (E) Interferon-gamma production in the presence of PSMA expressing cells. Monovalent Humabody® VH did not stimulate IL2 or IFN-gamma production in the assay. Bispecific molecules in the presence of PSMA expressing cells were able to effectively enhance cytokinelL-2 production. The anti CD137 antibody (soluble/non-cross linked) enhanced IL-2 production in a PSMA independent response.



FIG. 4. Mode of action of bispecific molecule. This figure illustrates the mode of action of a binding molecule that binds both CD137 and PSMA, leading to tumor selective T cell agonism.



FIG. 5: Enhancement of TNF alpha production. SEB Pre-stimulated PBMCs were treated for 3 days with 1 ng/ml SEB and Humabody constructs 4.1-6GS-1.1, 3.8-6GS-1.1 or a control VH in the presence of either (A) CHO PSMA cells or (B) CHO parent cells and 1 ng/ml SEB. TNF-alpha concentrations (Mean±standard deviation) were determined from 3 replicate wells.



FIG. 6: In vivo experiment: Effect of Humabody® in DU145 PSMA/hu PBMC engrafted NCG Mice Pooled tumour volume data from HuPBMC engrafted NCG mice implanted with DU145 PSMA prostate cell lines. Groups 4-6 (3 huPBMC donors, each group n=5 mice per donor) were treated on days 9-32 with PBS (BIW) and on days 33-45 with 4.1-6GS-1.1-VH (MSA) (3 mg/kg, daily). Groups 7-9 (3 huPBMC donors, n=5 mice per donor) were treated with control anti CD137 antibody (3 mg/kg, BIW) on days 9-32. Group 3 (non hPBMC engrafted group, n=5 mice) were untreated. Tumor volumes were measured at day 46 post-tumor implant. Statistical Significance (Mann-Whitney U test): **=P<0.01, compared to group 3.





DETAILED DESCRIPTION OF THE EMBODIMENTS

The embodiments of the invention will now be further described. In the following passages, different embodiments are described. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.


Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012); Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009); and Antibody Engineering, 2nd Ed., Vols 1 and 2, Ontermann and Dubel, eds., Springer-Verlag, Heidelberg (2010).


Enzymatic reactions and purification techniques are performed according to manufacturers specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. Suitable assays to measure the properties as set out above are also described in the examples.


The term “antibody” as used herein broadly refers to any immunoglobulin (Ig) molecule, or antigen binding portion thereof, comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule.


In a full-length antibody, each heavy chain is comprised of a heavy chain variable region or domain (abbreviated herein as HCVR) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain has a light chain variable region or domain (abbreviated herein as LCVR) and a light chain constant region. The light chain constant region is comprised of one domain, CL.


The heavy chain and light chain variable regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each heavy chain and light chain variable region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.


Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgAI and IgA2) or subclass.


The term “CDR” refers to the complementarity-determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term “CDR set” refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs can be defined differently according to different systems known in the art.


The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain). Another system is the ImMunoGeneTics (IMGT) numbering scheme. The IMGT numbering scheme is described in Lefranc et al., Dev. Comp. Immunol., 29, 185-203 (2005).


The system described by Kabat is used herein. The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling” are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion.


A chimeric antibody is a recombinant protein that contains the variable domains including the complementarity determining regions (CDRs) of an antibody derived from one species, preferably a rodent antibody, while the constant domains of the antibody molecule are derived from those of a human antibody.


A humanized antibody is a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent antibody, are transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains (e.g., framework region sequences). The constant domains of the antibody molecule are derived from those of a human antibody. In certain embodiments, a limited number of framework region amino acid residues from the parent (rodent) antibody may be substituted into the human antibody framework region sequences.


The term “antigen binding site” refers to the part of the antibody or antibody fragment that comprises the area that specifically binds to an antigen. An antigen binding site may be provided by one or more antibody variable domains. An antigen binding site is typically comprised within the associated VH and VL of an antibody or antibody fragment.


An antibody fragment is a portion of an antibody, for example as F(ab′)2, Fab, Fv, scFv, heavy chain, light chain, heavy (VH), variable light (VL) chain domain and the like. Functional fragments of a full length antibody retain the target specificity of a full antibody. Recombinant functional antibody fragments, such as Fab (Fragment, antibody), scFv (single chain variable chain fragments) and single domain antibodies (dAbs) have therefore been used to develop therapeutics as an alternative to therapeutics based on mAbs.


scFv fragments (˜25 kDa) consist of the two variable domains, VH and VL. Naturally, VH and VL domain are non-covalently associated via hydrophobic interaction and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create a single chain Fv (scFv).


The smallest antigen binding fragment is the single variable fragment, namely the single variable heavy (VH) or single variable light (VL) chain domain. VH and VL domains respectively are capable of binding to an antigen. Binding to a light chain/heavy chain partner respectively or indeed the presence of other parts of the full antibody is not required for target binding. The antigen-binding entity of an antibody, reduced in size to one single domain (corresponding to the VH or VL domain), is generally referred to as a “single domain antibody” or “single immunoglobulin variable domain”. A single domain antibody (˜12 to 15 kDa) thus consists of either the VH or VL domain, but it does not comprise other parts of a full length antibody. Single domain antibodies derived from camelid heavy chain only antibodies that are naturally devoid of light chains as well as single domain antibodies that have a human heavy chain domain have been described (Muyldermans 2001, Holliger 2005). 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” or “sdAb” generally refers to a single immunoglobulin variable domain (VH, VHH or VL) polypeptide that specifically binds antigen. Such a molecule only has the VH or VL binding domain respectively, but does not comprise other parts of a full length antibody. Unless otherwise specified, as used herein, the term refers to a single domain antibody that has a VH domain. For use in therapy, human single domain antibodies are preferred, primarily because they are not as likely to provoke an immune response when administered to a patient.


The terms “single domain antibody”, “VH domain antibody”, “single VH domain antibody”, “VH single domain antibody”, “single variable domain”, “single variable domain antibody”, “single variable heavy chain domain antibody” or immunoglobulin single variable domain (ISV)” are thus all well known in the art and describe the single variable fragment of an antibody that binds to a target antigen. These terms are used interchangeably herein. These terms above and specifically “single heavy chain domain antibody”, “single variable heavy chain domain antibody” “single VH domain antibody”, “ISV”, and “VH single domain” as used herein describe a part of an antibody, i.e. the single heavy chain variable fragment of an antibody, e.g. the VH domain, which retains binding specificity to the antigen in the absence of light chain or other antibody fragments. Such a molecule, e.g. a single variable heavy chain domain antibody is capable of binding to an antigen in the absence of light chain. A single variable heavy chain domain antibody does not comprise other parts of a full length antibody; it only includes the VH domain. Thus, as used herein, these terms and specifically a single domain antibody, specify a binding moiety that is solely made up of the VH domain and does not have other parts of an antibody. As explained herein, the CD137 binding entity illustrated below is a VH single domain antibody and in preferred embodiments, the PSMA binding entity is also a VH single domain antibody.


As explained below, the embodiments relate to isolated binding molecules which comprise or consist of a single variable heavy chain domain antibody/immunoglobulin single variable heavy chain domain which bind a CD137 antigen and also comprise a moiety that binds to PSMA. Thus, the single variable heavy chain domain antibody (i.e. a VH domain) is capable of binding to CD137 in the absence of light chain. Human single variable heavy chain domain antibodies (“VH domain antibody”) are particularly preferred. Such binding molecules are also termed Humabody® herein. Humabody® is a registered trademark of Crescendo Biologics Ltd.


The term “isolated” refers to a moiety that is isolated from its natural environment. For example, the term “isolated” refers to a single domain antibody or binding molecule that is substantially free of other single domain antibodies or binding molecule, antibodies or antibody fragments. Moreover, an isolated single domain antibody may be substantially free of other cellular material and/or chemicals.


Each VH domain antibody comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Thus, in one embodiment of the invention, the domain is a human variable heavy chain (VH) domain with the following formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.


Modifications to the C or N-terminal VH framework sequence may be made to the single domain antibodies of the invention to improve their properties. For example, the VH domain may comprise C or N-terminal extensions. C-terminal extensions can be added to the C-terminal end of a VH domain which terminates with the residues VTVSS (SEQ ID No. 788).


In one embodiment, the single domain antibodies of the invention comprise C-terminal extensions of from 1 to 50 residues, for example 1 to 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, 1-20, 1-30 or 1-40 additional amino acids. In one embodiment, the single domain antibodies of the invention comprise additional amino acids of the human CH1 domain thus that the C terminal end extends into the CH1 domain. For example, C-terminal extensions may comprise neutral, nonpolar amino acids, such as A, L, V, P, M, G, I, F or W or neutral polar amino acids, such as S or T. C-terminal extensions may also be selected from peptide linkers or tags, e.g. SEQ ID Nos. 790-797.


Additional C or N-terminal residues can be peptide linkers that are for example used to conjugate the single domain antibodies of the invention to another moiety, or tags that aid the detection of the molecule. Such tags are well known in the art and include for, example linker His tags, e.g., hexa-His (HHHHHH, SEQ ID No. 789) or myc tags.


As used herein, the term “homology” or “identity” generally refers to the percentage of amino acid residues in a sequence that are identical with the residues of the reference polypeptide with which it is compared, after aligning the sequences and in some embodiments after introducing gaps, if necessary, to achieve the maximum percentage homology, and not considering any conservative substitutions as part of the sequence identity. Thus, the percentage homology between two amino acid sequences is equivalent to the percentage identity between the two sequences. Neither N- or C-terminal extensions, tags or insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known. The percentage identity between two amino acid sequences can be determined using well known mathematical algorithms.


According to some embodiments of the various aspects of the invention, the variable domain of the single domain antibodies as described herein is a human variable domain (as used herein VH refers to a human domain), a camelid variable domain (VHH), a humanised VHH domain, a camelized VH domain, a sequence modified VH or VHH domain. In one embodiment, the variable domain of the single domain antibodies as described herein is a VH domain.


As used herein, a human VH domain includes a fully human or substantially fully human VH domain. As used herein, the term human VH domain also includes VH domains that are isolated from heavy chain only antibodies made by transgenic mice expressing fully human immunoglobulin heavy chain loci, in particular in response to an immunisation with an antigen of interest, for example as described in WO2016/062990 and in the examples below. In one embodiment, a human VH domain can also include a VH domain that is derived from or based on a human VH domain amino acid or produced from a human VH nucleic acid sequence. Thus, the term human VH domain includes variable heavy chain regions derived from or encoded by human germline immunoglobulin sequences and for example obtained from heavy chain only antibodies produced in transgenic mice expressing fully human VH genes. In some embodiments, a substantially human VH domain or VH domain that is derived from or based on a human VH domain may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro, e.g. by random or site-specific mutagenesis, or introduced by somatic mutation in vivo). The term “human VH domain” therefore also includes a substantially human VH domain, i.e. human VH domain wherein one or more amino acid residue has been modified, for example to remove sequence liabilities. For example, a substantially human VH domain the VH domain may include up to 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or up to 20 amino acid modifications compared to a germline human sequence.


However, the term “human VH domain” or “substantially human VH domain”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. In one embodiment, the term “human VH domain”, as used herein, is also not intended to include camelized VH domains, that is human VH domains that have been specifically modified, for example in vitro by conventional mutagenesis methods to select predetermined positions in the VH domains sequence and introduce one or more point mutation at the predetermined position to change one or more predetermined residue to a specific residue that can be found in a camelid VHH domain.


The term “KD” refers to the “equilibrium dissociation constant” and refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (Koff) by the association rate constant (Kon). “KA” refers to the affinity constant. The association rate constant, the dissociation rate constant and the equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. Using fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental approaches and instruments such as a BIAcore® assay can be used.


The term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used in this disclosure can be exhibited, for example, by a molecule having a KD for the target of at least about 10-6 M, alternatively at least about 10-7 M, alternatively at least about 10-8 M, alternatively at least about 10-9 M, alternatively at least about 10-10 M, alternatively at least about 10-11 M, alternatively at least about 10-12 M, or greater affinity. In one embodiment, the term “specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.


In some embodiments, there is provided binding molecule that comprises a VH single domain antibody that is a variant of any of the single VH domain antibodies described herein having one or more amino acid substitutions, deletions, insertions or other modifications, and which retains a biological function of the single domain antibody. Thus, variant VH single domain antibody can be sequence engineered.


Modifications may include one or more substitution, deletion or insertion of one or more codons encoding the single domain antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence VH single domain antibody or polypeptide. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 25, for example 1 to 5, 1 to 10, 1 to 15 or 1 to 20 amino acids, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence. A variant of a VH single domain antibody described herein has at least 50%, for example at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 8 0%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the non-variant molecule, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.


In one embodiment, the modification is a conservative sequence modification. As used herein, the term “conservative sequence modifications” is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an sdAb of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of a single domain antibody of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., CD137 binding) using the functional assays described herein.


Thus, these amino acid changes can typically be made without altering the biological activity, function, or other desired property of the polypeptide, such as its affinity or its specificity for antigen. In some instances these changes are made to improve the affinity of the antibody, e.g., single VH domain antibody, for its target antigen. In general, single amino acid substitutions in nonessential regions of a polypeptide do not substantially alter biological activity. Furthermore, substitutions of amino acids that are similar in structure or function are less likely to disrupt the polypeptides' biological activity. Abbreviations for the amino acid residues that comprise polypeptides and peptides described herein, and conservative substitutions for these amino acid residues are shown in Table 1 below.









TABLE 1







Amino Acid Residues and Examples of Conservative


Amino Acid Substitutions








Original residue



Three letter code, single letter code
Conservative substitution





Alanine, Ala, A
Gly, Ser


Arginine, Arg, R
Lys, His


Asparagine, Asn, N
Gln, His


Aspartic acid Asp, D
Glu, Asn


Cysteine, Cys, C
Ser, Ala


Glutamine, Gln, Q
Asn


Glutamic acid, Glu, E
Asp, Gln


Glycine, Gly, G
Ala


Histidein, His, H
Asn, Gln


Isoleucine, Ile, I
Leu, Val


Leucine, Leu, L
Ile, Val


Lysine, lys, K
Ar, His


Methionine, Met, M
Leu, Ile, Tyr


Phenylalanine, Phe, F
Tyr, Met, Leu


Proline, Pro, P
Ala


Serine, Ser, S
Thr


Threonine, Thr, T
Ser


Tryptophan, Trp, W
Tyr, Phe


Tyrosine, Tyr, Y
Try, Phe


Valine, Val, V
Ile, Leu









In some embodiments, the binding molecule includes a VH single domain antibody that is a variant of a single domain antibody selected from those shown in Tables 2, 3 and 4 that comprises one or more sequence modification and has improvements in one or more of a property such as binding affinity, specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility as compared to the unmodified single domain antibody.


A skilled person will know that there are different ways to identify, obtain and optimise the antigen binding molecules as described herein, including in vitro and in vivo expression libraries. This is further described in the examples. Optimisation techniques known in the art, such as display (e.g., ribosome and/or phage display) and/or mutagenesis (e.g., error-prone mutagenesis) can be used. The invention therefore also comprises sequence optimised variants of the single domain antibodies described herein.


In one embodiment, modifications can be made to decrease the immunogenicity of the single domain antibody. For example, one approach is to revert one or more framework residues to the corresponding human germline sequence. More specifically, a single domain antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the single domain antibody is derived. Such residues can be identified by comparing the single domain antibody framework sequences to the germline sequences from which the single domain antibody is derived. In one embodiment, all framework residues are germline sequence.


To return one or more of the amino acid residues in the framework region sequences to their germline configuration, the somatic mutations can be “backmutated” to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.


Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody.


In still another embodiment, the glycosylation is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for the antigen.


In one embodiment, the one or more substitution is in the CDR1, 2 or 3 region. For example, there may be 1, 2, 3, 4 or 5 amino acid substitutions in the CDR1, 2 or 3. In another example, there may be 1 or 2 amino acid deletions. In one embodiment, the one or more substitution is in the framework region. For example, there may be 1 to 10 or more amino acid substitutions in the CDR1, 2 or 3. In another example, there may be 1 to 10 or more amino acid deletions.


The term “epitope” or “antigenic determinant” refers to a site on the surface of an antigen (e.g., PSMA or CD137) to which an immunoglobulin, antibody or antibody fragment, including a VH single domain antibody specifically binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and conformational epitopes. Epitopes within protein antigens can be formed both from contiguous amino acids (usually a linear epitope) or non-contiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody or antibody fragment (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from are tested for reactivity with a given antibody or antibody fragment. An antibody binds “essentially the same epitope” as a reference antibody, when the two antibodies recognize identical or sterically overlapping epitopes. The most widely used and rapid methods for determining whether two epitopes bind to identical or sterically overlapping epitopes are competition assays, which can be configured in different formats, using either labelled antigen or labelled antibody.


The inventors have surprisingly identified single variable heavy chain domain antibodies that, when targeted to CD137 in a monospecific format, that is without being linked to another moiety specific to a second antigen, bind specifically to CD137, but do not induce clustering of the CD137 receptor. Binding of the single variable heavy chain domain antibodies described herein in a monovalent or monospecific format does therefore not activate CD137 signalling and does not lead to CD137 signalling. Binding of the single variable heavy chain domain antibodies described herein does not agonise CD137 signalling unless they are provided together with another moiety specific to a second antigen, for example as a bispecific fusion protein wherein a single variable heavy chain domain antibody described herein is linked to a moiety that binds to a tumor specific antigen, for example a single variable heavy chain domain antibody that binds to a tumor specific antigen.


When a single variable heavy chain domain antibody as described herein is provided as part of a binding molecule, for example as fusion protein together with a moiety that binds to PSMA, such as a single variable heavy chain domain antibody that binds to PSMA, binding to CD137 and the target moiety results in clustering of the CD137 receptor and CD137 signalling. Induction of CD137 signalling thus requires dual engagement of both targets, i.e. CD137 and PSMA. This leads to localised CD137 signalling in the tumor microenvironment. Only simultaneous engagement of both targets by the bispecific molecule results in CD137 activation. Target specific activation in the vicinity of the tumor potentially avoids systemic CD137 effects leading to uncontrollable side effects. The binding molecules effectively engage CD137 on the surface of cells through mechanisms other than binding to Fc-receptors thus also avoiding unwanted liver toxicity. Simultaneously, they engage cells that express PSMA.


We describe a binding molecule that binds to both CD137 and PSMA. The terms “binding molecule” and “binding agent” are used interchangeably herein. A binding molecule as used herein refers to a binding molecule that specifically binds at least two targets, i.e. CD137 and PSMA, wherein one subunit/entity/moiety that binds to CD137 is conjugated/linked a second subunit/entity/moiety that binds PSMA. As described herein, in some embodiments, the binding molecule is a fusion protein wherein one polypeptide that binds to CD137 is conjugated/linked to a second polypeptide that binds to PSMA.


In one aspect, the invention relates to an isolated multispecific binding molecule that binds to both CD137 and PSMA and comprises a single variable heavy chain domain antibody that binds to CD137. In some embodiments, the single variable heavy chain domain antibody that binds to CD137 is as described herein.


The properties of the multispecific binding molecules of the invention can be exploited in therapeutic methods and uses as well as in pharmaceutical formulations as described herein.


In one aspect, the invention relates an isolated binding molecule comprising or consisting of a) a single variable heavy chain domain antibody that binds to CD137 and

    • b) a moiety that binds to PSMA.


Thus, the entity that binds to CD137 is not a full antibody that comprises light and heavy chains, but a single variable heavy chain domain, i.e. a VH domain only. In some embodiments, the single variable heavy chain domain antibody that binds to CD137 is selected from one of the single variable heavy chain domain antibodies that bind to CD137 having a SEQ ID No. as described herein as listed in the tables below (tables 2 and 3) wherein the CDRs are defined according to Kabat.


The single variable heavy chain domain antibody that binds to CD137 and the moiety that binds to PSMA are linked, for example by a peptide linker. For example, the single variable heavy chain domain antibody that binds to CD137 can be linked at its N or C terminus to the moiety that binds to PSMA.


The moiety that binds PSMA can be selected from an antibody, antibody mimetic, antibody scaffold, antibody fragment or other polypeptide. An antibody fragment can be selected from a portion of an antibody, for example a F(ab′)2, Fab, Fv, scFv, heavy chain, light chain, heavy or variable light chain domain heavy or variable light chain domain, or part thereof, such as a CDR.


In one embodiment, the entity that binds PSMA is a single variable heavy chain domain antibody that binds to PSMA, such as a human VH domain antibody. In some embodiments, the single variable heavy chain domain antibody that binds to PSMA is selected from one of the single variable heavy chain domain antibodies that bind to PSMA having a SEQ ID No. as described herein and in the tables below.


In one embodiment, there is provided a binding molecule comprising or consisting of


a) a single variable heavy chain domain antibody that binds to CD137 and


b) a single variable heavy chain domain antibody that binds to PSMA.


Thus, the binding molecule has two single variable heavy chain domains, one that binds to CD137 and one that binds to PSMA. No other domains/chains of a full antibody are present. In some embodiments, the single variable heavy chain domain antibody that binds CD137 and the single variable heavy chain domain antibody that binds PSMA is a human VH domain antibody.


In one aspect, we provide a binding molecule comprising a single variable heavy chain domain antibody that binds to CD137, for example having a SEQ ID No. as described herein (e.g. as set out in table 2 or 3), linked to another moiety that binds to PSMA, for example having a SEQ ID No. as described herein wherein the binding molecule exhibits one or more of the following properties:


(a) binds to human CD137 with a KD as measured in the examples;


(b) inhibits the interaction between human CD137 ligand and human CD137 expressed on the surface of cells. This can be measured as shown in example 4;


(c) does not bind to mouse CD137;


(d) binds to cells expressing CD137 and simultaneously binds to cells expressing PSMA. This can be measured as shown in example 5;


(e) increases reporter gene activity. This can be measured as shown in example 5;


(f) inhibits tumor cell growth in vivo;


(g) promotes CD8+ T cell expansion;


(h) induces activation of cytotoxic T lymphocytes (CTL);


(i) stimulates IL-2 production from CD8+ cells. This can be measured as shown in example 5;


(j) induces tumor specific T cell activation;


(k) activates CD137 signalling in T cells as measured in the examples;


(l) inhibits activation induced cell death;


(m) enhances T cell survival;


(n) limits systemic T cell activation;


(o) enhances the cytotoxic effector function of T cells;


(p) promotes local activation of anti-tumor cells in tumor antigen positive tumors;


(q) enhances of antibody-dependent cellular cytotoxicity;


(r) binds simultaneously to CD137 and PSMA;


(s) binds to cyno CD137;


(t) reverses the regulator function of T-reg cells;


(u) activates NK cells and/or


(v) reduces tumour volume compared to control groups in mice as shown in example 6.


In one embodiment, the binding molecule exhibits more than 1 of the properties above, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or all of the properties selected from the above list, including any combination of properties. In one embodiment, the binding agent inhibits the interaction between human CD137 ligand and human CD137 expressed on the surface of cells.


In one embodiment, the binding molecule is a fusion protein comprising at least two subunits, i.e. a CD137-binding subunit fused to a PSMA-binding subunit wherein the CD137-binding subunit is a single variable heavy chain domain antibody that binds to CD137. In one embodiment, the binding molecule is a fusion protein comprising a single variable heavy chain domain antibody that binds to CD137 linked to a single variable heavy chain domain antibody that binds to PSMA. The linker is, for example, a peptide linker with GS residues such as (Gly4Ser)n, where n=from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 as further described below.


The binding agent is multispecific, for example bispecific or trispecific. A bispecific molecule binds to 2 different targets. A trispecific molecule binds to 2 different targets. A monovalent molecule has one binding entity. A bivalent molecule has two binding entities which bind to the same or different target.


In one embodiment, the binding molecule comprises a first VH single domain antibody that binds to CD137 (VH (A)) and a second VH single domain antibody (VH (B)) that binds to PSMA and thus has the following formula: VH (A)-L-VH (B). VH (A) is conjugated to VH (B), that is linked, for example with a peptide linker. L denotes a linker.


Each VH comprises CDR and FR regions. Thus, the binding molecule may have the following formula: FR1(A)-CDR1(A)-FR2(A)-CDR2(A)-FR3(A)-CDR3(A)-FR4(A)-L-FR1(B)-CDR1(B)-FR2(B)-CDR2(B)-FR3(B)-CDR3(B)-FR4(B). The order of the single VH domains A and B is not particularly limited, so that, within a polypeptide of the invention, single variable domain A may be located N-terminally and single variable domain B may be located C-terminally, or vice versa.


The term “peptide linker” as used in the various embodiments described herein refers to a peptide comprising one or more amino acids. A peptide linker comprises 1 to 44 amino acids, more particularly 2 to 20 amino acids. Peptide linkers are known in the art or are described herein. Suitable, non-immunogenic linker peptides are, for example, linkers that include G and/or S residues, (G4S)n, (SG4)n or G4(SG4)n peptide linkers, wherein “n” is generally a number between 1 and 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the peptide is for example selected from the group consisting of GGGGS (SEQ ID NO: 790), GGGGSGGGGS (SEQ ID NO:791), SGGGGSGGGG (SEQ ID NO:792), GGGGSGGGGSGGGG (SEQ ID NO:793), GSGSGSGS (SEQ ID NO:794), GGSGSGSG (SEQ ID NO:795), GGSGSG (SEQ ID NO:796), GGSG (SEQ ID NO:797).


The fusion protein described above is capable of dual, e.g. simultaneous, engagement/binding to CD137 on the surface of effector cells and to PSMA displayed on the cell surface of tumor cells. The dual, e.g. simultaneous binding leads to clustering of the CD137 receptor resulting in CD137 signalling. This leads to T cell activation. In some embodiments, dual, e.g. simultaneous binding leads to tumor antigen specific effector cell activation and results in tumor cell killing.


In some embodiments, the fusion protein is capable of binding CD137 with an EC50 value that is similar or at least equivalent to the EC50 value by which the monovalent single heavy chain domain antibody binds to CD137. In some embodiments, the fusion protein binds CD137 with an EC50 value as shown in the examples.


In some embodiments, the fusion protein described herein may be capable of co-stimulating T cell responses in a functional T cell activation essentially described as in the examples. In some embodiments, the fusion protein described herein may be able to induce IL-2 and/or IFN gamma secretion and T cell proliferation in a functional T cell activation. The fusion polypeptide as described herein is, in some embodiments, also capable of local induction of IL-2 and/or IFN gamma secretion in the vicinity of the targeted tumor that is cells that are positive for the tumor antigen to which the fusion protein binds.


In some embodiments, the fusion protein may be capable of producing a synergistic effect through dual targeting of the CD137 expressing cell and the tumor antigen expressing cell.


Dual, e.g. simultaneous targeting of CD137 and PSMA in the microenvironment of the tumor may enhance anti-tumor activity and reduce tumor growth. Moreover, by eliciting CD137 signalling locally, side effects may be reduced. CD137 signalling results in the recruitment of TRAF family members and activation of kinases. T cell mediated signalling protects CD8+ cells from activation induced death. Also provided is the use of the fusion protein as described herein for co-stimulating T cells.


In one embodiment, a binding molecule as described herein binds to CD137 with a KD of at least about 10-6 M, alternatively at least about 10-7 M, alternatively at least about 10-8 M, alternatively at least about 10-9 M, alternatively at least about 10-10 M, alternatively at least about 10-11 M, alternatively at least about 10-12 M, or greater affinity as measured according to the methods shown in the examples. In one embodiment, a binding molecule as described herein binds to PSMA with a KD of at least about 10-6 M, alternatively at least about 10-7 M, alternatively at least about 10-8 M, alternatively at least about 10-9 M, alternatively at least about 10-10 M, alternatively at least about 10-11 M, alternatively at least about 10-12 M, or greater affinity as measured according to the methods shown in the examples. Binding can be measured as in the examples. In some embodiments, the binding molecules of the invention have IC50 and/or EC50 values as further described herein and as shown in the examples. Binding molecules described herein have shown excellent stability.


In another aspect, a nucleic acid molecule encoding a fusion protein described herein is provided. For example, the nucleic acid molecule comprises a nucleic acid encoding a single variable heavy chain domain antibody that binds to CD137 as specified herein and a nucleic acid encoding a single variable heavy chain domain antibody that binds to PSMA as specified herein.


A nucleic acid may comprise DNA or RNA and may be wholly or partially synthetic or recombinantly produced. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.


Furthermore, the invention relates to a nucleic acid construct comprising at least one nucleic acid as defined above. The construct may be in the form of a plasmid, vector, transcription or expression cassette.


The invention also relates to an isolated recombinant host cell comprising one or more nucleic acid construct as described above. The host cell may be a bacterial, viral, plant, mammalian or other suitable host cell. In one embodiment, the cell is an E. coli cell. In another embodiment, the cell is a yeast cell. In another embodiment, the cell is a Chinese Hamster Ovary (CHO) cell.


In one embodiment, a method of making the fusion protein as described herein is provided, wherein the method comprises culturing the host cell under conditions suitable for expression of the polynucleotide encoding the fusion protein, and isolating the single domain antibody.


Exemplary Immunoglobulins Included in the Binding Molecule


As described above, the binding molecules comprise a single variable heavy chain domain antibody that binds CD137 (CD137-binding subunit) and a moiety that binds PSMA (PSMA binding subunit). For example, the binding molecule can be a fusion protein wherein a single variable heavy chain domain antibody that binds CD137 is linked to another polypeptide that binds to PSMA. The below provides examples of the CD137-binding subunit and the PSMA binding subunit.


Exemplary Immunoglobulins Included in the Binding Molecule and which Bind CD137


Examples of single variable heavy chain domain antibodies that bind to CD137 and that may form one of the subunits of the binding molecule that bind to both, CD137 and PSMA, are described and can be used in the various embodiments of the invention.


CD137 is an important regulator of immune responses and therefore an important target in cancer therapy. The T cell costimulatory receptor CD137 is induced on activated T cells and plays a variety of crucial roles: preventing activation-induced cell death (AICD), promoting cell cycle progression, enhancing cytotoxicity and the production of type 1 cytokines such as IL-2, IFN-γ, and TNF-α, and increasing the memory CD8+ T cells. In vivo CD137 triggering with agonistic antibodies enhances CD8+ T cell responses against tumors. CD137 mediated anti-cancer effects are based on its ability to induce activation of cytotoxic T lymphocytes (CTL), and among others, high amounts of IFN-γ. CD137/CD137L interactions are also considered positive regulators of CD8+ T cell responses against viruses such as influenza virus, lymphocytic choriomeningitis virus (LCMV), and herpes simplex virus (HSV). CD137 is involved in sustaining the T cell responses after initial T-cell activation.


Importantly, CD137 signalling requires clustering of the CD137 receptor. Such clustering is mediated by the interaction of the trimeric CD137 ligand with the CD137 receptor resulting in recruitment of signalling molecules such as the TRAF family of proteins. This in turn leads to kinase modulation and activation of the Nf-KB signalling pathway. The NF-KB family of transcription factors has an essential role in inflammation and innate immunity. Furthermore, NF-KB is increasingly recognized as a crucial player in many steps of cancer initiation and progression.


Single domain antibodies described herein bind specifically to wild type human CD137 (UniProt Accession No. Q07011, GenBank Accession No. NM_001561). The amino acid sequence (SEQ ID No. 786) and nucleotide sequences for wild type human CD137 are shown below (SEQ ID No. 787).









(SEQ ID No. 786)


MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPP





NSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCS





MCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNG





TKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALL





FLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE





GGCEL





(SEQ ID No. 787)


ATGGGAAACAGCTGTTACAACATAGTAGCCACTCTGTTGCTGGTCCTCAA





CTTTGAGAGGACAAGATCATTGCAGGATCCTTGTAGTAACTGCCCAGCTG





GTACATTCTGTGATAATAACAGGAATCAGATTTGCAGTCCCTGTCCTCCA





AATAGTTTCTCCAGCGCAGGTGGACAAAGGACCTGTGACATATGCAGGCA





GTGTAAAGGTGTTTTCAGGACCAGGAAGGAGTGTTCCTCCACCAGCAATG





CAGAGTGTGACTGCACTCCAGGGTTTCACTGCCTGGGGGCAGGATGCAGC





ATGTGTGAACAGGATTGTAAACAAGGTCAAGAACTGACAAAAAAAGGTTG





TAAAGACTGTTGCTTTGGGACATTTAACGATCAGAAACGTGGCATCTGTC





GACCCTGGACAAACTGTTCTTTGGATGGAAAGTCTGTGCTTGTGAATGGG





ACGAAGGAGAGGGACGTGGTCTGTGGACCATCTCCAGCCGACCTCTCTCC





GGGAGCATCCTCTGTGACCCCGCCTGCCCCTGCGAGAGAGCCAGGACACT





CTCCGCAGATCATCTCCTTCTTTCTTGCGCTGACGTCGACTGCGTTGCTC





TTCCTGCTGTTCTTCCTCACGCTCCGTTTCTCTGTTGTTAAACGGGGCAG





AAAGAAACTCCTGTATATATTCAAACACCATTTATGAGACCAGTACAAAC





TACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAG





GAGGATGTGAACTGTGA






Unless otherwise specified, the term CD137 as used herein refers to human CD137. CD137 is also known as “4-1BB”, “TNF receptor superfamily member 9”, “TNFRS9”, “induced by lymphocyte activation” and “ILA” these terms are used interchangeably, and include variants, isoforms of human CD137.


The terms “CD137 binding molecule/protein/polypeptide/agent/moiety”, “CD137 antigen binding molecule molecule/protein/polypeptide/agent/moiety”, “anti-CD137 single domain antibody”, “anti-CD137 single immunoglobulin variable domain”, “anti-CD137 heavy chain only antibody” or “anti-CD137 antibody” all refer to a molecule capable of specifically binding to the human CD137 antigen. The binding reaction may be shown by standard methods, for example with reference to a negative control test using an antibody of unrelated specificity.


A multispecific binding agent described herein, “which binds” or is “capable of binding” an antigen of interest, e.g. human CD137, is one that binds the antigen with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen CD137. Binding is to the extracellular domain of CD137.


Binding molecules of the invention bind specifically to human CD137. In other words, binding to the CD137 antigen is measurably different from a non-specific interaction. They do not cross react with mouse CD137. In one embodiment, the sdAb binds to human CD137 and also binds to monkey (e.g., cynomolgous) CD137.


In one aspect, the monovalent single domain antibody used in the multispecific molecule exhibits one or more of the following properties as a monovalent entity (i.e. when it is not provided in a multispecific format together with an entity that binds to PSMA):


(a) binds to human CD137 with a KD as measured in the examples;


(b) binds to cells expressing CD137, but does not bind to cells that do not express CD137. This can be measured in a FMAT assay;


(c) shows minimal cell internalisation. This can be measured as shown in the examples;


(d) inhibits the interaction between CD137 ligand and CD137 expressed on the surface of cells. This can be measured in a FMAT assay.


(e) does not activate CD137 signalling in T cells. This can be measured as shown in the examples;


(f) does not stimulate IL-2 production from CD8+ cells. This can be measured as shown in example 5;


(g) does not bind to mouse CD137;


(h) provides good stability as shown in the examples and/or


(i) does not increase reporter gene activity and thus does not elicit CD137 signalling. This can be measured as shown in example 5.


Exemplary Sequence Features


In one aspect, the single variable heavy chain domain antibody comprises a CDR1, CDR2 or CDR3 as shown for one of the single domain antibodies as shown in Table 2 or a set of CDRs (i.e. CDR1, CDR2, CDR3) wherein said set is as shown for one of the single domain antibodies as shown in Table 2. In one aspect, it comprises a CDR1, CDR2, CDR3 at least 40% or 75% homology thereto. For example, the single variable heavy chain domain antibody comprises a CDR1 comprising SEQ ID NO. 1 or a sequence with at least 40%, 75% or 80% homology thereto, a CDR2 comprising SEQ ID NO. 2 or a sequence with at least 40%, 75% or 80% homology thereto and a CDR3 comprising SEQ ID NO. 3 or a sequence with at least 40%, 75% or 80% homology, or a CDR1 comprising SEQ ID NO. 5 or a sequence with at least 75% homology thereto, a CDR2 comprising SEQ ID NO. 6 or a sequence with at least 40%, 75% or 80% homology thereto and a CDR3 comprising SEQ ID NO. 7 or a sequence with at least 40%, 75% or 80% homology thereto and so forth.


Sequence homology as above and as used generally herein can be at least 40%, 50%, 60%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% for example at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.


In one embodiment, the single variable heavy chain domain antibody comprises human framework regions.


In another aspect, the single variable heavy chain domain antibody according to the invention comprises or consists of a full length sequence as shown in Table 2 or a sequence with at least 50% homology thereto. For example, the single variable heavy chain domain antibody has a sequence selected from the sequences listed in Table 2, i.e. SEQ ID NO. 4, 8, 12, 16, 20 and so forth or a sequence with at least 50% homology thereto. Sequence homology can be at least 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% for example at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology. In one embodiment, the single variable heavy chain domain antibody comprises a CDR1, 2, and 3 as shown for VH single domain antibodies 1.1 to 1.89 or 1.90 to 1.106 or comprises or consists of a full length sequence as shown for VH single domain antibodies 1.1 to 1.89 or 1.90 to 1.106 (i.e. SEQ ID NOs. 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420 or 424). In one embodiment, the single variable heavy chain domain antibody comprises a CDR1, 2, and 3 as shown for VH single domain antibodies VH 1.107 to 1.114 as shown in table 2, e.g. SEQ ID No. 873, 874, 875) or a sequence with at least 75%, 80% or 90% homology thereto. In one embodiment, the single variable heavy chain domain antibody is selected from VH 1.107 to 1.114 as shown in table 2, i.e. VH 1.107, 1.108, 1.109, 1.110, 1.111, 1.112, 1.113 or 1.114, that is SEQ ID NOs. 852, 856, 860, 864, 868, 872, 876 or 880 or a sequence with at least 75%, 80% or 90% homology thereto. In one embodiment, the single variable heavy chain domain antibody is VH 1.113 or a sequence with at least 75%, 80% or 90% homology thereto.









TABLE 2







Full length sequences and CDR sequences of VH single domain antibodies (Family 1)











Name
CDR1
CDR2
CDR3
Full Length





1.1
SEQ ID NO:
SEQ ID NO: 2
SEQ ID NO: 3
SEQ ID NO: 4



1
HIKEDGSEKY
GGDGYSDSHF
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLE



SHWMT
YEDSVEG
GVDV
WVAHIKEDGSEKYYEDSVEGRFTVSRDNAKNSVYLQMNSLRAEDTAV






YYCARGGDGYSDSHFGVDVWGQGTTVTVSS





1.2
SEQ ID NO:
SEQ ID NO: 6
SEQ ID NO: 7
SEQ ID NO: 8



5
HIKEDGSEKY 
GGDGYSDSHY
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMTWFRQAPGKGLE



SYWMT
YVDSVEG
GVDV
WVAHIKEDGSEKYYVDSVEGRFTISRDNANNSLYLQMNSLRAEDTAV






YYCARGGDGYSDSHYGVDVWGQGTTVTVSS





1.3
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 11
SEQ ID NO: 12



9
10
GGLGYGDSHY
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMTWFRQAPGRGLE



SYWMT
NINQDGSEK
GMDV
WVANINQDGSEKYYVDSVEGRFTVSRDNAKNSLYLQMNSLRAEDTA




YYVDSVEG

VYYCARGGLGYGDSHYGMDVWGQGTTVTVSS





1.4
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 15
SEQ ID NO: 16



13
14
GGLGYGDSHY
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMTWFRQAPGGGLE



NYWMT
NINQDGSEK
GMDV
WVANINQDGSEKYYVDSVEGRFTVSRDNAKNSLYLQMNSLRAEDTA




YYVDSVEG

VYYCARGGLGYGDSHYGMDVWGQGTTVTVSS





1.5
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 19
SEQ ID NO: 20



17
18
GGDGYSGSHH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMIWFRQAPGKGLE



NYWMI
NINQDGSEK
GTDV
WVANINQDGSEKYYVDSVEGRFTISRDNAKNSLYLQMNSLRAEDTAV




YYVDSVEG

YYCARGGDGYSGSHHGTDVWGQGTTVTVSS





1.6
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 23
SEQ ID NO: 24



21
22
GGEGYSTSHYG
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLE



SYWMS
NIKQDGSEKY
MDV
WVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV




YVDSVKG

YYCARGGEGYSTSHYGMDVWGQGTTVTVSS





1.7
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 27
SEQ ID NO: 28



25
26
GGDGYSDSHF
EVQLVESGGGLVQPGGSLRLSCGASGFTFSSYWMLWFRQAPGKGLE



SYWML
NINQDGSEK
GTDV
WVANINQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNTLRAEDTAI




YYVDSVKG

YYCARGGDGYSDSHFGTDVWGQGTTVTVSS





1.8
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 31
SEQ ID NO: 32



29
30
GGDGYSDSHY
EVQLVESGGGLVQPGGSLRLSCGASGFTFSSYWMFWFRQAPGEGLE



SYWMF
NINQDGSEK
GTDV
WVANINQDGSEKYYVDSVEGRFTISRDNAKNSLYLQMNSLRAEDTAIY




YYVDSVEG

YCARGGDGYSDSHYGTDVWGQGTTVTVSS





1.9
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 35
SEQ ID NO: 36



33
34
GGLGYGDSHY
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMTWFRQAPGGGLE



NYWMT
NINQDGSEK
GMDV
WVANINQDGSEKYYVDSVEGRFTVSRDNAKNSLDLQMNSLRAEDTA




YYVDSVEG

VYYCARGGLGYGDSHYGMDVWGQGTTVTVSS





1.10
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 39
SEQ ID NO: 40



37
38
GGAGYSMSHY
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMNWARQAPGKGLE



DYWMN
NIKEDGSEKY
GMDV
WVANIKEDGSEKYYVDSVEGRFTISRDNAKNSTYLQMNSLRVEDTAV




YVDSVEG

YYCARGGAGYSMSHYGMDVWGQGTTVTVSS





1.11
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 43
SEQ ID NO: 44



41
42
GGAGYSMSHY
EVQLVESGGGLVQPGGSLRLSCEASGFTFSDYWMNWARQAPGKGLE



DYWMN
NIKEDGSEKY
GMDV
WVANIKEDGSEKYYVDSVEGRFTISRDNAKNSTYLQMNSLRAEDTAV




YVDSVEG

YYCARGGAGYSMSHYGMDVWGQGTTVTVSS





1.12
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 47
SEQ ID NO: 48



45
46
GGDGYSNSHF
EVQLVESGGGLVQPGGSLRLSCGASGFTFSSYWMLWFRQAPGKGLE



SYWML
NINQDGSEK
GTDV
WVANINQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNTLRAEDTAI




YYVDSVKG

YYCARGGDGYSNSHFGTDVWGQGTTVTVSS





1.13
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 51
SEQ ID NO: 52



49
50
GGDGYSDSHY
EVQLVESGGGLVKPGGSLRLSCGASGFTFSSYWMFWFRQAPGEGLE



SYWMF
NINQDGSEK
GTDV
WVANINQDGSEKYYVDSVEGRFTISRDNAKNSLYLQMNSLRAEDTAIY




YYVDSVEG

YCARGGDGYSDSHYGTDVWGQGTTVTVSS





1.14
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 55
SEQ ID NO: 56



53
54
GGEGYSTSHYG
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLE



NYWMN
NIKEDGSENY
MDV
WVANIKEDGSENYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV




YVDSVKG

YYCARGGEGYSTSHYGMDVWGQGTTVTVSS





1.15
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 59
SEQ ID NO: 60



57
58
GGDGYSDSHF
EVQLVESGGGLVQPGGSLRLSCGASGFTFSTYWMLWFRQAPGKGLE



TYWML
NINQDGSEK
GTDV
WVANINQDGSEKYYVDSVKGRFTISRDNAKNSLSLQMNSLRAEDTAT




YYVDSVKG

YYCARGGDGYSDSHFGTDVWGQGTTVTVSS





1.16
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 63
SEQ ID NO: 64



61
62
GGDGYSSSHY
EVQLVESGGGLVQPGGSLRLSCVASGFTFSNYWMMWFRQAPGKGL



NYWMM
NINQDGSEK
GTDV
EWVANINQDGSEKYFVDSVEGRFTISRDNAKNSLYLQMNSLRAEDTA




YFVDSVEG

VYYCARGGDGYSSSHYGTDVWGQGTTVTVSS





1.17
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 67
SEQ ID NO: 68



65
66
GGDSYGYRDY
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMNWVRQAPGKGLE



SYWMN
NIKEDGSEKY
GMDV
WVANIKEDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY




YVDSVKG

YCARGGDSYGYRDYGMDVWGQGTTVTVSS





1.18
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 71
SEQ ID NO: 72



69
70
GGVGYGDSHF
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTHWMNWARQAPGKELE



THWMN
NINQDGSEK
GMDV
WVANINQDGSEKYYVDSVEGRFTISRDNANNSLYLQMNSLRAEDTAV




YYVDSVEG

YYCARGGVGYGDSHFGMDVWGLGTTVTVSS





1.19
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 75
SEQ ID NO: 76



73
74
GGDDYSNSHY
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMIWVRQAPGKGLE



SYWMI
NINQDGSEK
GMDV
WVANINQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV




YYVDSVKG

YYCARGGDDYSNSHYGMDVSGQGTTVTVSS





1.20
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 79
SEQ ID NO: 80



77
78
GGFGYGDSHY
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMNWVRQAPGKGLE



SYWMN
NINQDGSEK
GMDV
WVANINQDGSEKYYVDSVQGRFTISRDNANNSLYLQMNSLRAEDTA




YYVDSVQG

VYYCARGGFGYGDSHYGMDVWGQGTTVTVSS





1.21
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 83
SEQ ID NO: 84



81
82
GGEGYSDSHY
EVQLVESGGGLVQPGGSLRLSCGASGFTFSSYWMFWFRQAPGKELE



SYWMF
NVNQDGSEK
GTDV
WVANVNQDGSEKYYVDSVEGRFTISRDNAKNSLYLQMNSLRAEDTAI




YYVDSVEG

YYCARGGEGYSDSHYGTDVWGQGTTVTVSS





1.22
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 87
SEQ ID NO: 88



85
86
GGEGYGDSHY
QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGL



NYWMN
NIKEDGSEKY
GMDV
EWVANIKEDGSEKYYVDSVEGRFTISRDNARNSLYLQMNSLRAEDTAV




YVDSVEG

YYCARGGEGYGDSHYGMDVSGQGTTVTVSS





1.23
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 91
SEQ ID NO: 92



89
90
GGEGYGDDHY
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMNWVRQAPGKGLE



SYWMN
NIKQDGSEKY
GMDV
WVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLTAEDTAV




YVDSVKG

YYCARGGEGYGDDHYGMDVWGQGTTVTVSS





1.24
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 95
SEQ ID NO: 96



93
94
GGEGYGDYHY
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLE



SYWMS
NIKQDGSEKY
GLDV
WVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV




YVDSVKG

YYCARGGEGYGDYHYGLDVSGQGTTVTVSS





1.25
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 99
SEQ ID NO: 100



97
98
GGDSYGYRDY
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMNWVRQAPGKGLE



SYWMN
NIKQDGSEKY
GMDV
WVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV




YVDSVKG

YYCARGGDSYGYRDYGMDVWGQGTTVTVSS





1.26
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 104



101
102
103
EVQLVESGGGLVQPGGSLRLSCVASGFTFSTHWMNWARQAPGKELE



THWMN
NINQDGSEK
GGVGYGDSHF
WVANINQDGSEKYYVDSVEGRFTISRDNANNSLYLQMNSLRAEDTAV




YYVDSVEG
GMDV
YYCARGGVGYGDSHFGMDVWGLGTTVTVSS





1.27
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 108



105
106
107
EVQLVESGGGLVQPGGSLRLSCGASGFTFSSYWMLWFRQAPGEGLE



SYWML
NINQDGSEK
GGEGYSDSHH
WVANINQDGSEKYYVDSVEGRLTISRDNAKNALYLQMNSLRAEDTAI




YYVDSVEG
GTDV
YYCARGGEGYSDSHHGTDVWGQGTTVTVSS





1.28
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 112



109
110
111
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLE



NYWMN
NIKQDGSEKY
GGDNYAYRDF
WVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV




YVDSVKG
GMDV
YYCARGGDNYAYRDFGMDVWGQGTTVTVSS





1.29
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 116



113
114
115
EVQLVESGGGLVQPGGSLRLSCGASGFTFSNYWMFWFRQAPGKELE



NYWMF
NVNQDGSEK
GGEGYSDSHY
WVANVNQDGSEKYYVDSVEGRFTISRDNAKNSLYLQMNSLRAEDTAI




YYVDSVEG
GTDV
YYCARGGEGYSDSHYGTDVWGQGTTVTVSS





1.30
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 120



117
118
119
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMNWVRQAPGKGLE



SYWMN
NINQDGSEK
GGEEYGSSHYG
WVANINQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV




YYVDSVKG
MDV
YYCARGGEEYGSSHYGMDVWGLGTTVTVSS





1.31
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 124



121
122
123
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMNWVRQAPGKGLE



SYWMN
NINQDGSEK
GGDSYGYRDY
WVANINQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV




YYVDSVKG
GMDV
YYCARGGDSYGYRDYGMDVWGQGTTVTVSS





1.32
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 128



125
126
127
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMNWVRQTPGKGLE



SYWMN
NINQNGSEK
GGFGYGDSHY
WVANINQNGSEKYYVDSVEGRFNISRDNAKNSLYLQMSSLRAEDTAV




YYVDSVEG
GMDV
YYCARGGFGYGDSHYGMDVWGQGTTVTVSS





1.33
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 132



129
130
131
EVQLVESGGGLVQAGGSLRLSCVASGFTFSNYWMTWFRQAPGKGLE



NYWMT
NINQDESEEY
GGDGYSDSHY
WVANINQDESEEYYVDSVKGRFTISRDNAKNSLFLQMNSLRAEDTAIY




YVDSVKG
GTDV
YCARGGDGYSDSHYGTDVWGQGTTVTVSS





1.34
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 136



133
134
135
QVQLQESGGGLVQPGGSLRLSCTASGFTFSNYWMNWVRQAPGKGL



NYWMN
NIKEDGSENY
GGEGYSTSHYG
EWVANIKEDGSENYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTA




YVDSVKG
MDV
VYYCARGGEGYSTSHYGMDVWGQGTTVTVSS





1.35
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 140



137
138
139
QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGL



NYWMN
NIKQDGSEKY
GGEGYGESHY
EWVANIKQDGSEKYYVDSVEGRFTISRDNAKNSLYLQMDSLRAEDTA




YVDSVEG
GMDV
VYYCARGGEGYGESHYGMDVSGQGTTVTVSS





1.36
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 144



141
142
143
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYWMNWVRQAPGKGLE



TYWMN
NIKQDGSEKY
GGDSYGYRDY
WVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV




YVDSVKG
GMDV
YYCARGGDSYGYRDYGMDVWGQGTTVTVSS





1.37
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 148



145
146
147
EVQLVESGGGLVQPGGSLRLSCAASGFTFSYYWMIWFRQAPGEELE



YYWMI
NINQDGSEK
GGDGYSNSHF
WVANINQDGSEKYYVDSVKGRFIISRDNATNSLFLQMNSLRAEDTAVY




YYVDSVKG
GMDV
YCARGGDGYSNSHFGMDVWGQGTTVTVSS





1.38
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 152



149
150
151
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMIWYRQAPGEELE



NYWMI
NINQDGSEK
GGEGYSDSHY
WVANINQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAIY




YYVDSVKG
GTDV
YCARGGEGYSDSHYGTDVWGQGTTVTVSS





1.39
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 156



153
154
155
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKELE



NYWMN
NINQDESEKY
GGFGYGDSHF
WVANINQDESEKYYVDSVKGRFTVSRDNAKNSLFLQMNSLRADDTA




YVDSVKG
GMDV
VYYCARGGFGYGDSHFGMDVWGQGTTVTVSS





1.40
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 160



157
158
159
EVQLVESGGGLVQPGGSLRLSCGASGFTFSNYWMFWFRQAPGKELE



NYWMF
NVNQDGSEK
GGEGYSDSHY
WVANVNQDGSEKYYVDSVEGRFTISRDDAKNSLYLQMNSLRAEDTAI




YYVDSVEG
GTDV
YYCARGGEGYSDSHYGTDVWGQGTTVTVSS





1.41
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 164



161
162
163
EVQLVESGGGLVQPGGSLRLSCGASGFTFSNYWMFWFRQAPGKELE



NYWMF
NVNQNGSEK
GGEGYSDSHY
WVANVNQNGSEKYYVDSVEGRFTISRDNAKNSLYLQMNSLRAEDTAI




YYVDSVEG
GTDV
YYCARGGEGYSDSHYGTDVWGQGTTVTVSS





1.42
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 168



165
166
167
QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGL



NYWMN
NIKQDGSEKY
GGEGYGDSHY
EWVANIKQDGSEKYYVDSVKGRFTISRDNAKDSLYLQMNSLRAEDTAI




YVDSVKG
GMDV
YYCARGGEGYGDSHYGMDVSGQGTTVTVSS





1.43
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 172



169
170
171
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMIWYRQAPGEELE



NYWMI
NINQDGSEK
GGDGYSNSHY
WVANINQDGSEKYYVDSVKGRFTISRDNATNSLFLQMNSLRAEDTAV




YYVDSVKG
GMDV
YYCARGGDGYSNSHYGMDVWGQGTTVTVSS





1.44
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 176



173
174
175
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMIWYRQAPGEELE



DYWMI
NINQDGSEK
GGDGYSNSHY
WVANINQDGSEKYYVDSVKGRFTISRDNATNSLFLQMNSLRAEDTAV




YYVDSVKG
GMDV
YYCARGGDGYSNSHYGMDVWGQGTTVTVSS





1.45
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 180



177
178
179
EVQLVESGGGLVQPGGSLRLSCAASGFTFSKYWMIWVRQAPEKGLE



KYWMI
NINQDGSEK
GGDDYSNSHY
WVANINQDGSEKYYVDSVEGRFTISRDNVNNSLYLQMNSLRAEDTAV




YYVDSVEG
GMDV
YYCARGGDDYSNSHYGMDVSGQGTTVTVSS





1.46
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 184



181
182
183
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGRGLE



NYWMS
NINQDGSEK
GGEEYSSSHYG
WVANINQDGSEKYYVDSVKGRFTISRDNAKSSLYLQMNSLRAEDTAV




YYVDSVKG
MDV
YYCARGGEEYSSSHYGMDVWGQGTTVTVSS





1.47
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 188



185
186
187
EVQLVESGGGLVQPGGSLRLSCIASGFSFSNYWMNWVRQAPGKGLE



NYWMN
NIKQDGSEN
GGEGYSTSHYG
WVANIKQDGSENYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV




YYVDSVKG
MDV
YYCARGGEGYSTSHYGMDVWGQGTAVTVSS





1.48
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 192



189
190
191
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLE



SYWMS
NIKQDGSEKY
GGEGYGVDHY
WVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAVY




YVDSVKG
GLDV
FCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.49
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 196



193
194
195
EVQLVESGGGLVQPGGSLRLSCGASGFTFSSYWMLWFRQAPGKELE



SYWML
NVNQDGSEN
GGEDYGNSHF
WVANVNQDGSENYYVDSVEGRFTISRDNAKNSLYLQMHSLRAEDTA




YYVDSVEG
GMDV
VYYCARGGEDYGNSHFGMDVWGQGTMVTVSS





1.50
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 200



197
198
199
EVQLVESGGGLVQPGRSLRLSCAASGFTFSNYWMIWYRQAPGEELE



NYWMI
NINQDGSEK
GGDGYSNSHY
WVANINQDGSEKYYVDSVKGRFTISRDNATNSLFLQMNSLRAEDTAV




YYVDSVKG
GMDV
YYCARGGDGYSNSHYGMDVWGQGTTVTVSS





1.51
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 204



201
202
203
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYWMIWYRQAPGEELE



NYWMI
NINQDGSEK
GGDGYSNSHY
WVANINQDGSEKYYVDSVKGRFTISRDNATNSLFLQMNSLRAEDTAV




YYVDSVKG
GMDV
YYCARGGDGYSNSHYGMDVWGQGTTVTVSS





1.52
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 208



205
206
207
EVQLVESGGGLVQIGGSLRLSCAASGFTFSKYWMIWVRQAPEKGLEW



KYWMI
NINQDGSEK
GGDDYSISHFG
VANINQDGSEKYYVDSVEGRFTISRDNANNSLFLQMNSLRAEDTAVYY




YYVDSVEG
MDV
CARGGDDYSISHFGMDVSGQGTRVTVSS





1.53
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 212



209
210
211
EVQLVESGGGLVQIGGSLRLSCVASGFTFSKYWMIWVRQAPEKGLEW



KYWMI
NINQDGSEK
GGDDYSHSHY
VANINQDGSEKYYVDSVEGRFTISRDNANNSLYLQMNSLRAEDTAVYY




YYVDSVEG
GMDV
CARGGDDYSHSHYGMDVSGQGTTVTVSS





1.54
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 216



213
214
215
EVQLVESGGGLVQPGGSLRLSCAASGFNFSNYWMNWVRQAPGKELE



NYWMN
NINQDGSEK
GGFGYGDSHY
WVANINQDGSEKYYVDSVKGRFTISRDNAKNSLFLQMNSLRADDTAV




YYVDSVKG
GMDV
YYCARGGFGYGDSHYGMDVWGQGTTVTVSS





1.55
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 220



217
218
219
EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWLNWVRQAPGKGLE



SYWLN
NINQDGSEN
GGEDYGNSHF
WVANINQDGSENYYVDSVEGRFTISRDNAKNSLYLQMHSLRAEDTAV




YYVDSVEG
GMDV
YYCARGGEDYGNSHFGMDVWGQGTMVTVSS





1.56
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 224



221
222
223
QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLE



SYWMS
NIKQDGSEKY
GGEGYGVDHY
WVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAVY




YVDSVKG
GLDV
FCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.57
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 228



225
226
227
QVQLVESGGGLVQPGGSLRLSCTASGFTFSDYWMNWVRQAPGKGL



DYWMN
NIKEDGSEKY
GGEGYGDNHY
EWVANIKEDGSEKYYVDSVEGRFTISRDNARNSLYLQMTSLREEDTA




YVDSVEG
GMDV
MYYCARGGEGYGDNHYGMDVSGQGTTVTVSS





1.58
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 232



229
230
231
EVQLVESGGGLVQPGGSLRLSCAASGFTFRSYWMNWVRQAPGKEAE



SYWMN
NINQDGSEK
GGPDYGDLHY
WVANINQDGSEKYYVDSVEGRFTISRDNAKNSLFLQMNSLRDEDTAV




YYVDSVEG
GMDV
YYCARGGPDYGDLHYGMDVWGQGTTVTVSS





1.59
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 236



233
234
235
QVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLE



RYWMS
NINQDGREK
GGEGYGDYHY
RVANINQDGREKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV




YYVDSVKG
GMDV
YYCARGGEGYGDYHYGMDVSGQGTTVTVSS





1.60
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 240



237
238
239
EVQLVESGGGLVQPGGSPRLSCAASGFTLSNYWMIWYRQAPGEKLE



NYWMI
NINQDGSEK
GGDGYSNSHY
WVANINQDGSEKYYVDSVKGRFTISRDNATNSLFLQMNSLRAEDTAV




YYVDSVKG
GMDV
YYCARGGDGYSNSHYGMDVWGQGTTVTVSS





1.61
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 244



241
242
243
EVQLVESGGGLVQPGGSLRLSCVASGFNFSNYWMNWVRQAPGKELE



NYWMN
NINQDESEKY
GGFGYGDSHF
WVANINQDESEKYYVDSVKGRFTISRDNAKNSLFLQMNSLRADDTAV




YVDSVKG
GMDV
YYCARGGFGYGDSHFGMDVWGQGTTVTVSS





1.62
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 248



245
246
247
EVQLVESGGGLVQPGGSLRLSCAASGFNFSNYWMNWVRQAPGKELE



NYWMN
NINQDESEKY
GGFGYGDSHF
WVANINQDESEKYYVDSVKGRFTIFRDNAKNSLFLQMNSLRADDTAV




YVDSVKG
GMDV
YYCARGGFGYGDSHFGMDVWGQGTTVTVSS





1.63
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 252



249
250
251
EVQLVESGGGLVQPGGSLSLSCAASGFTFRSFWMNWVRQAPGKEAE



SFWMN
NINQDGSEK
GGPDYGDLHY
WVANINQDGSEKYYVDSVKGRFTISRDNAKNSLFLQMNSLRAEDTAV




YYVDSVKG
GMDV
YYCARGGPDYGDLHYGMDVWGQGTTVTVSS





1.64
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 256



253
254
255
EVQLVESGGGLVQPGGSLRLSCAASGFTFRSYWMNWVRQAPGKEAE



SYWMN
NINQDGSEK
GGPDYGDLHY
WVANINQDGSEKYYVDSVKGRFTISRDNAKNSLFLQMNSLRDEDTAV




YYVDSVKG
GMDV
YYCARGGPDYGDLHYGMDVWGQGTTVTVSS





1.65
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 260



257
258
259
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMIWYRQAPGEELE



NYWMI
NINQDGSEK
GGEDYGNSHY
WVANINQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMHSLRAEDTAV




YYVDSVKG
GMDV
YYCARGGEDYGNSHYGMDVWGQGTMVTVSS





1.66
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 264



261
262
263
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLE



NYWMN
NINQDGSER
GGEGYGIDHY
WVANINQDGSERYYVDSVKGRFTISRDNAKSSLYLQMNSLRAEDTAV




YYVDSVKG
GLDV
YFCARGGEGYGIDHYGLDVSGQGTTVTVSS





1.67
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 268



265
266
267
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLE



SYWMS
NINQDGSER
GGEGYGVDHY
WVANINQDGSERYYVDSVKGRFTISRDNAKSSLYLQMSSLRAEDTAVY




YYVDSVKG
GLDV
FCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.68
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 272



269
270
271
QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMIWVRQAPGKGLE



NYWMI
NINQDGSEK
GGEGYGVDHY
WVANINQDGSEKYYVDSVEGRFTISRDNAKSSLYLQMSNLRAEDTAV




YYVDSVEG
GLDV
YFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.69
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 276



273
274
275
QVQLVESGGGLVQPGGSLRLSCAATGFTLSNYWMNWVRQAPGKGL



NYWMN
NINQDGSEK
GGTGYGSDHY
EWVANINQDGSEKYYVDSVKGRFTISRDNAKNSLFLQMNSLRAEDTA




YYVDSVKG
GMDV
VYYCARGGTGYGSDHYGMDVSGQGTTVTVSS





1.70
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 280



277
278
279
EVQLVESGGGLVQPGGSLRLSCAASGFNFSNYWMNWVRQAPGKELE



NYWMN
NINQDGSEN
GGFGYGDSHY
WVANINQDGSENYYVDSVKGRFTISRDNVKNSLFLQMNRLRADDTA




YYVDSVKG
GMDV
VYYCARGGFGYGDSHYGMDVWGQGTTVTVSS





1.71
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 284



281
282
283
EVQLVESGGGLVQPGGSLRLSCAASGFTFGNYWMIWVRQAPGKELE



NYWMI
NINQNGSER
GGADYSNSHY
WLANINQNGSERYYVDSVQGRFTISRDNAKNSLYLQMNSLRAEDTAV




YYVDSVQG
GMDV
YYCARGGADYSNSHYGMDVSGQGTTVTVSS





1.72
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 288



285
286
287
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLE



SYWMS
NINQDGSER
GGEGYGVDHY
WVANINQDGSERYYVDSVKGRFTISRDNANNSLHLQMSSLRAEDTAV




YYVDSVKG
GLDV
YFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.73
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 292



289
290
291
EVQLVESGGGLVQPGGSLRLSCAASGFTFRSYWMNWVRQAPGKEAE



SYWMN
NINPDGSEKY
GGPGYGDLHY
WVANINPDGSEKYYVDSVQGRHTISRDNAKNSLFLEMNSLRVEDTAL




YVDSVQG
GMDV
YYCARGGPGYGDLHYGMDVWGQGTTVTVSS





1.74
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 296



293
294
295
QVQLVESGGGLVQPGGSLRLSCAATGFTLSNYWMNWVRQAPGKGL



NYWMN
NINQDGSEK
GGEGYGVDHY
EWVANINQDGSEKYYVDSVEGRFTISRDNAKSSLYLQMSSLRAEDTAV




YYVDSVEG
GLDV
YFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.75
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 300



297
298
299
QVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLE



DYYMS
NINQDGSER
GGEGYGVDHY
WVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAV




YYVDSVKG
GLDV
YFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.76
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 304



301
302
303
EVQLVESGGGLVQPGGSLRLSCAATGFTLSNYWMNWVRQAPGKGLE



NYWMN
NINQDGSER
GGEGYGVDHY
WVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAV




YYVDSVKG
GLDV
YFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.77
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 308



305
306
307
QVQLVESGGGLVQPGGSLRLSCAASGFTLSNYWMNWVRQAPGKGL



NYWMN
NINQDGSER
GGEGYGVDHY
EWVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMSSLRAEDTA




YYVDSVKG
GLDV
VYFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.78
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 312



309
310
311
QVQLVESGGGLVQPGGSLRLSCAATGFTLSNYWMNWVRQAPGKGL



NYWMN
NINQDGSER
GGEGYGVDHY
EWVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMSSLRAEDTA




YYVDSVKG
GLDV
VYFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.79
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 316



313
314
315
QVQLVESGGGLVQPGGSLRLSCAATGFTLSNYWMNWVRQAPGKGL



NYWMN
NINQDGSER
GGEGYGVNHY
EWVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMSSLRAEDTA




YYVDSVKG
GLDV
VYFCARGGEGYGVNHYGLDVSGQGTTVTVSS





1.80
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 320



317
318
319
QVQLVESGGGLVKPGGSLRLSCVASGFTFSDYYMSWIRQAPGKGLEW



DYYMS
NIKQDGSERY
GGEGYGVDHY
VANIKQDGSERYYVDSVKGRFTISRDNAKSSLYLQMSSLRAEDTAVYFC




YVDSVKG
GLDV
ARGGEGYGVDHYGLDVSGQGTTVTVSS





1.81
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 324



321
322
323
QVQLVESGGGLVQPGGSLRLSCAATGFTLSNYWMNWVRQAPGKGL



NYWMN
NINQDGSER
GGEGYGVDHY
EWVANINQDGSERYYVDSVEGRFTISRDNAKSSLYLQMSNLRAEDTA




YYVDSVEG
GLDV
VYFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.82
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 328



325
326
327
QVQLVESGGGLVQPGGSLRLSCAATGFTLSNYWMNWVRQAPGKGL



NYWMN
NINQDGSER
GGEGYGVDHY
EWVANINQDGSERYYVDSVKGRFTISRDNAKSSLYLQMSSLRAEDTAV




YYVDSVKG
GLDV
YFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.83
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 332



329
330
331
QVQLQESGGGLVQPGGSLRLSCAATGFTLSNYWMNWVRQAPGKGL



NYWMN
NINQDGSER
GGEGYGVDHY
EWVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMSSLRAEDTA




YYVDSVKG
GLDV
VYFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.84
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 336



333
334
335
QVQLVESGGGLVKPGGSLRLSCAATGFTLSNYWMNWVRQAPGKGL



NYWMN
NINQDGSER
GGEGYGVDHY
EWVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMSSLRAEDTA




YYVDSVKG
GLDV
VYFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.85
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 340



337
338
339
QVQLVESGGGLVQPGGSLRLSCAATGFTLSNYWMNWVRQAPGKGL



NYWMN
NINQDGSER
GGEGYGVDHY
EWVANINQDGSERYYVDSVKGRFTISRDNANNSLHLQMSSLRAEDTA




YYVDSVKG
GLDV
VYFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.86
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 344



341
342
343
QVQLGESGGGLVQPGGSLRLSCAATGFTLSNYWMNWVRQAPGKGL



NYWMN
NINQDGSER
GGEGYGVDHY
EWVANINQDGSERYYVDSVEGRFTISRDNAKSSLYLQMSNLRAEDTA




YYVDSVEG
GLDV
VYFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.87
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 348



345
346
347
QVQLVESGGGLVQPGGSLKLSCAATGFTLSNYWMNWVRQAPGKGL



NYWMN
NINQDGSER
GGEGYGVDHY
EWVANINQDGSERYYVDSVKGRFTISRDNAKSSLYLQMSSLRAEDTAV




YYVDSVKG
GLDV
YFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.88
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 352



349
350
351
QVQLQESGGGLVQPGGSLRLSCAATGFTLSNYWMNWVRQAPGKGL



NYWMN
NINQDGSER
GGEGYGVDHY
EWVANINQDGSERYYVDSVKGRFTISRGNAKNSLYLQMSSLRAEDTA




YYVDSVKG
GLDV
VYFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.89
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 356



353
354
355
QVQLVESGGGVVQPGRSLRLSCAASGFTFDDYGMSWVRQAPGKGLE



DYGMS
NINQDGSER
GGEGYGVDHY
WVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAV




YYVDSVKG
GLDV
YFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.90
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 360



357
358
359
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWVRQAPGKGLE



SHWMT
HIKEDGSEKY
GGDGYSDSHF
WVAHIKEDGSEKYYEDSVEGRFTVSRDNAKNSVYLQMNSLRAEDTAV




YEDSVEG
GVDV
YYCARGGDGYSDSHFGVDVWGQGTTVTVSS





1.91
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 364



361
362
363
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLE



SHWMT
HIKEDGSEKY
GGDGYSDSHF
WVAHIKEDGSEKYYVDSVKGRFTVSRDNAKNSVYLQMNSLRAEDTAV




YVDSVKG
GVDV
YYCARGGDGYSDSHFGVDVWGQGTTVTVSS





1.92
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 368



365
366
367
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLE



SHWMT
HIKEDGSEKY
GGDGYSDSHF
WVAHIKEDGSEKYYEDSVKGRFTVSRDNAKNSVYLQMNSLRAEDTAV




YEDSVKG
GVDV
YYCARGGDGYSDSHFGVDVWGQGTTVTVSS





1.93
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 372



369
370
371
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLE



SHWMT
HIKEDGSEKY
GGDGYSDSHF
WVAHIKEDGSEKYYEDSVEGRFTISRDNAKNSVYLQMNSLRAEDTAVY




YEDSVEG
GVDV
YCARGGDGYSDSHFGVDVWGQGTTVTVSS





1.94
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 376



373
374
375
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLE



SHWMT
HIKEDGSEKY
GGDGYSDSHF
WVAHIKEDGSEKYYEDSVEGRFTVSRDNAKNSLYLQMNSLRAEDTAV




YEDSVEG
GVDV
YYCARGGDGYSDSHFGVDVWGQGTTVTVSS





1.95
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 380



377
378
379
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWVRQAPGKGLE



SHWMT
HIKEDGSEKY
GGDGYSDSHF
WVAHIKEDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY




YVDSVKG
GVDV
YCARGGDGYSDSHFGVDVWGQGTTVTVSS





1.96
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 384



381
382
383
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLE



SHWMT
HIKEDGSEKY
GGDGYSDSHF
WVAHIKEDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY




YVDSVKG
GVDV
YCARGGDGYSDSHFGVDVWGQGTTVTVSS





1.97
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 388



385
386
387
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWVRQAPGKGLE



SHWMT
HIKEDESEKY
GGVGYSISHFG
WVAHIKEDESEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY




YVDSVKG
VDV
YCARGGVGYSISHFGVDVWGQGTTVTVSS





1.98
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 392



389
390
391
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWVRQAPGKGLE



SHWMT
HIKEDESEKY
GGEGYSISHFG
WVAHIKEDESEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY




YVDSVKG
VDV
YCARGGEGYSISHFGVDVWGQGTTVTVSS





1.99
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 396



393
394
395
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLE



SHWMT
HIKEDESEKY
GGDGYSDSHF
WVAHIKEDESEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY




YVDSVKG
GVDV
YCARGGDGYSDSHFGVDVWGQGTTVTVSS





1.100
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 400



397
398
399
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLE



SHWMT
HIKEDESEKY
GGDGYSISHFG
WVAHIKEDESEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY




YVDSVKG
VDV
YCARGGDGYSISHFGVDVWGQGTTVTVSS





1.101
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 404



401
402
403
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLE



SHWMT
HIKEGGSEKY
GGDGYSDSHF
WVAHIKEGGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY




YVDSVKG
GVDV
YCARGGDGYSDSHFGVDVWGQGTTVTVSS





1.102
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 408



405
406
407
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLE



SHWMT
HIKEEGSEKY
GGDGYSDSHF
WVAHIKEEGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY




YVDSVKG
GVDV
YCARGGDGYSDSHFGVDVWGQGTTVTVSS





1.103
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 412



409
410
411
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLE



SHWMT
HIKEDGSEKY
GGEGYSDSHF
WVAHIKEDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY




YVDSVKG
GVDV
YCARGGEGYSDSHFGVDVWGQGTTVTVSS





1.104
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 416



413
414
415
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLE



SHWMT
HIKEDGSEKY
GGVGYSDSHF
WVAHIKEDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY




YVDSVKG
GVDV
YCARGGVGYSDSHFGVDVWGQGTTVTVSS





1.105
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 420



417
418
419
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLE



SHWMT
HIKEDESEKY
GGVGYSISHFG
WVAHIKEDESEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY




YVDSVKG
VDV
YCARGGVGYSISHFGVDVWGQGTTVTVSS





1.106
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 424



421
422
423
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLE



SHWMT
HIKEDESEKY
GGEGYSISHFG
WVAHIKEDESEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY




YVDSVKG
VDV
YCARGGEGYSISHFGVDVWGQGTTVTVSS





1.107
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 852



849
850
851
EVQLVESGGGLVQPGGSLRLSCAASGFTLSNYWMNWVRQAPGKGLE



NYWMN
NINQDGSER
GGEGYGVDHY
WVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAV




YYVDSVKG
GLDV
YFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.108
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 856



853
854
855
EVQLVESGGGLVQPGGSLRLSCAATGFTLSNYWMNWVRQAPGKGLE



NYWMN
NINQDGSER
GGEGYGVDHY
WVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV




YYVDSVKG
GLDV
YFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.109
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 860



857
858
859
EVQLVESGGGLVQPGGSLRLSCAATGFTLSNYWMNWVRQAPGKGLE



NYWMN
NINQDGSER
GGEGYGVDHY
WVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAV




YYVDSVKG
GLDV
YYCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.110
SEQ ID NO:
SEQ ID
SEQ ID NO:
SEQ ID NO: 864



861
NO:862
863
EVQLVESGGGLVQPGGSLRLSCAASGFTLSNYWMNWVRQAPGKGLE



NYWMN
NINQDGSER
GGEGYGVDHY
WVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV




YYVDSVKG
GLDV
YFCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.111
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 868



865
866
867
EVQLVESGGGLVQPGGSLRLSCAASGFTLSNYWMNWVRQAPGKGLE



NYWMN
NINQDGSER
GGEGYGVDHY
WVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAV




YYVDSVKG
GLDV
YYCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.112
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 872



869
870
871
EVQLVESGGGLVQPGGSLRLSCAATGFTLSNYWMNWVRQAPGKGLE



NYWMN
NINQDGSER
GGEGYGVDHY
WVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV




YYVDSVKG
GLDV
YYCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.113
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 876



873
874
875
EVQLVESGGGLVQPGGSLRLSCAASGFTLSNYWMNWVRQAPGKGLE



NYWMN
NINQDGSER
GGEGYGVDHY
WVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV




YYVDSVKG
GLDV
YYCARGGEGYGVDHYGLDVSGQGTTVTVSS





1.114
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 880



877
878
879
EVQLVESGGGLVQPGGSLRLSCAASGFTLSNYWMNWVRQAPGKGLE



NYWMN
NINQDESERY
GGEGYGVDHY
WVANINQDESERYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV




YVDSVKG
GLDV
YYCARGGEGYGVDHYGLDVSGQGTTVTVSS









In one aspect, the single variable heavy chain domain antibody comprises a CDR1, CDR2 or CDR3 as shown for one of the single domain antibodies as shown in Table 3 or comprising a CDR1, CDR2, CDR3 at least 75% homology thereto. For example, the single variable heavy chain domain antibody comprises a CDR1 comprising SEQ ID NO. 425 or a sequence with at least 80% homology thereto, a CDR2 comprising SEQ ID NO. 426 or a sequence with at least 75% homology thereto and a CDR3 comprising SEQ ID NO. 427 or a sequence with at least 75% homology, or a CDR1 comprising SEQ ID NO. 429 or a sequence with at least 75% homology thereto, a CDR2 comprising SEQ ID NO. 430 or a sequence with at least 75% homology thereto and a CDR3 comprising SEQ ID NO. 431 and so forth.


Sequence homology can be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% for example at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.


In one embodiment, the single variable heavy chain domain antibody comprises human framework regions.


In one embodiment, the single variable heavy chain domain antibody comprising or consisting of a full length sequence as shown in Table 3 or a sequence with at least 50% homology thereto. For example, the single variable heavy chain domain antibody has a sequence selected from those shown in Table 3, e.g. SEQ ID NO. 428, 432, 436, 440 and so forth or a sequence with at least 50% homology thereto. Sequence homology can be at least 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% for example at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.


In one embodiment, the single variable heavy chain domain antibody comprises CDR1, 2, and 3 as shown for VH single domain antibodies 2.41 to 2.51 or comprises or consists of a full length sequence as shown for VH single domain antibodies 2.41 to 2.51 (i.e. SEQ ID NOs. 588, 592, 596, 600, 604, 608, 612, 616, or 620).









TABLE 3







Full length sequences and CDR sequences of VH single domain antibodies (Family 2)











Name
CDR1
CDR2
CDR3
Full Length





2.1
SEQ ID NO:
SEQ ID NO: 426
SEQ ID NO: 427
SEQ ID NO: 428



425
YISGSGDIIDYA
EDSRLIGTTDFD
EVQLVESGGGLVKPGGSLRVSCAASGFTFSDYYMSWFRQAP



DYYMS
DSVKG
N
GKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQM






NNLRAEDTAVYHCAREDSRLTGTTDFDNWGQGTLVTVSS





2.2
SEQ ID NO:
SEQ ID NO: 430
SEQ ID NO: 431
SEQ ID NO: 432



429
YISGSGDIIDYA
EDSRIPGTTDFD
EVQLVESGGGLVKPGGSLRVSCAASGFTFSDYYMSWFRQAP



DYYMS
DSVKG
N
GKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYHCAKEDSRIPGTTDFDNWGQGTLVTVSS





2.3
SEQ ID NO:
SEQ ID NO: 434
SEQ ID NO: 435
SEQ ID NO: 436



433
YISGSGDIIDYA
EDSRIPGTTDFD
EVQLVESGGGLVKPGGSLRVSCAASGFTFSDYYMSWFRQAP



DYYMS
DSVKG
N
GKGLEWISYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQMN






SLRAEDTAVYHCAKEDSRIPGTTDFDNWGQGTLVTVSS





2.4
SEQ ID NO:
SEQ ID NO: 438
SEQ ID NO: 439
SEQ ID NO: 440



437
YISGSGDVIDYA
EDSRIPGTTDFD
EVQLVESGGGLVKPGGSLRVSCAASGFTFSDYYMSWFRQAP



DYYMS
DSVKG
N
GKGLEWVSYISGSGDVIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYHCAKEDSRIPGTTDFDNWGQGTLVTVSS





2.5
SEQ ID NO:
SEQ ID NO: 442
SEQ ID NO: 443
SEQ ID NO: 444



441
YISGSGDIIDYA
EDSRIPGTTDFD
EVQLVESGGGLVKPGGSLRLSCAVSGFTFSDYYMSWFRQAP



DYYMS
DSVKG
N
GKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYHCAKEDSRIPGTTDFDNWGQGTLVTVSS





2.6
SEQ ID NO:
SEQ ID NO: 446
SEQ ID NO: 447
SEQ ID NO: 448



445
YISGSGDIIDYA
EDSRIPGTTDFDS
EVQLVESGGGLVKPGGSLRVSCAASGFTFSDYYMSWFRQAP



DYYMS
DSVKG

GKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYHCAKEDSRIPGTTDFDSWGQGTMVTVSS





2.7
SEQ ID NO:
SEQ ID NO: 450
SEQ ID NO: 451
SEQ ID NO: 452



449
YISGSGTTIDYA
EDIRMTGTTDFD
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWFRQAP



DYYMS
DSVKG
N
GKGLEWVSYISGSGTTIDYADSVKGRFTISRDNARNSLYLQM






NSLRAEDTAVYYCAREDIRMTGTTDFDNWGQGTLVTVSS





2.8
SEQ ID NO:
SEQ ID NO: 454
SEQ ID NO: 455
SEQ ID NO: 456



453
HISGSGTTIDYA
EDSRMPGTTDFD
EVQLVESGGGLVKPGGSLRLSCAASGFAFSDYYMSWFRQAP



DYYMS
DSVKG
N
GKGLEWVSHISGSGTTIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYHCAREDSRMPGTTDFDNWGQGTLVTVSS





2.9
SEQ ID NO:
SEQ ID NO: 458
SEQ ID NO: 459
SEQ ID NO: 460



457
YISGSGDTIDYA
EDSRIAGTTDFD
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWFRQAP



DYYMT
ESVKG
N
GKGLEWISYISGSGDTIDYAESVKGRFTISRDNAKNSLYLQMN






SLRAEDTAVYHCAREDSRIAGTTDFDNWGPGTLVTVSS





2.10
SEQ ID NO:
SEQ ID NO: 462
SEQ ID NO: 463
SEQ ID NO: 464



461
YISSSGSNIDYA
EDSRLSGTTDFDY
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWFRQAP



DYYMT
DSVKG

GKGLEWVSYISSSGSNIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCAREDSRLSGTTDFDYWGQGTLVTVSS





2.11
SEQ ID NO:
SEQ ID NO: 466
SEQ ID NO: 467
SEQ ID NO: 468



465
YISGSGDTIDYA
EDSRIAGTTDFD
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWFRQAP



DYYMT
ESVKG
N
GKGLEWISYISGSGDTIDYAESVKGRFTISRDNAKNSLYLQMN






SLRAEDTAVYHCAREDSRIAGTTDFDNWGPGTLVTVSS





2.12
SEQ ID NO:
SEQ ID NO: 470
SEQ ID NO: 471
SEQ ID NO: 472



469
HISGSGTTIDYA
EDIRMTGTTDFD
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWFRQAP



DYYMS
DSVKG
H
GKGLEWVSHISGSGTTIDYADSVKGRFTISRDNARKSLYLQM






NSLRAEDTAVYYCAREDIRMTGTTDFDHWGQGTLVTVSS





2.13
SEQ ID NO:
SEQ ID NO: 474
SEQ ID NO: 475
SEQ ID NO: 476



473
HISSSGNTIDYA
EDPRLPGTTDFD
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWFRQAP



DYYMS
DSVKG
Y
GKGLEWVSHISSSGNTIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCAREDPRLPGTTDFDYWGQGTLVTVSS





2.14
SEQ ID NO:
SEQ ID NO: 478
SEQ ID NO: 479
SEQ ID NO: 480



477
YISGSGDTIDYA
EDIRMPGTTDFD
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWFRQAP



DYYMT
ESVKG
H
GKGLEWISYISGSGDTIDYAESVKGRFTISRDNAKNSLYLQMN






SLRAEDTAVYYCAREDIRMPGTTDFDHWGQGTLVTVSS





2.15
SEQ ID NO:
SEQ ID NO: 482
SEQ ID NO: 483
SEQ ID NO: 484



481
HISGSGTTIDYA
EDIRMPGTTDFD
EVQLVESGGGLVKPGGSLRLSCAVSGFTFSDYYMSWFRQAP



DYYMS
DSVKG
H
GKGLEWVSHISGSGTTIDYADSVKGRFTISRDNARNSLYLQM






NSLRAEDTAVYYCAREDIRMPGTTDFDHWGQGTLVTVSS





2.16
SEQ ID NO:
SEQ ID NO: 486
SEQ ID NO: 487
SEQ ID NO: 488



485
HISSSGSTIDYA
EDPRLIGTTDFD
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAP



DYYMS
DSVKG
Y
GKGLEWVSHISSSGSTIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCAREDPRLIGTTDFDYWGQGALVTVSS





2.17
SEQ ID NO:
SEQ ID NO: 490
SEQ ID NO: 491
SEQ ID NO: 492



489
YISSSGSTISYAD
EDPRISGTTDFD
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAP



DYYMS
SVKG
N
GKGLEWVSYISSSGSTISYADSVKGRFTISRDNAKNSLYLQMN






SLRAEDTAVYYCAREDPRISGTTDFDNWGQGTLVTVSS





2.18
SEQ ID NO:
SEQ ID NO: 494
SEQ ID NO: 495
SEQ ID NO: 496



493
HISSSGNTIDYA
EDPRLPGTTDFD
QVQLQESGGGLVKPGGSLRLSCAASGFTFSDYYMSWFRQAP



DYYMS
DSVKG
Y
GKGLEWVSHISSSGNTIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCAREDPRLPGTTDFDYWGQGTLVTVSS





2.19
SEQ ID NO:
SEQ ID NO: 498
SEQ ID NO: 499
SEQ ID NO: 500



497
YISGTGITTDYA
EDPRLPGTSEFD
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWFRQAP



DYYMS
DSVKG
N
GKGLEWVSYISGTGITTDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCAREDPRLPGTSEFDNWGQGTLVTVSS





2.20
SEQ ID NO:
SEQ ID NO: 502
SEQ ID NO: 503
SEQ ID NO: 504



501
HISSSGSTIDYA
EDPRMPGTFDFD
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAP



DYYMS
DSVKG
N
GKGLEWVSHISSSGSTIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCAREDPRMPGTFDFDNWGQGTLVTVSS





2.21
SEQ ID NO:
SEQ ID NO: 506
SEQ ID NO: 507
SEQ ID NO: 508



505
HISGSGTTIDYA
EDIRMPGTTDFD
EVQLVESGGGLVKPGGSLRLSCAASGFAFSDYYMSWFRQAP



DYYMS
DSVKG
H
GKGLEWVSHISGSGTTIDYADSVKGRFTISRDNARNSLYLQM






NSLRAEDTAVYYCAREDIRMPGTTDFDHWGQGTLVTVSS





2.22
SEQ ID NO:
SEQ ID NO: 510
SEQ ID NO: 511
SEQ ID NO: 512



509
HISGSGTTIDYA
EDIRMPGTTDFD
EVQLVESGGGLVKPGGSLRLSCAVSGFTFSDYYMSWFRQAP



DYYMS
DSVKG
H
GKGLEWVSHISGSGTTIDYADSVKGRFTISRDNARDSLYLQM






NSLRAEDTAVYYCAREDIRMPGTTDFDHWGQGTLVTVSS





2.23
SEQ ID NO:
SEQ ID NO: 514
SEQ ID NO: 515
SEQ ID NO: 516



513
HISGSGTTIDYA
EDIRMPGTTDFD
EVQLVESGGGLVTPGGSLRLSCAVSGFTFSDYYMSWFRQAP



DYYMS
DSVKG
H
GKGLEWVSHISGSGTTIDYADSVKGRFTISRDNARNSLYLQM






NSLRAEDTAVYYCAREDIRMPGTTDFDHWGQGTLVTVSS





2.24
SEQ ID NO:
SEQ ID NO: 518
SEQ ID NO: 519
SEQ ID NO: 520



517
HISGSGTTIDYA
EDIRMPGTTDFD
EVQLVESGGGLVKPGGSLRLSCAVSGFTFSDYYMSWFRQAP



DYYMS
DSVKG
H
GKGLEWVSHISGSGTTIDYADSVKGRFTISRDNARNSLYLQM






NSLRAEDTAMYYCAREDIRMPGTTDFDHWGQGTLVTVSS





2.25
SEQ ID NO:
SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 524



521
HISGSGTTIDYA
EDIRMPGTTDFD
EVQLVESGGGLVKPGGSLRLSCAASGFAFSDYYMSWFRQAP



DYYMS
DSVKD
H
GKGLEWVSHISGSGTTIDYADSVKDRFTISRDNARNSLYLQM






NSLRAEDTAVYYCAREDIRMPGTTDFDHWGQGTLVTVSS





2.26
SEQ ID NO:
SEQ ID NO: 526
SEQ ID NO: 527
SEQ ID NO: 528



525
HISSSGTTIDYA
EDIRMPGTTDFD
EVQLVESGGGLVKPGGSLRLSCTASGFTFTDYYMSWFRQAP



DYYMS
DSVKG
N
GKGLEWVSHISSSGTTIDYADSVKGRFTISRDNAKNSLYLQM






NSLRADDTAVYYCAREDIRMPGTTDFDNWGQGTLVTVSS





2.27
SEQ ID NO:
SEQ ID NO: 530
SEQ ID NO: 531
SEQ ID NO: 532



529
YISSSGSTISYAD
EDIRMSGTTDFD
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWFRQAP



DYYMT
SVKG
Y
GKGLEWVSYISSSGSTISYADSVKGRFTISRDNANNSLYLQM






NSLRAEDTAVYHCAREDIRMSGTTDFDYWGQGTLVTVSS





2.28
SEQ ID NO:
SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 536



533
HISSSGSSIDYA
EDPRLSGTIDFDS
QVQLVESGGGLVKPGGSLRLSCAASGFIFSDYYMSWFRQAP



DYYMS
DSVKG

GKGLEWVSHISSSGSSIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCAREDPRLSGTIDFDSWGQGTLVTVSS





2.29
SEQ ID NO:
SEQ ID NO: 538
SEQ ID NO: 539
SEQ ID NO: 540



537
HIGGSGTTIDYA
EDIRMPGTTDFD
EVQLVESGGGLVKPGGSLRLSCAASGFAFSDYYMSWFRQAP



DYYMS
DSVKG
H
GKGLEWVSHIGGSGTTIDYADSVKGRFTISRDNARNSLYLQM






NSLRAEDTAVYYCAREDIRMPGTTDFDHWGQGTLVTVSS





2.30
SEQ ID NO:
SEQ ID NO: 542
SEQ ID NO: 543
SEQ ID NO: 544



541
YISSSGSTIYYAD
EDPRVPGTTNFD
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAP



DYYMS
SVKG
Y
GKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMN






SLRAEDTAVYYCAREDPRVPGTTNFDYWGQGTLVTVSS





2.31
SEQ ID NO:
SEQ ID NO: 546
SEQ ID NO: 547
SEQ ID NO: 548



545
YISGSGSTIDYA
EDGRIPGTTDFD
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWMRQA



DYYMT
DSVKG
H
PGKGLEWVSYISGSGSTIDYADSVKGRFTISRDNAKNSLYLQ






MNSLRPEDTAVYYCAKEDGRIPGTTDFDHWGQGTLVTVSS





2.32
SEQ ID NO:
SEQ ID NO: 550
SEQ ID NO: 551
SEQ ID NO: 552



549
HISGSGTTIDYA
EDIRMPGTTDFD
EVQLVESGGGLVQPGGSLRLSCAASGFAFSDYYMSWFRQAP



DYYMS
DSVKD
H
GKGLEWVSHISGSGTTIDYADSVKDRFTISRDNARNSLYLQM






NSLRAEDTAVYYCAREDIRMPGTTDFDHWGQGTLVTVSS





2.33
SEQ ID NO:
SEQ ID NO: 554
SEQ ID NO: 555
SEQ ID NO: 556



553
HISSSGNSIDYA
EDPRLPGTTDFD
QVQLVESGGGLVKPGGSLRLSCAASGFPFSDYFMSWFRQAP



DYFMS
DSVKG
Y
GKGLEWVSHISSSGNSIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCAKEDPRLPGTTDFDYWGQGTLVTVSS





2.34
SEQ ID NO:
SEQ ID NO: 558
SEQ ID NO: 559
SEQ ID NO: 560



557
HISNSGSTISYA
EDPRLPGTSDFD
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDSYMSWIRQAP



DSYMS
DSVKG
Y
GKGLEWVSHISNSGSTISYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCAREDPRLPGTSDFDYWGQGTLVTVSS





2.35
SEQ ID NO:
SEQ ID NO: 562
SEQ ID NO: 563 
SEQ ID NO: 564



561
HISSSGSSIDYA
EDPRLSGTTDFD
QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYYMSWIRQAP



DYYMS
DSVKG
Q
GKGLEWVSHISSSGSSIDYADSVKGRFTISRDNAKNSLYLQM






NSLRDEDTAVYYCAREDPRLSGTTDFDQWGQGTLVTVSS





2.36
SEQ ID NO:
SEQ ID NO: 566
SEQ ID NO: 567
SEQ ID NO: 568



565
HISSSGSTIDYA
EEDPRMTGTTDFD
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQA



SYWMS
SVKG
Y
PGKGLEWVSHISSSGSTIDYAESVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCAREDPRMIGTTDFDYWGQGTLVTVSS





2.37
SEQ ID NO:
SEQ ID NO: 570
SEQ ID NO: 571
SEQ ID NO: 572



569
HISSSGNTIDYA
EDPRLPGTTDFD
QVQLQESGGGLVKPGGSLRLSCAASGFTFSNYFMSWIRQAP



NYFMS
DSVKG
Y
GKGLEWVSHISSSGNTIDYADSVKGRFTISRDNAKNSLYLQM






DSLRAEDTAVYYCSREDPRLPGTTDFDYWGQGTLVTVSS





2.38
SEQ ID NO:
SEQ ID NO: 574
SEQ ID NO: 575
SEQ ID NO: 576



573
YISSGGSTIHYA
ENPRLPGTMDFD
EVQLVESGGGVVKPGGSLRLSCAASGFTFSDYYMTWIRQGP



DYYMT
DSVKG
Y
GKGQEWISYISSGGSTIHYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCARENPRLPGTMDFDYWGQGTLVTVSS





2.39
SEQ ID NO:
SEQ ID NO: 578
SEQ ID NO: 579
SEQ ID NO: 580



577
NIKQDGSEKYY
EDPRLIGTTDFD
QVQLVESGGGLVQPGGSLRLSCAASGFTFSDHFMSWFRQA



DHFMS
VDSVKG
N
PGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLFLQ






MNSLRAEDTAMYYCAREDPRLIGTTDFDNWGQGTLVTVSS





2.40
SEQ ID NO:
SEQ ID NO: 582
SEQ ID NO: 583
SEQ ID NO: 584



581
HISSTGSTIDYA
EDPRLPGTMDFD
EVQLVESGGGLVQAGGSLRLSCVASGFTFSNYWMTWFRQA



NYWMT
DSVKG
Y
PGRGLEWVSHISSTGSTIDYADSVKGRFTISRDNAENSLYLQ






MNSLRAEDTAVYYCAREDPRLPGTMDFDYWGQGTLVTVSS





2.41
SEQ ID NO:
SEQ ID NO: 586
SEQ ID NO: 587
SEQ ID NO: 588



585
YISGSGDIIDYA
EDSRLIGTTDFD
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWFRQAP



DYYMS
DSVKG
N
GKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQM






NNLRAEDTAVYHCAREDSRLTGTTDFDNWGQGTLVTVSS





2.42
SEQ ID NO:
SEQ ID NO: 590
SEQ ID NO: 591
SEQ ID NO: 592



589
YISGSGDIIDYA
EDSRLIGTTDFD
EVQLVESGGGLVKPGGSLRVSCAASGFTFSDYYMSWIRQAP



DYYMS
DSVKG
N
GKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQM






NNLRAEDTAVYHCAREDSRLTGTTDFDNWGQGTLVTVSS





2.43
SEQ ID NO:
SEQ ID NO: 594
SEQ ID NO: 595
SEQ ID NO: 596



593
YISGSGDIIDYA
EDSRLIGTTDFD
EVQLVESGGGLVKPGGSLRVSCAASGFTFSDYYMSWFRQAP



DYYMS
DSVKG
N
GKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQM






NNLRAEDTAVYYCAREDSRLIGTTDFDNWGQGTLVTVSS





2.44
SEQ ID NO:
SEQ ID NO: 598
SEQ ID NO: 599
SEQ ID NO: 600



597
YISGSGDIIDYA
EDSRLIGTTDFD
EVQLVESGGGLVKPGGSLRVSCAASGFTFSDYYMSWFRQAP



DYYMS
DSVKG
N
GKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYHCAREDSRLTGTTDFDNWGQGTLVTVSS





2.45
SEQ ID NO:
SEQ ID NO: 602
SEQ ID NO: 603
SEQ ID NO: 604



601
YISGSGDIIDYA
EDSRLIGTTDFD
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWFRQAP



DYYMS
DSVKG
N
GKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQM






NNLRAEDTAVYYCAREDSRLIGTTDFDNWGQGTLVTVSS





2.46
SEQ ID NO:
SEQ ID NO: 606
SEQ ID NO: 607
SEQ ID NO: 608



605
YISGSGDIIDYA
EDSRLIGTTDFD
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAP



DYYMS
DSVKG
N
GKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCAREDSRLIGTTDFDNWGQGTLVTVSS





2.47
SEQ ID NO:
SEQ ID NO: 610
SEQ ID NO: 611
SEQ ID NO: 612



609
YISGSGDIIDYA
EDSRLIGTTDFD
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWFRQAP



DYYMS
DSVKG
N
GKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCAREDSRLIGTTDFDNWGQGTLVTVSS





2.48
SEQ ID NO:
SEQ ID NO: 614
SEQ ID NO: 615
SEQ ID NO: 616



613
YISGSGDIIDYA
EDARLIGTTDFD
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAP



DYYMS
DSVKG
N
GKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCAREDARLIGTTDFDNWGQGTLVTVSS





2.49
SEQ ID NO:
SEQ ID NO: 618
SEQ ID NO: 619
SEQ ID NO: 620



617
YISGSGDIIDYA
EDPRLIGTTDFD
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAP



DYYMS
DSVKG
N
GKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCAREDPRLIGTTDFDNWGQGTLVTVSS





2.50
SEQ ID NO:
SEQ ID NO: 622
SEQ ID NO: 623
SEQ ID NO: 624



621
YISGSGDIIDYA
EDARLIGTTDFD
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWFRQAP



DYYMS
DSVKG
N
GKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCAREDARLIGTTDFDNWGQGTLVTVSS





2.51
SEQ ID NO:
SEQ ID NO: 626
SEQ ID NO: 627
SEQ ID NO: 628



625
YISGSGDIIDYA
EDPRLIGTTDFD
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWFRQAP



DYYMS
DSVKG
N
GKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCAREDPRLIGTTDFDNWGQGTLVTVSS









In one embodiment, the binding molecule may comprise one or more single domain antibodies that bind to CD137, for example one or two single domain antibodies as described above in Tables 2 and 3.


In some embodiments, there is provided a VH single domain antibody that is a variant of any of the above single VH domain antibodies shown in Table 2 or Table 3 having one or more amino acid substitutions, deletions, insertions or other modifications, and which retains a biological function of the single domain antibody. Thus, variant VH single domain antibodies can be sequence engineered. Modifications are described elsewhere herein. In one embodiment, there is provided a variant of VHS 1.07 to 1.113, for example VH1.113 which has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.


Examples of Substitutions


In one embodiment, the variant comprises one or more the following substitutions with reference to SEQ ID NO. 4 (VH1.1) or combinations thereof:

    • F37V
    • E61V+E65K
    • E65K
    • V70I
    • V79L
    • F37V+E61V+E65K+V70I+V79L
    • E61V+E65K+V70I+V79L
    • F37V+E61V+E65K+V70I+V79L+G55E+D101V+D105I
    • F37V+E61V+E65K+V70I+V79LFGL+G55E+D101E+D105I
    • E61V+E65K+V70I+V79L+G55E
    • E61V+E65K+V70I+V79L+G55E+D105I
    • E61V+E65K+V70I+V79L+D54G, E61V+E65K+V70I+V79L+D54E,
    • E61V+E65K+V70I+V79L+D101E
    • E61V+E65K+V70I+V79L+D101V or
    • E61V+E65K+V70I+V79L+G55E+D101V+D105I, E61V+E65K+V70I+V79L+G55E+D101E+D105I


In one embodiment, the variant comprises one or more the following substitutions with reference to SEQ ID NO. 4 (VH1.1) or combinations thereof:


a) E61V+E65K+V70I+V79L+G55E+D101→any amino acid selected from the following F, L, I, M, V, S, P, T, A, Y, H, Q, K, D, W, R, G;


b) E61V+E65K+V70I+V79L+G55E+D105→any amino acid selected from the following F, L, M, S, P, T, A, Y, H, Q, N, K, D, E, W, R, G or


c) E61V+E65K+V70I+V79L+G55E, D101→any amino acid selected from the following F, L, I, M, V, S, P, T, A, Y, H, Q, K, D, W, R, G+D105→any amino acid selected from the following F, L, M, S, P, T, A, Y, H, Q, N, K, D, E, W, R, G.


In one embodiment, the variant comprises one or more the following substitutions with reference to SEQ ID NO. 312 (VH1.78) or combinations thereof:

    • Q1E+T25S
    • Q1E+S84N
    • Q1E+F95Y
    • Q1E+T25S+S84N
    • Q1E+T25S+F95Y
    • Q1E+S84N+F95Y
    • Q1E+T25S+S84N+F95Y or
    • Q1E+T25S+G55E+S84N+F95Y


In one embodiment, the variant comprises one or more the following substitutions with reference to SEQ ID NO. 428 (VH2.1) or combinations thereof:

    • V20L,
    • F371,
    • N85S,
    • N95Y,
    • V20L+H95Y,
    • V20L+F371+N85S+H95Y, V20L+N85S+H95Y,
    • V20L+F371+N85S+H95Y+S101A,
    • V20L+F371+N85S+H95Y+S101P or
    • V20L+N85S+H95Y+S101A, V20L+N85S+H95Y+S101A.


In one embodiment, the variant comprises one or more the following substitutions with reference to SEQ ID NO. 624 (VH2.50) or combinations thereof:

    • S35T+V48I+I57T+L103M+T104R+T107I
    • S35T+A101 T+L103V+T104R+T107V
    • S35T+V48I+I57T+A101E+L103L+T104W+T107V
    • S35T+V48I+L103T+T104R+T107V
    • Y32H+S35T+V48I+S52G+S54D+D56A+158L+A101L+T104P+T107I+N111H
    • S35T+A101T+L103V+T104R+T107V+N111S
    • A28T+S30T+Y33W+S35T+V48I+G53S+S54D+D56K+157T+L103M+T104P+T107I+N111Y
    • S35T+V48I+L103T+T104R+T107V+N111S
    • S35T+V48I+I57T+S101E+T104W+T107V+N111S or
    • S35T+V48I+I57T+L103M+T104R+T107I+N111S


Exemplary Nucleic Acids Encoding CD137 Polypeptide


An isolated nucleic acid encoding a single domain antibody as shown above is set out below. Nucleic acid may include DNA and/or RNA. In one aspect, the present invention provides a nucleic acid that codes for a CDR, for example CDR3, a set of two or three CDRs or a VH single domain antibody of the invention as shown above.









TABLE 4







Nucleic acids encoding VH 1.1 to 1.114








Name
Nucleotide Sequence





1.1
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGG



ATTCACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATAAA



GGAAGACGGAAGTGAGAAATACTATGAGGACTCTGTGGAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGAACTC



GGTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGCTACAG



TGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 629





1.2
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGG



ATTCACCTTTAGTAGTTATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATAAAG



GAAGACGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAATAACTCG



CTGTATCTACAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGCTACAGT



GACTCCCACTACGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 630





1.3
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAGTTATTGGATGACCTGGTTCCGCCAGGCTCCAGGGAGGGGGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGAACTCAC



TGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGATTAGGCTACGGTG



ACTCCCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCCTCA



SEQ ID NO. 631





1.4
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGG



ATTCACCTTTAGTAACTATTGGATGACCTGGTTCCGCCAGGCTCCAGGGGGGGGGCTGGAGTGGGTGGCCAACATAAA



CCAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGAACTC



ACTGTATCTACAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGATTAGGCTACGG



TGACTCCCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCCTCA



SEQ ID NO. 632





1.5
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAACTATTGGATGATCTGGTTCCGCCAGGCTCCAGGAAAGGGGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCAC



TGTATCTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGCTACAGTG



GCTCCCACCACGGTACGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 633





1.6
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAGCTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAG



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGGAAGGCTATAG



CACCTCGCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 634





1.7
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGGAGCCTCTGGA



TTCACCTTTAGTAGCTATTGGATGCTCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACACCCTGAGAGCCGAGGACACGGCTATTTATTATTGTGCGAGAGGGGGTGATGGCTACAGTGA



CTCCCACTTCGGTACGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCCTCA



SEQ ID NO. 635





1.8
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGGAGCCTCTGGA



TTCACCTTTAGTAGCTATTGGATGTTCTGGTTCCGCCAGGCTCCAGGAGAGGGGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCTCT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTATTTATTACTGTGCGAGAGGAGGTGATGGCTACAGTGA



TTCCCACTACGGTACGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 636





1.9
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGG



ATTCACCTTTAGTAACTATTGGATGACCTGGTTCCGCCAGGCTCCAGGGGGGGGGCTGGAGTGGGTGGCCAACATAAA



CCAAGATGGGAGTGAGAAGTACTATGTGGACTCTGTGGAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGAACTC



ACTGGATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGATTAGGCTACG



GTGACTCCCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 637





1.10
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTGACTATTGGATGAACTGGGCCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAG



GAGGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATATCCAGAGACAACGCCAAGAACTCA



ACGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGAGGAGGGGCCGGGTATAG



CATGTCTCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 638





1.11
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGAAGCCTCTGGA



TTCACCTTTAGTGACTATTGGATGAACTGGGCCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAG



GAGGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATATCCAGAGACAACGCCAAGAACTCA



ACGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGAGGGGCCGGGTATAG



TATGTCTCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCCTCA



SEQ ID NO. 639





1.12
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGGAGCCTCTGGA



TTCACCTTTAGTAGCTATTGGATGCTCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACACCCTGCGAGCCGAGGACACGGCTATTTATTATTGTGCGAGAGGGGGTGATGGCTACAGTAA



CTCCCACTTCGGTACGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTTCA



SEQ ID NO. 640





1.13
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGGAGCCTCTGG



ATTCACCTTTAGTAGCTATTGGATGTTCTGGTTCCGCCAGGCTCCAGGAGAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCTC



TGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTATTTATTACTGTGCGAGAGGAGGTGATGGCTACAGTG



ATTCCCACTACGGTACGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 641





1.14
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAATTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAG



GAAGATGGAAGTGAGAATTACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAGGGGGGGAAGGCTATAGC



ACCTCGCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCCTCA



SEQ ID NO. 642





1.15
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGGAGCCTCTGGA



TTCACCTTTAGTACCTATTGGATGCTCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACT



GTCTCTACAAATGAACAGCCTGAGAGCCGAGGACACGGCAACTTATTACTGTGCGAGAGGAGGTGATGGCTACAGTGA



CTCCCACTTCGGTACGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 643





1.16
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGA



TTCACCTTTAGTAACTATTGGATGATGTGGTTCCGCCAGGCTCCAGGAAAGGGGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTTTGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGCTACAGTAG



CTCTCACTACGGTACGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTTCA



SEQ ID NO. 644





1.17
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAGCTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAG



GAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGGACAGCTATGGT



TACAGGGACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCCTCA



SEQ ID NO. 645





1.18
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTACCCATTGGATGAACTGGGCCCGCCAGGCTCCAGGGAAGGAGCTGGAATGGGTGGCCAACATAAAC



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAACAATTCAC



TGTATCTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCTGTATATTACTGTGCGAGAGGGGGGGTTGGCTACGGTG



ACTCCCACTTCGGTATGGACGTCTGGGGCCTAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 646





1.19
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAGCTATTGGATGATCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGACTACAGT



AACTCCCACTACGGTATGGACGTCTCGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 647





1.20
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAGCTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGCAGGGCCGATTCACCATCTCCAGAGACAATGCCAATAACTCAC



TGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGTTTGGCTACGGTG



ACTCCCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 648





1.21
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGGAGCCTCTGGA



TTCACCTTTAGTAGTTATTGGATGTTCTGGTTCCGCCAGGCTCCAGGAAAGGAGCTGGAGTGGGTGGCCAATGTTAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCAC



TGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTATTTATTACTGTGCGAGAGGAGGTGAGGGCTACAGTG



ATTCCCACTACGGTACGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 649





1.22
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAATATAAAG



GAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAGGAACTCA



CTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGGAGGGCTACGG



TGACTCCCACTACGGTATGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 650





1.23
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAGTTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAG



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTTTATCTGCAAATGAACAGCCTGACAGCCGAGGACACGGCTGTGTATTATTGTGCGAGAGGGGGGGAGGGCTACGGT



GACGACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO.651





1.24
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAGCTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAG



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGGAGGGCTACGG



TGACTACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 652





1.25
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAGCTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAG



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGGACAGCTATGGT



TACAGGGACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 653





1.26
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGA



TTCACCTTTAGTACCCATTGGATGAACTGGGCCCGCCAGGCTCCAGGGAAGGAGCTGGAATGGGTGGCCAACATAAAC



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAACAATTCAC



TGTATCTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCTGTATATTACTGTGCGAGAGGGGGGGTTGGCTACGGTG



ACTCCCACTTCGGTATGGACGTCTGGGGCCTAGGGACCACGGTCACTGTCTCCTCA



SEQ ID NO. 654





1.27
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGGAGCCTCTGGA



TTCACCTTTAGTAGTTATTGGATGCTCTGGTTCCGCCAGGCTCCAGGAGAGGGGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGACTCACCATCTCCAGAGACAACGCCAAGAACGCTC



TGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTATTTATTACTGTGCGAGAGGAGGTGAAGGCTACAGTG



ATTCCCACCACGGTACGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 655





1.28
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAG



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGGACAACTATGCT



TACAGGGACTTCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO.656





1.29
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGGAGCCTCTGGA



TTCACCTTTAGTAATTATTGGATGTTCTGGTTCCGCCAGGCTCCAGGAAAGGAGCTGGAGTGGGTGGCCAATGTTAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCAC



TGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTATTTATTACTGTGCGAGAGGAGGTGAGGGCTACAGTG



ATTCCCACTACGGTACGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 657





1.30
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAGCTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGGAAGAGTATGG



GAGCTCGCACTACGGTATGGACGTCTGGGGCCTGGGGACCACGGTCACTGTCTCCTCA



SEQ ID NO. 658





1.31
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAGCTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTATTGTGCGAGAGGGGGGGACAGCTATGGT



TACAGGGACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 659





1.32
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTAAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAGTTATTGGATGAACTGGGTCCGCCAGACTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAT



CAAAATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCAACATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTACAAATGAGTAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGTTTGGCTACGGT



GATTCCCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 660





1.33
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGGCGGGGGGGTCCCTAAGACTCTCCTGTGTAGCCTCTGG



ATTCACCTTTAGTAATTATTGGATGACCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGAAAGTGAGGAATACTATGTGGACTCTGTGAAGGGCCGTTTCACCATCTCCAGAGACAACGCCAAGAACTCAC



TGTTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTATTTATTACTGTGCGAGAGGAGGTGATGGCTACAGTG



ACTCCCACTACGGTACGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 661





1.34
CAGGTGCAGCTGCAGGAGTCGGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGA



TTCACCTTTAGTAATTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAG



GAAGATGGAAGTGAGAATTACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAGGGGGGGAAGGCTATAGC



ACCTCGCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTTCA



SEQ ID NO. 662





1.35
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATTAAG



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGGACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAGGGGGGGAGGGCTACGG



TGAATCCCACTACGGTATGGACGTCTCGGGCCAAGGGACCACGGTCACCGTCTCTTCA



SEQ ID NO. 663





1.36
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTACCTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAA



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGGACAGCTATGGT



TACAGGGACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCCTCA



SEQ ID NO. 664





1.37
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTTACTATTGGATGATCTGGTTCCGCCAGGCTCCAGGTGAGGAGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCATTATCTCCAGAGACAACGCCACGAACTCACT



GTTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAGGAGGTGATGGCTACAGTAA



TTCCCACTTCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTTCA



SEQ ID NO. 665





1.38
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAACTATTGGATGATCTGGTACCGCCAGGCTCCAGGTGAGGAGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTATTTATTACTGTGCGAGAGGAGGTGAGGGCTACAGTGA



TTCCCACTACGGTACGGACGTCTGGGGCCAGGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 666





1.39
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGAGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGAAAGTGAAAAATACTATGTTGACTCTGTGAAGGGCCGTTTCACCGTCTCCAGAGACAACGCCAAGAACTCAC



TGTTTCTGCAAATGAACAGCCTGAGAGCCGACGACACGGCTGTATATTACTGTGCGAGAGGGGGGTTTGGCTACGGTG



ACTCCCACTTCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTTCA



SEQ ID NO.667





1.40
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGGAGCCTCTGGA



TTCACCTTTAGTAATTATTGGATGTTCTGGTTCCGCCAGGCTCCAGGAAAGGAGCTGGAGTGGGTGGCCAATGTTAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACTCAC



TGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTATTTATTACTGTGCGAGAGGAGGTGAGGGCTACAGTG



ATTCCCACTACGGTACGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO.668





1.41
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGGAGCCTCTGGA



TTCACCTTTAGTAATTATTGGATGTTCTGGTTCCGCCAGGCTCCAGGAAAGGAGCTGGAGTGGGTGGCCAATGTTAACC



AAAATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTATTTATTACTGTGCGAGAGGAGGTGAGGGCTACAGTGA



TTCCCACTACGGTACGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCCTCA



SEQ ID NO. 669





1.42
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAG



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGGACTCA



CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTATTTATTATTGTGCGAGAGGGGGGGAGGGTTACGGT



GACTCCCACTACGGTATGGACGTCTCGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 670





1.43
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAACTATTGGATGATCTGGTACCGCCAGGCTCCAGGTGAGGAGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCACGAACTCACT



GTTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAGGAGGTGATGGCTACAGTAA



TTCCCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCCTCA



SEQ ID NO. 671





1.44
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTGACTATTGGATGATCTGGTACCGCCAGGCTCCAGGTGAGGAGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCACGAACTCACT



GTTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAGGAGGTGATGGCTACAGTAA



TTCCCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 672





1.45
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAAATATTGGATGATCTGGGTCCGCCAGGCTCCAGAAAAGGGGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATTTCCAGAGACAATGTCAATAACTCATT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTACTACTGTGCGAGAGGAGGTGATGACTACAGTAA



CTCCCACTACGGTATGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCCTCA



SEQ ID NO. 673





1.46
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAACTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAGGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAGCTCA



CTGTATCTGCAAATGAACAGCCTTAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAGGGGGGGAAGAATATAGC



AGCTCCCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCCTCA



SEQ ID NO. 674





1.47
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTATAGCCTCTGGA



TTCAGCTTTAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAG



CAAGATGGAAGTGAGAATTACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCAC



TGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGGAAGGGTATAGC



ACCTCGCACTACGGTATGGACGTCTGGGGCCAAGGGACCGCGGTCACTGTCTCTTCA



SEQ ID NO. 675





1.48
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAGCTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAG



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 676





1.49
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGGAGCCTCTGGA



TTCACCTTTAGTAGCTATTGGATGCTCTGGTTCCGCCAGGCTCCAGGAAAGGAGCTGGAGTGGGTGGCCAATGTTAACC



AAGATGGCAGTGAGAATTACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACT



GTATCTGCAAATGCACAGCCTGAGAGCCGAGGACACGGCTGTATATTACTGTGCGAGAGGAGGTGAAGACTACGGTAA



CTCCCACTTCGGCATGGACGTCTGGGGCCAAGGGACCATGGTCACCGTCTCCTCA



SEQ ID NO. 677





1.50
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGCAGATCCCTGAGACTCTCTTGTGCAGCCTCTGGA



TTCACCTTTAGTAACTATTGGATGATCTGGTACCGCCAGGCTCCAGGTGAGGAGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCACGAACTCACT



GTTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAGGAGGTGATGGCTACAGTAA



TTCCCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 678





1.51
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAACTATTGGATGATCTGGTACCGCCAGGCTCCAGGTGAGGAGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCACGAACTCACT



GTTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAGGAGGTGATGGCTACAGTAA



TTCCCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 679





1.52
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGATTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGA



TTCACCTTTAGTAAATATTGGATGATCTGGGTCCGCCAGGCTCCAGAAAAGGGGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATTTCCAGAGACAACGCCAATAACTCACT



GTTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTACTACTGTGCGAGAGGAGGTGATGACTACAGTAT



CTCCCACTTCGGTATGGACGTCTCGGGCCAAGGGACCAGGGTCACCGTCTCCTCA



SEQ ID NO. 680





1.53
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGATTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGA



TTCACCTTTAGTAAATATTGGATGATCTGGGTCCGCCAGGCTCCAGAAAAGGGGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATTTCCAGAGACAACGCCAATAATTCATT



GTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTACTACTGTGCGAGAGGAGGTGATGACTACAGTCA



CTCCCACTACGGTATGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 681





1.54
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCAACTTTAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGAGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTTTCTGCAAATGAACAGCCTGAGAGCCGACGACACGGCTGTGTATTACTGTGCGAGAGGGGGGTTTGGCTACGGT



GACTCCCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 682





1.55
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTGGTAGTTATTGGCTGAATTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGCAGTGAGAATTACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCAC



TGTATCTGCAAATGCACAGCCTGAGAGCCGAGGACACGGCTGTATATTACTGTGCGAGAGGAGGTGAAGACTACGGTA



ACTCCCACTTCGGCATGGACGTCTGGGGCCAAGGGACCATGGTCACTGTCTCTTCA



SEQ ID NO. 683





1.56
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAGCTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAG



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 684





1.57
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGA



TTCACCTTTAGTGACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGTAAGGGGCTGGAGTGGGTGGCCAATATAAAG



GAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAGGAACTCA



CTGTATCTGCAAATGACCAGCCTGAGAGAAGAAGACACGGCTATGTATTACTGTGCGAGAGGGGGGGAGGGCTACGG



TGACAACCACTACGGTATGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 685





1.58
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGAAGCTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGAGGCGGAATGGGTGGCCAACATAAAC



CAAGATGGAAGTGAGAAATATTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTTTCTGCAAATGAACAGCCTGAGAGACGAGGACACGGCTGTTTATTACTGTGCGAGAGGAGGCCCCGACTACGGT



GACCTCCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 686





1.59
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTAAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAGGTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGCGGGTGGCCAACATAAAC



CAAGATGGACGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCAC



TGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTATTGTGCGAGAGGGGGGGAGGGCTACGGT



GACTACCACTACGGTATGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 687





1.60
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCCGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCCTTAGTAACTATTGGATGATCTGGTACCGCCAGGCTCCAGGTGAGAAGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCACGAACTCACT



GTTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAGGAGGTGATGGCTACAGTAA



TTCCCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 688





1.61
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGA



TTCAACTTCAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGAGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGAAAGTGAAAAATACTATGTAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCAC



TGTTTCTGCAAATGAACAGCCTGAGAGCCGACGACACGGCTGTGTATTACTGTGCGAGAGGGGGGTTTGGCTACGGTG



ACTCCCACTTCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 689





1.62
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCAATTTTAGTAACTATTGGATGAACTGGGTCCGTCAGGCTCCAGGGAAGGAGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGAAAGTGAAAAATACTATGTAGACTCTGTGAAGGGCCGATTCACCATTTTCAGAGACAACGCCAAGAACTCAC



TGTTTCTGCAAATGAACAGCCTGAGAGCCGACGACACGGCTGTGTATTACTGTGCGAGAGGGGGGTTTGGCTACGGTG



ACTCCCACTTCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 690





1.63
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGTCTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGAAGCTTTTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGAGGCGGAATGGGTGGCCAACATAAAT



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAGGAGGCCCCGACTACGGT



GACCTCCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 691





1.64
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGAAGCTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGAGGCGGAATGGGTGGCCAACATAAAC



CAAGATGGAAGTGAGAAATATTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCAC



TGTTTCTGCAAATGAACAGCCTGAGAGACGAGGACACGGCTGTTTATTACTGTGCGAGAGGAGGCCCCGACTACGGTG



ACCTCCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 692





1.65
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAACTATTGGATGATCTGGTACCGCCAGGCTCCAGGTGAGGAGCTGGAGTGGGTGGCCAACATAAACC



AAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACT



GTATCTGCAAATGCACAGCCTGAGAGCCGAGGACACGGCTGTATATTACTGTGCGAGAGGAGGTGAAGACTACGGTAA



CTCCCACTACGGCATGGACGTCTGGGGCCAAGGGACCATGGTCACCGTCTCTTCA



SEQ ID NO. 693





1.66
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAGCTCA



CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



ATCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 694





1.67
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAGCTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAT



CAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAGCTCA



CTGTATCTGCAAATGAGTAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCCTCA



SEQ ID NO. 695





1.68
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAACTATTGGATGATCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAGCTCA



CTGTATCTGCAAATGAGCAACCTGAGAGCCGAGGACACGGCTGTATATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 696





1.69
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCACTGGA



TTCACCTTAAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAAAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCAC



TGTTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGAACAGGCTATGGTT



CCGACCACTACGGTATGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 697





1.70
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCAATTTTAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGAGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAGAATTACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGTCAAGAACTCAC



TGTTTCTGCAAATGAACCGCCTGAGAGCCGACGACACGGCTGTGTATTACTGTGCGAGAGGGGGGTTTGGCTACGGTG



ACTCCCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCCTCA



SEQ ID NO. 698





1.71
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTGGTAACTATTGGATGATCTGGGTCCGCCAGGCTCCAGGCAAGGAGTTGGAGTGGCTGGCCAACATAAACC



AAAATGGAAGTGAGAGATACTATGTGGACTCTGTGCAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTATATTACTGTGCGAGAGGGGGTGCTGACTACAGTAA



CTCCCACTACGGTATGGACGTCAGCGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 699





1.72
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAGCTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAACAACTCAC



TGCATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 700





1.73
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGG



ATTCACCTTTAGAAGTTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAAGAGGCGGAATGGGTGGCCAACATAAA



CCCAGATGGAAGTGAGAAATACTATGTGGACTCTGTGCAGGGCCGACACACCATCTCCAGAGACAACGCCAAGAACTC



ACTGTTTCTGGAAATGAACAGCCTGAGAGTCGAGGACACGGCTCTTTATTACTGTGCGAGAGGAGGCCCCGGCTACGG



TGACCTCCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCCTCA



SEQ ID NO. 701





1.74
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCACTGGA



TTCACCTTAAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAT



CAAGATGGAAGTGAAAAATACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAGCTCA



CTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCCTCA



SEQ ID NO. 702





1.75
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACCGTCTCTTCA



SEQ ID NO. 703





1.76
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCACTGGA



TTCACCTTAAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACCGTCTCTTCA



SEQ ID NO. 704





1.77
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTAAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 705





1.78
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCACTGGA



TTCACCTTAAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 706





1.79
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCACTGGA



TTCACCTTAAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCAACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACCGTCTCTTCA



SEQ ID NO. 707





1.80
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGA



TTCACCTTCAGTGACTACTATATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAG



CAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAGCTCA



CTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO.708





1.81
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCACTGGA



TTCACCTTAAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAGCTCA



CTGTATCTGCAAATGAGCAACCTGAGAGCCGAGGACACGGCTGTATATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 709





1.82
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCACTGGA



TTCACCTTAAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGAAGGGCCGATTCACCATTTCCAGAGACAACGCCAAGAGCTCA



CTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 710





1.83
CAGGTGCAGCTGCAGGAGTCGGGGGGAGGCTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCACTGG



ATTCACCTTAAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAA



CCAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTC



ACTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATG



GTGTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 711





1.84
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCACTGGA



TTCACCTTAAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO.712





1.85
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCACTGGA



TTCACCTTAAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAACAACTCAC



TGCATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 713





1.86
CAGGTGCAGCTGGGGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCACTGG



ATTCACCTTAAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAA



CCAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAGCTC



ACTGTATCTGCAAATGAGCAACCTGAGAGCCGAGGACACGGCTGTATATTTCTGTGCGAGAGGGGGGGAAGGCTATGG



TGTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 714





1.87
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAAACTCTCCTGTGCAGCCACTGGA



TTCACCTTAAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGAAGGGCCGATTCACCATTTCCAGAGACAACGCCAAGAGCTCA



CTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 715





1.88
CAGGTGCAGCTGCAGGAGTCGGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCACTGG



ATTCACCTTAAGTAACTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAA



CCAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGGCAACGCCAAGAACTC



ACTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATG



GTGTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 716





1.89
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAC



CAAGATGGAAGTGAAAGATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGGGGGGAAGGCTATGGT



GTCGACCACTACGGTTTGGACGTCTCGGGCCAAGGGACCACGGTCACTGTCTCTTCA



SEQ ID NO. 717





1.90
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTTAGTAGCCATTGGATGACTTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATA



AAGGAAGACGGAAGTGAGAAATACTATGAGGACTCTGTGGAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGA



ACTCGGTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGC



TACAGTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 718





1.91
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATA



AAGGAAGACGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGA



ACTCGGTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGC



TACAGTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 719





1.92
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATA



AAGGAAGACGGAAGTGAGAAATACTATGAGGACTCTGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGA



ACTCGGTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGC



TACAGTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 720





1.93
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATA



AAGGAAGACGGAAGTGAGAAATACTATGAGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA



ACTCGGTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGC



TACAGTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 721





1.94
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATA



AAGGAAGACGGAAGTGAGAAATACTATGAGGACTCTGTGGAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGA



ACTCGTTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGC



TACAGTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 722





1.95
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTTAGTAGCCATTGGATGACTTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATA



AAGGAAGACGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA



ACTCGTTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGC



TACAGTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 723





1.96
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATA



AAGGAAGACGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA



ACTCGTTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGC



TACAGTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 724





1.97
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTTAGTAGCCATTGGATGACTTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATA



AAGGAAGACGAAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA



ACTCGTTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGTTGGC



TACAGTATCTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 725





1.98
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTTAGTAGCCATTGGATGACTTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATA



AAGGAAGACGAAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA



ACTCGTTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGAGGGC



TACAGTATCTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 726





1.99
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATA



AAGGAAGACGAAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA



ACTCGTTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGC



TACAGTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 727





1.10
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG


0
GATTCACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATA



AAGGAAGACGAAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA



ACTCGTTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGC



TACAGTATCTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 728





1.10
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG


1
GATTCACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATA



AAGGAAGGCGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA



ACTCGTTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGC



TACAGTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 729





1.10
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG


2
GATTCACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATA



AAGGAAGAGGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA



ACTCGTTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGC



TACAGTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 730





1.10
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG


3
GATTCACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATA



AAGGAAGACGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA



ACTCGTTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGAGGGC



TACAGTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 731





1.10
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG


4
GATTCACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATA



AAGGAAGACGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA



ACTCGTTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGTTGGC



TACAGTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 732





1.10
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG


5
GATTCACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATA



AAGGAAGACGAAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA



ACTCGTTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGTTGGC



TACAGTATCTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO.733





1.10
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG


6
GATTCACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATA



AAGGAAGACGAAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA



ACTCGTTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGAGGGC



TACAGTATCTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



SEQ ID NO. 734





1.1
GAGGTGCAATTAGTCGAATCGGGGGGTGGACTGGTTCAGCCGGGAGGTAGCCTGCGCCTGTCCTGTGCCGCATCTG


07
GTTTTACATTAAGTAACTACTGGATGAATTGGGTTCGTCAAGCGCCTGGAAAGGGCTTAGAGTGGGTGGCTAATATT



AACCAGGACGGGTCAGAGCGCTACTATGTGGATTCAGTAAAAGGTCGCTTCACTATCAGCCGCGATAATGCTAAAAA



TTCGCTGTACCTTCAGATGTCATCACTTCGTGCAGAGGATACAGCTGTGTATTTCTGCGCGCGTGGAGGCGAGGGGT



ACGGGGTAGACCACTATGGGTTGGATGTCTCGGGACAAGGCACGACCGTCACTGTCAGTAGC



SEQ ID NO. 881





1.1
GAGGTCCAGTTGGTTGAGTCCGGCGGCGGCTTGGTCCAACCAGGGGGGTCGCTTCGCTTATCTTGCGCTGCCACAGG


08
GTTTACCCTGAGCAACTACTGGATGAACTGGGTGCGCCAAGCGCCTGGGAAGGGGTTAGAGTGGGTCGCCAACATC



AACCAAGACGGTTCGGAGCGTTACTATGTCGACAGCGTGAAGGGCCGTTTCACGATCTCCCGCGATAACGCTAAGAA



CTCCCTGTATTTGCAAATGAATAGCCTTCGTGCGGAGGATACTGCGGTTTATTTCTGTGCTCGTGGCGGTGAAGGATA



TGGGGTTGACCATTATGGGTTGGATGTCTCCGGGCAAGGGACAACGGTGACCGTGTCATCC



SEQ ID NO. 882





1.1
GAGGTTCAACTTGTTGAATCGGGTGGCGGATTAGTACAACCCGGCGGCTCGCTGCGTTTATCGTGTGCGGCAACCGG


09
ATTTACTTTATCAAACTATTGGATGAATTGGGTGCGCCAGGCTCCAGGGAAAGGTCTGGAATGGGTAGCGAATATCA



ACCAAGACGGCTCAGAACGCTACTACGTGGACTCCGTAAAAGGTCGTTTCACCATCTCTCGTGACAATGCTAAAAATT



CTTTGTATTTGCAAATGAGTTCACTTCGTGCTGAGGATACTGCGGTCTATTACTGTGCTCGCGGGGGGGAAGGCTACG



GAGTAGACCACTACGGGTTGGATGTTTCTGGACAGGGAACGACGGTTACTGTAAGCAGC



SEQ ID NO. 883





1.1
GAGGTTCAGTTAGTTGAGTCCGGCGGGGGATTAGTTCAACCTGGCGGAAGCCTTCGTCTGAGTTGTGCCGCGAGCG


10
GGTTTACCCTTAGCAATTACTGGATGAACTGGGTACGTCAAGCTCCAGGTAAAGGTTTAGAATGGGTCGCTAACATTA



ATCAAGATGGTTCTGAACGCTATTATGTAGACTCGGTAAAGGGTCGTTTTACAATTTCTCGCGACAACGCCAAAAACT



CTTTGTACCTTCAAATGAATTCCTTACGCGCTGAGGACACTGCTGTCTATTTCTGTGCGCGTGGAGGGGAGGGATACG



GAGTTGACCACTATGGGCTGGACGTTTCAGGACAGGGCACTACGGTAACTGTGTCTTCG



SEQ ID NO. 884





1.1
GAGGTTCAGTTAGTAGAGTCCGGGGGAGGACTGGTACAACCTGGGGGTAGTTTGCGTCTGTCTTGTGCAGCCAGCG


11
GTTTCACATTGTCTAACTATTGGATGAATTGGGTTCGTCAAGCGCCTGGCAAGGGACTGGAGTGGGTTGCAAACATT



AATCAAGATGGCAGCGAGCGTTATTACGTGGACTCAGTAAAAGGGCGCTTCACGATTAGCCGCGATAATGCTAAGAA



CTCCTTATATCTGCAGATGTCATCTTTGCGTGCCGAGGACACGGCAGTTTACTATTGCGCACGTGGTGGCGAGGGATA



CGGCGTGGATCACTATGGTTTGGACGTATCGGGCCAAGGGACTACCGTGACTGTGTCCTCT



SEQ ID NO. 885





1.1
GAGGAGGTACAGCTTGTCGAGTCTGGCGGTGGCCTTGTGCAACCGGGGGGTTCTTTACGTTTATCCTGTGCCGCTAC


12
AGGATTTACGTTAAGCAACTATTGGATGAACTGGGTACGTCAAGCTCCGGGGAAGGGGCTGGAATGGGTTGCCAAT



ATCAATCAGGATGGGTCTGAACGCTACTACGTTGATTCTGTTAAGGGTCGCTTTACTATTTCACGTGACAATGCCAAG



AACAGTCTTTACCTTCAAATGAACTCGTTACGCGCTGAGGATACTGCTGTGTACTACTGTGCGCGCGGCGGAGAGGG



ATACGGTGTCGATCATTATGGGCTTGACGTAAGCGGGCAGGGTACGACGGTGACGGTATCATCA



SEQ ID NO. 886





1.1
GAGGTGCAGTTAGTTGAGAGCGGAGGTGGTTTAGTTCAGCCGGGGGGCTCGCTTCGCCTGTCGTGCGCCGCCTCGG


13
GATTCACATTATCAAACTACTGGATGAATTGGGTCCGCCAGGCTCCGGGCAAAGGTCTTGAGTGGGTGGCGAACATT



AATCAGGACGGGAGCGAGCGTTATTACGTTGATTCGGTAAAAGGACGTTTCACTATCAGTCGTGACAACGCTAAAAA



TTCCTTGTACTTACAGATGAACTCACTTCGTGCTGAGGACACCGCAGTGTACTACTGTGCTCGCGGTGGTGAAGGATA



CGGCGTCGATCACTACGGCCTTGATGTATCAGGACAGGGGACTACAGTTACCGTCTCTTCC



SEQ ID NO. 887





1.1
GAGGTGCAGTTGGTAGAGAGTGGGGGTGGCCTGGTCCAACCAGGTGGGTCCCTTCGTTTGTCTTGCGCCGCCTCTGG


14
GTTTACTCTGTCAAATTATTGGATGAACTGGGTGCGCCAAGCTCCCGGCAAGGGGTTGGAGTGGGTTGCCAACATTA



ATCAGGACGAATCCGAGCGTTACTATGTTGATTCTGTAAAAGGGCGCTTCACTATCTCTCGTGATAATGCTAAGAACA



GTTTGTACCTGCAAATGAATTCACTGCGTGCCGAGGATACCGCGGTGTACTATTGTGCCCGTGGAGGAGAGGGATAC



GGGGTCGATCACTATGGCTTAGACGTATCGGGCCAGGGAACAACCGTCACCGTATCCTCA



SEQ ID NO. 888
















TABLE 5 







Nucleic acids encoding VH 2.1 to 2.51








Name
Nucleotide Sequence











2.1
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTG



GTAGTGGTGATATCATAGACTACGCAGACTCTGTAAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCTCT



GTATCTGCAGATGAACAACCTGAGAGCCGAGGACACGGCCGTGTATCACTGTGCGAGAGAAGATTCCCGTCTAACTGG



AACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 735





2.2
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCGTACATTAGTG



GTAGTGGTGATATCATAGACTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATCACTGTGCGAAAGAAGATTCCCGTATACCTGG



AACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA



SEQ ID NO. 736





2.3
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTATATGAGTTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATTTCGTACATTAGTG



GTAGTGGTGATATCATAGACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATCACTGTGCGAAAGAAGATTCCCGTATACCTGG



AACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCTTCA



SEQ ID NO. 737





2.4
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCGTACATTAGTG



GTAGTGGTGATGTCATTGACTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAATTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATCACTGTGCGAAAGAAGATTCCCGTATACCTGG



AACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCACTGTCTCTTCA



SEQ ID NO. 738





2.5
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGTCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCGTACATTAGTG



GTAGTGGTGATATCATAGACTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATCACTGTGCGAAAGAAGATTCCCGTATACCTGG



AACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA



SEQ ID NO. 739





2.6
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCGTACATTAGTG



GTAGTGGTGATATCATAGACTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATCACTGTGCGAAAGAAGATTCCCGTATACCTGG



AACTACGGACTTTGACAGTTGGGGCCAAGGGACAATGGTCACCGTCTCCTCA



SEQ ID NO. 740





2.7
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCAGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTG



GTAGTGGTACTACCATAGACTACGCAGACTCTGTGAAGGGCCGCTTCACCATCTCCAGGGACAACGCCAGGAACTCACT



ATATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCCAGAGAAGATATCAGGATGACTGG



AACTACGGACTTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCTTCA



SEQ ID NO. 741





2.8
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCGCCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTG



GTAGTGGAACTACCATAGACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACT



ATATCTACAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATCACTGTGCGAGAGAAGATTCCCGCATGCCTGG



AACTACGGACTTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 742





2.9
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTATATGACCTGGTTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGATTTCATACATTAGTG



GTAGTGGTGATACCATAGACTACGCAGAGTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAATTCACT



GTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATCACTGTGCGAGAGAAGATTCGCGTATAGCCGG



AACTACGGACTTTGACAACTGGGGCCCGGGAACCCTGGTCACTGTCTCTTCA



SEQ ID NO. 743





2.10
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGACCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTA



GTAGTGGTAGTAACATAGATTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCTAGGGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATTCCCGTTTAAGTGG



AACTACGGACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCTTCA



SEQ ID NO. 744





2.11
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTATATGACCTGGTTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGATTTCATACATTAGTG



GTAGTGGTGATACCATAGACTACGCAGAGTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAATTCACT



GTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATCACTGTGCGAGAGAAGATTCGCGTATAGCCGG



AACTACGGACTTTGACAACTGGGGCCCGGGAACCCTGGTCACTGTCTCCTCA



SEQ ID NO. 745





2.12
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTG



GTAGTGGTACTACCATAGACTACGCAGACTCTGTGAAGGGCCGCTTCACCATCTCCAGGGACAACGCCAGGAAGTCACT



ATATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTCTATTACTGTGCCAGAGAAGATATCAGGATGACTGG



AACTACGGACTTTGACCACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 746





2.13
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTA



GTAGTGGTAATACCATAGACTACGCAGACTCTGTGAAGGGCCGCTTCACCATCTCCAGGGACAACGCCAAGAACTCACT



TTATCTGCAAATGAATAGTCTGAGAGCCGAGGACACGGCCGTTTATTACTGTGCGAGAGAAGATCCTCGTTTACCTGGA



ACTACAGATTTTGACTACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA



SEQ ID NO. 747





2.14
GAGGTGCAGTTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTATATGACCTGGTTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGATTTCATACATTAGTG



GTAGTGGTGATACCATAGACTACGCAGAGTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAATTCACT



GTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCCAGAGAAGATATCAGGATGCCTGG



AACTACGGACTTTGACCACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 748





2.15
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGTCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTG



GTAGTGGAACTACCATAGACTACGCAGACTCTGTGAAGGGCCGCTTCACCATCTCCAGGGACAACGCCAGGAATTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCCAGAGAAGATATCAGGATGCCTGG



AACTACGGATTTTGACCACTGGGGCCAAGGAACCCTGGTCACTGTCTCTTCA



SEQ ID NO. 749





2.16
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTA



GTAGTGGGAGTACCATAGACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATCCTCGTTTAACTGG



AACTACAGATTTTGACTACTGGGGCCAGGGAGCCCTGGTCACTGTCTCCTCA



SEQ ID NO. 750





2.17
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTA



GTAGTGGTAGTACCATATCCTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATCCTCGTATAAGTGG



AACTACAGATTTTGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 751





2.18
CAGGTGCAGCTGCAGGAGTCGGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTA



GTAGTGGTAATACCATAGACTACGCAGACTCTGTGAAGGGCCGCTTCACCATCTCCAGGGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTTTATTACTGTGCGAGAGAAGATCCTCGTTTACCTGGA



ACTACAGATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 752





2.19
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTG



GTACTGGTATTACCACAGACTACGCAGACTCTGTGAAGGGCCGCTTCACCATCTCCAGGGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATCCTCGTTTACCTGG



AACTTCAGAATTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 753





2.20
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTA



GTAGTGGTAGTACCATAGATTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTCTATTACTGTGCGAGAGAAGATCCCCGTATGCCTGG



AACTTTTGACTTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 754





2.21
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCGCCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTG



GTAGTGGAACTACCATAGACTACGCAGACTCTGTGAAGGGCCGCTTCACCATCTCCAGGGACAACGCCAGGAATTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCCAGAGAAGATATCAGGATGCCTGG



AACTACGGACTTTGACCACTGGGGCCAAGGAACCCTGGTCACTGTCTCTTCA



SEQ ID NO. 755





2.22
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGTCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTG



GTAGTGGAACTACCATAGACTACGCAGACTCTGTGAAGGGCCGCTTCACCATCTCCAGGGACAACGCCAGGGATTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCCAGAGAAGATATCAGGATGCCTGG



AACTACGGATTTTGACCACTGGGGCCAAGGAACCCTGGTCACTGTCTCTTCA



SEQ ID NO. 756





2.23
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCACGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGTCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTG



GTAGTGGAACTACCATAGACTACGCAGACTCTGTGAAGGGCCGCTTCACCATCTCCAGGGACAACGCCAGGAATTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCCAGAGAAGATATCAGGATGCCTGG



AACTACGGATTTTGACCACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 757





2.24
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGTCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTG



GTAGTGGAACTACCATAGACTACGCAGACTCTGTGAAGGGCCGCTTCACCATCTCCAGGGACAACGCCAGGAATTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCATGTATTACTGTGCCAGAGAAGATATCAGGATGCCTGG



AACTACGGATTTTGACCACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 758





2.25
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTTAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCGCCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTG



GTAGTGGAACTACCATAGACTACGCAGACTCTGTGAAGGACCGCTTCACCATCTCCAGGGACAACGCCAGGAATTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCCAGAGAAGATATCAGGATGCCTGG



AACTACGGACTTTGACCACTGGGGCCAAGGAACCCTGGTCACTGTCTCTTCA



SEQ ID NO. 759





2.26
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGA



TTCACCTTCACTGACTATTATATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGTTTCACACATTAGTA



GTAGTGGTACTACAATAGACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGACGACACGGCCGTATATTACTGTGCGAGAGAAGATATCAGGATGCCTGG



AACTACGGACTTTGACAACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA



SEQ ID NO.760





2.27
CAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGACCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTA



GTAGTGGTAGTACCATTTCCTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAACAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATCACTGTGCGAGAGAAGATATACGTATGAGTGG



GACTACGGACTTTGACTACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA



SEQ ID NO. 761





2.28
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCATCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTA



GTAGTGGTAGTTCCATAGACTACGCAGACTCTGTGAAGGGCCGATTCACCATTTCGAGGGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATCCTCGTTTAAGTGG



AACTATAGATTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCTTCA



SEQ ID NO. 762





2.29
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCGCCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTGGTG



GTAGTGGAACTACCATAGACTACGCAGACTCTGTGAAGGGCCGCTTCACCATCTCCAGGGACAACGCCAGGAATTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCCAGAGAAGATATCAGGATGCCTGG



AACTACGGACTTTGACCACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 763





2.30
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTA



GTAGTGGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATCCTCGTGTGCCTGG



AACTACGAACTTTGACTACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA



SEQ ID NO. 764





2.31
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGACCTGGATGCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTG



GCAGTGGTAGTACCATTGACTATGCAGACTCTGTGAAGGGCCGATTCACGATCTCCAGGGACAACGCCAAGAACTCACT



GTACCTGCAAATGAACAGCCTGAGACCCGAGGACACGGCCGTGTATTACTGTGCGAAAGAAGATGGCCGTATACCTGG



AACTACGGACTTTGACCACTGGGGCCAGGGAACCCTGGTCACTGTCTCTTCA



SEQ ID NO. 765





2.32
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCGCCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCGGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTG



GTAGTGGAACTACCATAGACTACGCAGACTCTGTGAAGGACCGCTTCACCATCTCCAGGGACAACGCCAGGAATTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCCAGAGAAGATATCAGGATGCCTGG



AACTACGGACTTTGACCACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 766





2.33
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCCCCTTCAGTGACTACTTCATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTA



GTAGTGGTAATTCCATAGACTACGCAGACTCTGTGAAGGGCCGCTTCACCATCTCCAGGGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTTTATTACTGTGCGAAAGAAGATCCTCGTTTACCTGGA



ACTACAGATTTTGACTACTGGGGCCAGGGAACCCTGGTCACTGTCTCTTCA



SEQ ID NO. 767





2.34
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTCCTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACATATTAGTA



ATTCTGGTAGTACCATAAGCTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATCCTCGTTTACCTGG



AACTTCAGATTTTGACTACTGGGGCCAGGGAACCCTGGTCACTGTCTCTTCA



SEQ ID NO. 768





2.35
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTA



GTAGTGGTAGTTCCATAGACTACGCAGACTCTGTGAAGGGCCGATTCACCATTTCGAGGGACAACGCCAAGAATTCACT



GTATCTGCAAATGAACAGCCTGAGAGACGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATCCTCGTTTAAGTGG



AACTACAGATTTTGACCAGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 769





2.36
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTTAGTAGCTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTA



GTAGTGGTAGTACCATAGACTACGCAGAGTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACT



GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATCCTCGTATGACTGG



AACTACAGATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCTTCA



SEQ ID NO. 770





2.37
CAGGTGCAGCTGCAGGAGTCGGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTAACTACTTCATGAGTTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCACACATTAGTA



GTAGTGGTAATACCATAGACTACGCAGACTCTGTGAAGGGCCGCTTCACCATCTCCAGGGACAACGCCAAGAACTCACT



TTATCTGCAAATGGATAGTCTGAGAGCCGAGGACACGGCCGTTTATTACTGTTCGAGAGAAGATCCTCGTTTACCTGGA



ACTACAGATTTTGACTACTGGGGCCAGGGAACCCTGGTCACTGTCTCTTCA



SEQ ID NO. 771





2.38
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGG



ATTCACTTTCAGTGACTACTACATGACCTGGATCCGCCAGGGTCCAGGGAAGGGACAGGAATGGATTTCATACATTAGT



AGTGGTGGTAGCACCATACACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCAC



TGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAAATCCCCGTTTACCTGG



AACTATGGACTTTGACTATTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA



SEQ ID NO.772





2.39
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



TTCACCTTCAGTGACCACTTCATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAA



CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCA



CTGTTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTATGTATTACTGTGCGAGAGAGGATCCTCGTTTAACTG



GAACTACAGATTTTGACAACTGGGGCCAGGGAACCCTGGTCACTGTCTCTTCA



SEQ ID NO. 773





2.40
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCCAGGCGGGGGGGTCCCTAAGACTCTCCTGTGTAGCCTCTGG



ATTCACCTTTAGTAATTATTGGATGACCTGGTTCCGCCAGGCTCCAGGGAGGGGGCTGGAGTGGGTTTCACACATTAGT



AGTACTGGATCTACCATAGACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCGAGAACTCAC



TATATTTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATCCCCGTTTACCTGG



AACTATGGACTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 774





2.41
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTA



GTGGTAGTGGTGATATCATAGACTACGCAGACTCTGTAAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAAC



TCTCTGTATCTGCAGATGAACAACCTGAGAGCCGAGGACACGGCCGTGTATCACTGTGCGAGAGAAGATTCCCGTCT



AACTGGAACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 775





2.42
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTG



GATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATT



AGTGGTAGTGGTGATATCATAGACTACGCAGACTCTGTAAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAA



CTCTCTGTATCTGCAGATGAACAACCTGAGAGCCGAGGACACGGCCGTGTATCACTGTGCGAGAGAAGATTCCCGTCT



AACTGGAACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 776





2.43
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTG



GATTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTA



GTGGTAGTGGTGATATCATAGACTACGCAGACTCTGTAAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAAC



TCTCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATCACTGTGCGAGAGAAGATTCCCGTCT



AACTGGAACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 777





2.44
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTG



GATTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTA



GTGGTAGTGGTGATATCATAGACTACGCAGACTCTGTAAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAAC



TCTCTGTATCTGCAGATGAACAACCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATTCCCGTCTA



ACTGGAACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 778





2.45
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTA



GTGGTAGTGGTGATATCATAGACTACGCAGACTCTGTAAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAAC



TCTCTGTATCTGCAGATGAACAACCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATTCCCGTCTA



ACTGGAACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 779





2.46
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATT



AGTGGTAGTGGTGATATCATAGACTACGCAGACTCTGTAAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAA



CTCTCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATTCCCGTCT



AACTGGAACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 780





2.47
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTA



GTGGTAGTGGTGATATCATAGACTACGCAGACTCTGTAAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAAC



TCTCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATTCCCGTCT



AACTGGAACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 781





2.48
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATT



AGTGGTAGTGGTGATATCATAGACTACGCAGACTCTGTAAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAA



CTCTCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATGCCCGTC



TAACTGGAACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 782





2.49
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATT



AGTGGTAGTGGTGATATCATAGACTACGCAGACTCTGTAAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAA



CTCTCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATCCCCGTC



TAACTGGAACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 783





2.50
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTA



GTGGTAGTGGTGATATCATAGACTACGCAGACTCTGTAAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAAC



TCTCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATGCCCGTCT



AACTGGAACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 784





2.51
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTG



GATTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTA



GTGGTAGTGGTGATATCATAGACTACGCAGACTCTGTAAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAAC



TCTCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAAGATCCCCGTCT



AACTGGAACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



SEQ ID NO. 785









In one embodiment, the nucleic acid sequence has at least 50% sequence homology to one of the sequences selected above. In one embodiment, said sequence homology is at least 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In one embodiment, the nucleic acid is selected from one SEQ ID Nos. 629, 706, 881, 882, 883, 884, 885, 886, 887 or 735.


Exemplary Immunoglobulins Included in the Binding Molecule and which Bind PSMA


Examples of moieties for example, single variable heavy chain domain antibodies, that bind to PSMA and that may form one of the subunits of the binding molecules that bind to both, CD137 and PSMA, are described and can be used in the various embodiments of the invention.


The PSMA binding molecules bind to wild type human PSMA (UniProt Accession NO. Q04609). The sequence for the monomer is shown below (SEQ ID No. 842).










  1 MWNLLHETDS AVATARRPRW LCAGALVLAG GFFLLGFLFG WFIKSSNEAT NITPKHNMKA






 61 FLDELKAENI KKFLYNFTQI PHLAGTEQNF QLAKQIQSQW KEFGLDSVEL AHYDVLLSYP





121 NKTHPNYISI INEDGNEIFN TSLFEPPPPG YENVSDIVPP FSAFSPQGMP EGDLVYVNYA





181 RTEDFFKLER DMKINCSGKI VIARYGKVFR GNKVKNAQLA GAKGVILYSD PADYFAPGVK





241 SYPDGWNLPG GGVQRGNILN LNGAGDPLTP GYPANEYAYR RGTAEAVGLP SIPVHPTGY





301 DAQKLLEKMG GSAPPDSSWR GSLKVPYNVG PGFTGNFSTQ KVKMHIHSTN EVTRIYNVTG





361 TLRGAVEPDR YVILGGHRDS WVFGGTDPQS GAAVVHEIVR SFGTLKKEGW RPRRTILFAS





421 WDAEEFGLLG STEWAEENSR LLQERGVAYI NADSSIEGNY TLRVDCTPLM YSLVHNLTKE





481 LKSPDEGFEG KSLYESWTKK SPSPEFSGMP RISKLGSGND FEVFFQRLGT ASGRARYTKN





541 WETNKFSGYP LYHSVYETYE LVEKFYDPMF KYHLTVAQVR GGMVFELANS IVLPFDCRDY





601 AVVLRKYADK IYSISMKHPQ EMKTYSVSFD SLFSAVKNFT EIASKFSERL QDFDKSNPIV





661 LRMMNDQLMF LERAFIDPLG LPDRPFYRHV IYAPSSHNKY AGESFPGTYD ALFDIESKVD





721 PSKAWGEVKR QIYVAAFTVQ AAAETLSEVA






In one embodiment, the PSMA binding molecules of the invention bind to wild type human PSMA and/or cyno PSMA. The terms “PSMA binding molecule”, “PSMA binding protein” “anti-PSMA single domain antibody” or “anti-PSMA antibody” as used herein all refer to a molecule capable of binding to the human PSMA antigen. The term “PSMA binding molecule” includes a PSMA binding protein. The binding reaction may be shown by standard methods (qualitative assays) including, for example, a binding assay, competition assay or a bioassay for determining the inhibition of PSMA binding to its receptor or any kind of binding assays, with reference to a negative control test in which an antibody of unrelated specificity.


Suitable assays are shown in the examples.


A binding molecule of the invention, including a single domain antibody and multivalent or multispecific binding agent described herein, “which binds” or is “capable of binding” an antigen of interest, e.g. PSMA, is one that binds, i.e. targets, the PSMA antigen with sufficient affinity such that it is useful in therapy in targeting a cell or tissue expressing the antigen.


Exemplary Sequences


A single VH domain antibody that binds to PSMA may be as described in WO2017/122017 incorporated herein by reference. In one embodiment, the single variable heavy chain domain antibody comprises a CDR1 comprising SEQ ID NO. 812 or a sequence with at least 75% homology thereto, a CDR2 comprising SEQ ID NO. 813 or a sequence with at least 75% homology thereto and a CDR3 comprising SEQ ID NO. 814 or a sequence with at least 75% homology thereto. In one embodiment, the single variable heavy chain domain antibody comprises a CDR1 comprising SEQ ID NO. 837 or a sequence with at least 75% homology thereto, a CDR2 comprising SEQ ID NO. 838 or a sequence with at least 75% homology thereto and a CDR3 comprising SEQ ID NO. 839 or a sequence with at least 75% homology thereto.


Sequence homology can be 75, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% for example at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.


In one embodiment, the single variable heavy chain domain antibody comprises human framework regions. In one embodiment, the single variable heavy chain domain antibody is selected from single variable heavy chain domain antibody as shown in table 6 or 7, or from a sequence with at least 80% homology thereto. The PSMA binding entity can also be selected form a part thereof, such as a CDR3.


In one embodiment, the single variable heavy chain domain antibody comprises a sequence selected from SEQ ID NOs. 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, or 840. In one embodiment, the sequence is SEQ ID NO. 822 or from a sequence with at least 50%, 60%, 70% or 75% homology thereto. Sequence homology can be 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% for example at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology. In one embodiment, the single variable heavy chain domain antibody comprises a sequence selected from SEQ ID NOs. 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, or 840 or a variant thereof, e.g. a variant with 1 to 10 or 1 to 20 substitutions.









TABLE 6a 







PSMA binding single VH domain antibodies











Name
CDR1
CDR2
CDR3
SEQ ID full VH





3.1
SEQ ID
SEQ ID 
SEQ ID 
SEQ ID NO. 815



NO. 812
NO. 813
NO. 814
EVQLLESGGGLVQPGGSLRLS



SYAMS
STGENDG
DGVH
CAASGFSFSSYAMSWVRQAPG




TTDYADS

KGLEWVSSTGENDGTTDYADS




VKG

VKGRFTISRDNSKSMLYLQMN






SLRVEDTAVYYCVKDGVHWGQ






GTLVTVSS
















TABLE 6b 







PSMA binding single VH domain antibodies








Clone



number
VH Full length sequence











3.2
SEQ ID NO. 816



EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYALSWVR



QAPGKGLEWVSSTGENDGTTDYADFVKGRFTISRDNSK



NTLYLQMNSLRVEDTAVYYCVKDGVHWGQGTLVTVSS





3.3
SEQ ID NO. 817



EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYALSWVR



QAPGKGLEWVSSTGENDGTTDYADNVKGRFTISRDNS



KNTLYLQMNSLRVEDTAVYYCVKDGVHWGQGTLVTVSS





3.4
SEQ ID NO. 818



EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYALSWVR



QAPGKGLEWVSSTGENDGTTDYAADVKGRFTISRDNS



KNTLYLQMNSLRVEDTAVYYCVKDGVHWGQGTLVTVSS





3.5
SEQ ID NO. 819



EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYALSWVR



QAPGKGLEWVSSTGENDGTTDYADVVKGRFTISRDNS



KNTLYLQMNSLRVEDTAVYYCVKDGVHWGQGTLVTVSS





3.6
SEQ ID NO. 820



EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYALSWVR



QAPGKGLEWVSSTGENDGTTDYAAFVKGRFTISRDNSK



NTLYLQMNSLRVEDTAVYYCVKDGVHWGQGTLVTVSS





3.7
SEQ ID NO. 821



EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYALSWVR



QAPGKGLEWVSSTGENDGTTDYADTVKGRFTISRDNSK



NTLYLQMNSLRVEDTAVYYCVKDGVHWGQGTLVTVSS





3.8
SEQ ID NO. 822



EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYALSWVR



QAPGKGLEWVSSTGENDGTTDYADAVKGRFTISRDNS



KNTLYLQMNSLRVEDTAVYYCVKDGVHWGQGTLVTVSS





3.9
SEQ ID NO. 823



EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYALSWVR



QAPGKGLEWVSSTGENDGTTDYAASVKGRFTISRDNSK



NTLYLQMNSLRVEDTAVYYCVKDGVHWGQGTLVTVSS





3.10
SEQ ID NO. 824



EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYALSWVR



QAPGKGLEWVSSTGENDGTTDYAAYVKGRFTISRDNSK



NTLYLQMNSLRVEDTAVYYCVKDGVHWGQGTLVTVSS





3.11
SEQ ID NO. 825



EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYALSWVR



QAPGKGLEWVSSTGENDGTTDYAATVKGRFTISRDNSK



NTLYLQMNSLRVEDTAVYYCVKDGVHWGQGTLVTVSS
















TABLE 7 





PSMA binding single VH domain antibody



















4.1
SEQ ID 
SEQ ID
SEQ ID 
SEQ ID NO. 840



NO. 837
NO. 838
NO. 839
EVQLVESGGGVVQPGRSLRL



GYGMH
YISYDGSN
DPAWGLR
SCAASGFSFSGYGMHWVRQA




KYYADSVK
LGESSSY
PGKGLEWVAYISYDGSNKYY




G
DFDI
ADSVKGRFTISRDNSKNTLY






LQMNSLRAEDTAVYYCAKDP






AWGLRLGESSSYDFDIWGQG






TMVTVSS









In one embodiment, the variant comprises substitutions. Typical modifications are explained elsewhere herein.


Exemplary Nucleic Acids


Nucleic acids that encode the single domain antibodies shown above are set out below in table 7b.














3.1 SEQ ID NO. 826


GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


AGTTTTAGCAGCTATGCCATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAAT


GATGGTACCACAGACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAGTATGCTGTATCTGC


AAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGG


TCACCGTCTCCTCA





3.2 SEQ ID NO. 827


GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


AGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAAT


AACGCTACCACAGACTACGCAGACTTCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTATCTGC


AAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGG


TCACCGTCTCCTCA





3.3 SEQ ID NO. 828


GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


AGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAAT


GATGGTACCACAGACTACGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTATCTGC


AAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGG


TCACCGTCTCCTCA





3.4 SEQ ID NO. 829


GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


AGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAAT


GATGGTACCACAGACTACGCAGCCGACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTATCTGC


AAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGG


TCACCGTCTCCTCA





3.5 SEQ ID NO. 830


GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


AGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAAT


GATGGTACCACAGACTACGCAGACGTCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTATCTGC


AAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGG


TCACCGTCTCCTCA





3.6 SEQ ID NO. 831


GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


AGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAAT


GATGGTACCACAGACTACGCAGCCTTCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTATCTGC


AAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGG


TCACCGTCTCCTCA





3.7 SEQ ID NO. 832


GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


AGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAAT


GATGGTACCACAGACTACGCAGACACCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTATCTGC


AAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGG


TCACCGTCTCCTCA





3.8 SEQ ID NO. 833


GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


AGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAAT


GATGGTACCACAGACTACGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTATCTGC


AAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGG


TCACCGTCTCCTCA





3.9 SEQ ID NO. 834


GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


AGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAAT


GATGGTACCACAGACTACGCAGCCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTATCTGC


AAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGG


TCACCGTCTCCTCA





3.10 SEQ ID NO. 835


GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


AGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAAT


GATGGTACCACAGACTACGCAGCCTACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTATCTGC


AAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGG


TCACCGTCTCCTCA





3.11 SEQ ID NO. 836


GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


AGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAAT


GATGGTACCACAGACTACGCAGCCACCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTATCTGC


AAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGG


TCACCGTCTCCTCA





4.1 SEQ ID NO. 841


GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


TCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATG


GAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCA


AATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTC


ATCGTCCTATGATTTTGATATCTGGGGCCAAGGGACAATGGTCACTGTCTCTTCA









In one embodiment, the binding molecule may comprise one or more single domain antibodies that bind to PSMA, for example one or two single domain antibodies as described above.


In one embodiment, the binding molecule may comprise one or more single domain antibodies that bind to PSMA, for example one or two single domain antibodies as described above. In one embodiment the binding molecule comprises two single domain antibodies that bind to PSMA wherein each binds to a different epitope of PSMA hus providing a biparatopic PSMA binder.


Exemplary binding molecules Any combination of the aforesaid single variable heavy chain domain antibodies that bind to CD137 and PSMA respectively can be used in a binding agent for dual engagement of CD137 and PSMA expressing cells. Thus, in one embodiment, any of the single variable heavy chain domain antibodies disclosed above, for example as listed in any of Tables 2 or 3 can be combined in a fusion protein with any of the single variable heavy chain domain antibodies as listed in any of Tables 6a, 6b or 7.


Thus, in one embodiment, the fusion protein comprises a single domain antibody having a sequence selected from SEQ ID Nos 4, 8, 12, 16, 20, 24, 28, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 448, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, 852, 856, 860, 864, 868, 872, 876 or 880 or a sequence with at least 75% homology thereto and a single domain antibody having a sequence selected from SEQ ID Nos 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825 or 840 or a sequence with at least 75% homology thereto. In one embodiment, the sequence is selected from SEQ ID Nos 852, 856, 860, 864, 868, 872, 876 or 880 or a sequence with at least 75% homology thereto.


In another embodiment, the fusion protein comprises a single domain antibody having a sequence selected from SEQ ID Nos 428, 432, 436, 440, 444, 448, 452, 456, 460, 464, 468, 472, 476, 480, 484, 488, 492, 496, 500, 508, 512, 516, 520, 524, 528, 532, 536, 540, 544, 548, 552, 556, 560, 564, 568, 572, 576, 580, 584, 588, 592, 596, 600, 604, 608, 612, 616, 620, 624 or 628 or a sequence with at least 75% homology thereto and a single domain antibody having a sequence selected from SEQ ID Nos 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825 or 840 or a sequence with at least 75% homology thereto. In one embodiment, a single domain antibody 1.1 or 2.1 having SEQ ID No. 4 or 428 or a single domain antibody having SEQ ID No. 312, 852, 856, 860, 864, 868, 872, 876, 880 or 428 is linked to a single domain antibody 3.1, 3.8 or 4.1 having SEQ ID No. 815, 822 or 840 respectively. In one embodiment, a single domain 1.113 having SEQ ID No. 876 is linked to a single domain antibody 3.1, 3.8 or 4.1 having SEQ ID No. 815, 822 or 840 respectively. In one embodiment, a single domain 1.113 having SEQ ID No. 876 is linked to a single domain antibody 4.1 having SEQ ID No. 840. In one embodiment, the fusion protein comprises or consists of single domain antibody 1.113 linked to single domain antibody 4.1 or 3.8 and single domain antibody as shown in SEQ ID No. 901. The order of the single domain antibodies can vary, for example the CD137 binding single domain antibody can located be 3′ or 5′ of the PSMA binding molecule. The single domain antibody as shown in SEQ ID No. 901 is preferably located at the C terminal end.


The two single domain antibodies can be linked in the fusion protein with a peptide linker, such as a G4S linker as described herein. In one embodiment the invention relates to one of the fusion proteins and nucleic acids encoding such fusion proteins as listed in table 8 below (i.e. selected from one of the protein and (i.e. selected from one of the protein and nucleic acid constructs of SEQ ID NO. 806-811) or fusion proteins and nucleic acids encoding such fusion proteins as listed in table 10 in the examples (i.e. selected from one of the protein and nucleic acid constructs of SEQ ID NO. 889-900). In one embodiment, the protein comprises the single domain antibodies defined as 4.1 or 1.113 as described herein linked with a (G4S)n peptide linker wherein n is as defined herein, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (i.e. 4.1-(G4S)n-1.113). In one embodiment, the protein is selected from SEQ ID Nos. 844, 846, 890 or 894. In one embodiment, the protein comprises or consists of SEQ ID NO. 890 optionally including a HSA binding sdAb. In one embodiment, the protein is encoded by the nucleic acid sequence of SEQ ID NO. 891.









TABLE 8 







SEQ ID NO: 806 4.1(PSMA-binding VH)-6GS-1.1 Protein


EVQLVESGGGVVQPGRSLRLSCAASGFSFSGYGMHWVRQAPGKGLEWVAYISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNS


LRAEDTAVYYCAKDPAWGLRLGESSSYDFDIWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGG


LVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLEWVAHIKEDGSEKYYEDSVEGRFTVSRDNAKNSVYLQMNSLRAEDTAVYY


CARGGDGYSDSHFGVDVWGQGTTVTVSS





SEQ ID NO: 807 4.1(PSMA-binding VH)-6GS-1.1 Nucleotide


GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCT


CCTTCAGTGGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATGGA


AGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATG


AACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGT


CCTATGATTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGA


GGTGGAGGTTCAGGAGGTGGTGGTTCTGGCGGCGGTGGATCGGGTGGAGGTGGTAGTGAGGTGCAGCTGGTGGAGTCTGG


GGGAGGCTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATCACC1TAGTAGCCATTGGATGACTT


GGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATAAAGGAAGACGGAAGTGAGAAATACTATGAGGACT


CTGTGGAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGAACTCGGTATATCTGCAAATGAACAGTCTGAGAGCCGAAGAC


ACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGCTACAGTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCA


CGGTCACTGTCTCTTCA





SEQ ID NO: 808 4.1(PSMA-binding VH)-6GS-2.1


EVQLVESGGGVVQPGRSLRLSCAASGFSFSGYGMHWVRQAPGKGLEWVAYISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNS


LRAEDTAVYYCAKDPAWGLRLGESSSYDFDIWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGG


LVKPGGSLRVSCAASGFTFSDYYMSWFRQAPGKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQMNNLRAEDTAVYH


CAREDSRLTGTTDFDNWGQGTLVTVSS





SEQ ID NO: 809 4.1(PSMA-binding VH)-6GS-2.1 Nucleotide


GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCT


CCTTCAGTGGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATGGA


AGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATG


AACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGT


CCTATGATTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGA


GGTGGAGGTTCAGGAGGTGGTGGTTCTGGCGGCGGTGGATCGGGTGGAGGTGGTAGTGAGGTGCAGCTGGTGGAGTCTGG


GGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCT


GGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTGGTAGTGGTGATATCATAGACTACGCAGACTCT


GTAAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCTCTGTATCTGCAGATGAACAACCTGAGAGCCGAGGACAC


GGCCGTGTATCACTGTGCGAGAGAAGATTCCCGTCTAACTGGAACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCA


CCGTCTCCTCA





SEQ ID NO: 810 Protein 3.8-6GS-1.1


EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYALSWVRQAPGKGLEWVSSTGENDGTTDYADAVKGRFTISRDNSKNTLYLQMNS


LRVEDTAVYYCVKDGVHWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASG


FTFSSHWMTWFRQAPGKGLEWVAHIKEDGSEKYYEDSVEGRFTVSRDNAKNSVYLQMNSLRAEDTAVYYCARGGDGYSDSHFGV


DVWGQGTTVTVSS





SEQ ID NO: 811 3.8-6GS-1. Nucleotide


GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCA


GTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATGAT


GGTACCACAGACTACGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTATCTGCAAAT


GAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACC


GTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGAGGTGGAGGTTCAGGAGGTGGTGGTTCTGGCGGCGGTGGA


TCGGGTGGAGGTGGTAGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCC


TGTGCAGCCTCTGGATTCACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGC


CCACATAAAGGAAGACGGAAGTGAGAAATACTATGAGGACTCTGTGGAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAG


AACTCGGTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGCTACA


GTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA









Exemplary Modifications of the Binding Molecule


In one embodiment, a binding agent described above comprises further binding molecules. Thus, the binding agent can, for example, be trispecific or tetraspecific.


In one embodiment, the binding molecule comprises a first VH single domain antibody that binds to CD137 (VH (A)) and a second moiety, for example a VH single domain antibody, that binds to PSMA (VH (B)). It further comprises a third, fourth, fifth etc moiety, for example a VH single domain antibody (i.e. VH (C), VH (D), VH (E)) that binds to another antigen. Thus, in one embodiment, the binding molecule has the following formula (wherein VH stands for a single domain antibody as defined herein, that is the single VH domain that does not comprise other parts of a full antibody and retains binding to the antigen): VH (A)-L-VH (B)-L-VH (X)n wherein X denotes a VH binding to a target other than the target VH (A) and VH (B) bind to and wherein X is 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. L denotes a linker, for example a peptide linker. As explained elsewhere, a moiety that binds to PSMA or another target can be selected from an antibody or fragment thereof or other polypeptide.


In another embodiment, the further moiety may serve to prolong the half-life of the binding molecule. The further moiety may comprise a protein, for example a peptide, antibody, or part thereof, such as a VH or CDR, that binds a serum albumin, e.g., human serum albumin (HSA) or mouse serum albumin (MSA). In one embodiment, the further moiety may comprise a VH domain that binds serum albumin, e.g., human serum albumin (HSA) or mouse serum albumin (MSA). In one embodiment, the VH domain that binds HSA is SEQ ID NO. 901 or a sequence with at least 90% sequence homology thereto. The further moiety may comprise a serum albumin, e.g. a HSA or a variant thereof such as HSA C34S. Further provided is a binding molecule as described herein comprising a VH domain and an Fc domain, e.g., wherein the VH domain is fused to an Fc domain.


The term “half-life” as used herein refers to the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and/or clearance or sequestration of the sequence or compound by natural mechanisms. Half-life may be increased by at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding VH single domain antibodies of the invention. For example, increased half-life may be more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding VH single domain antibodies or fusion protein of the invention. The in vivo half-life of an amino acid sequence, compound or polypeptide of the invention can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art. Half life can for example be expressed using parameters such as the t1/2-alpha t1/2-beta and the area under the curve (AUC).


In one embodiment, the binding agents are labelled with a detectable or functional label. A label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorophores, fluorescers, radiolabels, enzymes, chemiluminescers, a nuclear magnetic resonance active label or photosensitizers. Thus, the binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance.


In still other embodiments binding agents are coupled to at least one therapeutic moiety, such as a drug, an enzyme or a toxin. In one embodiment, the therapeutic moiety is a toxin, for example a cytotoxic radionuclide, chemical toxin or protein toxin.


In another aspect, the binding agents of the invention are modified to increase half-life, for example by a chemical modification, especially by PEGylation, or by incorporation in a liposome or using a serum albumin protein. Increased half life can also be conferred by conjugating the molecule to a n antibody fragment, for example a VH domain that increases half life.


To generate multivalent binding agents as described above, two binding molecules can be connected by a linker, for example a polypeptide linker. Suitable linkers include for example a linker with GS residues such as (Gly4Ser)n, where n=from 1 to 10 or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and specific sequences of examples are provided elsewhere.


Exemplary Methods for Making Single Domain Antibody and Binding Molecules


A single domain antibody described herein for use in the multispecifc molecule can be obtained from a transgenic rodent that expresses heavy chain only antibodies upon stimulation with a CD137 or PSMA antigen respectively. The transgenic rodent, for example a mouse, preferably has a reduced capacity to express endogenous antibody genes. Thus, in one embodiment, the rodent has a reduced capacity to express endogenous light and/or heavy chain antibody genes. The rodent may therefore comprise modifications to disrupt expression of endogenous kappa and lambda light and/or heavy chain antibody genes so that no functional light and/or heavy chains are produced, for example as further explained below.


A method for producing a human heavy chain only antibody capable of binding the target antigen comprises

    • a) immunising a transgenic rodent, e.g. a mouse, with target antigen (i.e. PSMA or CD137) wherein said rodent expresses a nucleic acid construct comprising unrearranged human heavy chain V genes and is not capable of making functional endogenous light or heavy chains,
    • b) isolating human heavy chain only antibodies.


Further steps can include isolating a VH domain from said heavy chain only antibody, for example by generating a library of sequences comprising VH domain sequences from said rodent, e.g. mouse, and isolating sequences comprising VH domain sequences from said libraries.


A method for producing a single VH domain antibody capable of binding to the target antigen comprises

    • a) immunising a transgenic rodent, e.g. a mouse with a target antigen wherein said rodent, e.g. mouse, expresses a nucleic acid construct comprising unrearranged human heavy chain V genes and is not capable of making functional endogenous light or heavy chains,
    • b) generating a library of sequences comprising VH domain sequences from said rodent, e.g. mouse, and
    • c) isolating sequences comprising VH domain sequences from said libraries.


Further steps may include identifying a single VH domain antibody or heavy chain only antibody that binds to the target antigen, for example by using functional assays as shown in the examples.


Methods for preparing or generating the polypeptides, nucleic acids, host cells, products and compositions described herein using in vitro expression libraries can comprise the steps of:


a) providing a set, collection or library of nucleic acid sequences encoding amino acid sequences; and


b) screening said set, collection or library for amino acid sequences that can bind to/have affinity for the target antigen and


c) isolating the amino acid sequence(s) that can bind to/have affinity for target antigen.


In the above method, the set, collection or library of amino acid sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) amino acid sequences will be clear to the person skilled in the art (see for example Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press; 1st edition (Oct. 28, 1996) Brian K. Kay, Jill Winter, John McCafferty).


Libraries, for example phage libraries, are generated by isolating a cell or tissue expressing an antigen-specific, heavy chain-only antibody, cloning the sequence encoding the VH domain(s) from mRNA derived from the isolated cell or tissue and displaying the encoded protein using a library. The VH domain(s) can be expressed in bacterial, yeast or other expression systems.


In the various aspects and embodiments as out herein, the term rodent may relate to a mouse or a rat. In one embodiment, the rodent is a mouse. The mouse may comprise a non-functional endogenous lambda light chain locus. Thus, the mouse does not make a functional endogenous lambda light chain. In one embodiment, the lambda light chain locus is deleted in part or completely or rendered non-functional through insertion, inversion, a recombination event, gene editing or gene silencing. For example, at least the constant region genes C1, C2 and C3 may be deleted or rendered non-functional through insertion or other modification as described above. In one embodiment, the locus is functionally silenced so that the mouse does not make a functional lambda light chain.


Furthermore, the mouse may comprise a non-functional endogenous kappa light chain locus. Thus, the mouse does not make a functional endogenous kappa light chain. In one embodiment, the kappa light chain locus is deleted in part or completely or rendered non-functional through insertion, inversion, a recombination event, gene editing or gene silencing. In one embodiment, the locus is functionally silenced so that the mouse does not make a functional kappa light chain.


The mouse having functionally-silenced endogenous lambda and kappa L-chain loci may, for example, be made as disclosed in WO 2003/000737, which is hereby incorporated by reference in its entirety.


Furthermore, the mouse may comprise a non-functional endogenous heavy chain locus. Thus, the mouse does not make a functional endogenous heavy chain. In one embodiment, the heavy chain locus is deleted in part or completely or rendered non-functional through insertion, inversion, a recombination event, gene editing or gene silencing. In one embodiment, the locus is functionally silenced so that the mouse does not make a functional heavy chain.


For example, as described in WO 2004/076618 (hereby incorporated by reference in its entirety), all 8 endogenous heavy chain constant region immunoglobulin genes (μ, δ, γ3, γ1, γ2a, γ2b, ε and α) are absent in the mouse, or partially absent to the extent that they are non-functional, or genes δ, γ3, γ1, γ2a, γ2b and are absent and the flanking genes μ and α are partially absent to the extent that they are rendered non-functional, or genes μ, δ, γ3, γ1, γ2a, γ2b and ε are absent and α is partially absent to the extent that it is rendered non-functional, or δ, γ3, γ1, γ2a, γ2b, ε and α are absent and μ is partially absent to the extent that it is rendered non-functional. By deletion in part is meant that the endogenous locus gene sequence has been deleted or disrupted, for example by an insertion, to the extent that no functional endogenous gene product is encoded by the locus, i.e., that no functional product is expressed from the locus. In another embodiment, the locus is functionally silenced.


In one embodiment, the mouse comprises a non-functional endogenous heavy chain locus, a non-functional endogenous lambda light chain locus and a non-functional endogenous kappa light chain locus. The mouse therefore does not produce any functional endogenous light or heavy chains. Thus, the mouse is a triple knockout (TKO) mouse.


The transgenic mouse may comprise a vector, for example a Yeast Artificial Chromosome (YAC) for expressing a heterologous, preferably a human, heavy chain locus. YACs are vectors that can be employed for the cloning of very large DNA inserts in yeast. As well as comprising all three cis-acting structural elements essential for behaving like natural yeast chromosomes (an autonomously replicating sequence (ARS), a centromere (CEN) and two telomeres (TEL)), their capacity to accept large DNA inserts enables them to reach the minimum size (150 kb) required for chromosome-like stability and for fidelity of transmission in yeast cells. The construction and use of YACs is well known in the art (e.g., Bruschi, C. V. and Gjuracic, K. Yeast Artificial Chromosomes, Encyclopedia of Life Sciences, 2002 Macmillan Publishers Ltd, Nature Publishing Group).


For example, the YAC may comprise a plethora of unrearranaged human VH, D and J genes in combination with mouse immunoglobulin constant region genes lacking CH1 domains, mouse enhancer and regulatory regions. The human VH, D and J genes are human VH, D and J loci and they are unrearranged genes that are fully human.


Alternative methods known in the art may be used for deletion or inactivation of endogenous mouse or rat immunoglobulin genes and introduction of human VH, D and J genes in combination with mouse immunoglobulin constant region genes lacking CH1 domains, mouse enhancer and regulatory regions.


Transgenic mice can be created according to standard techniques as illustrated in the examples. The two most characterised routes for creating transgenic mice are via pronuclear microinjection of genetic material into freshly fertilised oocytes or via the introduction of stably transfected embryonic stem cells into morula or blastocyst stage embryos. Regardless of how the genetic material is introduced, the manipulated embryos are transferred to pseudo-pregnant female recipients where pregnancy continues and candidate transgenic pups are born. The main differences between these broad methods are that ES clones can be screened extensively before their use to create a transgenic animal. In contrast, pronuclear microinjection relies on the genetic material integrating to the host genome after its introduction and, generally speaking, the successful incorporation of the transgene cannot be confirmed until after pups are born.


There are many methods known in the art to both assist with and determine whether successful integration of transgenes occurs. Transgenic animals can be generated by multiple means including random integration of the construct into the genome, site-specific integration, or homologous recombination. There are various tools and techniques that can be used to both drive and select for transgene integration and subsequent modification including the use of drug resistance markers (positive selection), recombinases, recombination-mediated cassette exchange, negative selection techniques, and nucleases to improve the efficiency of recombination. Most of these methods are commonly used in the modification of ES cells. However, some of the techniques may have utility for enhancing transgenesis mediated via pronuclear injection.


Further refinements can be used to give more efficient generation of the transgenic line within the desired background. As described above, in preferred embodiments, the endogenous mouse immunoglobulin expression is silenced to permit sole use of the introduced transgene for the expression of the heavy-chain only repertoire that can be exploited for drug discovery. Genetically-manipulated mice, for example TKO mice that are silenced for all endogenous immunoglobulin loci (mouse heavy chain, mouse kappa chain and mouse lambda chain) can be used as described above. The transfer of any introduced transgene to this TKO background can be achieved via breeding, either conventional or with the inclusion of an IVF step to give efficient scaling of the process. However, it is also possible to include the TKO background during the transgenesis procedure. For example, for microinjection, the oocytes may be derived from TKO donors. Similarly, ES cells from TKO embryos can be derived for use in transgenesis.


Triple knock-out mice into which transgenes have been introduced to express immunoglobulin loci are referred to herein as TKO/Tg. In one embodiment, the mouse is as described in WO2016/062990.


Fusion proteins as described herein can be generated by linking a nucleic acid encoding a single variable heavy chain domain antibody that binds to CD137 to a nucleic acid encoding a single variable heavy chain domain antibody that binds to PSMA, for example using a nucleic acid sequence that encodes a peptide linker. Such fusion nucleic acid molecules are then expressed in suitable host cells.


Exemplary therapeutic applications of the binding molecules In another aspect, there is provided a pharmaceutical composition comprising a binding molecule as described herein and optionally a pharmaceutically acceptable carrier. A binding molecule as described herein or the pharmaceutical composition of the invention can be administered by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin or by inhalation.


Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably, the compositions are administered parenterally.


The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The term “carrier” refers to a diluent, adjuvant or excipient, with which a drug antibody of the present invention is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to an animal, the single domain antibody of the present invention or compositions and pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the drug antibody of the present invention are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.


The pharmaceutical composition of the invention can be in the form of a liquid, e.g., a solution, emulsion or suspension. The liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or sub-cutaneously.


When intended for oral administration, the composition is preferably in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.


As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, corn starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the composition is in the form of a capsule (e. g. a gelatin capsule), it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.


The composition can be in the form of a liquid, e. g. an elixir, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection. When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.


Compositions can take the form of one or more dosage units.


In specific embodiments, it can be desirable to administer the composition locally to the area in need of treatment, or by intravenous injection or infusion.


The amount of the therapeutic that is effective/active in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.


Typically, the amount is at least about 0.01% of a single domain antibody of the present invention by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1% to about 80% by weight of the composition. Preferred oral compositions can comprise from about 4% to about 50% of the single domain antibody of the present invention by weight of the composition.


Preferred compositions of the present invention are prepared so that a parenteral dosage unit contains from about 0.01% to about 2% by weight of the single domain antibody of the present invention. The invention also relates to a device, such as a pre-filled syringe which comprises a binding molecule of the invention.


For administration by injection, the composition can comprise from about typically about 0.1 mg/kg to about 250 mg/kg of the subject's body weight, preferably, between about 0.1 mg/kg and about 20 mg/kg of the subject's body weight, and more preferably about 1 mg/kg to about 10 mg/kg of the subject's body weight. In one embodiment, the composition is administered at a dose of about 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, or about 3 mg/kg. The dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks.


As used herein, “treat”, “treating” or “treatment” means inhibiting or relieving a disease or disorder. For example, treatment can include a postponement of development of the symptoms associated with a disease or disorder, and/or a reduction in the severity of such symptoms that will, or are expected, to develop with said disease. The terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result is being conferred on at least some of the mammals, e.g., human patients, being treated. Many medical treatments are effective for some, but not all, patients that undergo the treatment.


The term “subject” or “patient” refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.


As used herein, the term “effective amount” means an amount of the binding molecule as described herein, that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to achieve the desired therapeutic or prophylactic effect under the conditions of administration.


The invention furthermore relates to a method for the prevention and/or treatment of cancer, in particular prostate cancer, comprising administering a binding molecule of the invention to a subject, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of a binding molecule and/or of a pharmaceutical composition of the invention. In particular, the invention relates to a method for the prevention and/or treatment of cancer, in particular prostate cancer, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of a binding molecule or a pharmaceutical composition of the invention.


The invention also relates to a binding molecule of the invention for use in the treatment of a disease. The invention also relates to a binding molecule of the invention for use in the treatment of cancer, in particular prostate cancer or a prostatic disorder. “Prostate cancer” refers to all stages and all forms of cancer arising from the tissue of the prostate gland. The invention also relates to the treatment of a disease characterized by aberrant expression of PSMA.


In another aspect, the invention relates to the use of a binding molecule of the invention in the treatment of disease. In another aspect, the invention relates to the use of a binding molecule of the invention in the manufacture of a medicament for the treatment of cancer, such as prostate cancer or a prostatic disorder.


The binding molecules of the invention are also useful for the treatment, prevention, or amelioration of a disease. In one embodiment, the disease is associated with PSMA positive cells. In one embodiment, the disease is cancer. In one embodiment, the cancer associated with a PSMA positive tumor.


Expression of PSMA has been detected in other cancers, more specifically in the neovasculature associated with these cancers. A wide range of carcinomas, including conventional (clear cell) renal cell, transitional cell of the bladder, testicular-embryonal, neuroendocrine, colon, and breast, and the different types of malignancies were found consistently and strongly to express PSMA in their neovasculature. In one embodiment, the cancer to be treated is selected from a tumor in the neovasculature that expresses PSMA, for example selected from renal, bladder, testicular, neuroendocrine, colon, and breast cancer.


In one aspect, the cancer is locally advanced unresectable, metastatic, or recurrent cancer.


In one embodiment, the cancer is selected from the following non limiting list: prostate cancer, lung cancer or glioblastoma. In one embodiment, the disease is a prostatic disorder which refers to any disease that afflicts the prostate gland in the male reproductive system. The prostate gland is dependent on the hormonal secretions of the testes.


The binding molecule of the invention may be administered as the sole active ingredient or in combination with one or more other therapeutic and/or cytotoxic moiety. In one embodiment, the binding molecule may be conjugated to a toxic moiety.


In one embodiment, the single domain antibody is used in combination with surgery.


Exemplary Combinations with Other Agents


The molecules or pharmaceutical composition of the invention, including monovalent or multivalent molecules, may be administered as the sole active ingredient or in combination with one or more other therapeutic agent. A therapeutic agent is a compound or molecule which is useful in the treatment of a disease. Examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptotic agents, anti-angiogenic agents, boron compounds, photoactive agents or dyes and radioisotopes. An antibody molecule includes a full antibody or fragment thereof (e.g., a Fab, F(ab′)2, Fv, a single chain Fv fragment (scFv) or a single domain antibody, for example a VH domain, antibody mimetic protein or a protein that mimics the natural ligand of CD137.


The anti-cancer therapy may include a therapeutic agent or radiation therapy and includes gene therapy, viral therapy, RNA therapy bone marrow transplantation, nanotherapy, targeted anti-cancer therapies or oncolytic drugs. Examples of other therapeutic agents include other checkpoint inhibitors, antineoplastic agents, immunogenic agents, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor-derived antigen or nucleic acids, immune stimulating cytokines (e.g., IL-2, IFNa2, GM-CSF), targeted small molecules and biological molecules (such as components of signal transduction pathways, e.g. modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and agents that bind to tumor-specific antigens, including EGFR antagonists), an anti-inflammatory agent, a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent and cells transfected with a gene encoding an immune stimulating cytokine (e.g., GM-CSF), chemotherapy. In one embodiment, administration is in combination with surgery. The binding molecule of the invention may be administered at the same time or at a different time as the other therapy, e.g., simultaneously, separately or sequentially.


Exemplary methods for modulating immune response, inhibiting tumor growth etc In yet another aspect, there is provided a method of modulating an immune response in a subject comprising administering to the subject the binding molecule or pharmaceutical composition described herein such that the immune response in the subject is modulated. Preferably, the binding molecule enhances, stimulates or increases the immune response in the subject.


In a further aspect, there is provided a method of inhibiting growth of tumor cells or promoting tumor regression in a subject, comprising administering to a subject a therapeutically effective amount of a binding molecule or a pharmaceutical composition described herein.


In a further aspect, there is provided a method for activating the downstream signalling pathway of CD137 comprising administering to a subject a binding molecule or a pharmaceutical composition described herein.


In a further aspect, there is provided a method for inducing T lymphocyte activation and/or proliferation comprising administering to a subject a binding molecule or a pharmaceutical composition described herein.


In a further aspect, there is provided a method for dual targeting of a CD137 expressing cell and a tumor antigen, i.e. PSMA, expressing cell comprising administering to a subject a binding molecule comprising a single variable heavy chain domain antibody that binds to CD137 or a pharmaceutical composition described herein.


In a further aspect, there is provided a binding molecule comprising a single variable heavy chain domain antibody that binds to CD137 or a pharmaceutical composition described herein for dual targeting of a CD137 expressing cell and a tumor antigen, i.e. PSMA, expressing cell. For example, the binding molecule comprises a single variable heavy chain domain antibody that binds to CD137 described herein and a single variable heavy chain domain antibody that binds to PSMA described herein


Exemplary Immunoconjugates


In another aspect, there is provided an immunoconjugate comprising a binding molecule described herein conjugated to at least one therapeutic and/or diagnostic agent i.e. an imagining agent.


Exemplary kits In another aspect, the invention provides a kit for detecting prostate cancer for diagnosis, treatment, prognosis or monitoring comprising a binding molecule of the invention. The kit may also comprise instructions for use. The kits may include a labeled binding molecule of the invention as described above and one or more compounds for detecting the label. Also provided is a binding molecule of the invention packaged in lyophilized form, or packaged in an aqueous medium. The kits may include reagent, (e.g. for reconstituting) and/or instructions for use and/or a device for administration.


Exemplary non-therapeutic applications The invention also relates to detection methods using the binding molecule of the invention. Given their ability to bind to human PSMA, the human-PSMA-binding molecules as disclosed herein can be used to detect PSMA (e.g., in a biological sample, such as serum or plasma), using a conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry. In particular, the invention also relates to in vitro or in vivo methods for diagnosing or monitoring progression of a cancer, in particular prostate cancer. In vitro methods comprise detecting the presence of a PSMA protein in a test sample and comparing this with control sample from a normal subject or with a standard value or standard value range for a normal subject. The sample may be selected from blood, plasma, serum, semen, urine or a tissue biopsy.


The method may include: (a) contacting the sample (and optionally, a reference, e.g., a positive and/or negative control sample) with a PSMA binding molecule of the invention and (b) detecting either the binding molecule bound to PSMA or unbound binding molecule in the sample, to thereby detect PSMA in the biological sample. The binding molecule can be directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.


In vivo methods may comprise detecting the presence of PSMA in vivo, for example by imaging in a subject. In this method, a PSMA binding molecule of the invention is labeled to detect binding. A labelled molecule of the invention may thus be used as an imaging agent.


As an alternative to labeling the binding molecule of the invention, human PSMA can be assayed in biological fluids by a competition immunoassay utilizing PSMA standards labeled with a detectable substance and an unlabeled human PSMA binding molecule. In this assay, the biological sample, the labeled PSMA standards and the human PSMA binding molecule are combined and the amount of labeled PSMA standard bound to the unlabeled binding molecule is determined. The amount of human PSMA in the biological sample is inversely proportional to the amount of labeled PSMA standard bound to the PSMA binding molecule. Similarly, human PSMA can also be assayed in biological fluids by a competition immunoassay utilizing PSMA standards labeled with a detectable substance and an unlabeled human PSMA binding molecule.


Binding molecules disclosed herein can be used to inhibit PSMA activity, e.g., in a cell culture containing PSMA, in human subjects or in other mammalian subjects having PSMA with which a binding molecule disclosed herein cross-reacts. In one embodiment, a method for inhibiting or increasing PSMA activity is provided comprising contacting PSMA with a binding molecule disclosed herein such that PSMA activity is inhibited or increased. For example, in a cell culture containing, or suspected of containing PSMA, a binding molecule disclosed herein can be added to the culture medium to inhibit PSMA activity in the culture.


Therefore, in one embodiment, the invention also relates to a method of ablating or killing a cell that expresses PSMA, e.g., a cancerous or non-cancerous prostatic cell. Methods of the invention include contacting the cell, with PSMA binding molecule of the invention, in an amount sufficient to ablate or kill the cell. The methods can be used on cells in culture, e.g., in vitro or ex vivo.


Also provided is a binding molecule or pharmaceutical composition described herein with reference to the figures and examples. Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present disclosure, including methods, as well as the best mode thereof, of making and using this disclosure, the following examples are provided to further enable those skilled in the art to practice this disclosure. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present disclosure will be apparent to those skilled in the art in view of the present disclosure.


All documents mentioned in this specification are incorporated herein by reference in their entirety, including references to gene accession numbers, scientific publications and references to patent publications.


“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.


The invention is now further described in the non-limiting examples.


EXAMPLES
Example 1. Isolation of Humabody® VH Specific for CD137 and PSMA

Humabody® VH specific for CD137 or PSMA were generated by immunisation of the Crescendo's proprietary transgenic mouse that has silenced murine immunoglobulin loci and a transgene containing human heavy chain antibody genes.


Mice carrying a human heavy-chain antibody transgenic locus in germline configuration within a background that is silenced for endogenous heavy and light chain antibody expression (triple knock-out, or TKO) were created as previously described (WO2004/076618, WO2003/000737, Ren et al., Genomics, 84, 686, 2004; Zou et al., J. Immunol., 170, 1354, 2003 and WO2016/062990). Briefly, transgenic mice were derived following pronuclear microinjection of freshly fertilised oocytes with a yeast artificial chromosome (YAC) comprising a plethora of human VH, D and J genes in combination with mouse immunoglobulin constant region genes lacking CH1 domains, mouse enhancer and regulatory regions. The YAC used comprised multiple human heavy chain V genes, multiple human heavy chain D and J genes, a murine CH1 gene and a murine 3′ enhancer gene. It lacks the CH1 exon.


Mice were immunised with recombinant human CD137 or PSMA proteins. At the end of the immunisation schedule, lymph nodes and spleens were harvested for RNA extraction. Heavy chain variable domains (VH) sequences were amplified by PCR and cloned into a phagemid vector. Standard phage display techniques were used to isolate the VH that bound to the target proteins, including generation of libraries from immunised mice described above followed standard protocols of library generation, standard screening procedures of bacterial periplasmic extracts, optimisation, sequencing and purification methods. Amino acid and nucleic acid sequences for clones 1.1, 2.1, 3.1 and 4.1 are shown in tables 2, 3, 5, 6 and 7.


For example, the PSMA-binding subunit may be generated as disclosed in WO2017/122017. For the generation of VH that bind CD137 Tg/TKO mice aged 8-12 weeks were immunised with human CD137-human Fc chimeric protein (Acro Biosystems cat no. 41B-H5258), human CD137-His tagged protein (R&D Systems, custom product), CHO cells over-expressing human CD137 (cell line produced in-house using standard methods) or a combination of recombinant protein and CHO human CD137 expressing cells. Serum was then collected from mice before and after immunisation and checked by ELISA for the presence of serum human CD137 reactive heavy chain antibodies in response to immunisation with CD137 antigen.


Generation of libraries from immunised mice described above followed standard protocols of library generation as summarised below. Tissue, including total spleen, inguinal and brachial lymph nodes was collected into RNAlater® from several immunised mice. Total RNA was extracted from supernatants and


VH sequences were mined from the RNA samples using Superscript III RT-PCR high-fidelity kit (Invitrogen cat. no. 12574-035) according to the manufacturer's protocol. Preparation of library phage stocks and phage display selections were performed according to published methods (Antibody Engineering, edited by Benny Lo, chapter 8, p 161-176, 2004). In most cases, phage display combined with a panning approach was used to isolate binding VH domains. However, a variety of different selection methods are well described in the art, including soluble protein selections, cell based selections and selections performed under stress (e.g., heat). Following selections of the libraries, specific VH that bound to CHO cells expressing human CD137, did not bind to CHO parental cells and inhibited the interaction between human CD137 expressed on the surface of CHO cells and recombinant human CD137 Ligand protein were identified by single point screening of bacterial periplasmic extracts. Binding of His-tagged VH in the supernatants to CHO human CD137 cells and to CHO parent cells for determination of non CD137 specific binding was assessed using Fluorescence Microvolume Assay Technology (FMAT). In parallel to the binding assay, periplasmic extracts were tested for their ability to inhibit the interaction of human CD137 ligand protein with CHO human CD137 cells in an FMAT format. Families of VH were identified that bound to the CHO human CD137 cells, did not bind CHO parental cells and that inhibited CD137 binding to CD137 Ligand. Each individual VH clone as identified above was sequenced from the phagemid and grouped based on VH germline and CDR3 amino acid similarity. Representative clones were further characterised. Further clones were generated by sequence optimisation of clone Humabody® VH 1.1 and Humabody® VH 2.1 respectively to improve binding activity, revert sequence to germline or remove biophysical sequence liabilities such as isomerisation or deamidation sites. Purified VH were obtained by using standard procedures. Table 2 shows the sequences of Family 1 Vs and table 3 those of Family 2 VHs.


Purified Humabody® VH were tested for binding to human CD137 protein, rhesus CD137Fc recombinant protein, mouse CD137 protein, tumour necrosis factor receptor family members OX40 and GITR (Glucocorticoid-induced TNFR-related), CHO human CD137 cells, CHO parent cells and human T-cells. VH bound to human and rhesus CD137 but not to mouse CD137 protein. Binding of serially diluted VH to CHO human CD137 cells and CHO parent cells were performed using an FMAT assay format. VH bound to CHO human CD137 expressing cells but did not bind to CHO parental cells. Binding of monovalent single domain antibodies to primary T cells was measured using flow cytometry. Humabody® VH as described herein bound to pre-stimulated CD8+ cells. The ability of purified Humabody VH to inhibit the binding of CD137 Ligand to CHO human CD137 cells was also measured in the FMAT ligand inhibition assay. VH inhibited the binding of human CD137 Ligand to human CD137. A functional assay was also carried out to assess the ability of monovalent VH that bind to CD137, to act as CD137 agonists was assessed in a reporter gene assay using Jurkat cells expressing CD137 and an NF-kB luciferase reporter gene. Their activity was compared to bivalent and trivalent molecules which have increased potential for avid interactions and to bispecific molecules consisting of CD137 VH linked to a VH that bound to the tumour antigen PSMA. In the bispecific molecule, CD137 agonism resulted from co-engagement of both CD137 and the cell expressed PSMA.


Example 2. Generation of Bispecific Constructs and Stability Testing

Multivalent constructs were generated by linking isolated Humabody® VH nucleic acid sequences using linkers of encoding glycine/serine rich sequences (G4S)x where x is the number of G4S repeats and ranges from 2 to 12 repeat units. DNA sequences encoding each Humabody® were amplified by PCR. The products were assembled into larger fragments with the VH single domain antibody sequences flanked by the (G4S)x linkers and ligated into an expression vector by a restriction enzyme-based method. Plasmids were transformed into microbial expression systems as per standard molecular biology techniques. The presence of inserts were verified by standard colony PCR technique and sequence confirmed by Sanger sequencing using vector-specific and internal primers to ensure complete sequence coverage. For generation of bispecific molecules the first and second sequence were not the same with one sequence corresponding to a Humabody® VH specific for CD137 and the other to a Humabody® that binds the tumour associated antigen PSMA. In another example monovalent and bispecific binding agents were optionally linked to a half-life extending Humabody® nucleic acid sequence (MSA binder). Bispecific and trispecific constructs were expressed by microbial cell systems. For stability testing purified bispecific and trispecific VH were subjected to size exclusion chromatography. Example data for representative constructs is shown in table 9.











TABLE 9









Timepoint (days)










Antibody

% Monomer by SEC 4° C.
% Monomer by SEC 40° C.

















clone
Conc.
1
4
7
14
0
1
4
7
14





4.1-6GS-2.1
5 mg/ml
99.44
99.37
99.41
99.38
99.44
99.14
98.95
98.90
98.64


3.8-6GS-1.1
5 mg/ml
98.58
98.53
98.55
98.52
98.58
97.54
96.83
96.63
92.66


4.1-6GS-1.1-
5 mg/ml
99.39
99.36
99.34
99.33
99.39
99.37
99.22
99.16
99.16


6GS-VH (MSA)









Examples of trispecific fusion proteins and nucleic acids encoding such fusion proteins are shown below. Examples of bivalent molecules are also shown below (table 10).














SEQ ID NO: 798 1.1-6GS-1.1 Protein


EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLEWVAHIKEDGSEKYYEDSVEGRFTVSRDNAKNSVYLQM


NSLRAEDTAVYYCARGGDGYSDSHFGVDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGL


VQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLEWVAHIKEDGSEKYYEDSVEGRFTVSRDNAKNSVYLQMNSLRAEDTAVY


YCARGGDGYSDSHFGVDVWGQGTTVTVSS





SEQ ID NO: 799 1.1-6GS-1.1 Nucleotide


GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


ACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATAAAGGAAGAC


GGAAGTGAGAAATACTATGAGGACTCTGTGGAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGAACTCGGTATATCTG


CAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGCTACAGTGACTCCCACTTC


GGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCAGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGAGG


TGGAGGTTCAGGAGGTGGTGGTTCTGGCGGCGGTGGATCGGGTGGAGGTGGTAGTGAGGTGCAGCTGGTGGAGTCTGGG


GGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCCATTGGATGACTT


GGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATAAAGGAAGACGGAAGTGAGAAATACTATGAGGAC


TCTGTGGAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGAACTCGGTATATCTGCAAATGAACAGTCTGAGAGCCGAA


GACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGCTACAGTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGG


ACCACGGTCACTGTCTCTTCA





SEQ ID NO: 800 2.1-6GS-2.1 Protein


EVQLVESGGGLVKPGGSLRVSCAASGFTFSDYYMSWFRQAPGKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQMNN


LRAEDTAVYHCAREDSRLTGTTDFDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKP


GGSLRVSCAASGFTFSDYYMSWFRQAPGKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQMNNLRAEDTAVYHCARE


DSRLTGTTDFDNWGQGTLVTVSS





SEQ ID NO: 801 2.1-6GS-2.1 Nucleotide


GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTCAAGCCTGGAGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTGGATTC


ACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTGGTAGTG


GTGATATCATAGACTACGCAGACTCTGTAAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCTCTGTATCTGCA


GATGAACAACCTGAGAGCCGAGGACACGGCCGTGTATCACTGTGCGAGAGAAGATTCCCGTCTAACTGGAACTACGGACTT


TGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGAGGTGGAG


GTTCAGGAGGTGGTGGTTCTGGCGGCGGTGGATCGGGTGGAGGTGGTAGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGG


CTTGGTCAAGCCTGGAGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGTTCC


GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTGGTAGTGGTGATATCATAGACTACGCAGACTCTGTAA


AGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCTCTGTATCTGCAGATGAACAACCTGAGAGCCGAGGACACGG


CCGTGTATCACTGTGCGAGAGAAGATTCCCGTCTAACTGGAACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCAC


CGTCTCCTCA





SEQ ID NO: 802 1.1-6GS-1.1-6GS-1.1 Protein


EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLEWVAHIKEDGSEKYYEDSVEGRFTVSRDNAKNSVYLQM


NSLRAEDTAVYYCARGGDGYSDSHFGVDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGL


VQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLEWVAHIKEDGSEKYYEDSVEGRFTVSRDNAKNSVYLQMNSLRAEDTAVY


YCARGGDGYSDSHFGVDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSC


AASGFTFSSHWMTWFRQAPGKGLEWVAHIKEDGSEKYYEDSVEGRFTVSRDNAKNSVYLQMNSLRAEDTAVYYCARGGDGYS


DSHFGVDVWGQGTTVTVSS





SEQ ID NO: 803 1.1-6GS-1.1-6GS-1.1 Nucleotide


GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


ACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATAAAGGAAGAC


GGAAGTGAGAAATACTATGAGGACTCTGTGGAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGAACTCGGTATATCTG


CAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGCTACAGTGACTCCCACTTC


GGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCAGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGAGG


TGGAGGTTCAGGAGGTGGTGGTTCTGGCGGCGGTGGATCGGGTGGAGGTGGTAGTGAGGTGCAGCTGGTGGAGTCTGGG


GGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCCATTGGATGACTT


GGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATAAAGGAAGACGGAAGTGAGAAATACTATGAGGAC


TCTGTGGAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGAACTCGGTATATCTGCAAATGAACAGTCTGAGAGCCGAA


GACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGCTACAGTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGG


ACCACGGTCACTGTCTCTTCAGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGAGGTGGAGGTTCAGGAGGTGGTGGTTCT


GGCGGCGGTGGATCGGGTGGAGGTGGTAGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGT


CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGG


CTGGAGTGGGTGGCCCACATAAAGGAAGACGGAAGTGAGAAATACTATGAGGACTCTGTGGAGGGCCGATTCACCGTCTCC


AGAGACAACGCCAAGAACTCGGTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGA


GGAGGTGATGGCTACAGTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA





SEQ ID NO: 804 2.1-6GS-2.1-6GS-2.1 Protein





EVQLVESGGGLVKPGGSLRVSCAASGFTFSDYYMSWFRQAPGKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQMNN


LRAEDTAVYHCAREDSRLTGTTDFDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKP


GGSLRVSCAASGFTFSDYYMSWFRQAPGKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQMNNLRAEDTAVYHCARE


DSRLTGTTDFDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRVSCAASGFTF


SDYYMSWFRQAPGKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQMNNLRAEDTAVYHCAREDSRLTGTTDFDNWG


QGTLVTVSS





SEQ ID NO: 805 2.1-6GS-2.1-6GS-2.1 Nucleotide


GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTGGATTC


ACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTGGTAGTG


GTGATATCATAGACTACGCAGACTCTGTAAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCTCTGTATCTGCA


GATGAACAACCTGAGAGCCGAGGACACGGCCGTGTATCACTGTGCGAGAGAAGATTCCCGTCTAACTGGAACTACGGACTT


TGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGAGGTGGAG


GTTCAGGAGGTGGTGGTTCTGGCGGCGGTGGATCGGGTGGAGGTGGTAGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGG


CTTGGTCAAGCCTGGAGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGTTCC


GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTGGTAGTGGTGATATCATAGACTACGCAGACTCTGTAA


AGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCTCTGTATCTGCAGATGAACAACCTGAGAGCCGAGGACACGG


CCGTGTATCACTGTGCGAGAGAAGATTCCCGTCTAACTGGAACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCAC


CGTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGAGGTGGAGGTTCAGGAGGTGGTGGTTCTGGCGGCGGTG


GATCGGGTGGAGGTGGTAGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGAGTC


TCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGG


TTTCATACATTAGTGGTAGTGGTGATATCATAGACTACGCAGACTCTGTAAAGGGCCGATTCACCATCTCCAGGGACAACGCC


AAGAACTCTCTGTATCTGCAGATGAACAACCTGAGAGCCGAGGACACGGCCGTGTATCACTGTGCGAGAGAAGATTCCCGTC


TAACTGGAACTACGGACTTTGACAATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA





SEQ ID NO. 843 4.1-6GS-1.1-6GS-VH (MSA) Nucleotide


GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


TCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATG


GAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCA


AATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTC


ATCGTCCTATGATTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGC


TCTGGAGGTGGAGGTTCAGGAGGTGGTGGTTCTGGCGGCGGTGGATCGGGTGGAGGTGGTAGTGAGGTGCAGCTGGTGG


AGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCCATTG


GATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCCACATAAAGGAAGACGGAAGTGAGAAATACT


ATGAGGACTCTGTGGAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGAACTCGGTATATCTGCAAATGAACAGTCTGAG


AGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAGGTGATGGCTACAGTGACTCCCACTTCGGTGTGGACGTCTGGGG


CCAAGGGACCACGGTCACTGTCTCTTCAGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGAGGTGGAGGTTCAGGAGGTG


GTGGTTCTGGCGGCGGTGGATCGGGTGGAGGTGGTAGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTGGTACAGCC


GGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCA


GGGAAGGGGCTGGAGTGGGTCGCAACTATTAGTGATAGTGGTAGTAGTGCAGACTACGCAGATTCCGTGAAGGGACGGTT


CACCATCTCCAGAGACAACTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCTGAAGACACGGCCGTGTATTAC


TGTGCGAGAGGCCGGTATAACTGGAACCCCCGAGCTTTGGGTATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCA





SEQ ID NO. 844 4.1-6GS-1.1-6GS-MSA Protein





EVQLVESGGGVVQPGRSLRLSCAASGFSFSGYGMHWVRQAPGKGLEWVAYISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMN


SLRAEDTAVYYCAKDPAWGLRLGESSSYDFDIWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG


GGLVQPGGSLRLSCAASGFTFSSHWMTWFRQAPGKGLEWVAHIKEDGSEKYYEDSVEGRFTVSRDNAKNSVYLQMNSLRAEDT


AVYYCARGGDGYSDSHFGVDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSL


RLSCAASGFTFSSYAMSWVRQAPGKGLEWVATISDSGSSADYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRYN


WNPRALGIWGQGTMVTVSS





SEQ ID NO. 845 4.1-6GS-1.1-6GS-MSA Nucleotide


GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


TCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATG


GAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCA


AATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTC


ATCGTCCTATGATTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGC


TCTGGAGGTGGAGGTTCAGGAGGTGGTGGTTCTGGCGGCGGTGGATCGGGTGGAGGTGGTAGTGAGGTGCAGCTGGTGG


AGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA


CATGAGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTGGTAGTGGTGATATCATAGACTA


CGCAGACTCTGTAAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCTCTGTATCTGCAGATGAACAACCTGAGA


GCCGAGGACACGGCCGTGTATCACTGTGCGAGAGAAGATTCCCGTCTAACTGGAACTACGGACTTTGACAATTGGGGCCAG


GGAACCCTGGTCACCGTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGAGGTGGAGGTTCAGGAGGTGGTGG


TTCTGGCGGCGGTGGATCGGGTGGAGGTGGTAGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCGGGG


GGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGA


AGGGGCTGGAGTGGGTCGCAACTATTAGTGATAGTGGTAGTAGTGCAGACTACGCAGATTCCGTGAAGGGACGGTTCACCA


TCTCCAGAGACAACTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCTGAAGACACGGCCGTGTATTACTGTGC


GAGAGGCCGGTATAACTGGAACCCCCGAGCTTTGGGTATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCA





SEQ ID NO. 846 4.1-6GS-2.1-6GS-MSA Protein





EVQLVESGGGVVQPGRSLRLSCAASGFSFSGYGMHWVRQAPGKGLEWVAYISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMN


SLRAEDTAVYYCAKDPAWGLRLGESSSYDFDIWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG


GGLVKPGGSLRVSCAASGFTFSDYYMSWFRQAPGKGLEWVSYISGSGDIIDYADSVKGRFTISRDNAKNSLYLQMNNLRAEDTAV


YHCAREDSRLTGTTDFDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSC


AASGFTFSSYAMSWVRQAPGKGLEWVATISDSGSSADYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRYNWNPR


ALGIWGQGTMVTVSS





SEQ ID NO. 847 3.8-6GS-1.1-6GS-MSA nucleotide


GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCICTGGATTC


AGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAAT


GATGGTACCACAGACTACGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTATCTGC


AAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGG


TCACCGTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGAGGTGGAGGTTCAGGAGGTGGTGGTTCTGGCGGC


GGTGGATCGGGTGGAGGTGGTAGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGA


GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCCATTGGATGACTTGGTTCCGTCAGGCTCCAGGGAAGGGGCTGGA


GTGGGTGGCCCACATAAAGGAAGACGGAAGTGAGAAATACTATGAGGACTCTGTGGAGGGCCGATTCACCGTCTCCAGAG


ACAACGCCAAGAACTCGGTATATCTGCAAATGAACAGTCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGAG


GTGATGGCTACAGTGACTCCCACTTCGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCAGGTGGTGGCG


GTTCAGGCGGAGGTGGCTCTGGAGGTGGAGGTTCAGGAGGTGGTGGTTCTGGCGGCGGTGGATCGGGTGGAGGTGGTAG


TCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATT


CACCTTTAGTAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGCAACTATTAGTGATAG


TGGTAGTAGTGCAGACTACGCAGATTCCGTGAAGGGACGGTTCACCATCTCCAGAGACAACTCCAAGAACACGCTGTATCTT


CAAATGAACAGCCTGAGAGCTGAAGACACGGCCGTGTATTACTGTGCGAGAGGCCGGTATAACTGGAACCCCCGAGCTTTG


GGTATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCA





SEQ ID NO. 848 3.8-6GS-1.1-6GS-MSA Protein


EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYALSWVRQAPGKGLEWVSSTGENDGTTDYADAVKGRFTISRDNSKNTLYLQMNS


LRVETAVYYCVKDGVHWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCA


ASGFTFSSHWMTWFRQAPGKGLEWVAHIKEDGSEKYYEDSVEGRFTVSRDNAKNSVYLQMNSLRAEDTAVYYCARGGDGYSDS


HFGVDVWGQGTIVIVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAM


SWVRQAPGKGLEWVATISDSGSSADYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRYNWNPRALGIWGQGTM


VTVSS





SEQ ID NO. 889 4.1-6GS-1.113-4G5 Nucleotide


GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


TCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATG


GAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCA


AATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTC


ATCGTCCTATGATTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGC


TCTGGAGGTGGAGGTTCAGGAGGTGGTGGTTCTGGCGGCGGTGGATCGGGTGGAGGTGGTAGTGAGGTGCAGTTAGTTGA


GAGCGGAGGTGGTTTAGTTCAGCCGGGGGGCTCGCTTCGCCTGTCGTGCGCCGCCTCGGGATTCACATTATCAAACTACTGG


ATGAATTGGGTCCGCCAGGCTCCGGGCAAAGGTCTTGAGTGGGTGGCGAACATTAATCAGGACGGGAGCGAGCGTTATTAC


GTTGATTCGGTAAAAGGACGTTTCACTATCAGTCGTGACAACGCTAAAAATTCCTTGTACTTACAGATGAACTCACTTCGTGC


TGAGGACACCGCAGTGTACTACTGTGCTCGCGGTGGTGAAGGATACGGCGTCGATCACTACGGCCTTGATGTATCAGGACA


GGGGACTACAGTTACCGTCTCTTCCGGCGGAGGTGGCTCTGGAGGAGGAGGTTCAGGAGGTGGTGGATCTGGCGGCGGTG


GTAGT





SEQ ID NO. 890 4.1-6GS-1.113-4G5 Protein


EVQLVESGGGVVQPGRSLRLSCAASGFSFSGYGMHWVRQAPGKGLEWVAYISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMN


SLRAEDTAVYYCAKDPAWGLRLGESSSYDFDIWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG


GGLVQPGGSLRLSCAASGFTLSNYWMNWVRQAPGKGLEWVANINQDGSERYYDSVKGRFTISRDNAKNSLYLQMNSLRAEDTA


VYYCARGGEGYGVDHYGLDVSGQGTTVTVSSGGGGSGGGGSGGGGSGGGG





SEQ ID NO. 891 3.8-6GS-1.113-4G5 Nucleotide


GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCICTGGATTC


AGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAAT


GATGGTACCACAGACTACGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTATCTGC


AAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGG


TCACCGTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGAGGTGGAGGTTCAGGAGGTGGTGGTTCTGGCGGC


GGTGGATCGGGTGGAGGTGGTAGTGAGGTGCAGTTAGTTGAGAGCGGAGGTGGTTTAGTTCAGCCGGGGGGCTCGCTTCG


CCTGTCGTGCGCCGCCTCGGGATTCACATTATCAAACTACTGGATGAATTGGGTCCGCCAGGCTCCGGGCAAAGGTCTTGAG


TGGGTGGCGAACATTAATCAGGACGGGAGCGAGCGTTATTACGTTGATTCGGTAAAAGGACGTTTCACTATCAGTCGTGACA


ACGCTAAAAATTCCTTGTACTTACAGATGAACTCACTTCGTGCTGAGGACACCGCAGTGTACTACTGTGCTCGCGGTGGTGAA


GGATACGGCGTCGATCACTACGGCCTTGATGTATCAGGACAGGGGACTACAGTTACCGTCTCTTCCGGCGGAGGTGGC


TCTGGAGGAGGAGGTTCAGGAGGTGGTGGATCTGGCGGCGGTGGTAGT





SEQ ID NO. 892 3.8-6GS-1.113-4G5 Protein


EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYALSWVRQAPGKGLEWVSSTGENDGTTDYADAVKGRFTISRDNSKNTLYLQMNS


LRVEDTAVYYCVKDGVHWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCA


ASGFTLSNYWMNWVRQAPGKGLEWVANINQDGSERYYVDSVKGRFTISRDNAKN


SLYLQMNSLRAEDTAVYYCARGGEGYGVDHYGLDVSGQGTTVTVSSGGGGSGGGGSGGGGSGGGGS





SEQ ID NO. 893 4.1-6GS-1.113-6GS-VH (HSA)-4G5 Nucleotide


GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


TCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATG


GAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCA


AATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTC


ATCGTCCTATGATTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGC


TCTGGAGGTGGAGGTTCAGGAGGTGGTGGTTCTGGCGGCGGTGGATCGGGTGGAGGTGGTAGTGAGGTGCAGTTAGTTGA


GAGCGGAGGTGGTTTAGTTCAGCCGGGGGGCTCGCTTCGCCTGTCGTGCGCCGCCTCGGGATTCACATTATCAAACTACTGG


ATGAATTGGGTCCGCCAGGCTCCGGGCAAAGGTCTTGAGTGGGTGGCGAACATTAATCAGGACGGGAGCGAGCGTTATTAC


GTTGATTCGGTAAAAGGACGTTTCACTATCAGTCGTGACAACGCTAAAAATTCCTTGTACTTACAGATGAACTCACTTCGTGC


TGAGGACACCGCAGTGTACTACTGTGCTCGCGGTGGTGAAGGATACGGCGTCGATCACTACGGCCTTGATGTATCAGGACA


GGGGACTACAGTTACCGTCTCTTCCGGCGGAGGTGGCTCTGGAGGAGGCGGATCGGGGGGTGGAGGAAGTGGCGGCGGT


GGTAGTGGAGGAGGTGGTTCTGGAGGCGGTGGCTCTGAAGTACAACTGGTTGAATCGGGTGGTGGATTGGTCCAACCTGG


AAGATCATTGAGGCTTTCTTGTGCAGCTTCCGGATTCACCTTTCATCACTATGCTATGCACTGGGTGAGACAAGCCCCTGGTA


AGGGCTTGGAATGGGTGTCCGGAATCTCCTGGAATGGTAACAAAATAACATATGCAGATTCCGTTAAGGGTAGATTTACTAT


TAGCCGTGATAATGCAAAAAACAGTTTATACTTGCAGATGAATTCCTTGAGGGCTGAGGATACAGCTCTTTACTATTGTGTGC


GTGACTCATCGTTGTTCATTGTCGGAGCCCCAACTTTCGAACATTGGGGTAGAGGTACCCTAGTTACGGTTAGCTCAGGCGG


AGGTGGCTCTGGAGGAGGCGGATCGGGGGGTGGAGGAAGTGGCGGCGGTGGTAGT





SEQ ID NO. 894 4.1-6GS-1.113-6GS-VH (HSA)-4G5 Protein


EVQLVESGGGVVQPGRSLRLSCAASGFSFSGYGMHWVRQAPGKGLEWVAYISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMN


SLRAEDTAVYYCAKDPAWGLRLGESSSYDFDIWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG


GGLVQPGGSLRLSCAASGFTLSNYWMNWVRQAPGKGLEWVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDT


AVYYCARGGEGYGVDHYGLDVSGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLR


LSCAASGFTFHHYAMHWVRQAPGKGLEWVSGISWNGNKITYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCVRDSSLFI


VGAPTFEHWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGS





SEQ ID NO. 895 3.8-6GS-VH (HSA)-1.113-4G5 Nucleotide


GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


AGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCAAGTATTGGTGAGAAT


GATGGTACCACAGACTACGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTATCTAC


AAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGG


TCACCGTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGAGGTGGAGGTTCAGGAGGTGGTGGTTCTGGCGGC


GGTGGATCGGGTGGAGGTGGTAGTGAAGTACAACTGGTTGAATCGGGTGGTGGATTGGTCCAACCTGGAAGATCATTGAG


GCTTTCTTGTGCAGCTTCCGGATTCACCTTTCATCACTATGCTATGCACTGGGTGAGACAAGCCCCTGGTAAGGGCTTGGAAT


GGGTGTCCGGAATCTCCTGGAATGGTAACAAAATAACATATGCAGATTCCGTTAAGGGTAGATTTACTATTAGCCGTGATAA


TGCAAAAAACAGTTTATACTTGCAGATGAATTCCTTGAGGGCTGAGGATACAGCTCTTTACTATTGTGTGCGTGACTCATCGT


TGTTCATTGTCGGAGCCCCAACTTTCGAACATTGGGGTAGAGGTACCCTAGTTACGGTTAGCTCAGGCGGAGGTGGCTCTGG


AGGAGGAGGTTCAGGAGGTGGTGGATCTGGAGGAGGCGGATCGGGGGGTGGAGGAAGTGGCGGCGGTGGTAGTGAGGT


GCAGTTAGTTGAGAGCGGAGGTGGTTTAGTTCAGCCGGGGGGCTCGCTTCGCCTGTCGTGCGCCGCCTCGGGATTCACATTA


TCAAACTACTGGATGAATTGGGTCCGCCAGGCTCCGGGCAAAGGTCTTGAGTGGGTGGCGAACATTAATCAGGACGGGAGC


GAGCGTTATTACGTTGATTCGGTAAAAGGACGTTTCACTATCAGTCGTGACAACGCTAAAAATTCCTTGTACTTACAGATGAA


CTCACTTCGTGCTGAGGACACCGCAGTGTACTACTGTGCTCGCGGTGGTGAAGGATACGGCGTCGATCACTACGGCCTTGAT


GTATCAGGACAGGGGACTACAGTTACCGTCTCTTCCGGCGGAGGTGGCTCTGGAGGAGGCGGATCGGGGGGTGGAGGAAG


TGGCGGCGGTGGTAGTGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCT


GTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTC


AAGTATTGGTGAGAATGATGGTACCACAGACTACGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAA


GAATACGCTGTATCTACAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGAAAGATGGTGTCCACTGG


GGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGAGGTGGAGGTTCAGGAGG


TGGTGGTTCTGGCGGCGGTGGATCGGGTGGAGGTGGTAGTGAAGTACAACTGGTTGAATCGGGTGGTGGATTGGTCCAAC


CTGGAAGATCATTGAGGCTTTCTTGTGCAGCTTCCGGATTCACCTTTCATCACTATGCTATGCACTGGGTGAGACAAGCCCCT


GGTAAGGGCTTGGAATGGGTGTCCGGAATCTCCTGGAATGGTAACAAAATAACATATGCAGATTCCGTTAAGGGTAGATTTA


CTATTAGCCGTGATAATGCAAAAAACAGTTTATACTTGCAGATGAATTCCTTGAGGGCTGAGGATACAGCTCTTTACTATTGT


GTGCGTGACTCATCGTTGTTCATTGTCGGAGCCCCAACTTTCGAACATTGGGGTAGAGGTACCCTAGTTACGGTTAGCTCAGG


CGGAGGTGGCTCTGGAGGAGGAGGTTCAGGAGGTGGTGGATCTGGAGGAGGCGGATCGGGGGGTGGAGGAAGTGGCGG


CGGTGGTAGTGAGGTGCAGTTAGTTGAGAGCGGAGGTGGTTTAGTTCAGCCGGGGGGCTCGCTTCGCCTGTCGTGCGCCGC


CTCGGGATTCACATTATCAAACTACTGGATGAATTGGGTCCGCCAGGCTCCGGGCAAAGGTCTTGAGTGGGTGGCGAACATT


AATCAGGACGGGAGCGAGCGTTATTACGTTGATTCGGTAAAAGGACGTTTCACTATCAGTCGTGACAACGCTAAAAATTCCT


TGTACTTACAGATGAACTCACTTCGTGCTGAGGACACCGCAGTGTACTACTGTGCTCGCGGTGGTGAAGGATACGGCGTCGA


TCACTACGGCCTTGATGTATCAGGACAGGGGACTACAGTTACCGTCTCTTCCGGCGGAGGTGGCTCTGGAGGAGGCGGATC


GGGGGGTGGAGGAAGTGGCGGCGGTGGTAGT





SEQ ID NO. 896 3.8-6GS-VH (HSA)-1.113-4G5 Protein


EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYALSWVRQAPGKGLEWVSSTGENDGTTDYADAVKGRFTISRDNSKNTLYLQMNS


LRVEDTAVYYCVKDGVHWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCA


ASGFTFHHYAMHWVRQAPGKGLEWVSGISWNGNKITYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCVRDSSLFIVGA


PTFEHWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYWMN


WVRQAPGKGLEWVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGEGYGVDHYGLDVSGQGT


TVTVSSGGGGSGGGGSGGGGSGGGGS





SEQ ID NO. 897 3.8-6GS-1.113-6GS-VH (HSA)-4G5 Nucleotide


GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCICTGGATTC


AGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCAAGTATTGGTGAGAAT


GATGGTACCACAGACTACGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTATCTAC


AAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTGAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGG


TCACCGTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGAGGTGGAGGTTCAGGAGGTGGTGGTTCTGGCGGC


GGTGGATCGGGTGGAGGTGGTAGTGAGGTGCAGTTAGTTGAGAGCGGAGGTGGTTTAGTTCAGCCGGGGGGCTCGCTTCG


CCTGTCGTGCGCCGCCTCGGGATTCACATTATCAAACTACTGGATGAATTGGGTCCGCCAGGCTCCGGGCAAAGGTCTTGAG


TGGGTGGCGAACATTAATCAGGACGGGAGCGAGCGTTATTACGTTGATTCGGTAAAAGGACGTTTCACTATCAGTCGTGACA


ACGCTAAAAATTCCTTGTACTTACAGATGAACTCACTTCGTGCTGAGGACACCGCAGTGTACTACTGTGCTCGCGGTGGTGAA


GGATACGGCGTCGATCACTACGGCCTTGATGTATCAGGACAGGGGACTACAGTTACCGTCTCTTCCGGCGGAGGTGGCTCTG


GAGGAGGCGGATCGGGGGGTGGAGGAAGTGGCGGCGGTGGTAGTGGAGGAGGTGGTTCTGGAGGCGGTGGCTCTGAAG


TACAACTGGTTGAATCGGGTGGTGGATTGGTCCAACCTGGAAGATCATTGAGGCTTTCTTGTGCAGCTTCCGGATTCACCTTT


CATCACTATGCTATGCACTGGGTGAGACAAGCCCCTGGTAAGGGCTTGGAATGGGTGTCCGGAATCTCCTGGAATGGTAACA


AAATAACATATGCAGATTCCGTTAAGGGTAGATTTACTATTAGCCGTGATAATGCAAAAAACAGTTTATACTTGCAGATGAAT


TCCTTGAGGGCTGAGGATACAGCTCTTTACTATTGTGTGCGTGACTCATCGTTGTTCATTGTCGGAGCCCCAACTTTCGAACAT


TGGGGTAGAGGTACCCTAGTTACGGTTAGCTCAGGCGGAGGTGGCTCTGGAGGAGGCGGATCGGGGGGTGGAGGAAGTG


GCGGCGGTGGTAGT





SEQ ID NO. 898 Protein 3.8-6GS-1.113-6GS-VH (HSA)-4G5


EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYALSWVRQAPGKGLEWVSSTGENDGTTDYADAVKGRFTISRDNSKNTLYLQMNS


LRVEDTAVYYCVKDGVHWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCA


ASGFTLSNYWMNWVRQAPGKGLEWVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGEGYGV


DHYGLDVSGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFHHYA


MHWVRQAPGKGLEWVSGISWNGNKITYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCVRDSSLFIVGAPTFEHWGRG


TLVTVSSGGGGSGGGGSGGGGSGGGGS





SEQ ID NO. 899 4.1-6GS-VH (HSA)-1.113-4G5 Nucleotide


GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC


TCCTTCAGTGGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATG


GAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCA


AATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTC


ATCGTCCTATGATTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGC


TCTGGAGGTGGAGGTTCAGGAGGTGGTGGTTCTGGCGGCGGTGGATCGGGTGGAGGTGGTAGTGAAGTACAACTGGTTGA


ATCGGGTGGTGGATTGGTCCAACCTGGAAGATCATTGAGGCTTTCTTGTGCAGCTTCCGGATTCACCTTTCATCACTATGCTA


TGCACTGGGTGAGACAAGCCCCTGGTAAGGGCTTGGAATGGGTGTCCGGAATCTCCTGGAATGGTAACAAAATAACATATG


CAGATTCCGTTAAGGGTAGATTTACTATTAGCCGTGATAATGCAAAAAACAGTTTATACTTGCAGATGAATTCCTTGAGGGCT


GAGGATACAGCTCTTTACTATTGTGTGCGTGACTCATCGTTGTTCATTGTCGGAGCCCCAACTTTCGAACATTGGGGTAGAGG


TACCCTAGTTACGGTTAGCTCAGGCGGAGGTGGCTCTGGAGGAGGAGGTTCAGGAGGTGGTGGATCTGGAGGAGGCGGAT


CGGGGGGTGGAGGAAGTGGCGGCGGTGGTAGTGAGGTGCAGTTAGTTGAGAGCGGAGGTGGTTTAGTTCAGCCGGGGGG


CTCGCTTCGCCTGTCGTGCGCCGCCTCGGGATTCACATTATCAAACTACTGGATGAATTGGGTCCGCCAGGCTCCGGGCAAA


GGTCTTGAGTGGGTGGCGAACATTAATCAGGACGGGAGCGAGCGTTATTACGTTGATTCGGTAAAAGGACGTTTCACTATCA


GTCGTGACAACGCTAAAAATTCCTTGTACTTACAGATGAACTCACTTCGTGCTGAGGACACCGCAGTGTACTACTGTGCTCGC


GGTGGTGAAGGATACGGCGTCGATCACTACGGCCTTGATGTATCAGGACAGGGGACTACAGTTACCGTCTCTTCCGGCGGA


GGTGGCTCTGGAGGAGGCGGATCGGGGGGTGGAGGAAGTGGCGGCGGTGGTAGT





SEQ ID NO. 900 4.1-6GS-VH (HSA)-1.113-4G5 Protein





EVQLVESGGGVVQPGRSLRLSCAASGFSFSGYGMHWVRQAPGKGLEWVAYISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMN


SLRAEDTAVYYCAKDPAWGLRLGESSSYDFDIWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG


GGLVQPGRSLRLSCAASGFTFHHYAMHWVRQAPGKGLEWVSGISWNGNKITYADSVKGRFTISRDNAKNSLYLQMNSLRAEDT


ALYYCVRDSSLFIVGAPTFEHWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS


CAASGFTLSNYWMNWVRQAPGKGLEWVANINQDGSERYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGEGY


GVDHYGLDVSGQGTTVTVSSGGGGSGGGGSGGGGSGGGGS





SEQ ID NO. 901 VH that binds to HSA as used in constructs above protein


EVQLVESGGGLVQPGRSLRSCAASGFTFHHYAMHWVRQAPGKGLEWVSGISWNGNKITYADSVKGRFTISRDNAKNSLYLQMN


SLRAEDTALYYCVRDSSLFIVGAPTFEHWGRGTLVTVSS








SEQ ID NO. 902 VH that binds to HSA as used in constructs above nucleotide


GGCTTTGTGAGCGGATACAATTATAATATGTGGAATTGTGAGCGCTCACAATTCCACAACGGTTTCCCTCTAGAAATAAT


TTTGTTTAACTTTTAGGAGGTAAAACATATGAAGAAAACGGCAATCGCAATCGCAGTCGCTCTGGCGGGTTTCGCAACTG


TAGCGCAAGCCGAGGTGCAACTGGTCGAGTCTGGTGGTGGTTTGGTGCAACCTGGTAGAAGCTTGCGTTTGAGTTGTGCC


GCTTCCGGCTTCACTTTCCATCATTATGCTATGCACTGGGTTCGTCAAGCTCCCGGAAAAGGTTTGGAGTGGGTTTCCGG


AATTTCCTGGAATGGCAATAAGATTACGTACGCTGATTCAGTGAAAGGAAGGTTTACAATCAGTAGAGATAATGCTAAAA


ACTCATTGTATCTACAAATGAACAGCCTAAGAGCAGAAGATACCGCTCTGTACTACTGTGTTAGAGATAGCTCGTTATTC


ATTGTAGGTGCACCAACTTTTGAACATTGGGGTCGGGGTACTCTTGTGACTGTCTCATCCGCGGCCGCACACCACCATCA


TCACCACTAACTCGAGCGCCTAATGAAAGCTTCCCCAAGGGCGACACCCCCTAATTAGCCCGGGCGAAAGGCCCAGTCTT


TCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCAGTTCCCTACTCTCGCATGGGGAGTCCCCACACTACCATCGGCGCT


ACGGCGTTTCACTTCTGAGTTCGGCATGGA









Example 3: Binding to CD137 and PSMA

3.1 Cell Binding


Binding of His-tagged molecules to CHO human CD137, CHO parent, CHO human PSMA, DU145 PSMA and DU145 parent cells was assessed using Fluorescence Microvolume Assay Technology (FMAT). All reagents were prepared in FMAT assay buffer (pH 7.4) containing PBS, 0.1% Bovine Serum Albumin, 0.05% Sodium Azide. Serially diluted samples were transferred into 384 well black clear-bottomed assay plates (Costar cat. no. 3655) and incubated for a minimum of 2 hours at room temperature with 1.5 nM Anti-His (Millipore cat. no. 05-949), 3 nM Goat Anti-Mouse Alexa Fluor-488 (Jackson ImmunoResearch cat. no. 115-545-071) and 2000 cells/well pre-stained with DRAQ5 (Thermo Scientific cat. no. 62251). Fluorescence emission was then measured on the TTP Mirrorball plate reader in the FL2 (502 nm-537 nm) and FL5 (677-800 nm) channels following excitation at 488 nm and 640 nm. Data was gated on FL5 perimeter and peak intensity and the FL2 median mean fluorescence intensity of the gated data used for determination of VH binding. Example EC50 values for binding are shown in table 11. Monovalent CD137 specific Humabody® VH, bispecific and trispecific molecules with a CD137 binding arm bound to CHO CD137 expressing cells. Monovalent PSMA specific Humabody® VH, bispecific and trispecific molecules with a PSMA binding arm bound to PSMA expressing cells.











TABLE 11








FMAT
Flow














CHO
CHO
CHO
DU145
DU145
Cytometry


Humabody ®
huCD137
Parent
PSMA
PSMA
Parent
CD8+
















1.78
3.2E−10




6.1E−10


1.1
2.2E−10




2.2E−10


2.1
6.5E−10




2.4E−09


4.1


1.7E−10
1.9E−10

nd


3.8


2.3E−10
2.9E−10

nd


4.1-6GS-1.1
4.3E−10

1.9E−10
2.1E−10

7.3E−10


4.1-6GS-2.1
9.7E−10

2.8E−10
3.9E−10

3.0E−09


3.8-6GS-1.1
5.6E−10

6.0E−10
6.8E−10

1.1E−09


1.1-6GS-3.8
5.6E−10

6.3E−10
8.1E−10

nd


4.1-6GS-1.1-
5.8E−10

3.2E−10
3.6E−10

nd


VH (MSA)








4.1-6GS-1.1-
1.9E−09

3.0E−10
4.2E−10

nd


VH (MSA)








3.8-6GS-1.1-
5.9E−10

5.9E−10
8.2E−10

nd


VH (MSA)









Binding of monovalent single domain antibodies to primary T cells was measured using flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated from human blood by density gradient centrifugation then CD8+ T cells purified using a negative selection isolation kit according to the manufacturer's protocol (Miltenyi Biotech cat no 130-042-401). CD8+ T cells were stimulated PMA/lonomycin for 48-72 hours in RPMI media supplemented with 10% FBS, 2 mM Glutamine, 1× Pen/Strep. Cells were transferred into 96 well plates, blocked for 10 mins with staining buffer (PBS/1% BSA/0.05% Sodium Azide) then incubated with serially diluted VH in staining buffer (PBS/1% BSA) for 30 mins-1 hour at 4° C. Cells were washed by centrifugation then VH binding detected using Anti His antibody (Millipore 05-949) and Goat Anti Mouse Alexa Fluor-488 (Jackson ImmunoResearch cat no. 115-545-071). A Live Dead near IR stain (Molecular Probes cat no. L10119) was used for discrimination of live cells. After further washing cells were fixed and fluorescence measured by flow cytometry. Average EC50 values for binding (2-3 donors) are shown in Table 9. Monovalent CD137 specific Humabody® VH and bispecific molecules with a CD137 binding arm bound to pre-stimulated CD8+ cells.


3.2 Affinity Kinetics


Binding kinetics of purified monovalent VH, and PSMA-CD137 targeting bispecific molecules were determined on a ForteBio Octet RED 384 instrument. To study the interaction with the antigens, CD137-Fc tag protein (Acro Biosystems cat no. 41B-H5258) or PSMA-his (R&D Systems cat no. 4234-ZN) was immobilised onto AR2G biosensors (ForteBio cat no. 18-5082) by amine coupling. Monovalent VH and bispecific molecules were serially diluted (typically 1:2 dilution series starting between 12-25 nM, at the highest concentration) in kinetics buffer (0.1% BSA, 0.02% Tween, 1×PBS) and binding to the immobilised proteins was studied during the association and dissociation phases. PSMA binding was measured using 180 seconds association and 600 seconds dissociation phases. CD137 binding was measured 180 seconds association and 600 seconds dissociation phases. Reference subtracted data were fitted to a 1:1 binding model using the ForteBio Octet Data Analysis software. Example kinetic and binding affinity data obtained are shown in Table 12.











TABLE 12








Human CD137
Human PSMA















Kon


kon



Humabody ®
KD (M)
(1/Ms)
kdis (1/s)
KD (M)
(1/Ms)
kdis (1/s)





1.1
5.2E−10
7.1E+05
3.6E−04





2.1
6.7E−09
7.7E+05
5.2E−03





4.1



5.3E−10
3.1E+05
1.6E−04


3.8



9.0E−10
3.9E+05
3.5E−04


4.1-6GS-1.1
7.3E−10
4.1E+05
3.1E−04
1.3E−09
4.3E+05
5.4E−04


4.1-6GS-2.1
4.8E−09
5.8E+05
2.6E−03
6.6E−10
5.0E+05
3.3E−04


3.8-6GS-1.1
5.6E−10
4.9E+05
2.9E−04
5.7E−09
3.0E+05
1.7E−03









The Biacore T200 instrument was used to study the interaction between VH with human and rhesus CD137-human IgG1 Fc tagged protein by surface plasmon resonance (SPR). Single cycle kinetics assays used to evaluate the kinetics and affinity of the interaction. Experiments were performed at 25° C. in HBS-EP+ assay buffer with a flow rate of 30 μl/minute. A Protein G chip was used to capture the Fc tagged recombinant CD137 diluted to 2 μg/ml to one of the flow cells over 7 seconds. A second flow cell without any captured CD137 was used as the reference cell. A five point, three-fold dilution series of VH was made with a top concentration of 60 nM. The binding kinetics were followed by flowing these over the chip surface. The contact time for each of the binding steps was 180 seconds and the dissociation step was 1800 and 3600 seconds for rhesus and human CD137 respectively. After each run, the sensors were regenerated with glycine pH 1.5 to remove the captured CD137. The data was fitted to a 1:1 binding model after double reference subtraction using the Biacore T200 Evaluation software. Average kinetic constants (±Standard deviation) for Humabody® 1.113 for binding to human CD137Fc were ka 3.6E+06±1.6E+06 (1/Ms), Kd is 3.0E-04±1.1E-04 (1/s) and KD 8.5E-11±7.8E-12 (M) and for binding to rhesus CD137Fc were ka 1.1E+06±2.2E+05 (1/Ms), Kdis 2.8E-04±6.8E-06 (1/s) and KD 2.7E-10±5.2E-11. Average kinetic constants (±Standard deviation) for construct 4.1-6GS-1.113-6GS-VH that binds HSA-4GS for binding to human CD137Fc were ka 5.6E+05 (1/Ms), Kdis 1.7E-05 (1/s) and KD 3.1E-11 (M) and for binding to rhesus CD137Fc were ka 4.4E+05 (1/Ms), Kdis 5.2E-05 (1/s) and KD 1.2E-10 (M). Humabody® 1.113 showed better binding to cyno compared to other molecules and also showed better developability characteristics (stability and expression) compared to other molecules.


3.3 Co-Engagement


Engagement of 3 targets was also demonstrated using ForteBio Octet instrument. Association 1—Binding of the trispecific molecule to CD137Fc captured on protein G biosensors. Association 2—binding of Human serum Albumin, Association 3—Binding of PSMA in the presence of Human serum albumin to minimise HSA dissociation. This demonstrated that trispecific molecules can simultaneously bind targets. Dual target engagement of CD137 and PSMA by the bispecific molecules was assessed using an ELISA format and by flow cytometry with T cells. For ELISA, CHO-PSMA cells (20000/well) were seeded into 96 well plates (Greiner cat no. 353872) in Hams F12 supplemented with L-Glutamine+Blasticidin+Tetracycline and incubated at 37° C. with 5% CO2 overnight. All subsequent steps were performed at room temperature and included washes with PBS between each step. Plates were blocked with PBS/0.1% BSA for 1 hour then serially diluted Humabody® VH were added and allowed to bind for 1 hour. Following removal of unbound VH, 1 nM CD137huFc (Acro Biosystems cat no. 41B-H5258) was added to the wells and incubated for 1 hour. A 1:3000 dilution of Anti-huFc-HRP (Jackson ImmunoResearch cat no. 109-035-098) was subsequently added for 1 hour and plates developed by addition of TMB. The reaction was stopped by addition of 0.5M sulphuric acid and plates read on BMG PheraStar at Absorbance 450 nm. FIG. 1 shows representative data demonstrating that bispecific molecules can simultaneously bind both human CD137 and human PSMA. Human CD8+ T cells pre-stimulated for 48 hours with PMA/ionomycin were washed with FACS buffer (PBS supplemented with 10% human serum and 0.05% sodium azide) by centrifugation. Serially diluted bispecific and trispecific Humabody VH construct were added to the cells for 1 hour on ice. Plates were washed with FACS buffer then detection mix containing 5 nM biotinylated-PSMA, 20 nM Streptavidin AlexaFluor488 and Live/Dead NearIR cell stain dilution added. Plates were incubated at 4° C. for 1 hour then washed twice with FACS buffer by centrifugation. Cells were fixed by addition 1× BD Cell fix solution, then washed twice with PBS by centrifugation and the cell pellets resuspended in PBS. Fluorescence was measured in the RL2-H channels (Live/Dead stain) and BL1-H channel (AlexaFluor488 stain) on the iQue Plus Screener (IntelliCyt). The bispecific and trispecific molecules were able to simultaneously bind human CD8+ T cells and PSMA protein.


Binding to cell lines was also measured using Fluorescence Microvolume Assay technology as per section 3.1. CD137huFc/anti humanFc-Alexa Fluor-488 was used for detection of binding to PSMA expressing cells.


ELISA data is the average EC50 (±SD) for n=3 determinations. Flow cytometry data shown is the average EC50 (±SD) for a minimum of 6 different human T-cell donors (Table 13). Construct 4.1-6GS-1.113-6GS-VH HSA-4GS bound CHO cynomolgus PSMA cells in the FMAT assay (Average EC50±SD (M) 5.7E-10±4.6E-11, 0.57 nM n=3).











TABLE 13






ELISA EC50 ± SD
Flow Cytometry







3.8-6GS-VH
2.5E−09 ± 9.6E−10
4.0E−09 ± 1.5E−09


HSA-6GS-1.113-4GS




3.8-6GS-1.113-6GS-VH
1.6E−09 ± 4.2E−10
3.4E−09 ± 1.8E−09


HSA 4GS




3.8-6GS-1.113
1.3E−09 ± 2.6E−10
8.7E−10 ± 4.5E−10


4.1-6GS-VH HSA
1.1E−09 ± 3.4E−10
3.7E−09 ± 1.7E−09


6GS-1.113-4GS




4.1-6GS-1.113-6GS-VH
2.2E−09 ± 2.0E−10
4.2E−09 ± 1.3E−09


HSA 4GS




4.1-6GS-1.113
5.7E−10 ± 2.0E−10
1.3E−09 ± 8.8E−10









Example 4. Inhibition of CD137L Binding

Humabody® VHS were tested for their ability to inhibit the interaction of human CD137-ligand protein with CHO human CD137 cells in an FMAT format. Serially diluted VH in FMAT assay buffer were incubated for a minimum of 2 hours at room temperature with 0.2 nM 0.4 nM CD137L-huFc (Sino Biologicals cat no. 15693-101H), 3 nM Alexa Fluor® 488 Goat Anti-Human IgG, Fc gamma fragment specific goat anti human IgG (Jackson ImmunoResearch cat no. 109-545-098) and 2000 cells pre-stained with DRAQ5. Total binding controls containing FMAT assay buffer and non-specific binding controls containing excess non-Fc tagged competitor were set up on each plate for data normalisation. Fluorescence signal was measured using the TTP Mirrorball and the FL2 median mean fluorescence intensity of gated used for the data normalisation. The data was expressed as a % of the total binding control (% control) after subtraction of the background signal determined from the non-specific binding control wells. VH in monovalent and bispecific formats inhibited CD137 binding to CD137 Ligand (Table 14).












TABLE 14







Humabody ® VH
IC50 (M)









1.78
1.5E−09



1.1
1.1E−09



2.1
5.7E−09



4.1




3.8




4.1-6GS-1.1
2.5E−09



4.1-6GS-2.1
1.3E−08



4.1-6GS-1.78
3.7E−09



1.78-6GS-4.1
2.8E−09



3.8-6GS-1.1
1.7E−09



1.1-6GS-3.8
1.3E−09










Example 5 Functional Activity

5.1 Co-Culture Functional Activity Assays


The ability of bispecific molecules to act as CD137 agonists was assessed in a co-culture reporter gene assay using Jurkat cells expressing CD137 and an NF-kB luciferase reporter gene and cells expressing human PSMA. Assays were performed using high PSMA expressing cells (CHO PSMA) and low PSMA expressing cells (DU145 PSMA). PSMA expressing cells or parental cells (5000/well) were plated overnight into 384 well, white clear bottomed tissue culture treated plates. For CHO cells media was removed the next day and replace by assay media (RPMI 1640 supplemented with 10% FBS, 2 mM L-Glutamine, 1× Pen/Strep). Serially diluted monomer VH, bispecific and Jurkat reporter cells were prepared in assay media (RPMI 1640 supplemented with 10% FBS, 2 mM L-Glutamine, 1× Pen/Strep) and added to the wells. After a 5-6 hour incubation at 37° C. in a CO2 incubator the level of luciferase reporter expression was determined by addition of BioGlo reagent (Promega G7940) and measurement of luminescent signal on the BMG Pherastar. FIG. 2 exemplifies the concentration and PSMA dependent stimulation of the NF-kB pathway by CD137-PSMA VH bispecific molecules. The level of PSMA expression determines the maximum response with higher maximal levels obtained for the CHO PSMA high PSMA expressing cell line compared to the low expressing DU145 PSMA cells (FIG. 2b). Anti CD137 antibody stimulates NF-kB driven reporter gene activity in a PSMA independent manner (FIG. 2c). The experiments show that in the bispecific molecule, CD137 agonism resulted from co-engagement of both CD137 and the cell expressed PSMA.


5.2 IL-2 Release


Bispecific molecules were tested for their ability to induce IL-2 release from T cells in a co-culture assay. DU145 PSMA or parental DU145 cells were resuspended in media (RPMI 1640 supplemented with 10% FBS, 2 mM L-Glutamine, 1× Pen/Strep) and seeded at a density of 20000 per well onto 96 well flat bottom plates that had been pre-coated with 5 ug/ml anti CD3 antibody (e-Bioscience cat no. 14-0037-82). Cells were allowed to adhere overnight at 37° C., 5% CO2. Peripheral blood mononuclear cells (PBMCs) were isolated from human blood by density gradient centrifugation then CD8+ T cells purified using a negative selection isolation kit according to the manufacturer's protocol (Miltenyi Biotech cat no 130-042-401). Humabody® VH, bispecifics and benchmark antibodies were prepared in media and added together with the T cells (100000 cells/well) to the assay plates. Supernatants were harvested after a 48 hour incubation at 37° C., 5% CO2 and IL-2 levels quantified using a human IL-2 assay kit according to the manufacturer's instructions (Cisbio Cat no. 641L2PEB). Stimulation of IL-2 production by CD8+ T cells was independent of PSMA for the anti CD137 benchmark antibody but was PSMA dependent (FIG. 3a) and concentration dependent (FIG. 3b, 3d). Maximum responses levels were T cell donor dependent for both antibody and bispecific molecules (FIG. 3c). Interferon gamma was also induced (FIG. 3e).


5.3. Stimulation of Superantigen-Activated Cells


PBMC from healthy donor were stimulated with 10 ng/ml SEB (Staphylococcal enterotoxin B) for 16 hours prior to treatment. CHO cells or CHO cells expressing PSMA were plated into 96-well plates at 10,000 per well. Humabody® constructs were added to a final concentration of 50 nM and a 4-fold dilution series. SEB-stimulated PBMC were added at 75,000 per well in media with 1 ng/ml SEB. Plates were incubated at 37° C. 5% CO2 for 3 days. Supernatants were harvested for cytokine measurement. TNF-alpha was measured using Cisbio HTRF kit (62HTNFAPEG) according to manufacturer's instructions. TNF-alpha increased in a bispecific Humabody® dependant dose-response manner in the presence of cells expressing PSMA. There was no induction in the absence of PSMA (FIG. 5).


Example 6. Effect of Humabody® in DU145 PSMA/Hu PBMC Engrafted NCG Mice

Male NCG mice (NOD-Prkdcem26Cd52112rgem26Cd22/NjuCrl, Charles River) were injected sub-cutaneously in the right flank with 1×10 7 DU145 PSMA cells in 50% matrigel. On Day 8, hPBMCs (HemaCare BioResearch Products) were engrafted via tail vein. Non engrafted mice were used as control groups. Mice were then treated with Humabody® or control CD137 agonist antibody administered intraperitoneally and body weights, clinical observations, and tumour volumes recorded. Study was performed at Charles River Discovery Services North Carolina (CR Discovery Services) which specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care, and is accredited by AAALAC. Half-life extended bispecific Humabody® treated groups showed a reduced tumour volume compare to the controls group (FIG. 6).


Example 7 Pharmacokinetics Analysis of Single Intravenous Dose of a Half Life Extended Molecule in Double Transgenic Humanised FcRn/HSA Mouse

Experiments were conducted using a Human Neonatal Fc Receptor/Albumin Mouse model by genOway®. This double humanized neonatal Fc receptor (FcRn)/albumin mouse model maintains an autologous receptor-ligand interaction and mimics the physiological drug clearance in humans, and therefore represents a unique and reliable tool to measure and optimize albumin-linked small molecules and conventional drug pharmacokinetics and study and predict the half-life of circulating biologics and biosimilar drugs (Viuff D, Antunes F, Evans L, Cameron J, Dyrnesli H, Thue Ravn B, Stougaard M, Thiam K, Andersen B, Kjærulff S, Howard K A. 2016. Generation of a double transgenic humanized neonatal Fc receptor (FcRn)/albumin mouse to study the pharmacokinetics of albumin-linked drugs. J Control Release).


The hFcRn/HSA humanized mouse provides more predictable “human-like” pharmacokinetic results than WT mice. This model is well suited for in vivo assessment of HSA-binding drugs' pharmacokinetic, distribution and toxicity.


A trispecific molecule (4.1-6GS-1.113-6GS-VH HSA-4GS) was used in these experiments to test pharmacokinetics.


Briefly, male genOway® Human HSA/FcRn Tg mice were dosed with a single intravenous injection of trispecific (n=3) at 2 mg/kg via tail vein. Blood samples were collected at Pre-dose and at 0.083 h, 1 h, 8 h, 24 h, 48 h, 72 h and 96 h post drug administration via the saphenous vein. At 168 h post dose all animals were euthanised and blood was collected. Plasma was separated and stored at −80° C. until an assay was carried out. Plasma samples were analysed on Gyrolab immunoassay platform, using biotinylated human PSMA as capture and human CD137Dylight650 as detection. Data was analysed using Gyros to obtain concentrations in plasma. Pharmacokinetic analysis of data was done using PK Solver 2.0.


Results of study show that the molecule has a half-life of 18.13±0.412 hours (n=3) when dosed at 2 mg/kg intravenously in human HSA/FcRn Tg mice.


REFERENCES



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  • Dass S. Vinay and Byoung S. Kwon. 4-1BB (CD137), an inducible costimulatory receptor, as a specific target for cancer therapy. BMB Rep. 2014 March; 47(3): 122-129.

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  • Holliger P, Hudson P J. Engineered antibody fragments and the rise of single domains. Nat Biotechnol. September; 23(9):1126-36. (2005)

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  • Sanchez-Paulete A. R, Labiano S., Rodriguez-Ruiz M. E., Azpilikueta A., Etxeberria I., Bolanos E., Lang V., Rodriguez M., Aznar M. A., Jure-Kunkel M. and Melero I. Deciphering CD137 (4-1BB) signaling in T-cell costimulation for translation into successful cancer. Immunotherapy. Eur. J. Immunol. 2016. 46: 513-522 Vinay D. S. and Kwon B. S. Immunotherapy of Cancer with 4-1BB Mol Cancer Ther; 11(5) May 2012 Yannick Bulliard, Rose Jolicoeur, Jimin Zhang, Glenn Dranoff, Nicholas S Wilson, and Jennifer L Brogdon OX40 engagement depletes intratumoral Tregs via activating FcγRs, leading to antitumor efficacy. Immunology and Cell Biology (2014) 92, 475-480; published online 15 Apr. 2014


Claims
  • 1. An isolated binding molecule comprising a) a single variable heavy chain domain antibody that binds to CD137 andb) a moiety that binds to PSMA.
  • 2. The isolated binding molecule according to claim 1 wherein the single variable heavy chain domain antibody that binds to CD137 comprises a CDR1 comprising SEQ ID NO. 1 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 2 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 3 or a sequence with at least 40% homology thereto or a CDR1 comprising SEQ ID NO. 425 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 426 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 427 or a sequence with at least 40% homology thereto or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 427 or a sequence with at least 75% homology thereto.
  • 3. The isolated binding molecule according to claim 2 wherein the single variable heavy chain domain antibody that binds to CD137 comprises human framework regions.
  • 4. The isolated binding molecule according to a preceding claim wherein the single variable heavy chain domain antibody that binds to CD137 comprises a full length sequence as listed in table 2 or 3 or a sequence with at least 75% homology thereto.
  • 5. The isolated binding molecule according to a preceding claim wherein the single variable heavy chain domain antibody that binds to CD137 comprises SEQ ID NO. 4, 312, 852, 856, 860, 864, 868, 872, 876, 880 or 428 or a sequence with at least 75% homology thereto.
  • 6. The isolated binding molecule according to a preceding claim wherein the moiety that binds to PSMA is selected from an antibody, an antibody fragment, an antibody mimetic or other polypeptide.
  • 7. The isolated binding molecule according to claim 6 wherein said antibody fragment is selected from a Fab, F(ab′)2, Fv, a single chain Fv fragment (scFv), a single domain antibody or fragment thereof.
  • 8. The isolated binding molecule according to claim 7 wherein said single domain antibody is a VH single domain antibody.
  • 9. The isolated binding molecule according to claim 8 wherein said VH single domain antibody comprises a CDR1 comprising SEQ ID NO. 812 or a sequence with at least 75% homology thereto, a CDR2 comprising SEQ ID NO. 813 or a sequence with at least 75% homology thereto and a CDR3 comprising SEQ ID NO. 814 or a sequence with at least 75% homology thereto or wherein said VH single domain antibody comprises a CDR1 comprising SEQ ID NO. 837 or a sequence with at least 75% homology thereto, a CDR2 comprising SEQ ID NO. 838 or a sequence with at least 75% homology thereto and a CDR3 comprising SEQ ID NO. 839 or a sequence with at least 75% homology thereto.
  • 10. The isolated binding molecule according to any preceding claim wherein the single variable heavy chain domain antibody that binds to PSMA comprises a full length sequence as listed in table 6 or 7 or a sequence with at least 75% homology thereto.
  • 11. The isolated binding molecule according to any preceding claim wherein the single variable heavy chain domain antibody that binds to PSMA comprises SEQ ID NO. 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825 or 840 or a sequence with at least 75% homology thereto.
  • 12. The isolated binding molecule according to any preceding claim wherein a single domain antibody 1.1 or 2.1 having SEQ ID No. 4 or 428, or a single domain antibody having SEQ ID No 312, 852, 856, 860, 864, 868, 872 or 876 or a sequence with at least 75% sequence homology thereto is linked to a single domain antibody 3.1, 3.8 or 4.1 having SEQ ID No. 815, 822 or 840 respectively.
  • 13. The isolated binding molecule according to any preceding claim capable of binding CD137 with an affinity with a Kd of at least about 10-6 M to about 10-12M.
  • 14. The isolated binding molecule according to any preceding claim capable of binding PSMA with an affinity with a Kd of about 10-6 M to about 10-12M.
  • 15. The isolated binding molecule according to any preceding claim wherein the single variable heavy chain domain antibody that binds to CD137 is linked to the moiety that binds PSMA by a peptide linker.
  • 16. The binding molecule according to claim 15 wherein said linker is selected from a (G4S)n linker wherein n is an integer selected from 1 to 10.
  • 17. The isolated binding molecule according to any preceding claim conjugated to one or more moiety selected from a toxin, enzyme, radioisotope, half-life extending moiety, label, therapeutic molecule and other chemical moiety.
  • 18. The isolated binding molecule according to claim 17 wherein said half-life extending moiety is selected from the group consisting of an albumin binding moiety, a transferrin binding moiety, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of human serum albumin, and an albumin binding peptide and single domain antibody that binds to human serum albumin.
  • 19. The isolated binding molecule according to any preceding claim wherein the single variable heavy chain domain antibody which binds to human CD137 does not cause CD137 signalling when bound to CD137 as a monospecific entity.
  • 20. The isolated binding molecule according to any preceding claim wherein the single variable heavy chain domain antibody which binds to human CD137 is obtained or obtainable from a transgenic rodent that expresses a transgene comprising human V, D and J regions.
  • 21. The single variable heavy chain domain antibody according to claim 20 wherein said rodent does not produce functional endogenous light and heavy chains.
  • 22. A pharmaceutical composition comprising a binding molecule according to preceding claim and a pharmaceutical carrier.
  • 23. A binding molecule according to any of claims 1 to 21 or a pharmaceutical composition according to claim 23 for use in the treatment of disease.
  • 24. A binding molecule according to any of claims 1 to 21 or a pharmaceutical composition according to claim 22 wherein said disease is a cancer.
  • 25. A method for treating prostate cancer or a prostatic disorder comprising administering a therapeutically effective amount of a binding molecule according to any of claims 1 to 21 or a pharmaceutical composition according to claim 22.
  • 26. A binding molecule according to claim 24 or a method according to claim 25 wherein said cancer is prostate cancer lung cancer, glioblastoma, renal, bladder, testicular, neuroendocrine, colon, and breast cancer.
  • 27. A nucleic acid molecule comprising a nucleic acid sequence encoding the binding molecule according to any of claims 1 to 21.
  • 28. A vector comprising nucleic acid molecule according to claim 27.
  • 29. A host cell comprising a nucleic acid molecule according to claim 27 or a vector according to claim 28.
  • 30. The host cell according to claim 29 wherein said host cell is a bacterial, yeast, viral or mammalian cell.
  • 31. A method for producing a binding molecule according to any of claims 1 to 21 comprising expressing a nucleic acid encoding said binding molecule in a host cell and isolating the binding molecule from the host cell.
  • 32. A method for promoting CD8+ T cell expansion, inducing activation of cytotoxic T lymphocytes (CTL) and/or cytokine release comprising administering to a binding molecule according to any of claims 1 to 21 or a pharmaceutical composition according to claim 22.
  • 33. An in vivo or in vitro method for reducing human PSMA activity comprising contacting human PSMA with a binding molecule according to any of claims 1 to 21.
  • 34. A kit comprising a binding molecule according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 22.
  • 35. A use of a binding molecule according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 22 for simultaneously activating downstream signalling pathways of CD137 and PSMA.
  • 36. A method for co-stimulating downstream signalling pathways of CD137 and PSMA comprising administering a binding molecule according to any of claims 1 to 21 or a pharmaceutical composition according to claim 22.
  • 37. A use of a binding molecule according to any of claims 1 to 21 or a pharmaceutical composition according to claim 22 for inducing a local T cell response in the vicinity of a PSMA positive tumor cell or tissue.
  • 38. A method for inducing a local T cell response in the vicinity of a PSMA positive tumor cell or tissue comprising administering a binding molecule according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 22.
Priority Claims (3)
Number Date Country Kind
1718734.5 Nov 2017 GB national
1718735.2 Nov 2017 GB national
1808589.4 May 2018 GB national
PCT Information
Filing Document Filing Date Country Kind
PCT/GB2018/053280 11/13/2018 WO 00