Multispecific antibody constructs

Abstract

The present invention provides a multispecific antibody having at least one conditional activity and a method for producing the same. [Solution] A multispecific antibody comprises at least one binding site for a cell-specific antigen and at least one binding site for a tumor-reactive lymphocyte antigen, and the multispecific antibody comprises an IgG antibody or fragment thereof that binds to a first antigen; and at least one scFv antibody that binds to a second antigen different from the first antigen and is ligated to the C-terminus of at least one light chain or heavy chain of the IgG antibody or fragment. This multispecific antibody reversibly binds to the cell-specific antigen and the tumor-reactive lymphocyte antigen or at least one of the first antigen and the second antigen with greater affinity under abnormal conditions than under normal physiological conditions. Conjugates of multispecific antibodies and methods for producing multispecific antibodies are also provided.

Description

[Technical Field]

[0001] This disclosure relates to the field of multispecific antibodies. In particular, this disclosure relates to multispecific antibodies having at least one conditional activity and methods for producing them. [Background technology]

[0002] Proteins can be modified to possess various properties, such as high activity or improved stability, in order to function under different conditions. For example, enzymes have evolved to be stable at high temperatures while maintaining varying levels of activity. In situations where there is an increase in activity at high temperatures, a significant portion of this increase may be due to high kinetic activity, generally described by the Q10 rule (which estimates that in the case of enzymes, the metabolic turnover rate doubles for every 10 degrees Celsius increase). Mutations introduced into enhanced proteins typically result in a decrease in the protein's activity under normal functional conditions. Mutant enzymes designed to function at high temperatures may be active at normal functional temperatures, but their activity levels may be typically lower compared to wild-type enzymes.

[0003] Antibodies are a major class of therapeutic proteins. Conventional antibodies typically bind to a single epitope on an antigen. Novel antibody structures, called multispecific antibodies, have been developed to bind to two or more antigens or two or more epitopes on the same antigen. Multispecific antibodies can be, for example, bispecific, triplicate, or quadruplicate antibodies. Multispecific antibodies have shown potential in a wide range of clinical and diagnostic applications. There are two bispecific antibody drugs approved in the European Union and the United States for the treatment of oncological diseases (Catumaxomab® and Blinatumab®). Due to their unique characteristics, multispecific antibodies are attractive as next-generation antibody therapies.

[0004] U.S. Patent Application Publication No. 2013 / 0017200 discloses a method for synthesizing multispecific antibodies. A first antibody fragment is obtained from a first parent antibody having a first monospecificity, which, having free sulfhydryl groups, can react with a thioreactive crosslinking agent to form an antibody fragment-crosslinking moiety. When this antibody fragment-crosslinking moiety reacts in pairs with two or more further antibody fragments, each having free sulfhydryl groups, obtained from other parent antibodies having a different monospecificity than the first antibody fragment, a multispecific antibody is produced. This multispecific antibody may be suitable as a novel therapeutic and diagnostic agent.

[0005] Brinkmann and Kontermann ("The making of bispecific antibodies," MABS, 2017, vol.9, pp.182-212, 2017) investigated the formats of bispecific antibodies, including small molecules composed only of the antigen-binding sites of two antibodies, molecules with IgG structures, and large complex molecules composed of different antigen-binding sites often combined with dimerization modules. Depending on the application, bispecific antibodies can vary in the size, arrangement, valency, mobility, and geometry of their binding modules, as well as their distribution and pharmacokinetic properties. Overall, bispecific formats increase the diversity of antibodies that can be applied to the development of therapeutics for various indications.

[0006] Furthermore, it is desirable to generate useful antibodies with conditional activity. For example, antibodies that are virtually inactive under normal physiological conditions but significantly more active under conditions other than normal physiological conditions (e.g., abnormal conditions), antibodies that are activated or inactivated in certain microenvironments (e.g., activated in the tumor microenvironment), or antibodies that are activated or inactivated over time. In addition to temperature, other inducing conditions that can evolve or optimize antibodies include pH, osmotic pressure, osmolality, oxidative stress, oxygen concentration, and electrolyte concentration. In addition to activity, other desirable properties of antibodies that can be optimized during evolution include stability, half-life, chemical resistance, and proteolytic resistance.

[0007] Many strategies have been published for evolving or modifying parent antibodies into mutant antibodies with desired properties. However, to modify or evolve a parent antibody so that it is inactive or virtually inactive (less than 10% activity, and especially less than 5% activity) under normal physiological conditions, but exhibits activity equal to or better than the original parent antibody under normal physiological conditions under abnormal conditions, one or more destabilizing mutations must coexist with activity-enhancing mutations that do not counteract the destabilizing effect. The destabilizing mutations are expected to result in a decrease in antibody activity that exceeds the magnitude predicted by standard laws such as the Q10 rule. Therefore, if it is possible to evolve a protein that functions efficiently (has higher activity) under specific abnormal conditions, such as lower temperature or pH, compared to an evolved protein that is in or even substantially inactive under normal physiological conditions, a remarkable new class of antibodies called conditionally active proteins can be created. For example, a conditionally active protein may have a higher ratio of activity under abnormal conditions to activity under normal physiological conditions than the same ratio for the parent protein. In some cases, this ratio for conditionally active proteins may be at least about 2:1, or at least about 4:1, or at least about 6:1, or at least about 10:1, or at least about 20:1, or at least about 50:1, or at least about 80:1, or at least about 100:1. [Overview of the project] [Means for solving the problem]

[0008] The present invention provides a novel class of multispecific antibodies that are conditionally active against at least one epitope or antigen. This novel class of multispecific antibodies leverages the versatility and adaptability of conventional multispecific antibodies while simultaneously directing the activity, affinity, and / or avidity of the multispecific antibody to the desired site, tissue, or organ.

[0009] In one embodiment, the present disclosure provides a multispecific antibody comprising at least one binding site for a cell antigen; and at least one binding site for a tumor-reactive lymphocyte antigen. The multispecific antibody binds to at least one of the cell antigen and the tumor-reactive lymphocyte antigen with greater activity, affinity, and / or avidity under first physiological conditions than under second physiological conditions.

[0010] In the embodiments described above, the cell antigen may be a cancer cell-associated antigen, or the cancer cell-associated antigen may be a neoantigen.

[0011] In any one of the embodiments described above, the cell antigen may be a senescent cell-associated antigen.

[0012] In any one of the embodiments described above, the first physiological condition may be an abnormal condition, and the second physiological condition may be a normal physiological condition.

[0013] In the embodiments described above, the abnormal conditions may be conditions in the tumor microenvironment or conditions in the senescent cell microenvironment.

[0014] In any of the embodiments described above, the binding of a multispecific antibody to at least one of the cell antigen and the tumor-reactive lymphocyte antigen may be reversible.

[0015] In any one of the previous embodiments, the multispecific antibody may be composed in a format selected from: (1) bispecific antibody conjugate; (2) hybrid bispecific IgG2; (3) bispecific antibody molecule of only variable domains; (4) CH1 / CL fusion protein; (5) Fab fusion protein; (6) non-immunoglobulin fusion protein; (7) Fc-modified IgG; (8) additional and Fc-modified IgG; (9) modified Fc and CH3 fusion protein; (10) additional IgG-HC fusion; (11) additional IgG-LC fusion; (12) additional IgG-HC and LC fusion; (13) Fc fusion; (14) CH3 fusion; (15) IgE / IgM CH2 fusion; (16) F(ab’)2 fusion; (17) CH1 / CL fusion protein; (18) modified IgG; and (19) non-immunoglobulin fusion.

[0016] In any one of the previous embodiments, the multispecific antibody may be conjugated to a polymer. The polymer may be selected from at least one of protein, fatty acid, and polymer, or the polymer may be albumin or polyethylene glycol.

[0017] In any one of the previous embodiments, at least one binding site for a cell antigen may be an IgG antibody or a fragment thereof, and the cell antigen may be a tumor cell antigen.

[0018] In any one of the previous embodiments, at least one binding site for a tumor-reactive lymphocyte antigen may be a single-chain antibody. The single-chain antibody may be a scFv antibody. The scFv antibody may be linked via a linker to the C-terminus of at least one light chain of the IgG antibody or a fragment thereof. In any one of the previous embodiments where the antibody is a scFv antibody, the scFv antibody may include a light chain variable region selected from light chain variable regions having the amino acid sequences of SEQ ID NOs: 1-10, and a heavy chain variable region selected from heavy chain variable regions having the amino acid sequences of SEQ ID NOs: 11-15. In any one of the previous embodiments, the scFv antibody may have an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 26-71.

[0019] In any one of the previous embodiments, the IgG antibody comprises a light chain variable region selected from the light chain variable regions having the amino acid sequences of SEQ ID NOs: 16-17, 20, 22, and 88-95, which binds to an antigen selected from Axl, Her2, B7-H3, and EpCAM; and a heavy chain variable region selected from the heavy chain variable regions having the amino acid sequences of SEQ ID NOs: 18-19, 21, 23-25, and 80-87, which binds to the same antigen.

[0020] In any one of the previous embodiments, the tumor-reactive lymphocyte antigen can be a CD3 antigen.

[0021] In any one of the previous embodiments, the tumor-reactive lymphocyte antigen can be on lymphocytes selected from T cells, macrophages, Jurkat cells, monocytes, NK cells, neutrophils, eosinophils, basophils, and lymphokine-activated killer cells.

[0022] In any one of the previous embodiments, the cellular antigen can be selected from Axl, EpCAM, Ror2, Her2, and B7-H3.

[0023] In any one of the previous embodiments, the multispecific antibody can bind to the cellular antigen with a greater affinity under the first physiological condition than under the second physiological condition.

[0024] In any one of the previous embodiments, the multispecific antibody can bind to the tumor-reactive lymphocyte antigen with a greater affinity under the first physiological condition than under the second physiological condition.

[0025] In any one of the previous embodiments, the multispecific antibody can bind to both the cellular antigen and the tumor-reactive lymphocyte antigen with a greater affinity under the first physiological condition than under the second physiological condition.

[0026] In any one of the embodiments described above, the multispecific antibody can bind to a combination of cell antigen and tumor-reactive lymphocyte antigen with greater avidity under the first physiological conditions than under the second physiological conditions.

[0027] In another embodiment, the Disclosure provides a second embodiment of a multispecific antibody comprising an IgG antibody or fragment thereof that binds to a first antigen; and at least one scFv antibody that binds to a second antigen different from the first antigen, the scFv antibody being ligated to the C-terminus of at least one light chain of the IgG antibody or fragment, the multispecific antibody reversibly binds to at least one of the first and second antigens with greater affinity and / or avidity under abnormal conditions than under normal physiological conditions.

[0028] In the second embodiment, the second antigen may be a CD3 antigen.

[0029] In any one of the second embodiments described above, the scFv antibody may include a light chain variable region selected from light chain variable regions having the amino acid sequences of SEQ ID NOs. 1 to 10, and a heavy chain variable region selected from heavy chain variable regions having the amino acid sequences of SEQ ID NOs. 11 to 15. In any one of the embodiments described above, the scFv antibody may have an amino acid sequence selected from amino acid sequences of SEQ ID NOs. 26 to 71.

[0030] In any one of the second embodiments described above, the IgG antibody or fragment may include a light chain variable region selected from light chain variable regions having amino acid sequences of SEQ ID NOs. 16-17, 20, 22 and 88-95 that binds to an antigen selected from Axl, Her2, B7-H3 and EpCAM; and a heavy chain variable region selected from heavy chain variable regions having amino acid sequences of SEQ ID NOs. 18-19, 21, 23-25 ​​and 80-87 that binds to the same antigen.

[0031] In any one of the second embodiments described above, the multispecific antibody can bind to the first antigen with greater affinity under abnormal conditions than under normal physiological conditions.

[0032] In any one of the second embodiments described above, the multispecific antibody may bind to the second antigen with greater affinity under abnormal conditions than under normal physiological conditions.

[0033] In any one of the second embodiments described above, the multispecific antibody may bind to both the first and second antigens with greater affinity under abnormal conditions than under normal physiological conditions.

[0034] In any one of the second embodiments described above, the multispecific antibody can bind to the combination of the first and second antigens with greater avidity under the first physiological conditions than under the second physiological conditions.

[0035] In any of the second embodiments described above, the first antigen may be a cell surface antigen, and such cell surface antigen may be a cancer cell surface antigen. In any of the second embodiments described above, the first antigen may be a neoantigen. In any of the second embodiments described above, the first antigen may be selected from Axl, EpCAM, Ror2, Her2, and B7-H3.

[0036] In yet another embodiment, the Disclosure provides a method for producing a multispecific antibody, comprising: a) obtaining an IgG antibody or fragment thereof that binds to a first antigen; b) ligating at least one scFv antibody that binds to a second antigen to the C-terminus of at least one light chain of the IgG antibody or fragment to produce one or more structures; c) screening one or more structures from b) for binding to at least one of the first antigen and the second antigen under abnormal and normal physiological conditions; and d) selecting from one or more structures a multispecific antibody that reversibly binds to at least one of the first antigen and the second antigen with greater affinity under abnormal conditions than under normal physiological conditions.

[0037] In the method described above, the first antigen may be a tumor cell antigen or neoantigen that can be selected from Axl, EpCAM, Ror2, Her2, and B7-H3.

[0038] In any of the methods described above, the second antigen may be a tumor-reactive lymphocyte antigen such as CD3. Tumor-reactive lymphocyte antigens may be present on lymphocytes selected from T cells, macrophages, Jurkat cells, monocytes, NK cells, neutrophils, eosinophils, basophils, and lymphokine-activated killer cells.

[0039] In any one of the methods described above, the scFv antibody may include a light chain variable region selected from the light chain variable regions having the amino acid sequences of SEQ ID NOs. 1 to 10, and a heavy chain variable region selected from the heavy chain variable regions having the amino acid sequences of SEQ ID NOs. 11 to 15. In any one of the embodiments described above, the scFv antibody may have an amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 26 to 71.

[0040] In any of the methods described above, the IgG antibody may include a light chain variable region selected from light chain variable regions having amino acid sequences of SEQ ID NOs. 16-17, 20, 22 and 88-95, which binds to an antigen selected from Axl, Her2, B7-H3 and EpCAM; and a heavy chain variable region selected from heavy chain variable regions having amino acid sequences of SEQ ID NOs. 18-19, 21, 23-25 ​​and 80-87, which binds to the same antigen.

[0041] In any of the methods described above, the multispecific antibody may bind to the first antigen with greater affinity under abnormal conditions than under normal physiological conditions.

[0042] In any of the methods described above, the multispecific antibody may bind to the second antigen with greater affinity under abnormal conditions than under normal physiological conditions.

[0043] In any of the methods described above, the multispecific antibody may bind to both the first and second antigens with greater affinity under abnormal conditions than under normal physiological conditions.

[0044] In any of the methods described above, the multispecific antibody may bind to both the first and second antigens in combination with greater avidity under abnormal conditions than under normal physiological conditions.

[0045] In yet another embodiment, the present disclosure provides a method for treating a target tumor, comprising administering a multispecific antibody of any one of the embodiments described above.

[0046] In this treatment method, multispecific antibodies may be administered in combination with the cancer neoantigen vaccine, or they may be administered after the administration of the cancer neoantigen vaccine.

[0047] In yet another embodiment, the Disclosure relates to a method for producing a multispecific antibody, comprising: a) obtaining an IgG antibody or fragment thereof that binds to a first antigen; b) obtaining an scFv antibody that binds to a second antigen; c) evolving one or both of the antibodies from a) and b) to produce one or more evolved antibodies; d) screening one or more evolved antibodies from c) to select an antibody that binds to each antigen with greater affinity under abnormal conditions than under normal physiological conditions; e) ligating the scFv antibody that binds to the second antigen to the C-terminus of at least one light chain of the IgG antibody or fragment that binds to the first antigen; The present invention provides a method comprising the steps of: producing one or more structures, wherein at least one of the scFv antibody and the IgG antibody is selected in d), and if present, the scFv antibody or IgG antibody not selected in d) is from step a) or b); f) screening one or more structures from step e) for binding to at least one of a first antigen and a second antigen under abnormal and normal physiological conditions; and g) selecting a multispecific antibody from one or more structures that binds to at least one of the first antigen and the second antigen with greater affinity under abnormal conditions than under normal physiological conditions.

[0048] In yet another embodiment, the Disclosure provides a method for producing a multispecific antibody, comprising: a) obtaining an IgG antibody or fragment thereof that binds to a first antigen; b) producing one or more structures by ligating at least one scFv antibody that binds to a second antigen to the C-terminus of at least one light chain of the IgG or fragment thereof, wherein the at least one scFv antibody binds to the second antigen with greater affinity under abnormal conditions than under normal physiological conditions; c) screening one or more structures for binding to the first antigen and the second antigen under normal physiological conditions and abnormal conditions; and d) selecting a multispecific antibody that binds to the first antigen and reversibly binds to the second antigen with greater affinity under abnormal conditions than under normal physiological conditions.

[0049] In yet another embodiment, the Disclosure provides a multispecific antibody comprising an IgG antibody or fragment thereof that binds to a cell-specific antigen and at least one scFv antibody that binds to a T lymphocyte antigen, wherein the at least one scFv antibody binds to the T lymphocyte antigen with greater affinity under abnormal conditions than under normal physiological conditions. [Brief explanation of the drawing]

[0050] [Figure 1] This is a schematic structure of a bivalent multispecific antibody, which is a heterodimer in which one arm binds to the antigen (Ag) and the other arm binds to CD3.

[0051] [Figure 2] This is a schematic structure of a tetravalent multispecific antibody, in which each arm is a homodimer with a binding site for the antigen (Ag) and a binding site for CD3.

[0052] [Figure 3A] Figure 2 illustrates a common method used to measure the simultaneous binding of a tetravalent multispecific antibody to both the antigen and CD3.

[0053] [Figure 3B] This study demonstrates the unconditional binding activity of a multispecific antibody against CD3 / Axl to CD3 at pH 6.0 and 7.4. The antibody was added to a CD3-coated ELISA plate, and binding was detected using an HRP-labeled anti-IgG antibody (secondary antibody 1 in Figure 3A).

[0054] [Figure 3C] The study demonstrates conditional binding activity to Axl by multispecific antibodies at pH 6.0 and 7.4. Antibodies were added to CD3-coated ELISA plates, and binding was detected using AXL protein (antigen in Figure 3A) and HRP-labeled anti-AXL antibody (secondary antibody 2 in Figure 3A).

[0055] [Figure 4A] This demonstrates the conditional and unconditional binding activity of multispecific antibodies against CD3 / Axl to immobilized CD3 at pH 6.0 and 7.4.

[0056] [Figure 4B] Figure 4A shows the conditional and unconditional binding activity of some of the multispecific antibodies to CD3 at pH 6.0 and 7.4 when Axl is immobilized.

[0057] [Figure 5A] This study demonstrates the conditional and unconditional binding activity of multispecific antibodies against CD3 / Her2 to immobilized CD3 at pH levels of 6.0 and 7.4.

[0058] [Figure 5B] Figure 5A shows the conditional and unconditional binding activity of the multispecific antibody to CD3 at pH 6.0 and 7.4 when Her2 is immobilized.

[0059] [Figure 6] This demonstrates the conditional binding activity of a multispecific antibody against CD3 / B7-H3 to immobilized CD3 at pH 6.0 and 7.4.

[0060] [Figure 7] A schematic diagram of a working model of the multispecific antibody of the present invention for binding to both CD3 on modified Jurkat effector cells and antigens on tumor target cells is shown.

[0061] [Figure 8A] This study demonstrates the stimulating activity of effector cells by a bispecific antibody that has non-conditional binding activity to both CD3 and Axl.

[0062] [Figure 8B]This study demonstrates the stimulating activity of Jurkat effector cells by a bispecific antibody that has unconditional binding activity to Axl and conditional binding activity to CD3.

[0063] [Figure 9] This shows the conditional binding activity of the multispecific antibodies of the present invention to CD3 at pH values ​​of 6.0 to 7.4, as measured by ELISA. These multispecific antibodies bind to both CD3 and EpCAM. [Figure 10] This shows the average tumor volume obtained by treating tumor xenograft mice with the bispecific antibody (EpCAM×CAB-CD3) of the present invention.

[0064] [Figure 11] This shows the reduction in T cell activation in the peripheral circulatory system by the bispecific antibody (EpCAM×CAB-CD3) of the present invention compared to the vehicle, isotype control, and unconditional activation benchmark antibody. [Modes for carrying out the invention]

[0065] definition To facilitate understanding of the examples provided herein, certain frequently occurring methods and / or terms are defined herein.

[0066] Terms such as "about", "activity", "drug", "ambiguous base requirements", "amino acid", "amplification", "chimeric properties", "cognite", "comparison window", "conservative amino acid substitution", "corresponding to", "degradation efficacy", "defined sequence framework", "digestion", "directional ligation", "DNA shuffling", "drug" or "drug molecule", "effective amount", "electrolyte", "epitope", "enzyme", "evolution" or "evolving", "fragment", "derivative", "analog", "total single amino acid substitution range", "gene", "genetic instability", "heterogeneous", "homogeneous" or "homogeneous", "industrial application", "identical" or "identity", "identity range", "isolated", "isolated nucleic acid", "ligand", "ligation", "linker" or "spacer", "microenvironment", "evolving molecular properties", "mutation", "naturally occurring", "normal physiological conditions" or "wild-type functional conditions", "nucleic acid molecule", "nucleic acid sequence encoding ~", or "DNA coding sequence of ~", or "nucleotide sequence encoding ~", "promoter sequence", "nucleic acid encoding enzyme (protein)", or "enzyme (protein) encoding "DNA encoding enzymes (proteins)", "polynucleotides encoding enzymes (proteins)", "specific nucleic acid molecular species", "assemble working nucleic acid samples into a nucleic acid library", "nucleic acid library", "nucleic acid construct", or "nucleotide construct", or "DNA construct", "construct", "oligonucleotide", or "oligo", "allogene", "operably linked", "operably linked to ~", "parent polynucleotide set", "patient", or "subject", "physiological conditions", "population", "pro-type", "pre-pro-type", "pseudo-random", "quasi-repeating unit", "random peptide library", "random peptide sequence", "receptor", "recombination", "synthesis", "related polynucleotide", "decreasing recombination", "reference sequence", "comparison window", "sequence identity", "sequence identity percentage", "substantial identity", "reference sequence", "repetition index (RI)", "restriction site", "selectable polynucleotide", "sequence identity", "similarity", "specifically binds", "specific hybridization", "specific polynucleotide", "stringent hybridization conditions",The definitions of "substantially identical," "substantially pure enzyme," "substantially pure," "to treat," "variable segment," "mutant," "working," "conditionally activated antibody," "antibody-dependent cell-mediated cytotoxicity" or "ADCC," "cancer" and "cancerous," "multispecific antibody," "full-length antibody," "library," "recombinant antibody," and "individual" or "subject" are the same as those in International Publication No. 2016 / 138071, and are therefore incorporated herein by reference in whole.

[0067] The term "abnormal condition," as used herein, refers to a condition that deviates from the normal acceptable range for that condition in the subject. The term "normal physiological condition," as used herein, refers to a condition in a tissue or organ at a certain location or site of action in the subject, such as the administration site, that is considered to be within the normal range in the subject. In relation to this definition, senescent cells and tumor cells are not considered normal cells, and therefore conditions caused by, occurring within or near, senescent cells and tumor cells are considered abnormal conditions.

[0068] The term “antibody,” as used herein, refers to intact immunoglobulin molecules as well as fragments of immunoglobulin molecules that have the ability to bind to an antigen epitope, such as Fab, Fab', (Fab')2, Fv, and single-chain antibody (SCA) fragments. These antibody fragments retain some ability to selectively bind to the antigen of the original antibody from which they are derived (e.g., polypeptide antigens) and can be prepared using methods well known in the art (see, for example, Harlow and Lane, cited above), and are further described below. Antibodies useful in the claimed implementation of the present invention may be IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, sIgA, IgD, or IgE. Antibodies can be used to isolate a preparation amount of antigen by immunoaffinity chromatography. Various other uses of such antibodies include diagnosing and / or staging diseases (e.g., neoplasia) and therapeutic applications for treating diseases such as neoplasia, autoimmune diseases, AIDS, cardiovascular diseases, and infections. Chimeric, human-like, humanized, or fully human antibodies are particularly useful for administration to human patients.

[0069] Fab fragments consist of monovalent antigen-binding fragments of antibody molecules and can be produced by digesting the entire antibody molecule with the enzyme papain, which yields fragments consisting of intact light chains and parts of the heavy chain.

[0070] The Fab' fragments of antibody molecules can be obtained by treating the entire antibody molecule with pepsin and then reducing it, which yields molecules consisting of intact light chains and portions of heavy chains. Two Fab' fragments are obtained for each antibody molecule treated in this way.

[0071] The (Fab')2 fragment of an antibody can be obtained by treating the entire antibody molecule with the enzyme pepsin and then omitting the subsequent reduction. The (Fab')2 fragment is a dimer of two Fab' fragments held together by two disulfide bonds.

[0072] An Fv fragment is defined as a genetically modified fragment containing a variable light chain region and a variable heavy chain region, expressed as two strands.

[0073] A single-chain antibody ("SCA") is a genetically engineered single-chain molecule containing a variable region of the light chain and a variable region of the heavy chain linked by a suitable mobile polypeptide linker, which may include additional amino acid sequences at the amino-terminus and / or carboxyl-terminus. For example, scFv antibodies are single-chain antibodies. For instance, a single-chain antibody may include a tether segment for linking to a coding polynucleotide. Functional single-chain antibodies generally contain a portion of the variable region of the light chain and a portion of the variable region of the heavy chain sufficient to retain the binding properties to specific target molecules or epitopes of a full-length antibody. Single-chain antibodies are generally proteins consisting of one or more polypeptide segments of at least 10 consecutive amino acids, encoded by genes of the immunoglobulin superfamily (see, for example, *The Immunoglobulin Gene Superfamily*, AFWilliams and ANBarclay, in *Immunoglobulin Genes*, T. Honjo, FWAlt, and THE. Rabbits, eds., (1989) Academic press: San Diego, Calif., pp. 361-368), most frequently rodents, non-human primates, birds, pigs, cattle, sheep, goats, or human heavy or light chain gene sequences. Functional single-chain antibodies generally contain a portion of an immunoglobulin superfamily gene product sufficient to retain binding properties to a specific target molecule, typically a receptor or antigen (epitope).

[0074] The terms “antigen” or “Ag” are defined as, as used herein, as molecules that induce an immune response. This immune response may involve antibody production, activation of specific immune-qualified cells, or both. Those skilled in the art will understand that virtually all proteins or peptides and any macromolecules, including polysaccharides, nucleic acids, or lipids, can act as antigens. Furthermore, antigens may be derived from recombinant DNA or genomic DNA. Thus, those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response will encode an “antigen” as the term is used herein. Furthermore, those skilled in the art will understand that antigens do not have to be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent, though not limited to, the use of partial nucleotide sequences of two or more genes and that these nucleotide sequences can be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that antigens may not be encoded by any “gene” at all. It is readily apparent that antigens can be generated and synthesized, or that antigens can be derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0075] The term "avidity," as used herein, refers to the combined strength of multiple binding sites between two molecules, such as between multiple antigen-binding sites of a multispecific antibody that interacts with two targets simultaneously. When two or more binding interactions exist, the two molecules will only dissociate when all binding sites have dissociated. Therefore, the dissociation rate is slower than that of individual binding sites, resulting in a higher combined binding strength (avidity) compared to the binding strength (affinity) of individual binding sites. Thus, avidity is related to both the affinity and specific epitopes of individual binding sites, as well as the valence of the multispecific antibody and antigen. For example, the interaction between a bispecific antibody and an antigen with a repeating epitope structure, such as a polymer, can be considered to have high avidity because the binding may be to multiple epitopes on the antigen.

[0076] The terms “cancer” and “malignant,” as used herein, refer to a physiological condition in mammals typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, B-cell lymphoma (Hodgkin lymphoma and / or non-Hodgkin lymphoma), brain tumors, breast cancer, colon cancer, lung cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer, brain cancer, and, but are not limited to, prostate cancer, including androgen-dependent and androgen-independent prostate cancer.

[0077] The terms “cellular antigen” or “cell-associated antigen,” as used herein, refer to any protein, carbohydrate, or other component derived from or expressed by a cell capable of eliciting an immune response. For example, the cell may be any cell of interest, particularly cancer cells and senescent cells. A cellular antigen may be an antigen located on the surface of a cell or inside a cell. This definition is intended to include, but is not limited to, proteins purified from the cell surface or cell membrane, or unique carbohydrate moieties associated with the cell surface of a cell. This definition also includes antigens from the cell surface, which require special processing of the cell for the antibodies of the present invention to reach.

[0078] The term "conditional activity" or "conditionally active" refers to the higher affinity or avidity of a multispecific antibody under one or more abnormal conditions compared to normal physiological conditions. This conditionally active multispecific antibody can exhibit activity in selected regions of the organism and / or exhibit increased or decreased activity under abnormal or tolerable physiological conditions. In one embodiment, a conditionally active multispecific antibody is virtually inactive under normal physiological conditions but active under abnormal conditions. For example, in one embodiment, a conditionally active multispecific antibody is virtually inactive at normal physiological pH but active at lower pH in the dementia brain or tumor microenvironment. In another embodiment, a conditionally active multispecific antibody can be reversibly or irreversibly inactivated at normal physiological pH. In a further embodiment, a conditionally active multispecific antibody is derived from a therapeutic protein. In another embodiment, a conditionally active multispecific antibody is used as a drug or therapeutic agent.

[0079] The terms “epitope” or “antigenic determinant” as used herein refer to the site on an antigen to which an antibody binds. Epitopes can be formed from either continuous amino acids (linear epitopes) or discontinuous amino acids aligned by tertiary folding of a protein (stereoepitopes). Epitopes formed from continuous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes may contain three or more amino acids. Typically, epitopes consist of at least 5-7 amino acids (e.g., 5, 6, or 7 amino acids in the epitope), or at least 8-11 amino acids (e.g., 8, 9, 10, or 11 amino acids in the epitope), or more than 11 amino acids (e.g., 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in the epitope), or more than 20 amino acids (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in the epitope), and less frequently, 31-40 amino acids. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996). A preferred method for epitope mapping on antigens is surface plasmon resonance.

[0080] The term "full-length antibody" refers to an antibody with a variable antigen-binding region (V H or V L ) and light chain constant domain (C LThis refers to antibodies containing the heavy chain constant domains CH1, CH2, and CH3. The constant domains may be the natural sequence constant domain (e.g., the human natural sequence constant domain) or amino acid sequence variants thereof. Full-length antibodies can be assigned to different "classes" depending on the amino acid sequence of the heavy chain constant domain. There are five main classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be subdivided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different antibody classes are called α, δ, ε, γ, and μ, respectively.

[0081] "Individual," "patient," or "subject" can be a human or an animal. For example, a subject may be a mammal selected from domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0082] The term “library,” as used herein, refers to a collection of nucleic acids or proteins in a single pool. Libraries can be created using DNA recombination techniques. For example, a protein library can be created by inserting a collection of cDNA or any other protein-coding DNA into an expression vector. A collection of cDNA or protein-coding DNA can also be inserted into a phage genome to create a bacteriophage display library of wild-type proteins. Collections of cDNA can be prepared from selected cell populations or tissue samples, such as by the method disclosed by Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). Collections of cDNA from selected cell types are also commercially available from suppliers such as Stratagene®. A library of wild-type proteins as used herein is not a collection of biological samples.

[0083] The term “multispecific antibody,” as used herein, refers to a full-length antibody, antibody fragment, or structure comprising one or more full-length antibodies and antibody fragments having at least two distinct binding sites, each capable of binding to the same or different epitopes on the same antigen. The structure may be a modified antibody having two, three or more (e.g., four, five, six, or seven) functional antigen-binding sites, which are also included within the scope of multispecific antibodies (see, for example, U.S. Patent Application Publication No. 2002 / 0004587 A1 and Brinkman and Kontermann, MAbs, vol. 9, pp. 182–212, 2017).

[0084] The term “pharmaceutically acceptable salt” as used herein refers to the salt form of the conditionally active multispecific antibody of the present invention. Salt forms may be formed with acid addition salts (e.g., with a free amino group) and with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, oxalic acid, tartaric acid, and maleic acid. Salts formed with a free carboxyl group may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, as well as organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, and procaine.

[0085] As used herein, the term “preventive effective dose” means any amount, without limitation, that results in the prevention or improvement of a disease, disorder or adverse event, or a reduction in the rate of progression of a disease or disorder, compared to a corresponding subject who did not receive such a dose. Within that scope, the term also includes amounts that are effective in enhancing normal physiological function, and amounts that are effective in producing physiological functions in a patient that enhance or contribute to the therapeutic or preventive effect of the second drug.

[0086] The term "prevention," as used herein, refers to preventing or avoiding the occurrence of a particular pathological condition. In some embodiments, prevention relates to the improvement of damage associated with pathological conditions, such as tumors or age-related conditions, using the multispecific antibodies of the present invention.

[0087] The term "small molecule," as used herein, refers to a molecule or ion with a molecular weight of less than 900 Da, less than 500 Da, less than 200 Da, or less than 100 Da. In the assays and environments of the present invention, small molecules may often exist as a mixture of molecules and their deprotonated ions, primarily depending on the pH of the assay or environment.

[0088] When used herein, the term “therapeutic dose” means any amount of the multispecific antibody of the present invention that, when compared to a corresponding subject that did not receive such dose, results in the cure, prevention or improvement of a disease, disorder or adverse effect, or a reduction in the rate of progression of the disease or disorder, but is not limited to such amount. Within that scope, the term also includes amounts effective in enhancing normal physiological function and amounts effective in producing physiological functions in a patient that enhance or contribute to the therapeutic effect of the second drug.

[0089] The term “to treat” or “treatment” includes reducing the number of symptoms of a disease or disorder (e.g., cancer) in a subject, or reducing the severity, duration, or frequency of one or more symptoms. The term to treat may also mean delaying the onset or progression of the disorder or symptoms of the disorder in a subject, or the progression of the severity of those symptoms, or extending the lifespan of the subject with the disorder or disorder.

[0090] The term “tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The term “tumor microenvironment” refers to all elements of the tumor environment, including those that create a structural and / or functional environment for malignant processes to survive and / or grow and / or spread.

[0091] The term “unit dosage form,” as used herein, refers to a physically distinct unit suitable as a unit-by-unit dosage for a subject, each unit containing a predetermined amount of the conditionally activated multispecific antibody of the present invention, calculated in an amount sufficient to produce the desired therapeutic effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle.

[0092] When used herein and in the appended claims, it should be noted that the singular forms “a,” “an,” and “it” include a reference to the plural unless the context specifically indicates otherwise. Furthermore, the terms “a” (or “an”), “one or more,” and “at least one” can be used synonymously herein. The terms “contain,” “include,” “have,” and “compose” can also be used synonymously.

[0093] Unless otherwise indicated, all numerical values ​​used in this specification and in the claims to represent the quantities, molecular weights, percentages, ratios, reaction conditions, and other properties of the components shall be understood to be modified by the term "approximately," whether or not the term "approximately" is present. Accordingly, unless otherwise indicated, the numerical parameters shown in this specification and in the claims are approximations that may vary depending on the desired properties to be achieved by this disclosure. At a minimum, and without attempting to limit the application of the doctrine of equivalents to the claims, each numerical parameter should be interpreted by applying ordinary rounding techniques, at least in light of the number of significant figures reported. Although the ranges and parameters of numerical values ​​representing the broad scope of this disclosure are approximations, the numerical values ​​shown in specific examples are reported as accurately as possible. However, each numerical value inherently contains some degree of error that inevitably arises from the standard deviation found in its respective test measurements.

[0094] It should be understood that each component, compound, substituent, or parameter disclosed herein is disclosed for use alone or in combination with one or more of the other components, compounds, substituents, or parameters disclosed herein.

[0095] Furthermore, each quantity / value or quantity / value range for each component, compound, substituent, or parameter disclosed herein should be interpreted as also being disclosed in combination with any other quantity / value or quantity / value range disclosed herein for any other component, compound, substituent, or parameter disclosed herein. Accordingly, any combination of quantities / values ​​or quantity / value ranges for two or more components, compounds, substituents, or parameters disclosed herein should also be understood to be disclosed in combination with each other for the purposes of this description.

[0096] Furthermore, it is understood that each range disclosed herein should be interpreted as a disclosure of each specific value having the same number of significant figures within the disclosed range. Thus, the range 1–4 should be interpreted as an explicit disclosure of the values ​​1, 2, 3, and 4. Furthermore, it is understood that each lower limit of each range disclosed herein should be interpreted as being disclosed in combination with each upper limit and each specific value within each range disclosed herein for the same component, compound, substituent, or parameter. Thus, this disclosure should be interpreted as a disclosure of all ranges derived by combining each lower limit of each range with each upper limit or each specific value within each range, or by combining each upper limit of each range with each specific value within each range.

[0097] Furthermore, any specific amounts / values ​​of components, compounds, substituents, or parameters disclosed in this description or examples should be interpreted as disclosures of either a lower or upper limit of a range, and can therefore be combined with any other lower or upper limit or specific amounts / values ​​of the same component, compound, substituent, or parameter disclosed in other parts of this application to form a range for that component, compound, substituent, or parameter.

[0098] In one embodiment, the present invention provides a multispecific antibody comprising at least one binding site for a cell antigen and at least one binding site for a tumor-reactive lymphocyte antigen. This multispecific antibody binds to at least one cell antigen and tumor-reactive lymphocyte antigen with greater affinity under first physiological conditions than under second physiological conditions. In some embodiments, the first physiological conditions are abnormal conditions, and the second physiological conditions are normal physiological conditions. For example, the abnormal conditions may be conditions in the tumor microenvironment. The multispecific antibody of the present invention may be referred to as a conditionally active multispecific antibody. As another example, the abnormal conditions may be conditions in the senescent cell microenvironment. In this regard, senescent cells are characterized as abnormal because they are not the same as normal cells that previously existed in the same location before senescence occurred. Abnormal conditions in the senescent cell microenvironment may be an acidic pH, a lower oxygen concentration, and / or a senescence-associated secretory phenotype (SASP).

[0099] In some embodiments, conditionally activated multispecific antibodies are virtually inactive under normal physiological conditions but active under abnormal conditions, and optionally have a higher activity level compared to the activity of the conditionally activated multispecific antibody under normal physiological conditions or the activity of the original parent antibody from which it is derived under normal physiological conditions. In another embodiment, conditionally activated multispecific antibodies are virtually inactive at pH 7.2–7.4 but active at a lower pH of 6.0–6.8. In some cases, conditionally activated multispecific antibodies are reversibly or irreversibly inactivated under normal physiological conditions. In another example, conditionally activated multispecific antibodies may be more or less active in high-oxygen blood or low-pH environments found in the tumor microenvironment, for example, after passing through the lungs. Conditionally activated multispecific antibodies may be used as drugs, therapeutic agents or diagnostic agents.

[0100] In some embodiments, the binding of multispecific antibodies to cell antigens and / or tumor-reactive lymphocyte antigens is reversible. That is, the multispecific antibody binds to the cell antigen and / or tumor-reactive lymphocyte antigen, and subsequently, these two can separate. The separated multispecific antibody has the ability to re-bind to the cell antigen and / or tumor-reactive lymphocyte antigen.

[0101] In some embodiments, the cell antigen may be a cell surface antigen or an intracellular antigen. Cells may be targeted for inhibition, damage, destruction, or killing by tumor-reactive lymphocytes. These cells may be referred to as target cells. Thus, cells may be targets in the treatment with the multispecific antibodies of the present invention. Specifically, for the treatment of certain diseases or conditions, cells may be targets for elimination. For example, cancer cells and senescent cells may be targets for elimination. In such cases, the cell antigen may be a cancer cell antigen or a senescent cell antigen.

[0102] In some embodiments, the cell antigens are preferentially associated with target cells but are less commonly found in other cell types. Thus, the multispecific antibodies of the present invention can preferentially interact with target cells. Target cells may be cancer cells. Examples of cancer cell-specific antigens include 4-IBB, 5T4, adenocarcinoma antigen, α-fetoprotein, BAFF, B-lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, and CD44. v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, Fibronectin Extradomain-B, Folate Receptor 1, GD2, GD3 Ganglioside, Glycoprotein 75, GPNMB, HER2 / neu, B7-H3, Axl, Ror2, HGF, Human Scattering Factor Receptor Kinase, IGF-1 Receptor, IGF-I, IgGl, LI-CAM, IL-13, IL-6, Insulin-like Growth Factor I Receptor, Integrin α5β1, Integrin ανβ3, MORAb-009, MS4A1, MUC1, Mucin CanAg, N-Glycolylneuraminic Acid, NPC-1C, PDGF-Ra, PDL192, Phosphatidylserine, Prostate Cancer Cells, RANKL, RON, ROR1, SCH Examples include 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, and vimentin.

[0103] In one embodiment, the cancer cell-specific antigen is selected from CD3, Axl, EpCAM, Her2, Ror2, and B7-H3.

[0104] In one embodiment, the targeted cancer cells are breast cancer cells, in which case the breast cancer cell-specific antigen may be one of the following: EpCAM (epithelial cell adhesion molecule), Her2 / neu (human epidermal growth factor receptor 2), MUC-1, EGFR (epidermal growth factor receptor), TAG-12 (tumor-associated glycoprotein 12), IGFlR (insulin-like growth factor 1 receptor), TACSTD2 (tumor-associated calcium signaling transducer 2), CD318, CD340, CD104, and N-cadherin.

[0105] In another embodiment, the cancer cells are prostate cancer cells, in which case the prostate cancer cell-specific antigen may be one of EpCAM, MUC-1, EGFR, PSMA (prostate-specific membrane antigen), PSA (prostate-specific antigen), TACSTD2, PSCA (prostate stem cell antigen), PCSA (prostate cell surface antigen), CD318, CD104, and N-cadherin.

[0106] In yet another embodiment, the cancer cells are colorectal cancer cells, in which case the colorectal cancer cell-specific antigen may be one of EpCAM, CD66c, CD66e, CEA (carcinoembryonic antigen), TACSTD2, CK20 (cytokeratin 20), CD104, MUC-1, CD318, and N-cadherin.

[0107] In yet another embodiment, the cancer cells are lung cancer cells, in which case the lung cancer cell-specific antigen may be one of CK18, CK19, CEA, EGFR, TACSTD2, CD318, CD104, and EpCAM.

[0108] In another embodiment, the cancer cells are pancreatic cancer cells, in which case the pancreatic cancer cell-specific antigen may be one of HSP70, mHSP70, MUC-1, TACSTD2, CEA, CD104, CD318, N-cadherin, and EpCAM.

[0109] In a further embodiment, the cancer cells are ovarian cancer cells, in which case the ovarian cancer cell-specific antigen may be one of MUC-1, TACSTD2, CD318, CD104, N-cadherin, and EpCAM.

[0110] In yet another embodiment, the cancer cells are bladder cancer cells, in which case the bladder cancer cell-specific antigens may be CD34, CD146, CD62, CD105, CD106, VEGF receptor (vascular endothelial growth factor receptor), MUC-1, TACSTD2, EpCAM, CD318, EGFR, 6B5, and one of the folate-binding receptors.

[0111] In some cases, cancer cells are cancer stem cells, in which case the cancer stem cell-specific antigen may be one of CD133, CD135, CD117, or CD34.

[0112] In other cases, the cancer cells are melanoma cancer cells, in which case the melanoma cancer cell-specific antigen may be one of the melanocyte differentiation antigens, carcinoembryonic antigens, or SEREX antigens. Examples of melanocyte differentiation antigens include, but are not limited to, tyrosinase, gp75, gplOO, MART1, or TRP-2. Examples of carcinoembryonic antigens include antigens of the MAGE family (MAGE-Al, MAGE-A4), BAGE family, GAGE ​​family, and NY-ESOl. Examples of SEREX antigens include Dl and SSX-2. Further examples of tumor-specific antigens include CDK4 and 13-catenin.

[0113] In some embodiments, the cancer cell antigen is a neoantigen. Examples are given in Table 1. [Table 1] JPEG2026123122000002.jpg171170

[0114] In some embodiments, the cell-specific antigens are APC, ARHGAP1, ARMCX-3, AXL, B2MG, BCL2L1, CAPNS2, CD261, CD39, CD54, CD73, CD95, CDC42, CDKN2C, CLYBL, COPG1, CRKL, DCR1, DCR2, DCR3, DEP1, DGKA, EBP, EBP50, FASL, FGF1, GBA3, GIT2, ICAM1, ICAM3, IGF1, ISG20, ITGAV, KITLG, Lamin B1, LANCL1, LCMT2, LPHN1, MADCAM1, MAG, MAP3K14, MAPK The antigens selected may include senescent cell-specific antigens such as MEF2C, miR22, MMP3, MTHFD2, NAIP, NAPG, NCKAP1, Nectin 4, NNMT, NOTCH3, NTAL, OPG, OSBPL3, p16, p16INK4a, p19, p21, p53, PAI1, PARK2, PFN1, PGM, PLD3, PMS2, POU5F1, PPP1A, PPP1CB, PRKRA, PRPF19, PRTG, RAC1, RAPGEF1, RET, Smurf2, STX4, VAMP3, VIT, VPS26A, WEE1, YAP1, YH2AX, and YWHAE.

[0115] Other antigens to which the multispecific antibodies of the present invention bind may be tumor-reactive lymphocyte antigens, which are antigens of lymphocytes that target tumor cells. In particular, lymphocytes are tumor-reactive when they attack, inhibit, or destroy tumor cells. Tumor-reactive lymphocytes may be selected from T cells, macrophages, Jurkat cells, monocytes, NK cells, activated NK cells, neutrophils, eosinophils, basophils, B cells, and lymphokine-activated killer (LAK) cells. T cells may be naive T cells, helper T cells, effector T cells, memory T cells, cytotoxic T cells, antigen-specific T cells, and CD28-CD27-CD4-positive T cells.

[0116] Antigens of tumor-reactive lymphocytes typically include markers on T cells such as CD2, CD3, CD4, CD8, CD25, CD28, CD27, CD45RA, CD45RO, CD62L, CD95, CD127, CD137, α / βTCR, γ / δTCR, CCR7, PD-1, and Lag3. Some examples of antigens on macrophages include CCR2, CD14, CD68, CD163, CSFIR, and MSR1.

[0117] While we do not wish to be limited by theory, the multispecific antibodies of the present invention bind to both target cells and tumor-reactive lymphocytes, thereby bringing the target cells into very close proximity to the tumor-reactive lymphocytes. This is thought to promote the attack of the tumor-reactive lymphocytes on the target cells, thereby inhibiting, damaging, or destroying them. The therapeutic effect of inhibiting or eliminating tumor cells and / or senescent cells can be achieved by using the multispecific antibodies of the present invention to deliver reactive lymphocytes to tumor cells and / or senescent cells for inhibition, destruction, and elimination of tumor cells and / or senescent cells from the target.

[0118] The first and second physiological conditions are different numerical values ​​of the same condition, which can be selected from temperature, pH, osmotic pressure, osmolality, oxidative stress, oxygen concentration, and electrolyte concentration. For example, the first physiological condition may be an acidic pH in the tumor microenvironment, in the range of 5.2–7.0, 5.8–7.0, or 6.0–6.8. The second physiological condition may be a normal physiological pH in the subject's blood, in the range of 7.0–7.8 or 7.2–7.6.

[0119] In some embodiments, the first physiological condition is a hypoxic concentration in the tumor microenvironment, and the second physiological condition is a normal physiological oxygen concentration in the subject's blood. In some other embodiments, the first physiological condition is an abnormal condition in the environment surrounding senescent cells (senescent cell microenvironment), such as an acidic pH and / or hypoxic concentration. In addition, abnormal conditions in the senescent cell microenvironment also include senescence-associated secretory phenotypes (SASPs). Senescent cells in the senescent cell microenvironment are metabolically active and secrete proteins that form a signature of the senescent cell microenvironment, producing senescence-associated secretory phenotypes (Coppe et al., Annu Rev Pathol., vol.5, pp.99-118, 2010).

[0120] SASP, also known as the aging messenger secretome, may include the expression / secretion of the following biologically active factors (Pawlikowski et al., J Cell Sci, vol.126, pp.4061-4067, 2013): i. Interleukins, e.g., IL-1α, IL-1β, IL-6, IL-7, IL-13, IL-15; ii. Chemokines, e.g., IL-8, MCP2, MCP4, GROα, GROβ, GROγ; iii. Growth factors, e.g., EGF, HGF, VEGF; iv. Receptors and ligands, e.g., ICAM1, ICAM3, TRAIL-R3, Fas, uPAR, sTNFRI, sTNFRIII; v. Proteases and regulatory factors, e.g., MMP1, MMP3, MMP10, MMP12, TIMP1, TIMP2, PAI1, PAI2; and vi. Extracellular insoluble molecules, such as collagen, fibronectin, and laminin. One or more of these factors may be used as conditions to produce the conditioned activity of the present invention.

[0121] In addition, SASP can be further characterized by the following features (Pawlikowski et al., J Cell Sci, vol.126, pp.4061-4067, 2013): a. Enlarged, flattened form, b.p16 INK4a Expression, c. Increased lysosomal activity (aging-related β-galactosidase; SA β-gal), d. DNA damage response, e. Chromatin remodeling, and f. Autophagy.

[0122] In some embodiments, SASP is the following factors: IL-1α, IL-1β, IL-6, IL-7, IL-8, IL-10, IL-13, IL-15, IL-18, MCP1, MCP2, MCP4, MIF, MIP-1a, MIP-3a, HCC-4, Eotaxin-3, TECK, ENA-78, I-309, I-TAC, GROα, GROβ, GROγ, VEGF It contains one or more of the following: EGF, HGF, FGF, bFGF, KGF, amphiregulin, epiregulin, heregulin, SCF, SDF-1α, PIGF, IGFBP-2, -3, -4, -6, -7, GM-CSF, PDGF-BB, TGF-α, TGF-β1, TGF-β2, TGF-β3, ICAM1, ICAM3, TRAIL-R3, Fas, OPG, SGP130, EGF-R uPAR, sTNFRI, sTNFRIII, MMP1, MMP3, MMP7, MMP9, MMP10, MMP12, MMP13, MMP14, TIMP1, TIMP2, PAI1, PAI2, SLPI, endothelin, collagen, fibronectin, and laminin. In some embodiments, SASP comprises one or more of IL-8, GROα, VEGF, endothelin, MMP7, MMP9, MMP10, MMP12, MMP13, TIMP1, TIMP2, and TGF-β1. In some other embodiments, SASP comprises at least IL-8, GROα, VEGF, endothelin, MMP7, MMP9, MMP10, MMP12, MMP13, TIMP1, TIMP2, and TGF-β1. One or more of these factors can be used as conditions to produce the conditioned activity of the present invention.

[0123] In some other embodiments, the first physiological condition is an abnormal condition in the diseased site, tissue, or organ. The second physiological condition is typically a normal physiological condition in the blood of the subject, such as a normal physiological pH.

[0124] A multispecific antibody binds to at least one cell-specific antigen and a reactive lymphocyte antigen with increased affinity under first physiological conditions compared to affinity under second physiological conditions. In some embodiments, the multispecific antibody binds to at least one of the cell-specific antigen and the reactive lymphocyte antigen with increased affinity under first physiological conditions compared to affinity under second physiological conditions. For example, a multispecific antibody may bind to a cell-specific antigen with increased binding affinity under first physiological conditions compared to binding affinity under second physiological conditions, while still binding to a reactive lymphocyte antigen with unconditional activity. In another example, the multispecific antibody binds to a reactive lymphocyte antigen with increased binding affinity under first physiological conditions compared to binding affinity under second physiological conditions, while still binding to a cell-specific antigen with unconditional activity. In some embodiments, the multispecific antibody binds to both the cell-specific antigen and the reactive lymphocyte antigen with higher avidity under first physiological conditions compared to avidity under second physiological conditions.

[0125] The structure / format of a bispecific antibody may be one of the structures / formats described in Brinkmann and Kontermann, “The making of bispecific antibodies,” MABs, vol.9, pp.182-212, 2017. Specifically, Figure 2 in Brinkmann and Kontermann's work describes 19 different structures / formats of bispecific antibodies. These structures / formats include (1) bispecific antibody conjugates; (2) hybrid bispecific IgG2; (3) bispecific antibody molecules with "variable domain only"; (4) CH1 / CL fusion proteins; (5) Fab fusion proteins; (6) non-immunoglobulin fusion proteins; (7) Fc-modified IgG; (8) added and Fc-modified IgG; (9) modified Fc and CH3 fusion proteins; (10) added IgG-HC fusions; (11) added IgG-LC fusions; (12) added IgG-HC and LC fusions; (13) Fc fusions; (14) CH3 fusions; (15) IgE / IgM CH2 fusions; (16) F(ab')2 fusions; (17) CH1 / CL fusion proteins; (18) modified IgG; and (19) non-immunoglobulin fusions.

[0126] In detailed embodiments, the multispecific antibody may be a bivalent scFv-Fc heterodimer as shown in Figure 1 or a tetravalent homodimer "butterfly" as shown in Figure 2. In these two structures, the reactive lymphocyte antigen is not limited to CD3, which is merely depicted as a representative example of a tumor-reactive lymphocyte antigen. The multispecific antibody in Figure 1 has a first binding site for a cellular antigen (Ag) linked to a first heavy chain constant region (e.g., IgG), and a second binding site for a reactive lymphocyte antigen (e.g., CD3) linked to a second heavy chain constant region (e.g., IgG). The two heavy chains are modified, for example, using a knob-in-hole technique, so that only heterodimers can be formed. The first and second binding sites are scFv antibodies that bind to the cellular antigen and the reactive lymphocyte antigen, respectively. Either or both of the first and second binding sites have conditionally active binding activity for their respective antigens.

[0127] The multispecific antibody shown in Figure 2 may have a full-length IgG antibody that binds to a cell-specific antigen (Ag) and an scFv antibody that binds to a reactive lymphocyte antigen (e.g., CD3). The scFv antibody is linked to the C-terminus of the light chain of the IgG antibody via a linker. The linker is a short alanine linker (Ala). n Serine Linker (Ser) n The linker may be hydrophilic or glycine-serine rich. The heavy chain of the IgG antibody combines with the light chain of the IgG antibody linked to the scFv antibody, thus forming half of the homodimer. This multispecific antibody has a "butterfly" configuration.

[0128] In some embodiments, the multispecific antibody comprises an IgG antibody or fragment thereof that binds to a tumor-reactive lymphocyte antigen and a single-chain antibody that binds to a tumor cell antigen, similarly forming a “butterfly” configuration as shown in Figure 2. The single-chain antibody may be an scFv antibody. The scFv antibody may be linked to the C-terminus of the IgG antibody via a linker as described herein.

[0129] The binding sites of the multispecific antibodies of the present invention each include a light chain variable region and a heavy chain variable region. The light chain variable region and the heavy chain variable region may be in a single-chain antibody format or in a double-chain format as formed by the combination of the light chain and the heavy chain (Figures 1-2). In a conditionally active binding site, either the light chain and the heavy chain variable region may be conditionally active, or both may be conditionally active. Exemplary light chain variable regions that bind to CD3 include an unconditionally active light chain variable region of the amino acid sequence of SEQ ID NO: 1 and a conditionally active light chain variable region of an amino acid sequence selected from SEQ ID NOs: 2-10. Exemplary heavy chain variable regions that bind to CD3 include an unconditionally active heavy chain variable region of the amino acid sequence of SEQ ID NO: 11 and a conditionally active heavy chain variable region of an amino acid sequence selected from SEQ ID NOs: 12-15.

[0130] Exemplary light chain variable regions that bind to Axl include the unconditionally activated light chain variable region of the amino acid sequence of SEQ ID NO: 16 and the conditionally activated light chain variable region of the amino acid sequence of SEQ ID NO: 17. Exemplary heavy chain variable regions that bind to Axl include the unconditionally activated heavy chain variable region of the amino acid sequence of SEQ ID NO: 18 and the conditionally activated heavy chain variable region of the amino acid sequence of SEQ ID NO: 19.

[0131] The exemplary light chain variable region that binds to Her2 is the unconditionally active light chain variable region of the amino acid sequence of SEQ ID NO: 20. The exemplary heavy chain variable region that binds to Her2 is the unconditionally active heavy chain variable region of the amino acid sequence of SEQ ID NO: 21.

[0132] The exemplary light chain variable region that binds to B7-H3 is the unconditionally activated light chain variable region of the amino acid sequence of SEQ ID NO: 22. The exemplary heavy chain variable regions that bind to B7-H3 include the unconditionally activated heavy chain variable region of the amino acid sequence of SEQ ID NO: 23 and the conditionally activated heavy chain variable region of an amino acid sequence selected from SEQ ID NOs: 24-25.

[0133] The exemplary light chain variable region that binds to EpCAM is the unconditionally active light chain variable region of the amino acid sequences of SEQ ID NOs. 88-95. The exemplary heavy chain variable region that binds to EpCAM includes the unconditionally active heavy chain variable region of the amino acid sequences of SEQ ID NOs. 80-87. One light chain variable region combines with one heavy chain variable region to form a binding site for EpCAM. This EpCAM binding site is linked to a single-chain anti-CD3 antibody having an amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 26-71 to form a multispecific antibody that binds to both EpCAM and CD3.

[0134] Some examples of multispecific antibodies that bind to EpCAM and CD3 are shown in Table 2 below. [Table 2]

[0135] In some other embodiments, a multispecific antibody can be constructed having two variable regions that form binding sites for cell-specific antigens and two other variable regions that form binding sites for reactive lymphocyte antigens, as shown in Figure 1. These variable regions can be selected from light and heavy chain variable regions having amino acid sequences of SEQ ID NOs: 1-25 and 80-95. One or both of the binding sites must be conditionally active for their respective antigens. At each conditionally active binding site, at least one of the light chain variable region and the heavy chain variable region has increased affinity for its antigen under first physiological conditions (e.g., abnormal conditions) compared to affinity under second physiological conditions (e.g., normal physiological conditions). Thus, a person skilled in the art can select appropriate light chain and heavy chain variable regions from those having amino acid sequences of SEQ ID NOs: 1-25 and 80-95 to construct a multispecific antibody as shown in Figure 1. The heavy chain fragment in Figure 1 is selected from the constant region of an IgG antibody, including any subclass of IgG: IgG1, IgG2, IgG3, IgG4.

[0136] In some other embodiments, multispecific antibodies can be constructed as shown in Figure 2. Similarly, the light chain variable regions and heavy chain variable regions of scFv antibodies and full-length IgG antibodies can be selected from light chain and heavy chain variable regions having amino acid sequences of SEQ ID NOs. 1-25 and 80-95. At each binding site exhibiting conditional activity, at least one of the light chain variable region and heavy chain variable region has increased affinity for its antigen under first physiological conditions (e.g., abnormal conditions) compared to affinity under second physiological conditions (e.g., normal physiological conditions). Thus, those skilled in the art can select appropriate light chain and heavy chain variable regions from those having amino acid sequences of SEQ ID NOs. 1-25 and 80-95 to construct the multispecific antibodies shown in Figure 2. The constant region in Figure 2 is selected from the constant region of an IgG antibody, including any subclass of IgG: IgG1, IgG2, IgG3, IgG4.

[0137] In some embodiments, the multispecific antibody binds to CD3 as a tumor-reactive lymphocyte antigen and to another tumor-associated antigen (TAA) as a cell-specific antigen. The multispecific antibody has a binding site for CD3 that includes a light chain variable region having the amino acid sequence of SEQ ID NOs. 1-10 and a heavy chain variable region having the amino acid sequence of SEQ ID NOs. 11-15. Alternatively, the binding site for CD3 includes an anti-CD3 single-chain antibody having an amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 26-71. The multispecific antibody has a binding site for TAA that includes a light chain variable region having the amino acid sequence of SEQ ID NOs. 16-17, 20, and 22 that binds to one of Axl, Her2, and B7-H3, and a light chain variable region selected from the heavy chain variable regions having the amino acid sequence of SEQ ID NOs. 18-19, 21, and 23-25 ​​that binds to one of Axl, Her2, and B7-H3. The binding site for EpCAM includes a light chain variable region of an amino acid sequence selected from SEQ ID NOs. 88-95 and a heavy chain variable region of an amino acid sequence selected from SEQ ID NOs. 80-87. At least one of the binding sites for CD3 and the binding sites for Axl, EpCAM, Her2, or B7-H3 is conditionally active.

[0138] Table 3 shows some examples of multispecific antibodies. These examples have binding sites for TAA and CD3, respectively. "WT" indicates that the affinity of the binding site is unconditional, meaning that the multispecific antibody does not have a significant difference in affinity for the antigen between the first condition (e.g., abnormal condition) and the second condition (e.g., normal physiological condition). "CAB" indicates that the affinity of the binding site is conditional, meaning that the multispecific antibody has a greater affinity for the antigen under the first condition (e.g., abnormal condition) than under the second condition (e.g., normal physiological condition). [Table 3]

[0139] In some embodiments, one or both of the IgG antibody and / or single-chain antibody are therapeutic or prophylactic antibodies administered to a subject to treat or prevent a disease or condition, or to improve the subject's health. Therapeutic or prophylactic antibodies may be approved for therapeutic or prophylactic use in humans or animals by national or regional regulatory bodies such as the U.S. Food and Drug Administration and the European Medicines Agency.

[0140] In some other embodiments, one or both of the IgG antibody and / or single-chain antibody are biosimilars, which are biopharmaceuticals (as defined in 42 U.S.C., 262(i)) that are considered equivalent in terms of quality, safety, and efficacy to a reference biopharmaceutical marketed by the original pharmaceutical company. There may be slight differences in clinically inactive components between the biosimilar and the reference biopharmaceutical.

[0141] In one embodiment, a multispecific antibody binds to cancer cell-specific antigens with greater affinity under abnormal conditions than under normal physiological conditions. In another embodiment, a multispecific antibody binds to reactive lymphocyte antigens with greater affinity under abnormal physiological conditions than under normal physiological conditions. In yet another embodiment, a multispecific antibody binds to both cancer cell-specific antigens and reactive lymphocyte antigens with greater affinity under abnormal conditions than under normal physiological conditions. In yet another embodiment, a multispecific antibody binds to a combination of cancer cell-specific antigens and reactive lymphocyte antigens with greater avidity under abnormal conditions than under normal physiological conditions. Abnormal and normal physiological conditions may be selected from pH, oxygen concentration, or any other conditions that distinguish the tumor microenvironment from the target blood.

[0142] In one embodiment, a multispecific antibody binds to senescent cell-specific antigens with greater affinity under abnormal conditions of the senescent cell microenvironment than under normal physiological conditions. In another embodiment, a multispecific antibody binds to reactive lymphocyte antigens with greater affinity under abnormal conditions of the senescent cell microenvironment than under normal physiological conditions. In yet another embodiment, a multispecific antibody binds to both cancer cell-specific antigens and reactive lymphocyte antigens with greater affinity under abnormal conditions of the senescent cell microenvironment than under normal physiological conditions. In yet another embodiment, a multispecific antibody binds to a combination of cancer cell-specific antigens and reactive lymphocyte antigens with greater avidity under abnormal conditions of the senescent cell microenvironment than under normal physiological conditions. The abnormal and normal physiological conditions may be selected from pH, oxygen concentration, or any other conditions that distinguish the senescent cell microenvironment from the target blood or normal tissue.

[0143] In another embodiment, the present invention provides a multispecific antibody comprising an IgG antibody that binds to a first antigen (e.g., a cell-specific antigen) and at least one scFv antibody that binds to a second antigen different from the first antigen (e.g., a reactive lymphocyte antigen). The scFv antibody may be linked at the C-terminus of the IgG antibody via a linker as described herein. The multispecific antibody reversibly binds to at least one of the first and second antigens with greater affinity under abnormal conditions than under normal physiological conditions.

[0144] The first and second antigens are not limited to specific antigens, but rather can be any antigen pair having some relationship that facilitates the achievement of the desired result. In one embodiment, the first antigen is a cell surface antigen. The first antigen may be a cancer cell-specific antigen such as Axl, Ror2, Her2, EpCAM, or B7-H3. In another embodiment, the second antigen is an antigen of tumor-reactive lymphocytes, such as CD3. Further examples of suitable cancer cell antigens and reactive lymphocyte antigens are described in other parts of this application.

[0145] In some embodiments, the multispecific antibody is in the format shown in Figure 2, where the scFv antibody includes a light chain variable region and a heavy chain variable region selected from those that bind to CD3 and have the amino acid sequences of SEQ ID NOs. 1-10 and SEQ ID NOs. 11-15. Alternatively, the binding site to CD3 includes an anti-CD3 single-chain antibody having an amino acid sequence selected from those that bind to SEQ ID NOs. 26-71. The IgG antibody includes a light chain variable region that binds to TAA and binds to one of Axl, Her2, and B7-H3 and has the amino acid sequences of SEQ ID NOs. 16-17, 20, and 22, and a heavy chain variable region selected from those that bind to one of Axl, Her2, and B7-H3 and have the amino acid sequences of SEQ ID NOs. 18-19, 21, and 23-25. The binding site for EpCAM includes a light chain variable region of an amino acid sequence selected from SEQ ID NOs: 88-95 and a heavy chain variable region of an amino acid sequence selected from SEQ ID NOs: 80-87. The constant regions of the light and heavy chains of the IgG antibody may be selected from the constant regions of the IgG antibody, including any subclass of IgG: IgG1, IgG2, IgG3, and IgG4. Among these multispecific antibodies, a multispecific antibody is considered to be conditionally active and within the scope of the present invention as long as at least one of the binding sites for CD3 and the binding sites for Axl, EpCAM, Her2, or B7-H3 is conditionally active.

[0146] The present invention provides a platform for producing multispecific antibodies, which can significantly reduce development time. This platform may be called "plug and play," in which a single conditionally activated antitumor-reactive lymphocyte antigen antibody or antibody fragment (e.g., anti-CD3 antibody) can be covalently bound to another antibody or antibody fragment against another antigen (e.g., cancer cell surface antigen) to produce a conditionally activated multispecific antibody. The antibody or antibody fragment against the other antigen may or may not be conditionally activated.

[0147] The advantage of this "plug-and-play" platform is that, for example, once a conditionally activated antitumor-reactive lymphocyte antigen antibody or antibody fragment (e.g., anti-CD3 antibody) becomes available, a conditionally activated multispecific antibody can be produced by linking it to another antibody or antibody fragment. The conditionally activated antitumor-reactive lymphocyte antibody or antibody fragment is a full-length antibody, V H and V L The antibody fragments or single-chain antibodies containing the region are possible. Since the two components of the conditionally active multispecific antibody produced are known to possess conditional binding activity and binding affinity to their respective antigens, it is reasonably expected that the production of conditionally active multispecific antibodies or antibody fragments will be successful with minimal development time. As demonstrated by the present invention, this platform has been successfully applied to the production of multispecific antibodies that bind to both CD3 and each of the antigens AXL, EpCAM, HER2, and B7-H3.

[0148] In some embodiments, abnormal conditions are acidic pH in the range of about 5.0 to about 7.0, or about 5.2 to about 6.8, or about 5.4 to about 6.8, or about 5.6 to about 6.8, or about 5.8 to about 6.8, or about 6.0 to about 6.8, or about 6.2 to about 6.8, or about 6.4 to about 6.8, or about 6.6 to about 6.8. In some embodiments, the acidic pH may be in the range of about 6.4 to about 7.0, or about 6.6 to about 7.0, or about 6.8 to about 7.0. Normal physiological conditions may be the normal physiological pH in the blood, which is well established in the art. In some embodiments, the normal physiological pH in the blood may be in the range of about 7.0 to about 7.8, or about 7.1 to about 7.7, or about 7.2 to about 7.6, or about 7.2 to about 7.5, or about 7.2 to about 7.4.

[0149] In certain embodiments, the multispecific antibodies of the present invention have a ratio of affinity or avidity to cell antigens and / or tumor-reactive lymphocyte antigens under abnormal conditions to the same affinity or avidity under normal physiological conditions of at least about 1.3:1, or at least about 2:1, or at least about 3:1, or at least about 4:1, or at least about 5:1, or at least about 6:1, or at least about 7:1, or at least about 8:1, or at least about 9:1, or at least about 10:1, or at least about 11 The ratio is 1, or at least about 12:1, or at least about 13:1, or at least about 14:1, or at least about 15:1, or at least about 16:1, or at least about 17:1, or at least about 18:1, or at least about 19:1, or at least about 20:1, or at least about 30:1, or at least about 40:1, or at least about 50:1, or at least about 60:1, or at least about 70:1, or at least about 80:1, or at least about 90:1, or at least about 100:1.

[0150] In one embodiment, the multispecific antibody consists solely of naturally occurring amino acids. There are 20 naturally occurring amino acids, which are referred to as alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine ​​(cys or C), glutamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y), and valine (val or V).

[0151] In some embodiments, the multispecific antibody contains one or more amino acids that do not exist in nature. For example, amino acids that do not exist in nature include carbonyl groups, acetyl groups, aminooxy groups, hydrazine groups, hydrazide groups, semicarbazide groups, azide groups, or alkyne groups. For example, see U.S. Patent No. 7,632,924 for suitable amino acids. The term “amino acids that do not exist in nature” also includes amino acids that are produced by modification (e.g., post-translational modification) of naturally occurring amino acids, but which are not naturally incorporated into the growing polypeptide chain by the biological translation complex. Examples of such amino acids that do not exist in nature include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

[0152] In some embodiments, the multispecific antibodies are in the form of “mimetic” or “peptide mimetic,” which either include those composed entirely of unnatural analogs of amino acid synthesis, or are chimeric molecules in which some are naturally occurring amino acids and some are unnatural analogs of amino acids. The mimetic may also incorporate any amount of naturally occurring amino acid-conserving substitutions, as long as such substitutions do not substantially alter the antibody’s structure and / or activity.

[0153] The mimetic type may include any combination of non-natural structural components. In one embodiment, the mimetic of the present disclosure includes three structural groups: a) residue linking groups other than natural amide bonds ("peptide bonds"); b) non-natural residues in place of naturally occurring amino acid residues; or c) residues that induce secondary structure mimicry, i.e., residues that induce or stabilize secondary structures such as β-turns, γ-turns, β-sheets, α-helix conformations, etc. For example, a multispecific antibody can be characterized as a mimetic when all or some of its residues are linked by chemical means other than natural peptide bonds. Individual peptide mimetic residues can be linked by peptide bonds, other chemical bonds or coupling means, such as glutaraldehyde, N-hydroxysuccinimide esters, difunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC), or N,N'-diisopropylcarbodiimide (DIC). Examples of linking groups that can replace conventional amide bonds ("peptide bonds") include ketomethylene (e.g., -C(=O)~NH- instead of ~C(=O)~CH2~), aminomethylene (CH2-NH), ethylene, olefin (CH=CH), ether (CH2~O), thioether (CH2~S), tetrazole, thiazole, retroamide, thioamide, or ester (see, for example, Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, vol.7, pp.267-357, “Peptide Backbone Modifications,” in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, vol.7, B. Weistein, ed., New York: Marcell Dekker, pp.257-267).

[0154] Further examples of amino acid residues that do not exist in nature include D- or L-naphthylalanine; D- or L-phenylglycine; D- or L-2-thienylalanine; D- or L-1,-2,3- or 4-pyrenylalanine; D- or L-3-thienylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyradinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine; Examples of non-natural amino acid aromatic rings include D-(trifluoromethyl)-phenylalanine; Dp-fluoro-phenylalanine; D- or Lp-biphenylphenylalanine; D- or Lp-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and D- or L-alkylamines (where alkyl may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-isobutyl, isopentyl, or a non-acidic amino acid). Examples of non-natural amino acid aromatic rings include thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

[0155] Acidic non-natural amino acids can be produced by substitution with non-carboxylate amino acids, such as (phosphono)alanine or sulfated threonine, while maintaining a negative charge. Carboxyl side groups (e.g., aspartyl or glutamyl) can also be selectively modified by reaction with carbodiimides (R'~NC--N--R'), such as 1-cyclohexyl-3(2-morpholinyl-(4-ethyl)carbodiimide or l-ethyl-3(4-azonia-4,4-dimethylpentyl)carbodiimide. Aspartyl or glutamyl can also be converted to asparaginyl and glutamyl residues by reaction with ammonium ions.

[0156] Basic non-natural amino acids can be produced, for example, by substitution with ornithine, citrulline, or (guanidino)-acetic acid or (guanidino)alkyl-acetic acid (where alkyl is as defined above), in addition to lysine and arginine. Nitrile derivatives (e.g., containing a CN- moiety instead of COOH) can be substituted for asparagine or glutamine. Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues. Arginine residue mimetic can be produced by reacting arginyl with, for example, one or more conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin (which may be under alkaline conditions). Tyrosine residue mimetic can be produced by reacting tyrosyl with, for example, an aromatic diazonium compound or tetranitromethane. Using N-acetylimidazole and tetranitromethane, O-acetyltyrosyl species and 3-nitro derivatives can be formed, respectively. Cysteine ​​residue mimics can be produced by reacting cysteinyl residues with, for example, α-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines to produce carboxymethyl or carboxyamidemethyl derivatives. Cysteine ​​residue mimics can also be produced by reacting cysteinyl residues with, for example, bromo-trifluoroacetone, α-bromo-β-(5-imidazoyl)propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercrinebenzoate; 2-chloromercrine-4-nitrophenol; or chloro-7-nitrobenzo-oxa-l,3-diazole. Lysine mimics can be produced (and the amino-terminal residue can be altered) by reacting ricinyl with, for example, succinic acid or other carboxylic acid anhydrides.Lysine and other α-amino acid-containing residue mimics can also be produced by reactions with imide esters such as methyl picolinimide, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4,pentanedione, and by transamidase-catalyzed reactions with glyoxylic acid. For example, a methionine mimic can be produced by reaction with methionine sulfoxide. Examples of proline mimics include pipecolic acid, thiazolidinediolic acid, 3- or 4-hydroxyproline, dehydroproline, 3- or 4-methylproline, or 3,3-dimethylproline. A histidine residue mimic can be produced by reacting histidyl with, for example, diethyl pyrocarbonate or p-bromophenacyl bromide. Other mimics include, for example, those produced by hydroxylation of proline and lysine; phosphorylation of hydroxyl groups of ceryl or threonyl residues; methylation of α-amino groups of lysine, arginine, and histidine; acetylation of N-terminal amines; methylation or substitution of main-chain amide residues with N-methyl amino acids; or amidation of C-terminal carboxyl groups.

[0157] Mimic-type multispecific antibodies may also contain one or more amino acids with the opposite chirality. Therefore, any naturally occurring amino acid in the L configuration (sometimes referred to as R or S depending on the chemical structure) can be replaced by amino acids of the same chemical structure type or peptide mimetic but with the opposite chirality, referred to as D-amino acids (though sometimes referred to as R or S configurations).

[0158] Mimic-type multispecific antibodies can be synthesized using any protein chemosynthesis technique. In a typical in vitro protein synthesis process, the length of a peptide is extended by one amino acid at a time through the formation of peptide bonds between peptides and amino acids. Peptide bond formation is carried out using ligation reactions that can use natural or non-natural amino acids. Therefore, mimics can be created by introducing non-natural amino acids into the multispecific antibodies of the present invention in this way.

[0159] In some embodiments, naturally occurring amino acids in multispecific antibodies can provide linkage to macromolecules such as polymers, proteins, or fatty acids. In some embodiments, multispecific antibodies are linked to polymers (e.g., polymers other than polypeptides) (e.g., covalently). Suitable polymers include, for example, biocompatible polymers and water-soluble biocompatible polymers. Suitable polymers include synthetic polymers and naturally occurring polymers. Examples of polymers include substituted or unsubstituted linear or branched polyalkylenes, polyalkenes, or polyoxyalkylene polymers, or branched or unbranched polysaccharides, such as homo or heteropolysaccharides.Further examples of polymers include ethylene vinyl alcohol copolymer (commonly known as EVOH or EVAL); polybutyl methacrylate; poly(hydroxyvalerate); poly(L-lactic acid); polycaprolactone; poly(lactide-co-glycolide); poly(hydroxybutyrate); poly(hydroxybutyrate-co-valerate); polydioxanone; polyorthoester; polyanhydride; poly(glycolic acid); poly(D,L-lactic acid); poly(glycolic acid-co-trimethylene carbonate); polyphosphoester; poly Phosphoesterurethanes; poly(amino acids); cyanoacrylates; poly(trimethylene carbonate); poly(iminocarbonates); copolymers (ether-esters) (e.g., poly(ethylene oxide)-poly(lactic acid)(PEO / PLA) copolymers); polyoxalate alkylenes; polyphosphazenes; biomolecules, e.g., fibrin, fibrinogen, cellulose, starch, collagen and hyaluronic acid; polyurethanes; silicones; polyesters; polyolefins; polyisobutylene and ethylene-α-olefin copolymers Examples include polymers; acrylic polymers and copolymers; halogenated vinyl polymers and copolymers such as polyvinyl chloride; polyvinyl methyl ethers such as polyvinyl ether; halogenated polyvinylidenes such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile; polyvinyl ketones; polyvinyl aromatics such as polystyrene; polyvinyl acetate and other polyvinyl esters; copolymers of vinyl monomers and olefins, such as ethylene-methyl methacrylate copolymer, acrylonitrile-styrene copolymer, ABS resin and ethylene-vinyl acetate copolymer; polyamides, such as nylon 66 and polycaprolactam; alkyd resins; polycarbonates; polyoxymethylenes; polyimides; polyethers; epoxy resins; polyurethanes; rayon; rayon triacetate; cellulose; cellulose acetate; cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ethers; amorphous teflon; poly(ethylene glycol); and carboxymethylcellulose.

[0160] Examples of synthetic polymers include unsubstituted and substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol), and their derivatives, as well as substituted poly(ethylene glycol) such as methoxypoly(ethylene glycol) and their derivatives. Suitable naturally occurring polymers include, for example, albumin, amylose, dextran, glycogen, and their derivatives.

[0161] The polymers to be linked may have an average molecular weight in the range of 500 Da to 50000 Da, for example, 5000 Da to 40000 Da or 25000 to 40000 Da. For example, in some embodiments, if the multispecific antibody contains poly(ethylene glycol) (PEG) or methoxypoly(ethylene glycol) polymer, the PEG or methoxypoly(ethylene glycol) polymer may have a molecular weight in the range of about 0.5 kilodaltons (kDa) to 1 kDa, about 1 kDa to 5 kDa, 5 kDa to 10 kDa, 10 kDa to 25 kDa, 25 kDa to 40 kDa or 40 kDa to 60 kDa.

[0162] For example, a water-soluble polymer (e.g., PEG) can be linked to a multispecific antibody by reacting a water-soluble polymer containing a carbonyl group with a multispecific antibody having amino acids that do not exist in nature and contain aminooxy, hydrazine, hydrazide, or semicarbazide groups. Alternatively, a multispecific antibody containing an alkyne-containing amino acid can be linked to a water-soluble polymer by reacting it with a water-soluble polymer containing an azide moiety. In some cases, the azide or alkyne group is linked to the PEG molecule via an amide bond.

[0163] In some embodiments, the polymer linked to the multispecific antibody is albumin. The albumin may be, for example, the albumin of the subject receiving the multispecific antibody. For instance, if the multispecific antibody is intended for use in humans, human albumin is linked to it. If the multispecific antibody is intended for use in dogs, canine albumin is linked to it. Generally speaking, for multispecific antibodies intended for use in a particular species, albumin from that species is linked to the multispecific antibody.

[0164] Examples of linkers for conjugating polymers to multispecific antibodies include glutaraldehyde, homobifunctional crosslinkers, or heterobifunctional crosslinkers. Glutaraldehyde crosslinks polypeptides via their amino moieties. Homobifunctional crosslinkers (e.g., homobifunctional imide esters, homobifunctional N-hydroxysuccinimidyl (NHS) esters, or homobifunctional sulfhydryl reactive crosslinkers) contain two or more identical reactive moieties and can be used in a one-step reaction procedure by adding the crosslinker to a solution containing a mixture of the polymer to be linked and the multispecific antibody. At a weakly alkaline pH, imide esters react only with primary amines to form imidoamides, and the overall charge of the crosslinked polymer and multispecific antibody remains unaffected. Examples of homobifunctional sulfhydryl reactive crosslinking agents include bismaleimidehexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and 1,4-di-(3',2'-pyridyldithio)propionamidobutane (DPDPB).

[0165] Heterobifunctional crosslinkers have two or more different reactive moieties (e.g., an amine-reactive moiety and a sulfhydryl-reactive moiety), and crosslink with one of the polymers and multispecific antibodies via the amine or sulfhydryl-reactive moiety, and then react with the other polymer and multispecific antibody via the unreacted moiety. Similar to pyridyl disulfide crosslinkers, multiple heterobifunctional haloacetyl crosslinkers are available. Carbodiimides are a classic example of heterobifunctional crosslinking reagents used to couple carboxyl groups to amines to form amide bonds.

[0166] Multispecific antibodies can be glycosylated, for example, by covalent linking to a carbohydrate or polysaccharide moiety. Glycosylation of multispecific antibodies is typically N-linked or O-linked. N-linked glycosylation refers to the linkage of the carbohydrate moiety to the side chain of an asparagine residue in the multispecific antibody. The tripeptide sequence "asparagine-X-serine" or "asparagine-X-threonine" (where X is any amino acid other than proline) is the recognition sequence for the enzymatic linkage of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in a multispecific antibody creates a potential glycosylation site. O-linked glycosylation refers to the linkage of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine (however, 5-hydroxyproline or 5-hydroxylysine may also be used).

[0167] The addition of glycosylation sites to multispecific antibodies can be achieved by altering their amino acid sequence to include one or more of the aforementioned tripeptide sequences (for N-linked glycosylation sites). This alteration can also be achieved by adding or substituting one or more serine or threonine residues into the original antibody sequence (for O-linked glycosylation sites). Conversely, the removal of glycosylation sites can be achieved by altering the amino acid sequence within the range of the native glycosylation sites of multispecific antibodies.

[0168] Multispecific antibodies can be covalently linked to another polymer (e.g., lipids, polypeptides, synthetic polymers, carbohydrates, etc.) using a linker selected from glutaraldehyde, a homobifunctional crosslinker, or a heterobifunctional crosslinker. Glutaraldehyde crosslinks the multispecific antibodies via their amino moieties. Homobifunctional and heterobifunctional crosslinkers are described herein.

[0169] In the construction of multispecific antibodies, conditionally active antibodies or their fragments, as described in International Publication No. 2017 / 078839, may be used. These conditionally active antibodies (full-length antibodies, fragments, or single-chain antibodies) exhibit increased affinity for their antigens under abnormal conditions compared to normal physiological conditions. Multispecific antibodies can be constructed by linking a conditionally active antibody (full-length antibody, fragment, or single-chain antibody) with one or more antibodies (full-length antibodies, fragments, or single-chain antibodies) that may or may not have conditional activity.

[0170] Linkers used in the construction of multispecific antibodies can be mobile peptides that ensure proper folding of the multispecific antibody. Exemplary linkers include (Ser)n, (Ser-Ala)n, and (Ala)n.

[0171] In some embodiments, multipurpose conditionally activated antibodies (full-length antibodies, fragments, or single-chain antibodies) can be created that bind to antigens on reactive lymphocytes and are linked to antigens on different target cells (e.g., different tumors) with various antibodies. Such multispecific antibodies can deliver the same reactive lymphocytes to each of their different target cells (e.g., different types of tumors). Therefore, such multispecific antibodies have the ability to be used to target multiple different types of tumor cells, for example, when the subject has multiple different tumors or a single, unidentified tumor. This can be particularly useful when the tumor is located in a position that makes biopsy difficult.

[0172] In some embodiments, a multipurpose conditionally active antibody (full-length or single-chain antibody) that binds to an antigen on cancer cells (e.g., breast cancer cells) can be linked with various antibodies that bind to antigens of different reactive lymphocytes, thereby generating a multispecific antibody that delivers different reactive lymphocytes to the same target cancer cells. These multipurpose multispecific antibodies have a higher conditional affinity for cancer cell antigens under the conditions of the tumor microenvironment. Therefore, such multispecific antibodies have the ability to deliver different reactive lymphocytes (e.g., T cells, macrophages, NK cells) to the same tumor (breast tumor) to increase therapeutic efficacy.

[0173] To generate a conditionally activated antibody against a first antigen (e.g., a cellular antigen) or a second antigen (e.g., a tumor-reactive lymphocyte antigen) whose affinity for the antigen (first or second antigen) is greater under abnormal conditions than under normal physiological conditions, a method as described in International Publication No. 2016 / 138071 may be used, starting from a parent antibody that binds to either or both of the first and second antigens. This conditionally activated antibody can be used to construct the multispecific antibody of the present invention.

[0174] Parental antibodies may be monoclonal or polyclonal antibodies produced by immunizing animals with an antigen. Methods for immunization, production of antibodies (polyclonal and monoclonal), and isolation are known to those skilled in the art and are described in scientific literature and patent documents. See, for example, Coligan, Current Protocols In Immunology, Wiley / Greene, NY (1991); Stites (eds.), Basic And Clinical Immunology (7th ed.), Lange Medical Publications, Los Altos, Calif. ("Stites"); Goding, Monoclonal Antibodies: Principles And Practice (2nd ed.), Academic Press, New York, NY (1986); Kohler (1975), "Continuous cultures of fused cells secreting antibody of predefined specificity," Nature 256:495; and Harlow (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York. Antibodies can be generated in vitro, for example, using recombinant antibody binding sites that express phage display libraries, in addition to conventional in vivo methods using animals. See, for example, Hoogenboom (1997) “Designing and optimizing library selection strategies for generating high-affinity antibodies”, Trends Biotechnol. 15:62-70; and Katz (1997) “Structural and mechanistic determinants of affinity and specificity of ligands discovered or engineered by phage display”, Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

[0175] Examples of assay solutions for the first and second assays include buffers selected from citrate buffers such as sodium citrate, phosphate buffers, bicarbonate buffers such as Krebs buffer, phosphate-buffered saline (PBS) buffer, Hanks buffer, Tris buffer, HEPES buffer, and the like. Other buffers known to those skilled in the art to be suitable for the assay may also be used.

[0176] The assay solutions of the first and second assays may contain at least one molecule selected from inorganic compounds, ions, and organic molecules, or one commonly found in the bodily fluids of mammals or animals such as humans. These inorganic compounds, ions, and organic molecules are described in detail in International Publication No. 2016 / 138071.

[0177] Conditionally activated antibodies can interact with molecules or ions selected from inorganic compounds, ions, and organic molecules. Interactions between conditionally activated antibodies and molecules or ions may include hydrogen bonding, hydrophobic interactions, and van der Waals interactions.

[0178] For example, molecules or ions such as bicarbonates can reduce the binding activity of a conditionally active antibody to its antigen by forming salt bridges in the antibody. For instance, at pH levels below its pKa of 6.4, bicarbonates are protonated and therefore uncharged. Uncharged bicarbonates do not have the ability to form salt bridges and therefore have little effect on the binding of a conditionally active antibody to its antigen. Consequently, this conditionally active antibody exhibits high binding activity to its antigen at low pH levels. On the other hand, at high pH levels above the pKa of bicarbonates, bicarbonates are ionized by losing protons and therefore become negatively charged. Negatively charged bicarbonates stabilize the structure of the conditionally active antibody by forming salt bridges between positively charged or polarized portions on the antibody. This blocks or reduces the binding of the conditionally active antibody to its antigen. Consequently, this conditionally active antibody exhibits low activity at high pH levels. Thus, conditionally active antibodies exhibit pH-dependent activity in the presence of bicarbonate, with higher binding activity at lower pH levels compared to higher pH levels.

[0179] When molecules or ions such as bicarbonates are not present in the assay solution, conditionally activated antibodies may lose their conditional activity. This is thought to be due to the lack of salt bridges that stabilize (fix) the protein structure of conditionally activated antibodies. Therefore, the partner antibody will reach the binding site on the conditionally activated antibody in the same way at any pH, and will exhibit similar activity at both the first and second pH levels.

[0180] While salt bridges (ionic bonds) are the most powerful and common way in which molecules or ions influence the activity of conditionally active antibodies, it should be understood that the other interactions between such molecules or ions and conditionally active antibodies, as described above, can also contribute to stabilizing (fixing) the structure of conditionally active antibodies.

[0181] Exemplary molecules and ions are selected from disulfides, hydrogen sulfide, histidine, histamine, citrate, bicarbonate, acetate, and lactate. Each of these small molecules has a pKa of 6.2–7.0. For other suitable small molecules, refer to textbooks using the principles of this application, such as the CRC Handbook of Chemistry and Physics, 96th Edition, by CRC press, 2015; and the Chemical Properties Handbook, McGraw-Hill Education, 1998.

[0182] Molecules or ions have low molecular weights and / or relatively small conformations to minimize steric hindrance and maximize their reach into small pockets on conditionally active proteins. For this reason, small molecules or ions with molecular weights typically less than 900 Da, less than 500 Da, less than 200 Da, or less than 100 Da are often used. For example, hydrogen sulfide, disulfides, and bicarbonates all have low molecular weights and small structures, allowing them to reach pockets on conditionally active proteins.

[0183] In one embodiment, human serum may be added to both the normal physiological and abnormal assay solutions at substantially the same concentration. Since human serum contains numerous inorganic compounds, ions, and organic molecules (including proteins), the assay solution will contain a number of components selected from these inorganic compounds, ions, and organic molecules, provided at substantially the same concentration between the two assay solutions.

[0184] In some embodiments, certain serum components may be intentionally minimized or omitted from the assay solution. For example, during antibody screening, serum components that bind to or adsorb proteins may be minimized or omitted from the assay solution. Such protein binding may lead to false positives, and therefore may include the binding of mutant proteins that are not conditionally active but rather simply bind to components present in serum under various different conditions. Therefore, by carefully selecting assay components to minimize or omit such molecules that may potentially bind to mutant proteins in the assay, the number of false-positive mutant proteins that may be incorrectly identified as positive for conditional activity due to binding to molecules in the assay other than the desired binding partner can be reduced. For example, in some embodiments where mutant proteins that tend to bind to components in human serum are being screened, bovine serum albumin may be used in the assay solution to reduce or eliminate the possibility of false positives caused by mutant proteins that bind to human serum components. In specific examples, other similar substitutions may be made to achieve the same objective, which will be well understood by those skilled in the art.

[0185] In another embodiment, the present invention provides a method for producing multispecific antibodies. This method is a) A step of obtaining an IgG antibody that binds to the first antigen; and b) A step of forming one or more structures by linking at least one single-chain antibody that binds to a second antigen to the C-terminus of at least one light chain of an IgG antibody via a linker; c)b) A step of screening one or more structures for binding to at least one of the first or second antigens under abnormal and normal physiological conditions; and d) A step of selecting from those structures a multispecific antibody that reversibly binds to at least one of the first or second antigen with greater affinity under abnormal conditions than under normal physiological conditions. Includes.

[0186] In some embodiments, the first antigen may be a cell-specific antigen, particularly a cancer cell-specific antigen or a senescent cell-specific antigen as described herein. In one embodiment, the first antigen is selected from Axl, EpCAM, Ror2, Her2, and B7-H3. The second antigen may be a tumor-reactive lymphocyte antigen, such as CD3. Further examples of suitable reactive lymphocyte antigens are also described herein.

[0187] In some other embodiments, the second antigen is a neoantigen as described herein.

[0188] In some embodiments, the multispecific antibody binds to CD3 as a tumor-reactive lymphocyte antigen and to another tumor-associated antigen (TAA) as a cell-specific antigen. The multispecific antibody has a binding site for CD3 that includes a light chain variable region having the amino acid sequence of SEQ ID NOs. 1-10 and a heavy chain variable region having the amino acid sequence of SEQ ID NOs. 11-15. Alternatively, the binding site for CD3 includes an anti-CD3 single-chain antibody having an amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 26-71. The multispecific antibody has a binding site for TAA that includes a light chain variable region having the amino acid sequence of SEQ ID NOs. 16-17, 20, and 22 that binds to one of Axl, Her2, and B7-H3, and a light chain variable region selected from the heavy chain variable regions having the amino acid sequence of SEQ ID NOs. 18-19, 21, and 23-25 ​​that binds to one of Axl, Her2, and B7-H3. The binding site for EpCAM includes a light chain variable region of an amino acid sequence selected from SEQ ID NOs. 88-95 and a heavy chain variable region of an amino acid sequence selected from SEQ ID NOs. Among these multispecific antibodies, as long as at least one of the binding site for CD3 and the binding site for Axl, EpCAM, Her2, or B7-H3 is conditionally active, the multispecific antibody is considered conditionally active and within the scope of the present invention.

[0189] In one example, a multispecific antibody binds to a first antigen with greater affinity under abnormal conditions than under normal physiological conditions. In another example, a multispecific antibody binds to a second antigen with greater affinity under abnormal conditions than under normal physiological conditions. In yet another example, a multispecific antibody binds to both the first and second antigens with greater affinity under abnormal conditions than under normal physiological conditions. In yet another example, a multispecific antibody binds to a combination of the first and second antigens with greater avidity under abnormal conditions than under normal physiological conditions.

[0190] In some embodiments, abnormal conditions are acidic pH in the range of about 5.0 to about 7.0, or about 5.2 to about 6.8, or about 5.4 to about 6.8, or about 5.6 to about 6.8, or about 5.8 to about 6.8, or about 6.0 to about 6.8, or about 6.2 to about 6.8, or about 6.4 to about 6.8, or about 6.6 to about 6.8. In some embodiments, the acidic pH may be in the range of about 6.4 to about 7.0, or about 6.6 to about 7.0, or about 6.8 to about 7.0. Normal physiological conditions may be the normal physiological pH in the blood, which is well established in the art. In some embodiments, the normal physiological pH in the blood may be in the range of about 7.0 to about 7.8, or about 7.1 to about 7.7, or about 7.2 to about 7.6, or about 7.2 to about 7.5, or about 7.2 to about 7.4.

[0191] Production of multispecific antibodies In another embodiment, a method for generating multispecific antibodies is provided. This method generates multispecific antibodies from two starting materials: an IgG antibody or a fragment thereof that binds to a first antigen and an scFv antibody that binds to a second antigen. By evolving one or both of these two antibodies, evolved antibodies are produced and screened for IgG and / or scFv antibodies that bind to their respective first or second antigens with greater affinity under abnormal conditions than under normal physiological conditions. One or more structures are produced by ligating at least one scFv antibody that binds to the second antigen to the C-terminus of at least one light chain of the IgG antibody or fragment. At least one of the scFv antibody and the IgG antibody is an antibody screened from antibodies evolved from one or both of the starting IgG and scFv antibodies. When a starting antibody is evolved, that starting antibody may be called the “parent antibody,” and one or more antibodies evolved from it may be called “mutant antibodies” or “evolved antibodies.”

[0192] The structure is further screened for binding to at least one of the first and second antigens under both abnormal and normal physiological conditions in order to select a multispecific antibody that binds to at least one of the first and second antigens with greater affinity under abnormal conditions than under normal physiological conditions. The binding of the multispecific antibody to the first or second antigen may be reversible.

[0193] Suitable methods for evolving starting material IgG antibodies and scFv antibodies are described, for example, in International Publication No. 2012 / 009026. Suitable methods for screening evolved antibodies or structures are described, for example, in International Publication No. 2017 / 078839.

[0194] In another embodiment, a method for generating a multispecific antibody is provided. This method starts with an IgG antibody or fragment thereof that binds to a first antigen and an scFv antibody that binds to a second antigen with greater affinity under abnormal conditions than under normal physiological conditions. This method includes the steps of: producing one or more structures by ligating an scFv antibody that binds to a second antigen to the C-terminus of at least one light chain of an IgG antibody or fragment thereof; screening one or more structures for their binding activity to the first and second antigens under normal physiological conditions and abnormal conditions; and selecting a multispecific antibody that binds to the first antigen and reversibly binds to the second antigen with greater affinity under the abnormal conditions than under normal physiological conditions.

[0195] Multispecific antibodies produced by the method described above are also provided. Such multispecific antibodies comprise an IgG antibody or fragment thereof that binds to a cell-specific antigen and at least one scFv antibody that binds to a T lymphocyte antigen, which is ligated to the C-terminus of at least one light chain or at least one heavy chain of the IgG antibody or fragment thereof. The at least one scFv antibody reversibly binds to the T lymphocyte antigen with greater affinity under abnormal conditions than under normal physiological conditions.

[0196] The starting materials for creating multispecific antibodies include an IgG antibody or fragment thereof that binds to a first antigen as described herein, and an scFv antibody that binds to a second antigen. Other characteristics of these multispecific antibodies are also described in other parts of this specification.

[0197] Conjugation with drugs In some embodiments, the multispecific antibody may be conjugated with a drug, which may be a therapeutic agent, prophylactic agent, diagnostic agent, detectable label, chelator, or contrast agent. In some embodiments, the conjugated drug on the multispecific antibody may optionally be released from the multispecific antibody after the multispecific antibody has reached the site of action (e.g., a tumor). In these embodiments, the multispecific antibody may serve as a delivery vehicle for transporting the conjugated drug (such as a therapeutic agent, prophylactic agent, or diagnostic agent) to the target site of action.

[0198] Multispecific antibodies can be conjugated to drugs by covalent or non-covalent conjugation. Covalent conjugation may be direct or linker-mediated. In certain embodiments, direct conjugation is by constructing a fusion protein of the drug and the multispecific antibody (i.e., by gene fusion of two genes encoding the multispecific antibody and the drug, and expression as a single protein). In certain embodiments, direct conjugation is by the formation of a covalent bond between a reactive group on the multispecific antibody and a corresponding group on the drug. In certain embodiments, direct conjugation is by modification (i.e., gene modification) of the multispecific antibody to include a reactive group (as a non-limiting example, a sulfhydryl group or a carboxyl group) that forms a covalent bond with the drug under appropriate conditions, or vice versa. For example, a disulfide bond that forms with the drug can be formed by introducing an amino acid having the desired reactive group (i.e., a cysteine ​​residue) into the multispecific antibody. Methods for covalently conjugating drugs to multispecific antibodies are also known in the art (i.e., photocrosslinking, see, for example, Zatsepin et al. Russ. Chem. Rev., 74:77-95 (2005)).

[0199] Non-covalent conjugations can be formed by any non-covalent bonding means, including hydrophobic bonds, ionic bonds, and electrostatic interactions, as will be readily understood by those skilled in the art.

[0200] Conjugation can also be carried out using various linkers. For example, multispecific antibodies and drugs can be conjugated using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imide esters (such as dimethylHCl adipimidoate), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazide compounds (such as bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bisactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Peptide linkers consisting of 1 to 20 amino acids linked together by peptide bonds may also be used. In certain such embodiments, the amino acids are selected from 20 naturally occurring amino acids. In certain other such embodiments, one or more amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine.

[0201] The linker may be a "cleavable linker" that facilitates the release of the drug upon delivery to the site of action. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res., 52:127-131 (1992); U.S. Patent No. 5,208,020) may be used.

[0202] The therapeutic or prophylactic agent to be conjugated can be toxic to the body, such as radioactive particles, chemotherapeutic drugs or cytotoxins (i.e., cytotoxin). When the conjugated therapeutic agent is delivered to the site of action using the multispecific antibody of the present invention, the toxic effects of such therapeutic agents in the body within an undesirable range of its activity will be significantly reduced. The technique of conjugating radioactive particles to antibodies is known in the art. Ibritumomab tiuxetan (Zevalin®) and tositumomab (Bexxar®) are examples of monoclonal antibodies conjugated with radioactive particles. Both are antibodies against the CD20 antigen conjugated with different radioactive particles. Similarly, the technique of conjugating chemotherapeutic drugs to antibodies is also known in the art. There are at least two commercially available antibodies conjugated with chemotherapeutic drugs: brentuximab vedotin (Adcetris®) and ado-trastuzumab emtansine (Kadcyla™). The technique of conjugating cytotoxins to antibodies is also known in the art. For example, denileukin diftitox (Ontak™, an anticancer drug) consists of an immune system protein known as interleukin-2 (IL-2) conjugated to a toxin derived from the pathogen that causes diphtheria.

[0203] It is contemplated that the multispecific antibodies of the present invention can conjugate all kinds of radioactive particles, chemotherapeutic drugs and cytotoxins in order to reduce the side effects of the drugs during delivery to the site of action or the diseased site.

[0204] In some embodiments, the radioactive particles conjugated to the multispecific antibody include particles impregnated with one or more radioisotopes and have sufficient radioactivity for ablation in a local region of the cell. The particles can include glass, metal, resin, albumin or one or more polymers. The metal in the radioactive particles can be selected from iron, gadolinium and calcium. Examples of one or more radioisotopes in the radioactive particles are gallium-67 ( 67 Ga), yttrium-90 ( 90 Y), gallium-68 (68 Ga), Thallium-201 ( 201 T1), Strontium-89 ( 89 Sr), Indium-III ( 111 In), Iodine-131( 131 I) Samarium-153 ( 153 Sm), Technetium-99m( 99m Tc), Rhenium-186( 186 Re), Rhenium-188 188 Re), copper-62( 62 Cu) and copper-64( 64 Selected from (Cu). One or more radioactive isotopes in the composition may emit beta rays, gamma rays and / or positrons.

[0205] In some embodiments, the chemotherapeutic agent conjugated to the multispecific antibody is selected from anthracyclines, topoisomerase I and / or II inhibitors, spindle toxin plant alkaloids, alkylating agents, antimetabolites, ellipticin, and harmine.

[0206] Anthracyclines (or anthracycline antibiotics) are derived from bacteria of the genus Streptomyces. These compounds are used, for example, to treat a wide range of cancers, including hepatocellular carcinoma, leukemia, lymphoma, and breast, uterine, ovarian, and lung cancer. Examples of anthracyclines include, but are not limited to, doxorubicin, daunorubicin, epirubicin, idarubicin, barurubicin, pirarubicin, zorubicin, akurarubicin, detrubicin, carminomycin, morpholinodoxorubicin, morpholinodaunorubicin, methoxymorpholinyldoxorubicin, and pharmaceutically acceptable salts thereof.

[0207] Topoisomerases are essential enzymes that maintain the topology of DNA. Inhibition of type I or type II topoisomerase interferes with both DNA transcription and replication by disrupting proper DNA supercoil formation. Some type I topoisomerase inhibitors include camptothecin derivatives. Camptothecin derivatives refer to camptothecin analogs such as irinotecan, topotecan, hexatecan, siratecan, lutoltecan, calenitecan (BNP1350), gimatecan (ST1481), berotecan (CKD602), or pharmaceutically acceptable salts thereof. Examples of type II topoisomerase inhibitors include, but are not limited to, amsacrine, etoposide, etoposide phosphate, and teniposide. These are semi-synthetic derivatives of epipodophyllotoxin, an alkaloid naturally found in the roots of Podophyllum peltatum.

[0208] Spindle toxins, plant alkaloids, are derived from plants and inhibit cell division by interfering with microtubule function, which is essential for cell division. Examples of such alkaloids include, but are not limited to, vinca alkaloids (such as vinblastine, vincristine, vindesine, vinorelbine, and vinpocetine) and taxanes. Examples of taxanes include, but are not limited to, paclitaxel, docetaxel, larotaxel, cabazitaxel, ortataxel, tesetaxel, and pharmaceutically acceptable salts thereof.

[0209] Examples of alkylating agents, though not limited to them, include mechloretamine, cyclophosphamide, chlorambucil, ifosfamide, and platinum compounds, such as oxaliplatin, cisplatin, or carboplatin.

[0210] Antimetabolites are chemicals that inhibit the use of metabolites that are part of normal metabolism. The presence of antimetabolites alters cell growth and cell division. Purines or pyrimidine analogs interfere with the uptake of nucleotides into DNA, stopping DNA synthesis and therefore cell division. They also affect RNA synthesis. Examples of purine analogs include azathioprine, mercaptopurine, thioguanine, fludarabine, pentostatin, and cladribine. Examples of pyrimidine analogs include 5-fluorouracil (5FU), phloxuridine (FUDR), and cytosine arabinoside (cytarabine), which inhibit thymidylate synthase.

[0211] Antifolic acid agents are chemotherapeutic drugs that impair the function of folic acid. A well-known example is methotrexate, a folic acid analog that inhibits the enzyme dihydrofolate reductase (DHFR) and thus prevents the formation of tetrahydrofolate. This leads to the inhibition of DNA, RNA, and protein production (since tetrahydrofolate is also involved in the synthesis of the amino acids serine and methionine). Other antifolic acid agents include, but are not limited to, trimethoprim, larcitrexed, pyrimethamine, and pemetrexed.

[0212] Furthermore, multispecific antibodies can be conjugated with other chemotherapeutic agents such as ellipticin and harmine. Ellipticin and its derivatives, such as 9-hydroxyellipticinium, N2-methyl-9-hydroxyellipticinium, 2-(diethylamino-2-ethyl)9-hydroxyellipticinium acetate, 2-(diisopropylamino-ethyl)9-hydroxyellipticinium acetate, and 2-(β-piperidino-2-ethyl)9-hydroxyellipticinium, are all effective chemotherapeutic agents.

[0213] Harmine is a natural plant alkaloid product isolated from the seeds of Peganum harmala. Harmine-based chemotherapeutic agents include harmine, harmanine, harmol, harmalol, and harman, as well as quinazoline derivatives: bacicin and bacicinone.

[0214] In some embodiments, cytotoxins conjugated to multispecific antibodies include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracendione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogs or homologs. Other toxins include, for example, lysine, CC-1065 and its analogs, and duocalmycin. Further toxins include diphtheria toxin and snake venom (e.g., cobra venom).

[0215] In some embodiments, multispecific antibodies can be conjugated to diagnostic agents. Examples of diagnostic agents used in the present invention include, for example, Armstrong et al, Diagnostic Imaging, 5 thThis may include any diagnostic agents known in the art, as provided in Ed., Blackwell Publishing (2004); Torchilin, VP, Ed., Targeted Delivery of Imaging Agents, CRC Press (1995); Vallabhajosula, S., Molecular Imaging: Radiopharmaceuticals for PET and SPECT, Springer (2009). Diagnostic agents can be detected by a variety of methods, including, but are not limited to, using agents that provide and / or enhance detectable signals, such as gamma-ray emission signals, radioactive signals, echogenic signals, optical signals, fluorescence signals, absorption signals, magnetic signals, or tomographic signals. Imaging techniques for diagnostic agents include, but are not limited to, single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, fluorescence imaging, positron emission tomography (PET), computed tomography (CT), X-ray imaging, and gamma-ray imaging.

[0216] In some embodiments, multispecific antibodies can be conjugated to chelators that bind to metal ions, for example, used in various imaging techniques. Examples of chelators include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), [4-(l,4,8,11-tetraazacyclotetradeca-l-yl)methylbenzoic acid (CPTA), cyclohexanediaminetetraacetic acid (CDTA), ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), citric acid, hydroxyethylethylenediaminetriacetic acid (HEDTA), iminodiacetic acid (IDA), triethylenetetraaminehexaacetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-l,4,7,10-tetra(methylenephosphonic acid) (DOTP), 1,4,8,1l-tetraazacyclododecane-l,4,8,ll-tetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), and their derivatives.

[0217] Multispecific antibodies can be conjugated to detectable labels. Suitable detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Suitable detectable labels include, but are not limited to, magnetic beads (e.g., Dynabeads®), fluorescent dyes (e.g., fluorescein isothiocyanate, TEXAS RED®, rhodamine, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.), and radioactive labels (e.g., 3 H, 125 I, 35 S, 14 C or 32 Examples include P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, and others commonly used in enzyme-linked immunosorbent assays (ELISA)), and colorimetric labels such as colloidal gold or colored glass, or plastic beads (e.g., lutistyrene, multipropylene, latex, etc.).

[0218] In other embodiments, the detectable label is selected from optical agents such as fluorescent agents, phosphors, and chemiluminescent agents. Numerous agents (e.g., dyes, probes, labels, or indicators) are known in the art and can be used in the present invention (see, for example, Invitrogen, The Handbook: A Guide to Fluorescent Probes and Labeling Technologies, Tenth Edition (2005)). Fluorescent agents may include various organic and / or inorganic small molecules or various fluorescent proteins and their derivatives. For example, fluorescent agents include, but are not limited to, cyanines, phthalocyanines, porphyrins, indocyanines, rhodamines, phenoxazines, phenylxanthenes, phenothiazines, phenoselenazines, fluoresceins, benzoporphyrins, squalanes, dipyrrolopyrimidones, tetracenes, quinolines, pyrazines, cholines, croconiums, acridons, phenanthridines, rhodamines, acridines, anthraquinones, and chalcones. Examples include genopyryllium analogs, chlorines, naphthalocyanines, methine dyes, indolenium dyes, azo compounds, azulenes, azaazulenes, triphenylmethane dyes, indoles, benzoindoles, indocarbocyanines, benzoindocarbocyanines, and BODIPY(trademark) derivatives having the general structure of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, and / or conjugates and / or derivatives of any of these.

[0219] Other detectable agents include fluorescein, fluorescein-polyaspartate conjugate, fluorescein-polyglutamate conjugate, fluorescein-polyarginine conjugate, indocyanine green, indocyanine-dodecaaspartate conjugate, indocyanine (NIRD)-polyaspartate conjugate, isosulfan blue, indole disulfonate, benzoindole disulfonate, bis(ethylcarboxymethyl)indocyanine, bis(pentylcarboxymethyl)indocyanine, polyhydroxyindole sulfonate, polyhydroxybenzoindole sulfonate, rigid heteroatom indole sulfonate, indocyanine bispropanoate, indocyanine bishexanoate, 3,6-dicyano-2,5-[( Examples include N,N,N',N'-tetrakis(carboxymethyl)amino]pyrazine, 3,6-[(N,N,N',N'-tetrakis(2-hydroxyethyl)amino]pyrazine-2,5-dicarboxylic acid, 3,6-bis(N-azatedino)pyrazine-2,5-dicarboxylic acid, 3,6-bis(N-morpholino)pyrazine-2,5-dicarboxylic acid, 3,6-bis(N-piperazino)pyrazine-2,5-dicarboxylic acid, 3,6-bis(N-thiomorpholino)pyrazine-2,5-dicarboxylic acid, 3,6-bis(N-thiomorpholino)pyrazine-2,5-dicarboxylic acid S-oxide, 2,5-dicyano-3,6-bis(N-thiomorpholino)pyrazine S,S-dioxide, indocarbocyanine tetrasulfonate, chloroindocarbocyanine, and 3,6-diaminopyrazine-2,5-dicarboxylic acid.

[0220] In some embodiments, multispecific antibodies may be conjugated with a contrast agent, which is suitable for use in imaging, such as imaging procedures performed on humans. Non-limiting examples of contrast agents include gadolinium (Gd), dysprosium, and iron. Multispecific antibodies can be conjugated with contrast agents using standard techniques. For example, multispecific antibodies can be iodized with chloramine T or 1,3,4,6-tetrachloro-3α,6α-diphenylglycoluryl. In fluorination, fluorine is conjugated to the multispecific antibody during synthesis by a fluoride ion substitution reaction. For a review of protein synthesis with such radioisotopes, see Muller-Gartner, H., TIB Tech., 16:122-130 (1998) and Saji, H., Crit. Rev. Ther. Drug Carrier Syst., 16(2):209-244 (1999). For example, multispecific antibodies can be conjugated to Gd by conjugating the antibody with a low-molecular-weight Gd chelate such as Gd-diethylenetriaminepentaacetic acid (GdDTPA) or Gd-tetraazacyclododecanetetraacetic acid (GdDOTA). See Caravan et al., Chem. Rev. 99:2293-2352 (1999) and Lauffer et al., J. Magn. Reson. Imaging, 3:11-16 (1985). Multispecific antibodies can also be conjugated to Gd by conjugating the antibody with a polylysine-Gd chelate, for example. See, for example, Curtet et al., Invest. Radiol., 33(10):752-761 (1998). Alternatively, multispecific antibodies can be conjugated to Gd by incubating paramagnetic polymerized liposomes containing Gd chelator lipids with avidin and biotinylated antibodies. For example, see Sipkins et al., Nature Med., 4:623-626 (1998).

[0221] In further embodiments, the contrast agent may be an X-ray contrast agent as described in the following references: HS Thomsen, R. Muller and R. F Mattrey, Eds., Trends in Contrast Media, (Berlin: Springer-Verlag, 1999); P. Dawson, D. Cosgrove and R. Grainger, Eds., Textbook of Contrast Media (ISIS Medical Media 1999); Torchilin, VP, Curr. Pharm. Biotech., vol.1, pages 183-215 (2000); Bogdanov, AA et al, Adv. Drug Del. Rev., Vol.37, pages 279-293 (1999); Sachse, A. et al, Investigative Radiology, vol.32, pages 44-50 (1997). Examples of X-ray contrast agents include, without limitation, iopamidol, iomeprole, iohexol, iopentol, iopromide, iosimide, ioversol, iotrolane, iotasul, iodixanol, iodecimol, ioglucamide, iogrunide, yoglamide, iosalcol, ioxiran, iopamilon, metrizamide, iobitridol, and iosimenol. In specific embodiments, examples of X-ray contrast agents include iopamidol, iomeprole, iopromide, iohexol, iopentol, ioversol, iobitridol, iodixanol, iotrolane, and iosimenol.

[0222] In some embodiments, multispecific antibodies can be conjugated with labels that are easily visible using, for example, "radiopaque" labels, such as X-ray labels. Radiopaque materials are well known to those skilled in the art. The most common radiopaque materials include iodides, bromides, or barium salts. Other radiopaque materials are also known and not limited to, organobismuth derivatives (see, for example, U.S. Patent No. 5,939,045), radiopaque multiurethanes (see, for example, U.S. Patent No. 5,346,981), organobismuth complexes (see, for example, U.S. Patent No. 5,256,334), and radiopaque barium polymer complexes (see, for example, U.S. Patent No. 4,866,132).

[0223] Suitable fluorescent proteins that can be conjugated to multispecific antibodies include, but are not limited to, green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria, or its mutants or derivatives, as described in U.S. Patent Nos. 6,066,476; 6,020,192; 5,985,577; 5,976,796; 5,968,750; 5,968,738; 5,958,713; 5,919,445; and 5,874,304. Examples of GFP include, for example, those commercially available from Clontech, Inc.; red fluorescent protein; yellow fluorescent protein; and any of the various fluorescent and colored proteins derived from anthozoan species, as described, for example, Matz et al. (1999) Nature Biotechnol. 17:969-973.

[0224] In some embodiments, the conjugation is on the Fc region of a multispecific antibody. The conjugate molecules, compounds, or drugs described above may be conjugated to the Fc region as described in U.S. Patent No. 8,362,210. For example, the Fc region may be conjugated to a therapeutic, prophylactic, or diagnostic agent that the multispecific antibody intends to deliver to a site with abnormal conditions in which it preferentially exhibits activity. Further methods for conjugating to the Fc region of an antibody are known in the art. For example, U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095 and 5,112,9 Specification No. 46; European Patent No. 307,434; European Patent No. 367,166; European Patent No. 394,827; International Publication Brochures No. 91 / 06570, No. 96 / 04388, No. 96 / 22024, No. 97 / 34631 and No. 99 / 04813; Ashkenazi See et al., Proc. Natl. Acad. Sci. USA, vol. 88, pages 10535-10539, 1991; Traunecker et al., Nature, vol. 331, pages 84-86, 1988; Zheng et al., J. Immunol., vol. 154, pages 5590-5600, 1995; and Vie et al., Proc. Natl. Acad. Sci. USA, vol. 89, pages 11337-11341, 1992.

[0225] Compositions, formulations, kits The multispecific antibodies of the present invention may be included in pharmaceutical compositions, medical devices, kits, or products for therapeutic, prophylactic, or diagnostic applications. Suitable pharmaceutical compositions, medical devices, kits, or products are described in detail in International Publication No. 2016 / 138071.

[0226] In some embodiments, the pharmaceutical composition may be in liquid form, lyophilized form, or liquid form reconstituted from a lyophilized form. Lyophilized preparations are typically reconstituted with a sterile solution before administration. A standard reconstitution procedure for lyophilized compositions involves adding a certain volume of pure water (typically approximately equal to the volume removed during lyophilization). Solutions containing antibacterial agents may also be used in the preparation of pharmaceutical compositions for parenteral administration. See also Chen, Drug Dev Ind Pharm, vol. 18, pp. 1311-54, 1994.

[0227] The composition may contain pharmaceutically acceptable isotonic agents to adjust the tonicity of the formulation. Exemplary isotonic agents include sodium chloride, potassium chloride, glycerin, and any component from the group of amino acids and sugars, as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, but hypertonic or hypotonic solutions may also be preferred. The term “isotonic” means a solution that has the same tonicity as any other solution to which it is compared, such as physiological saline or serum. Isotonic agents may be used in amounts of about 5 mM to about 350 mM, for example, 100 mM to 350 nM.

[0228] The composition may contain pharmaceutically acceptable surfactants to reduce aggregation of formulated multispecific antibodies and / or minimize the formation of particulate matter in the formulation and / or reduce adsorption. Exemplary surfactants include polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, alkylphenyl polyoxyethylene ethers (Triton-X®), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic®), and sodium dodecyl sulfate (SDS). Examples of preferred polyoxyethylene sorbitan fatty acid esters include polysorbate 20 (marketed under the trademark Tween 20®) and polysorbate 80 (marketed under the trademark Tween 80®). Examples of preferred polyethylene-polypropylene copolymers are those marketed under the trade names Pluronic® F68 or Poloxamer 188®. Examples of preferred polyoxyethylene alkyl ethers are those marketed under the trademark Brij®. The exemplary concentration of the surfactant in the composition may be in the range of about 0.001% to about 1% w / v.

[0229] A lyophilization protectant may be added to the composition to protect unstable active ingredients (e.g., proteins) from destabilizing conditions during the freeze-drying process. For example, known lyophilization protectants include sugars (including glucose and sucrose), polyols (including mannitol, sorbitol, and glycerol), and amino acids (including alanine, glycine, and glutamic acid). The lyophilization protectant may be included in an amount of about 10 nM to 500 nM.

[0230] In some embodiments, compositions containing one or more surfactants, buffers, stabilizers, and isotonic agents essentially do not contain one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propylparabens, benzalkonium chloride, or combinations thereof. In other embodiments, preservatives selected from ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propylparabens, benzalkonium chloride, or combinations thereof may be included in the formulation at concentrations ranging, for example, from about 0.001% to about 2% (w / v).

[0231] Unit dosage forms for oral administration may be provided, such as syrups, elixirs, and suspensions, where each dosage unit, e.g., a teaspoon, a spoonful, a tablet, or a vial, contains a predetermined amount of the composition. Similarly, unit dosage forms for injection or intravenous administration may contain multispecific antibodies in the composition as a solution in sterile water, standard saline, or another pharmaceutically acceptable carrier.

[0232] Multispecific antibodies can be formulated as injectable formulations. Typically, the injectable composition is prepared as a liquid solution or suspension, and a solid form suitable for solution or suspension in a liquid vehicle before injection may also be prepared. The preparation may also be emulsified, and the multispecific antibodies may be encapsulated in a liposome vehicle.

[0233] In some embodiments, multispecific antibodies may be formulated as aerosols and intranasal compositions. For suppositories, the composition will include conventional binders and carriers, such as polyalkylene glycols or triglycerides. Such compositions may be formed from a mixture containing multispecific antibodies in an amount ranging from about 0.5% to about 10% (w / w), for example, from about 1% to about 2%.

[0234] Multispecific antibodies can be formulated as intranasal preparations containing a vehicle that does not cause irritation to the nasal mucosa or significantly impair ciliary function. In this invention, diluents such as water, physiological saline solution, or other known substances can be used. The intranasal preparation may also contain preservatives such as chlorobutanol and benzalkonium chloride, although this is not limited to these preservatives. Surfactants may be present to enhance the absorption of multispecific antibodies by the nasal mucosa.

[0235] In some embodiments, multispecific antibodies are formulated into controlled-release formulations. Within the scope of the present invention, controlled-release means one of several sustained-release drug formulations. For the purposes of the present invention, the following types of controlled-release can be used: continuous release, delayed release, stepwise release, long-term release, planned release, long-term release, proportional release, prolonged release, slow release, intervald release, sustained release, timed release, delayed action, long-term action, multi-layered time action, long-acting type, long-term action, repeated action, sustained action and sustained release. For further consideration of these terms and how they are formulated, refer to Lesczek Krowczynski, Extended-Release Dosage Forms, 1987 (CRC Press, Inc.).

[0236] Controlled-release compositions can be prepared using methods known in the art. Examples of controlled-release preparations include semipermeable solid hydrophobic polymer matrices containing multispecific antibodies, where the matrix is ​​in the form of a molded article, such as a film or microcapsules. Examples of sustained-release matrices include polyesters, copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers, and poly-D-(-)-3-hydroxybutyric acid. Potential loss of biological activity and potential changes in immunogenicity of multispecific antibodies contained in sustained-release formulations can be reduced or prevented by controlling the water content using appropriate additives and by developing special polymer matrix compositions.

[0237] Controlled release technologies include both physical and chemical systems. Physical systems include reservoir systems with rate-limiting membranes, such as microencapsulation, macroencapsulation, and membrane systems; reservoir systems without rate-limiting membranes, such as hollow fibers, ultraporous cellulose triacetate, and porous polymer substrates and foams; monolithic systems including systems in which substances are physically dissolved in non-porous, polymer, or elastomer matrices (e.g., impermeable, erodable, environmentally drug-permeable, and degradable) and systems in which substances are physically dispersed in non-porous, polymer, or elastomer matrices (e.g., impermeable, erodable, environmentally drug-permeable, and degradable); laminate structures including an outer control layer and a chemically similar or different reservoir layer; and other physical methods such as adsorption to osmotic pumps or ion exchange resins.

[0238] Chemical systems include chemical erosion of the polymer matrix (e.g., heterogeneous or homogeneous erosion) or biological erosion of the polymer matrix (e.g., heterogeneous or homogeneous erosion). For further consideration of the types of controlled release systems, see Agis F. Kydonieus, Controlled Release Technologies: Methods, Theory and Applications, 1980 (CRC Press, Inc.).

[0239] Several controlled-release drug formulations for oral administration are available and can be used to formulate multispecific antibodies. These controlled-release formulations include osmotically controlled gastrointestinal delivery systems; hydrodynamically controlled gastrointestinal delivery systems; membrane permeability controlled gastrointestinal delivery systems, including microporous membrane permeability controlled gastrointestinal delivery devices; gastric acid-tolerant intestinal targeted controlled-release gastrointestinal delivery devices; gel diffusion controlled gastrointestinal delivery systems; and ion exchange controlled gastrointestinal delivery systems, including cationic and anionic drugs. For further information on controlled-release drug delivery systems, refer to Yie W. Chien, Novel Drug Delivery Systems, 1992 (Marcel Dekker, Inc.).

[0240] Multispecific antibodies may be administered to patients / subjects using any available method and route suitable for drug delivery, including in vivo and exovivo methods, as well as systemic and topical administration routes. Conventional pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical, intravenous, intra-arterial, rectal, nasal, oral, and other enteral and parenteral administration routes. The routes of administration may be combined as needed or modified depending on the multispecific antibody and / or the desired effect. Multispecific antibodies may be administered in single or multi-dose doses. In some embodiments, multispecific antibodies are administered orally. In some embodiments, multispecific antibodies are administered by inhalation. In some embodiments, multispecific antibodies are administered intranasally. In some embodiments, multispecific antibodies are administered topically. In some embodiments, multispecific antibodies are administered intracranially. In some embodiments, multispecific antibodies are administered intravenously.

[0241] In another embodiment, the present invention provides a method for treating cancer (tumor) using the multispecific antibodies described herein. This method comprises administering the multispecific antibodies to a subject having cancer or a tumor.

[0242] In some embodiments, the multispecific antibody is administered in combination with the cancer neoantigen vaccine or after the administration of the cancer neoantigen vaccine. The neoantigen vaccine and its production are described in U.S. Patent Application Publication No. 2017 / 0202939. The following examples are illustrative and not limiting to the methods of this disclosure. Other suitable variations and adaptations of various conditions and parameters commonly encountered in the art and obvious to those skilled in the art are within the scope of this disclosure. [Examples]

[0243] Examples 1-15 of the preparation of conditionally activated antibodies are described in International Publication No. 2017 / 078839.

[0244] Example 16: Multispecific antibody that binds to CD3 and Axl

[0245] Two multispecific antibodies were constructed. One multispecific antibody, combined with an unconditional active binding site (IgG antibody) for Axl(WT-Axl), provided a butterfly-type configuration WT-CD3-WT-Axl using an unconditional active binding site (scFv antibody) for CD3(WT-CD3) (Figures 2 and 3A-3C). Similarly, the second multispecific antibody, combined with a conditional active binding site (IgG antibody) for Axl(CAB-Axl), formed a butterfly-type configuration WT-CD3-CAB-Axl using an unconditional active binding site (scFv antibody) for CD3(WT-CD3) (Figures 2 and 3A-3C).

[0246] These two multispecific antibodies were assayed using ELISA to assess their affinity for CD3 and Axl at pH 6.0 and pH 7.4, respectively (Figures 3B-3C). The ELISA assay described herein followed the protocol: 1. The day before ELISA, 96-well plates were coated overnight with 100 μl of 0.5 μg / ml recombinant CD3 or Axl in ELISA coating buffer at 4°C. 2. Dilute the sample with ELISA assay buffer. 3. Shake off the buffer solution from the antigen-coated plate and wipe it dry with a paper towel. 4. Block the plate with 200 μl of ELISA assay buffer at room temperature for 1 hour. 5. Add 100 μl of diluted sample to each well. 6. Incubate the plate at room temperature for 1 hour. 7. Prepare the secondary antibody in the screening buffer according to the plate layout. 8. Shake off the buffer solution from the plate and wipe it dry with a paper towel. 9. Wash the plate a total of three times with ELISA washing buffer. 10. Add 100 μl of the diluted corresponding secondary antibody to the well in the ELISA assay buffer. - For wells coated with recombinant CD3, add an anti-human HRP secondary antibody. - For wells coated with Axl, recombinant CD3 is added, and then detected with anti-His antibody and anti-mouse HRP secondary antibody. 12. Incubate the plate at room temperature for 1 hour. 13. Shake off the buffer solution from the plate and wipe it dry with a paper towel. 14. Wash the plate a total of three times with ELISA washing buffer. 15. Shake off the buffer solution from the plate and wipe it dry with a paper towel. 16. Add 50 μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate according to the plate layout. Stop the color development with 17.50 μl of 1N HCl. 18. Read at OD450nm using a plate reader.

[0247] Negative controls (negative controls controlled by wild-type IgG antibodies for CD3, wild-type IgG antibodies for Axl, and isotype) did not show a significant difference in their affinity for CD3 at two different pH levels, 6.0 and 7.4 (Figure 3B). Similarly, negative controls did not show a significant difference in their affinity for Axl at two different pH levels, 6.0 and 7.4 (Figure 3C).

[0248] For these two multispecific antibodies, neither WT-CD3-WT-Axl nor WT-CD3-CAB-Axl showed a significant difference in their affinity for CD3 at two different pH levels, 6.0 and 7.4, because their CD3 binding sites are not in a conditionally active form (Figure 3B). Furthermore, WT-CD3-WT-Axl also showed no significant difference in its affinity for Axl at two different pH levels, 6.0 and 7.4, because its Axl binding site is not in a conditionally active form (Figure 3C). However, for WT-CD3-CAB-Axl, this multispecific antibody contains a conditionally active binding site for Axl, and therefore showed a significant increase in affinity for Axl at pH 6.0 compared to its affinity for Axl at pH 7.4 (Figure 3C).

[0249] Example 17: Multispecific antibody that binds to CD3 and Axl

[0250] In this example, further multispecific antibodies that bind to CD3 and Axl were constructed. The multispecific antibodies were prepared as described in Example 16 and named in the same manner as in Example 16: Axl-WT×CD3-CAB1: A conditional active binding site for Axl combined with a conditional active binding site for CD3; Axl-WT×CD3-CAB3: A conditional active binding site for Axl combined with a conditional active binding site for CD3; Axl-WT×CD3-CAB4: A conditional active binding site for Axl combined with a conditional active binding site for CD3; Axl-WT × CD3-WT: an unconditional active binding site for Axl combined with an unconditional active binding site for CD3; Axl-CAB×CD3-CAB1: A conditional active binding site for Axl combined with a conditional active binding site for CD3; Axl-CAB×CD3-CAB3: an unconditional active binding site for Axl combined with a conditional active binding site for CD3; and Axl-CAB×CD3-CAB4: An unconditional active binding site for Axl combined with a conditional active binding site for CD3.

[0251] For the aforementioned multispecific antibodies, their affinity for CD3 was assayed by immobilizing CD3 in an ELISA assay at pH 6.0 and 7.4 (Figure 4A). When the conditional active variable regions having binding sites to CD3 were included in the multispecific antibodies, it was observed that those multispecific antibodies showed an increase in affinity for CD3 at pH 6.0 as compared to their affinity for CD3 at pH 7.4. The two negative controls were an isotype control antibody that did not bind to CD3 and a buffer without added antibody.

[0252] For some of the multispecific antibodies, their binding to CD3 was assayed by immobilizing Axl. In the multispecific antibodies containing the conditional active variable regions of the CD3 antibody, the affinity for CD3 at pH 6.0 increased as compared to their affinity for CD3 at pH 7.4 (Figure 4B).

[0253] Example 18: Multispecific Antibodies That Bind to CD3 and Her2

[0254] In this example, multispecific antibodies that bind to CD3 and Her2 were constructed. Multispecific antibodies were created as described in Example 16 and named in the same manner as in the case of Example 16: Her2-WT×CD3-CAB1: An unconditional active binding site for Her2 combined with a conditional active binding site for CD3; Her2-WT×CD3-CAB3: An unconditional active binding site for Her2 combined with a conditional active binding site for CD3; and Her2-WT×CD3-CAB4: An unconditional active binding site for Her2 combined with a conditional active binding site for CD3; Her2-WT×CD3-WT: An unconditional active binding site for Her2 combined with an unconditional active binding site for CD3.

[0255] For the above-mentioned multispecific antibody, its affinity for CD3 was assayed by immobilizing CD3 in an ELISA assay at pH 6.0 and 7.4. Two negative controls were antibodies (B12 and NC) that did not bind to CD3. When the multispecific antibody contained a conditional active variable region of a CD3 antibody, it was observed that the antibody showed an increase in affinity for CD3 at pH 6.0 compared to its affinity for CD3 at pH 7.4 (Figure 5A).

[0256] In addition, for the multispecific antibody, its binding to CD3 was assayed by immobilizing Her2 in the assay. The multispecific antibody containing the conditional active variable region of the CD3 antibody showed an increase in affinity for CD3 at pH 6.0 compared to its affinity for CD3 at pH 7.4 (Figure 5B).

[0257] Example 19: Multispecific antibodies bind to CD3 and B7-H3

[0258] In this example, a multispecific antibody that binds to CD3 and B7-H3 was constructed. Multispecific antibodies were created as described in Example 16 and named in the same manner as in Example 16: B7-H3-WT×CD3-WT: Unconditional active binding site for B7-H3 combined with an unconditional active binding site for CD3; B7-H3-WT×CD3-CAB3: Unconditional active binding site for B7-H3 combined with a conditional active binding site for CD3; and B7-H3-WT×CD3-CAB4: Unconditional active binding site for B7-H3 combined with a conditional active binding site for CD3; B7-H – 3-CAB1×CD3-CAB4: Conditional active binding site for B7-H3 combined with a conditional active binding site for CD3; and B7-H3-CAB2×CD3-CAB4: Conditional active binding site for B7-H3 combined with a conditional active binding site for CD3.

[0259] The affinity of the above multispecific antibodies for CD3 was assayed in ELISA assays at pH 6.0 and 7.4 with CD3 immobilized on the phase. The two negative controls were an isotype control antibody that does not bind to CD3 and a buffer without antibodies. When the conditionally active variable region of the CD3 antibody was included in the multispecific antibody, it was observed that these multispecific antibodies showed an increased affinity for CD3 at pH 6.0 compared to their affinity for CD3 at pH 7.4 (Figure 6).

[0260] Example 20: Functional assay of a multispecific antibody that binds to CD3 and Axl

[0261] In this example, two multispecific antibodies were assayed for their function in stimulating Jurkat cells, which are tumor-reactive lymphocytes (Figure 7). This assay used modified Jurkat cells with constructs expressing luciferase driven by the IL-2 promoter or an NFAT regulatory element (RE). When the multispecific antibodies bound to Axl on tumor cells and CD3 on Jurkat cells, the luciferase construct in Jurkat cells was activated, and luciferase was expressed. The amount of luciferase was then measured. In this example, tumor cells were represented by target CHO cells modified to express Axl. The measured amount of luciferase indicated the level of stimulation of Jurkat cells by the multispecific antibodies after binding to the modified target CHO cells.

[0262] The functional assay (Promega CD3 assay) used the following protocol: 1. Add target cells to the wells (5000 cells / 100 μL medium, Costar #3917 96-well white plate) and incubate overnight at 37°C. 2. Remove the culture medium from the well. 3. Add 25 μL / well of 2X Ab dilution to pH 10% RPMI medium (final concentration should follow the plate layout). Add 4.25 μL / well of NFAT-Luc2PJurkat cells (1.2 mL frozen cells => 6 mL pH 10% RPMI). Incubate at 5.37°C for 5 hours. Add 6.50 μL / well of Bio-Glo at room temperature for 5 minutes. 7. Measure the luminescence using the Promega Bio-Glo program.

[0263] In Figure 8A, the multispecific antibody assayed was Axl-WT×CD3-WT from Example 17. The measured luciferase levels (relative luciferase units, RLU) were similar at both pH levels as the target cell count increased (the concentration of CHO-Axl cells is shown on the X-axis), indicating that the multispecific antibody produced similar levels of Jurkat cell stimulation at both pH 6.0 and pH 7.4. Therefore, this multispecific antibody was not conditionally active at pH 6.0 and 7.4.

[0264] Figure 8B shows that the multispecific antibody Axl-WT×CD3-CAB1 significantly increases the stimulation of Jurkat cells at pH 6.0 compared to the stimulation at pH 7.4. Therefore, this multispecific antibody was conditionally active in terms of Jurkat cell stimulation, as indicated by the significant increase in the amount of luciferase produced by Jurkat cells stimulated at pH 6.0 compared to the amount produced by Jurkat cells stimulated at pH 7.4.

[0265] Table 4 shows the luciferase levels measured at pH 6.0 and 7.4 for CHO-Axl cells at various concentrations. [Table 4]

[0266] In this example, the negative control was CHO cells that do not express Axl (i.e., non-target cells). When non-target cells were present, the multispecific antibody did not activate Jurkat cells (Figures 8A - 8B), indicating that the activation of Jurkat cells was dependent on the presence of target cells.

[0267] Example 21: Multispecific Antibodies That Bind CD3 and EpCAM

[0268] In this example, a multispecific antibody that binds CD3 and EpCAM was constructed. The multispecific antibody was prepared as described in Example 16 and named in the same manner as in Example 16: EpCAM-WT×CD3-BF1: An unconditioned active binding site for EpCAM combined with an unconditioned active binding site for CD3 (BAP150-07-BF1); EpCAM-WT×CD3-BF3: An unconditioned active binding site for EpCAM combined with a conditioned active binding site for CD3 (BAP150-07-BF3); EpCAM-WT×CD3-BF5: An unconditioned active binding site for EpCAM combined with a conditioned active binding site for CD3 (BAP150-07-BF5); EpCAM-WT×CD3-BF11: An unconditioned active binding site for EpCAM combined with a conditioned active binding site for CD3 (BAP150-07-BF11); EpCAM-WT×CD3-BF36: An unconditioned active binding site for EpCAM combined with a conditioned active binding site for CD3 (BAP150-07-BF36); EpCAM-WT×CD3-BF37: An unconditioned active binding site for EpCAM combined with a conditioned active binding site for CD3 (BAP150-07-BF37); EpCAM-WT×CD3-BF38: An unconditioned active binding site for EpCAM combined with a conditioned active binding site for CD3 (BAP150-07-BF38); EpCAM-WT×CD3-BF39: A conditional active binding site for EpCAM combined with a conditional active binding site for CD3 (BAP150-07-BF39); EpCAM-WT×CD3-BF40: an unconditional active binding site for EpCAM combined with a conditionally active binding site for CD3 (BAP150-07-BF40); and EpCAM-WT × CD3-BF41: A conditional active binding site for EpCAM combined with a conditional active binding site for CD3 (BAP150-07-BF41).

[0269] The EpCAM-WT portion of each multispecific antibody is identical and contains a full light chain including an anti-EpCAM heavy chain variable region (SEQ ID NO: 85) and an anti-EpCAM light chain variable region (SEQ ID NO: 93). The C-terminus of the full light chain is linked to a CD3 binding site. Among the CD3 binding sites, CD3-BF1 (SEQ ID NO: 26) is an unconditionally activated single-chain anti-CD3 antibody, while CD3-BF3 (SEQ ID NO: 28), CD3-BF5 (SEQ ID NO: 30), CD3-BF11 (SEQ ID NO: 36), CD3-BF36 (SEQ ID NO: 61), CD3-BF37 (SEQ ID NO: 62), CD3-BF38 (SEQ ID NO: 63), CD3-BF39 (SEQ ID NO: 64), CD3-BF40 (SEQ ID NO: 65), and CD3-BF41 (SEQ ID NO: 66) are conditionally activated single-chain anti-CD3 antibodies.

[0270] The affinity of the aforementioned multispecific antibodies for CD3 was assayed in an ELISA assay with CD3 immobilized at pH values ​​of 6.0 to 7.4 (Figure 9). When conditionally active anti-CD3 single-chain antibodies were included in the multispecific antibodies, it was observed that these multispecific antibodies showed an increased affinity for CD3 at pH 6.0 compared to their affinity for CD3 at pH 7.4. In the negative control (BAP150-07-BF1) whose CD3 binding site is not conditionally active, the binding affinity for CD3 in the pH range of 6.0 to 7.4 was not pH-dependent.

[0271] Example 22: Multispecific antibodies against CD3 and EpCAM for tumor treatment

[0272] A multispecific antibody (EpCAM×CAB-CD3) containing both an unconditional binding site for EpCAM and a conditional binding site for CD3 was used to treat a tumor xenograft mouse model of the MiXeno mouse model manufactured by Crown Bioscience (San Diego, CA). Specifically, HCT116 colon cancer cell line (EpCAM-positive) was transplanted into triple-immunodeficient mice engrafted with human peripheral blood mononuclear cells to induce tumors in the mouse model. The tumor volume was approximately 150 mm². 3 At this point, tumor-bearing animals were randomized into four treatment groups. These four treatment groups were treated with either a vehicle as a negative control (Group 1), a non-CAB-CD3 benchmark antibody as a positive control (Group 2), a multispecific antibody EpCAM×CAB-CD3 (Group 3), or an isotype-matching antibody as a negative control (Group 4). Antibodies were administered every other week for four weeks at a dose of 2.5 mg / kg. The non-CAB-CD3 benchmark antibody included an unconditional active binding site for EpCAM and an unconditional active binding site for CD3.

[0273] The multispecific antibody EpCAM×CAB-CD3 was as effective as the positive control non-CAB-CD3 benchmark antibody in inducing complete tumor regression in a xenograft mouse model. However, two negative controls failed to induce tumor regression, as tumor size continued to increase in mice in these negative control groups. See Figure 10.

[0274] Anti-CD3 antibodies have the side effect of inducing T cell activation in the peripheral circulatory system, which can be measured by serum INF-γ levels using the Meso Scale Discovery (MSD) assay. The multispecific antibody EpCAM×CAB-CD3 resulted in a significant reduction in T cell activation compared to the positive control non-CAB-CD3 benchmark antibody. See Figure 11. Thus, because the multispecific antibody EpCAM×CAB-CD3 contains a conditionally activated anti-CD3 antibody component, it resulted in a significant reduction in side effects compared to the positive control non-CAB-CD3 benchmark antibody, but the therapeutic effect was equivalent.

[0275] All documents referenced herein are incorporated herein by reference, both in whole and at least in part, to provide disclosures on which they are specifically relied or cited. The applicants do not intend to provide any disclosed embodiments to the public, and any disclosed variations or modifications may not be literally included within the claims, so they shall be considered part thereof under the doctrine of equivalents.

[0276] However, even though many features and advantages of the present invention are shown in the foregoing description in conjunction with details of the structure and function of the present invention, it should be understood that this disclosure is merely illustrative and that modifications may be made within the scope of the principles of the present invention to reach the full scope indicated by the broad general meaning of the terms used to express the appended claims, particularly in terms of the shape, size and arrangement of the parts.

[0277] SEQUENCE LISTING <110> BioAtla LLC <120> MULTI-SPECIFIC ANTIBODY CONSTRUCTS <130> BIAT-1025 <160> 95 <170> PatentIn version 3.5 <210> 1 <211> 110 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 1 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 2 <211> 110 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 2 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Pro Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 3 <211> 110 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 3 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Pro Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 4 <211> 110 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 4 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Leu Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 5 <211> 110 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 5 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Ser Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 6 <211> 110 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 6 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Phe Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Thr Gly 100 105 110 <210> 7 <211> 110 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 7 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Gln Gly 100 105 110 <210> 8 <211> 110 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 8 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Asp Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 9 <211> 110 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 9 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Glu Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 10 <211> 110 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 10 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Lys Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 11 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 12 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Lys Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 13 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 14 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 15 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Glu His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 16 <211> 107 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 16 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ser Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Glu His Phe Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 17 <211> 107 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Val Ser Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Trp Gln Asp Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Glu His Phe Ser Pro Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 18 <211> 120 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 18 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Ser Phe Thr Gly His 20 25 30 Thr Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Lys Pro Ser Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala His Gly His Tyr Glu Ser Tyr Phe Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 19 <211> 120 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 19 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Ser Phe Trp Gly Ala 20 25 30 Thr Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Lys Pro Ser Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala His Gly His Tyr Glu Ser Tyr Glu Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 107 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 20 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 21 <211> 120 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 21 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 22 <211> 112 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 22 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp His Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Ile His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 23 <211> 116 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 23 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly His Asn Tyr Gly Met Asn 20 25 30 Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile 35 40 45 Asn Pro Tyr Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe Lys Gly Arg 50 55 60 Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile 65 70 75 80 Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp 85 90 95 Asp Gly Tyr Phe Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser 115 <210> 24 <211> 116 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 24 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly His Asn Tyr Gly Asp Asn 20 25 30 Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile 35 40 45 Asn Pro Tyr Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe Lys Gly Arg 50 55 60 Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile 65 70 75 80 Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp 85 90 95 Asp Gly Tyr Phe Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser 115 <210> 25 <211> 116 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 25 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly His Asn Tyr Gly Met Asn 20 25 30 Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile 35 40 45 Asn Pro Tyr Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe Lys Gly Arg 50 55 60 Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile 65 70 75 80 Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp 85 90 95 Asp Gly Tyr Phe Tyr Asp Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser 115 <210> 26 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 27 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 27 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 28 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 28 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 29 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 29 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Pro Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 30 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 31 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 31 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 32 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 32 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Lys Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 33 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 33 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn His Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 34 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 34 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Tyr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 35 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 35 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Pro Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 36 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 36 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Gly Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 37 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 37 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 38 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 38 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Pro Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 39 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 39 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Pro Asn Ser Tyr Val Ser Trp Phe 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 40 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 40 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Lys Val Ser Trp Phe 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 41 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 41 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Pro Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 42 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Pro Asn Ser Tyr Val Ser Trp Phe 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 43 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 43 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Gly Asn Ser Lys Val Ser Trp Phe 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 44 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 44 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Pro Asn Ser Lys Val Ser Trp Phe 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 45 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 45 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Pro Asn Ser Lys Val Ser Trp Phe 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 46 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 46 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Lys Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 47 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 47 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Pro Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Lys Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 48 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 48 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Lys Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 49 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 49 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Lys Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 50 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 50 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Pro Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Lys Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 51 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 51 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Gly Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Lys Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 52 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 52 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Lys Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 53 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 53 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Pro Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Lys Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 54 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 54 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Pro Asn Ser Tyr Val Ser Trp Phe 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Lys Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 55 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 55 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Lys Val Ser Trp Phe 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Lys Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 56 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 56 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Pro Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Lys Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 57 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 57 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Pro Asn Ser Tyr Val Ser Trp Phe 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Lys Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 58 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 58 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Gly Asn Ser Lys Val Ser Trp Phe 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Lys Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 59 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 59 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Pro Asn Ser Lys Val Ser Trp Phe 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Lys Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 60 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 60 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Pro Asn Ser Lys Val Ser Trp Phe 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Lys Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 61 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 61 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Ala Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 62 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 62 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Trp Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 63 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 63 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Tyr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 64 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 64 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ala Asn Phe Gly Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 65 <211> 248 <212> PRT <213> Artificial <220> <223> Synehtic sequence <400> 65 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Pro Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 66 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 66 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Gly Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 67 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 67 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Thr Asn Phe Gly Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 68 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 68 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ala Asn Phe Gly Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Ala Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 69 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 69 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Pro Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Ala Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 70 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 70 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ser Asn Phe Gly Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Ala Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 71 <211> 248 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 71 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Thr Asn Phe Gly Asn Ser Lys Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr 130 135 140 Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 145 150 155 160 Cys Arg Ser Ser Ala Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp 165 170 175 Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr 180 185 190 Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 195 200 205 Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 210 215 220 Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 72 <211> 469 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 72 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Glu Ile Ala Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Asp Tyr Phe Cys His Gln Trp Ser Thr Tyr His Thr Phe 85 90 95 Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser 100 105 110 Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala 115 120 125 Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 130 135 140 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser 145 150 155 160 Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 165 170 175 Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys 180 185 190 Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 195 200 205 Arg Gly Glu Cys Ser Arg Ser Gly Gly Gly Gly Glu Val Gln Leu Val 210 215 220 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 225 230 235 240 Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val 245 250 255 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser 260 265 270 Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg 275 280 285 Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met 290 295 300 Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His 305 310 315 320 Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln 325 330 335 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 340 345 350 Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr Gln Glu Pro Ser Leu 355 360 365 Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr 370 375 380 Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp Val Gln Gln Lys Pro 385 390 395 400 Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro 405 410 415 Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala 420 425 430 Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 435 440 445 Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu 450 455 460 Thr Val Leu Ser Arg 465 <210> 73 <211> 469 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 73 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Glu Ile Ala Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Asp Tyr Phe Cys His Gln Trp Ser Thr Tyr His Thr Phe 85 90 95 Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser 100 105 110 Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala 115 120 125 Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 130 135 140 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser 145 150 155 160 Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 165 170 175 Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys 180 185 190 Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 195 200 205 Arg Gly Glu Cys Ser Arg Ser Gly Gly Gly Gly Glu Val Gln Leu Val 210 215 220 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 225 230 235 240 Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val 245 250 255 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser 260 265 270 Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg 275 280 285 Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met 290 295 300 Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His 305 310 315 320 Ser Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln 325 330 335 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 340 345 350 Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr Gln Glu Pro Ser Leu 355 360 365 Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr 370 375 380 Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp Val Gln Gln Lys Pro 385 390 395 400 Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro 405 410 415 Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala 420 425 430 Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 435 440 445 Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu 450 455 460 Thr Val Leu Ser Arg 465 <210> 74 <211> 469 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 74 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Glu Ile Ala Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Asp Tyr Phe Cys His Gln Trp Ser Thr Tyr His Thr Phe 85 90 95 Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser 100 105 110 Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala 115 120 125 Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 130 135 140 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser 145 150 155 160 Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 165 170 175 Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys 180 185 190 Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 195 200 205 Arg Gly Glu Cys Ser Arg Ser Gly Gly Gly Gly Glu Val Gln Leu Val 210 215 220 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 225 230 235 240 Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val 245 250 255 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser 260 265 270 Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg 275 280 285 Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met 290 295 300 Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His 305 310 315 320 Gly Asn Phe Pro Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln 325 330 335 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 340 345 350 Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr Gln Glu Pro Ser Leu 355 360 365 Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr 370 375 380 Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp Val Gln Gln Lys Pro 385 390 395 400 Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro 405 410 415 Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala 420 425 430 Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 435 440 445 Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu 450 455 460 Thr Val Leu Ser Arg 465 <210> 75 <211> 469 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 75 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Glu Ile Ala Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Asp Tyr Phe Cys His Gln Trp Ser Thr Tyr His Thr Phe 85 90 95 Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser 100 105 110 Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala 115 120 125 Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 130 135 140 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser 145 150 155 160 Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 165 170 175 Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys 180 185 190 Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 195 200 205 Arg Gly Glu Cys Ser Arg Ser Gly Gly Gly Gly Glu Val Gln Leu Val 210 215 220 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 225 230 235 240 Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val 245 250 255 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser 260 265 270 Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg 275 280 285 Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met 290 295 300 Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His 305 310 315 320 Gly Asn Phe Gly Asn Ser Lys Val Ser Trp Phe Ala Tyr Trp Gly Gln 325 330 335 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 340 345 350 Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr Gln Glu Pro Ser Leu 355 360 365 Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr 370 375 380 Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp Val Gln Gln Lys Pro 385 390 395 400 Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro 405 410 415 Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala 420 425 430 Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 435 440 445 Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu 450 455 460 Thr Val Leu Ser Arg 465 <210> 76 <211> 469 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 76 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Glu Ile Ala Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Asp Tyr Phe Cys His Gln Trp Ser Thr Tyr His Thr Phe 85 90 95 Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser 100 105 110 Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala 115 120 125 Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 130 135 140 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser 145 150 155 160 Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 165 170 175 Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys 180 185 190 Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 195 200 205 Arg Gly Glu Cys Ser Arg Ser Gly Gly Gly Gly Glu Val Gln Leu Val 210 215 220 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 225 230 235 240 Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val 245 250 255 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser 260 265 270 Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg 275 280 285 Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met 290 295 300 Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His 305 310 315 320 Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Gln Tyr Trp Gly Gln 325 330 335 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 340 345 350 Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr Gln Glu Pro Ser Leu 355 360 365 Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr 370 375 380 Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp Val Gln Gln Lys Pro 385 390 395 400 Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro 405 410 415 Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala 420 425 430 Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 435 440 445 Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu 450 455 460 Thr Val Leu Ser Arg 465 <210> 77 <211> 469 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 77 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Glu Ile Ala Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Asp Tyr Phe Cys His Gln Trp Ser Thr Tyr His Thr Phe 85 90 95 Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser 100 105 110 Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala 115 120 125 Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 130 135 140 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser 145 150 155 160 Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 165 170 175 Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys 180 185 190 Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 195 200 205 Arg Gly Glu Cys Ser Arg Ser Gly Gly Gly Gly Glu Val Gln Leu Val 210 215 220 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 225 230 235 240 Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val 245 250 255 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser 260 265 270 Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg 275 280 285 Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met 290 295 300 Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His 305 310 315 320 Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln 325 330 335 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 340 345 350 Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr Gln Glu Pro Ser Leu 355 360 365 Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr 370 375 380 Gly Ala Val Thr Thr Lys Asn Tyr Asp Asn Trp Val Gln Gln Lys Pro 385 390 395 400 Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro 405 410 415 Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala 420 425 430 Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 435 440 445 Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu 450 455 460 Thr Val Leu Ser Arg 465 <210> 78 <211> 469 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 78 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Glu Ile Ala Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Asp Tyr Phe Cys His Gln Trp Ser Thr Tyr His Thr Phe 85 90 95 Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser 100 105 110 Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala 115 120 125 Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 130 135 140 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser 145 150 155 160 Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 165 170 175 Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys 180 185 190 Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 195 200 205 Arg Gly Glu Cys Ser Arg Ser Gly Gly Gly Gly Glu Val Gln Leu Val 210 215 220 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 225 230 235 240 Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val 245 250 255 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser 260 265 270 Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg 275 280 285 Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met 290 295 300 Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His 305 310 315 320 Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln 325 330 335 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 340 345 350 Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr Gln Glu Pro Ser Leu 355 360 365 Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr 370 375 380 Gly Ala Val Thr Thr Ser Asn Tyr Asp Asn Trp Val Gln Gln Lys Pro 385 390 395 400 Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro 405 410 415 Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala 420 425 430 Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 435 440 445 Ala Leu Trp Tyr Ser Asn His Trp Val Phe Gly Gly Gly Thr Lys Leu 450 455 460 Thr Val Leu Ser Arg 465 <210> 79 <211> 469 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 79 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Glu Ile Ala Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Asp Tyr Phe Cys His Gln Trp Ser Thr Tyr His Thr Phe 85 90 95 Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser 100 105 110 Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala 115 120 125 Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 130 135 140 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser 145 150 155 160 Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 165 170 175 Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys 180 185 190 Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 195 200 205 Arg Gly Glu Cys Ser Arg Ser Gly Gly Gly Gly Glu Val Gln Leu Val 210 215 220 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 225 230 235 240 Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val 245 250 255 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser 260 265 270 Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg 275 280 285 Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met 290 295 300 Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His 305 310 315 320 Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln 325 330 335 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 340 345 350 Ser Gly Gly Ser Gly Gly Gln Ala Val Val Thr Gln Glu Pro Ser Leu 355 360 365 Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr 370 375 380 Gly Ala Val Thr Tyr Ser Asn Tyr Asp Asn Trp Val Gln Gln Lys Pro 385 390 395 400 Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro 405 410 415 Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala 420 425 430 Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 435 440 445 Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu 450 455 460 Thr Val Leu Ser Arg 465 <210> 80 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 80 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Arg Pro Ser Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Asp Asn Trp Val Gly Phe Ala Asn Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 81 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 81 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Arg Pro Ser Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Asp Asn Trp Val Gly Phe Ala Asn Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 82 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 82 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Arg Pro Ser Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Asp Asn Trp Val Gly Phe Ala Asn Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 83 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 83 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Arg Pro Ser Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Asp Asn Trp Val Gly Phe Ala Asn Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 84 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 84 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Arg Pro Ser Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Asp Asn Trp Val Gly Phe Ala Asn Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 85 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 85 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Arg Pro Ser Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Asp Asn Trp Val Gly Phe Ala Asn Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 86 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 86 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Arg Pro Ser Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Asp Asn Trp Val Gly Phe Ala Asn Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 87 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 87 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Arg Pro Ser Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Asp Asn Trp Val Gly Phe Ala Asn Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 88 <211> 105 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 88 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Glu Ile Ala Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Asp Tyr Phe Cys His Gln Trp Ser Thr Tyr His Thr Phe 85 90 95 Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 89 <211> 105 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 89 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Glu Ile Ala Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Asp Tyr Phe Cys His Gln Trp Ser Thr Tyr His Thr Phe 85 90 95 Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 90 <211> 105 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 90 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Glu Ile Ala Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Asp Tyr Phe Cys His Gln Trp Ser Thr Tyr His Thr Phe 85 90 95 Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 91 <211> 105 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 91 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Glu Ile Ala Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Asp Tyr Phe Cys His Gln Trp Ser Thr Tyr His Thr Phe 85 90 95 Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 92 <211> 105 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 92 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Glu Ile Ala Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Asp Tyr Phe Cys His Gln Trp Ser Thr Tyr His Thr Phe 85 90 95 Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 93 <211> 105 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 93 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Glu Ile Ala Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Asp Tyr Phe Cys His Gln Trp Ser Thr Tyr His Thr Phe 85 90 95 Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 94 <211> 105 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 94 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Glu Ile Ala Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Asp Tyr Phe Cys His Gln Trp Ser Thr Tyr His Thr Phe 85 90 95 Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 95 <211> 105 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 95 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Glu Ile Ala Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Asp Tyr Phe Cys His Gln Trp Ser Thr Tyr His Thr Phe 85 90 95 Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105

Claims

1. At least one binding site for a cell-specific antigen; and at least one binding site for tumor-reactive lymphocyte antigens A multispecific antibody comprising the above, wherein under first physiological conditions, it binds to at least one of the cell-specific antigen and the tumor-reactive lymphocyte antigen with greater affinity than under second physiological conditions.

2. The multispecific antibody according to claim 1, wherein the cell-specific antigen is a cancer cell-specific antigen.

3. The multispecific antibody according to claim 2, wherein the cancer cell-specific antigen is a neoantigen.

4. The multispecific antibody according to claim 1, wherein the cell-specific antigen is a senescent cell-specific antigen.

5. A multispecific antibody according to any one of claims 1 to 4, wherein the first physiological condition is an abnormal condition, and the second physiological condition is a normal physiological condition.

6. The multispecific antibody according to claim 5, wherein the aforementioned abnormal conditions are conditions in the tumor microenvironment.

7. The multispecific antibody according to claim 5, wherein the aforementioned abnormal conditions are conditions in the senescent cell microenvironment.

8. The multispecific antibody according to any one of claims 1 to 7, wherein the binding of the multispecific antibody to at least one of the cell-specific antigen and the tumor-reactive lymphocyte antigen is reversible.

9. (1) Bispecific antibody conjugate; (2) Hybrid bispecific IgG2; (3) Bispecific antibody molecule with only variable domain; (4) CH1 / CL fusion protein; (5) Fab fusion protein; (6) Non-immunoglobulin fusion protein; (7) Fc-modified IgG; (8) Additive and Fc-modified IgG; (9) Modified Fc and CH3 fusion protein; (10) Additive IgG-HC fusion; (11) Additive IgG-LC fusion; (12) Additive IgG-HC and LC fusion; (13) Fc fusion; (14) CH3 fusion; (15) IgE / IgM CH2 fusion; (16) F(ab') ２ A multispecific antibody according to any one of claims 1 to 8, comprising a fusion; (17) a CH1 / CL fusion protein; (18) a modified IgG; and (19) a non-immunoglobulin fusion in a format selected from these.

10. A conjugated multispecific antibody comprising a multispecific antibody according to any one of claims 1 to 9, which is conjugated to a polymer.

11. The conjugate-type multispecific antibody according to claim 10, wherein the polymer is selected from at least one of a protein, a fatty acid, and a polymer.

12. The conjugate-type multispecific antibody according to claim 10, wherein the polymer is albumin or polyethylene glycol.

13. The at least one binding site for the cell antigen is an IgG antibody or a fragment thereof, and the cell-specific antigen is a tumor cell antigen, and The multispecific antibody according to any one of claims 1 to 12, wherein the at least one binding site for the tumor-reactive lymphocyte antigen is a single-chain antibody.

14. The multispecific antibody according to claim 13, wherein at least one single-chain antibody is an scFv antibody.

15. The multispecific antibody according to claim 14, wherein at least one scFv antibody is linked via a linker to the C-terminus of at least one light chain or heavy chain of the IgG antibody or a fragment thereof.

16. The multispecific antibody according to claim 14 or 15, wherein the scFv antibody comprises a light chain variable region selected from light chain variable regions having the amino acid sequence of SEQ ID NOs: 1 to 10, and a heavy chain variable region selected from heavy chain variable regions having the amino acid sequence of SEQ ID NOs: 11 to 15.

17. The multispecific antibody according to claim 14 or 15, wherein the scFv antibody has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 26 to 71.

18. The IgG antibody A light chain variable region selected from light chain variable regions that bind to an antigen selected from Axl, Her2, B7-H3 and EpCAM and have amino acid sequences of SEQ ID NOs. 16-17, 20, 22 and 88-95; and A heavy chain variable region selected from heavy chain variable regions that bind to the same antigen as the light chain variable region and have the amino acid sequences of SEQ ID NOs: 18-19, 21, 23-25 ​​and 80-87. A multispecific antibody according to any one of claims 14 to 17, comprising:

19. The multispecific antibody according to any one of claims 1 to 18, wherein the tumor-reactive lymphocyte antigen is a CD3 antigen.

20. The multispecific antibody according to any one of claims 1 to 19, wherein the tumor-reactive lymphocyte antigen is located on lymphocytes selected from T cells, macrophages, Jurkat cells, monocytes, NK cells, neutrophils, eosinophils, basophils, and lymphokine-activated killer cells.

21. A multispecific antibody according to any one of claims 1 to 9 or 13 to 20, wherein the cell-specific antigen is selected from Axl, EpCAM, Ror2, Her2, and B7-H3.

22. A multispecific antibody according to any one of claims 1 to 9 or 13 to 21, which binds to the cell-specific antigen with greater affinity under the first physiological conditions than under the second physiological conditions.

23. A multispecific antibody according to any one of claims 1 to 9 or 13 to 22, which binds to the tumor-reactive lymphocyte antigen with greater affinity under the first physiological conditions than under the second physiological conditions.

24. A multispecific antibody according to any one of claims 1 to 9 or 13 to 23, which binds to both the cell antigen and the tumor-reactive lymphocyte antigen with greater affinity under the first physiological conditions than under the second physiological conditions.

25. A multispecific antibody according to any one of claims 1 to 9 or 13 to 24, which binds to the combination of the cell antigen and the tumor-reactive lymphocyte antigen with greater avidity under the first physiological conditions than under the second physiological conditions.

26. An IgG antibody or fragment thereof that binds to the first antigen; and At least one scFv antibody that binds to a second antigen different from the first antigen and is linked to the C-terminus of at least one light chain or heavy chain of the IgG antibody or fragment. A multispecific antibody comprising the above, which reversibly binds to at least one of the first antigen and the second antigen with greater affinity under abnormal conditions than under normal physiological conditions.

27. The multispecific antibody according to claim 26, wherein the second antigen is the CD3 antigen.

28. The multispecific antibody according to claim 26 or 27, wherein the scFv antibody comprises a light chain variable region selected from light chain variable regions having the amino acid sequence of SEQ ID NOs: 1 to 10, and a heavy chain variable region selected from heavy chain variable regions having the amino acid sequence of SEQ ID NOs: 11 to 15.

29. The multispecific antibody according to claim 26 or 27, wherein the scFv antibody has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 26 to 71.

30. The IgG antibody A light chain variable region selected from light chain variable regions that bind to an antigen selected from Axl, Her2, B7-H3 and EpCAM and have amino acid sequences of SEQ ID NOs. 16-17, 20, 22 and 88-95; and A heavy chain variable region selected from heavy chain variable regions that bind to the same antigen as the light chain variable region and have the amino acid sequences of SEQ ID NOs: 18-19, 21, 23-25 ​​and 80-87. A multispecific antibody according to any one of claims 26 to 29, comprising:

31. A multispecific antibody according to any one of claims 26 to 30, which binds to the first antigen with greater affinity under the abnormal conditions than under the normal physiological conditions.

32. A multispecific antibody according to any one of claims 26 to 31, which binds to the second antigen with greater affinity under the abnormal conditions than under the normal physiological conditions.

33. A multispecific antibody according to any one of claims 26 to 32, wherein under the abnormal conditions, it binds to both the first antigen and the second antigen with greater affinity than under the normal physiological conditions.

34. A multispecific antibody according to any one of claims 26 to 33, which, under the abnormal conditions, binds to the combination of the first antigen and the second antigen with greater avidity than under the normal physiological conditions.

35. The multispecific antibody according to any one of claims 26 to 34, wherein the first antigen is a cell surface antigen.

36. The multispecific antibody according to claim 35, wherein the cell surface antigen is a cancer cell-specific antigen.

37. The multispecific antibody according to any one of claims 26 to 36, wherein the first antigen is a neoantigen.

38. The multispecific antibody according to any one of claims 26 to 36, wherein the first antigen is selected from Axl, EpCAM, Ror2, Her2, and B7-H3.

39. A method for producing multispecific antibodies, a) A step of obtaining an IgG antibody or a fragment thereof that binds to a first antigen; b) A step of producing one or more structures by ligating at least one scFv antibody bound to a second antigen to the C-terminus of at least one light chain of the IgG antibody or fragment; c) A step of screening one or more of the structures from step b) for binding to at least one of the first antigen and the second antigen under abnormal and normal physiological conditions; and d) A step of selecting from one or more structures a multispecific antibody that reversibly binds to at least one of the first antigen and the second antigen with greater affinity under abnormal conditions than under normal physiological conditions. A method that includes this.

40. The method according to claim 39, wherein the first antigen is a tumor cell antigen.

41. The method according to claim 40, wherein the tumor cell antigen is selected from Axl, EpCAM, Ror2, Her2, and B7-H3.

42. The method according to claim 39, wherein the tumor cell antigen is a neoantigen.

43. The method according to any one of claims 39 to 42, wherein the second antigen is a tumor-reactive lymphocyte antigen.

44. The method according to claim 43, wherein the tumor-reactive lymphocyte antigen is CD3.

45. The method according to claim 43, wherein the tumor-reactive lymphocyte antigen is located on lymphocytes selected from T cells, macrophages, Jurkat cells, monocytes, NK cells, neutrophils, eosinophils, basophils, and lymphokine-activated killer cells.

46. The method according to any one of claims 39 to 45, wherein the scFv antibody comprises a light chain variable region selected from light chain variable regions having the amino acid sequence of SEQ ID NOs: 1 to 10, and a heavy chain variable region selected from heavy chain variable regions having the amino acid sequence of SEQ ID NOs: 11 to 15.

47. The method according to any one of claims 39 to 45, wherein the scFv antibody has an amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 26 to 71.

48. The IgG antibody A light chain variable region selected from light chain variable regions that bind to an antigen selected from Axl, Her2, B7-H3 and EpCAM and have amino acid sequences of SEQ ID NOs. 16-17, 20, 22 and 88-95; and A heavy chain variable region selected from heavy chain variable regions that bind to the same antigen as the light chain variable region and have the amino acid sequences of SEQ ID NOs: 18-19, 21, 23-25 ​​and 80-87. The method according to any one of claims 39 to 47, including the method described in any one of claims 39 to 47.

49. The method according to any one of claims 39 to 48, wherein the multispecific antibody binds to the first antigen with greater affinity under the abnormal conditions than under the normal physiological conditions.

50. The method according to any one of claims 39 to 49, wherein the multispecific antibody binds to the second antigen with greater affinity under the abnormal conditions than under the normal physiological conditions.

51. The method according to any one of claims 39 to 50, wherein the multispecific antibody binds to both the first antigen and the second antigen with greater affinity under the abnormal conditions than under the normal physiological conditions.

52. The method according to any one of claims 39 to 51, wherein the multispecific antibody binds to the combination of the first antigen and the second antigen with greater avidity under the abnormal conditions than under the normal physiological conditions.

53. A method for treating a target tumor, comprising administering a multispecific antibody according to any one of claims 1 to 9 or 13 to 38 or a conjugate multispecific antibody according to any one of claims 10 to 12.

54. The method according to claim 53, wherein the multispecific antibody or conjugate multispecific antibody is administered in combination with a cancer neoantigen vaccine.

55. The method according to claim 54, wherein the multispecific antibody or the conjugate multispecific antibody is administered after administration of a cancer neoantigen vaccine.

56. A method for producing multispecific antibodies, a) A step of obtaining an IgG antibody or a fragment thereof that binds to a first antigen; b) A step to obtain an scFv antibody that binds to a second antigen; c) A step of producing one or more evolved antibodies by evolving one or both of the antibodies in a) and b); d) A step of screening one or more evolved antibodies from c) and selecting antibodies that bind to each of their antigens with greater affinity under abnormal conditions than under normal physiological conditions; e) A step of producing one or more structures by ligating an scFv antibody bound to the second antigen to the C-terminus of at least one light chain of an IgG antibody or fragment bound to the first antigen, wherein at least one of the scFv antibody and the IgG antibody is selected in d), and if present, the scFv antibody or the IgG antibody not selected in d) is from either step a) or b); f) A step of screening one or more of the structures from step e) for binding to at least one of the first antigen and the second antigen under the abnormal conditions and the normal physiological conditions; and g) A step of selecting from one or more structures a multispecific antibody that binds to at least one of the first antigen and the second antigen with greater affinity under abnormal conditions than under normal physiological conditions. A method that includes this.

57. IgG antibodies or fragments thereof that bind to cell-specific antigens; and A multispecific antibody comprising at least one scFv antibody that binds to a T lymphocyte antigen, which is linked to the C-terminus of at least one light chain or at least one heavy chain of the IgG antibody or a fragment thereof, wherein the at least one scFv antibody binds to the T lymphocyte antigen with greater affinity under abnormal conditions than under normal physiological conditions.

58. A method for producing multispecific antibodies, a) A step of obtaining an IgG antibody or a fragment thereof that binds to a first antigen; b) A step of producing one or more structures by ligating at least one scFv antibody bound to a second antigen to the C-terminus of at least one light chain of IgG or a fragment thereof, wherein the at least one scFv antibody binds to the second antigen with greater affinity under abnormal conditions than under normal physiological conditions; c) A step of screening one or more structures for binding to the first antigen and the second antigen under normal physiological conditions and abnormal conditions; and d) A step of selecting a multispecific antibody that binds to the first antigen and reversibly binds to the second antigen with a greater affinity under abnormal conditions than under normal physiological conditions. A method that includes this.

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