Mesothelin-binding protein

Abstract

Disclosed herein are MSLN binding proteins having improved binding affinity and improved ability to mediate T cell-dependent killing of mesothelin-expressing cancer cells.Further provided are pharmaceutical compositions comprising the binding proteins disclosed herein and methods of using such formulations.

Description

[0001] This application is a divisional application of the Chinese patent application with an application date of May 11, 2018, application number 201880046583.8, and invention name “Mesothelin-binding protein” (the application date of the corresponding PCT application is May 11, 2018, and application number PCT / US2018 / 032418).

[0002] Cross-references

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 505,719, filed May 12, 2017, and U.S. Provisional Application No. 62 / 657,417, filed April 13, 2018, each of which is incorporated herein by reference in its entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on May 11, 2018, is named 47517-719_601_SL.txt, and is 145,039 bytes in size.

[0006] Incorporation by reference

[0007] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Background Art

[0008] The present disclosure provides mesothelin (MSLN) binding proteins that can be used to diagnose and treat indications related to the expression of MSLN. Mesothelin (MSLN) is a GPI-linked membrane-bound tumor antigen MSLN that is overexpressed in ovarian cancer, pancreatic cancer, lung cancer, triple-negative breast cancer, and mesothelioma. Normal tissue expression of MSLN is limited to the single-cell mesothelial layer lining the pleural, pericardial, and peritoneal cavities. Overexpression of MSLN is associated with a poor prognosis in lung adenocarcinoma and triple-negative breast cancer. MSLN has been used as a cancer antigen for a large number of approaches, including immunotoxins, vaccines, antibody drug conjugates, and CAR-T cells. Early signs of clinical efficacy have confirmed MSLN as a target, but there is a need for improved therapeutics to treat cancers expressing MSLN. Summary of the Invention

[0009] One embodiment provides a single-domain mesothelin binding protein, wherein the protein comprises one or more conserved regions comprising a sequence identical to, or comprising one or more amino acid residue substitutions relative to, SEQ ID NO: 41, 42, 43, or 44. In some embodiments, the protein comprises a conserved region comprising a sequence identical to, or comprising one or more amino acid residue substitutions relative to, SEQ ID NO: 41. In some embodiments, the protein comprises a conserved region comprising a sequence identical to, or comprising one or more amino acid residue substitutions relative to, SEQ ID NO: 42. In some embodiments, the protein comprises a conserved region comprising a sequence identical to, or comprising one or more amino acid residue substitutions relative to, SEQ ID NO: 43. In some embodiments, the protein comprises a conserved region comprising a sequence identical to, or comprising one or more amino acid residue substitutions relative to, SEQ ID NO: 44. In some embodiments, the protein comprises (i) a stretch of amino acids corresponding to SEQ ID NO:41; (ii) a stretch of amino acids corresponding to SEQ ID NO:42; (iii) a stretch of amino acids corresponding to SEQ ID NO:43; and (iv) a stretch of amino acids corresponding to SEQ ID NO:44.

[0010] One embodiment provides a single-domain mesothelin binding protein, wherein the protein comprises the formula:

[0011] f1-r1-f2-r2-f3-r3-f4

[0012] wherein r1 is identical to SEQ ID NO:51 or comprises one or more amino acid residue substitutions relative to SEQ ID NO:51; r2 is identical to SEQ ID NO:52 or comprises one or more amino acid residue substitutions relative to SEQ ID NO:52; and r3 is identical to SEQ ID NO:53 or comprises one or more amino acid residue substitutions relative to SEQ ID NO:53; and wherein f1, f2, f3, and f4 are framework residues. In some embodiments, the protein comprises a sequence that is at least 80% identical to a sequence selected from SEQ ID NOs:1-29, 30-40, 58, and 60-62. In some embodiments, the protein comprises one or more modifications that result in humanization of the binding protein. In some embodiments, the modifications comprise substitutions, additions, or deletions of amino acid residues. In some embodiments, the protein comprises 111 to 124 amino acids. In some embodiments, the protein comprises a VHH domain derived from a non-human source. In some embodiments, the protein comprises a llama VHH domain. In some embodiments, the epitope is located in Region I comprising amino acid residues 296-390 of SEQ ID NO:57, Region II comprising amino acid residues 391-486 of SEQ ID NO:57, or Region III comprising amino acid residues 487-598 of SEQ ID NO:57.

[0013] Some embodiments provide a single-domain mesothelin binding protein, wherein the protein comprises one or more conserved regions comprising a sequence identical to or comprising one or more amino acid residue substitutions relative to SEQ ID NO: 45, 46, 47, 48, 49, or 50. In some embodiments, the protein comprises a conserved region comprising a sequence identical to or comprising one or more amino acid residue substitutions relative to SEQ ID NO: 45. In some embodiments, the protein comprises a conserved region comprising a sequence identical to or comprising one or more amino acid residue substitutions relative to SEQ ID NO: 46. In some embodiments, the protein comprises a conserved region comprising a sequence identical to or comprising one or more amino acid residue substitutions relative to SEQ ID NO: 47. In some embodiments, the protein comprises a conserved region comprising a sequence identical to or comprising one or more amino acid residue substitutions relative to SEQ ID NO: 48. In some embodiments, the protein comprises a conserved region comprising a sequence that is identical to SEQ ID NO: 49 or comprises one or more amino acid residue substitutions relative to SEQ ID NO: 49. In some embodiments, the protein comprises a conserved region comprising a sequence that is identical to SEQ ID NO: 50 or comprises one or more amino acid residue substitutions relative to SEQ ID NO: 50. In some embodiments, the protein comprises (i) a stretch of amino acids corresponding to SEQ ID NO: 45; (ii) a stretch of amino acids corresponding to SEQ ID NO: 46; (iii) a stretch of amino acids corresponding to SEQ ID NO: 47; (iv) a stretch of amino acids corresponding to SEQ ID NO: 48; (v) a stretch of amino acids corresponding to SEQ ID NO: 49; and (vi) a stretch of amino acids corresponding to SEQ ID NO: 50.

[0014] One embodiment provides a single-domain mesothelin binding protein, wherein the protein comprises the formula:

[0015] f1-r1-f2-r2-f3-r3-f4

[0016] wherein r1 is identical to or comprises one or more amino acid residue substitutions relative to SEQ ID NO:54; r2 is identical to or comprises one or more amino acid residue substitutions relative to SEQ ID NO:55; and r3 is identical to or comprises one or more amino acid residue substitutions relative to SEQ ID NO:56; and wherein f1, f2, f3, and f4 are framework residues. In some embodiments, the protein comprises a sequence that is at least 80% identical to a sequence selected from SEQ ID Nos:30-40, 58, and 60-62. In some embodiments, the protein comprises 111 to 119 amino acids. In some embodiments, the protein comprises a VHH domain derived from a non-human source. In some embodiments, the protein comprises a llama VHH domain. In some embodiments, the protein binds to a human mesothelin protein comprising the sequence set forth in SEQ ID NO:57. In some embodiments, the protein binds to an epitope of mesothelin, wherein the epitope is located in Region I comprising amino acid residues 296-390 of SEQ ID NO: 57, Region II comprising amino acid residues 391-486 of SEQ ID NO: 57, or Region III comprising amino acid residues 487-598 of SEQ ID NO: 57. In some embodiments, the binding protein is a chimeric antibody or a humanized antibody. In some embodiments, the binding protein is a single domain antibody. In some embodiments, the binding protein is a humanized single domain antibody.

[0017] One embodiment provides a single-domain mesothelin-binding protein, wherein the protein comprises one or more CDRs selected from SEQ ID Nos: 51-56 and 63-179. In some embodiments, the protein comprises a CDR1 comprising the sequence of any one of SEQ ID Nos.: 51, 54, and 63-101. In some embodiments, the protein comprises a CDR2 comprising the sequence of any one of SEQ ID Nos.: 52, 55, and 102-140. In some embodiments, the protein comprises a CDR3 comprising the sequence of any one of SEQ ID Nos.: 53, 56, and 141-179. In some embodiments, the protein comprises a framework region 1 (f1) comprising the sequence of any one of SEQ ID Nos.: 180-218. In some embodiments, the protein comprises a framework region 2 (f2) comprising the sequence of any one of SEQ ID Nos: 219-257. In some embodiments, the protein comprises framework region 3 (f3), which comprises the sequence shown in any one of SEQ ID Nos: 258-296. In some embodiments, the protein comprises framework region 4 (f4), which comprises the sequence shown in any one of SEQ ID Nos: 297-335. In some embodiments, the protein comprises the amino acid sequence shown in any one of SEQ ID Nos.: 1-40 and 58.

[0018] One embodiment provides a polynucleotide encoding a single-domain mesothelin-binding protein according to any one of the above embodiments. Another embodiment provides a vector comprising the polynucleotide of the above embodiments. Another embodiment provides a host cell transformed with the vector according to the above embodiments.

[0019] One embodiment provides a pharmaceutical composition comprising (i) a single-domain mesothelin-binding protein according to any one of the above embodiments, a polynucleotide according to any one of the above embodiments, a vector according to any one of the above embodiments, or a host cell according to any one of the above embodiments, and (ii) a pharmaceutically acceptable carrier.

[0020] Another embodiment provides a method for producing a single-domain mesothelin-binding protein according to any one of the above embodiments, the method comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding the single-domain mesothelin-binding protein according to any one of the above embodiments under conditions that allow expression of the mesothelin-binding protein and recovery and purification of the produced protein from the culture.

[0021] One embodiment provides a method for treating or ameliorating a proliferative disease or a neoplastic disease, the method comprising administering a mesothelin-binding protein according to any one of the above embodiments to a subject in need thereof. In some embodiments, the subject is a human. In some embodiments, the method further comprises administering a pharmaceutical agent in combination with a single-domain mesothelin-binding protein according to any one of the above embodiments. In some embodiments, the single-domain mesothelin-binding protein selectively binds to tumor cells expressing mesothelin. In some embodiments, the single-domain mesothelin-binding protein mediates T cell killing of tumor cells expressing mesothelin. In some embodiments, the neoplastic disease comprises a solid tumor disease. In some embodiments, the solid tumor disease comprises mesothelioma, lung cancer, gastric cancer, ovarian cancer, or triple-negative breast cancer. In some embodiments, the solid tumor disease is metastatic. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description of illustrative embodiments utilizing the principles of the present invention and the accompanying drawings, in which:

[0023] Figure 1 Shown are the efficacy of exemplary MSLN-targeting trispecific molecules (2A2 and 2A4) containing anti-MSLN binding proteins according to the present disclosure in killing OVCAR8 cells expressing the target protein MSLN.

[0024] Figure 2 Shown is a trispecific MSLN-targeting antigen-binding protein containing an exemplary MSLN-binding domain (MH6T) of the present disclosure that directs T cells from five donors (donor 02; donor 86; donor 41; donor 81; and donor 35) to kill Caov3 cells. The figure also shows that a control trispecific protein (GFP TriTAC) was unable to direct T cells from these five donors (donor 02; donor 86; donor 41; donor 81; and donor 35) to kill Caov3 cells.

[0025] Figure 3 Shown is a trispecific MSLN-targeting antigen-binding protein containing an exemplary MSLN-binding domain (MH6T) of the present disclosure that directs T cells from five donors (donor 02; donor 86; donor 41; donor 81; and donor 35) to kill OVCAR3 cells. The figure also shows that a control trispecific protein (GFP TriTAC) was unable to direct T cells from these five donors (donor 02; donor 86; donor 41; donor 81; and donor 35) to kill OVCAR3 cells.

[0026] Figure 4It is shown that a trispecific MSLN-targeting antigen-binding protein containing an exemplary MSLN-binding domain (MH6T) of the present disclosure can guide T cells from healthy donors to kill cells expressing MSLN (OVCAR3 cells; Caov4 cells; OVCAR3 cells; and OVCAR8 cells). The figure also shows that a trispecific MSLN-targeting antigen-binding protein containing an exemplary MSLN-binding domain (MH6T) of the present disclosure cannot guide T cells from healthy donors to kill cells that do not express MSLN (MDAPCa2b cells; and NCI-H510A cells).

[0027] Figure 5 It shows that a trispecific MSLN-targeting antigen-binding protein containing an exemplary MSLN-binding domain (MH6T) of the present disclosure can guide T cells from cynomolgus monkeys to kill human ovarian cancer cell lines (OVCAR3 cells; Caov3 cells). The figure also shows that a control trispecific protein (GFP TriTAC) cannot guide T cells from cynomolgus monkeys to kill human ovarian cancer cell lines (OVCAR3 cells; Caov3 cells).

[0028] Figure 6 It was shown that a trispecific MSLN-targeting antigen binding protein containing an exemplary MSLN-binding domain (MH6T) of the present disclosure was able to direct T cells to kill MSLN-expressing NCI-H2052 mesothelioma cells in the presence or absence of human serum albumin (HSA).

[0029] Figure 7 It is shown that a trispecific MSLN-targeting antigen binding protein containing an exemplary MSLN-binding domain (MH6T) of the present disclosure is able to activate T cells from four healthy donors (Donor 2; Donor 86; Donor 35; and Donor 81) in the presence of Caov4 cells expressing MSLN, as demonstrated by T cell secretion of TNF-α.

[0030] Figure 8 It is shown that a trispecific MSLN-targeting antigen binding protein containing an exemplary MSLN-binding domain (MH6T) of the present disclosure is able to activate T cells from four healthy donors (Donor 2; Donor 86; Donor 35; and Donor 81) in the presence of MSLN-expressing OVCAR8 cells, as demonstrated by activation of CD69 expression on the T cells.

[0031] Figure 9A and Figure 9B Shown is the binding of a trispecific MSLN-targeting antigen binding protein containing an exemplary MSLN-binding domain (MH6T) of the present disclosure to a cell line that expresses MSLN or a cell line that does not express MSLN. Figure 9AShown is binding to cells expressing MSLN (Caov3 cells - upper left panel; Caov4 cells - upper right panel; OVCAR3 cells - lower left panel; OVCAR8 cells - lower right panel) bound to a trispecific MSLN-targeting antigen binding protein containing an exemplary MSLN-binding domain (MH6T) of the present disclosure; Figure 9A It is further shown that a control trispecific protein (GFP TriTAC) is unable to bind to the same cell line. Figure 9B It is shown that both the trispecific MSLN-targeting antigen binding protein containing an exemplary MSLN-binding domain (MH6T) of the present disclosure and the GFP TriTAC are unable to bind to cell lines that do not express MSLN (MDCA2b cells - left panel; NCI-H510A cells - right panel).

[0032] Figure 10 Binding of a trispecific MSLN-targeting antigen-binding protein containing an exemplary MSLN-binding domain (MH6T) of the present disclosure to T cells from four healthy donors (Donor 2 - upper left panel; Donor 35 - upper right panel; Donor 41 - lower left panel; Donor 81 - lower right panel) is shown.

[0033] Figure 11 It was shown that a trispecific MSLN-targeting antigen binding protein containing an exemplary MSLN-binding domain (MH6T) of the present disclosure was able to inhibit tumor growth in NCG mice implanted with MSLN-expressing NCI-H292 cells.

[0034] Figure 12 Shown is a pharmacokinetic profile of a trispecific MSLN-targeted antigen-binding protein containing an exemplary MSLN-binding domain (MH6T) of the present disclosure. The figure shows serum levels of a trispecific MSLN-targeted antigen-binding protein containing an exemplary MSLN-binding domain (MH6T) of the present disclosure at different time points after injection into two cynomolgus monkeys. DETAILED DESCRIPTION

[0035] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein can be used to implement the present invention. The following claims are intended to define the scope of the present invention and thus encompass methods and structures within the scope of these claims and their equivalents.

[0036] Certain definitions

[0037] The terminology used herein is for descriptive purposes only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "includes," "comprising," "including," "having," or variations thereof are used in the detailed description and / or claims, these terms are intended to be inclusive in a manner similar to the term "comprising."

[0038] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which acceptable error range will depend in part on how the value is measured or determined, for example, limitations of the measurement system. For example, depending on the practice of a given value, "about" can mean within 1 or more than 1 standard deviation. Where specific values ​​are described in this application and claims, unless otherwise indicated, the term "about" should be considered to mean an acceptable error range for that specific value.

[0039] The terms "individual," "patient," or "subject" are used interchangeably. None of these terms require or are limited to situations characterized by supervision (e.g., continuous or intermittent) by a healthcare practitioner (e.g., a physician, registered nurse, nurse practitioner, physician assistant, orderlies, or hospice workers).

[0040] The term "framework" or "FR" residues (or regions) refers to variable domain residues other than CDR or hypervariable region residues as defined herein. A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in selected human immunoglobulin VL or VH framework sequences.

[0041] As used herein, "variable region" or "variable domain" refers to the following: certain portions of the variable domain vary widely in sequence between antibodies and are used in the binding and specificity of each particular antibody to its specific antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable domains. The more highly conserved portions of the variable domain are called frameworks (FRs). The variable domains of native heavy and light chains each contain four FR regions, which primarily adopt a β-pleated sheet configuration, connected by three CDRs, which form loops connecting the β-pleated sheet structure and, in some cases, form part of the β-pleated sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). Although the constant domain does not directly participate in the binding of the antibody to the antigen, it exhibits various effector functions, such as the antibody's participation in the antibody-dependent cellular toxicity of the antibody. "According to the variable domain residue numbering of Kabat" or "according to the amino acid position numbering of Kabat" and its variations refer to the numbering system of the heavy chain variable domain or light chain variable domain compiled for antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, the fifth edition of Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, which correspond to the shortening of the FR or CDR of the variable domain or to the insertion thereof. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and an insertion residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). The Kabat numbering of residues for a given antibody can be determined by aligning the antibody sequence over regions of homology with "standard" Kabat numbered sequences. This does not mean that the CDRs disclosed herein necessarily correspond to the Kabat numbering convention.

[0042] As used herein, the term " amino acid sequence identity percentage (%) " that is used for sequence is defined as after sequence is compared and introducing room (if necessary) to reach maximum sequence identity percentage, the amino-acid residue in the candidate sequence is the same percentage as the amino-acid residue in the specific sequence, and does not think that any conservative substitution is a part for sequence identity.Comparison that is used to determine amino acid sequence identity percentage purpose can realize by the whole bag of tricks within the skill of the art, for example, use publicly available computer software, such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine to be used to measure the appropriate parameter of comparison, are included in and realize maximum comparison required any algorithm on the full length of the sequence compared.

[0043] As used herein, "elimination half-life" is used in its ordinary sense, as described in Goodman and Gillman, The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman and Alfred Gilman, eds., 6th ed. 1980). In short, the term is intended to include a quantitative measure of the time course of drug elimination. The elimination of most drugs is exponential (i.e., follows first-order kinetics) because drug concentrations do not usually approach the concentration required for saturation of the elimination process. The rate of an exponential process can be determined by its rate constant, k, or by its half-life, t. 1 / 2 The rate constant k represents the fractional change per unit time, and the half-life t 1 / 2 The units of these two constants are time and -1 and time. The first-order rate constant and half-life of this reaction are simply related (k×t 1 / 2 =0.693) and can be interchanged accordingly. Since first-order elimination kinetics indicate that a constant fraction of drug is lost per unit time, a plot of the logarithm of drug concentration versus time is linear after the initial distribution phase (i.e., after drug absorption and distribution are complete). The half-life of drug elimination can be accurately determined from such a plot.

[0044] As used herein, the term "binding affinity" refers to the affinity of a protein described in the present disclosure for its binding target and is expressed numerically using a "Kd" value. If two or more proteins are shown to have comparable binding affinities for their binding targets, then the Kd values ​​for each protein binding to its binding target are within ±2-fold of each other. If two or more proteins are shown to have comparable binding affinities for a single binding target, then the Kd values ​​for each protein binding to the single binding target are within ±2-fold of each other. If a protein is shown to bind two or more targets with comparable binding affinities, then the Kd values ​​for the protein binding to the two or more targets are within ±2-fold of each other. Generally, higher Kd values ​​correspond to weaker binding. In some embodiments, BIAcore is used to compare the binding affinity of the proteins to the binding targets. TM -2000 or BIAcore TM "Kd" is measured by radiolabeled antigen binding assay (RIA) or surface plasmon resonance assay using a BIAcore-3000 (BIAcore, Inc., Piscataway, NJ). In certain embodiments, BIAcore TM -2000 or BIAcore TM The "on rate" or "rate of association" or "on rate" or "kon" and the "off rate" or "rate of dissociation" or "off rate" or "koff" are measured using a surface plasmon resonance technique using a BIAcore-3000 (BIAcore, Inc., Piscataway, NJ). Systems (Pall Life Sciences) to determine "Kd", "kon" and "koff". In an exemplary method for measuring binding affinity using a ligand-binding system, a ligand (e.g., biotinylated human or cynomolgus monkey PSMA) is immobilized on The streptavidin capillary sensor tip is then activated with approximately 20-50 μg / ml of human or cynomolgus monkey MSLN protein according to the manufacturer's instructions. A PBS / casein solution is also introduced as a blocking agent. For association kinetic measurements, the MSLN-binding protein variants are introduced at a concentration of approximately 10 ng / mL to approximately 100 μg / mL, approximately 50 ng / mL to approximately 5 μg / mL, or approximately 2 ng / mL to approximately 20 μg / mL. In some embodiments, the MSLN-binding single-domain protein is used at a concentration of approximately 2 ng / mL to approximately 20 μg / mL. Complete dissociation is observed in the negative control, i.e., assay buffer without binding protein. The kinetic parameters of the binding reaction are then determined using appropriate tools, such as ForteBio software.

[0045] Described herein are MSLN-binding proteins, pharmaceutical compositions, and nucleic acids, recombinant expression vectors, and host cells for preparing such MSLN-binding proteins. Also provided are methods for preventing and / or treating diseases, conditions, and disorders using the disclosed MSLN-binding proteins. The MSLN-binding proteins are capable of specifically binding to MSLN. In some embodiments, the MSLN-binding proteins comprise additional domains, such as a CD3 binding domain and an albumin binding domain. Mesothelin (MSLN) and its role in neoplastic diseases

[0046] This article relates to mesothelin binding proteins. Mesothelin is a glycoprotein present on the surface of mesothelial lining cells of the peritoneal, pleural and pericardial cavities. The mesothelin gene (MSLN) encodes a 71 kilodalton (kDa) precursor protein that is processed into a 40-kDa protein called mesothelin, which is a glycosylphosphatidylinositol-anchored glycoprotein present on the cell surface (Chang et al., Proc Natl Acad Sci USA (1996) 93: 136-40). Mesothelin cDNA was cloned from a library prepared from the HPC-Y5 cell line (Kojima et al. (1995) J. Biol. Chem. 270: 21984-21990). The monoclonal antibody K1 that recognizes mesothelioma was also used to clone cDNA (Chang and Pastan (1996) Proc. Natl. Acad. Sci. USA 93: 136-40). Mesothelin is a differentiation antigen whose expression in normal human tissues is restricted to mesothelial cells lining body cavities such as the pleura, pericardium, and peritoneum. Mesothelin is also highly expressed in several different human cancers, including mesothelioma, pancreatic adenocarcinoma, ovarian cancer, gastric and lung adenocarcinoma. (Hassan et al., Eur J Cancer (2008) 44:46-53) (Ordonez, Am J Surg Pathol (2003) 27:1418-28; Ho et al., Clin Cancer Res (2007) 13:1571-5). Mesothelin is overexpressed in the vast majority of primary pancreatic adenocarcinomas, and rare and weak expression can be seen in benign pancreatic tissue. Argani P et al. Clin Cancer Res. 2001; 7(12):3862-3868. Epithelial malignant pleural mesothelioma (MPM) universally expresses mesothelin, while sarcomatoid MPM may not express mesothelin. Most serous epithelial ovarian cancers and related primary peritoneal cancers express mesothelin.

[0047] Compared to its native membrane-bound form, mesothelin is also shed from tumor cells as a soluble form of the protein (Hellstrom et al., Cancer Epidemiol Biomarkers Prev (2006) 15: 1014-20; Ho et al., Cancer Epidemiol Biomarkers Prev (2006) 15: 1751). Structurally, mesothelin is expressed on the cell surface as a 60 kDa precursor polypeptide that is proteolytically processed into a 31 kDa shed component (corresponding to MPF) and a 40 kDa membrane-bound component (Hassan et al. (2004) Clin. Cancer. Res. 10: 3937-3942). Mesothelin has been shown to interact with CA125 (also known as MUC-16), a mucin-like glycoprotein present on the surface of tumor cells that has previously been identified as an ovarian cancer antigen. In addition, the binding of CA125 to membrane-bound mesothelin mediates heterotypic cell adhesion, and CA125 and mesothelin are co-expressed in advanced ovarian adenocarcinoma (Rump, A. et al. (2004) J. Biol. Chem. 279: 9190-9198). Mesothelin expression in the peritoneal lining is associated with the preferred site of metastasis formation in ovarian cancer, and mesothelin-CA125 binding is thought to promote peritoneal metastasis of ovarian tumors (Gubbels, JA et al. (2006) Mol. Cancer. 5: 50).

[0048] Mesothelin is a target of the innate immune response in ovarian cancer and has been proposed as a target for cancer immunotherapy. Bracci L et al. Clin Cancer Res. 2007; 13(2Pt 1): 644-653; Moschella F et al. Cancer Res. 2011; 71(10): 3528-3539; Gross G et al. FASEB J. 1992; 6(15): 3370-3378; Sadelain M et al. Nat Rev Cancer. 2003; 3(1): 35-45; Muul LM et al. Blood. 2003; 101(7): 2563-2569; Yee C et al. Proc Natl Acad Sci USA. 2002; 99(25): 16168-16173. The presence of mesothelin-specific CTLs in pancreatic cancer patients is associated with overall survival. Thomas AM et al. J Exp Med. 2004; 200: 297-306. In addition, Pastan and colleagues used a soluble antibody fragment of an anti-mesothelin antibody conjugated to an immunotoxin to treat cancer patients with mesothelin-positive tumors. This approach showed adequate safety and some clinical activity in pancreatic cancer. Hassan R et al. Cancer Immun. 2007; 7: 20, and Hassan R et al. Clin Cancer Res. 2007; 13(17): 5144-5149. In ovarian cancer, this treatment strategy produced a minor response according to RECIST criteria and resulted in stable disease in a second patient with complete resolution of ascites.

[0049] Mesothelin can also be used as a marker for the diagnosis and prognosis of certain types of cancer, as trace amounts of mesothelin can be detected in the blood of some mesothelin-positive cancer patients (Cristaudo et al., Clin. Cancer Res. 13: 5076-5081, 2007). It has been reported that mesothelin can be released into the serum by deletion at its carboxyl terminus or by proteolytic cleavage from its membrane-bound form (Hassan et al., Clin. Cancer Res. 10: 3937-3942, 2004). Increased soluble forms of mesothelin can be detected years before the development of malignant mesothelioma in workers exposed to asbestos (Creaney and Robinson, Hematol. Oncol. Clin. North Am. 19: 1025-1040, 2005). In addition, soluble mesothelin is also elevated in the serum of patients with ovarian cancer, pancreatic cancer, and lung cancer (Cristaudo et al., Clin. Cancer Res. 13:5076-5081, 2007; Hassan et al., Clin. Cancer Res. 12:447-453, 2006; Croso et al., Cancer Detect. Prev. 30:180-187, 2006). Therefore, mesothelin is a suitable target for disease prevention or treatment methods, and effective mesothelin-specific antibodies are needed.

[0050] It has been shown that mature mesothelin on the cell surface contains three distinct domains, namely region I (containing residues 296-390), II (containing residues 391-486), and III (containing residues 487-598). (Tang et al., A human single-domain antibody elicits potent antitumor activity by targeting an epitope inmesothelin close to the cancer cell surface, Mol. Can. Therapeutics, 12(4):416-426, 2013).

[0051] The first anti-mesothelin antibodies generated for therapeutic intervention were designed to interfere with the interaction between mesothelin and CA-125. Fv SS was identified by phage display, affinity optimized, and used to generate the recombinant immunotoxin SS1P targeting mesothelin. The MORAb-009 antibody amatuximab, which also uses SS1, recognizes a nonlinear epitope within the amino-terminal 64 amino acids of region I of mesothelin. Chimeric antigen receptor-engineered T cells have also been generated using SS1 Fv. Recently, new anti-mesothelin antibodies that recognize other regions of the mesothelin protein have been reported.

[0052] There remains a need for additional available options for treating solid tumor diseases associated with mesothelin overexpression, such as ovarian cancer, pancreatic cancer, mesothelioma, lung cancer, gastric cancer, and triple-negative breast cancer.In certain embodiments, the present disclosure provides single domain proteins that specifically bind to MSLN on the surface of tumor target cells.

[0053] MSLN-binding protein

[0054] In certain embodiments, provided herein are binding proteins, such as anti-MSLN single domain antibodies or antibody variants, that bind to an epitope in an MSLN protein. In some embodiments, the MSLN binding protein binds to a protein comprising the sequence of SEQ ID NO: 57. In some embodiments, the MSLN binding protein binds to a protein comprising a truncated sequence compared to SEQ ID NO: 57.

[0055] In some embodiments, the MSLN-binding proteins disclosed herein recognize full-length mesothelin. In certain instances, the MSLN-binding proteins disclosed herein recognize an epitope within Region I (comprising amino acid residues 296-390 of SEQ ID NO: 57), Region II (comprising amino acid residues 391-486 of SEQ ID NO: 57), or Region III (comprising amino acid residues 487-598 of SEQ ID NO: 57) of mesothelin. It is contemplated that in some embodiments, the MSLN-binding proteins disclosed herein may recognize and bind to an epitope located outside of Regions I, II, or III of mesothelin. In yet other embodiments, MSLN-binding proteins are disclosed that recognize and bind to an epitope different from that of the MORAb-009 antibody.

[0056] In some embodiments, the MSLN-binding proteins of the present disclosure are expressed within multi-domain proteins that include additional immunoglobulin domains. Such multi-domain proteins can act through immunotoxin-based tumor growth inhibition and induction of antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, multi-domain proteins containing the MSLN-binding proteins of the present disclosure exhibit complement-dependent cytotoxicity (CDC) activity. In some embodiments, multi-domain proteins containing the MSLN-binding proteins of the present disclosure exhibit both ADCC and CDC activity against cancer cells expressing mesothelin.

[0057] In addition, in some embodiments, when the multi-domain protein containing the MSLN binding protein functions via CDC, the MSLN binding protein can recognize a conformational epitope at the C-terminus of the mesothelin protein near the cell surface. In some embodiments, the mesothelin protein comprises the sequence set forth in SEQ ID NO: 57, and the C-terminus comprises amino acid residues 539-588.

[0058] In some embodiments, the MSLN-binding protein is an anti-MSLN antibody or antibody variant. As used herein, the term "antibody variant" refers to variants and derivatives of the antibodies described herein. In certain embodiments, amino acid sequence variants of the anti-MSLN antibodies described herein are contemplated. For example, in certain embodiments, amino acid sequence variants of the anti-MSLN antibodies described herein are expected to improve the binding affinity and / or other biological properties of the antibodies. Exemplary methods for preparing amino acid variants include, but are not limited to, introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the antibody's amino acid sequence.

[0059] Can carry out any combination of deletion, insertion and replacement to obtain final construct, condition is that final construct has desired characteristic, for example antigen binding.In certain embodiments, provide antibody variant with one or more amino acid replacements.The site of interest for replacement mutagenesis comprises CDR and framework region.The example of this type of replacement is described below.Amino acid replacement can be introduced into the antibody of interest, and for required activity, for example, retain / improve antigen binding, the immunogenicity of reduction or the antibody dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) of improvement come screening product.Conservative and non-conservative amino acid replacement are all considered for the preparation of antibody variants.

[0060] In another example of substitutions used to generate variant anti-MSLN antibodies, one or more hypervariable region residues of a parent antibody are substituted. Typically, variants are then selected based on improvements in desired properties compared to the parent antibody, e.g., increased affinity, decreased affinity, decreased immunogenicity, increased pH dependence of binding. For example, affinity-matured variant antibodies can be generated using, for example, phage display-based affinity maturation techniques, as described herein and known in the art.

[0061] In some embodiments, the MSLN-binding proteins described herein are single domain antibodies, such as heavy chain variable domains (VH) specific for mesothelin, variable domains (VHH) of llama-derived sdAbs, peptides, ligands, or small molecule entities. In some embodiments, the mesothelin-binding domain of the MSLN-binding proteins described herein is any domain that binds to mesothelin, including but not limited to domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In certain embodiments, the MSLN-binding protein is a single domain antibody. In other embodiments, the MSLN-binding protein is a peptide. In further embodiments, the MSLN-binding protein is a small molecule.

[0062] In general, it should be noted that the term single domain antibody, as used herein in its broadest sense, is not limited to a specific biological source or a specific method of preparation. A single domain antibody is an antibody whose complementary determining region is part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies that are naturally free of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds different from those derived from antibodies. A single domain antibody can be any single domain antibody in the art, or any future single domain antibody. Single domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, and cattle. For example, in some embodiments, the single domain antibodies of the present disclosure are obtained by: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanization" of a naturally occurring VHH domain or by expressing a nucleic acid encoding such a humanized VHH domain; (4) by "camelization" of a naturally occurring VH domain from any animal species, in particular from a mammalian species, such as from humans, or by expressing a nucleic acid encoding such a camelized VH domain; (5) by "camelization" of a "domain antibody" or "Dab", or by expressing a nucleic acid encoding such a camelized VH domain; (6) by preparing a protein, polypeptide or other amino acid sequence using synthetic or semisynthetic techniques; (7) by preparing a nucleic acid encoding a single domain antibody using nucleic acid synthesis techniques known in the art and then expressing the nucleic acid obtained thereby; and / or (8) by any combination of one or more of the foregoing.

[0063] In one embodiment, the single domain antibody corresponds to the VHH domain of a naturally occurring heavy chain antibody against MSLN. As further described herein, such VHH sequences can generally be generated or obtained by suitably immunizing a llama species with MSLN (i.e., to generate an immune response and / or heavy chain antibodies against MSLN), obtaining a suitable biological sample (such as a blood sample, serum sample, or B cell sample) from the llama, and generating VHH sequences against MSLN starting from the sample using any suitable technique known in the art.

[0064] In another embodiment, such naturally occurring VHH domains against MSLN are obtained from a naive library of Camelidae VHH sequences, for example by screening such a library using MSLN or at least one portion, fragment, antigenic determinant or epitope thereof using one or more screening techniques known in the art. Such libraries and techniques are described, for example, in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020 and WO 03 / 035694. Alternatively, improved synthetic or semisynthetic libraries derived from naive VHH libraries are used, such as VHH libraries obtained from naive VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as described, for example, in WO 00 / 43507.

[0065] In a further embodiment, another technique for obtaining VHH sequences against MSLN involves appropriately immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e., to generate an immune response and / or heavy chain antibodies against MSLN), obtaining a suitable biological sample (e.g., a blood sample, a serum sample, or a B cell sample) from the transgenic mammal, and then generating VHH sequences against MSLN starting from the sample using any suitable technique known in the art. For example, rats or mice expressing heavy chain antibodies can be used for this purpose, as well as other methods and techniques described in WO 02 / 085945 and WO 04 / 049794.

[0066] In some embodiments, the anti-MSLN antibodies described herein include single domain antibodies having an amino acid sequence corresponding to the amino acid sequence of a naturally occurring VHH domain, but which has been "humanized", i.e., by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (particularly in the framework sequence) with one or more amino acid residues present at the corresponding positions in the VH domain of a conventional 4-chain antibody from a human (e.g., as described above). This can be done in a manner known in the art, which will be apparent to those skilled in the art, for example based on the further description herein. In addition, it should be noted that such humanized anti-MSLN single domain antibodies of the present disclosure are obtained in any suitable manner known per se (i.e., as indicated in points (1)-(8) above), and are therefore not strictly limited to polypeptides obtained using polypeptides comprising naturally occurring VHH domains as starting material. In some further embodiments, the single domain MSLN antibodies as described herein include single domain antibodies having an amino acid sequence corresponding to the amino acid sequence of a naturally occurring VH domain, but having been "camelized", i.e., by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody with one or more amino acid residues present at the corresponding positions in the VHH domain of a heavy chain antibody. Such "camelization" substitutions are preferably inserted at amino acid positions forming the VH-VL interface and / or present at the VH-VL interface and / or at the so-called Camelidae hallmark residues (see, e.g., WO 94 / 04678, and Davies and Riechmann (1994 and 1996)). Preferably, the VH sequence used as the starting material or starting point for generating or designing a camelized single domain is preferably a VH sequence from a mammal, more preferably a human VH sequence, such as a VH3 sequence. However, it should be noted that in certain embodiments, such camelized anti-MSLN single domain antibodies of the present disclosure are obtained in any suitable manner known in the art (i.e., as indicated in points (1)-(8) above), and are therefore not strictly limited to polypeptides obtained using polypeptides comprising naturally occurring VH domains as starting materials. For example, as further described herein, both "humanization" and "camelization" are performed by providing a nucleotide sequence encoding a naturally occurring VHH domain or VH domain, respectively, and then altering one or more codons in the nucleotide sequence in such a way that the new nucleotide sequence encodes a "humanized" or "camelized" single domain antibody, respectively. The nucleic acid can then be expressed to provide the desired anti-MSLN single domain antibody of the present disclosure. Alternatively, in other embodiments, the amino acid sequence of the desired humanized or camelized anti-MSLN single domain antibody of the present disclosure is designed based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using known peptide synthesis techniques.In some embodiments, a nucleotide sequence encoding the desired humanized or camelized anti-MSLN single domain antibody of the present disclosure is designed based on the amino acid sequence or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using known nucleic acid synthesis techniques, and the nucleic acid thus obtained is then expressed using known expression techniques to provide the desired anti-MSLN single domain antibody of the present disclosure.

[0067] Other suitable methods and techniques for obtaining the anti-MSLN single domain antibodies of the present disclosure and / or nucleic acids encoding the anti-MSLN single domain antibodies starting from naturally occurring VH sequences or VHH sequences include, for example, combining one or more parts of one or more naturally occurring VH sequences (such as one or more framework (FR) sequences and / or complementarity determining region (CDR) sequences), one or more parts of one or more naturally occurring VHH sequences (such as one or more FR sequences or CDR sequences) and / or one or more synthetic or semi-synthetic sequences in a suitable manner to provide the anti-MSLN single domain antibodies of the present disclosure or the nucleotide sequences or nucleic acids encoding the anti-MSLN single domain antibodies.

[0068] In some embodiments, it is contemplated that the MSLN binding protein is quite small, and in some embodiments is no greater than 25 kD, no greater than 20 kD, no greater than 15 kD, or no greater than 10 kD. In certain instances, the MSLN binding protein, if a peptide or small molecule entity, is 5 kD or less.

[0069] In some embodiments, the MSLN-binding protein is an anti-MSLN-specific antibody comprising a heavy chain variable complementary determining region CDR1, a heavy chain variable CDR2, a heavy chain variable CDR3, a light chain variable CDR1, a light chain variable CDR2, and a light chain variable CDR3. In some embodiments, the MSLN-binding protein comprises any domain that binds to MSLN, including but not limited to domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, or antigen-binding fragments such as single domain antibodies (sdAb), Fab, Fab', F(ab)2, and Fv fragments, fragments consisting of one or more CDRs, single-chain antibodies (e.g., single-chain Fv fragments (scFv)), disulfide-stabilized Fv (dsFv) fragments, heteroconjugate antibodies (e.g., bispecific antibodies), pFv fragments, heavy chain monomers or dimers, light chain monomers or dimers, and dimers consisting of one heavy chain and one light chain. In some embodiments, the MSLN-binding protein is a single domain antibody. In some embodiments, an anti-MSLN single domain antibody comprises a heavy chain variable complementarity determining region (CDR), CDR1, CDR2, and CDR3.

[0070] In some embodiments, the MSLN-binding protein of the present disclosure is a polypeptide comprising an amino acid sequence consisting of four framework regions / sequences (f1-f4) interrupted by three complementarity determining regions / sequences, as shown in the following formula: f1-r1-f2-r2-f3-r3-f4, wherein r1, r2, and r3 are complementarity determining regions CDR1, CDR2, and CDR3, respectively, and f1, f2, f3, and f4 are framework residues. The framework residues of the MSLN-binding protein of the present disclosure comprise, for example, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, or 94 amino acid residues, and the complementarity determining regions comprise, for example, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 amino acid residues. In some embodiments, the MSLN binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-40.

[0071] In some embodiments, the CDR1 comprises the amino acid sequence of SEQ ID NO: 51, or a variant having one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in SEQ ID NO: 51. In some embodiments, the CDR2 comprises the sequence of SEQ ID NO: 52, or a variant having one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in SEQ ID NO: 52. In some embodiments, the CDR3 comprises the sequence of SEQ ID NO: 53, or a variant having one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in SEQ ID NO: 53.

[0072] In some embodiments, the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 54, or a variant having one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in SEQ ID NO: 54. In some embodiments, the CDR2 comprises the sequence set forth in SEQ ID NO: 55, or a variant having one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in SEQ ID NO: 55. In some embodiments, the CDR3 comprises the sequence set forth in SEQ ID NO: 56, or a variant having one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in SEQ ID NO: 56.

[0073] In certain instances, the MSLN-binding proteins of the present disclosure comprise one or more conserved regions. The conserved regions comprise the sequences set forth in SEQ ID NOs: 41-50, or variants comprising one or more amino acid residue substitutions relative to the sequences. Exemplary embodiments include MSLN-binding proteins comprising one or more conserved regions selected from SEQ ID NOs: 41-44, or variants comprising one or more amino acid residue substitutions relative to the sequences. In some cases, the MSLN-binding proteins comprise (i) a stretch of amino acids corresponding to SEQ ID NO: 41, (ii) a stretch of amino acids corresponding to SEQ ID NO: 42, (iii) a stretch of amino acids corresponding to SEQ ID NO: 43, and (iv) a stretch of amino acids corresponding to SEQ ID NO: 44.

[0074] Other exemplary embodiments include MSLN binding proteins comprising one or more conserved regions selected from SEQ ID NOs: 45-50, or variants comprising one or more amino acid residue substitutions relative to said sequences. In some cases, the MSLN binding protein comprises (i) a stretch of amino acids corresponding to SEQ ID NO: 45, (ii) a stretch of amino acids corresponding to SEQ ID NO: 46, (iii) a stretch of amino acids corresponding to SEQ ID NO: 47, (iv) a stretch of amino acids corresponding to SEQ ID NO: 48, (v) a stretch of amino acids corresponding to SEQ ID NO: 49, and (vi) a stretch of amino acids corresponding to SEQ ID NO: 50.

[0075] In various embodiments, the MSLN-binding proteins of the present disclosure are at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 1-29, 58, and 60-62.

[0076] In various embodiments, the MSLN-binding proteins of the present disclosure are at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 30-40, 58, and 60-62.

[0077] In various embodiments, the complementarity determining regions of the MSLN binding proteins of the present disclosure are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:51 or SEQ ID NO:54.

[0078] In various embodiments, the complementarity determining regions of the MSLN binding proteins of the present disclosure are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:52 or SEQ ID NO:55.

[0079] In various embodiments, the complementarity determining regions of the MSLN binding proteins of the present disclosure are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:53 or SEQ ID NO:56.

[0080] In various embodiments, the complementarity determining region 1 (CDR1) of the MSLN binding proteins of the present disclosure is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 63-101.

[0081] In various embodiments, the complementarity determining region 2 (CDR2) of the MSLN binding proteins of the present disclosure is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 102-140.

[0082] In various embodiments, the complementarity determining region 3 (CDR3) of the MSLN binding proteins of the present disclosure is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 141-179.

[0083] In various embodiments, the framework region 1 (f1) of the MSLN binding proteins of the present disclosure is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 180-218.

[0084] In various embodiments, the framework region 1 (f1) of the MSLN binding proteins of the present disclosure is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 219-257.

[0085] In various embodiments, framework region 2 (f2) of an MSLN binding protein of the present disclosure is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 258-296.

[0086] In various embodiments, framework region 3 (f3) of an MSLN-binding protein of the present disclosure is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 297-335.

[0087] In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 1. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 2. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 3. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 4. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 5. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 6. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 7. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 8. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 9. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 10. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 11. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 12. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 13. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 14. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 15. In some embodiments, the MSLN binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 16. In some embodiments, the MSLN binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 17.In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 18. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 19. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 20. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 21. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 22. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 23. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 24. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 25. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 26. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 27. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 28. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 28.

[0088] In some embodiments, the MSLN-binding protein according to any of the above embodiments is a humanized single domain antibody comprising the sequence of SEQ ID NO: 30. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a humanized single domain antibody comprising the sequence of SEQ ID NO: 31. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a humanized single domain antibody comprising the sequence of SEQ ID NO: 32. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a humanized single domain antibody comprising the sequence of SEQ ID NO: 33. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a humanized single domain antibody comprising the sequence of SEQ ID NO: 34. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a humanized single domain antibody comprising the sequence of SEQ ID NO: 35. In some embodiments, the MSLN-binding protein according to any of the above embodiments is a humanized single domain antibody comprising the sequence of SEQ ID NO: 36. In some embodiments, the MSLN binding protein according to any of the above embodiments is a humanized single domain antibody comprising the sequence of SEQ ID NO: 37. In some embodiments, the MSLN binding protein according to any of the above embodiments is a humanized single domain antibody comprising the sequence of SEQ ID NO: 38. In some embodiments, the MSLN binding protein according to any of the above embodiments is a humanized single domain antibody comprising the sequence of SEQ ID NO: 39. In some embodiments, the MSLN binding protein according to any of the above embodiments is a humanized single domain antibody comprising the sequence of SEQ ID NO: 40. In some embodiments, the MSLN binding protein according to any of the above embodiments is a humanized single domain antibody comprising the sequence of SEQ ID NO: 58. In some embodiments, the MSLN binding protein according to any of the above embodiments is a humanized single domain antibody comprising the sequence of SEQ ID NO: 60. In some embodiments, the MSLN binding protein according to any of the above embodiments is a humanized single domain antibody comprising the sequence of SEQ ID NO: 61. In some embodiments, the MSLN binding protein according to any of the above embodiments is a humanized single domain antibody comprising the sequence of SEQ ID NO:62.

[0089] In some embodiments, the MSLN-binding proteins are cross-reactive for human and cynomolgus monkey mesothelin. In some embodiments, the MSLN-binding proteins are specific for human mesothelin. In certain embodiments, the MSLN-binding proteins disclosed herein bind to human mesothelin with a human Kd (hKd). In certain embodiments, the MSLN-binding proteins disclosed herein bind to cynomolgus monkey mesothelin with a cynomolgus monkey Kd (cKd). In certain embodiments, the MSLN-binding proteins disclosed herein bind to cynomolgus monkey mesothelin and human mesothelin with a cynomolgus monkey Kd (cKd) and a human Kd (hKd), respectively. In some embodiments, the MSLN-binding proteins bind to human and cynomolgus monkey mesothelin with comparable binding affinities (i.e., the hKd and cKd values ​​differ by no more than ±10%). In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 500 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 450 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 400 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 350 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 300 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 250 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 200 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 150 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 100 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 90 nM. In some embodiments, the hKd and cKd are in the range of about 0.2 nM to about 80 nM. In some embodiments, the hKd and cKd are in the range of about 0.3 nM to about 70 nM. In some embodiments, the hKd and cKd are in the range of about 0.4 nM to about 50 nM. In some embodiments, the hKd and cKd are in the range of about 0.5 nM to about 30 nM. In some embodiments, the hKd and cKd are in the range of about 0.6 nM to about 10 nM. In some embodiments, the hKd and cKd are in the range of about 0.7 nM to about 8 nM. In some embodiments, the hKd and cKd are in the range of about 0.8 nM to about 6 nM. In some embodiments, the hKd and cKd are in the range of about 0.9 nM to about 4 nM. In some embodiments, the hKd and cKd are in the range of about 1 nM to about 2 nM.

[0090] In some embodiments, any of the aforementioned MSLN binding proteins (e.g., anti-MSLN single domain antibodies of SEQ ID NOs: 1-40 and 58) is affinity peptide tagged for ease of purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also known as 6X-his.

[0091] In certain embodiments, an MSLN-binding protein according to the present disclosure can be incorporated into a trispecific protein targeting MSLN. In some examples, the trispecific binding protein comprises a CD3 binding domain, a human serum albumin (HSA) binding domain, and an anti-MSLN binding domain according to the present disclosure. In some cases, the trispecific binding protein comprises the above-mentioned domains in the following orientation: MSLN-HSA-CD3.

[0092] In certain embodiments, the MSLN-binding proteins of the present disclosure preferentially bind to membrane-bound mesothelin relative to soluble mesothelin. Membrane-bound mesothelin refers to mesothelin present in or on the cell membrane surface of cells expressing mesothelin. Soluble mesothelin refers to mesothelin that is no longer present in or on the cell membrane surface of cells expressing or previously expressing mesothelin. In certain instances, soluble mesothelin is present in the blood and / or lymphatic circulation of a subject. In one embodiment, the MSLN-binding protein binds to membrane-bound mesothelin at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold more preferentially than to soluble mesothelin. In one embodiment, the antigen-binding proteins of the present disclosure preferentially bind to membrane-bound mesothelin 30-fold more preferentially than to soluble mesothelin. Preferential binding of an antigen binding protein to membrane-bound MSLN relative to soluble MSLN can be readily determined using assays well known in the art.

[0093] Integration into chimeric antigen receptors (CARs)

[0094] In certain examples, the MSLN binding proteins of the present disclosure, such as anti-MSLN single domain antibodies, can be incorporated into chimeric antigen receptors (CARs). Engineered immune effector cells, such as T cells or NK cells, can be used to express CARs, which include anti-MSLN single domain antibodies as described herein. In one embodiment, the CAR including anti-MSLN single domain antibodies as described herein is connected to a transmembrane domain via a hinge region, and is further connected to a costimulatory domain, such as a functional signaling domain obtained from OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) or 4-1BB. In some embodiments, the CAR further includes a sequence encoding an intracellular signaling domain such as 4-1BB and / or CD3ζ.

[0095] Tumor growth reducing properties

[0096] In certain embodiments, when administered to a subject having tumor cells expressing mesothelin, the MSLN-binding proteins of the present disclosure reduce tumor cell growth in vivo. Measurement of reduced tumor cell growth can be determined by a variety of different methods known in the art. Non-limiting examples include direct measurement of tumor size, measurement of excised tumor mass and comparison with control subjects, measurement by imaging techniques (e.g., CT or MRI) that may or may not use isotopes or luminescent molecules (e.g., luciferase) to enhance analysis, and the like. In specific embodiments, administration of an antigen-binding agent of the present disclosure results in a reduction in tumor cell growth in vivo of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, compared to a control antigen-binding agent, with a reduction in tumor growth of about 100% indicating a complete response and tumor disappearance. In further embodiments, administration of an antigen-binding agent of the present disclosure results in a reduction in tumor cell growth in vivo of about 50-100%, about 75-100%, or about 90-100%, compared to a control antigen-binding agent. In further embodiments, administration of an antigen binding agent of the present disclosure results in about 50-60%, about 60-70%, about 70-80%, about 80-90%, or about 90-100% reduction in in vivo growth of tumor cells compared to a control antigen binding agent.

[0097] MSLN-binding protein modification

[0098] The MSLN-binding proteins described herein include derivatives or analogs in which (i) amino acids are replaced with amino acid residues not encoded by the genetic code, (ii) the mature polypeptide is fused to another compound, such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as a leader or secretory sequence or a sequence used to block immunogenic domains and / or to purify the protein.

[0099] Typical modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, such as arginylation, and ubiquitination.

[0100] Modifications can be made at any position in the MSLN-binding proteins described herein, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Certain common peptide modifications that can be used to modify MSLN-binding proteins include glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, blocking of amino or carboxyl groups, or both, in the polypeptide by covalent modification, and ADP-ribosylation.

[0101] Polynucleotide encoding MSLN binding protein

[0102] In some embodiments, polynucleotide molecules encoding the MSLN-binding proteins described herein are also provided. In some embodiments, the polynucleotide molecules are provided in the form of DNA constructs. In other embodiments, the polynucleotide molecules are provided in the form of messenger RNA transcripts.

[0103] The polynucleotide molecule is constructed by known methods, for example, by combining a gene encoding an anti-MSLN binding protein operably linked to a suitable promoter and optionally a suitable transcription terminator, and expressing it in bacteria or other suitable expression systems such as CHO cells.

[0104] In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, which represents a further embodiment. Such recombinant vectors can be constructed according to known methods. Vectors of particular interest include plasmids, phagemids, phage derivatives, viruses (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, etc.), and cosmids.

[0105] A variety of expression vector / host systems can be used to contain and express the polynucleotide encoding the polypeptide of the MSLN binding protein. Examples of expression vectors are pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1):111-27) for expression in E. coli, pcDNA5 (Invitrogen) for expression in mammalian cells, PICHIAPINK TM Yeast Expression Systems(Invitrogen)、BACUVANCE TM BaculovirusExpression System (GenScript).

[0106] Thus, in some embodiments, an MSLN binding protein as described herein is produced by introducing a vector encoding a protein as described above into a host cell and culturing the host cell under conditions that allow expression of the protein domain, which can be isolated and optionally further purified.

[0107] Pharmaceutical composition

[0108] In some embodiments, pharmaceutical compositions are also provided, comprising an MSLN-binding protein described herein, a vector comprising a polynucleotide encoding a polypeptide encoding the MSLN-binding protein, or a host cell transformed with the vector, and at least one pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the component and is non-toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil-in-water emulsions, various types of wetting agents, sterile solutions, and the like. Such carriers can be formulated by conventional methods and administered to a subject at an appropriate dose. Preferably, the composition is sterile. These compositions may also contain excipients such as preservatives, emulsifiers, and dispersants. Various antibacterial and antifungal agents may be included to ensure protection against the action of microorganisms. Another embodiment provides one or more of the above-described binding proteins, such as anti-MSLN single-domain antibodies or antigen-binding fragments thereof, packaged in lyophilized form or in an aqueous medium.

[0109] In some embodiments of the pharmaceutical composition, the MSLN-binding protein described herein is encapsulated in a nanoparticle. In some embodiments, the nanoparticle is a fullerene, a liquid crystal, a liposome, a quantum dot, a superparamagnetic nanoparticle, a dendrimer, or a nanorod. In other embodiments of the pharmaceutical composition, the MSLN-binding protein is attached to a liposome. In some cases, the MSLN-binding protein is conjugated to the surface of the liposome. In some cases, the MSLN-binding protein is encapsulated within the shell of the liposome. In some cases, the liposome is a cationic liposome.

[0110] The MSLN-binding proteins described herein are contemplated for use as pharmaceuticals. Administration can be achieved in various ways, for example, by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In some embodiments, the route of administration depends on the type of therapy and the type of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician based on other clinical factors. The dosage for any one patient depends on many factors, including the patient's size, body surface area, age, sex, the specific compound to be administered, the timing and route of administration, the type of therapy, overall health, and other concurrently administered medications. An "effective dose" refers to the amount of active ingredient sufficient to affect the course and severity of the disease, thereby resulting in a reduction or alleviation of such pathology, and can be determined using known methods.

[0111] In some embodiments, the MSLN conjugates of the present disclosure are administered at a frequency of up to 10 mg / kg at a dose of once weekly. In some cases, the dosage range is from about 1 ng / kg to about 10 mg / kg. In some embodiments, the dosage is from about 1 ng / kg to about 10 ng / kg, from about 5 ng / kg to about 15 ng / kg, from about 12 ng / kg to about 20 ng / kg, from about 18 ng / kg to about 30 ng / kg, from about 25 ng / kg to about 50 ng / kg, from about 35 ng / kg to about 60 ng / kg, from about 45 ng / kg to about 70 ng / kg, from about 65 ng / kg to about 85 ng / kg, from about 80 ng / kg to about 1 μg / kg, from about 0.5 μg / kg, or from about 1 μg / kg to about 2 μg / kg. g / kg to about 5 μg / kg, about 2 μg / kg to about 10 μg / kg, about 7 μg / kg to about 15 μg / kg, about 12 μg / kg to about 25 μg / kg, about 20 μg / kg to about 50 μg / kg, about 35 μg / kg to about 70 μg / kg, about 45 μg / kg to about 80 μg / kg, about 65 μg / kg to about 90 μg / kg, about 85 μg / kg to about 0.1 mg / kg, about 0.095 mg / kg to about 10 mg / kg. In some cases, the dosage is about 0.1 mg / kg to about 0.2 mg / kg, about 0.25 mg / kg to about 0.5 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.75 mg / kg to about 3 mg / kg, about 2.5 mg / kg to about 4 mg / kg, about 3.5 mg / kg to about 5 mg / kg, about 4.5 mg / kg to about 6 mg / kg, about 5.5 mg / kg to about 7 mg / kg, about 6.5 mg / kg to about 8 mg / kg, about 7.5 mg / kg to about 9 mg / kg, or about 8.5 mg / kg to about 10 mg / kg. In some embodiments, the dosage is about less than once a day, once every other day, less than once a day, twice a week, once a week, once every 7 days, once every two weeks, once every two weeks, once every three weeks, once every four weeks, or once a month. In some cases, the dosage is once a week. In some cases, the dosage is once a week, with a dose of up to 10 mg / kg. In some cases, administration duration is from about 1 day to about 4 weeks or longer

[0112] Treatment

[0113] In some embodiments, methods and uses for stimulating the immune system of an individual in need thereof are also provided herein, comprising administering an MSLN-binding protein as described herein. In some cases, administration of an MSLN-binding protein as described herein induces and / or maintains cytotoxicity against cells expressing a target antigen. In some cases, cells expressing a target antigen are cancer cells or tumor cells, virally infected cells, bacterially infected cells, autoreactive T or B cells, damaged red blood cells, arterial plaque, or fibrotic tissue.

[0114] Also provided herein are methods and uses for treating a disease, disorder, or condition associated with a target antigen, comprising administering to a subject in need thereof an MSLN-binding protein described herein or a multispecific binding protein comprising the MSLN-binding protein. Diseases, disorders, or conditions associated with a target antigen include, but are not limited to, viral infections, bacterial infections, autoimmune diseases, transplant rejection, atherosclerosis, or fibrosis. In other embodiments, the disease, disorder, or condition associated with a target antigen is a proliferative disease, a neoplastic disease, an inflammatory disease, an immune disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease, or a host-versus-graft disease. In one embodiment, the disease, disorder, or condition associated with a target antigen is cancer. Cancers that can be treated, prevented, or controlled using the MSLN-binding proteins of the present disclosure and methods of use thereof include, but are not limited to, cancers of epithelial origin. Examples of such cancers include the following: leukemias, such as, but not limited to, acute leukemias, acute lymphocytic leukemias, acute myeloid leukemias, such as myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemias, and myelodysplastic syndromes; chronic leukemias, such as, but not limited to, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas, such as, but not limited to, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, such as, but not limited to, smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas, such as, but not limited to, skeletal sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, and soft tissue sarcoma. Histiosarcoma, angiosarcoma, fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma; brain tumors, such as but not limited to glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, non-glioma, acoustic neurotheca, craniopharyngioma, medulloblastoma, meningioma, pineal cell tumor, pineoblastoma, primary brain lymphoma; breast cancer, including but not limited to ductal carcinoma, adenocarcinoma, lobular carcinoma (Small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammatory breast cancer; adrenal cancers, such as, but not limited to, pheochromocytoma and adrenocortical carcinoma; thyroid cancers, such as, but not limited to, papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer; pancreatic cancers, such as, but not limited to, insulinomas, gastrinomas, glucagonomas, viromas, somatostatin-secreting tumors, and carcinoid or islet cell tumors;Pituitary cancers, such as, but not limited to, Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; eye cancers, such as, but not limited to, ocular melanomas, such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers, such as, but not limited to, squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancers, such as, but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers, such as, but not limited to, squamous cell carcinoma and adenocarcinoma; uterine cancers, such as, but not limited to, endometrial cancer and uterine sarcoma; ovarian cancer, Examples include, but are not limited to, ovarian epithelial cancer, borderline tumors, germ cell tumors, and stromal tumors; esophageal cancer, such as, but not limited to, squamous carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; gastric cancer, such as, but not limited to, adenocarcinoma, fungoid (polypoid), ulcerative, superficial spreading, diffuse spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancer, such as, but not limited to, hepatocellular carcinoma and hepatoblastoma; and gallbladder cancer. , such as adenocarcinoma; bile duct cancer, such as, but not limited to, papillary, nodular, and diffuse; lung cancer, such as, but not limited to, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer; testicular cancer, such as, but not limited to, germ cell tumor, seminoma, anaplastic, classic (typical), spermatoma, non-seminoma, embryonal carcinoma, teratoma, choriocarcinoma (yolk sac tumor); prostate cancer, such as, but not limited to, prostatic intraepithelial neoplasia, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; pineal cancer; oral cancer; Cavity cancer, such as but not limited to squamous cell carcinoma; basal carcinoma; salivary gland cancer, such as but not limited to adenocarcinoma, mucoepidermoid carcinoma and adenoid cystic carcinoma; pharyngeal cancer, such as but not limited to squamous cell carcinoma and verrucous; skin cancer, such as but not limited to basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, nevus malignant melanoma, acral lentiginous melanoma; kidney cancer, such as but not limited to renal cell carcinoma, adenocarcinoma, adrenal tumor, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or uterus); Wilms' tumor;Bladder cancer, such as, but not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma. In addition, cancer includes myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinoma (for a review of such conditions, see Fishman et al., 1985, Medicine, 2nd Edition, JB Lippincott Co., Philadelphia, and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America). ;

[0115] The MSLN binding proteins of the present disclosure can also be used to treat or prevent a variety of cancers or other abnormal proliferative diseases, including but not limited to the following: carcinomas, including bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer and skin cancer; including squamous cell carcinoma; hematological tumors of the lymphoid lineage, including leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B cell lymphoma, T cell lymphoma, Burkitt lymphoma; hematological tumors of the myeloid lineage, including ... and chronic myeloid leukemia and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, tetracarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous systems, including astrocytoma, neuroblastoma, glioma and schwannoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratomyoma, seminoma, follicular thyroid carcinoma and teratoma. It is also contemplated that cancers caused by apoptosis abnormalities may also be treated by the methods and compositions of the present disclosure. Such cancers may include, but are not limited to, follicular lymphoma, carcinomas with p53 mutations, hormone-dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndrome. In specific embodiments, malignant or dysplastic changes (such as metaplasia and dysplasia) or hyperproliferative diseases are treated or prevented in the skin, lung, colon, breast, prostate, bladder, kidney, pancreas, ovary or uterus. In other specific embodiments, sarcoma, melanoma or leukemia are treated or prevented.

[0116] In some embodiments, as used herein, "treatment" or "treatment" refers to therapeutic treatment, wherein the purpose is to slow down (mitigate) an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desired clinical result. For the purposes described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; reduction in the extent of the condition, disorder or disease; stabilization (i.e., no worsening) of the condition, disorder or disease state; delayed onset or slowed progression of the condition, disorder or disease; improvement of the condition, disorder or disease state; and detectable or undetectable relief (partial or complete) or enhancement or improvement of the condition, disorder or disease. Treatment includes inducing a clinically significant response without excessive levels of side effects. Treatment also includes extending survival compared to the expected survival if not receiving treatment. In other embodiments, "treatment" or "treatment" refers to preventive measures, wherein the purpose is to delay the onset of an undesirable physiological condition, disorder or disease or reduce its severity, for example, in a person susceptible to the disease (e.g., an individual carrying genetic markers for a disease such as breast cancer).

[0117] In some embodiments of the methods described herein, the MSLN-binding proteins described herein are administered in combination with agents used to treat specific diseases, disorders, or conditions. Agents include, but are not limited to, therapies involving antibodies, small molecules (e.g., chemotherapeutics), hormones (steroids, peptides, etc.), radiation therapy (gamma rays, X-rays, and / or targeted delivery of radioisotopes, microwaves, UV radiation, etc.), gene therapy (e.g., antisense, retroviral therapy, etc.), and other immunotherapies. In some embodiments, the MSLN-binding proteins described herein are administered in combination with antidiarrheals, antiemetics, analgesics, opioids, and / or nonsteroidal anti-inflammatory agents. In some embodiments, the MSLN-binding proteins described herein are administered in combination with anticancer agents. Non-limiting examples of anticancer agents that can be used in various embodiments of the present disclosure, including pharmaceutical compositions and dosage forms and kits of the present disclosure, include: acivicin; aclarubicin; acodazole hydrochloride; aclonine; adolesin; aldesleukin; hexamethylmelamine; ambomycin; ametrine acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; trilin; azacitidine; azatepa; azotocin; batimastat; Benzodepa; bicalutamide; bisantrene hydrochloride; binefad dimesylate; bisezolesine; bleomycin sulfate; brequinar sodium; bropiridamine; busulfan; actinomycin C; carlotestosterone; caracetamide; carbetimer; carboplatin; carmustine; carrubicin hydrochloride; carzelesin; cedifingol; chlorambucil; sirolimus; cisplatin; cladribine; crestofenol mesylate; cyclophosphamide; cytarabine; dacarbazine; actinomycin D; daunorubicin hydrochloride decitabine; dextromaplatin; dezaguanine; dezaguanine mesylate; diazaquinone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; drolostanone propionate; dazomicin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enprobamate; epiripridine; epirubicin hydrochloride; erbulazole; esorubicin hydrochloride; estramustine; estramustine sodium phosphate; etanidazole; etoposide; etoposide phosphate cefotaxime; etobanid; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flucitabine; fosquamol; fostramoxine sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; imofosine; interleukin II (including recombinant interleukin II or rIL2); interferon alpha-2a; interferon alpha-2b; interferon alpha-n1; interferon alpha-n3; interferon beta-I a; interferon gamma-Ib; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprorelin acetate; liarole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprofen; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menolide; mercaptopurine; methotrexate; methotrexate sodium; chlorpheniramine; metodepa; mibutamide; mitocarcin;Mitoclopramide; Mitoxetine; Mitomycin; Mitospe; Mitotane; Mitoxantrone hydrochloride; Mycophenolic acid; Nocodazole; Nogamycin; Ormaplatin; Oxysulam; Paclitaxel; Pegaspargase; Pelimycin; Pentamidine; Pelimycin sulfate; Perfosfamide; Pipobroman; Piposulfan; Pyroxantrone hydrochloride; Plicamycin; Promestane; Porfimer sodium; Porfiromycin; Prednimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride; Pyrazofuranoside; Liboadenosine; Roglulimide; Safingol; Safingol hydrochloride; Semustine; Simtrazine; Spadronate sodium; Sparamycin; Spirogermanium hydrochloride; Spiromustine; Spiroplatin; Streptomelanocin; Streptomelanocin Cytoxan; sulfaquinoxaline; tacrimycin; tecogalan sodium; tegafur; tiloxantrone hydrochloride; temoporfin; teniposide; tiroxilon; testolactone; thiamidine; thioguanine; thiotepa; thiazolamide nucleoside; tirapazamine; toremifene citrate; triptolon acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronide; triptorelin; tobradazole hydrochloride; uramustine; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinpoxetine sulfate; vinpoxetine sulfate; vinsorbate sulfate; vinorelbine tartrate; vinprodine sulfate; vinblastine sulfate; vorozole; zeniplatin; zirastatin; zorubicin hydrochloride. Other examples of anticancer drugs include, but are not limited to, 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adenosine; adolesin; aldesleukin; ALL-TK antagonists; hexamethylmelamine; aminostine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; anrelix; anti-dorsalizing morphogenetic protein-1 (ADMP-1); protein-1); antiandrogen; prostate cancer; antiestrogens; antineoplastons; antisense oligonucleotides; glycine aphidicolin; apoptosis gene regulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; amustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR / ABL antagonists; benzochlorins; benzoylstaurosporine; β-lactam derivatives; β-alethine; betaclamycin B; betulinic acid; bFGF inhibitors; bicalutamide; bisantrene; diazinonespermine; binefad;Bistratene A; bizolexin; breflate; bropirimine; budotentan; buthionine sulfate imide; calcipotriol; calcitropin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxamide triazole; CaRest M3; CARN 700; cartilage-derived inhibitors; carzelesin; casein kinase inhibitor (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomiphene analogs; clotrimazole; collismycin A; collismycin B; congenin A4; congenin analogs; conagenin; crambescidin 816; crestol; candidin 8; candidin A derivatives; curacin A; cyclopentanthrone; cycloplatam; cepirimycin; cytarabine octadecyl phosphate; cytolytic factor; estradiol phosphate (cytostatin); daclizumab; decitabine; dehydroepirimycin B; deslorelin; dexamethasone; dextroifosfamide; dexrazoxane; dexverapamil; diazocone; epirimycin B; didox; diethylnorspermine; dihydro-5-azacytidine; 9-dihydrotaxol; dioxamycin ; diphenylspiromustine; docetaxel; docosinol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; eticomustine; edelfosine; edrecolomab; eflornithine; elemene; ethiprofen; epirubicin; aprepitant; estramustine analogs; estrogen agonists; estrogen antagonists; etanercept; etoposide phosphate; exemestane; fadrozoles; fazarabine; fenretinide; filgrastim; finasteride; fulamicin Fludarabine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; fostramexane; formestane; fostramexine; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylenebisacetamide; gold Ibandronic acid; Idarubicin; Idoxifen; Idemanone; Imofosine; Ilomastat; Imidazoloacridone; Imiquimod; Immunostimulatory peptides; Insulin-like growth factor-I receptor inhibitors; Interferon agonists; Interferon; Interleukin; Iodobenzylguanidine; Iodoxorubicin; 4-Amylovorol; Iropraxine; Isoladine; Isobutrazol; Isohalichondrine B; Itasetron; Jasplakinolide; Kahalalide F; Triacetin-N; Lanreotide;Ranamycin; lenograstim; lentinan sulfate; rabotoxetine; letrozole; leukemia inhibitory factor; interferon-alpha; leuprolide + estrogen + progesterone; leuprolide; levamisole; lirazole; linear polyamine analogs; lipophilic disaccharide peptides; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; earthworm phospholipids; lometrexol; lonidamine; loxantrone; HMG-CoA reductase inhibitors (such as, but not limited to, lovastatin, pravastatin, fluvastatin, statins, simvastatin, and atorvastatin); loxoribine; lutetium texaphyrin; lysofylline; lytic peptide; maytansine; manostatin A; marimastat; masorofol; mamsopeptin; matrix lytic factor inhibitor; matrix metalloproteinase inhibitor; menolipin; merbarone; avorelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; miriproxyprogesterone; mismatched double-stranded RNA; mitoxantrone; dibromodulanol; mitomycin analog; mitonadamide; mytotoxin fibroblast growth factor-saporin; mitoxantrone; mofaradine; morasporastin; human chorionic gonadotropin monoclonal antibody; monophosphoryl lipid A+ mycobacterial cell wall sk; mopidarol; multidrug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; Indian Ocean sponge B (mycaperoxide) B); mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone + pentazocine; napavin; naphterpin; narostim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidants; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducers; ormaplatin; oxatron; oxatron oxaunomycin; paclitaxel; paclitaxel analogs; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronate; panaxatriol; panomifen; parabactin; pozeptin; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentozole; perflurane; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; pirtrexine; placetin A; placetin B; plasminogen activator inhibitors; platinum complexes; platinum compounds; platinum-triamine complexes; porfimer sodium; porfinomycin; prednisone;Propyldiacridone; prostaglandin J2; proteasome inhibitors; protein A-based immunomodulators; protein kinase C inhibitors; protein kinase C inhibitors; microalgae; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurin; methoxypyrazoline acridinium; pyridoxaline hemoglobin polyoxyethylene conjugates; Raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitors; demethylated reteptine; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII isotretinoin; roglulimide; roxitomine; romotide; roquineme; rubiginone B1; ruboxyl; safengo; saintopin; SarCNU; inositol A; sargramostim; Sdi 1 mimetic; semustine; senescence-derived inhibitor 1; sense oligonucleotide; signal transduction inhibitor; signal transduction regulator; single-chain antigen binding protein; sizolan; sobuzolan; borcarbazine sodium; sodium phenylacetate; solverol; somatomedin binding protein; sonamyl; fospartate; spicamycin D; spiromustine; spongistatin 1 1); squalamine; stem cell inhibitor; stem cell division inhibitor; stipiamide; stromelysin inhibitor; sulfinosine; potent vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycan; tamustine; tamoxifen methylthiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitor; temoporfin; temozolomide; teniposide; tetrachlorodecaoxid; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymofasin; thymopoietin receptor agonist; thymotrexone; thyroid-stimulating hormone; tin ethyletiopurpurin; tirapazamine; titanocene dichloride bichloride); topsentin; toremifene; totipotent stem cell factor; translation inhibitor; retinoic acid; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turotelene; tyrosine kinase inhibitor; tyrosine phosphorylation inhibitor (tyrphostin); UBC inhibitor; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonist; vapreotide; variolin B; vector system; erythrocyte gene therapy; vilaresol; veratramine; verdins; verteporfin; vinorelbine; vinxaltine; Vorozole; zanotron; zeniplatin; zilascorb; and fenstatin. Other anticancer drugs are 5-fluorouracil and folinic acid. These two agents are particularly useful when used in conjunction with thalidomide and a topoisomerase inhibitor. In some embodiments, the anti-MSLN single-domain binding proteins of the present disclosure are used in combination with gemcitabine.

[0118] In some embodiments, an MSLN-binding protein described herein is administered before, during, or after surgery.

[0119] Detection methods of mesothelin expression and diagnosis of mesothelin-related cancers

[0120] According to another embodiment of the present disclosure, a kit for detecting mesothelin expression in vitro or in vivo is provided. The kit includes the aforementioned MSLN-binding protein (e.g., a labeled anti-MSLN single-domain antibody or antigen-binding fragment thereof) and one or more compounds for detecting a marker. In some embodiments, the marker is selected from a fluorescent marker, an enzyme marker, a radioactive marker, a nuclear magnetic resonance-active marker, a luminescent marker, and a chromophore marker.

[0121] In some cases, mesothelin expression is detected in a biological sample. The sample can be any sample, including but not limited to tissue from a biopsy, autopsy, and pathology specimens. Biological samples also include tissue sections, e.g., frozen sections for histological purposes. Biological samples also include body fluids, such as blood, serum, plasma, sputum, spinal fluid, or urine. Biological samples are typically obtained from a mammal, such as a human or non-human primate.

[0122] In one embodiment, a method for determining whether a subject has cancer is provided by contacting a sample from the subject with an anti-MSLN single domain antibody disclosed herein and detecting binding of the single domain antibody to the sample. Increased binding of the antibody to the sample compared to binding of the antibody to a control sample determines that the subject has cancer.

[0123] In another embodiment, a method for confirming a diagnosis of cancer in a subject is provided by contacting a sample from a subject diagnosed with cancer with an anti-MSLN single domain antibody disclosed herein and detecting binding of the antibody to the sample. Increased binding of the antibody to the sample compared to binding of the antibody to a control sample confirms a diagnosis of cancer in the subject.

[0124] In some examples of the disclosed methods, the single domain antibody is directly labeled.

[0125] In some examples, the method further comprises contacting the sample with a second antibody that specifically binds to the single domain antibody; and detecting binding of the second antibody. Increased binding of the second antibody to the sample compared to binding of the second antibody to a control sample detects cancer in the subject or confirms a diagnosis of cancer in the subject.

[0126] In some instances, the cancer is mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, triple-negative breast cancer, or ovarian cancer, or any other type of cancer that expresses mesothelin.

[0127] In some instances, the control sample is a sample from a subject without cancer. In specific instances, the sample is a blood or tissue sample.

[0128] In some cases, the antibody that binds (e.g., specifically binds) mesothelin is directly labeled with a detectable label. In another embodiment, the antibody that binds (e.g., specifically binds) mesothelin (the first antibody) is not labeled, and the second antibody or other molecule that can bind to the antibody that specifically binds mesothelin is labeled. The second antibody is selected so that it can specifically bind to a particular type and class of the first antibody. For example, if the first antibody is llama IgG, the second antibody can be anti-llama IgG. Other molecules that can bind to the antibody include, but are not limited to, protein A and protein G, both of which are commercially available. Suitable labels for the antibody or second antibody are described above and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary magnetic agent is gadolinium, and non-limiting exemplary radiolabels include 125I, 131I, 35S or 3H.

[0129] In an alternative embodiment, mesothelin can be measured in a biological sample using a competitive immunoassay that utilizes a mesothelin standard labeled with a detectable substance and an unlabeled antibody that specifically binds to mesothelin. In this assay, the biological sample, the labeled mesothelin standard, and the antibody that specifically binds to mesothelin are mixed together, and the amount of the labeled mesothelin standard bound to the unlabeled antibody is measured. The amount of mesothelin in the biological sample is inversely proportional to the amount of the labeled mesothelin standard bound to the antibody that specifically binds to mesothelin.

[0130] The immunoassays and methods disclosed herein can be used for a variety of purposes. In one embodiment, an antibody that specifically binds to mesothelin can be used to detect the production of mesothelin in cells in cell culture. In another embodiment, the antibody can be used to detect the amount of mesothelin in a biological sample, such as a tissue sample or a blood or serum sample. In some examples, the mesothelin is cell surface mesothelin. In other examples, the mesothelin is soluble mesothelin (e.g., mesothelin in cell culture supernatant or mesothelin in a body fluid sample, such as a blood or serum sample).

[0131] In one embodiment, a kit for detecting mesothelin in a biological sample, such as a blood sample or a tissue sample, is provided. For example, to confirm a diagnosis of cancer in a subject, a biopsy can be performed to obtain a tissue sample for histological examination. Alternatively, a blood sample can be obtained to detect the presence of a soluble mesothelin protein or fragment. According to the present disclosure, a kit for detecting a polypeptide will generally contain a single domain antibody that specifically binds to mesothelin. In some embodiments, the kit includes an antibody fragment, such as an scFv fragment, a VH domain, or a Fab. In further embodiments, the antibody is labeled (e.g., with a fluorescent, radioactive, or enzymatic marker).

[0132] In one embodiment, the kit includes instructional materials that disclose means for using the antibody that binds to mesothelin. The instructional materials can be written in electronic form (e.g., on a computer floppy disk or compact disk) or can be visual (e.g., a video file). The kit can also include other components to facilitate the specific application for which the kit is designed. Thus, for example, the kit can additionally include means for detecting the label (e.g., an enzyme substrate for an enzyme label, a filter set for detecting a fluorescent label, a suitable secondary label, such as a secondary antibody, etc.). The kit can additionally include buffers and other reagents conventionally used to practice a particular method. Such kits and appropriate contents are well known to those skilled in the art.

[0133] In one embodiment, the diagnostic kit comprises an immunoassay. Although the details of the immunoassay may vary depending on the particular format employed, methods for detecting mesothelin in a biological sample generally include the steps of contacting the biological sample with an antibody that specifically reacts with a mesothelin polypeptide under immunoreactive conditions. The antibody is allowed to specifically bind under immunoreactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0134] Methods for determining the presence or absence of cell surface markers are well known in the art. For example, antibodies can be conjugated to other compounds including, but not limited to, enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorescent dyes, metal compounds, radioactive compounds, or drugs. Antibodies can also be used in immunoassays, such as, but not limited to, radioimmunoassays (RIA), ELISA, or immunohistochemistry assays. Antibodies can also be used in fluorescence-activated cell sorting (FACS). FACS uses multiple color channels, low-angle and obtuse-angle light scattering detection channels, and impedance channels, as well as other more complex detection levels, to separate or classify cells (see U.S. Patent No. 5,061,620). As disclosed herein, any single domain antibody that binds to mesothelin can be used in these assays. Therefore, these antibodies can be used in conventional immunoassays, including, but not limited to, ELISA, RIA, FACS, tissue immunohistochemistry, Western blotting, or immunoprecipitation.

[0135] The present invention provides embodiments including but not limited to the following:

[0136] 1. A single-domain mesothelin-binding protein, wherein the protein comprises one or more conserved regions comprising a sequence identical to SEQ ID NO: 41, 42, 43 or 44 or comprising one or more amino acid residue substitutions relative to SEQ ID NO: 41, 42, 43 or 44.

[0137] 2. The single-domain mesothelin binding protein of embodiment 1, wherein the protein comprises a conserved region comprising a sequence identical to SEQ ID NO: 41 or comprising one or more amino acid residue substitutions relative to SEQ ID NO: 41.

[0138] 3. The single-domain mesothelin binding protein of embodiment 1 or 2, wherein the protein comprises a conserved region comprising a sequence identical to SEQ ID NO: 42 or comprising one or more amino acid residue substitutions relative to SEQ ID NO: 42.

[0139] 4. The single-domain mesothelin binding protein of embodiment 1, 2 or 3, wherein the protein comprises a conserved region comprising a sequence identical to SEQ ID NO: 43 or comprising one or more amino acid residue substitutions relative to SEQ ID NO: 43.

[0140] 5. The single-domain mesothelin binding protein of embodiment 1, 2 or 3, wherein the protein comprises a conserved region comprising a sequence identical to SEQ ID NO: 44 or comprising one or more amino acid residue substitutions relative to SEQ ID NO: 44.

[0141] 6. A single-domain mesothelin-binding protein according to embodiment 1, wherein the protein comprises (i) a stretch of amino acids corresponding to SEQ ID NO:41; (ii) a stretch of amino acids corresponding to SEQ ID NO:42; (iii) a stretch of amino acids corresponding to SEQ ID NO:43; and (iv) a stretch of amino acids corresponding to SEQ ID NO:44.

[0142] 7. A single-domain mesothelin binding protein, wherein the protein comprises the formula:

[0143] f1-r1-f2-r2-f3-r3-f4

[0144] wherein r1 is identical to SEQ ID NO:51 or comprises one or more amino acid residue substitutions relative to SEQ ID NO:51; r2 is identical to SEQ ID NO:52 or comprises one or more amino acid residue substitutions relative to SEQ ID NO:52; and r3 is identical to SEQ ID NO:53 or comprises one or more amino acid residue substitutions relative to SEQ ID NO:53; and wherein f1, f2, f3 and f4 are framework residues.

[0145] 8. The single-domain mesothelin binding protein of any one of embodiments 1-7, wherein the protein comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-40, 58, and 60-62.

[0146] 9. The single-domain mesothelin binding protein of any one of embodiments 1-8, wherein the protein comprises one or more modifications that result in humanization of the binding protein.

[0147] 10. The single-domain mesothelin binding protein according to embodiment 9, wherein the modification comprises substitution, addition or deletion of amino acid residues.

[0148] 11. The single-domain mesothelin binding protein of any one of embodiments 1-10, wherein the protein comprises 111 to 124 amino acids.

[0149] 12. The single-domain mesothelin binding protein of any one of embodiments 1-11, wherein the protein comprises a VHH domain derived from a non-human source.

[0150] 13. The single-domain mesothelin binding protein of any one of embodiments 1-12, wherein said protein comprises a llama VHH domain.

[0151] 14. The single-domain mesothelin binding protein of any one of embodiments 1-13, wherein the protein binds to an epitope of mesothelin, wherein the epitope is located in Region I comprising amino acid residues 296-390 of SEQ ID NO: 57, Region II comprising amino acid residues 391-486 of SEQ ID NO: 57, or Region III comprising amino acid residues 487-598 of SEQ ID NO: 57.

[0152] 15. A single-domain mesothelin binding protein, wherein the protein comprises one or more conserved regions comprising a sequence identical to SEQ ID NO: 45, 46, 47, 48, 49 or 50 or comprising one or more amino acid residue substitutions relative to SEQ ID NO: 45, 46, 47, 48, 49 or 50.

[0153] 16. The single-domain mesothelin binding protein of embodiment 15, wherein the protein comprises a conserved region comprising a sequence that is identical to SEQ ID NO: 45 or comprises one or more amino acid residue substitutions relative to SEQ ID NO: 45.

[0154] 17. The single-domain mesothelin binding protein of embodiment 15 or 16, wherein the protein comprises a conserved region comprising a sequence that is identical to SEQ ID NO: 46 or comprises one or more amino acid residue substitutions relative to SEQ ID NO: 46.

[0155] 18. The single-domain mesothelin binding protein of embodiment 15, 16 or 17, wherein the protein comprises a conserved region comprising a sequence that is identical to SEQ ID NO: 47 or comprises one or more amino acid residue substitutions relative to SEQ ID NO: 47.

[0156] 19. The single-domain mesothelin binding protein of any one of embodiments 15-18, wherein the protein comprises a conserved region comprising a sequence that is identical to SEQ ID NO: 48 or comprises one or more amino acid residue substitutions relative to SEQ ID NO: 48.

[0157] 20. The single-domain mesothelin binding protein of any one of embodiments 15-19, wherein the protein comprises a conserved region comprising a sequence that is identical to SEQ ID NO: 49 or comprises one or more amino acid residue substitutions relative to SEQ ID NO: 49.

[0158] 21. The single-domain mesothelin binding protein of any one of embodiments 15-20, wherein the protein comprises a conserved region comprising a sequence that is identical to SEQ ID NO: 50 or comprises one or more amino acid residue substitutions relative to SEQ ID NO: 50.

[0159] 22. A single-domain mesothelin binding protein according to embodiment 1, wherein the protein comprises (i) a stretch of amino acids corresponding to SEQ ID NO:45; (ii) a stretch of amino acids corresponding to SEQ ID NO:46; (iii) a stretch of amino acids corresponding to SEQ ID NO:47; (iv) a stretch of amino acids corresponding to SEQ ID NO:48; (v) a stretch of amino acids corresponding to SEQ ID NO:49; and (vi) a stretch of amino acids corresponding to SEQ ID NO:50.

[0160] 23. A single-domain mesothelin binding protein, wherein the protein comprises the formula:

[0161] f1-r1-f2-r2-f3-r3-f4

[0162] wherein r1 is identical to SEQ ID NO:54 or comprises one or more amino acid residue substitutions relative to SEQ ID NO:54; r2 is identical to SEQ ID NO:55 or comprises one or more amino acid residue substitutions relative to SEQ ID NO:55; and r3 is identical to SEQ ID NO:56 or comprises one or more amino acid residue substitutions relative to SEQ ID NO:56; and wherein f1, f2, f3 and f4 are framework residues.

[0163] 24. The single-domain mesothelin binding protein of any one of embodiments 15-23, wherein the protein comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID Nos: 30-40, 58, and 60-62.

[0164] 25. The single-domain mesothelin binding protein of any one of embodiments 15-24, wherein the protein comprises 111 to 119 amino acids.

[0165] 26. The single-domain mesothelin binding protein of any one of embodiments 15-25, wherein the protein comprises a VHH domain derived from a non-human source.

[0166] 27. The single domain mesothelin binding protein of any one of embodiments 15-26, wherein said protein comprises a llama VHH domain.

[0167] 28. The single-domain mesothelin binding protein of any one of embodiments 15-27, wherein said protein binds to a human mesothelin protein comprising the sequence shown in SEQ ID NO:57.

[0168] 29. The single-domain mesothelin binding protein of any one of embodiments 15-28, wherein the protein binds to an epitope of mesothelin, wherein the epitope is located in Region I comprising amino acid residues 296-390 of SEQ ID NO: 57, Region II comprising amino acid residues 391-486 of SEQ ID NO: 57, or Region III comprising amino acid residues 487-598 of SEQ ID NO: 57.

[0169] 30. The single-domain mesothelin binding protein of any one of embodiments 1-29, wherein the binding protein is a chimeric or humanized antibody.

[0170] 31. The single domain binding protein of any one of embodiments 1-30, wherein the binding protein is a single domain antibody.

[0171] 32. A single domain binding protein according to any one of embodiments 15-31, wherein the binding protein is a humanized single domain antibody.

[0172] 33. The single domain binding protein of any one of embodiments 1-32, wherein the binding protein comprises the sequence shown in SEQ ID NO:58.

[0173] 34. A polynucleotide encoding the single-domain mesothelin binding protein of any one of embodiments 1-33.

[0174] 35. A vector comprising the polynucleotide according to embodiment 34.

[0175] 36. A host cell transformed with the vector according to embodiment 35.

[0176] 37. A pharmaceutical composition comprising (i) the single-domain mesothelin binding protein of any one of embodiments 1-33, the polynucleotide of embodiment 33, the vector of embodiment 35 or the host cell of embodiment 36, and (ii) a pharmaceutically acceptable carrier.

[0177] 38. A method for producing a single-domain mesothelin-binding protein according to any one of embodiments 1-33, the method comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding the single-domain mesothelin-binding protein according to any one of embodiments 1-33 under conditions that allow expression of the mesothelin-binding protein and recovery and purification of the produced protein from the culture.

[0178] 39. A method for treating or ameliorating a proliferative disease or a tumor disease, comprising administering the mesothelin-binding protein according to any one of embodiments 1-33 to a subject in need thereof.

[0179] 40. The method of embodiment 39, wherein the subject is human.

[0180] 41. The method of embodiment 40, wherein the method further comprises administering an agent in conjunction with the single-domain mesothelin binding protein of any one of embodiments 1-33.

[0181] 42. The method of any one of embodiments 39-41, wherein the single-domain mesothelin binding protein selectively binds to tumor cells expressing mesothelin.

[0182] 43. A method according to embodiment 42, wherein the single-domain mesothelin binding protein mediates T cell killing of tumor cells expressing mesothelin.

[0183] 44. The method of any one of embodiments 39-43, wherein the neoplastic disease comprises a solid tumor disease.

[0184] 45. The method of embodiment 44, wherein the solid tumor disease comprises mesothelioma, lung cancer, gastric cancer, ovarian cancer, or triple-negative breast cancer.

[0185] 46. ​​The method of embodiment 45, wherein the solid tumor disease is metastatic.

[0186] 47. A single domain mesothelin binding protein, wherein said protein comprises one or more CDRs selected from the group consisting of SEQ ID Nos.: 51-56 and 63-179.

[0187] 48. The single-domain mesothelin binding protein of embodiment 47, wherein the protein comprises a CDR1 comprising the sequence shown in any one of SEQ ID Nos.: 51, 54, and 63-101.

[0188] 49. The single-domain mesothelin binding protein of embodiment 47 or 48, wherein the protein comprises a CDR2 comprising the sequence shown in any one of SEQ ID Nos.: 52, 55, and 102-140.

[0189] 50. The single-domain mesothelin binding protein of any one of embodiments 47-49, wherein the protein comprises a CDR3 comprising the sequence shown in any one of SEQ ID Nos.: 53, 56, and 141-179.

[0190] 51. The single-domain mesothelin binding protein of any one of embodiments 47-50, wherein the protein comprises a framework region 1 (f1) comprising the sequence shown in any one of SEQ ID No.: 180-218.

[0191] 52. The single-domain mesothelin binding protein of any one of embodiments 47-51, wherein the protein comprises a framework region 2 (f2) comprising the sequence shown in any one of SEQ ID No.: 219-257.

[0192] 53. The single-domain mesothelin binding protein of any one of embodiments 47-52, wherein the protein comprises a framework region 3 (f3) comprising the sequence shown in any one of SEQ ID No.: 258-296.

[0193] 54. The single-domain mesothelin binding protein of any one of embodiments 47-53, wherein the protein comprises a framework region 4 (f4) comprising the sequence shown in any one of SEQ ID No.: 297-335.

[0194] 55. The single-domain mesothelin binding protein of any one of embodiments 47-54, wherein the protein comprises the amino acid sequence shown in any one of SEQ ID No.: 1-40 and 58.

[0195] 56. A polynucleotide encoding the single-domain mesothelin binding protein of any one of embodiments 47-55.

[0196] 57. A vector comprising the polynucleotide according to embodiment 56.

[0197] 58. A host cell transformed with the vector according to embodiment 57.

[0198] 59. A pharmaceutical composition comprising (i) the single-domain mesothelin binding protein of any one of embodiments 47-55, the polynucleotide of embodiment 56, the vector of embodiment 57 or the host cell of embodiment 58, and (ii) a pharmaceutically acceptable carrier.

[0199] 60. A method for producing a single-domain mesothelin-binding protein according to any one of embodiments 47-55, the method comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding the single-domain mesothelin-binding protein according to any one of embodiments 47-55 under conditions that allow expression of the mesothelin-binding protein and recovery and purification of the produced protein from the culture.

[0200] 61. A method for treating or ameliorating a proliferative disease or a tumor disease, comprising administering the mesothelin-binding protein according to any one of embodiments 47-55 to a subject in need thereof.

[0201] 62. The method of embodiment 61, wherein the subject is human.

[0202] 63. The method of embodiment 62, wherein the method further comprises administering an agent in combination with the single-domain mesothelin binding protein of any one of embodiments 1-33.

[0203] 64. The method of any one of embodiments 61-63, wherein the single-domain mesothelin binding protein selectively binds to tumor cells expressing mesothelin.

[0204] 65. A method according to embodiment 64, wherein the single-domain mesothelin binding protein mediates T cell killing of tumor cells expressing mesothelin.

[0205] 66. A method according to any one of embodiments 61-65, wherein the neoplastic disease comprises a solid tumor disease.

[0206] 67. The method of embodiment 66, wherein the solid tumor disease comprises mesothelioma, lung cancer, gastric cancer, ovarian cancer, or triple-negative breast cancer.

[0207] 68. The method of embodiment 67, wherein the solid tumor disease is metastatic.

[0208] Example

[0209] The following examples further illustrate the described embodiments but do not limit the scope of the invention.

[0210] Example 1: Exemplary anti-MSLN single domain antibodies of the present disclosure mediate T cell killing of mesothelin-expressing cancer cells Cell capacity

[0211] The exemplary anti-MSLN single domain antibody sequences were transfected into Expi293 cells (Invitrogen). The amount of exemplary anti-MSLN antibodies in the conditioned medium from the transfected Expi293 cells was quantified using an Octet instrument with a protein A tip and a standard curve using a control anti-MSLN antibody.

[0212] Titrates of conditioned medium were added to a TDCC assay (T cell-dependent cytotoxicity assay) to assess whether anti-MSLN single-domain antibodies were able to form synapses between T cells and the mesothelin-expressing ovarian cancer cell line OVCAR8. The viability of the OVCAR8 cells was measured 48 hours later. As can be seen, the exemplary anti-MSLN single-domain antibodies mediated T cell killing.

[0213] Furthermore, it can be seen that the TDCC activity of the exemplary anti-MSLN single domain antibody is specific for cells expressing mesothelin, as the exemplary antibody does not mediate T cell killing of LNCaP cells that do not express mesothelin.

[0214] Example 2: Evaluation of several MSLN-targeting trispecific antigen binding proteins containing MSLN-binding domains according to the present disclosure Protein Binding and Cytotoxic Activity Methods

[0215] Protein production

[0216] The sequence of the MSLN-targeting trispecific molecule containing the MSLN-binding protein according to the present disclosure was cloned into the mammalian expression vector pCDNA 3.4 (Invitrogen), preceded by a leader sequence and followed by a 6x histidine tag. Expi293F cells (Life Technologies A14527) were suspended in Expi 293 medium in Optimum Growth Flasks (Thomson) at 0.2-8×1e6 cells / mL. Purified plasmid DNA was transfected into Expi293 cells according to the Expi293 Expression System Kit (Life Technologies, A14635) protocol and maintained for 4-6 days after transfection. The amount of the measured exemplary trispecific protein in the conditioned medium from the transfected Expi293 cells was quantified using an Octet instrument with a Protein A tip and a standard curve generated using a control trispecific protein.

[0217] Cytotoxicity assay

[0218] The ability of T cell engagers (including trispecific molecules) to guide T cells to kill tumor cells was measured using the human T cell-dependent cytotoxicity (TDCC) assay (Nazarian et al., 2015. J Biomol Screen. 20: 519-27). In this assay, T cells and target cancer cell line cells were mixed together in a 10: 1 ratio in a 384-well plate and different amounts of the tested trispecific protein were added. The tumor cell line was engineered to express luciferase protein. After 48 hours, the remaining viable tumor cells were quantified using Steady- Luminescent Assay (Promega).

[0219] In this study, titrations of conditioned medium were added to a TDCC assay (T cell-dependent cytotoxicity assay) to assess whether anti-MSLN single-domain antibodies could form synapses between T cells and the mesothelin-expressing ovarian cancer cell line OVCAR8. The viability of the OVCAR8 cells was measured 48 hours later. As can be seen, the trispecific protein mediated T cell killing. Figure 1 An exemplary cell viability assay performed with the tested trispecific proteins 2A2 and 2A4 is shown. Table 1 below lists the EC values ​​for TDCC activity of several other tested trispecific proteins. 50 .

[0220] Table 1: TDCC activity of MSLN-targeting trispecific proteins containing MSLN-binding proteins according to the present disclosure

[0221]

[0222] Furthermore, the TDCC activity of the tested trispecific proteins targeting MSLN was observed to be specific for cells expressing mesothelin, as the tested trispecific proteins did not mediate T cell killing of LNCaP cells that do not express mesothelin. In particular, the trispecific proteins 2A2, 11F3, 9H2, 5C2, 10B3, 2F4, 5F2, 7F1, 2F4, 5H1, 3B4, and 7H2 did not show any TDCC activity against LnCaP cells.

[0223] Example 3: ADCC activity of exemplary anti-MSLN single domain antibodies of the present disclosure

[0224] This study was designed to determine the ability of exemplary anti-MSLN single domain antibodies of the present disclosure to mediate ADCC compared to a comparative llama anti-MSLN antibody without sequence modifications or substitutions as an exemplary antibody of the present disclosure. Both antibodies were expressed as multidomain proteins containing additional immunoglobulin domains.

[0225] Material

[0226] The donors underwent leukapheresis and NK cells were isolated from the leukopack using the Milteni AutoMacs Pro negative selection system by the cell purification team. NK cells were kept overnight at 4°C on a shaker and then washed, counted, and plated at 4×10 6 Cells / mL were resuspended in complete RPMI for ADCC assay.

[0227] Targets: Tumor cell targets were selected based on mesothelin expression. Targets were washed and counted. 6×10 6 Targets were resuspended in complete RPMI and labeled with a final concentration of 10 μM calcein (Sigma #C1359-00UL CALCEIN AM 4MM IN ANHYDROUSDMSO) at 37°C, 5% CO2 for 40 minutes. Cells were washed twice in PBS, resuspended in complete RPMI, and incubated for 2 hours at 37°C, 5% CO2. After labeling, target cells were washed, re-counted, and counted at 0.2×10 6 Cells were resuspended in complete RPMI at a concentration of 10 cells / mL for use in ADCC assays.

[0228] method

[0229] ADCC assays were performed in 96-well round-bottom tissue culture plates (Corning 3799). The test protein was titrated from 20 μg / mL to 0.0002 μg / mL by including 10 μL (1:10 dilution) in 1000 μL of complete RPMI containing 10% FCS. 50 μL of calcein-labeled target was added to include 10,000 cells. Target cells and various concentrations of multidomain proteins containing exemplary anti-MSLN single-domain antibodies or comparative antibodies were incubated at 4°C for 40 minutes, followed by the addition of 50 μL of NK cell effectors to include 100,000 cells (10:1E:T ratio). The culture was incubated at 37°C for 4 hours, the supernatant was then removed, and the release of calcein was determined by measuring the fluorescence at 485-535 nm on a Wallac Victor II 1420 Multilable HTS counter. 100% lysis values ​​were determined by lysing six wells of labeled target with Igepal 630 detergent (3 μL per well), and spontaneous lysis values ​​were determined by measuring fluorescence in the supernatant from individual targets.

[0230] Statistical analysis

[0231] Percent specific lysis (%) is defined as (sample fluorescence) - (autogenous lysis fluorescence) / (100% lysis - autogenous lysis fluorescence). Spontaneous lysis is determined by wells containing only target, while 100% lysis is determined by wells in which the target is lysed with IGEPAL CA 630 detergent. Raw data were entered into an Excel spreadsheet with formulas embedded to calculate % specific lysis, and the resulting values ​​were transferred to a graphing program (GraphPad Prism) where the data were converted into a curve fit graph. Subsequent analysis (linear regression calculation) was then performed in GraphPad to generate EC values. 50 value.

[0232] Results and Discussion

[0233] Effector NK cells in wells incubated with multi-domain proteins containing comparative anti-MSLN antibodies were unable to mediate killing of calcein-labeled target cells, whereas effectors in wells with multi-domain proteins containing exemplary anti-MSLN single domain antibodies of the present disclosure were able to mediate antibody-dependent cellular cytotoxicity as measured by specific lytic activity (% specific lysis).

[0234] in conclusion

[0235] Exemplary anti-MSLN single domain antibodies of the present disclosure mediate significantly higher levels of killing of mesothelin-expressing target cells compared to comparable llama anti-MSLN single domain antibodies without sequence substitutions, modifications, or humanization.

[0236] Example 4: CDC activity of exemplary anti-MSLN single domain antibodies of the present disclosure

[0237] To evaluate the anti-tumor activity of exemplary anti-MSLN single-domain antibodies according to the present disclosure against cancer cells, cytotoxic activity was tested in A431 / H9 and NCI-H226 cell models in the presence of human serum as a complement source. The exemplary anti-MSLN single-domain antibodies were expressed as multi-domain proteins containing additional immunoglobulin domains. It can be seen that the multi-domain proteins containing the exemplary anti-MSLN single-domain antibodies of the present disclosure exerted potent CDC activity by killing approximately 40% of the A431 / H9 and more than 30% of the NCI-H226 mesothelioma cell lines, and showed no activity against the mesothelin-negative A431 cell line. Comparative llama anti-MSLN antibodies without sequence modifications or substitutions as in the exemplary antibodies of the present disclosure showed no activity at the same concentration.

[0238] To analyze the role of complement in the anti-tumor activity of exemplary anti-MSLN single-domain antibodies, flow cytometry was used to determine the binding of C1q to cancer cells reactive with anti-mesothelin human mAbs, following a well-established protocol for characterizing rituximab, ofatumumab, and other anti-CD20 therapeutic mAbs (Pawluczkowycz et al., J Immunol 183:749-758, 2009; Li et al., Cancer Res 68:2400-2408, 2008). It has been previously demonstrated that, like MORAb-009, the HN1 human mAb, which is specific for region I (away from the cell surface) of cell surface mesothelin, does not exhibit any CDC activity against mesothelin-expressing cancer cells (Ho et al., Int J Cancer 128:2020-2030, 2011).

[0239] However, it can be seen that in the presence of the exemplary anti-MSLN single-domain antibodies, C1q complement bound to A431 / H9 or NCI-H226 cells. In contrast, no C1q binding was observed in the presence of the comparative llama anti-MSLN antibody. Moreover, C1q binding to cancer cells correlated with cell binding of the exemplary anti-MSLN single-domain antibodies in a dose-response manner. These results demonstrate that the exemplary anti-MSLN single-domain antibodies exhibit improved CDC activity relative to the comparative llama anti-MSLN antibody.

[0240] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein can be used to implement the present invention. The following claims are intended to define the scope of the present invention and thus encompass methods and structures within the scope of these claims and their equivalents.

[0241] Example 5: Trispecific antigen binding targeting MSLN containing the MSLN binding domain (MH6T) according to the present disclosure Protein guides T cells to kill MSLN-expressing ovarian cancer cells

[0242] The ability of T cell engagers (including trispecific molecules) to guide T cells to kill tumor cells was measured using human T cell-dependent cytotoxicity (TDCC) assays (Nazarian et al. 2015. J Biomol Screen. 20: 519-27). The Caov3 cells used in this assay were engineered to express luciferase. T cells and target cancer cells Caov3 from 5 different healthy donors (donor 02, donor 86, donor 41, donor 81, and donor 34) were mixed together, and different amounts of MSLN-targeting trispecific antigen-binding proteins (SEQ ID NO: 58) containing the MSLN binding domain (MH6T) were added, and the mixture was incubated at 37°C for 48 hours. Caov3 cells and T cells were also incubated at 37°C for 48 hours with a control trispecific molecule GFP TriTAC (SEQ ID NO: 59) targeting GFP. After 48 hours, the remaining live tumor cells were quantified by luminescence assay.

[0243] It was observed that the trispecific antigen binding protein targeting MSLN containing the MSLN binding domain (MH6T) was able to guide T cells from all five healthy donors to kill the target cancer cells Caov3 (e.g. Figure 2 ), whereas the control GFP TriTAC molecule failed to direct T cells from any of the five healthy donors to kill Caov3 cells (also shown in Figure 2 middle).

[0244] Further assays were performed using the same protocol as above using OVCAR3 cells. It was observed that the MSLN-targeting trispecific antigen binding protein containing the MSLN binding domain (MH6T) was able to direct T cells from all five healthy donors to kill the target cancer cells, OVCAR3 (e.g. Figure 3 ), whereas the control GFP TriTAC molecule failed to direct T cells from any of the five healthy donors to kill OVCAR3 cells (also shown in Figure 3 middle).

[0245] ECs for killing target cells expressing MSLN are listed in Table II below. 50 value.

[0246] Table II: Trispecific antigen binding proteins targeting MSLN (containing the MH6 T domain) direct T cells from five different healthy donors to kill ECs of MSLN-expressing ovarian cancer cell lines 50 The raw data are represented in the Figure 2 and Figure 3 Available in.

[0247]

[0248] Example 6: Trispecific antigen binding targeting MSLN containing the MSLN binding domain (MH6T) according to the present disclosure Protein guides T cells to kill cells that express MSLN but not cells that do not

[0249] In this test, T cells from healthy donors were incubated with target cancer cells (Caov3 cells, Caov4 cells, OVCAR3 cells and OVCAR8 cells) expressing MSLN or target cancer cells (NCI-H510A cells, MDAPCa2b cells) that did not express MSLN. Each target cell used in this study was engineered to express luciferase. Different amounts of tri-specific antigen binding proteins (SEQ ID NO: 58) targeting MSLN containing the MH6T domain were added to the mixture of T cells and target cancer cells listed above. The mixture was incubated at 37°C for 48 hours. After 48 hours, the remaining live target cancer cells were quantified using luminescence assay.

[0250] It was observed that the trispecific antigen binding protein targeting MSLN containing the MH6T domain was able to guide T cells to kill target cancer cells expressing MSLN (i.e., Caov3, Caov4, OVCAR3, and OVCAR8 cells, such as Figure 4 However, the MSLN-targeting trispecific antigen binding protein containing the MH6T domain could not guide T cells to kill target cancer cells (MDAPCa2b and NCI-H510A cells) that did not express MSLN, as also shown in Figure 2A. Figure 4 shown.

[0251] The ECs for killing MSLN-expressing cancer cells are listed in Table III below. 50 value.

[0252] Table III: Trispecific antigen binding proteins targeting MSLN (containing the MH6T domain) guide T cells to kill ECs of cancer cell lines expressing MSLN 50 value.

[0253]

[0254]

[0255] Example 7: Trispecific antigen binding targeting MSLN containing the MSLN binding domain (MH6T) according to the present disclosure Protein guides T cells from cynomolgus macaques to kill human ovarian cancer cell lines

[0256] In this assay, peripheral blood mononuclear cells (PBMCs; T cells are part of PBMCs) from cynomolgus macaque donors were mixed with target cancer cells expressing MSLN (CaOV3 cells and OVCAR3 cells), and varying amounts of a trispecific antigen-binding protein targeting MSLN (containing the MH6T domain, SEQ ID NO: 58) were added to the mixture and incubated at 37°C for 48 hours. In parallel, as described above, a mixture of cynomolgus macaque PBMCs and cells expressing MSLN was incubated at 37°C for 48 hours with varying amounts of a control TriTAC molecule, GFP TriTAC (SEQ ID NO: 59), targeting GFP. The target cancer cells used in this assay were engineered to express luciferase. After 48 hours, the remaining live target cells were quantified using a luminescence assay.

[0257] like Figure 5 As shown in Figure 3, it was observed that the trispecific antigen binding protein targeting MSLN (containing the MH6T domain) was able to effectively guide cynomolgus macaque PBMCs to kill cells expressing MSLN (i.e., Caov3 and OVCAR), while the control GFP TriTAC molecule was unable to guide cynomolgus macaque PBMCs to kill the cells (also shown in Figure 3). Figure 5 EC of a trispecific antigen-binding protein targeting MSLN (containing the MH6T domain) 50 The values ​​were 2.9 pM for OVCAR3 cells and 3.0 pM for Caov3 cells, which were consistent with the EC values ​​observed using human T cells. 50 There were no significant differences in the values, as shown in Table II.

[0258] Example 8: Trispecific antigen binding targeting MSLN in the presence or absence of human serum albumin Protein (containing the MH6T domain) directs T cells to kill MSLN-expressing NCI-H2052 mesothelioma cells

[0259] The purpose of this study is to evaluate whether the binding of a trispecific antigen-binding protein targeting MSLN (containing the MH6T domain; SEQ ID NO: 58) to human serum albumin (HSA) affects the ability of the protein to guide T cells to kill cells expressing MSLN. The NCI-H2052 mesothelioma cells used in this study were engineered to express luciferase. T cells from healthy donors and cells expressing MSLN (NCI-H2052) were mixed, and different amounts of trispecific antigen-binding proteins targeting MSLN (containing the MH6T domain) were added to the mixture. The mixture was incubated at 37°C for 48 hours in the presence or absence of HSA. Also in the presence or absence of HSA, a mixture of NCI-H2052 cells and T cells was incubated at 37°C for 48 hours with a control trispecific molecule GFP TriTAC (SEQ ID NO: 59) targeting GFP. After 48 hours, the remaining live target cells were quantified using a luminescence assay.

[0260] It was observed that the trispecific antigen binding protein targeting MSLN (containing the MH6T domain) could effectively guide T cells to kill NCI-H2052 cells (e.g., Figure 6 ), whereas the control GFP TriTAC molecule could not achieve this (also as Figure 6 It was also observed that in the presence of HSA, cell killing of EC 50 The value increased by about 3.2 times (as shown in Table IV)

[0261] Further TDCC assays were performed with additional MSLN-expressing cell lines using a trispecific antigen binding protein targeting MSLN (containing the MH6T domain) in the presence or absence of 15 mg / ml HSA. 50 The values ​​are presented in Table IV.

[0262] Table IV: ECs of MSLN-targeting trispecific antigen binding proteins (containing the MH6 T domain) directing T cells to kill MSLN-expressing cancer cells in the presence or absence of HSA 50 value

[0263]

[0264] Example 9: Trispecific antigen binding protein targeting MSLN (containing MH6T domain) and expressing MSLN T cells from four different donors secreted TNF-α in the presence of Caov4 cells

[0265] The target cancer cell CaOv4 used in this experiment is engineered to express luciferase. In this experiment, T cells and Caov4 cells from 4 different healthy donors (donor 02, donor 86, donor 35 and donor 81) were mixed together, different amounts of trispecific antigen binding proteins targeting MSLN (containing MH6T domain; SEQ ID NO: 58) were added, and the mixture was incubated at 37 ° C for 48 hours. Caov4 cells and T cells were also incubated at 37 ° C for 48 hours with a control trispecific molecule GFPTriTAC (SEQ ID NO: 59) targeting GFP. The conditioned medium determined from the TDCC was collected at 48 hours, and the viability of the target cancer cells was measured using a luminescence assay. The concentration of TNF-α in the conditioned medium was determined using an AlphaLISA assay kit (PerkinElmer).

[0266] It was observed that TNF-α was secreted into the culture medium in the presence of Caov4 cells and the MSLN-targeting trispecific antigen binding protein (containing the MH6T domain), but not in the presence of Caov4 cells and the control GFP TriTAC molecule, as shown in FIG. Figure 7shown.

[0267] In addition, effective killing was observed for T cells from all four healthy donors in the presence of a trispecific antigen binding protein targeting MSLN (containing the MH6T domain), but no effective killing was observed in the presence of a control GFP TriTAC molecule. TDCC assays were also established for other cell lines expressing MSLN (Caov3 cells, OVCAR3 cells, and OVCAR8 cells), and similar TNF-α expression was observed. Trispecific antigen binding protein targeting MSLN (containing the MH6T domain) induced TNF-α-expressing ECs. 50 The values ​​are presented in Table V. However, when the assay was performed using cancer cells that do not express MSLN (NCI-H510A cells or MDAPCa2b cells), no TNF-α secretion was observed with the trispecific antigen-binding protein targeting MSLN (containing the MH6T domain) (data not shown). Therefore, this study demonstrates that the trispecific antigen-binding protein targeting MSLN (containing the MH6T domain) is able to activate T cells in the presence of target cancer cells expressing MSLN.

[0268] Table V: Trispecific antigen binding proteins targeting MSLN (containing the MH6 T domain) induce TNF-α-expressing ECs in T cells from four different T cell donors and four different MSLN-expressing cell lines 50 value

[0269]

[0270]

[0271] Example 10: Trispecific antigen binding protein targeting MSLN (containing MH6T domain) and expressing MSLN Activation of CD69 expression on T cells from four different donors in the presence of OVCAR8 cells

[0272] The OVCAR8 cells used in this test are engineered to express luciferase. In this test, T cells and OVCAR8 cells from 4 different healthy donors (donor 02, donor 86, donor 35 and donor 81) are mixed together, different amounts of tri-specific antigen binding proteins (containing MH6T domains; SEQ ID NO: 58) targeting MSLN are added, and the mixture is incubated at 37 DEG C for 48 hours. OVCAR8 cells and T cells are also incubated at 37 DEG C for 48 hours with the control tri-specific molecule GFP TriTAC (SEQ ID NO: 59) targeting GFP. After 48 hours, T cells are collected and the CD69 expression on T cells is measured by flow cytometry.

[0273] like Figure 8As shown in Figure 6, in the presence of OVCAR8 cells and a trispecific antigen binding protein targeting MSLN (containing the MH6T domain), CD69 expression was detected on T cells from all four healthy donors, but no CD69 expression was detected in the presence of the negative control GFPTriTAC and OVCAR8 cells. TDCC assays were also established for other cells expressing MSLN (Caov3 cells, OVCAR3 cells, and OVCAR8 cells), and similar CD69 expression was observed. Table VI shows the CD69 activation ECs induced by the trispecific antigen binding protein targeting MSLN (containing the MH6T domain) in Caov3 cells and OVCAR8 cells. 50 value.

[0274] Table VI: EC50 values ​​for activation of CD69 expression on T cells from 4 different donors in the presence of trispecific antigen binding proteins targeting MSLN (containing the MH6 T domain) and MSLN-expressing OVCAR8 cells or Caov3 cells.

[0275]

[0276] When the assay was performed using cancer cells that did not express MSLN (NCI-H510A cells or MDAPCa2b cells), no CD69 activation induced by the trispecific antigen-binding protein targeting MSLN (containing the MH6T domain) was observed (data not shown). Therefore, this study demonstrates that the trispecific antigen-binding protein targeting MSLN (containing the MH6T domain) is able to activate T cells in the presence of target cancer cells expressing MSLN.

[0277] Example 11: Trispecific antigen binding protein targeting MSLN (containing MH6T domain) and expression of MSLN / Measurement of binding in cell lines not expressing MSLN

[0278] For this study, certain target cancer cells expressing MSLN (Caov3 cells, CaOV4 cells, OVCAR3 cells, and OVCAR8 cells) and certain cancer cells that do not express MSLN (MDAPCa2b cells and NCI-H510A cells) were incubated with a trispecific antigen-binding protein targeting MSLN (containing the MH6T domain; SEQ ID NO: 58) or a control GFP TriTAC molecule (SEQ ID NO: 59). After incubation, the cells were washed to remove unbound MH6T or GFP TriTAC molecules and further incubated with a second antibody conjugated to Alexa Fluor 647 that recognizes the anti-albumin domain in the TriTAC molecule. The binding of the trispecific antigen-binding protein targeting MSLN (containing the MH6T domain) or GFP TriTAC to cells expressing or not expressing MSLN was measured by flow cytometry.

[0279] Strong binding of the MSLN-targeting trispecific antigen-binding protein (containing the MH6T domain) to MSLN-expressing cell lines (Caov3, Caov4, OVCAR3, and OVCAR8) was observed. Figure 9A As shown (the upper left figure shows the binding of the trispecific target antigen binding protein targeting MSLN containing the MH6T domain to Caov3 cells; the upper right figure shows the binding of the trispecific target antigen binding protein targeting MSLN containing the MH6T domain to Caov4 cells; the lower left figure shows the binding of the trispecific target antigen binding protein targeting MSLN containing the MH6T domain to OVCAR3 cells; the lower right figure shows the binding of the trispecific target antigen binding protein targeting MSLN containing the MH6T domain to OVCAR8 cells); and as Figure 9B As shown in the figure, no binding was observed in cell lines that do not express MSLN (the left figure shows that the trispecific antigen binding protein targeting MSLN (containing the MH6T domain) cannot bind to MDAPCa2b cells, and the right figure shows that the trispecific antigen binding protein targeting MSLN (containing the MH6T domain) cannot bind to NCI-H510A cells). In addition, no binding was observed when any cell type was incubated with the GFPTriTAC molecule, as shown in Figure 2. Figure 9A and Figure 9B shown.

[0280] Example 12: Trispecific antigen binding protein targeting MSLN (containing MH6T domain) and donor-derived Measurement of T cell binding

[0281] For this study, T cells from four healthy donors were incubated with a trispecific antigen-binding protein targeting MSLN (containing the MH6T domain; SEQ ID NO: 58) or buffer (as a negative control). After incubation, the cells were washed to remove unbound trispecific antigen-binding protein targeting MSLN (containing the MH6T domain) and further incubated with a secondary antibody conjugated to Alexa Fluor 647 that recognizes the anti-albumin domain in the trispecific antigen-binding protein targeting MSLN (containing the MH6T domain). Binding was measured by flow cytometry.

[0282] Strong binding was observed in T cells from all four donors treated with a trispecific antigen binding protein targeting MSLN (containing the MH6 T domain), as shown in Figure 5. Figure 10 As shown (the upper left figure shows the binding of the trispecific antigen-binding protein (containing the MH6T domain) targeting MSLN to T cells from donor 2; the upper right figure shows the binding of the trispecific antigen-binding protein (containing the MH6T domain) targeting MSLN to T cells from donor 35; the lower left figure shows the binding of the trispecific antigen-binding protein (containing the MH6T domain) targeting MSLN to T cells from donor 41; the lower right figure shows the binding of the trispecific antigen-binding protein (containing the MH6T domain) targeting MSLN to T cells from donor 81).

[0283] Example 13: Mice treated with a trispecific antigen binding protein targeting MSLN (containing the MH6T domain) Inhibition of tumor growth

[0284] For this study, 10 7 NCI-H292 cells and 10 7 Human PBMCs were subcutaneously implanted into two groups of NCG mice (8 mice per group). After 5 days, one group of mice was injected with a trispecific antigen-binding protein targeting MSLN (containing the MH6T domain; SEQ ID NO: 58) at a dose of 0.25 mg / kg per day for 10 days (days 5-14), while the other group of mice was injected with a vehicle control. Tumor volume was measured every few days and the study was terminated on day 36. Significant tumor growth inhibition was observed in mice injected with a trispecific antigen-binding protein targeting MSLN (containing the MH6T domain) compared to mice injected with a vehicle control, as shown in Figure 3. Figure 11 shown.

[0285] Example 12: Trispecific antigen binding protein targeting MSLN (containing MH6T domain) in cynomolgus monkeys Pharmacokinetics

[0286] For this study, two cynomolgus monkeys were injected intravenously with a 10 mg / kg dose of a trispecific antigen-binding protein targeting MSLN (containing the MH6T domain; SEQ ID NO: 58), and serum samples were collected at different time points after injection. The amount of trispecific antigen-binding protein targeting MSLN (containing the MH6T domain) in serum was measured using an anti-idiotypic antibody that recognizes the trispecific antigen-binding protein targeting MSLN (containing the MH6T domain) in an electrochemiluminescence assay. Figure 12 Shown are graphs of serum levels of trispecific antigen-binding proteins targeting MSLN (containing MH6T domains) at different time points. These data were then used to calculate the pharmacokinetic properties of trispecific antigen-binding proteins targeting MSLN (containing MH6T domains), as shown in Table VII.

[0287] Table VII: Pharmacokinetic parameters of trispecific antigen binding proteins targeting MSLN (containing MH6T domain)

[0288]

[0289] Sequence Listing

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296] CDR1 sequences of various exemplary MSLN-binding domains disclosed herein

[0297]

[0298]

[0299] CDR2 sequences of various exemplary MSLN-binding domains disclosed herein

[0300]

[0301]

[0302] CDR3 sequences of various exemplary MSLN-binding domains disclosed herein

[0303]

[0304]

[0305] Framework region 1 (f1) sequences of various exemplary MSLN binding domains

[0306]

[0307]

[0308] Framework region 2 (f2) sequences of various exemplary MSLN binding domains

[0309]

[0310]

[0311] Framework region 3 (f3) sequences of various exemplary MSLN binding domains

[0312]

[0313]

[0314] Framework region 4 (f4) sequences of various exemplary MSLN binding domains

[0315]

[0316] Sequence Listing <110> Harpurn Therapeutics <120> Mesothelin-binding protein <130> 47517-719.7111 <140> <141> <150> 62 / 657,417 <151> 2018-04-13 <150> 62 / 505,719 <151> 2017-05-12 <160> 336 <170> PatentIn version 3.5 <210> 1 <211> 110 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 1 Gln 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 Arg Thr Phe Ser Val Arg 20 25 30 Gly Met Ala Trp Tyr Arg Gln Ala Gly Asn Asn Arg Ala Leu Val Ala 35 40 45 Thr Met Asn Pro Asp Gly Phe Pro Asn Tyr Ala Asp Ala Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Trp Asp Ile Ala Glu Asn Thr Val Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Asn Ser Glu Asp Thr Thr Val Tyr Tyr Cys Asn Ser 85 90 95 Gly Pro Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 100 105 110 <210> 2 <211> 123 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 2 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Pro Ser Ile Glu 20 25 30 Gln Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Ala Leu Thr Ser Gly Gly Arg Ala Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gly Asp Asn Val Arg Asn Met Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ser 85 90 95 Ala Gly Arg Phe Lys Gly Asp Tyr Ala Gln Arg Ser Gly Met Asp Tyr 100 105 110 Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 3 <211> 116 <212> PRT <213> artificial sequence <220> <223> Description of artificial sequence: synthesis Polypeptide <400> 3 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly Thr Thr Tyr Thr Phe Asp 20 25 30 Leu Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Thr Val Val 35 40 45 Ala Ser Ile Ser Ser Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Arg 50 55 60 Gly Arg Phe Thr Ile Ser Gly Glu Asn Gly Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys Leu 85 90 95 Gly Gln Arg Ser Gly Val Arg Ala Phe Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115 <210> 4 <211> 123 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 4 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Thr Ser Asn Ile Asn 20 25 30 Asn Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Val Ile Thr Arg Gly Gly Tyr Ala Ile Tyr Leu Asp Ala Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Ala Ile Tyr Leu 65 70 75 80 Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Val Cys Asn 85 90 95 Ala Asp Arg Val Glu Gly Thr Ser Gly Gly Pro Gln Leu Arg Asp Tyr 100 105 110 Phe Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 5 <211> 116 <212> PRT <213> artificial sequence <220> <223> Description of artificial sequence: synthesis Polypeptide <400> 5 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Gly Ile Asn 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Val Ile Ser Arg Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Ser Leu 65 70 75 80 Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Asn 85 90 95 Ala Arg Thr Tyr Thr Arg His Asp Tyr Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115 <210> 6 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 6 Gln Val Arg Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Ile Ser Ala Phe Arg Leu Met 20 25 30 Ser Val Arg Trp Tyr Arg Gln Asp Pro Ser Lys Gln Arg Glu Trp Val 35 40 45 Ala Thr Ile Asp Gln Leu Gly Arg Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Ala Ile Ser Lys Asp Ser Thr Arg Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Met Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Gly Gly Gly Pro Leu Gly Ser Arg Trp Leu Arg Gly Arg His Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 7 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 7 Gln Val Arg Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Pro Phe Ser Ile Asn 20 25 30 Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Ser Ile Ser Ser Ser Gly Asp Phe Thr Tyr Thr Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Arg Arg Thr Tyr Leu Pro Arg Arg Phe Gly Ser Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 8 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 8 Gln Val Gln Pro Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser Gly Ser Asp Phe Thr Glu Asp 20 25 30 Ala Met Ala Trp Tyr Arg Gln Ala Ser Gly Lys Glu Arg Glu Ser Val 35 40 45 Ala Phe Val Ser Lys Asp Gly Lys Arg Ile Leu Tyr Leu Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Ile Asp Lys Lys Thr Val Tyr 65 70 75 80 Leu Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Asn Ser Ala Pro Gly Ala Ala Arg Asn Tyr Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 <210> 9 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 9 Gln Val Gln Pro Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser Gly Ser Asp Phe Thr Glu Asp 20 25 30 Ala Met Ala Trp Tyr Arg Gln Ala Ser Gly Lys Glu Arg Glu Ser Val 35 40 45 Ala Phe Val Ser Lys Asp Gly Lys Arg Ile Leu Tyr Leu Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Ile Tyr Lys Lys Thr Val Tyr 65 70 75 80 Leu Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Asn Ser Ala Pro Gly Ala Ala Arg Asn Val Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 <210> 10 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 11 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 11 Gln Val Gln Ile Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Leu Thr Tyr Ser Ile Val 20 25 30 Ala Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val 35 40 45 Ala Asp Ile Ser Pro Val Gly Asn Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Glu Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His 85 90 95 Ile Val Arg Gly Trp Leu Asp Glu Arg Pro Gly Pro Gly Pro Ile Val 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 12 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 12 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Gly Val Tyr 20 25 30 Gly Met Glu Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Trp Val 35 40 45 Ala Ser His Thr Ser Thr Gly Tyr Val Tyr Tyr Arg Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Lys 85 90 95 Ala Asn Arg Gly Ser Tyr Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> 13 <211> 114 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 13 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Thr Thr Ser Ser Ile Asn Ser 20 25 30 Met Ser Trp Tyr Arg Gln Ala Gln Gly Lys Gln Arg Glu Pro Val Ala 35 40 45 Val Ile Thr Asp Arg Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Thr Cys His Val 85 90 95 Ile Ala Asp Trp Arg Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser <210> 14 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 14 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Leu Ser Arg Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Gln Phe Val 35 40 45 Ala Ala Ile Ser Arg Ser Gly Gly Thr Thr Arg Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Ala Asn Thr Phe Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Arg Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Val Arg Arg Arg Gly Trp Gly Arg Thr Leu Glu Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 15 <211> 117 <212> PRT <213> artificial sequence <220> <223> Description of artificial sequence: synthesis Polypeptide <400> 15 Gln Val Gln Leu Gly Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Pro Asn 20 25 30 Ala Met Ile Trp His Arg Gln Ala Pro Gly Lys Gln Arg Glu Pro Val 35 40 45 Ala Ser Ile Asn Ser Ser Gly Ser Thr Asn Tyr Gly Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Val Ser Arg Asp Ile Val Lys Asn Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ser 85 90 95 Tyr Ser Asp Phe Arg Arg Gly Thr Gln Tyr Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 <210> 16 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 16 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Pro Ser Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Ser Ala Ile Thr 20 25 30 Asn Leu Gly Trp Tyr Arg Arg Ala Pro Gly Gln Val Arg Glu Met Val 35 40 45 Ala Arg Ile Ser Val Arg Glu Asp Lys Glu Asp Tyr Glu Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Gln Asn Leu Val Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Gln Pro His Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Gly Ala Gln Arg Trp Gly Arg Gly Pro Gly Thr Thr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 17 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 17 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Arg Ile Arg 20 25 30 Val Met Arg Trp Tyr Arg Gln Ala Pro Gly Thr Glu Arg Asp Leu Val 35 40 45 Ala Val Ile Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95 Asp Asp Ser Gly Ile Ala Arg Asp Tyr Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115 <210> 18 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 18 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu 1 5 10 15 Ser Arg Arg Leu Ser Cys Ala Val Ser Gly Asp Thr Ser Lys Phe Lys 20 25 30 Ala Val Gly Trp Tyr Arg Gln Ala Pro Gly Ala Gln Arg Glu Leu Leu 35 40 45 Ala Trp Ile Asn Asn Ser Gly Val Gly Asn Thr Ala Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Arg Leu Thr Pro Glu Asp Thr Asp Val Tyr Tyr Cys Arg 85 90 95 Phe Tyr Arg Arg Phe Gly Ile Asn Lys Asn Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 19 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 19 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Gly Asn Lys 20 25 30 Pro Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Val Ile Ser Ser Asp Gly Gly Ser Thr Arg Tyr Ala Ala Leu Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Glu Ser Leu Val Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Ala Leu Arg Thr Tyr Tyr Leu Asn Asp Pro Val Val Phe Ser Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 20 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 20 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Ser Ile Asn 20 25 30 Thr Met Tyr Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Phe Ile Ser Ser Gly Gly Ser Thr Asn Val Arg Asp Ser Val Lys 50 55 60 Gly Arg Phe Ser Val Ser Arg Asp Ser Ala Lys Asn Ile Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Thr Tyr Ile Pro Leu Arg Gly Thr Leu His Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> twenty one <211> 115 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> twenty one Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Asp Arg Ile Thr 20 25 30 Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Thr Ile Ser Asn Arg Gly Thr Ser Asn Tyr Ala Asn Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Arg Lys Trp Gly Arg Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> twenty two <211> 119 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> twenty two Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Arg Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Thr Ile Gly Ile Asn 20 25 30 Asp Met Ala Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu Leu Val 35 40 45 Ala Thr Ile Thr Lys Gly Gly Thr Thr Asp Tyr Ala Asp Ser Val Asp 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Thr Lys Arg Arg Glu Trp Ala Lys Asp Phe Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> twenty three <211> 114 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> twenty three Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Ala Ile Gly Ser Ile Asn Ser 20 25 30 Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Pro Val Ala 35 40 45 Val Ile Thr Asp Arg Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Thr Cys His Val 85 90 95 Ile Ala Asp Trp Arg Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser <210> twenty four <211> 119 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> twenty four Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Ser Ile Asn 20 25 30 Thr Met Tyr Trp Phe Arg Gln Ala Pro Gly Glu Glu Arg Glu Leu Val 35 40 45 Ala Thr Ile Asn Arg Gly Gly Ser Thr Asn Val Arg Asp Ser Val Lys 50 55 60 Gly Arg Phe Ser Val Ser Arg Asp Ser Ala Lys Asn Ile Val Tyr Leu 65 70 75 80 Gln Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Thr Tyr Ile Pro Tyr Gly Gly Thr Leu His Asp Phe Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 25 <211> 114 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 25 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Thr Ser Thr Thr Phe Ser Ile Asn Ser 20 25 30 Met Ser Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu Pro Val Ala 35 40 45 Val Ile Thr Asn Arg Gly Thr Thr Ser Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Val Tyr Leu Gln 65 70 75 80 Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Thr Cys His Val 85 90 95 Ile Ala Asp Trp Arg Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser <210> 26 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 26 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Ile Arg 20 25 30 Ala Met Arg Trp Tyr Arg Gln Ala Pro Gly Thr Glu Arg Asp Leu Val 35 40 45 Ala Val Ile Tyr Gly Ser Ser Thr Tyr Tyr Ala Asp Ala Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95 Asp Thr Ile Gly Thr Ala Arg Asp Tyr Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115 <210> 27 <211> 117 <212> PRT <213> artificial sequence <220> <223> Description of artificial sequence: synthesis Polypeptide <400> 27 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Ser Thr Ile Asp 20 25 30 Thr Met Tyr Trp His Arg Gln Ala Pro Gly Asn Glu Arg Glu Leu Val 35 40 45 Ala Tyr Val Thr Ser Arg Gly Thr Ser Asn Val Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ser 85 90 95 Val Arg Thr Thr Ser Tyr Pro Val Asp Phe Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 <210> 28 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 28 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Ser Ile Asn 20 25 30 Thr Met Tyr Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Phe Ile Ser Ser Gly Gly Ser Thr Asn Val Arg Asp Ser Val Lys 50 55 60 Gly Arg Phe Ser Val Ser Arg Asp Ser Ala Lys Asn Ile Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Thr Tyr Ile Pro Tyr Gly Gly Thr Leu His Asp Phe Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 29 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 29 Gln 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 Gly Asp Trp Ser Ala Asn 20 25 30 Phe Met Tyr Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Arg Ile Ser Gly Arg Gly Val Val Asp Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Val Ala Ser Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 100 105 110 <210> 30 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <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 Gly Asp Trp Ser Ala Asn 20 25 30 Phe Met Tyr Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Arg Ile Ser Gly Arg Gly Val Val Asp Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Val Ala Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 31 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <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 Gly Asp Trp Ser Ala Asn 20 25 30 Phe Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Ser Gly Arg Gly Val Val Asp Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Val Ala Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 32 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 32 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Ser Ile Asn 20 25 30 Thr Met Tyr Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Phe Ile Ser Ser Gly Gly Ser Thr Asn Val Arg Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Thr Tyr Ile Pro Tyr Gly Gly Thr Leu His Asp Phe Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 33 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic Polypeptide <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 Ser Thr Ser Ser Ile Asn 20 25 30 Thr Met Tyr Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Phe Ile Ser Ser Gly Gly Ser Thr Asn Val Arg Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Thr Tyr Ile Pro Tyr Gly Gly Thr Leu His Asp Phe Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 34 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <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 Ser Thr Ser Ser Ile Asn 20 25 30 Thr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ser Ser Gly Gly Ser Thr Asn Val Arg Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Thr Tyr Ile Pro Tyr Gly Gly Thr Leu His Asp Phe Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 35 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 35 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Ile Arg 20 25 30 Ala Met Arg Trp Tyr Arg Gln Ala Pro Gly Thr Glu Arg Asp Leu Val 35 40 45 Ala Val Ile Tyr Gly Ser Ser Thr Tyr Tyr Ala Asp Ala Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95 Asp Thr Ile Gly Thr Ala Arg Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 36 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 36 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Ile Arg 20 25 30 Ala Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Val Ile Tyr Gly Ser Ser Thr Tyr Tyr Ala Asp Ala Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95 Asp Thr Ile Gly Thr Ala Arg Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly 115 <210> 37 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 37 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Ile Arg 20 25 30 Ala Met Arg Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Gly Ser Ser Thr Tyr Tyr Ala Asp Ala Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95 Asp Thr Ile Gly Thr Ala Arg Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly 115 <210> 38 <211> 118 <212> PRT <213> artificial sequence <220> <223> Description of artificial sequence: synthesis Polypeptide <400> 38 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Ser Thr Ile Asp 20 25 30 Thr Met Tyr Trp His Arg Gln Ala Pro Gly Asn Glu Arg Glu Leu Val 35 40 45 Ala Tyr Val Thr Ser Arg Gly Thr Ser Asn Val Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser 85 90 95 Val Arg Thr Thr Ser Tyr Pro Val Asp Phe Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Gly Gly 115 <210> 39 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <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 Arg Thr Ser Thr Ile Asp 20 25 30 Thr Met Tyr Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Tyr Val Thr Ser Arg Gly Thr Ser Asn Val Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser 85 90 95 Val Arg Thr Thr Ser Tyr Pro Val Asp Phe Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 40 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <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 Arg Thr Ser Thr Ile Asp 20 25 30 Thr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Val Thr Ser Arg Gly Thr Ser Asn Val Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser 85 90 95 Val Arg Thr Thr Ser Tyr Pro Val Asp Phe Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 41 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 41 Glu Ser Gly Gly Gly Leu Val 1 5 <210> 42 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 42 Leu Ser Cys 1 <210> 43 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 43 Gly Arg Phe 1 <210> 44 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 44 Val Thr Val Ser Ser 1 5 <210> 45 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 45 Gln Leu Val Glu Ser Gly Gly Gly 1 5 <210> 46 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 46 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 1 5 10 <210> 47 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 47 Ala Ser Gly 1 <210> 48 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 48 Arg Gln Ala Pro Gly 1 5 <210> 49 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 49 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 1 5 10 15 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 20 25 30 Cys <210> 50 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 50 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 51 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 51 Gly Arg Thr Phe Ser Val Arg Gly Met Ala 1 5 10 <210> 52 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 52 Ile Asn Ser Ser Gly Ser Thr Asn Tyr Gly 1 5 10 <210> 53 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 53 Asn Ala Gly Gly Gly Pro Leu Gly Ser Arg 1 5 10 <210> 54 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 54 Gly Gly Asp Trp Ser Ala Asn Phe Met Tyr 1 5 10 <210> 55 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 55 Ile Ser Ser Gly Gly Ser Thr Asn Val Arg 1 5 10 <210> 56 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 56 Asn Ala Asp Thr Ile Gly Thr Ala Arg Asp 1 5 10 <210> 57 <211> 630 <212> PRT <213> Homo sapiens <400> 57 Met Ala Leu Pro Thr Ala Arg Pro Leu Leu Gly Ser Cys Gly Thr Pro 1 5 10 15 Ala Leu Gly Ser Leu Leu Phe Leu Leu Phe Ser Leu Gly Trp Val Gln 20 25 30 Pro Ser Arg Thr Leu Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu 35 40 45 Asp Gly Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser Pro Arg 50 55 60 Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr Glu 65 70 75 80 Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys Asn Val Lys Leu 85 90 95 Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro Pro 100 105 110 Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu Leu Leu Phe Leu Asn Pro 115 120 125 Asp Ala Phe Ser Gly Pro Gln Ala Cys Thr Arg Phe Phe Ser Arg Ile 130 135 140 Thr Lys Ala Asn Val Asp Leu Leu Pro Arg Gly Ala Pro Glu Arg Gln 145 150 155 160 Arg Leu Leu Pro Ala Ala Leu Ala Cys Trp Gly Val Arg Gly Ser Leu 165 170 175 Leu Ser Glu Ala Asp Val Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu 180 185 190 Pro Gly Arg Phe Val Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu 195 200 205 Val Ser Cys Pro Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg 210 215 220 Ala Ala Leu Gln Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp 225 230 235 240 Ser Val Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Val Leu Gly 245 250 255 Gln Pro Ile Ile Arg Ser Ile Pro Gln Gly Ile Val Ala Ala Trp Arg 260 265 270 Gln Arg Ser Ser Arg Asp Pro Ser Trp Arg Gln Pro Glu Arg Thr Ile 275 280 285 Leu Arg Pro Arg Phe Arg Arg Glu Val Glu Lys Thr Ala Cys Pro Ser 290 295 300 Gly Lys Lys Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys 305 310 315 320 Trp Glu Leu Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met 325 330 335 Asp Arg Val Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu 340 345 350 Lys His Lys Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro Glu Ser Val 355 360 365 Ile Gln His Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp Ile 370 375 380 Arg Lys Trp Asn Val Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu 385 390 395 400 Val Asn Lys Gly His Glu Met Ser Pro Gln Ala Pro Arg Arg Pro Leu 405 410 415 Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val Lys Gly Arg Gly Gln 420 425 430 Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr Pro Gly Tyr 435 440 445 Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val Pro Pro Ser Ser 450 455 460 Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr Cys Asp Pro Arg Gln 465 470 475 480 Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gln Asn Met Asn 485 490 495 Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe Leu Gly Gly Ala Pro 500 505 510 Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn Val Ser Met Asp Leu 515 520 525 Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val Leu Pro Leu Thr Val 530 535 540 Ala Glu Val Gln Lys Leu Leu Gly Pro His Val Glu Gly Leu Lys Ala 545 550 555 560 Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile Leu Arg Gln Arg Gln 565 570 575 Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln Gly Gly Ile Pro Asn 580 585 590 Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu Ala Leu Ser Gly Thr 595 600 605 Pro Cys Leu Leu Gly Pro Gly Pro Val Leu Thr Val Leu Ala Leu Leu 610 615 620 Leu Ala Ser Thr Leu Ala 625 630 <210> 58 <211> 116 <212> PRT <213> artificial sequence <220> <223> Description of artificial sequence: synthesis Polypeptide <400> 58 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Ile Arg 20 25 30 Ala Met Arg Trp Tyr Arg Gln Ala Pro Gly Thr Glu Arg Asp Leu Val 35 40 45 Ala Val Ile Tyr Gly Ser Ser Thr Tyr Tyr Ala Asp Ala Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95 Asp Thr Ile Gly Thr Ala Arg Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 59 <211> 503 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 59 Gln Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Val Asn Arg Tyr 20 25 30 Ser Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ala Gly Met Ser Ser Ala Gly Asp Arg Ser Ser Tyr Glu Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Arg Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Val Asn Val Gly Phe Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Phe Gly Met Ser Trp 145 150 155 160 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser 165 170 175 Gly Ser Gly Arg Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe 180 185 190 Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn 195 200 205 Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly 210 215 220 Ser Leu Ser Val Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser Gly 225 230 235 240 Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly 245 250 255 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala 260 265 270 Ser Gly Phe Thr Phe Asn Lys Tyr Ala Ile Asn Trp Val Arg Gln Ala 275 280 285 Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn 290 295 300 Asn Tyr Ala Thr Tyr Tyr Ala Asp Gln Val Lys Asp Arg Phe Thr Ile 305 310 315 320 Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu 325 330 335 Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Ala Asn Phe 340 345 350 Gly Asn Ser Tyr Ile Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr Leu 355 360 365 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 370 375 380 Gly Gly Gly Ser Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val 385 390 395 400 Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Ala Ser Ser Thr Gly Ala 405 410 415 Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln 420 425 430 Ala Pro Arg Gly Leu Ile Gly Gly Thr Lys Phe Leu Val Pro Gly Thr 435 440 445 Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr 450 455 460 Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Thr Leu 465 470 475 480 Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val 485 490 495 Leu His His His His His His 500 <210> 60 <211> 116 <212> PRT[[ID=3'7]] <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic Polypeptide <400> 60 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Ile Arg 20 25 30 Ala Met Arg Trp Tyr Arg Gln Ala Pro Gly Thr Glu Arg Asp Leu Val 35 40 45 Ala Val Ile Tyr Gly Ser Ser Thr Tyr Tyr Ala Asp Ala Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95 Asp Thr Ile Gly Thr Ala Arg Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 61 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 61 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Ile Arg 20 25 30 Ala Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Val Ile Tyr Gly Ser Ser Thr Tyr Tyr Ala Asp Ala Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95 Asp Thr Ile Gly Thr Ala Arg Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 62 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 62 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Ile Arg 20 25 30 Ala Met Arg Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Gly Ser Ser Thr Tyr Tyr Ala Asp Ala Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95 Asp Thr Ile Gly Thr Ala Arg Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 63 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 63 Gly Arg Thr Phe Ser Val Arg Gly Met Ala 1 5 10 <210> 64 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 64 Gly Ser Ile Pro Ser Ile Glu Gln Met Gly 1 5 10 <210> 65 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 65 Gly Thr Thr Tyr Thr Phe Asp Leu Met Ser 1 5 10 <210> 66 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 66 Gly Ser Thr Ser Asn Ile Asn Asn Met Arg 1 5 10 <210> 67 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 67 Gly Ser Thr Phe Gly Ile Asn Ala Met Gly 1 5 10 <210> 68 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 68 Ile Ser Ala Phe Arg Leu Met Ser Val Arg 1 5 10 <210> 69 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 69 Gly Arg Pro Phe Ser Ile Asn Thr Met Gly 1 5 10 <210> 70 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 70 Gly Ser Asp Phe Thr Glu Asp Ala Met Ala 1 5 10 <210> 71 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 71 Gly Ser Asp Phe Thr Glu Asp Ala Met Ala 1 5 10 <210> 72 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 72 Gly Phe Thr Phe Ser Ser Phe Gly Met Ser 1 5 10 <210> 73 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 73 Gly Leu Thr Tyr Ser Ile Val Ala Val Gly 1 5 10 <210> 74 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 74 Gly Leu Thr Phe Gly Val Tyr Gly Met Glu 1 5 10 <210> 75 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 75 Thr Thr Ser Ser Ile Asn Ser Met Ser 1 5 <210> 76 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 76 Gly Arg Thr Leu Ser Arg Tyr Ala Met Gly 1 5 10 <210> 77 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 77 Gly Ser Ile Phe Ser Pro Asn Ala Met Ile 1 5 10 <210> 78 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 78 Gly Ala Thr Ser Ala Ile Thr Asn Leu Gly 1 5 10 <210> 79 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 79 Gly Ser Thr Phe Arg Ile Arg Val Met Arg 1 5 10 <210> 80 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 80 Gly Asp Thr Ser Lys Phe Lys Ala Val Gly 1 5 10 <210> 81 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 81 Gly Ser Thr Phe Gly Asn Lys Pro Met Gly 1 5 10 <210> 82 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 82 Gly Ser Thr Ser Ser Ile Asn Thr Met Tyr 1 5 10 <210> 83 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 83 Gly Arg Thr Asp Arg Ile Thr Thr Met Gly 1 5 10 <210> 84 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 84 Gly Arg Thr Ile Gly Ile Asn Asp Met Ala 1 5 10 <210> 85 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 85 Ala Ile Gly Ser Ile Asn Ser Met Ser 1 5 <210> 86 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 86 Gly Ser Thr Ser Ser Ile Asn Thr Met Tyr 1 5 10 <210> 87 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 87 Thr Thr Phe Ser Ile Asn Ser Met Ser 1 5 <210> 88 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 88 Gly Ser Thr Phe Ser Ile Arg Ala Met Arg 1 5 10 <210> 89 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 89 Gly Arg Thr Ser Thr Ile Asp Thr Met Tyr 1 5 10 <210> 90 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 90 Gly Ser Thr Ser Ser Ile Asn Thr Met Tyr 1 5 10 <210> 91 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 91 Gly Gly Asp Trp Ser Ala Asn Phe Met Tyr 1 5 10 <210> 92 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 92 Gly Gly Asp Trp Ser Ala Asn Phe Met Tyr 1 5 10 <210> 93 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 93 Gly Ser Thr Ser Ser Ile Asn Thr Met Tyr 1 5 10 <210> 94 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 94 Gly Ser Thr Ser Ser Ile Asn Thr Met Tyr 1 5 10 <210> 95 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 95 Gly Ser Thr Ser Ser Ile Asn Thr Met Tyr 1 5 10 <210> 96 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 96 Gly Ser Thr Phe Ser Ile Arg Ala Met Arg 1 5 10 <210> 97 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 97 Gly Ser Thr Phe Ser Ile Arg Ala Met Arg 1 5 10 <210> 98 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 98 Gly Ser Thr Phe Ser Ile Arg Ala Met Arg 1 5 10 <210> 99 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 99 Gly Arg Thr Ser Thr Ile Asp Thr Met Tyr 1 5 10 <210> 100 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 100 Gly Arg Thr Ser Thr Ile Asp Thr Met Tyr 1 5 10 <210> 101 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 101 Gly Arg Thr Ser Thr Ile Asp Thr Met Tyr 1 5 10 <210> 102 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 102 Thr Met Asn Pro Asp Gly Phe Pro Asn Tyr Ala Asp Ala Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 103 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 103 Ala Leu Thr Ser Gly Gly Arg Ala Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 104 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 104 Ser Ile Ser Ser Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Arg Gly 1 5 10 15 Arg Phe Thr <210> 105 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 105 Val Ile Thr Arg Gly Gly Tyr Ala Ile Tyr Leu Asp Ala Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 106 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 106 Val Ile Ser Arg Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 107 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 107 Thr Ile Asp Gln Leu Gly Arg Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Ala <210> 108 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 108 Ser Ile Ser Ser Ser Gly Asp Phe Thr Tyr Thr Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 109 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 109 Phe Val Ser Lys Asp Gly Lys Arg Ile Leu Tyr Leu Asp Ser Val Arg 1 5 10 15 Gly Arg Phe Thr 20 <210> 110 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 110 Phe Val Ser Lys Asp Gly Lys Arg Ile Leu Tyr Leu Asp Ser Val Arg 1 5 10 15 Gly Arg Phe Thr 20 <210> 111 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 111 Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly Arg Phe Thr 20 <210> 112 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 112 Asp Ile Ser Pro Val Gly Asn Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 113 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 113 Ser His Thr Ser Thr Gly Tyr Val Tyr Tyr Arg Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 114 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 114 Val Ile Thr Asp Arg Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 115 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 115 Ala Ile Ser Arg Ser Gly Gly Thr Thr Arg Tyr Ser Asp Ser Val Lys 1 5 10 15 Gly Arg Phe Thr 20 <210> 116 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 116 Ser Ile Asn Ser Ser Gly Ser Thr Asn Tyr Gly Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 117 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 117 Arg Ile Ser Val Arg Glu Asp Lys Glu Asp Tyr Glu Asp Ser Val Lys 1 5 10 15 Gly Arg Phe Thr 20 <210> 118 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 118 Val Ile Ser Gly Ser Ser Thr Tyr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 1 5 10 15 Phe Thr <210> 119 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 119 Trp Ile Asn Asn Ser Gly Val Gly Asn Thr Ala Glu Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 120 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 120 Val Ile Ser Ser Asp Gly Gly Ser Thr Arg Tyr Ala Ala Leu Val Lys 1 5 10 15 Gly Arg Phe Thr 20 <210> 121 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 121 Phe Ile Ser Ser Gly Gly Ser Thr Asn Val Arg Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Ser <210> 122 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 122 Thr Ile Ser Asn Arg Gly Thr Ser Asn Tyr Ala Asn Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 123 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 123 Thr Ile Thr Lys Gly Gly Thr Thr Asp Tyr Ala Asp Ser Val Asp Gly 1 5 10 15 Arg Phe Thr <210> 124 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 124 Val Ile Thr Asp Arg Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 125 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 125 Thr Ile Asn Arg Gly Gly Ser Thr Asn Val Arg Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Ser <210> 126 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 126 Val Ile Thr Asn Arg Gly Thr Thr Ser Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 127 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 127 Val Ile Tyr Gly Ser Ser Thr Tyr Tyr Ala Asp Ala Val Lys Gly Arg 1 5 10 15 Phe Thr <210> 128 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 128 Tyr Val Thr Ser Arg Gly Thr Ser Asn Val Ala Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 129 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 129 Phe Ile Ser Ser Gly Gly Ser Thr Asn Val Arg Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Ser <210> 130 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 130 Arg Ile Ser Gly Arg Gly Val Val Asp Tyr Val Glu Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 131 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 131 Arg Ile Ser Gly Arg Gly Val Val Asp Tyr Val Glu Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 132 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 132 Phe Ile Ser Ser Gly Gly Ser Thr Asn Val Arg Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 133 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 133 Phe Ile Ser Ser Gly Gly Ser Thr Asn Val Arg Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 134 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 134 Phe Ile Ser Ser Gly Gly Ser Thr Asn Val Arg Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 135 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 135 Val Ile Tyr Gly Ser Ser Thr Tyr Tyr Ala Asp Ala Val Lys Gly Arg 1 5 10 15 Phe Thr <210> 136 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 136 Val Ile Tyr Gly Ser Ser Thr Tyr Tyr Ala Asp Ala Val Lys Gly Arg 1 5 10 15 Phe Thr <210> 137 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 137 Val Ile Tyr Gly Ser Ser Thr Tyr Tyr Ala Asp Ala Val Lys Gly Arg 1 5 10 15 Phe Thr <210> 138 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 138 Tyr Val Thr Ser Arg Gly Thr Ser Asn Val Ala Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 139 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 139 Tyr Val Thr Ser Arg Gly Thr Ser Asn Val Ala Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 140 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 140 Tyr Val Thr Ser Arg Gly Thr Ser Asn Val Ala Asp Ser Val Lys Gly 1 5 10 15 Arg Phe Thr <210> 141 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 141 Gly Pro Tyr 1 <210> 142 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 142 Gly Arg Phe Lys Gly Asp Tyr Ala Gln Arg Ser Gly Met Asp Tyr 1 5 10 15 <210> 143 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 143 Gln Arg Ser Gly Val Arg Ala Phe 1 5 <210> 144 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 144 Asp Arg Val Glu Gly Thr Ser Gly Gly Pro Gln Leu Arg Asp Tyr 1 5 10 15 <210> 145 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 145 Arg Thr Tyr Thr Arg His Asp Tyr 1 5 <210> 146 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 146 Gly Gly Gly Pro Leu Gly Ser Arg Trp Leu Arg Gly Arg His 1 5 10 <210> 147 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 147 Arg Arg Thr Tyr Leu Pro Arg Arg Phe Gly Ser 1 5 10 <210> 148 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 148 Ala Pro Gly Ala Ala Arg Asn Tyr 1 5 <210> 149 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 149 Ala Pro Gly Ala Ala Arg Asn Val 1 5 <210> 150 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 150 Gly Gly Ser Leu Ser Arg Ser Ser 1 5 <210> 151 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 151 Val Arg Gly Trp Leu Asp Glu Arg Pro Gly Pro Gly Pro Ile Val Tyr 1 5 10 15 <210> 152 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 152 Asn Arg Gly Ser Tyr Glu Tyr 1 5 <210> 153 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 153 Ile Ala Asp Trp Arg Gly Tyr 1 5 <210> 154 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 154 Arg Arg Arg Gly Trp Gly Arg Thr Leu Glu Tyr 1 5 10 <210> 155 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 155 Ser Asp Phe Arg Arg Gly Thr Gln Tyr 1 5 <210> 156 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 156 Gln Arg Trp Gly Arg Gly Pro Gly Thr Thr 1 5 10 <210> 157 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 157 Asp Asp Ser Gly Ile Ala Arg Asp Tyr 1 5 <210> 158 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 158 Tyr Arg Arg Phe Gly Ile Asn Lys Asn Tyr 1 5 10 <210> 159 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 159 Leu Arg Thr Tyr Tyr Leu Asn Asp Pro Val Val Phe Ser 1 5 10 <210> 160 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 160 Tyr Ile Pro Leu Arg Gly Thr Leu His Asp Tyr 1 5 10 <210> 161 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 161 Arg Lys Trp Gly Arg Asn Tyr 1 5 <210> 162 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 162 Lys Arg Arg Glu Trp Ala Lys Asp Phe Glu Tyr 1 5 10 <210> 163 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 163 Ile Ala Asp Trp Arg Gly Tyr 1 5 <210> 164 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 164 Tyr Ile Pro Tyr Gly Gly Thr Leu His Asp Phe 1 5 10 <210> 165 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 165 Ile Ala Asp Trp Arg Gly Tyr 1 5 <210> 166 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 166 Asp Thr Ile Gly Thr Ala Arg Asp Tyr 1 5 <210> 167 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 167 Arg Thr Thr Ser Tyr Pro Val Asp Phe 1 5 <210> 168 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 168 Tyr Ile Pro Tyr Gly Gly Thr Leu His Asp Phe 1 5 10 <210> 169 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 169 Ala Ser Tyr 1 <210> 170 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 170 Ala Ser Tyr 1 <210> 171 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 171 Tyr Ile Pro Tyr Gly Gly Thr Leu His Asp Phe 1 5 10 <210> 172 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 172 Tyr Ile Pro Tyr Gly Gly Thr Leu His Asp Phe 1 5 10 <210> 173 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 173 Tyr Ile Pro Tyr Gly Gly Thr Leu His Asp Phe 1 5 10 <210> 174 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 174 Asp Thr Ile Gly Thr Ala Arg Asp Tyr 1 5 <210> 175 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 175 Asp Thr Ile Gly Thr Ala Arg Asp Tyr 1 5 <210> 176 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 176 Asp Thr Ile Gly Thr Ala Arg Asp Tyr 1 5 <210> 177 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 177 Arg Thr Thr Ser Tyr Pro Val Asp Phe 1 5 <210> 178 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 178 Arg Thr Thr Ser Tyr Pro Val Asp Phe 1 5 <210> 179 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 179 Arg Thr Thr Ser Tyr Pro Val Asp Phe 1 5 <210> 180 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 180 Gln 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 20 25 <210> 181 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 181 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 182 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 182 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Phe Ser 20 25 <210> 183 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 183 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser 20 25 <210> 184 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 184 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 185 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 185 Gln Val Arg Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 186 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 186 Gln Val Arg Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 187 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 187 Gln Val Gln Pro Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser 20 25 <210> 188 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 188 Gln Val Gln Pro Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser 20 25 <210> 189 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 189 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 190 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 190 Gln Val Gln Ile Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser 20 25 <210> 191 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 191 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 192 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 192 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 193 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 193 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 194 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 194 Gln Val Gln Leu Gly Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 195 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 195 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Pro Ser Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 196 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 196 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 197 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 197 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu 1 5 10 15 Ser Arg Arg Leu Ser Cys Ala Val Ser 20 25 <210> 198 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 198 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 199 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 199 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 200 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 200 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser 20 25 <210> 201 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 201 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Arg Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser 20 25 <210> 202 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 202 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 203 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 203 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 204 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 204 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Thr Ser 20 25 <210> 205 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 205 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser 20 25 <210> 206 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 206 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser 20 25 <210> 207 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 207 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 208 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 208 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 20 25 <210> 209 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 209 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 20 25 <210> 210 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 210 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 211 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 211 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 20 25 <210> 212 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 212 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 20 25 <210> 213 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 213 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 214 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 214 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 215 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 215 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 216 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 216 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser 20 25 <210> 217 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 217 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 20 25 <210> 218 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 218 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 20 25 <210> 219 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 219 Trp Tyr Arg Gln Ala Gly Asn Asn Arg Ala Leu Val Ala 1 5 10 <210> 220 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 220 Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 <210> 221 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 221 Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Thr Val Val Ala 1 5 10 <210> 222 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 222 Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 1 5 10 <210> 223 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 223 Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 <210> 224 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 224 Trp Tyr Arg Gln Asp Pro Ser Lys Gln Arg Glu Trp Val Ala 1 5 10 <210> 225 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 225 Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 <210> 226 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 226 Trp Tyr Arg Gln Ala Ser Gly Lys Glu Arg Glu Ser Val Ala 1 5 10 <210> 227 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 227 Trp Tyr Arg Gln Ala Ser Gly Lys Glu Arg Glu Ser Val Ala 1 5 10 <210> 228 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 228 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 <210> 229 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 229 Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val Ala 1 5 10 <210> 230 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 230 Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Trp Val Ala 1 5 10 <210> 231 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 231 Trp Tyr Arg Gln Ala Gln Gly Lys Gln Arg Glu Pro Val Ala 1 5 10 <210> 232 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 232 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Gln Phe Val Ala 1 5 10 <210> 233 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 233 Trp His Arg Gln Ala Pro Gly Lys Gln Arg Glu Pro Val Ala 1 5 10 <210> 234 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 234 Trp Tyr Arg Arg Ala Pro Gly Gln Val Arg Glu Met Val Ala 1 5 10 <210> 235 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 235 Trp Tyr Arg Gln Ala Pro Gly Thr Glu Arg Asp Leu Val Ala 1 5 10 <210> 236 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 236 Trp Tyr Arg Gln Ala Pro Gly Ala Gln Arg Glu Leu Leu Ala 1 5 10 <210> 237 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 237 Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 <210> 238 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 238 Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 1 5 10 <210> 239 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 239 Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 <210> 240 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 240 Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu Leu Val Ala 1 5 10 <210> 241 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 241 Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Pro Val Ala 1 5 10 <210> 242 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 242 Trp Phe Arg Gln Ala Pro Gly Glu Glu Arg Glu Leu Val Ala 1 5 10 <210> 243 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 243 Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu Pro Val Ala 1 5 10 <210> 244 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 244 Trp Tyr Arg Gln Ala Pro Gly Thr Glu Arg Asp Leu Val Ala 1 5 10 <210> 245 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 245 Trp His Arg Gln Ala Pro Gly Asn Glu Arg Glu Leu Val Ala 1 5 10 <210> 246 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 246 Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 1 5 10 <210> 247 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 247 Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 <210> 248 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 248 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 <210> 249 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 249 Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 1 5 10 <210> 250 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 250 Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 1 5 10 <210> 251 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 251 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 <210> 252 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 252 Trp Tyr Arg Gln Ala Pro Gly Thr Glu Arg Asp Leu Val Ala 1 5 10 <210> 253 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 253 Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 1 5 10 <210> 254 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 254 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 <210> 255 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 255 Trp His Arg Gln Ala Pro Gly Asn Glu Arg Glu Leu Val Ala 1 5 10 <210> 256 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 256 Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 1 5 10 <210> 257 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 257 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 <210> 258 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 258 Ile Ser Trp Asp Ile Ala Glu Asn Thr Val Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Asn Ser Glu Asp Thr Thr Val Tyr Tyr Cys Asn Ser 20 25 <210> 259 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 259 Ile Ser Gly Asp Asn Val Arg Asn Met Val Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ser Ala 20 25 <210> 260 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 260 Ile Ser Gly Glu Asn Gly Lys Asn Thr Val Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys Leu Gly 20 25 <210> 261 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 261 Ile Ser Arg Asp Asn Ala Asn Asn Ala Ile Tyr Leu Glu Met Asn Ser 1 5 10 15 Leu Lys Pro Glu Asp Thr Ala Val Tyr Val Cys Asn Ala 20 25 <210> 262 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 262 Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Ser Leu Gln Met Asn Thr 1 5 10 15 Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Asn Ala 20 25 <210> 263 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 263 Ile Ser Lys Asp Ser Thr Arg Asn Thr Val Tyr Leu Gln Met Asn Met 1 5 10 15 Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 20 25 <210> 264 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 264 Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 20 25 <210> 265 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 265 Ile Ser Arg Asp Ile Asp Lys Lys Thr Val Tyr Leu Gln Met Asp Asn 1 5 10 15 Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Asn Ser 20 25 <210> 266 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 266 Ile Ser Arg Asp Ile Tyr Lys Lys Thr Val Tyr Leu Gln Met Asp Asn 1 5 10 15 Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Asn Ser 20 25 <210> 267 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 267 Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile 20 25 <210> 268 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 268 Ile Ser Lys Glu Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His Ile 20 25 <210> 269 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 269 Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Lys Ala 20 25 <210> 270 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 270 Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Lys Pro Glu Asp Thr Ala Ile Tyr Thr Cys His Val 20 25 <210> 271 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 271 Ile Ser Arg Asp Asn Ala Ala Asn Thr Phe Tyr Leu Gln Met Asn Asn 1 5 10 15 Leu Arg Pro Asp Asp Thr Ala Val Tyr Tyr Cys Asn Val 20 25 <210> 272 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 272 Val Ser Arg Asp Ile Val Lys Asn Thr Met Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ser Tyr 20 25 <210> 273 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 273 Ile Ser Arg Asp Asn Thr Gln Asn Leu Val Tyr Leu Gln Met Asn Asn 1 5 10 15 Leu Gln Pro His Asp Thr Ala Ile Tyr Tyr Cys Gly Ala 20 25 <210> 274 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 274 Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Asn 1 5 10 15 Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 20 25 <210> 275 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 275 Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Arg 1 5 10 15 Leu Thr Pro Glu Asp Thr Asp Val Tyr Tyr Cys Arg Phe 20 25 <210> 276 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 276 Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Glu Ser 1 5 10 15 Leu Val Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 20 25 <210> 277 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 277 Val Ser Arg Asp Ser Ala Lys Asn Ile Val Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Thr 20 25 <210> 278 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 278 Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 20 25 <210> 279 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 279 Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Thr 20 25 <210> 280 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 280 Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Lys Pro Glu Asp Thr Ala Ile Tyr Thr Cys His Val 20 25 <210> 281 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 281 Val Ser Arg Asp Ser Ala Lys Asn Ile Val Tyr Leu Gln Met Asn Arg 1 5 10 15 Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Thr 20 25 <210> 282 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 282 Ile Ser Arg Asp Asn Ala Arg Asn Thr Val Tyr Leu Gln Met Asp Ser 1 5 10 15 Leu Lys Pro Glu Asp Thr Ala Ile Tyr Thr Cys His Val 20 25 <210> 283 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 283 Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Asn 1 5 10 15 Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 20 25 <210> 284 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 284 Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ser Val 20 25 <210> 285 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 285 Val Ser Arg Asp Ser Ala Lys Asn Ile Val Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Thr 20 25 <210> 286 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 286 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Val 20 25 <210> 287 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 287 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Val 20 25 <210> 288 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 288 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Thr 20 25 <210> 289 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 289 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Thr 20 25 <210> 290 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 290 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Thr 20 25 <210> 291 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 291 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 20 25 <210> 292 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 292 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 20 25 <210> 293 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 293 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 20 25 <210> 294 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 294 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Val 20 25 <210> 295 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 295 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Val 20 25 <210> 296 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 296 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 1 5 10 15 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Val 20 25 <210> 297 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 297 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 298 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 298 Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 299 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 299 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 300 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 300 Phe Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 301 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 301 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 302 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 302 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 303 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 303 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 304 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 304 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 305 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 305 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 306 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 306 Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 <210> 307 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 307 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 308 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 308 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 309 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 309 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 310 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 310 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 311 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 311 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 312 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 312 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 313 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 313 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 314 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 314 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 315 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 315 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 316 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 316 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 317 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 317 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 318 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 318 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 319 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 319 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 320 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 320 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 321 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 321 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 322 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 322 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 323 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 323 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 324 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 324 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 325 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 325 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 326 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 326 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 327 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 327 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 328 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 328 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 329 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 329 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 330 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 330 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 1 5 10 <210> 331 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 331 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 1 5 10 <210> 332 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 332 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 1 5 10 <210> 333 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 333 Trp Gly Gln Gly Thr Leu Val Thr Val Ser 1 5 10 <210> 334 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 334 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 335 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis peptides <400> 335 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 336 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesis 6xHis tag <400> 336 His His His His His His 1 5

Claims

1. A single-domain mesothelin-binding protein, wherein the protein comprises the formula: f1-r1-f2-r2-f3-r3-f4 in, r1 is CDR1; r2 is CDR2; and r3 is CDR3; and wherein f1, f2, f3, and f4 are framework residues; and wherein: (i) the CDR1 consists of the sequence GSTSSINTMY, the CDR2 consists of the sequence FISSGGSTNVRDSVKGRFS, and the CDR3 consists of the sequence YIPLRGTLHDY; (ii) the CDR1 consists of the sequence GSTSSINTMY, the CDR2 consists of the sequence TINRGGSTNVRDSVKGRFS, and the CDR3 consists of the sequence YIPYGGTLHDF; (iii) the CDR1 consists of the sequence TTFSINSMS, the CDR2 consists of the sequence VITNRGTTSYADSVKGRFT, and the CDR3 consists of the sequence IADWRGY; (iv) the CDR1 consists of the sequence AIGSINSMS, the CDR2 consists of the sequence VITDRGSTSYADSVKGRFT, and the CDR3 consists of the sequence IADWRGY; (v) the CDR1 consists of the sequence GLTFGVYGME, the CDR2 consists of the sequence SHTSTGYVYYRDSVKGRFT, and the CDR3 consists of the sequence NRGSYEY; (vi) the CDR1 consists of the sequence GSTSNINMR, the CDR2 consists of the sequence VITRGGYAIYLDAVKGRFT, and the CDR3 consists of the sequence DRVEGTSGGPQLRDY; or (vii) the CDR1 consists of the sequence GTTYTFDLMS, the CDR2 consists of the sequence SISSDGRTSYADSVRGRFT, and the CDR3 consists of the sequence QRSGVRAF.

2. The single-domain mesothelin binding protein of claim 1, wherein the protein comprises a sequence selected from the group consisting of SEQ ID Nos: 3, 4, 12, 20, 23, 24, and 25.

3. The single-domain mesothelin binding protein of claim 2, wherein the EC 50 No more than 2.4×10 -12 M. 4 . A trispecific protein targeting MSLN, comprising the single-domain mesothelin-binding protein of claim 1 , a CD3-binding domain, and a human serum albumin (HSA)-binding domain.

5. Use of the single-domain mesothelin-binding protein of any one of claims 1 to 3 and the trispecific protein targeting MSLN of claim 4 in the preparation of a medicament for treating or alleviating a proliferative disease or a tumorous disease expressing mesothelin in an individual in need thereof, wherein the proliferative disease or the tumorous disease is ovarian cancer, pancreatic cancer, non-small cell lung cancer (NSCLC) or mesothelioma.

6. The use of claim 5, wherein the single-domain mesothelin binding protein is administered at a dose of up to 10 mg / kg.

7. The use of claim 6, wherein the single-domain mesothelin-binding protein is administered once a week, twice a week, once every other day, or once every three weeks.

8. The use according to claim 7, wherein the individual is a human.

9. The use of claim 5, wherein the single-domain mesothelin binding protein is administered in combination with another therapeutic agent.

10. The use of claim 5, wherein the single-domain mesothelin-binding protein selectively binds to tumor cells expressing mesothelin.

11. The use according to claim 10, wherein the single-domain mesothelin-binding protein mediates T cells killing mesothelin-expressing tumor cells.

12. The use according to claim 5, wherein the proliferative disease or tumor disease is ovarian cancer.

Images