Antigen binding polypeptides binding to CD47 and uses thereof

Abstract

Provided are an antigen-binding polypeptide or an antigen-binding portion thereof that binds to CD47, as well as a nucleic acid, a vector, a cell, and a pharmaceutical composition encoding the polypeptide. The polypeptide can reduce tumors or inhibit tumor cell growth, treat cancer, promote macrophage phagocytosis, etc.

Description

Technical Field

[0001] The present invention relates to antigen-binding polypeptides, in particular, to antigen-binding polypeptides and antigen-binding portions thereof that bind CD47, and to methods of using such antigen-binding polypeptides and antigen-binding portions thereof. Background Art

[0002] CD47 is a transmembrane glycoprotein widely expressed in multiple species and tissues, also known as integrin-associated protein, and a member of the immunoglobulin superfamily. CD47 is approximately 50 kD and contains an extracellular Ig-like variable domain, five highly hydrophobic transmembrane segments, and a short alternatively spliced ​​carboxyl-terminal cytoplasmic tail.

[0003] Inhibitory receptor signal regulatory protein α (SIRPα) is one of the ligands of CD47. CD47 binds to the NH 2 Terminal IgV-like domain. SIRPα is mainly expressed in cells of myeloid origin, including macrophages, granulocytes, DC cells, mast cells and their precursors, including hematopoietic stem cells. For example, CD47 can inhibit the phagocytic function of macrophages by binding to the protein SIRPα on the surface of macrophages. Studies have shown that blocking CD47-mediated binding to SIRPα on phagocytes, or lack of expression in CD47 knockout mice, can cause the removal of living cells and non-aging red blood cells. For those cells in which pre-phagocytic signals are also present, blocking SIRPα also allows the engulfment of targets that are not normally engulfed.

[0004] Programmed cell death (PCD) and phagocytic removal are common ways that organisms respond to remove damaged, precancerous or infected cells. Therefore, cells that survive this organism's response (cancerous cells, chronically infected cells, etc.) have ways to escape PCD and phagocytic removal. CD47 is constitutively upregulated in various types of diseased cells, cancer cells, and infected cells, allowing these cells to escape phagocytosis. Anti-CD47 compounds that block the interaction between CD47 on one cell (cancerous cells, chronically infected cells, etc.) and SIRPα on another cell (phagocyte) can counteract the increase in CD47 expression and promote phagocytosis of cancer cells and / or infected cells.

[0005] CD47 expression and / or activity has been implicated in many diseases and conditions. Different studies have shown that almost all tumor cells and tumor tissues highly express CD47. CD47 highly expressed on the surface of tumor cells binds to SIRPα on the surface of macrophages, releasing a "don't eat me" signal, which causes macrophages in the infiltrating area of ​​tumor tissue to not only live in harmony with tumor cells, but also promote the proliferation of blood vessels in the tumor, inhibit the function of effector T cells, and promote the proliferation and growth of tumor cells. Therefore, targeted CD47 therapy is needed. Summary of the invention

[0006] The present invention provides a novel separated antigen-binding polypeptide binding to CD47 and an antigen-binding portion thereof, providing a solution for CD47-targeted therapy.

[0007] In one aspect, the invention provides an isolated antigen-binding polypeptide, or antigen-binding portion thereof, that binds CD47, wherein the antigen-binding polypeptide comprises the following complementarity determining regions:

[0008] a heavy chain CDR1 comprising an amino acid sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4 or 5;

[0009] a heavy chain CDR2 comprising an amino acid sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NO: 6, 7, 8, 9 or 10;

[0010] a heavy chain CDR3 comprising an amino acid sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14 or 15;

[0011] a light chain CDR1 comprising an amino acid sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 19 or 20;

[0012] a light chain CDR2 comprising an amino acid sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NO: 21, 22, 23, 24 or 25; and

[0013] A light chain CDR3 comprising an amino acid sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NO: 26, 27, 28, 30 or 32.

[0014] Among them, the sequence shown in SEQ ID NO: 32 is QQFSX 2 STWT,X 2 It is D or E.

[0015] In another aspect, the invention provides an isolated antigen-binding polypeptide or antigen-binding portion thereof that binds CD47, wherein the antigen-binding polypeptide comprises the following complementarity determining regions:

[0016] a heavy chain CDR1 comprising an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4 or 5, or a conservative modification thereof;

[0017] a heavy chain CDR2 comprising an amino acid sequence selected from SEQ ID NO: 6, 7, 8, 9 or 10 or a conservative modification thereof;

[0018] a heavy chain CDR3 comprising an amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14 or 15 or a conservative modification thereof;

[0019] a light chain CDR1 comprising an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 19 or 20 or a conservative modification thereof;

[0020] A light chain CDR2 comprising an amino acid sequence selected from SEQ ID NO: 21, 22, 23, 24 or 25 or a conservative modification thereof; and

[0021] A light chain CDR3 comprising an amino acid sequence selected from SEQ ID NO: 26, 27, 28, 30 or 32 or a conservative modification thereof.

[0022] In another aspect, the present invention provides an isolated antigen-binding polypeptide or antigen-binding portion thereof that binds to CD47, wherein the antigen-binding polypeptide comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NO: 33, 35, 37, 39, 41, 83, 85 or 87, and the light chain variable region comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NO: 34, 36, 38, 40, 42, 84, 86 or 88.

[0023] Among them, the sequence shown in SEQ ID NO: 83 is:

[0024] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGEFIPGSDTTNYAQKFQGRVTITAX 3 X 4 STX 5 TAYMELSSLRSEDTAVYYCARGGLRRMDYWGQGTLVTVSS, where, X 3 For D or E, X4 I or E, X 5 is S or N;

[0025] The sequence shown in SEQ ID NO: 84 is:

[0026] DIQMTQSPSSSLSASVGDRVTITCRASSSVSSTYLHWYQQKPGKAPKLX 6 IYTTSTLASGVPSRFSGSGSGTX 7 X 8 TLTISSLQPEDFATYYCQQFSX 2 STWTFGQGTKLEIK, where X 6 L or W, X 7 D or S, X 8 F or Y, X 2 is D or E;

[0027] The sequence shown in SEQ ID NO: 85 is:

[0028] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLX 9 WMGWINTNTGEPTYAQX 10 LQGRVTMTX 11 DTSTX 12 TAYMELRSLRSDDTAVYYCX 13 RFSHLRGPMDYWGQGTLVTVSS, where X 9 For E or K, X 10 K or E, X 11 L or T, X 12 R or S, X 13 A or T;

[0029] The sequence shown in SEQ ID NO: 86 is:

[0030] DX 14 QMTQSPSSSLSASVGDRVTITCRSSQSLVHSNGYTYLHWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSQSTHVPPTFGQGTKLEIK, where, X 14 is I or A;

[0031] The sequence shown in SEQ ID NO: 87 is:

[0032] QVQLVQSGAEVKKPGASVKVSCKX 15 SGFNIEDDYIEWVRQAPGQGLEWMGRIDPANDKTKYAQKFQGRVTMTX 16 DTSTX 17 TVYMELSSLRSEDTAVYYCX 18 RPGLRRYYSMDYWGQGTLVTVSS, where X 15 A or V, X 16 R or G, X 17 S or N, X 18 A or T;

[0033] The sequence shown in SEQ ID NO: 88 is:

[0034] DIQMTQSPSSSLSASVGDRVTITCKASENVVSYVSWYQQKPGKAPKLLIYGASNRYTGVPSRFX 19 GSGSX 20 TDFTLTISSLQPEDFATYYCGQSYSYPLTFGQGTKLEIK, where, X 19 S or I, X 20 It is S or G.

[0035] In another aspect, the present invention provides an isolated antigen-binding polypeptide or antigen-binding portion thereof that binds CD47, wherein the antigen-binding polypeptide or antigen-binding portion thereof exhibits one or a combination of the following properties:

[0036] (a) With a K of 1.89E-08 or less (e.g., 1.53E-08 or less) D Values ​​bind to CD47;

[0037] (b) blocking the binding of CD47 to SIRPα;

[0038] (c) promoting macrophage-mediated phagocytosis of cells expressing CD47;

[0039] (d) no obvious induction of apoptosis of CD4 + T cells;

[0040] (e) does not cause substantial erythropenia, anemia or hemagglutination of red cells; and

[0041] (f) The antigen-binding polypeptide or the antigen-binding portion thereof has a melting temperature T≥62°C and a polymerization temperature Tagg≥61°C.

[0042] In another aspect, the invention provides an isolated antigen-binding polypeptide, or antigen-binding portion thereof, wherein the antigen-binding polypeptide, or antigen-binding portion thereof, binds to the same epitope on CD47 as any of the exemplary antigen-binding polypeptides, or antigen-binding portions thereof, provided herein.

[0043] In another aspect, the invention provides an isolated antigen-binding polypeptide, or antigen-binding portion thereof, wherein the antigen-binding polypeptide, or antigen-binding portion thereof, competes for binding to CD47 with any of the exemplary antigen-binding polypeptides, or antigen-binding portions thereof, provided herein, wherein the competition is measured by ELISA, flow cytometry, or surface plasmon resonance assay.

[0044] In this context, the antigen binding moiety may be selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab') 2 Fragments include, but are not limited to, Fd fragments, Fv fragments, dAb fragments, isolated complementarity determining regions and Nanobodies.

[0045] In another aspect, the present invention provides an immunoconjugate comprising an antigen-binding polypeptide or antigen-binding portion thereof that binds CD47 as described herein, and a therapeutic agent linked or conjugated to the antigen-binding polypeptide or antigen-binding portion thereof.

[0046] In this context, the antigen binding polypeptide or antigen binding portion thereof may be linked to the therapeutic agent via a linker. The linker may be cleavable or non-cleavable.

[0047] Herein, the therapeutic agent can be selected from cytotoxic drugs, radioisotopes or immunomodulators, such as chemotherapeutic agents, immunosuppressants, immunostimulants, antimetabolites, alkylating agents, antibiotics, anti-angiogenic agents, anti-mitotic agents, toxins, apoptotic agents, etc.

[0048] In another aspect, the present invention provides a composition comprising component A and a pharmaceutically acceptable carrier, wherein component A is an antigen-binding polypeptide or antigen-binding portion thereof that binds to CD47 as described herein, or an immunoconjugate as described herein.

[0049] In another aspect, the present invention provides an isolated hybridoma cell line selected from the group consisting of 2B2, 2H8, 3F10, 16E5 and 14A9.

[0050] In another aspect, the invention provides an isolated nucleic acid encoding an antigen binding polypeptide or antigen binding portion thereof that binds CD47 as described herein.

[0051] In another aspect, the present invention provides a vector comprising the isolated nucleic acid described herein.

[0052] In another aspect, the present invention provides a host cell comprising the vector described herein or having integrated into its genome the isolated nucleic acid described herein. Preferably, the vector is an expression vector.

[0053] In another aspect, the present invention provides a method for preparing an antigen-binding polypeptide that binds to CD47 or an antigen-binding portion thereof, comprising: culturing the host cell described herein under conditions suitable for expressing a nucleic acid encoding the antigen-binding polypeptide that binds to CD47 or an antigen-binding portion thereof described herein, and isolating the expressed antigen-binding polypeptide or antigen-binding portion thereof.

[0054] In yet another aspect, the present invention provides a method for reducing a tumor or inhibiting the growth of tumor cells in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of an antigen-binding polypeptide or antigen-binding portion thereof that binds to CD47 as described herein, an immunoconjugate as described herein, or the composition as described herein.

[0055] In yet another aspect, the present invention provides a method for treating cancer in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of an antigen-binding polypeptide or antigen-binding portion thereof that binds to CD47 as described herein, an immunoconjugate as described herein, or the composition as described herein.

[0056] In yet another aspect, the present invention provides a method for promoting phagocytosis of macrophages in a subject, wherein the method comprises administering to the subject an effective amount of an antigen-binding polypeptide or antigen-binding portion thereof that binds to CD47 as described herein, an immunoconjugate as described herein, or the composition as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is the effect of anti-CD47 chimeric antibody on red blood cell agglutination; among them, the samples in the same row (antibody or control) are of the same type but different concentrations, and the samples in the same column have the same concentration but different types; from left to right, the concentrations of samples in different columns are 10000ng / ml, 3333.33ng / ml, 1111.11ng / ml, 370.37ng / ml, 123.46ng / ml, 41.15ng / ml, 13.72ng / ml, and 4.57ng / ml, respectively; from top to bottom, the samples in different rows are Xi2B2, Xi2H8, Xi3F10, Xi16E5, Xi14A9, Xi16E5-2 (another repeat of Xi16E5), Hu5F9, IgG4, and PBS, respectively.

[0058] Figure 2The effect of some anti-CD47 humanized antibodies on erythrocyte agglutination; the antibodies in the same column are of the same type but different concentrations, and the antibodies in the same row are of the same concentration but different types; from top to bottom, the antibody concentrations in different rows are 10000ng / ml, 3333.33ng / ml, 1111.11ng / ml, 370.37ng / ml, 123.46ng / ml, 41.15ng / ml, 13.72ng / ml, 4 .57ng / ml; from left to right, the antibodies in columns 1-12 are hz3F10-1.1, hz3F10-2.1, hz3F10-3.1, hz3F10-4.1, hz3F10-5.1, hz3F10-6.1, hz3F10-1.2, hz3F10-2.2, hz3F10-3.2, hz3F10-4.2, hz3F10-5.2, and hz3F10-6.2.

[0059] Figure 3 This is the effect of some anti-CD47 humanized antibodies on red blood cell agglutination; among them, the antibodies in the same column are of the same type but different concentrations, and the antibodies in the same row are of the same concentration but different types; from top to bottom, the antibody concentrations in different rows are 10000ng / ml, 3333.33ng / ml, 1111.11ng / ml, 370.37ng / ml, 123.46ng / ml, 41.15ng / ml, 13.72ng / ml, and 4.57ng / ml, respectively; from left to right, the antibodies in columns 1-8 are hz16E5-1.1, hz16E5-3.1, hz16E5-1.2, hz16E5-3.2, hz16E5-1.3, hz16E5-3.3, hz14A9-2.3, and hz14A9-2.4, respectively.

[0060] Figure 4 This is the effect of anti-CD47 antibody on the survival rate of mice.

[0061] Figure 5 Figure 2 shows the changes in mouse body weight over time after a single administration of anti-CD47 antibody.

[0062] Figure 6 The weight change rate of mice at different time points after a single administration of anti-CD47 antibody.

[0063] Figure 7 The figure shows the changes of mouse RBC over time after a single administration of anti-CD47 antibody.

[0064] Figure 8 The change rate of mouse RBC at different time points after a single administration of anti-CD47 antibody.

[0065] Fig. 9The changes of HGB content in mice over time after a single administration of anti-CD47 antibody.

[0066] Fig.10 The change rate of HGB in mice at different time points after a single administration of anti-CD47 antibody.

[0067] Fig.11 The average tumor volume changes over time after administration of anti-CD47 antibody in the MOLM-16 cell mouse model of human acute myeloid leukemia cells. DETAILED DESCRIPTION

[0068] The following will describe exemplary embodiments of the present application, but those skilled in the art will understand that the scope of protection of the present application is not limited thereto. Instead, various modifications, changes or alterations may be made based on the spirit and concept of the present application, and the contents after such modifications, changes or alterations still fall within the scope of the present application.

[0069] the term

[0070] As used herein, the term "antigen-binding polypeptide" refers to polypeptides and proteins having at least one antigen-binding domain, examples of which include, but are not limited to, monoclonal antibodies, multispecific antibodies, or fusion proteins. As used herein, the term "antigen-binding portion" of an antigen-binding polypeptide refers to one or more fragments of an antigen-binding polypeptide that retain the ability to specifically bind to an antigen (e.g., CD47 protein). It has been shown that the antigen-binding function of an antigen-binding polypeptide can be performed by its fragments. Examples of "antigen-binding portions" of antigen-binding polypeptides include: (i) Fab fragments, consisting of V L 、V H , C L and C H1 (ii) "Fab' fragments" differ from Fab fragments in that the heavy chain C H1 A few residues are added to the carboxyl terminus of the domain, including one or more cysteines from the antibody hinge region; (iii) F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iv) an Fd fragment, consisting of V H and C H1 domains; (v) Fv fragments, consisting of the VL and VH domains of a single antibody arm; (vi) dAb fragments (Ward et al. (1989) Nature 341: 544-546), consisting of the V H (vii) a separate complementarity determining region (CDR); and (viii) a nanobody comprising a heavy chain variable region of a single variable domain and two constant domains. In addition, although the two domains of the Fv fragment, VL and V H Encoded by different genes, but they can be joined by synthetic linkers using recombinant methods so that they can be made into a single protein chain, where V L and V H The domains are paired to form monovalent molecules (called single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. USA 85: 5879-5883). Such single-chain antibodies are also intended to be encompassed within the term antigen-binding polypeptide. The antigen-binding portions herein can be obtained using conventional techniques well known to those skilled in the art, and the fragments can be screened for utility in the same manner as intact antigen-binding polypeptides.

[0071] The term "isotype" refers to the class of antibodies encoded by the heavy chain constant region gene. The antigen-binding polypeptides and antigen-binding portions thereof that bind to CD47 of the present invention may be derived from any species, including but not limited to mice, rats, rabbits, non-human primates (such as chimpanzees, cynomolgus monkeys, spider monkeys, macaques), llamas and humans. The antigen-binding polypeptides and antigen-binding portions thereof that bind to CD47 may be chimeric antibodies, humanized antibodies or complete human antibodies. In some embodiments, the antigen-binding polypeptides that bind to CD47 are antibodies produced by hybridoma cell lines derived from mice. Therefore, in some embodiments, the antigen-binding polypeptides that bind to CD47 are murine antibodies. In other embodiments, the antigen-binding polypeptides that bind to CD47 are chimeric antibodies. In some embodiments, the chimeric antibodies are mouse-human chimeric antibodies. In other embodiments, the antigen-binding polypeptides that bind to CD47 are humanized antibodies. In other embodiments, the antigen-binding polypeptides that bind to CD47 are derived from murine antibodies and are humanized.

[0072] The term "chimeric antibody" is an antibody having at least a portion of a heavy chain variable region and at least a portion of a light chain variable region derived from one species; and at least a portion of a constant region derived from another species. For example, in some embodiments, a chimeric antibody may comprise a murine variable region and a human constant region.

[0073] "Humanized antibodies" are antibodies that contain complementary determining regions (CDRs) derived from non-human antibodies; and framework regions and constant regions derived from human antibodies. For example, the humanized antibodies provided herein that bind to CD47 may comprise CDRs derived from one or more murine antibodies and human framework regions and constant regions. Thus, in some embodiments, the humanized antibodies provided herein bind to the same epitope on CD47 as the murine antibodies from which the CDRs of the antibodies are derived. Exemplary humanized antibodies are provided herein. Additional humanized antibodies or variants thereof that bind to CD47 comprising the heavy chain CDRs and light chain CDRs provided herein can be produced using any human framework sequence and are also included in the present invention. In some embodiments, framework sequences suitable for use in the present invention include those that are structurally similar to the framework sequences provided herein. Additional modifications may be made in the framework region to improve the properties of the antibodies provided herein. Such additional framework modifications may include chemical modifications, point mutations to reduce immunogenicity or remove T cell epitopes, or revert mutations to residues in the original germline sequence. In some embodiments, such modifications include those corresponding to the mutations exemplified herein, including the back mutations to germline sequences. For example, in some embodiments, one or more amino acids in the human framework regions of the VH and / or VL of the humanized antibodies provided herein are back mutated to the corresponding amino acids in the parent murine antibody. For example, for the VH and VL of humanized 3F10, 14A9 and 16E5, several sites of the framework amino acids of the above-mentioned template human antibodies are back mutated to the corresponding amino acid sequences in mouse 3F10, 14A9 and 16E5 antibodies. In some embodiments, the amino acids at positions 2 and / or 33 and / or 48 and / or 63 and / or 68 and / or 71 and / or 72 and / or 94 of the light chain variable region are back mutated to the corresponding amino acids found at the positions in mouse 3F10, 14A9 or 16E5 light chain variable regions. In other embodiments, the amino acids at positions 24 and / or 46 and / or 63 and / or 72 and / or 73 and / or 74 and / or 77 and / or 97 of the heavy chain variable region are backmutated to the corresponding amino acids found at said positions in the mouse 3F10, 14A9 or 16E5 heavy chain variable region.In some embodiments, the humanized 3F10 antibody comprises a heavy chain variable region, whose mutation type is selected from one or more of the following sites: the amino acid at position 73 mutates from Asp (D) to Glu (E); the amino acid at position 74 mutates from Glu (E) to Ile (I); and the amino acid at position 77 mutates from Ser (S) to Asn (N), and for the humanized 3F10 antibody comprises a light chain variable region, whose mutation type is selected from one or more of the following sites: the amino acid at position 48 mutates from Leu (L) to Trp (W); the amino acid at position 71 mutates from Asp (D) to Ser (S); the amino acid at position 72 mutates from Phe (F) to Tyr (Y); and the amino acid at position 94 mutates from Asp (D) to Glu (E). In some embodiments, the humanized 14A9 antibody comprises a heavy chain variable region, whose mutation type is selected from one or more of the following sites: the amino acid at position 24 mutates from Ala (A) to Val (V); the amino acid at position 72 mutates from Arg (R) to Gly (G); the amino acid at position 77 mutates from Ser (S) to Asn (N); and the amino acid at position 97 mutates from Ala (A) to Thr (T), and the humanized 14A9 comprises a light chain variable region, whose mutation type is selected from one or more of the following sites: the amino acid at position 63 mutates from Ser (S) to Ile (I); the amino acid at position 68 mutates from Gly (G) to Ser (S). In some embodiments, the humanized 16E5 antibody comprises a heavy chain variable region, whose mutation type is selected from one or more of the following sites: the amino acid at position 46 mutates from Glu (E) to Lys (K); the amino acid at position 63 mutates from Lys (K) to Glu (E); the amino acid at position 72 mutates from Thr (T) to Leu (L); the amino acid at position 77 mutates from Ser (S) to Arg (R); and the amino acid at position 97 mutates from Ala (A) to Thr (T), and the humanized 16E5 antibody comprises a light chain variable region, whose mutation type is selected from one or more of the following sites: the amino acid at position 2 mutates from Ile (I) to Ala (A); and the amino acid at position 33 mutates from Asn (N) to Gln (Q). Unless otherwise specified, the description of the mutation site number herein is counted sequentially starting from the first amino acid in the variable region. Additional or alternative back mutations can be performed in the framework region of the humanized antibodies provided herein to improve the properties of the antibodies. The invention also includes humanized antibodies that bind CD47 and contain framework modifications corresponding to the exemplary modifications described herein relative to any suitable framework sequence, as well as other framework modifications that otherwise improve antibody properties.

[0074] As used herein, the term "derived" when used in reference to a molecule or polypeptide relative to a reference antibody or other binding protein, means a molecule or polypeptide that is capable of specifically binding to the same epitope as the reference antibody or other binding protein.

[0075] The term "isolated" refers to a compound of interest (eg, an antibody or nucleic acid) that has been separated from its natural environment.

[0076] As used herein, the term "EC50" refers to the effective concentration, 50% of the maximum response of an antibody. As used herein, the term "IC50" refers to the inhibitory concentration, 50% of the maximum response of an antibody. Both EC50 and IC50 can be measured by ELISA or FACS analysis or any other method known in the art.

[0077] The term "K D "" as used herein refers to the dissociation constant, expressed as a molar concentration (M). The K D The value can be determined using methods known in the art. A preferred method for determining antibody K D A preferred method is to use surface plasmon resonance, more preferably a biosensor system such as a Biacore system.

[0078] The term "treatment" refers to measures used to cure, heal, alleviate, relieve, alter, remedy, improve, modify or affect a condition (e.g., a disease), symptoms of a condition, or prevent or delay the onset of symptoms, complications, biochemical markers, or otherwise arrest or inhibit the further development of a disease, condition or disorder in a statistically significant manner.

[0079] As used herein, the term "therapeutically effective amount" refers to the amount of a compound or composition necessary to provide a therapeutic and / or prophylactic benefit to a subject.

[0080] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Preferably, the subject according to the present invention is a human. Unless otherwise indicated, the terms "patient" or "subject" can be used interchangeably.

[0081] As used herein, "about" means within the acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation as practiced in the art. Alternatively, "about" may mean a range of up to ±5%, such as fluctuations within ±2%, within ±1%, or within ±0.5% of a given specific numerical range. When a specific value is given in the application or claims, unless otherwise indicated, the meaning of "about" should be considered to be within the acceptable error range for that specific value. In this document, unless otherwise indicated, the values ​​of step parameters or conditions are modified by "about" by default.

[0082] The term "identity" is also called consistency. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions × 100), wherein the number of gaps and the length of each gap that need to be introduced to produce the optimal alignment of the two sequences need to be considered. As shown in the following non-limiting examples, a mathematical algorithm can be used to complete the comparison of sequences and the determination of the percentage identity between two sequences. The percentage identity between two amino acid sequences can be determined using the algorithm of E.Meyers and W.Miller (Comput.Appl.Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), which uses the PAM120 residue weight table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity of two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 484-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6 or 4, and a length weight of 1, 2, 3, 4, 5 or 6.

[0083] The term "Xn" is equivalent to "Xaa" and refers to an unspecified amino acid, and its scope is specified by the subsequent definition in the relevant expression.

[0084] The terms “comprises,” “comprising,” or “including” and variations thereof should be understood as “including but not limited to,” meaning that in addition to the listed elements, components, and steps, other unspecified elements, components, and steps may also be included.

[0085] Herein, singular terms include plural referents and vice versa unless the context clearly dictates otherwise.

[0086] For the purpose of description and disclosure, all patents, patent applications and other confirmed publications are expressly incorporated herein by reference. These publications are provided only because they are disclosed earlier than the application date of the present application. All statements about the dates of these documents or the statements of the contents of these documents are based on the information available to the applicant and do not constitute any recognition of the correctness of the dates of these documents or the contents of these documents. Moreover, in any country, any reference to these publications in this article does not constitute an approval of the part of the common knowledge that becomes this area about these publications. Various aspects of the present invention will be described in further detail in the following parts.

[0087] Antigen binding polypeptides and antigen binding portions thereof that bind to CD47

[0088] Herein, the terms "CD47", "integrin-associated protein", "IAP (Integrin-associated protein)", "OA3", "MER6" are used interchangeably. The terms "human CD47" and "hCD47" are used interchangeably herein and refer to human CD47 and variants or isoforms of human CD47. Herein, "antibodies that bind to CD47" and "anti-CD47 antibodies" are used interchangeably.

[0089] The present invention provides antigen-binding polypeptides or antigen-binding portions thereof that bind to CD47, providing a new solution for CD47-targeted therapy. In addition to binding to human CD47 and / or cynomolgus monkey CD47, the antigen-binding polypeptides provided herein also exhibit many other desirable features for targeted therapy. Specifically, these desirable features may be, for example, a K of 1.53E-08 or less. D At least one of the following: binding to CD47, blocking the binding of CD47 to SIRPα, promoting macrophage-mediated phagocytosis of cells expressing CD47, not significantly inducing apoptosis of CD4+T cells, not causing substantial erythropenia, anemia or erythrocyte agglutination, the melting temperature T of the antigen-binding polypeptide or its antigen-binding portion is ≥62°C, and the polymerization temperature Tagg is ≥61°C. In some embodiments, the antigen-binding polypeptide that binds to CD47 exhibits better therapeutic effects; in some embodiments, the antigen-binding polypeptide that binds to CD47 exhibits reduced side effects.

[0090] In one aspect, the invention provides antigen-binding polypeptides and antigen-binding portions thereof that bind to CD47 and comprise specific sequences.

[0091] In some embodiments, provided herein are isolated antigen-binding polypeptides that bind CD47, or antigen-binding portions thereof, comprising one or more CDRs selected from the group consisting of SEQ ID NOs: 1-32.

[0092] In some embodiments, the isolated antigen-binding polypeptide, or antigen-binding portion thereof, that binds CD47 comprises a heavy chain CDR1 sequence comprising an amino acid sequence that is at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, or 5.

[0093] In some embodiments, the isolated antigen-binding polypeptide, or antigen-binding portion thereof, that binds CD47 comprises a heavy chain CDR2 comprising an amino acid sequence that is at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO: 6, 7, 8, 9, or 10.

[0094] In some embodiments, the isolated antigen-binding polypeptide, or antigen-binding portion thereof, that binds CD47 comprises a heavy chain CDR3 comprising an amino acid sequence that is at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14, or 15.

[0095] In some embodiments, the isolated antigen-binding polypeptide, or antigen-binding portion thereof, that binds CD47 comprises a light chain CDR1 comprising an amino acid sequence that is at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 19, or 20.

[0096] In some embodiments, the isolated antigen-binding polypeptide, or antigen-binding portion thereof, that binds CD47 comprises a light chain CDR2 comprising an amino acid sequence that is at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO: 21, 22, 23, 24, or 25.

[0097] In some embodiments, the isolated antigen-binding polypeptide, or antigen-binding portion thereof, that binds CD47 comprises a light chain CDR3 comprising an amino acid sequence that is at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO: 26, 27, 28, 30, or 32.

[0098] Those skilled in the art will appreciate that the heavy chain CDRs and light chain CDRs of the antigen-binding polypeptides provided herein can be independently selected or mixed and matched to form an antigen-binding polypeptide or antigen-binding portion thereof comprising any heavy chain CDR1, CDR2, and CDR3 from the antigen-binding polypeptides provided herein; and any light chain CDR1, CDR2, and CDR3. Therefore, in some embodiments, the present invention provides an isolated antigen-binding polypeptide that binds to CD47, comprising three heavy chain CDRs (heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3) and three light chain CDRs (light chain CDR1, light chain CDR2, and light chain CDR3), wherein:

[0099] a heavy chain CDR1 comprising an amino acid sequence that is at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, or 5;

[0100] a heavy chain CDR2 comprising an amino acid sequence that is at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO: 6, 7, 8, 9, or 10;

[0101] a heavy chain CDR3 comprising an amino acid sequence that is at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14, or 15;

[0102] a light chain CDR1 comprising an amino acid sequence that is at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 19, or 20;

[0103] a light chain CDR2 comprising an amino acid sequence that is at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO: 21, 22, 23, 24, or 25; and

[0104] A light chain CDR3 comprising an amino acid sequence that is at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO: 26, 27, 28, 30, or 32.

[0105] In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 comprises the following complementarity determining regions: a heavy chain CDR1 comprising an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4 or 5; a heavy chain CDR2 comprising an amino acid sequence selected from SEQ ID NO: 6, 7, 8, 9 or 10; a heavy chain CDR3 comprising an amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14 or 15; a light chain CDR1 comprising an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 19 or 20; a light chain CDR2 comprising an amino acid sequence selected from SEQ ID NO: 21, 22, 23, 24 or 25; and a light chain CDR3 comprising an amino acid sequence selected from SEQ ID NO: 26, 27, 28, 30 or 32.

[0106] In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 respectively comprise the residues corresponding to SEQ ID NO: The amino acid sequences shown in NOs: 1, 6 and 11 have an amino acid sequence that is at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical, and the light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the same amino acid sequence as SEQ ID The amino acid sequences shown in NOs: 16, 21 and 26 have amino acid sequences that are at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical. In some embodiments, the isolated antigen-binding polypeptide that binds CD47 comprises the following complementarity determining regions: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 21; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26.

[0107] In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 respectively comprise the residues corresponding to SEQ ID NO: The amino acid sequences shown in NOs: 2, 7 and 12 have an amino acid sequence that is at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical, and the light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the same amino acid sequence as SEQ ID The amino acid sequences shown in NOs: 17, 22 and 27 have amino acid sequences that are at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical. In some embodiments, the isolated antigen-binding polypeptide that binds CD47 comprises the following complementarity determining regions: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 22; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 27.

[0108] In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 respectively comprise the residues corresponding to SEQ ID NO: The amino acid sequences shown in NOs: 3, 8 and 13 have an amino acid sequence that is at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical, and the light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the same amino acid sequence as shown in SEQ ID The amino acid sequences shown in NOs: 18, 23 and 28 have amino acid sequences that are at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical. In some embodiments, the isolated antigen-binding polypeptide that binds CD47 comprises the following complementarity determining regions: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 18; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 23; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 28.

[0109] In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 respectively comprise the residues corresponding to SEQ ID NO: The amino acid sequences shown in NOs: 4, 9 and 14 have an amino acid sequence that is at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical, and the light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the same amino acid sequence as SEQ ID The amino acid sequences shown in NOs: 19, 24 and 32 have amino acid sequences that are at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical. In some embodiments, the isolated antigen-binding polypeptide that binds CD47 comprises the following complementarity determining regions: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 19; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 32.

[0110] In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 respectively comprise the residues corresponding to SEQ ID NO: The amino acid sequences shown in NOs: 5, 10 and 15 have an amino acid sequence that is at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical, and the light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the same amino acid sequence as SEQ ID The amino acid sequences shown in NOs: 20, 25 and 30 have amino acid sequences that are at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical. In some embodiments, the isolated antigen-binding polypeptide that binds CD47 comprises the following complementarity determining regions: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 20; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 30.

[0111] The present invention also provides antigen-binding polypeptides and antigen-binding portions thereof in conservatively modified forms. Those skilled in the art will recognize that conservative amino acid substitutions are substitutions of one amino acid with another amino acid having similar structure or chemical properties (e.g., like similar side chains). Exemplary conservative substitutions are described in the art, for example, in Watson et al., Molecular Biology of the Gene, The Bengamin / Cummings Publication Company, 4th edition (1987).

[0112] In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 comprises: a heavy chain CDR1 comprising an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4 or 5 or a conservative modification thereof; a heavy chain CDR2 comprising an amino acid sequence selected from SEQ ID NO: 6, 7, 8, 9 or 10 or a conservative modification thereof; a heavy chain CDR3 comprising an amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14 or 15 or a conservative modification thereof; a light chain CDR1 comprising an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 19 or 20 or a conservative modification thereof; a light chain CDR2 comprising an amino acid sequence selected from SEQ ID NO: 21, 22, 23, 24 or 25 or a conservative modification thereof; and a light chain CDR3 comprising an amino acid sequence selected from SEQ ID NO: 26, 27, 28, 30 or 32 or a conservative modification thereof. Conservative modifications are present in any one or more of the light chain CDRs or the heavy chain CDRs.

[0113] In some embodiments, the isolated antigen-binding polypeptide that binds CD47 comprises: a heavy chain CDR1 comprising an amino acid sequence selected from SEQ ID NO: 3, 4 or 5; a heavy chain CDR2 comprising an amino acid sequence selected from SEQ ID NO: 8, 9 or 10; a heavy chain CDR3 comprising an amino acid sequence selected from SEQ ID NO: 13, 14 or 15; a light chain CDR1 comprising an amino acid sequence selected from SEQ ID NO: 18, 19 or 20; a light chain CDR2 comprising an amino acid sequence selected from SEQ ID NO: 23, 24 or 25; and a light chain CDR3 comprising an amino acid sequence selected from SEQ ID NO: 28, 30 or 32.

[0114] In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 1 or a conservative modification thereof, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 6 or a conservative modification thereof, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 11 or a conservative modification thereof, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 16 or a conservative modification thereof, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 21 or a conservative modification thereof, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 26 or a conservative modification thereof.

[0115] In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 2 or a conservative modification thereof, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 7 or a conservative modification thereof, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 12 or a conservative modification thereof, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 17 or a conservative modification thereof, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 22 or a conservative modification thereof, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 27 or a conservative modification thereof.

[0116] In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 3 or a conservative modification thereof, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8 or a conservative modification thereof, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 13 or a conservative modification thereof, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 18 or a conservative modification thereof, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 23 or a conservative modification thereof, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 28 or a conservative modification thereof.

[0117] In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein the heavy chain CDR1 comprises an amino acid sequence of SEQ ID NO: 4 or a conservative modification thereof, the heavy chain CDR2 comprises an amino acid sequence of SEQ ID NO: 9 or a conservative modification thereof, the heavy chain CDR3 comprises an amino acid sequence of SEQ ID NO: 14 or a conservative modification thereof, the light chain CDR1 comprises an amino acid sequence of SEQ ID NO: 19 or a conservative modification thereof, the light chain CDR2 comprises an amino acid sequence of SEQ ID NO: 24 or a conservative modification thereof, and the light chain CDR3 comprises an amino acid sequence of SEQ ID NO: 32 or a conservative modification thereof.

[0118] In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein the heavy chain CDR1 comprises an amino acid sequence of SEQ ID NO: 5 or a conservative modification thereof, the heavy chain CDR2 comprises an amino acid sequence of SEQ ID NO: 10 or a conservative modification thereof, the heavy chain CDR3 comprises an amino acid sequence of SEQ ID NO: 15 or a conservative modification thereof, the light chain CDR1 comprises an amino acid sequence of SEQ ID NO: 20 or a conservative modification thereof, the light chain CDR2 comprises an amino acid sequence of SEQ ID NO: 25 or a conservative modification thereof, and the light chain CDR3 comprises an amino acid sequence of SEQ ID NO: 30 or a conservative modification thereof.

[0119] In some embodiments, the isolated antigen-binding polypeptides that bind to CD47 provided herein comprise a heavy chain variable region and a light chain variable region, the variable regions of the heavy chain and the light chain comprising CDR regions and framework regions FR (including FR1, FR2, FR3 and FR4), forming a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0120] Thus, in another aspect, the invention provides an isolated antigen-binding polypeptide or antigen-binding portion thereof that binds CD47, the antigen-binding polypeptide comprising a heavy chain variable region comprising an amino acid sequence that is at least 80%, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 33, 35, 37, 39, 41, 83, 85 or 87, and the light chain variable region comprising an amino acid sequence that is at least 80%, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 33, 35, 37, 39, 41, 83, 85 or 87 The amino acid sequence of NO:34, 36, 38, 40, 42, 84, 86 or 88 has an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical.

[0121] Among them, the sequence shown in SEQ ID NO: 83 is:

[0122] QVQLVQSGAEVKKPGSSVKVSCKAS GYTFSRYWIE WVRQAPGQGLEWMG EFIPGSDT TNYAQKFQGRVTITAX 3 X 4 STX 5 TAYMELSSLRSEDTAVYYCAR GGLRRMDY WGQGTLVTVSS, where X 3 For D or E, X 4 I or E, X 5 is S or N;

[0123] The sequence shown in SEQ ID NO: 84 is:

[0124] DIQMTQSPSSLSASVGDRVTITC RASSSVSSTYLH WYQQKPGKAPKLX 6 IY TTSTLAS GVPSRFSGSGSGTX 7 X 8 TLTISSLQPEDFATYYC QQFSX 2STWT FGQGTKLEIK, where X 6 L or W, X 7 D or S, X 8 F or Y, X 2 is D or E;

[0125] The sequence shown in SEQ ID NO: 85 is:

[0126] QVQLVQSGAEVKKPGASVKVSCKAS GYTFTNYGMN WVRQAPGQGLX 9 WMG WINTNTGEPT YQ 10 LQGRVTMTX 11 DTSTX 12 TAYMELRSLRSDDTAVYYCX 13 R FSHLRGPMDY WGQGTLVTVSS, where X 9 For E or K, X 10 K or E, X 11 L or T, X 12 R or S, X 13 A or T;

[0127] The sequence shown in SEQ ID NO: 86 is:

[0128] DX14 QMTQSPSSLSASVGDRVTITC RSSQSLVHSNGYTYLH WYQQKPGKAPKLLIY KVSNRFS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC SQSTHVPPT FGQGTKLEIK, where X 14 is I or A;

[0129] The sequence shown in SEQ ID NO: 87 is:

[0130] QVQLVQSGAEVKKPGASVKVSCKX 15 S GFNIEDDYIE WVRQAPGQGLEWMG RIDPANDKTK YAQKFQGRVTMTX 16 DTSTX 17 TVYMELSSLRSEDTAVYYCX 18 R PGLRRYYSMDY WGQGTLVTVSS, where X 15 A or V, X 16 R or G, X 17 S or N, X 18 A or T;

[0131] The sequence shown in SEQ ID NO: 88 is:

[0132] DIQMTQSPSSLSASVGDRVTITC KASENVVSYVS WYQQKPGKAPKLLIY GASNRYT GVPSRFX 19 GSGSX 20 TDFTLTISSLQPEDFATYYC GQSYSYPLT FGQGTKLEIK, where X 19 S or I, X 20 It is S or G.

[0133] In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region comprising or consisting of an amino acid sequence selected from SEQ ID NO: 33, 35, 37, 39, 41, 83, 85 or 87, and a light chain variable region comprising or consisting of an amino acid sequence selected from SEQ ID NO: 34, 36, 38, 40, 42, 84, 86 or 88.

[0134] In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 35, 39, 41, 83, 85, or 87, and a light chain variable region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 36, 40, 42, 84, 86, or 88. In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 79, and a light chain variable region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 80. In some embodiments, the isolated antigen-binding polypeptide that binds CD47 comprises a heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 81 and a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 82.

[0135] In some embodiments, the isolated antigen-binding polypeptide that binds CD47 comprises a heavy chain variable region comprising an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 33; and a light chain variable region comprising an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 33. NO:34 has an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical. In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:33 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:34.

[0136] In some embodiments, the isolated antigen-binding polypeptide that binds CD47 comprises a heavy chain variable region comprising an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 35 and a light chain variable region comprising an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 35. NO:36 has an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical. In some embodiments, the isolated antigen-binding polypeptide that binds to anti-CD47 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:35 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:36.

[0137] In some embodiments, the isolated antigen-binding polypeptide that binds CD47 comprises a heavy chain variable region comprising an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 37; and a light chain variable region comprising an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 37. NO:38 has an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical. In some embodiments, the isolated antigen-binding polypeptides that bind to CD47 provided herein comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:37 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:38.

[0138] In some embodiments, the isolated antigen-binding polypeptide that binds CD47 comprises a heavy chain variable region comprising an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 39; and a light chain variable region comprising an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 39. NO:40 has an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical. In some embodiments, the isolated antigen-binding polypeptides that bind to CD47 provided herein comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:39 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:40.

[0139] In some embodiments, the isolated antigen-binding polypeptide that binds CD47 comprises a heavy chain variable region comprising an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO:41; and a light chain variable region comprising an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO:41. NO:42 has an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical. In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 provided herein, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:41 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:42.

[0140] In some embodiments, the isolated antigen-binding polypeptide that binds CD47 comprises a heavy chain variable region comprising an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 83 and a light chain variable region comprising an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 83. NO:84 has an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical. In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:83 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:84.

[0141] In some embodiments, the isolated antigen-binding polypeptide that binds CD47 comprises a heavy chain variable region comprising an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 85 and a light chain variable region comprising an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 85. NO:86 has an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical. In some embodiments, the isolated antigen-binding polypeptide that binds CD47 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:85 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:86.

[0142] In some embodiments, the isolated antigen-binding polypeptide that binds CD47 comprises a heavy chain variable region comprising an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 87; and a light chain variable region comprising an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 87. NO:88 has an amino acid sequence that is at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical. In some embodiments, the isolated antigen-binding polypeptide that binds CD47 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:87 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:88.

[0143] In some embodiments, the heavy chain variable region and light chain variable region described herein contain the sequence features of the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 described herein.

[0144] In some embodiments, the isolated antigen-binding polypeptides that bind to CD47 provided herein comprise heavy chains and light chains, which, in addition to the heavy chain variable regions and light chain variable regions described herein, also comprise constant regions. In some embodiments, the constant regions are humanized. In some embodiments, the framework regions FR of the constant regions and variable regions are both humanized. Immunogenicity is reduced by constructing chimeric antibodies (e.g., humanized constant regions and non-human variable regions) or humanized antibodies (e.g., humanized constant regions and FR regions).

[0145] In some embodiments, the light chain constant region of the antigen binding polypeptide is a human kappa chain constant region. In some embodiments, the light chain constant region of the antigen binding polypeptide is a human lambda chain constant region.

[0146] The heavy chain constant region of the antigen-binding polypeptide can be from any type of constant region, such as IgG, IgM, IgD, IgA, and IgE; and any isotype, such as IgG1, IgG2, IgG3, and IgG4. In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 is of the IgG1 isotype. In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 is of the IgG4 isotype.

[0147] In some embodiments, the antigen binding polypeptide comprises a modified constant region. In some embodiments, the hinge region within the human IgG4 constant region is modified to avoid or reduce chain exchange, for example, an IgG4 antibody has a Ser228Pro (S228P) mutation according to the EU numbering index.

[0148] In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 is a chimeric antibody comprising a heavy chain variable region comprising or consisting of an amino acid sequence selected from SEQ ID NO: 33, 35, 37, 39 or 41, and a light chain variable region comprising or consisting of an amino acid sequence selected from SEQ ID NO: 34, 36, 38, 40 or 42.

[0149] In some embodiments, the isolated antigen-binding polypeptide that binds CD47 is a chimeric antibody comprising a heavy chain having an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 89, 93, 97, 101, or 105, and a light chain having an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 89, 93, 97, 101, or 105. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a chimeric antibody comprising a heavy chain according to SEQ ID NO: 89 and a light chain according to SEQ ID NO: 91. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a chimeric antibody comprising a heavy chain according to SEQ ID NO: 93 and a light chain according to SEQ ID NO: 95. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a chimeric antibody comprising a heavy chain according to SEQ ID NO: 97 and a light chain according to SEQ ID NO: 99. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a chimeric antibody comprising a heavy chain according to SEQ ID NO: 101 and a light chain according to SEQ ID NO: 103. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a chimeric antibody comprising a heavy chain according to SEQ ID NO: 105 and a light chain according to SEQ ID NO: 107.

[0150] In some embodiments, the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody comprising a heavy chain variable region comprising or consisting of an amino acid sequence selected from SEQ ID NO: 83, 85 or 87, and a light chain variable region comprising or consisting of an amino acid sequence selected from SEQ ID NO: 84, 86 or 88.

[0151] In some embodiments, the isolated antigen-binding polypeptide that binds CD47 is a humanized antibody, the heavy chain having an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, or 185, and the light chain having an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence set forth in SEQ ID NO: In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 109 and a light chain according to SEQ ID NO: 111. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 113 and a light chain according to SEQ ID NO: 115. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 117 and a light chain according to SEQ ID NO: 119. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 121 and a light chain according to SEQ ID NO: 123. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 125 and a light chain according to SEQ ID NO: 127. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 129 and a light chain according to SEQ ID NO: 131. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO:133 and a light chain according to SEQ ID NO:135.In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 137 and a light chain according to SEQ ID NO: 139. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 141 and a light chain according to SEQ ID NO: 143. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 145 and a light chain according to SEQ ID NO: 147. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 149 and a light chain according to SEQ ID NO: 151. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 153 and a light chain according to SEQ ID NO: 155. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 157 and a light chain according to SEQ ID NO: 159. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 161 and a light chain according to SEQ ID NO: 163. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 165 and a light chain according to SEQ ID NO: 167. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 169 and a light chain according to SEQ ID NO: 171. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 173 and a light chain according to SEQ ID NO: 175. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 177 and a light chain according to SEQ ID NO: 179. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 181 and a light chain according to SEQ ID NO: 183. In one embodiment, the isolated antigen binding polypeptide that binds CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 185 and a light chain according to SEQ ID NO: 187.

[0152] In one embodiment, the isolated antigen-binding polypeptide that binds CD47 comprises a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:93, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, or 185; and a light chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:95, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, or 187.

[0153] In some embodiments, the isolated antigen-binding polypeptide or / and antigen-binding portion thereof that binds CD47 has a K of 1.53E-08 M or less. D In some embodiments, the antigen binding polypeptide or / and its antigen binding portion binds to human CD47. In other embodiments, the antigen binding polypeptide or / and its antigen binding portion binds to cynomolgus monkey CD47. In other embodiments, the antigen binding polypeptide or / and its antigen binding portion binds to human CD47 and cynomolgus monkey CD47. In some embodiments, the antigen binding polypeptide or / and its antigen binding portion has the following binding affinity (K) for human CD47: D ): in the range of about 1E-12M to about 1E-05M, about 1E-12M to about 1.89E-06M, about 1E-12M to about 1.89E-07M, about 1E-11M to about 1.89E-07M, about 9.46E-10M to about 1.89E-07M, about 9.46E-10M to about 1.89E-08M, about 2.04E-10M to about 1.53E-07M or about 2.04E-10M to about 1.89E-08M. In some embodiments, the antigen binding polypeptide or / and antigen binding portion thereof has the following binding affinity (K) for human CD47: D ): about 1E-05M or less, about 1.89E-06M or less, about 1.89E-07M or less, about 1.89E-08M or less, about 1.53E-08M or less, about 9.31E-08M or less, about 9.31E-09M or less, about 8.55E-09M or less, about 2.35E-09M or less, or about 2.04E-010M or less. In some embodiments, the antigen-binding polypeptide or / and antigen-binding portion thereof has the following binding affinity (K) for human CD47: D): about 7.48E-09M, about 3.50E-10M, about 7.67E-10M, about 9.06E-09M, about 2.35E-09M, about 9.46E-10M, about 8.36E-10M, about 1.53E-09M, about 1.42E-09M, about 9.60E-10M, about 5.92E-10M, about 9.04E-10M, about 7.7 9E-10M, about 1.15E-09M, about 1.34E-09M, about 8.96E-10M, about 7.65E-10M, about 1.89E-08M, about 6.28E-09M, about 9.31E-09M, about 1.71E-08M, about 1.53E-08M, about 7.13E-09M, about 2.04E-10M, or about 8.55E-09M. In some embodiments, the binding affinity k of the antigen-binding polypeptides and antigen-binding portions thereof that bind to CD47 provided herein is measured by Biacore. D .

[0154] In some embodiments, the antigen binding polypeptide or / and antigen binding portion thereof that binds to CD47 has a binding EC50 of about 1 ng / mL to about 2000 ng / mL, about 1 ng / mL to about 1500 ng / mL, about 1 ng / mL to about 1106 ng / mL, about 1 ng / mL to about 699 ng / mL, about 1 ng / mL to about 340 ng / mL, about 30 ng / mL to about 340 ng / mL, about 50 ng / mL to about 340 ng / mL, or about 100 ng / mL to about 340 ng / mL for human CD47. In some embodiments, the antigen binding polypeptide or / and antigen binding portion thereof that binds to CD47 has a binding EC50 of about 2000 ng / mL or less, about 1500 ng / mL or less, about 1106 ng / mL or less, about 699 ng / mL or less, or about 340 ng / mL or less for human CD47. In some embodiments, the EC50 of the antigen-binding polypeptides, and antigen-binding portions thereof, provided herein that bind CD47 is measured by ELISA or FACS.

[0155] In some embodiments, the antigen binding polypeptide that binds to CD47 or / and its antigen binding portion blocks the binding of CD47 to SIRPα. Signal regulatory protein α (SIRPα) is one of the ligands that bind to CD47, and SIRPα is expressed on hematopoietic cells including macrophages and dendritic cells. The interaction of CD47 with SIRPα on macrophages sends a "don't eat me" signal to macrophages. In therapeutic situations, blocking the interaction of SIRPα with CD47 using an antigen binding polypeptide that binds to CD47 or its antigen binding portion can promote the host immune system to take up and eliminate cancer cells. Therefore, blocking the interaction of CD47 with SIRPα, thereby allowing macrophages to phagocytose tumor cells expressing CD47, is an important functional feature of some antigen binding polypeptides that bind to CD47. In some embodiments, based on Jurkat cells and flow cytometry detection, the blocking IC50 of the antigen-binding polypeptide (some examples are chimeric antibodies Xi2B2, Xi2H8, Xi3F10, Xi16E5, Xi14A9) or / and its antigen-binding portion is about 358.5 ng / mL to about 26966 ng / mL, about 358.5 ng / mL to about 4861 ng / mL, about 358.5 ng / mL to about 631.2 ng / mL or about 358.5 ng / mL to about 1283 ng / mL. In some embodiments, the blocking IC50 of the antigen-binding polypeptide or / and its antigen-binding portion is about 26966 ng / mL or less, about 15000 ng / mL or less, about 10000 ng / mL or less, about 8000 ng / mL or less, about 6000 ng / mL or less, about 4861 ng / mL or less, about 3000 ng / mL or less, about 2500 ng / mL or less, about 2000 ng / mL or less, about 1500 ng / mL or less, about 1283 ng / mL or less, about 1000 ng / mL or less, about 800 ng / mL or less, about 500 ng / mL or less, or about 358.5 ng / mL or less.

[0156] In some embodiments, based on ELISA analysis, the blocking IC50 of the antigen-binding polypeptide that binds to CD47 (some examples are chimeric antibodies Xi2B2, Xi2H8, Xi3F10 or humanized antibodies thereof, Xi16E5 or humanized antibodies thereof, Xi14A9 or humanized antibodies thereof, etc.) or / and the antigen-binding portion thereof is about 4.682 ng / mL to about 15246 ng / mL, about 621.1 ng / mL to about 15246 ng / mL, about 621.1 ng / mL to about 7910 ng / mL, about 621.1 ng / mL to about 2939 ng / mL, about 621.1 ng / mL to about 2500 ng / mL, about 621.1 ng / mL to about 15246 ng / mL, about 621.1 ng / mL to about 2939 ng / mL, about 621.1 ng / mL to about 250 ... L to about 2051 ng / mL, about 621.1 ng / mL to about 1738 ng / mL, about 621.1 ng / mL to about 1500 ng / mL, about 621.1 ng / mL to about 1400 ng / mL, about 621.1 ng / mL to about 1300 ng / mL, about 621.1 ng / mL to about 1200 ng / mL, about 621.1 ng / mL to about 1100 ng / mL, about 621.1 ng / mL to about 1000 ng / mL, about 621.1 ng / mL to about 900 ng / mL, about 621.1 ng / mL to about 800 ng / mL, or about 621.1 ng / mL to about 703.2 ng / mL. In some embodiments, the blocking IC50 of the antigen-binding polypeptide or / and antigen-binding portion thereof is about 15246 ng / mL or less, about 7910 ng / mL or less, about 2939 ng / mL or less, about 2500 ng / mL or less, about 2051 ng / mL or less, about 1738 ng / mL or less, about 1500 ng / mL or less, about 1400 ng / mL or less, about 1300 ng / mL or less, based on ELISA analysis. In some embodiments, the present invention relates to an aqueous phase or a mixture of about 1 ng / mL or less, about 2 ng / mL or less, about 40 ng / mL or less, about 30 ng / mL or less, about 20 ng / mL or less, about 10 ng / mL or less, about 15 ng / mL or less, about 20 ng / mL or less, about 10 ng / mL or less, about 25 ng / mL or less, about 30 ng / mL or less, about 40 ng / mL or less, about 30 ng / mL or less, about 20 ng / mL or less, about 10 ng / mL or less, or about 4.682 ng / mL or less.

[0157] In some embodiments, the antigen binding polypeptide or / and its antigen binding portion that binds to CD47 promotes macrophage-mediated phagocytosis of cells expressing CD47. As described above, the binding of SIRPα to CD47 is blocked by binding to the CD47 antigen binding polypeptide, thereby increasing the phagocytosis of macrophages on cells expressing CD47, such as cancer cells (specific examples such as HL60 cells, etc.). In some embodiments, at a concentration of about 1 μg / mL to about 10 μg / mL of the antigen binding polypeptide that binds to CD47, the macrophage phagocytosis rate is about 3.5% to about 65.7%, about 11.5% to about 65.7%, about 28.8% to about 65.7%, or about 44.67% to about 65.7%. In some embodiments, at a concentration of about 1 μg / mL to about 10 μg / mL of the antigen-binding polypeptide, the macrophage phagocytosis rate is about 1.34% to about 81.66%, about 2% to about 81.66%, about 11.95% to about 81.66%, about 24.19% to about 81.66%, or about 71.48% to about 81.66%.

[0158] In some embodiments, the antigen-binding polypeptide or / and its antigen-binding portion that binds to CD47 does not significantly induce apoptosis of CD4+T cells. In some embodiments, apoptosis detection is performed using a flow cytometry assay. Obvious apoptosis herein refers to being within an acceptable range (e.g., having no effect on the subject or within a controllable range) or being statistically insignificantly different (P≤0.05) from a negative control, for example, in some embodiments, being insignificantly different from a negative control IgG.

[0159] In some embodiments, the antigen binding polypeptide or / and antigen binding portion thereof that binds to CD47 has a Tm (melting temperature) of about 62°C or more, about 63°C or more, about 64°C or more, about 65°C or more, about 66°C or more, about 67°C or more, about 68°C or more, about 69°C or more, or about 70°C or more. In some embodiments, the antibody or / and fragment thereof has a Tagg (aggregation temperature) of about 61°C or more, about 62°C or more, about 63°C or more, about 64°C or more, about 65°C or more, about 66°C or more, about 67°C or more, about 68°C or more, about 69°C or more, about 70°C or more, about 71°C or more, about 72°C or more, or about 73°C or more. In some embodiments, the antigen binding polypeptide or / and the antigen binding portion thereof has a Tm of about 62°C to about 75°C, about 63°C to about 70.6°C, about 64°C to about 70.6°C, about 65°C to about 70.6°C, about 67°C to about 70.6°C, or about 68°C to about 70.6°C. In some embodiments, the antibody or / and its fragment has a Tagg of about 60°C to about 73.81°C, about 61°C to about 73.81°C, about 62°C to about 73.81°C, about 63°C to about 73.81°C, about 64°C to about 73.81°C, about 65°C to about 73.81°C, about 67°C to about 73.81°C, or about 68°C to about 73.81°C. In some embodiments, Tm, Tagg are detected by nano DSF.

[0160] In some preferred embodiments, the antigen-binding polypeptides or / and antigen-binding portions thereof that bind to CD47 do not cause substantial erythropenia, anemia or erythrocyte agglutination. The terms "red blood cells" and "erythrocytes" are synonymous and can be used interchangeably. The term "agglutination" refers to cell clumping. Since erythrocytes also express CD47, some antigen-binding polypeptides that bind to CD47 will bind to erythrocytes, thereby causing agglutination. Therefore, avoiding or reducing the damaging effects of antigen-binding polypeptides that bind to CD47 on erythrocytes will hopefully reduce the side effects of treatment.

[0161] Therefore, in another aspect, the present invention provides antigen-binding polypeptides and antigen-binding portions thereof that bind to CD47 and exhibit one or a combination of the following properties:

[0162] (a) With a K of 1.89E-08 (e.g. 1.53E-08) or less D Values ​​bind to CD47;

[0163] (b) blocking the binding of CD47 to SIRPα;

[0164] (c) promoting macrophage-mediated phagocytosis of cells expressing CD47;

[0165] (d) no obvious induction of apoptosis of CD4 + T cells;

[0166] (e) does not cause substantial erythropenia, anemia or hemagglutination of red cells; and

[0167] (f) The antigen-binding polypeptide or the antigen-binding portion thereof has a melting temperature T≥62°C and an aggregation temperature Tagg≥61°C.

[0168] In some embodiments, the antigen-binding polypeptide or / and antigen-binding portion thereof that binds to CD47 has one, two, three, four, five or six of the properties (a) to (f). In some embodiments, the antigen-binding polypeptide or its antigen-binding portion has the properties (a), (b) and (c). In some embodiments, the antigen-binding polypeptide or its antigen-binding portion has the properties (a), (b) and (e). In some embodiments, the antigen-binding polypeptide or its antigen-binding portion has the properties (a), (c) and (e). In some embodiments, the antigen-binding polypeptide or its antigen-binding portion has the properties (a), (b), (c) and (d). In some embodiments, the antigen-binding polypeptide or its antigen-binding portion has the properties (a), (b), (c) and (e). In some embodiments, the antigen-binding polypeptide or its antigen-binding portion has the properties (a), (b), (c) and (f). In some embodiments, the antigen-binding polypeptide or its antigen-binding portion has the properties (a), (b), (c) and (f). In some embodiments, the antigen-binding polypeptide or its antigen-binding portion has the properties (a), (b), (c), (d) and (f). In some embodiments, the antigen-binding polypeptide or antigen-binding portion thereof has properties (a), (b), (c), (d), (e), and (f).

[0169] In some embodiments, the antigen binding polypeptide or antigen binding portion thereof that binds to CD47 is an anti-CD47 antibody or an antigen binding portion thereof.

[0170] In some embodiments, the isolated antigen-binding polypeptide that binds CD47 provided herein is a monoclonal antibody.

[0171] In some embodiments, the isolated antibodies provided herein that bind CD47 are monospecific antibodies.

[0172] In some embodiments, the isolated antibodies that bind to CD47 provided herein are multispecific antibodies, such as bispecific antibodies or trispecific antibodies.

[0173] In some embodiments, the invention provides an antigen binding polypeptide or antigen binding portion thereof that binds to the same epitope on CD47 as any of the exemplary antibodies provided herein, such as a chimeric or humanized antibody of 16E5 that binds to the same epitope, such as a chimeric or humanized antibody of 3F10 that binds to the same epitope, such as a chimeric or humanized antibody of 14A9 that binds to the same epitope. In some embodiments, the antigen binding polypeptide or antigen binding portion thereof competes with any of the exemplary antibodies provided herein for binding to CD47, such as a chimeric or humanized antibody of 16E5 that competes for binding to CD47, such as a chimeric or humanized antibody of 3F10 that competes for binding to CD47, such as a chimeric or humanized antibody of 14A9 that competes for binding to CD47. Binding to CD47 can be measured by ELISA, flow cytometry, surface plasmon resonance (SPR) assay, or any other method known in the art.

[0174] The present invention provides some exemplary monoclonal antibodies that bind to CD47, including chimeric versions of 2B2, 2H8, 3F10, 16E5 and 14A9 antibodies (Xi2B2, Xi2H8, Xi3F10, Xi16E5 and Xi14A9) and humanized variants of 3F10, 16E5 and 14A9. The amino acid sequences of the heavy chain CDRs (heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3) of the exemplary antibodies that bind to CD47 provided herein are provided in Table S1 below, the amino acid sequences of the light chain CDRs (light chain CDR1, light chain CDR2 and light chain CDR3) are provided in Table S2 below, and the amino acid sequences of the variable regions and the full-length heavy and light chains are provided in Tables S3 and S4 below.

[0175] Table S1: CDR sequences of the heavy chain of anti-CD47 antibodies

[0176]

[0177] Table S2: Light chain CDR sequences of anti-CD47 antibodies

[0178]

[0179] Table S3: Variable region sequences of anti-CD47 antibodies

[0180]

[0181]

[0182]

[0183]

[0184] Table S4: Sequences of full-length heavy and light chains of anti-CD47 antibodies

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] Immunoconjugates

[0193] In some embodiments, the present invention provides immunoconjugates of antigen-binding polypeptides or antigen-binding portions thereof that bind to CD47. The therapeutic agent that can be linked or conjugated to the antigen-binding polypeptides or antigen-binding portions thereof that bind to CD47 can include, but is not limited to, cytotoxic drugs, radioisotopes, immunomodulators, or antibodies. In some embodiments, the antigen-binding polypeptides or antigen-binding portions thereof that bind to CD47 are directly conjugated to the therapeutic agent. In some embodiments, the antigen-binding polypeptides or antigen-binding portions thereof that bind to CD47 are conjugated to the therapeutic agent via a linker.

[0194] Composition

[0195] The present invention provides a pharmaceutical composition comprising an isolated antigen-binding polypeptide or antigen-binding portion thereof that binds to CD47, or a nucleic acid encoding the antibody or fragment, or an immunoconjugate as described herein, and further comprising one or more pharmaceutically acceptable carriers. In some embodiments, the composition comprises any monoclonal antibody of the chimeric antibody and humanized antibody of 2B2, 2H8, 3F10, 16E5 and 14A9 antibodies, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, excipients, diluents, encapsulating materials, fillers, buffers or other agents.

[0196] Hybridoma

[0197] The present invention provides an isolated hybridoma cell line selected from the group consisting of 2B2, 2H8, 3F10, 16E5 and 14A9. Hybridoma cell lines 2B2, 2H8, 3F10, 16E5 and 14A9 secrete monoclonal antibodies that bind to CD47.

[0198] Isolated nucleic acids

[0199] The present invention provides an isolated nucleic acid comprising a sequence encoding an antigen-binding polypeptide or an antigen-binding portion thereof that binds to CD47 as described herein, in some embodiments, the nucleic acid may encode an amino acid sequence comprising a heavy chain variable region or / and a light chain variable region of an antigen-binding polypeptide as described herein. In other embodiments, the nucleic acid may encode an amino acid sequence comprising a heavy chain of an antigen-binding polypeptide or / and a light chain of an antigen-binding polypeptide as described herein. The sequence table exemplifies the nucleotide sequences of some variable regions, heavy chains of antigen-binding polypeptides, and light chains.

[0200] Carrier

[0201] The present invention provides a vector comprising the isolated nucleic acid. In some embodiments, the vector is a cloning vector; in other embodiments, the vector is an expression vector.

[0202] The expression vector may be any expression vector capable of expressing the antigen-binding polypeptide or antigen-binding portion thereof described herein.

[0203] Host cells

[0204] In some embodiments, the present invention provides a host cell comprising the vector, which is a suitable host cell for cloning or encoding an antigen-binding polypeptide or an antigen-binding portion thereof that binds CD47. In some embodiments, the host cell is a prokaryotic cell. In other embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antigen-binding polypeptides or antigen-binding portions thereof. Mammalian cells are, for example, Chinese hamster (CHO) ovary cells.

[0205] Method for preparing CD47 antigen binding polypeptide

[0206] In some embodiments, the present invention provides a method for preparing an isolated antigen-binding polypeptide that binds to CD47, the method comprising: culturing a host cell containing a nucleic acid encoding the antigen-binding polypeptide under conditions suitable for expression of the antigen-binding polypeptide, and recovering the antigen-binding polypeptide from the host cell or the host cell culture medium. In order to produce an antigen-binding polypeptide that binds to CD47, the nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. The nucleic acid can be obtained by various methods known in the art, such as gene cloning, gene splicing, and chemical synthesis.

[0207] use

[0208] The present invention provides the use of an isolated antigen-binding polypeptide or an antigen-binding portion thereof that binds to CD47. In some embodiments, administering a therapeutically effective amount of an isolated antigen-binding polypeptide or an antigen-binding portion thereof that binds to CD47 to a subject can reduce or inhibit tumor cell growth. In some embodiments, administering a therapeutically effective amount of an isolated antigen-binding polypeptide or an antigen-binding portion thereof that binds to CD47 to a subject can treat cancer. Subjects in need of treatment include those who already have a disease or condition, as well as those who may have a disease or condition and whose purpose is to prevent, delay or attenuate a disease or condition. As used herein, "cancer" refers to a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, leukemia, lymphoma, ovarian cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, bladder cancer, urothelial cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, esophageal cancer, renal cell cancer, thyroid cancer, head and neck cancer, testicular cancer, endocrine gland cancer, adrenal gland cancer, pituitary cancer, skin cancer, soft tissue cancer, vascular cancer, brain cancer, neural cancer, eye cancer, meningeal cancer, oropharyngeal cancer, hypopharyngeal cancer, cervical cancer, and uterine cancer, glioblastoma, medulloblastoma, astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma, melanoma, acute myeloid leukemia, myelodysplastic syndrome, and sarcoma.

[0209] In some embodiments, administering to the subject an effective amount of an isolated antigen-binding polypeptide or antigen-binding portion thereof that binds CD47 herein promotes phagocytosis of the subject's macrophages.

[0210] Although the foregoing invention has been described in considerable detail by way of illustration and examples for the purpose of clear understanding, it will be apparent to those of ordinary skill in the art, based on the teachings of the present invention, that certain changes and modifications may be made to the present invention without departing from the spirit and scope of the appended claims. The following examples are provided by way of illustration only and are not intended to be limiting. Those skilled in the art will readily recognize a variety of non-critical parameters that may be changed or modified to produce substantially similar results. The positive control Hu5F9 used in the specific examples herein has the same sequence as the antibody "5F9" in U.S. Patent US2015 / 0183874A1, and is a humanized antibody.

[0211] Unless otherwise indicated, the practice of the present invention employs conventional molecular biology, cell biology, biochemistry, and immunology techniques that are well known in the art and described in, for example, Methods in Molecular Biology, Humana Press; Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Current Protocols in Immunology (JE Coligan et al., 1991); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Phage display: an academic manual (C. Barbas III et al., Cold Spring Harbor Laboratory Press, 2001); and Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999) et al.

[0212] Example 1: Generation of anti-CD47 monoclonal antibodies

[0213] Immunization of mice with CD47 protein:

[0214] To produce antibodies against CD47, recombinant hCD47-His Tag protein (ACRObiosystems, catalog number: CD7-H5227) (50 μg / mouse Balb / c) or hCD47-mFc (ACRObiosystems, catalog number: CD7-H52A5) emulsified with an equal volume of Freund's complete adjuvant (primary immunization) or Freund's incomplete adjuvant (boost immunization) was subcutaneously immunized every 2 weeks for three times for 6 weeks. The fourth immunization used hCD47-mFc (ACRObiosystems, catalog number: CD7-H52A5) protein, 20 μg / mouse, intramuscular injection (aqueous adjuvant). Three days before cell fusion, mice were boosted by intravenous injection of antigen without adjuvant. Splenocytes (1×10 8 ) and SP2 / 0 myeloma cells (1.5×10 7 After fusion, cells were dispensed into a 96-well plate at 0.1 ml / well and incubated at 37°C and 5% CO 2 Incubate in an incubator at 4 °C. On day 1, feed the cells by adding an additional 0.1 ml of HAT medium containing serum and supplemented with 2× methotrexate to each well. On days 3 and 7, replace 0.1 ml of medium from each well with 0.1 ml of fresh HAT medium. Screening is usually performed between days 9-14, and culture supernatants are tested by ELISA for antibodies reactive with hCD47-His Tag (ACRObiosystems, catalog number: CD7-H5227), screening positive wells.

[0215] The limited dilution method was used for hybridoma cell cloning. The selected positive mixed clones were subjected to limited dilution. One positive mixed clone was spread on two 96-well plates (100 cells / plate, 200 cells / plate). The hybridoma cells were cultured in RPMI 1640 (Hyclone, catalog number: SH30809.01) containing 10% fetal bovine serum. After 10 days, the subclones after limited dilution were also tested by ELISA test to detect the culture supernatant, and the positive subclones were picked. The cloned hybridoma cells were preserved and used for cDNA extraction.

[0216] cDNA extraction and variable region sequence acquisition of anti-human CD47 antibody:

[0217] Total RNA was isolated from the selected hybridoma cell lines producing hCD47 antibodies using an RNA extraction kit (Takara, catalog number: 9767), and the total RNA was used as a template to synthesize the first-strand cDNA using a reverse transcription kit (ThermoFisher, catalog number: K1652) according to the manufacturer's instructions. The synthesized cDNA was then used to amplify the antibody-related sequences in vitro using a PCR reaction using degenerate mouse IgG primers.

[0218] The PCR amplification products were separated by electrophoresis in 1% agarose / Tris-acetate gel. DNA fragments of expected size (about 450 bp for heavy and light chains) were cut from the gel and purified. 3 μl of the purified PCR product was cloned into pMD-19T vector (Takara, catalog number: 6013) and transformed into DH5α chemically competent Escherichia coli (Takara, catalog number: 9057). In each ligation reaction, 10 positive clones were randomly selected and DNA sequencing was performed using M13 forward primer.

[0219] The heavy chain variable region (VH) and light chain variable region (VL) sequences of five mouse monoclonal antibodies (2B2, 2H8, 3F10, 16E5, 14A9 antibodies) were amplified and sequenced from the corresponding hybridoma clones (2B2, 2H8, 3F10, 16E5, 14A9). The sequence information is as follows:

[0220]

[0221]

[0222] Construction and expression of chimeric antibodies:

[0223] The genes of the mouse antibody variable regions were chemically synthesized, and the VL region of each mouse antibody was connected to the human κ chain constant region to construct a chimeric light chain, and the mouse VH region was connected to the human IgG4 (EU number S228P) constant region to construct a chimeric heavy chain. The expression plasmids of the chimeric light chain and the chimeric heavy chain were constructed by conventional genetic engineering methods, and CHO cells (200 mL system, 10 6 Cells were transfected with 100 μg of each chimeric heavy chain expression plasmid and chimeric light chain expression plasmid at 100 cells / mL and cultured for 6 days. The chimeric antibody in the supernatant was then purified using a protein A column.

[0224] Five chimeric antibodies were constructed and expressed, and the full-length sequences of their chimeric heavy chains and chimeric light chains and their encoding nucleic acids are as follows: Xi2H8 (chimeric heavy chain and its encoding nucleic acid: SEQ ID NOs: 89-90; chimeric light chain and its encoding nucleic acid: SEQ ID NOs: 91-92), Xi2B2 (chimeric heavy chain and its encoding nucleic acid: SEQ ID NOs: 97-98; chimeric light chain and its encoding nucleic acid: SEQ ID NOs: 99-100), Xi3F10 (chimeric heavy chain and its encoding nucleic acid: SEQ ID NOs: 101-102; chimeric light chain and its encoding nucleic acid: SEQ ID NOs: 103-104), Xi16E5 (chimeric heavy chain and its encoding nucleic acid: SEQ ID NOs: 93-94; chimeric light chain and its encoding nucleic acid: SEQ ID NOs: 95-96), Xi14A9 (chimeric heavy chain and its encoding nucleic acid: SEQ ID NOs: 105-106; chimeric light chain and its encoding nucleic acid: SEQ ID NOs: NOs: 107~108).

[0225] Humanized antibody design:

[0226] The human-mouse chimeric antibodies of 3F10, 14A9 and 16E5 were humanized using the CDR transplantation method (see, for example, U.S. Patent No. 5,225,539). Homology modeling of the mouse antibodies 3F10, 14A9 and 16E5 was performed by MOE (Molecular Operating Environment) to generate a protein structure model of the Fv domain. The VH and VL amino acid sequences of the mouse 3F10, 14A9 and 16E5 antibodies were respectively input into the website of the International Immunogenetic Information System Related Tools (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi), and human germline antibody sequences with high homology to the variable regions of the mouse 3F10, 14A9 and 16E5 antibodies were screened out. The complementary determining regions (CDRs) in the VH and VL of the mouse 3F10, 14A9 and 16E5 antibodies were grafted to the template human antibodies. For 3F10, the selected template human VH is a combination of IGHV1-69*01 and IGHJ4*01, and the selected template human VL is a combination of IGKV1-39*01 and IGKJ2*01. For 14A9, the selected template human VH is a combination of IGHV1-46*01 and IGHJ4*01, and the selected template human VL is a combination of IGKV1-39*01 and IGKJ2*01. For 16E5, the selected template human VH is a combination of IGHV1-18*01 and IGHJ4*01, and the selected template human VL is a combination of IGKV1-39*01 and IGKJ2*01.

[0227] The CDR amino acid sequences of the above template human antibodies were replaced by the CDRs of hybridoma (mouse) 3F10, 14A9 and 16E5 antibodies. The essential amino acid sequences of VH and VL from mouse 3F10, 14A9 and 16E5 antibodies were grafted onto the above template human germline antibody VH and VL frameworks to obtain functional humanized antibodies. For VH and VL of 3F10, 14A9 and 16E5 antibodies, several positions of the framework amino acids of the above template human antibodies were backmutated to the corresponding amino acid sequences in mouse 3F10, 14A9 and 16E5 antibodies. Individual CDR regions were mutated.

[0228] For the heavy chain variable region of the humanized 3F10 antibody, the selection and matching of mutation types are from the following sites: the amino acid at position 73 mutates from Asp (D) to Glu (E); the amino acid at position 74 mutates from Glu (E) to Ile (I); the amino acid at position 77 mutates from Ser (S) to Asn (N), and for the light chain variable region of the humanized 3F10 antibody, the selection and matching of mutation types are from the following sites: the amino acid at position 48 mutates from Leu (L) to Trp (W); the amino acid at position 71 mutates from Asp (D) to Ser (S); the amino acid at position 72 mutates from Phe (F) to Tyr (Y); the amino acid at position 94 mutates from Asp (D) to Glu (E). For the heavy chain variable region of humanized 14A9, the selection and matching of mutation types are from the following sites: the amino acid at position 24 mutates from Ala (A) to Val (V); the amino acid at position 72 mutates from Arg (R) to Gly (G); the amino acid at position 77 mutates from Ser (S) to Asn (N); the amino acid at position 97 mutates from Ala (A) to Thr (T), and for the light chain variable region of humanized 14A9, the selection and matching of mutation types are from the following sites: the amino acid at position 63 mutates from Ser (S) to Ile (I); the amino acid at position 68 mutates from Gly (G) to Ser (S). For the heavy chain variable region of the humanized 16E5 antibody, the selection and matching of mutation types are from the following sites: the amino acid at position 46 mutates from Glu (E) to Lys (K); the amino acid at position 63 mutates from Lys (K) to Glu (E); the amino acid at position 72 mutates from Thr (T) to Leu (L); the amino acid at position 77 mutates from Ser (S) to Arg (R); the amino acid at position 97 mutates from Ala (A) to Thr (T), and for the light chain variable region of the humanized 16E5 antibody, the selection and matching of mutation types are from the following sites: the amino acid at position 2 mutates from Ile (I) to Ala (A); the amino acid at position 33 mutates from Asn (N) to Gln (Q). The description of the mutation site numbers in this paragraph is counted sequentially starting from the first amino acid in the variable region,

[0229] The sequences of humanized 3F10, 14A9 and 16E5 antibodies and their encoding nucleic acids are as follows: hz3F10-1.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs.109-110; light chain and its encoding nucleic acid: SEQ ID NOs.111-112), hz3F10-2.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs.113-114; light chain and its encoding nucleic acid: SEQ ID NOs.115-116), hz3F10-3.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs.117-118; light chain and its encoding nucleic acid: SEQ ID NOs.119-120), hz3F10-4.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs.121-122; light chain and its encoding nucleic acid: SEQ ID NOs.123-124), hz3F10-5.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs.126-127), ID NOs.125-126; light chain and its encoding nucleic acid: SEQ ID NOs.127-128), hz3F10-6.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs.129-130; light chain and its encoding nucleic acid: SEQ ID NOs.131-132), hz3F10-1.2 (heavy chain and its encoding nucleic acid: SEQ ID NOs.133-134; light chain and its encoding nucleic acid: SEQ ID NOs.135-136), hz3F10-2.2 (heavy chain and its encoding nucleic acid: SEQ ID NOs.137-138; light chain and its encoding nucleic acid: SEQ ID NOs.139-140), hz3F10-3.2 (heavy chain and its encoding nucleic acid: SEQ ID NOs.141-142; light chain and its encoding nucleic acid: SEQ ID NOs.143-144), hz3F10-4.2 (heavy chain and its encoding nucleic acid: SEQ ID NOs.145-146), hz3F10-5.2 (heavy chain and its encoding nucleic acid: SEQ ID NOs.147-148; light chain and its encoding nucleic acid: SEQ ID NOs.149-150), hz3F10-6.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs.129-130; light chain and its encoding nucleic acid: SEQ ID NOs.131-132), hz3F10-1.2 (heavy chain and its encoding nucleic acid: SEQ ID NOs.133-134; light chain and its encoding nucleic acid: SEQ ID NOs.135-136), hz3F10-2.2 (heavy chain and its encoding nucleic acid: SEQ ID NOs.137-138; light chain and its encoding nucleic acid: NOs.145-146; light chain and its encoding nucleic acid: SEQ ID NOs.147-148), hz3F10-5.2 (heavy chain and its encoding nucleic acid: SEQ ID NOs.149-150; light chain and its encoding nucleic acid: SEQ ID NOs.151-152), hz3F10-6.2 (heavy chain and its encoding nucleic acid: SEQ ID NOs.153-154; light chain and its encoding nucleic acid: SEQ ID NOs.155-156), hz16E5-1.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs.157-158; light chain and its encoding nucleic acid: SEQ ID NOs.159-160), hz16E5-1.3 (heavy chain and its encoding nucleic acid: SEQ ID NOs.161-162; light chain and its encoding nucleic acid: SEQ ID NOs.163-164), hz16E5-1.2 (heavy chain and its encoding nucleic acid: SEQ ID NOs.165-166; light chain and its encoding nucleic acid: SEQ ID NOs.167-168), hz16E5-3.2 (heavy chain and its encoding nucleic acid: SEQ ID NOs.169-170; light chain and its encoding nucleic acid: SEQ ID NOs.171-172), hz16E5-3.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs.173-174; light chain and its encoding nucleic acid: SEQ ID NOs.175-176), hz16E5-3.3 (heavy chain and its encoding nucleic acid: SEQ ID NOs.177-178; light chain and its encoding nucleic acid: SEQ ID NOs.179-180), hz14A9-2.3 (heavy chain and its encoding nucleic acid: SEQ ID NOs.181-182; light chain and its encoding nucleic acid: SEQ ID NOs.183-184), hz14A9-2.4 (heavy chain and its encoding nucleic acid: SEQ ID NOs.185-186; light chain and its encoding nucleic acid: SEQ ID NOs.187-188). .

[0230] Construction and expression of humanized 3F10, 14A9 and 16E5 antibodies:

[0231] DNA encoding the full-length light chain and full-length heavy chain of humanized 3F10, 14A9 and 16E5 antibodies was synthesized and cloned into expression vectors (including, for example, pcDNA3.1 vector disclosed in CN107001463A, pCHO1.0 vector disclosed in CN109422811A, etc.). CHO cells (200 ml system, at 10 6 Cells / ml) were transfected with 100 μg of each humanized antibody heavy chain expression plasmid and light chain expression plasmid and incubated with ExpiCHO medium (Gibco; Catalog No.: A29100-01) at 37°C and 5% CO 2 The cells were cultured in an incubator at 4 °C for 6 days. The supernatant was obtained by centrifugation and then purified using a protein A column to purify the humanized antibody in the supernatant.

[0232] Example 2: Biacore-based affinity analysis of anti-CD47 antibodies

[0233] The binding kinetics between human CD47 protein (Human CD47 Protein, His Tag; ACRObiosystems, catalog number: CD7-H5227) and cynomolgus / Rhesus macaque CD47 protein (Cynomolgus / Rhesus macaque CD47 Protein, His Tag; ACRObiosystems, catalog number: CD7-C52H1) and anti-CD47 antibodies (chimeric or humanized) were measured by Biacore to obtain the equilibrium dissociation constant K D (Unit: M); the Biacore analysis was performed at 25°C and recorded at a data collection rate of 1 Hz.

[0234] Polyclonal rabbit anti-mouse IgG (GE, BR-1008-38) was diluted with 10 mM sodium acetate pH 5.0 and immobilized to about 15000 RM on the reference flow cell and experimental flow cell of a CM5 biosensor chip using an amine coupling kit (GE, BR10050). At the beginning of each cycle, the diluted test antibody (1.5 μg / mL) was injected on the experimental flow cell for 1 minute to be captured. These data show that anti-CD47 antibodies (chimeric and humanized) bind to CD47 as measured by Biacore.

[0235] Table 1: Binding affinity of anti-CD47 chimeric antibodies to human CD47 and cynomolgus monkey CD47

[0236]

[0237] Table 2: Binding affinity of anti-CD47 humanized antibodies to human CD47 and cynomolgus monkey CD47

[0238]

[0239]

[0240] Example 3: ELISA-based binding analysis of anti-CD47 antibodies

[0241] ELISA binding analysis was performed based on human CD47 protein and anti-CD47 chimeric antibody or anti-CD47 humanized antibody. A 96-well plate (Costar, catalog number: 9018) was coated overnight at 4°C with 100 μl / well of 2 μg / ml CD47-His (Human CD47Protein, His Tag; ACRObiosystems, catalog number: CD7-H5227) in coating buffer PBS. The wells were aspirated and nonspecific binding sites were blocked by adding 100 μL / well blocking solution (PBS with 1% (w / v) bovine serum albumin (BSA, Sigma, catalog number: B2064-100G)) and incubating at 37°C for 1 hour. After washing the 96-well plate three times with washing buffer (PBS with 0.01% (w / v) Tween 20 (Sigma, catalog number: P9416-100ML)), add 100 μl / well of anti-CD47 antibody (chimeric antibodies, see Table 3, humanized antibodies, see Table 4) in a 1:4 serial dilution in blocking solution (starting from 30 μg / mL) and incubate at room temperature for 2 hours. Wash the 96-well plate and then incubate with the secondary antibody HRP-anti-Human IgG (BD, catalog number: 555788) in the dark for 20 minutes. After washing the 96-well plate, add 100 μl / well of substrate solution TMB (TIANGEN, catalog number: PA107-01) and incubate the 96-well plate at room temperature for 10 minutes. Add 100 μl / well of stop solution (1M H 2 SO 4 ) to stop the reaction. A colorimetric signal was generated and read at 450 nm using a multifunctional microplate reader (manufacturer: PerkinElmer; model specification EnVision). Data were analyzed using GraphPad Prism5, and EC50 was calculated. These data show that anti-CD47 antibodies bind to CD47 as measured by ELISA, and humanized antibodies have good performance.

[0242] Table 3: EC50 of binding of anti-CD47 chimeric antibodies to human CD47 protein based on ELISA

[0243] Antibody Name EC50(ng / mL) Xi2B2 349.5 Xi2H NA Xi3F10 1106 Xi16E5 87.89 Xi14A9 273.4

[0244] Note: NA means the detection limit was not reached.

[0245] Table 4: Binding EC50 of anti-CD47 humanized antibodies to human CD47 protein based on ELISA

[0246] Antibody Name EC50(ng / mL) Antibody Name EC50(ng / mL) hz3F10-1.1 70.02 hz3F10-5.2 105.6 hz3F10-2.1 80.94 hz3F10-6.2 129.3 hz3F10-3.1 126.9 hz16E5-1.1 306 hz3F10-4.1 115.1 hz16E5-3.1 139.7 hz3F10-5.1 99.33 hz16E5-1.2 159.5 hz3F10-6.1 56.33 hz16E5-3.2 215.1 hz3F10-1.2 124.8 hz16E5-1.3 138.1 hz3F10-2.2 105.8 hz16E5-3.3 240.9 hz3F10-3.2 165.9 hz14A9-2.3 156.8 hz3F10-4.2 180.7 hz14A9-2.4 339.3

[0247] Example 4: Cell-based binding assay of anti-CD47 antibodies

[0248] Cellular binding assays of anti-CD47 antibodies (chimeric or humanized) were performed based on binding to CHO-K1 cell lines stably expressing CD47. CHO-K1-CD47 cells (CHO-K1 cells expressing human CD47 protein) were added to each well of a 96-well plate (Eppendorf, catalog number: C030730.119) at 1.2 × 10 4 The cells were then washed three times with PBS buffer, and the secondary antibody Alexa Fluor 488 labeled goat anti-human IgG (Jackson ImmunoResearch Inc, catalog number: 109-545-088) was diluted 1:200 with PBS and added to the cells at 100 μl / well, and incubated at room temperature for 30 min. The cells were washed three times with PBS buffer, and the nuclear dye Hoechst (Sigma, catalog number: B2261) was diluted 1:500 with PBS buffer and added to each well at 100 μl / well for staining for 10 min. After washing 3 times with PBS, the cells were analyzed by high content screening instrument (Highcontent Screening System, manufacturer: PerkinElmer, model specification: Operetta). The results are shown in Tables 5 and 6. These data show that the anti-CD47 antibody binds to CD47 in the cell-based binding analysis.

[0249] Table 5: EC50 of cell-based binding of anti-CD47 chimeric antibodies to CD47

[0250] Antibody Name EC50(ng / mL) Xi2B2 30790 Xi2H NA Xi3F10 19803 Xi16E5 2026 Xi14A9 540.7

[0251] Note: NA means the detection limit was not reached.

[0252] Table 6: EC50 of anti-CD47 humanized antibodies binding to CD47 based on cells

[0253] Antibody Name EC50(ng / mL) Antibody Name EC50(ng / mL) hz3F10-1.1 875.7 hz3F10-6.2 1486 hz3F10-2.1 1068 hz16E5-1.1 3866 hz3F10-3.1 1121 hz16E5-3.1 5883 hz3F10-4.1 1162 hz16E5-1.2 NA hz3F10-5.1 1480 hz16E5-3.2 NA hz3F10-6.1 1184 hz16E5-1.3 18845 hz3F10-1.2 1267 Hz16E5-3.3 40479 hz3F10-2.2 1119 hz14A9-2.3 498.6 hz3F10-3.2 1195 hz14A9-2.4 470.7 hz3F10-4.2 1265 Hu5F9 1076 hz3F10-5.2 1260

[0254] Note: NA means the detection limit was not reached.

[0255] Example 5: Cell-based antibody blocking assay of anti-CD47 chimeric antibodies

[0256] The blocking effect of anti-CD47 chimeric antibody on the binding of CD47 to SIRPα was evaluated based on flow cytometry detection.

[0257] Jurkat cells (Cell Bank of the Chinese Academy of Sciences, catalog number: TCHU123) were added to a 96-well U-shaped plate, with 5×10 5 100 μl of 1 μg / mL CD47 ligand Human-SIRPα-mFC (Human SIRP alpha / CD172a Protein, Mouse IgG1Fc Tag, ACRObiosystems, catalog number: SIA-H52A8) diluted in PBS buffer was added to each well and incubated at room temperature for 1 hour. After washing twice with PBS buffer, 100 μl of anti-CD47 chimeric antibody (Table 7) was added to each well, the initial concentration was set to 20 μg / ml, and 4-fold dilution was performed with PBS buffer, 8 gradients, incubated at room temperature for 1-2 hours, and washed 3 times with PBS buffer. Alexa Fluor 488-labeled goat anti-mouse IgG (Alexa Fluor 488-Goat anti mouse IgG, Jackson ImmunoResearch Inc., catalog number: 115-545-062) was used as the detection secondary antibody, and 100 μl of detection secondary antibody diluted with PBS buffer at a dilution ratio of 1:200 was added to each well, room temperature for 1 hour, PBS buffer was washed 3 times, and the blocking effect of anti-CD47 chimeric antibody on the binding of CD47 to SIRPα was detected by flow cytometry (BD Company, model specification: C6). The results are shown in Table 7. Anti-CD47 chimeric antibody can block the binding of CD47 to SIRPα, among which Xi3F10, Xi16E5, and Xi14A9 have better blocking effects.

[0258] Table 7: Cell-based antibody blocking assay of anti-CD47 chimeric antibodies

[0259] Antibody Name Xi2B2 Xi2H Xi3F10 Xi16E5 Xi14A9 IC50(ng / mL) NA 26966 358.5 4861 1283

[0260] Note: NA means the detection limit was not reached.

[0261] Example 6: ELISA-based antibody blocking analysis of anti-CD47 antibodies

[0262] The blocking effect of anti-CD47 antibodies on the binding of CD47 to SIRPα was evaluated based on ELISA detection.

[0263] In a 96-well ELISA plate (Costar, catalog number: 9018), CD47-His (Human CD47 Protein, His Tag; ACRObiosystems, catalog number: CD7-H5227) was used as the capture antigen, 100 μl of PBS coating buffer containing the capture antigen at 2 μg / mL was added to each well, overnight at 2-8°C, the wells were aspirated to remove the coating buffer, and 100 μL / well of blocking solution (PBS with 1% (w / v) bovine serum albumin (BSA, Sigma, catalog number: B2064-100G)) was incubated at 37°C for 1 hour. The 96-well plate was washed with a washing buffer (PBS with 0.01% (w / v) Tween 20 (Sigma, catalog number: P9416-100ML)). Then add 100 μl / well of 1 μg / ml CD47 ligand Human-SIRPα-mFC (Human SIRPalpha / CD172a Protein, Mouse IgG1Fc Tag, ACRObiosystems, catalog number: SIA-H52A8), incubate at 25±2°C for 1.5h, wash the 96-well plate with PBS washing buffer, and then add: anti-CD47 chimeric antibody or anti-CD47 humanized antibody (chimeric antibodies see Table 8, humanized antibodies see Table 9), starting concentration 20 μg / mL, 100 μL / well, dilute 4-fold with PBS buffer, 8 gradients, and incubate at room temperature for 1.5h. After washing with PBS buffer, 100 μl / well of HRP-labeled goat anti-mouse IgG (HRP Goatanti mouse IgG, BD, catalog number: 554002) diluted 1:2000 with PBS buffer was added and incubated at 25±2°C for 1 h. After washing the 96-well plate with PBS buffer, 100 μl / well of substrate solution TMB (TIANGEN, catalog number: PA107-01) was added and incubated at room temperature for 10 min. 100 μl / well of 1M H 2 SO 4 The reaction was terminated. A colorimetric signal was generated and read at 450 nm using a multifunctional microplate reader (manufacturer: PerkinElmer; model specification EnVision). The data were analyzed using GraphPad Prism5, and the EC50 was calculated. The results are shown in Tables 8 and 9. Based on ELISA detection, the chimeric antibodies of 14A9, 16E5, 3F10 (Xi3F10, Xi16E5, Xi14A9) and the humanized antibodies had good blocking effects.

[0264] Table 8: ELISA-based antibody blocking analysis of anti-CD47 chimeric antibodies

[0265] Antibody Name IC50(ng / mL) Xi2B2 NA Xi2H 4.682 Xi3F10 7910 Xi16E5 946.1 Xi14A9 444.4

[0266] Note: NA means the detection limit was not reached.

[0267] Table 9: ELISA-based antibody blocking analysis of anti-CD47 humanized antibodies

[0268] Antibody Name IC50(ng / mL) Antibody Name IC50(ng / mL) hz3F10-1.1 1534 hz3F10-6.2 1839 hz3F10-2.1 1612 hz16E5-1.1 NA hz3F10-3.1 1678 hz16E5-3.1 15246 hz3F10-4.1 1952 hz16E5-1.2 2939 hz3F10-5.1 1701 hz16E5-3.2 2051 hz3F10-6.1 1768 hz16E5-1.3 2323 hz3F10-1.2 1689 hz16E5-3.3 1998 hz3F10-2.2 1725 hz14A9-2.3 703.2 hz3F10-3.2 1741 hz14A9-2.4 621.1 hz3F10-4.2 1738 Hu5F9 2398 hz3F10-5.2 1824

[0269] Note: NA means the detection limit was not reached.

[0270] Example 7: Analysis of anti-CD47 antibodies based on promoting macrophage phagocytosis

[0271] The ability of anti-CD47 antibodies to induce macrophages containing SIRPα on their cell surface to phagocytose tumor cells was determined based on flow cytometry.

[0272] Monocytes were isolated from human peripheral blood mononuclear cells (PBMC, Sai Li Biotechnology, catalog number: 190056) using a monocyte isolation kit (STEMCELL, catalog number: 19058) and plated into 24-well plates (Greiner bio-one, catalog number: 662-160), with 5 × 10 cells per well. 5 1×10 cells were induced into macrophages in an environment of 100 ng / mL stimulating factor M-CSF (R&D Systems, catalog number: 216-MC-010) for 7 days. 6 HL60 target cells (Shanghai Institute of Cell Biology, catalog number: TCHu23) were suspended in 5 μmol / L CFSE (BD, catalog number: 565082) fluorescent dye (fluorescent dye was diluted to the required concentration with PBS buffer) and incubated in a 37°C water bath in the dark for 15 min. The cells were washed with PBS buffer to prepare CFSE-labeled HL60 cells. CFSE-labeled HL60 cells were incubated with 10 μg / mL and 1 μg / mL anti-CD47 antibodies (chimeric antibodies are shown in Table 10, and humanized antibodies are shown in Table 11) at 37°C and 5% CO 2 Incubate in an incubator for 1-2 hours. Wash HL60 cells three times with PBS buffer and add them to macrophages at 37°C and 5% CO. 2After incubation for 2-4 hours in an incubator and washing three times with PBS buffer, 260 μl of a mixture of PBS, 20 μl of anti-APC-CD14 (APC Mouse Anti-Human CD14, BD, catalog number: 555399) and 20 μl of PE-CD11b (PE Mouse Anti-Human CD11b / Mac-1, BD, catalog number: 555388) antibodies were added to each well, incubated at 4°C in the dark for 30 minutes, washed three times with PBS buffer, digested with trypsin (Gibco, catalog number: 12604-013), and the phagocytic rate (%) of macrophages was detected by flow cytometer (BD, model specification: C6).

[0273] The effects of anti-CD47 chimeric antibodies and anti-CD47 humanized antibodies on macrophage phagocytosis are shown in Tables 10 and 11, among which the chimeric antibodies Xi3F10, Xi16E5, Xi14A9 and anti-CD47 humanized antibodies have good phagocytic effects, and the humanized antibodies hz14A9-2.3 and hz14A9-2.4 perform better.

[0274] Table 10: Analysis of anti-CD47 chimeric antibodies based on the promotion of macrophage phagocytosis (phagocytosis rate, %)

[0275] Antibody Name 10 μg / mL 1 μg / mL Xi2B2 4.1 7.3

[0276] Xi2H 3.5 7.1 Xi3F10 59.8 65.7 Xi16E5 28.8 11.5 Xi14A9 44.67 54.17

[0277] Table 11: Analysis of anti-CD47 humanized antibodies based on the promotion of macrophage phagocytosis (phagocytosis rate, %)

[0278] Antibody Name 10 μg / mL 1 μg / mL Antibody Name 10 μg / mL 1 μg / mL hz3F10-1.1 43.10 41.25 hz3F10-6.2 44.19 49.32 hz3F10-2.1 55.23 47.24 hz16E5-1.1 4.02 14.79 hz3F10-3.1 58.72 50.60 hz16E5-3.1 24.19 6.37 hz3F10-4.1 58.26 50.26 hz16E5-1.2 17.83 5.1 hz3F10-5.1 58.46 50.45 hz16E5-3.2 2.03 3.02 hz3F10-6.1 60.91 47.57 hz16E5-1.3 4.33 3.03 hz3F10-1.2 38.86 55.52 hz16E5-3.3 4.7 11.62 hz3F10-2.2 34.72 44.19 hz14A9-2.3 74.93 71.48 hz3F10-3.2 37.09 47.04 hz14A9-2.4 81.66 81.65 hz3F10-4.2 31.67 46.24 Hu5F9 72.35 67.5 hz3F10-5.2 29.6 47.06

[0279] Example 8: Effect of anti-CD47 antibodies on CD4+ T cells

[0280] The effect of anti-CD47 antibodies on CD4+ T cells was determined using a flow cytometry-based assay.

[0281] CD4+ T cells were isolated from human peripheral blood mononuclear cells (PBMC, Sai Li Biotech, catalog number: 190056) using a T cell isolation kit (Miltenyi Biotech, catalog number: 130-094-131) and plated into 24-well plates (Greinerbio-one, catalog number: 662106), with 5×10 per well. 5 cells, and anti-CD47 antibodies (chimeric antibodies are shown in Table 12, and humanized antibodies are shown in Table 13) were added to make the final concentrations 10 μg / mL and 1 μg / mL, respectively. 1640 complete medium was added and the cells were incubated at 37°C with 5% CO2 The cells were cultured in an incubator for 20 hours, and stained with an apoptosis kit (STEMCELL, catalog number: 556547) at room temperature for 15 minutes. The apoptosis rate (%) was detected by flow cytometry (BD, model specification: C6), and the apoptosis rate was calculated as the sum of early apoptosis and late apoptosis. The results showed that anti-CD47 antibodies (the chimeric antibodies are shown in Table 12 and the humanized antibodies are shown in Table 13) had no significant effect on T cell apoptosis.

[0282] Table 12: Effects of anti-CD47 chimeric antibodies on CD4+ T cells (apoptosis rate %)

[0283] Antibody Name 10 μg / mL 1 μg / mL Xi2B2 10.3 12.1 Xi2H 10.5 12.1 Xi3F10 13.6 12.6 Xi16E5 23.1 0.4 Xi14A9 1.34 1.42

[0284] Table 13: Effects of anti-CD47 humanized antibodies on CD4+ T cells (apoptosis rate %)

[0285] Antibody Name 10 μg / mL 1 μg / mL Antibody Name 10 μg / mL 1 μg / mL hz3F10-1.1 1.94 1.82 hz3F10-6.2 1.68 2.18 hz3F10-2.1 1.7 1.38 hz16E5-1.1 1.88 1.62 hz3F10-3.1 1.88 2 hz16E5-3.1 2.04 1.8 hz3F10-4.1 1.86 1.94 hz16E5-1.2 1.66 1.08 hz3F10-5.1 1.74 1.94 hz16E5-3.2 1.8 1.52 hz3F10-6.1 2.12 1.34 hz16E5-1.3 1.28 1.38 hz3F10-1.2 1.74 1.7 hz16E5-3.3 1.54 1.54 hz3F10-2.2 1.82 1.94 hz14A9-2.3 1.56 1.59 hz3F10-3.2 1.84 1.66 hz14A9-2.4 1.89 1.38 hz3F10-4.2 1.84 1.78 Hu5F9 1.35 1.58 hz3F10-5.2 1.94 1.78 IgG4 1.72 1.81

[0286] Example 9: Detection of thermal stability of anti-CD47 antibodies based on nano DSF

[0287] The high-throughput protein stability analyzer Prometheus NT.48 is a device that obtains various types of data such as protein molecular structure stability, aggregation stability, colloidal dispersion stability, etc. The device was used to detect the melting temperature (Tm) and aggregation temperature (Tagg) of anti-CD47 chimeric antibodies and anti-CD47 humanized antibodies. The results are shown in Tables 14 and 15, indicating that the anti-CD47 antibody has good thermal stability.

[0288] Table 14: Thermal stability of anti-CD47 chimeric antibodies

[0289] Antibody Name Tm(℃) Tagg(℃) Xi2B2 62.99 61.34

[0290] Xi2H 69.77 68.57 Xi3F10 70.51 70.59 Xi16E5 69.89 73.39 Xi14A9 66.73 75.75

[0291] Table 15: Thermal stability of anti-CD47 humanized antibodies

[0292] Antibody Name Tm(℃) Tagg(℃) Antibody Name Tm(℃) Tagg(℃) hz3F10-1.1 68.44 72.68 hz14A9-2.4 68.3 68.3 hz3F10-2.1 68.44 72.68 hz3F10-4.2 68.48 61.13 hz3F10-3.1 68.15 61.53 hz3F10-5.2 68.69 62.67 hz3F10-4.1 68.35 62.54 hz3F10-6.2 68.64 61.91 hz3F10-5.1 68.05 62.12 hz16E5-1.1 68.21 61.87 hz3F10-6.1 68.6 68.6 hz16E5-3.1 69.20 61.84 hz3F10-1.2 68.71 61.92 hz16E5-1.2 69.54 61.90 hz3F10-2.2 68.92 62.40 hz16E5-3.2 69.10 61.44 hz3F10-3.2 68.30 61.80 hz16E5-1.3 68.90 73.77 hz14A9-2.3 68.10 73.42 hz16E5-3.3 68.72 73.81

[0293] Example 10: Effect of anti-CD47 antibody on red blood cell agglutination

[0294] Some published studies have shown that there is a certain amount of CD47 protein on the surface of erythrocytes, which causes certain anti-CD47 antibodies to bind to erythrocytes after addition, thereby possibly causing agglutination of erythrocytes. Therefore, the effect of anti-CD47 antibodies on erythrocyte agglutination was tested.

[0295] Take anticoagulated blood, add an equal amount of physiological saline, mix well, centrifuge at 2000rpm / min for 5min, and remove the supernatant. Wash the red blood cells 3 times with Hank's balanced salt solution without calcium ions and magnesium ions (Thermo Fisher Scientific, catalog number: 14175-095), centrifuge at 2000rpm / min for 5min for the first 2 times, and centrifuge at 2000rpm / min for 10min for the last time, and remove the supernatant. Dilute the red blood cells 10 times with PBS buffer to make a 10% red blood cell suspension, and put 100μl / well into a 96-well U-shaped plate (eppendorf, catalog number: 003073119). Add 100μl / well of different concentrations of anti-CD47 antibody, the starting concentration of the antibody is set to 10μg / ml, diluted 3 times with PBS buffer, 8 concentration gradients, and incubate at 37°C for 2 to 6 hours. When observing and taking pictures, if all the red blood cells are agglutinated and sink to the bottom and spread out in a net-like shape, it is agglutination; if there is no agglutination, the red blood cells sink to the bottom of the hole in the form of dots.

[0296] The effect of anti-CD47 chimeric antibody on erythrocyte agglutination is shown in Figure 1 The effect of anti-CD47 humanized antibody on erythrocyte agglutination is shown in Figure 2 , Figure 3 Chimeric antibodies Xi3F10, Xi16E5 and Hu5F9 all showed coagulation phenomenon, among which Xi3F10 started to coagulate from 1111.1ng / ml, Xi16E5 and Hu5F9 started to coagulate from 123.46ng / ml, and chimeric antibodies Xi2B2, Xi2H8 and Xi14A9 showed no coagulation phenomenon at all tested concentrations. Humanized antibodies hz3F10-3.1, hz3F10-4.1, hz3F10-3.2, and hz3F10-4.2 began to coagulate at 3.3 μg / ml, and hz16E5-1.1, hz16E5-3.1, hz16E5-1.2, hz16E5-3.2, hz16E5-1.3, and hz16E5-3.3 began to coagulate at 3.3 μg / ml. Other humanized antibodies showed no coagulation at all tested concentrations.

[0297] Example 11: Analysis of binding of anti-CD47 antibodies to red blood cells

[0298] Based on a certain amount of CD47 on the surface of red blood cells, it may bind to certain anti-CD47 antibodies, so the binding of anti-CD47 antibodies to red blood cells is detected.

[0299] Take anticoagulated blood, add an equal amount of physiological saline, mix well, centrifuge at 2000 rpm / min for 5 min, and remove the supernatant. Wash red blood cells three times with Hank's balanced salt solution without calcium ions and magnesium ions (Thermo Fisher Scientific, catalog number: 14175-095), centrifuge at 2000 rpm / min for 5 min for the first two times, and centrifuge at 2000 rpm / min for 10 min for the last time, and remove the supernatant. The separated erythrocytes were fixed with 4% paraformaldehyde (Shanghai Bioengineering Co., Ltd., catalog number: E672002-0500) overnight, and incubated with 10 μg / ml or 1 μg / ml anti-CD47 antibody at room temperature for 1-2 hours the next day. After washing with PBS buffer, they were incubated with secondary antibody Alexa Fluor 488-labeled goat anti-human IgG (Jackson ImmunoResearch Inc, catalog number: 109-545-088) diluted 1:200 with PBS buffer at room temperature for 1 hour. After washing with PBS buffer, they were detected by flow cytometer (BD Company, model specification: C6), and the mean fluorescence intensity (MFI) was used to reflect the binding of erythrocytes to anti-CD47 chimeric antibodies (see Table 16) or humanized antibodies (see Table 17).

[0300] Table 16: Binding analysis of anti-CD47 chimeric antibodies to erythrocytes (MFI)

[0301] Antibody Name 10 μg / mL 1 μg / mL Xi2B2 608 656 Xi2H 5303 952 Xi3F10 61821 36114 Xi16E5 3318 1309 Xi14A9 32831 26477

[0302] Table 17: Binding analysis of anti-CD47 humanized antibodies to erythrocytes (MFI)

[0303] Antibody Name 10 μg / mL 1 μg / mL Antibody Name 10 μg / mL 1 μg / mL

[0304] hz3F10-1.1 50,463 4,449 hz3F10-6.2 55,886 3,435 hz3F10-2.1 53,218 8,454 hz16E5-1.1 6,461 945 hz3F10-3.1 41,362 6,703 hz16E5-3.1 5,145 945 hz3F10-4.1 41,900 5,976 hz16E5-1.2 4,729 913 hz3F10-5.1 50,634 7,403 hz16E5-3.2 4,204 860 hz3F10-6.1 53,048 7,583 hz16E5-1.3 4,885 894 hz3F10-1.2 56,720 6,247 hz16E5-3.3 3,874 848 hz3F10-2.2 56,086 5,612 hz14A9-2.3 10,979 2,506 hz3F10-3.2 45,305 5,096 hz14A9-2.4 14,954 5,237 hz3F10-4.2 45,616 4,814 IgG4 484 449 hz3F10-5.2 53,329 5,134

[0305] Example 12 Pharmacological activity of anti-CD47 antibodies in the lymphoma Raji cell mouse hematological tumor model

[0306] A mouse hematological tumor model was established using hematological tumor Raji cells, and anti-CD47 antibody was applied to this model to evaluate the pharmacological activity of the antibody.

[0307] The concentration of Raji cell suspension prepared in PBS buffer was 5×10 6 / mL, 0.1mL / mouse was injected into female NOD / SCID mice through the tail vein. After 7 days, they were randomly divided into 4 groups, 10 mice in each group, including group 1: IgG4 group, group 2: Hu5F9 group, group 3: hz3F10-6.1, group 4: hz14A9-2.3, intraperitoneal injection, the dosage was 10mg / kg, and the administration was continued for 21 days. The survival of the animals was observed.

[0308] like Figure 4 In the Raji hematoma model, anti-CD47 antibodies hz14A9-2.3 and hz3F10-6.1 have anti-tumor activity. The survival data of mice showed that all animals in the IgG4 group died on the 17th day, with a median survival of 13.5 days; all animals in the Hu5F9 group died on the 30th day, with a median survival of 18.5 days; all animals in the hz14A9-2.3 group died on the 34th day, with a median survival of 28.5 days; all animals in the hz3F10-6.1 group died on the 21st day, with a median survival of 17.5 days. These data show that the antibody hz14A9-2.3 is more effective than other groups in prolonging animal survival, and the effects of antibodies hz3F10-6.1 and Hu5F9 are basically the same.

[0309] Example 13 Toxicity testing of single administration of anti-CD47 antibody

[0310] Since CD47 is expressed on erythrocytes and plays a role in the clearance of aged erythrocytes, the toxic effects of anti-CD47 antibodies were compared in B-hCD47 mice.

[0311] B-hCD47 mice were randomly divided into 3 groups: 3 in the Hu5F9 group, 4 in the hz14A9-2.3 group, and 3 in the hz3F10-6.1 group. B-hCD47 mice were given a certain dose of Hu5F9 antibody, hz14A9-2.3 antibody, or hz3F10-6.1 antibody by a single tail vein injection. The dose was 10 mg / kg, and the changes in red blood cell count (RBC), hemoglobin (HGB), and body weight were detected 2 days, 4 days, 7 days, 10 days, and 14 days after administration.

[0312] Table 18: Change rate of mouse body weight after administration (vs before administration)

[0313] Days after drug administration Hu5F9 10mpk hz14A9-2.3 10mpk hz3F10-6.1 10mpk 0 0.0％ 0.0％ 0.0％ 2 -7.0％ 2.3％ -13.0％ 4 -3.2％ 6.3％ -15.8％ 7 -5.1％ 2.0％ -2.5％ 10 4.8％ 5.9％ -8.4％ 14 5.6％ 8.6％ 8.2％

[0314] Table 19: Change rate of RBC in mice after administration (vs before administration)

[0315] Days after drug administration Hu5F9 10mpk hz14A9-2.3 10mpk hz3F10-6.1 10mpk 0 0.0％ 0.0％ 0.0％ 2 -52.8％ -13.6％ -56.7％ 4 -61.8％ -16.7％ -75.6％ 7 -22.2％ -9.8％ -38.7％ 10 -21.6％ -8.5％ -6.6％ 14 -7.4％ -2.2％ -2.0％

[0316] Table 20: Change rate of HGB in mice after administration (vs before administration)

[0317] Days after drug administration Hu5F9 10mpk hz14A9-2.3 10mpk hz3F10-6.1 10mpk 0 0.0％ 0.0％ 0.0％ 2 -52.7％ -13.3％ -57.1％ 4 -57.8％ -18.5％ -74.7％

[0318] 7 -16.5％ -9.9％ -39.2％ 10 -12.8％ -10.0％ -6.0％ 14 -1.2％ -3.9％ -3.0％

[0319] As shown in Table 18, Figure 5 and Figure 6As shown in Table 19-20, only the weight of mice in the hz14A9-2.3 group continued to increase. The weight of mice in the Hu5F9 and hz3F10-6.1 groups first decreased and then increased after administration. The decrease in the hz3F10-6.1 group was greater than that in the Hu5F9 group. Figure 7-10 As shown, the RBC and HGB of the three groups of animals decreased after administration, reaching the lowest level 4 days after administration, and then began to rise. The RBC and HGB of the hz14A9-2.3 group basically returned to normal 7 days after administration, and the other two groups basically returned to normal at least 10 days after administration. The decrease in RBC and HGB 4 days after administration, from high to low, was: hz3F10-6.1>Hu5F9>hz14A9-2.3. It can be seen that the antibody hz14A9-2.3 has no significant toxicity and is superior to the positive antibody Hu5F9.

[0320] Embodiment 14

[0321] The anti-tumor activity of the antibody was tested using the MOLM-16 cell mouse model of human acute myeloid leukemia cells. Each NOD-SCID mouse was subcutaneously inoculated with MOLM-16 cells and the tumor was grown to 80-150 mm. 3 Afterwards, the animals were randomly divided into 5 groups: ① model group (negative control group, given 5 mg / kg human IgG4), n = 10; ② hz14A9-2.3 0.5 mg / kg group, n = 6; ③ hz14A9-2.3 1.5 mg / kg group, n = 6; ④ hz14A9-2.3 5 mg / kg group, n = 6; ⑤ Hu5F9 5 mg / kg group, n = 6. On day 0 (D0), the mice were divided into groups and intravenously injected, once every 3 days, for a total of 2 times, and the tumor diameter was measured twice a week. The efficacy was judged based on the tumor volume.

[0322] The tumor volume (V) is calculated as follows: V = (a × b 2 ) / 2, where a and b represent the length and width respectively.

[0323] T / C(%)=(TT 0 ) / (CC 0 )*100, where T and C are the average tumor volumes of the treatment group and negative control group at the end of the experiment, respectively; T 0 , C 0 These are the average tumor volumes of the treatment group and the negative control group at the beginning of the experiment, respectively.

[0324] Tumor inhibition rate (TGI) (%) = 100% - T / C (%).

[0325] The results are shown in Table 21 and Table 11. Antibody hz14A9-2.3 can effectively inhibit tumor growth at a dose of 1.5 mg / kg, and the tumor growth inhibition rate (or tumor inhibition rate) on day 17 (D17) reached 93%. At a dose of 5 mg / kg, the tumor growth inhibition rate of antibody hz14A9-2.3 reached 125%.

[0326] Table 21

[0327] Sequence Listing <110> Chia Tai Tianqing Pharmaceutical Group Co., Ltd. <120> Antigen binding polypeptides binding to CD47 and uses thereof <160> 198 <170> SIPOSequenceListing 1.0 <210> 1 <211> 10 <212> PRT <213> Mus musculus <400> 1 Gly Phe Ser Leu Thr Asn Tyr Gly Val His 1 5 10 <210> 2 <211> 10 <212> PRT <213> Mus musculus <400> 2 Gly Phe Thr Phe Ser Asn Tyr Tyr Met Ser 1 5 10 <210> 3 <211> 10 <212> PRT <213> Mus musculus <400> 3 Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn 1 5 10 <210> 4 <211> 10 <212> PRT <213> Mus musculus <400> 4 Gly Tyr Thr Phe Ser Arg Tyr Trp Ile Glu 1 5 10 <210> 5 <211> 10 <212> PRT <213> Mus musculus <400> 5 Gly Phe Asn Ile Glu Asp Asp Tyr Ile Glu 1 5 10 <210> 6 <211> 9 <212> PRT <213> Mus musculus <400> 6 Ile Ile Trp Ala Gly Gly Ser Thr Asn 1 5 <210> 7 <211> 10 <212> PRT <213> Mus musculus <400> 7 Tyr Ile Ser Ser Gly Gly Gly Ser Thr Tyr 1 5 10 <210> 8 <211> 10 <212> PRT <213> Mus musculus <400> 8 Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr 1 5 10 <210> 9 <211> 8 <212> PRT <213> Mus musculus <400> 9 Glu Phe Ile Pro Gly Ser Asp Thr 1 5 <210> 10 <211> 10 <212> PRT <213> Mus musculus <400> 10 Arg Ile Asp Pro Ala Asn Asp Lys Thr Lys 1 5 10 <210> 11 <211> 8 <212> PRT <213> Mus musculus <400> 11 Asp Asp Tyr Ala Ser Met Asp Tyr 1 5 <210> 12 <211> 3 <212> PRT <213> Mus musculus <400> 12 Phe Ala Tyr 1 <210> 13 <211> 10 <212> PRT <213> Mus musculus <400> 13 Phe Ser His Leu Arg Gly Pro Met Asp Tyr 1 5 10 <210> 14 <211> 8 <212> PRT <213> Mus musculus <400> 14 Gly Gly Leu Arg Arg Met Asp Tyr 1 5 <210> 15 <211> 11 <212> PRT <213> Mus musculus <400> 15 Pro Gly Leu Arg Arg Tyr Tyr Ser Met Asp Tyr 1 5 10 <210> 16 <211> 16 <212> PRT <213> Mus musculus <400> 16 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 17 <211> 16 <212> PRT <213> Mus musculus <400> 17 Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 18 <211> 16 <212> PRT <213> Mus musculus <400> 18 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Tyr Thr Tyr Leu His 1 5 10 15 <210> 19 <211> 12 <212> PRT <213> Mus musculus <400> 19 Arg Ala Ser Ser Ser Val Ser Ser Thr Tyr Leu His 1 5 10 <210> 20 <211> 11 <212> PRT <213> Mus musculus <400> 20 Lys Ala Ser Glu Asn Val Val Ser Tyr Val Ser 1 5 10 <210> 21 <211> 7 <212> PRT <213> Mus musculus <400> 21 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 22 <211> 7 <212> PRT <213> Mus musculus <400> 22 Leu Val Ser Lys Leu Asp Ser 1 5 <210> 23 <211> 7 <212> PRT <213> Mus musculus <400> 23 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 24 <211> 7 <212> PRT <213> Mus musculus <400> 24 Thr Thr Ser Thr Leu Ala Ser 1 5 <210> 25 <211> 7 <212> PRT <213> Mus musculus <400> 25 Gly Ala Ser Asn Arg Tyr Thr 1 5 <210> 26 <211> 9 <212> PRT <213> Mus musculus <400> 26 Ser Gln Ser Thr His Val Pro Tyr Thr 1 5 <210> 27 <211> 7 <212> PRT <213> Mus musculus <400> 27 Gly Thr His Phe Pro Leu Thr 1 5 <210> 28 <211> 9 <212> PRT <213> Mus musculus <400> 28 Ser Gln Ser Thr His Val Pro Pro Thr 1 5 <210> 29 <211> 9 <212> PRT <213> Mus musculus <400> 29 Gln Gln Phe Ser Asp Ser Thr Trp Thr 1 5 <210> 30 <211> 9 <212> PRT <213> Mus musculus <400> 30 Gly Gln Ser Tyr Ser Tyr Pro Leu Thr 1 5 <210> 31 <211> 9 <212> PRT <213> Artificial Sequence <400> 31 Gln Gln Phe Ser Glu Ser Thr Trp Thr 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> UNSURE <222> (5) <223> Xaa is D or E <400> 32 Gln Gln Phe Ser Xaa Ser Thr Trp Thr 1 5 <210> 33 <211> 116 <212> PRT <213> Mus musculus <400> 33 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Ile Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg Asp Asp Tyr Ala Ser Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser 115 <210> 34 <211> 112 <212> PRT <213> Mus musculus <400> 34 Asp Ala Phe Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Thr Leu Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 35 <211> 119 <212> PRT <213> Mus musculus <400> 35 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Val Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Arg Thr Ala Phe 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Thr Arg Phe Ser His Leu Arg Gly Pro Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ala Ser Val Thr Val Ser Ser 115 <210> 36 <211> 112 <212> PRT <213> Mus musculus <400> 36 Asp Ala Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Tyr Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Leu Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 37 <211> 112 <212> PRT <213> Mus musculus <400> 37 Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 110 <210> 38 <211> 112 <212> PRT <213> Mus musculus <400> 38 Asp Ala Val Val Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 39 <211> 117 <212> PRT <213> Mus musculus <400> 39 Glu Val Gln Leu Gln Gln Ser Val Ala Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Tyr Cys Lys Ala Thr Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Glu Ile Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 40 <211> 108 <212> PRT <213> Mus musculus <400> 40 Asp Ile Val Met Thr Gln Thr Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Asp Ser Thr 85 90 95 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 41 <211> 120 <212> PRT <213> Mus musculus <400> 41 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Val Ser Gly Phe Asn Ile Glu Asp Asp 20 25 30 Tyr Ile Glu Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Asp Lys Thr Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Asp Lys Ala Thr Ile Thr Gly Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Pro Gly Leu Arg Arg Tyr Tyr Ser Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 42 <211> 107 <212> PRT <213> Mus musculus <400> 42 Asn Ile Val Leu Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly 1 5 10 15 Glu Arg Val Thr Leu Ser Cys Lys Ala Ser Glu Asn Val Val Ser Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ile Gly 50 55 60 Ser Gly Ser Ser Thr Asp Phe Thr Leu Ile Ile Ser Ser Phe Gln Pro 65 70 75 80 Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Ser Tyr Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 43 <211> 117 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 43 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Ile Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 44 <211> 108 <212> PRT <213> Artificial Sequence <400> 44 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Asp Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 45 <211> 117 <212> PRT <213> Artificial Sequence <400> 45 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Ile Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 46 <211> 108 <212> PRT <213> Artificial Sequence <400> 46 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Glu Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 47 <211> 117 <212> PRT <213> Artificial Sequence <400> 47 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Ile Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 48 <211> 108 <212> PRT <213> Artificial Sequence <400> 48 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Asp Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 49 <211> 117 <212> PRT <213> Artificial Sequence <400> 49 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Ile Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 50 <211> 108 <212> PRT <213> Artificial Sequence <400> 50 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Glu Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 51 <211> 117 <212> PRT <213> Artificial Sequence <400> 51 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Ile Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 52 <211> 108 <212> PRT <213> Artificial Sequence <400> 52 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Asp Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 53 <211> 117 <212> PRT <213> Artificial Sequence <400> 53 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Ile Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 54 <211> 108 <212> PRT <213> Artificial Sequence <400> 54 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Glu Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 55 <211> 117 <212> PRT <213> Artificial Sequence <400> 55 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Glu Ile Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 56 <211> 108 <212> PRT <213> Artificial Sequence <400> 56 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Asp Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 57 <211> 117 <212> PRT <213> Artificial Sequence <400> 57 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Glu Ile Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 58 <211> 108 <212> PRT <213> Artificial Sequence <400> 58 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Glu Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 59 <211> 117 <212> PRT <213> Artificial Sequence <400> 59 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Glu Ile Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 60 <211> 108 <212> PRT <213> Artificial Sequence <400> 60 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Asp Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 61 <211> 117 <212> PRT <213> Artificial Sequence <400> 61 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Glu Ile Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 62 <211> 108 <212> PRT <213> Artificial Sequence <400> 62 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Glu Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 63 <211> 117 <212> PRT <213> Artificial Sequence <400> 63 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Glu Ile Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 64 <211> 108 <212> PRT <213> Artificial Sequence <400> 64 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Asp Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 65 <211> 117 <212> PRT <213> Artificial Sequence <400> 65 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Glu Ile Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 66 <211> 108 <212> PRT <213> Artificial Sequence <400> 66 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Glu Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 67 <211> 119 <212> PRT <213> Artificial Sequence <400> 67 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Leu Asp Thr Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Ser His Leu Arg Gly Pro Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 68 <211> 112 <212> PRT <213> Artificial Sequence <400> 68 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Tyr Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 69 <211> 119 <212> PRT <213> Artificial Sequence <400> 69 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Gln Glu Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Leu Asp Thr Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Ser His Leu Arg Gly Pro Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 70 <211> 112 <212> PRT <213> Artificial Sequence <400> 70 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Tyr Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 71 <211> 119 <212> PRT <213> Artificial Sequence <400> 71 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Gln Glu Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Leu Asp Thr Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Ser His Leu Arg Gly Pro Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 72 <211> 112 <212> PRT <213> Artificial Sequence <400> 72 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Tyr Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 73 <211> 119 <212> PRT <213> Artificial Sequence <400> 73 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Gln Glu Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Leu Asp Thr Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Ser His Leu Arg Gly Pro Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 74 <211> 112 <212> PRT <213> Artificial Sequence <400> 74 Asp Ala Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Tyr Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 75 <211> 119 <212> PRT <213> Artificial Sequence <400> 75 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Leu Asp Thr Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Ser His Leu Arg Gly Pro Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 76 <211> 112 <212> PRT <213> Artificial Sequence <400> 76 Asp Ala Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Tyr Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 77 <211> 119 <212> PRT <213> Artificial Sequence <400> 77 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Gln Glu Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Leu Asp Thr Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Ser His Leu Arg Gly Pro Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 78 <211> 112 <212> PRT <213> Artificial Sequence <400> 78 Asp Ala Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Tyr Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 79 <211> 120 <212> PRT <213> Artificial Sequence <400> 79 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Phe Asn Ile Glu Asp Asp 20 25 30 Tyr Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Asp Lys Thr Lys Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Gly Asp Thr Ser Thr Asn Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Pro Gly Leu Arg Arg Tyr Tyr Ser Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 80 <211> 107 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 80 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asn Val Val Ser Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ile Gly 50 55 60 Ser Gly Ser Ser Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gln Ser Tyr Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 81 <211> 120 <212> PRT <213> Artificial Sequence <400> 81 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Phe Asn Ile Glu Asp Asp 20 25 30 Tyr Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Asp Lys Thr Lys Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Gly Asp Thr Ser Thr Asn Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Pro Gly Leu Arg Arg Tyr Tyr Ser Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 82 <211> 107 <212> PRT <213> Artificial Sequence <400> 82 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asn Val Val Ser Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ile Gly 50 55 60 Ser Gly Ser Ser Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gln Ser Tyr Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 83 <211> 117 <212> PRT <213> Artificial Sequence <220> <221> UNSURE <222> (73) <223> Xaa is D or E <220> <221> UNSURE <222> (74) <223> Xaa is I or E <220> <221> UNSURE <222> (77) <223> Xaa is S or N <400> 83 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Xaa Xaa Ser Thr Xaa Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 84 <211> 108 <212> PRT <213> Artificial Sequence <220> <221> UNSURE <222> (48) <223> Xaa is L or W <220> <221> UNSURE <222> (71) <223> Xaa is D or S <220> <221> UNSURE <222> (72) <223> Xaa is F or Y <220> <221> UNSURE <222> (94) <223> Xaa is D or E <400> 84 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Xaa 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Xaa Xaa Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Xaa Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 85 <211> 119 <212> PRT <213> Artificial Sequence <220> <221> UNSURE <222> (46) <223> Xaa is E or K <220> <221> UNSURE <222> (63) <223> Xaa is E or K <220> <221> UNSURE <222> (72) <223> Xaa is L or T <220> <221> UNSURE <222> (77) <223> Xaa is R or S <220> <221> UNSURE <222> (97) <223> Xaa is A or T <400> 85 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Xaa Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Gln Xaa Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Xaa Asp Thr Ser Thr Xaa Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Xaa Arg Phe Ser His Leu Arg Gly Pro Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 86 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> UNSURE <222> (2) <223> Xaa is I or A <400> 86 Asp Xaa Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Tyr Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 87 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> UNSURE <222> (24) <223> Xaa is A or V <220> <221> UNSURE <222> (72) <223> Xaa is R or G <220> <221> UNSURE <222> (77) <223> Xaa is S or N <220> <221> UNSURE <222> (97) <223> Xaa is A or T <400> 87 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Xaa Ser Gly Phe Asn Ile Glu Asp Asp 20 25 30 Tyr Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Asp Lys Thr Lys Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Xaa Asp Thr Ser Thr Xaa Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Xaa Arg Pro Gly Leu Arg Arg Tyr Tyr Ser Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 88 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> UNSURE <222> (63) <223> Xaa is S or I <220> <221> UNSURE <222> (68) <223> Xaa is S or G <400> 88 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asn Val Val Ser Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Xaa Gly 50 55 60 Ser Gly Ser Xaa Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gln Ser Tyr Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 89 <211> 443 <212> PRT <213> Artificial Sequence <400> 89 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Ile Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg Asp Asp Tyr Ala Ser Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 210 215 220 Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 90 <211> 1332 <212> DNA <213> Artificial Sequence <400> 90 caggtgcagc tgaaggagtc tggaccagga ctggtggctc catctcagtc cctgagcatc 60 acctgcacag tgtccggctt cagcctgacc aactacggag tgcactgggt gaggcagcca 120 cctggcaagg gactggagtg gctgggcatc atctgggctg gcggctccac aaactataat 180 tctgccctga tgtcccggct gtctatctcc aaggacaaca gcaagtctca ggtgtttctg 240 aagatgaata gcctgcagac cgacgataca gccatgtact attgtgctag ggacgattac gccagcatgg attattgggg ccagggcacc tctgtgacag tgtccagcgc ctctacaaag ggcccttccg tgttcccact ggctccctgc tccagaagca catctgagtc caccgccgct 420 ctgggctgtc tggtgaagga ctacttccct gagccagtga ccgtgtcctg gaacagcggc 480 gccctgacat ctggcgtgca caccttcca gctgtgctgc agtccagcgg cctgtactcc 540 ctgtcttccg tggtgacagt gcccagctct tccctgggca ccaagacata tacctgcaac gtggaccata agccttccaa taccaaggtg gataagaggg tggagagcaa gtacggacca ccttgcccac catgtccagc tcctgagttt ctgggaggac catccgtgtt cctgtttcct 720 ccaaagccta aggacaccct gatgatcagc cggacacctg aggtgacctg cgtggtggtg 780 gacgtgtctc aggaggatcc agaggtgcag ttcaactggt acgtggatgg cgtggaggtg 840 cacaatgcta agaccaagcc aagagaggag cagtttaatt ccacataccg cgtggtgagc gtgctgaccg tgctgcatca ggattggctg aacggcaagg agtataagtg caaggtgtcc 960 aataagggcc tgcccagctc tatcgagaag acaatcagca aggctaaggg acagcctagg 1020 gagccacagg tgtacaccct gcccccttct caggaggaga tgacaaagaa ccaggtgtcc 1080 ctgacctgtc tggtgaaggg cttctatcca agcgacatcg ctgtggagtg ggagtctaat 1140 ggccagcccg agaacaatta caagaccaca ccacccgtgc tggactctga tggctccttc 1200 tttctgtatt ctaggctgac agtggataag tcccggtggc aggagggcaa cgtgtttagc 1260 tgctctgtga tgcacgaggc cctgcacaat cattataccc agaagtccct gagcctgtct 1320 ctgggcaagt ga 1332 <210> 91 <211> 219 <212> PRT <213> Artificial Sequence <400> 91 Asp Ala Phe Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Thr Leu Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 92 <211> 660 <212> DNA <213> Artificial Sequence <400> 92 60. gacgccttca tgacccagac accactgtcc ctgcctgtga gcctgggcga tcaggcttct atctcctgca gatccagcca gtctctggtg cactccaacg gcaatacata cctgcattgg tatctgcaga agccaggcca gtcccccag ctgctgatct acaaggtgag caacaggttc 240. tctggcgtgc ctgaccggtt tagcggctct ggctccggca ccgatttcac actgaagatc tccagggtgg aggctgagga cctggggcgtg tacttttgta gccagtctac ccacgtgcca 300 tatacactgg gcggcggcac caagctggag atcaagcgta cggtggccgc tcccagcgtg 360 ttcatctttc ccccttctga cgagcagctg aagtctggca ccgcttccgt ggtgtgcctg 420 ctgaacaatt tctaccccag agaggccag gtgcagtgga aggtggataa cgctctgcag tccggcaata gccaggagtc tgtgaccgag caggactcca aggatagcac atattctctg tcttccaccc tgacactgtc taaggccgac tacgagaagc acaaggtgta tgcttgcgag 600 gtgacccatc agggcctgag ctctcctgtg acaaagtcct ttaatcgcgg cgagtgttga 660 <210> 93 <211> 446 <212> PRT <213> Artificial Sequence <400> 93 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Val Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Arg Thr Ala Phe 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Thr Arg Phe Ser His Leu Arg Gly Pro Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ala Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 94 <211> 1341 <212> DNA <213> Artificial Sequence <400> 94 gaggtgcagc tgcagcagtc tggccccgag ctgaagaagc ctggcgagac cgtgaagatc 60 tcttgcaagg cctccggcta caccttcaca aactatggca tgaattgggt gaagcaggtg 120 ccaggcaagg gcctgaagtg gatgggctgg atcaacacca atacaggcga gcccacatac 180 gccgaggagt tcaagggcag attcgctttt agcctggaga cctctgcccg cacagctttt 240 ctgcagatca acaatctgaa gaacgaggac accgccacat acttctgtac caggttttcc 300 cacctgcggg gccccatgga ttattggggc cagggcgcta gcgtgacagt gtccagcgcc 360 tctacaaagg gcccttccgt gttcccactg gctccctgct ccagaagcac atctgagtcc 420 accgccgctc tgggctgtct ggtgaaggac tacttccctg agccagtgac cgtgtcctgg 480 aacagcggcg ccctgacatc tggcgtgcac acctttccag ctgtgctgca gtccagcggc 540 ctgtactccc tgtcttccgt ggtgacagtg cccagctt ccctgggcac caacacatat 600 acctgcaacg tggaccataa gccttccaat accaaggtgg atagaggtt ggagagcaag 660 tacggaccac cttgcccacc atgtccagct cctgagtttc tgggaggacc atccgtgttc 720 ctgtttccctc caaagcctaa ggacaccctg atgatcagcc ggacacctga ggtgaccctgc 780 gtggtggtgg acgtgtctca ggaggatcca gaggtgcagt tcactggta cgtggatggc 840 gtggaggtgc acatgctaa gaccaagcca agagaggagc agtttaattc cacataccgc 900 gtggtgagcg tgctgaccgt gctgcatcag gattggctga acggcaagga gtataagtgc 960 aaggtgtcca ataagggcct gcccagctct atcgagaga caatcagcaa ggctaaggga 1020 cagcctagggg agccacaggt gtacaccctg cccccttctc aggaggagat cakaagaac 1080 caggtgtccc tgactgtct ggtgaagggc ttctatccaa gcgacatcgc tgtggagtgg 1140 gagtctaatg gccacccga gacaattac agaccacac cacccgtgct ggactctgat 1200 ggctccttct ttctgtattc taggctgaca gtggataagt cccggtggca ggagggcac 1260 gtgtttagct gctctgtgat gcacgaggcc ctgcacaatc attataccca gaagtccctg 1320 agcctgtctc tgggcaagtg a 1341 <210> 95 <211> 219 <212> PRT <213> Artificial Sequence <400> 95 Asp Ala Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Tyr Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Leu Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 96 <211> 660 <212> DNA <213> Artificial Sequence <400> 96 gacgccgtga tgacccagac accactgtcc ctgcccgtga gcctgggcga tcaggcttct 60 atctcctgca gatccagcca gtctctggtg cactccaacg gctacacata tctgcattgg 120 180. tacctgcaga agcctggcca gtccccaaag ctgctgatct ataaggtgag caataggttc tctggagtgc cagaccggtt tagcggatct ggatccggca ccgatttcac actgaagatc tccaggctgg aggctgagga cctggggcgtg tacttctgta gccagtctac ccacgtgccc 300 cctacatttg gcggcggcac caagctggag atcaagcgta cggtggccgc tcccagcgtg 360 ttcatctttc ccccttctga cgagcagctg aagtctggca ccgcttccgt ggtgtgcctg 420 ctgaacaatt tctaccccag agaggccag gtgcagtgga aggtggataa cgctctgcag tccggcaata gccaggagtc tgtgaccgag caggactcca aggatagcac atattctctg tcttccaccc tgacactgtc taaggccgac tacgagaagc acaaggtgta tgcttgcgag 600. gtgacccatc agggcctgag ctctcctgtg acaaagtcct ttaatcgcgg cgagtgttga 660 <210> 97 <211> 439 <212> PRT <213> Artificial Sequence <400> 97 Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 115 120 125 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 180 185 190 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 195 200 205 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 210 215 220 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 225 230 235 240 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 245 250 255 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 260 265 270 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 275 280 285 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 290 295 300 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 305 310 315 320 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 325 330 335 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 340 345 350 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 355 360 365 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 370 375 380 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 385 390 395 400 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 405 410 415 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 420 425 430 Leu Ser Leu Ser Leu Gly Lys 435 <210> 98 <211> 1320 <212> DNA <213> Artificial Sequence <400> 98 gaagtgatgc tggtggagag cggaggagga ctggtgcagc caggaggcag cctgaagctg 60 tcttgcgccg cttccggctt cacctttagc aactactata tgtcttgggt gagacagaca 120 cccgagaagc gcctggagtg ggtggcttac atctccagcg gaggaggatc cacctactat 180 cctgacagcg tgaagggcag gttcaccatc tctcgggata acgccaagaa tacactgtac 240 ctgcagatgt cttccctgaa gtccgaggac acagctatgt actattgtgc ctggtttgct 300 tattggggcc agggcaccct ggtgacagtg tctgctgcct ctacaaaggg cccttccgtg 360 ttcccactgg ctccctgctc cagaagcaca tctgagtcca ccgccgctct gggctgtctg 420 gtgaaggact acttccctga gccagtgacc gtgtcctgga acagcggcgc cctgacatct 480 ggcgtgcaca cctttccagc tgtgctgcag tccagcggcc tgtactccct gtcttccgtg 540 gtgacagtgc ccagctcttc cctgggcacc aagacatata cctgcaacgt ggaccataag 600 ccttccaata ccaaggtgga taagagggtg gagagcaagt acggaccacc ttgcccacca 660 tgtccagctc ctgagtttct gggaggacca tccgtgttcc tgtttcctcc aaagcctaag 720 gacaccctga tgatcagccg gacacctgag gtgacctgcg tggtggtgga cgtgtctcag 780 gaggatccag aggtgcagtt caactggtac gtggatggcg tggaggtgca caatgctaag 840 accaagccaa gagaggagca gtttaattcc acataccgcg tggtgagcgt gctgaccgtg 900 ctgcatcagg attggctgaa cggcaaggag tataagtgca aggtgtccaa taagggcctg 960 cccagctcta tcgagaagac aatcagcaag gctaagggac agcctaggga gccacaggtg 1020 tacaccctgc ccccttctca ggaggagatg acaaagaacc aggtgtccct gacctgtctg 1080 gtgaagggct tctatccaag cgacatcgct gtggagtggg agtctaatgg ccagcccgag 1140 aacaattaca agaccacacc acccgtgctg gactctgatg gctccttctt tctgtattct 1200 aggctgacag tggataagtc ccggtggcag gagggcaacg tgtttagctg ctctgtgatg 1260 cacgaggccc tgcacaatca ttatacccag aagtccctga gcctgtctct gggcaagtga 1320 <210> 99 <211> 219 <212> PRT <213> Artificial Sequence <400> 99 Asp Ala Val Val Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 100 <211> 660 <212> DNA <213> Artificial Sequence <400> 100 gacgccgtgg tgacccagac accactgacc ctgtccgtga caatcggcca gcccgcttct 60 atctcctgca agtccagcca gtccctgctg gacagcgatg gcaagacata cctgaactgg 120 ctgctgcaga ggccaggaca gagccctaag cggctgatct atctggtgtc taagctggac 180 tccggcgtgc ctgatagatt caccggcagc ggctctggca ccgactttac actgaagatc 240 tctcgcgtgg aggccgagga tctgggcgtg tactattgtt ggcagggcac ccacttccca 300 ctgacatttg gcgctggcac caagctggag ctgaagcgta cggtggccgc tcccagcgtg 360 ttcatctttc ccccttctga cgagcagctg aagtctggca ccgcttccgt ggtgtgcctg 420 ctgaacaatt tctaccccag agaggccaag gtgcagtgga aggtggataa cgctctgcag 480 tccggcaata gccaggagtc tgtgaccgag caggactcca aggatagcac atattctctg 540 tcttccaccc tgacactgtc taaggccgac tacgagaagc acaaggtgta tgcttgcgag 600 gtgacccatc agggcctgag ctctcctgtg acaaagtcct ttaatcgcgg cgagtgttga 660 <210> 101 <211> 444 <212> PRT <213> Artificial Sequence <400> 101 Glu Val Gln Leu Gln Gln Ser Val Ala Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Tyr Cys Lys Ala Thr Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Glu Ile Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 102 <211> 1335 <212> DNA <213> Artificial Sequence <400> 102 gaggtgcagc tgcagcagtc tgtggccgag ctgatgaagc caggcgcttc cgtgaagatc 60 tactgcaagg ccaccggcta cacattcagc agatattgga tcgagtgggt gaagcagagg 120 ccaggacacg gactggagtg gatcggcgag ttcatccctg gctctgacac cacaaacttc 180 aatgagaagt ttaagggcaa ggccaccttt acagctgaga tctccagcaa caccgcttat 240 atgcagctgt cttccctgac atccgaggac agcgccgtgt acttttgtgc taggggcggc 300 ctgaggcgga tggattattg gggccagggc acctccgtga cagtgagctc tgcctctaca 360 aagggccctt ccgtgttccc actggctccc tgctccagaa gcacatctga gtccaccgcc 420 gctctgggct gtctggtgaa ggactacttc cctgagccag tgaccgtgtc ctggaacagc 480 ggcgccctga catctggcgt gcacaccttt ccagctgtgc tgcagtccag cggcctgtac 540 tccctgtctt ccgtggtgac agtgcccagc tcttccctgg gcaccaagac atatacctgc 600 aacgtggacc ataagccttc caataccaag gtggataaga gggtggagag caagtacgga 660 ccaccttgcc caccatgtcc agctcctgag tttctgggag gaccatccgt gttcctgttt 720 cctccaaagc ctaaggacac cctgatgatc agccggacac ctgaggtgac ctgcgtggtg 780 gtggacgtgt ctcaggagga tccagaggtg cagttcaact ggtacgtgga tggcgtggag 840 gtgcacaatg ctaagaccaa gccaagagag gagcagttta attccacata ccgcgtggtg 900 agcgtgctga ccgtgctgca tcaggattgg ctgaacggca aggagtataa gtgcaaggtg 960 tccaataagg gcctgcccag ctctatcgag aagacaatca gcaaggctaa gggacagcct 1020 agggagccac aggtgtacac cctgccccct tctcaggagg agatgacaaa gaaccaggtg 1080 tccctgacct gtctggtgaa gggcttctat ccaagcgaca tcgctgtgga gtgggagtct 1140 aatggccagc ccgagaacaa ttacaagacc acaccacccg tgctggactc tgatggctcc 1200 ttctttctgt attctaggct gacagtggat aagtcccggt ggcaggaggg caacgtgttt 1260 agctgctctg tgatgcacga ggccctgcac aatcattata cccagaagtc cctgagcctg 1320 tctctgggca agtga 1335 <210> 103 <211> 215 <212> PRT <213> Artificial Sequence <400> 103 Asp Ile Val Met Thr Gln Thr Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Asp Ser Thr 85 90 95 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 104 <211> 648 <212> DNA <213> Artificial Sequence <400> 104 gacatcgtga tgacccagac acccgccatc atgtccgcct ctcctggcga gaaggtgacc 60 atgacatgca gggcctccag ctccgtgtcc agcacctacc tgcactggta tcagcagaag 120 agcggcgctt ctccaaagct gtggatctac accacatcca cactggctag cggagtgcca 180 gctcggttct ccggaagcgg atctggcacc tcctatagcc tgacaatctc ttccgtggag 240 gccgaggacg ccgctaccta ctattgtcag cagttctctg attccacctg gacatttggc 300 ggcggcacaa agctggagat caagcgtacg gtggccgctc ccagcgtgtt catctttccc 360 ccttctgacg agcagctgaa gtctggcacc gcttccgtgg tgtgcctgct gaacaatttc 420 taccccagag aggccaaggt gcagtggaag gtggataacg ctctgcagtc cggcaatagc 480 caggagtctg tgaccgagca ggactccaag gatagcacat attctctgtc ttccaccctg 540 acactgtcta aggccgacta cgagaagcac aaggtgtatg cttgcgaggt gacccatcag 600 ggcctgagct ctcctgtgac aaagtccttt aatcgcggcg agtgttga 648 <210> 105 <211> 447 <212> PRT <213> Artificial Sequence <400> 105 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Val Ser Gly Phe Asn Ile Glu Asp Asp 20 25 30 Tyr Ile Glu Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Asp Lys Thr Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Asp Lys Ala Thr Ile Thr Gly Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Pro Gly Leu Arg Arg Tyr Tyr Ser Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 106 <211> 1344 <212> DNA <213> Artificial Sequence <400> 106 gaggtgcagc tgcagcagtc tggagctgag gtggctaggc caggagcttc cgtgaagctg 60 agctgcaccg tgtctggctt caacatcgag gacgattaca tcgagtgggt gaagcagaga 120 ccagagcagg gactggagtg gatcggaagg atcgaccccg ccaacgataa gacaaagtac 180 gctcctaagt ttcaggacaa ggccaccatc acaggcgata cctccagcaa tacagcttat 240 ctgcagctgt cttccctgac ctccgaggac acagccgtgt actattgtac cagacccggc 300 ctgaggcggt actattccat ggattattgg ggccagggca ccagcgtgac agtgagctct 360 gcctctacaa agggcccttc cgtgttccca ctggctccct gctccagaag cacatctgag 420 tccaccgccg ctctgggctg tctggtgaag gactacttcc ctgagccagt gaccgtgtcc 480 tggaacagcg gcgccctgac atctggcgtg cacacctttc cagctgtgct gcagtccagc 540 ggcctgtact ccctgtcttc cgtggtgaca gtgcccagct cttccctggg caccaagaca 600 tatacctgca acgtggacca taagccttcc aataccaagg tggataagag ggtggagagc 660 aagtacggac caccttgccc accatgtcca gctcctgagt ttctgggagg accatccgtg 720 ttcctgtttc ctccaaagcc taaggacacc ctgatgatca gccggacacc tgaggtgacc 780 tgcgtggtgg tggacgtgtc tcaggaggat ccagaggtgc agttcaactg gtacgtggat 840 ggcgtggagg tgcacaatgc taagaccaag ccaagagagg agcagtttaa ttccacatac 900 cgcgtggtga gcgtgctgac cgtgctgcat caggattggc tgaacggcaa ggagtataag 960 tgcaaggtgt ccaataaggg cctgcccagc tctatcgaga agacaatcag caaggctaag 1020 ggacagccta gggagccaca ggtgtacacc ctgccccctt ctcaggagga gatgacaaag 1080 aaccaggtgt ccctgacctg tctggtgaag ggcttctatc caagcgacat cgctgtggag 1140 tgggagtcta atggccagcc cgagaacaat tacaagacca caccacccgt gctggactct 1200 gatggctcct tctttctgta ttctaggctg acagtggata agtcccggtg gcaggagggc 1260 aacgtgttta gctgctctgt gatgcacgag gccctgcaca atcattatac ccagaagtcc 1320 ctgagcctgt ctctgggcaa gtga 1344 <210> 107 <211> 214 <212> PRT <213> Artificial Sequence <400> 107 Asn Ile Val Leu Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly 1 5 10 15 Glu Arg Val Thr Leu Ser Cys Lys Ala Ser Glu Asn Val Val Ser Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ile Gly 50 55 60 Ser Gly Ser Ser Thr Asp Phe Thr Leu Ile Ile Ser Ser Phe Gln Pro 65 70 75 80 Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Ser Tyr Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 108 <211> 645 <212> DNA <213> Artificial Sequence <400> 108 aacatcgtgc tgacccagtc ccctaagagc atgtctatgt ccgtgggcga gagggtgaca 60 ctgtcttgca aggcctccga gaacgtggtg tcctacgtga gctggtatca gcagaagccc 120 gagcagagcc ctaagctgct gatctacggc gcttctaata ggtataccgg agtgccagac 180 cggttcatcg gatccggctc cagcaccgat ttcacactga tcatctcttc ctttcagcca 240 gaggacctgg ccgattacca ctgtggccag agctactctt atcccctgac ctttggcgct 300 ggcacaaagc tggagctgaa gcgtacggtg gccgctccca gcgtgttcat ctttccccct 360 tctgacgagc agctgaagtc tggcaccgct tccgtggtgt gcctgctgaa caatttctac 420 cccagagagg ccaaggtgca gtggaaggtg gataacgctc tgcagtccgg caatagccag 480 gagtctgtga ccgagcagga ctccaaggat agcacatatt ctctgtcttc caccctgaca 540 ctgtctaagg ccgactacga gaagcacaag gtgtatgctt gcgaggtgac ccatcagggc 600 ctgagctctc ctgtgacaaa gtcctttaat cgcggcgagt gttga 645 <210> 109 <211> 444 <212> PRT <213> Artificial Sequence <400> 109 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Ile Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 110 <211> 1335 <212> DNA <213> Artificial Sequence <400> 110 caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggctccag cgtgaaggtg 60 tcttgcaagg cttccggcta caccttctct agatattgga tcgagtgggt gcgccaggct 120 ccaggacagg gactggagtg gatgggcgag ttcatccctg gctccgacac cacaaactac 180 gctcagaagt ttcagggcag ggtgaccatc acagccgata tctccaccag cacagcttat 240 atggagctgt cttccctgag gagcgaggac acagccgtgt actattgtgc tagaggcggc 300 ctgaggcgga tggattactg gggccagggc accctggtga cagtgagctc tgcctctaca 360 aagggccctt ccgtgttccc actggctccc tgctccagaa gcacatctga gtccaccgcc 420 gctctgggct gtctggtgaa ggactacttc cctgagccag tgaccgtgtc ctggaacagc 480 ggcgccctga catctggcgt gcacaccttt ccagctgtgc tgcagtccag cggcctgtac 540 tccctgtctt ccgtggtgac agtgcccagc tcttccctgg gcaccaagac atatacctgc 600 aacgtggacc ataagccttc caataccaag gtggataaga gggtggagag caagtacgga 660 ccaccttgcc caccatgtcc agctcctgag tttctgggag gaccatccgt gttcctgttt 720 cctccaaagc ctaaggacac cctgatgatc agccggacac ctgaggtgac ctgcgtggtg 780 gtggacgtgt ctcaggagga tccagaggtg cagttcaact ggtacgtgga tggcgtggag 840 gtgcacaatg ctaagaccaa gccaagagag gagcagttta attccacata ccgcgtggtg 900 agcgtgctga ccgtgctgca tcaggattgg ctgaacggca aggagtataa gtgcaaggtg 960 tccaataagg gcctgcccag ctctatcgag aagacaatca gcaaggctaa gggacagcct 1020 agggagccac aggtgtacac cctgccccct tctcaggagg agatgacaaa gaaccaggtg 1080 tccctgacct gtctggtgaa gggcttctat ccaagcgaca tcgctgtgga gtgggagtct 1140 aatggccagc ccgagaacaa ttacaagacc acaccacccg tgctggactc tgatggctcc 1200 ttctttctgt attctaggct gacagtggat aagtcccggt ggcaggaggg caacgtgttt 1260 agctgctctg tgatgcacga ggccctgcac aatcattata cccagaagtc cctgagcctg 1320 tctctgggca agtga 1335 <210> 111 <211> 215 <212> PRT <213> Artificial Sequence <400> 111 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Asp Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 112 <211> 648 <212> DNA <213> Artificial Sequence <400> 112 gacatccaga tgacacagag cccatccagc ctgtccgcca gcgtgggcga tagggtgacc 60 atcacatgcc gggcttcttc cagcgtgtct tccacctacc tgcactggta tcagcagaag 120 cccggcaagg cccctaagct gctgatctac accacaagca ccctggcttc tggcgtgcca 180 tccaggttct ctggctccgg cagcggcaca gactttaccc tgacaatcag ctctctgcag 240 cccgaggact tcgccaccta ctattgtcag cagttctctg attccacctg gacatttggc 300 cagggcacaa agctggagat caagcgtacg gtggccgctc ccagcgtgtt catctttccc 360 ccttctgacg agcagctgaa gtctggcacc gcttccgtgg tgtgcctgct gaacaatttc 420 taccccagag aggccaaggt gcagtggaag gtggataacg ctctgcagtc cggcaatagc 480 caggagtctg tgaccgagca ggactccaag gatagcacat attctctgtc ttccaccctg 540 acactgtcta aggccgacta cgagaagcac aaggtgtatg cttgcgaggt gacccatcag 600 ggcctgagct ctcctgtgac aaagtccttt aatcgcggcg agtgttga 648 <210> 113 <211> 444 <212> PRT <213> Artificial Sequence <400> 113 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Ile Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 114 <211> 1335 <212> DNA <213> Artificial Sequence <400> 114 caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggctccag cgtgaaggtg 60 tcttgcaagg cttccggcta caccttctct agatattgga tcgagtgggt gcgccaggct 120 ccaggacagg gactggagtg gatgggcgag ttcatccctg gctccgacac cacaaactac 180 gctcagaagt ttcagggcag ggtgaccatc acagccgata tctccaccag cacagcttat 240 atggagctgt cttccctgag gagcgaggac acagccgtgt actattgtgc tagaggcggc 300 ctgaggcgga tggattactg gggccagggc accctggtga cagtgagctc tgcctctaca 360 aagggccctt ccgtgttccc actggctccc tgctccagaa gcacatctga gtccaccgcc 420 gctctgggct gtctggtgaa ggactacttc cctgagccag tgaccgtgtc ctggaacagc 480 ggcgccctga catctggcgt gcacaccttt ccagctgtgc tgcagtccag cggcctgtac 540 tccctgtctt ccgtggtgac agtgcccagc tcttccctgg gcaccaagac atatacctgc 600 aacgtggacc ataagccttc caataccaag gtggataaga gggtggagag caagtacgga 660 ccaccttgcc caccatgtcc agctcctgag tttctgggag gaccatccgt gttcctgttt 720 cctccaaagc ctaaggacac cctgatgatc agccggacac ctgaggtgac ctgcgtggtg 780 gtggacgtgt ctcaggagga tccagaggtg cagttcaact ggtacgtgga tggcgtggag 840 gtgcacaatg ctaagaccaa gccaagagag gagcagttta attccacata ccgcgtggtg 900 agcgtgctga ccgtgctgca tcaggattgg ctgaacggca aggagtataa gtgcaaggtg 960 tccaataagg gcctgcccag ctctatcgag aagacaatca gcaaggctaa gggacagcct 1020 agggagccac aggtgtacac cctgccccct tctcaggagg agatgacaaa gaaccaggtg 1080 tccctgacct gtctggtgaa gggcttctat ccaagcgaca tcgctgtgga gtgggagtct 1140 aatggccagc ccgagaacaa ttacaagacc acaccacccg tgctggactc tgatggctcc 1200 ttctttctgt attctaggct gacagtggat aagtcccggt ggcaggaggg caacgtgttt 1260 agctgctctg tgatgcacga ggccctgcac aatcattata cccagaagtc cctgagcctg 1320 tctctgggca agtga 1335 <210> 115 <211> 215 <212> PRT <213> Artificial Sequence <400> 115 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Glu Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 116 <211> 648 <212> DNA <213> Artificial Sequence <400> 116 gacatccaga tgacacagag cccatccagc ctgtccgcca gcgtgggcga tagggtgacc 60 atcacatgcc gggcttcttc cagcgtgtct tccacctacc tgcactggta tcagcagaag 120 cccggcaagg cccctaagct gctgatctac accacaagca ccctggcttc tggcgtgcca 180 tccaggttct ctggctccgg cagcggcaca gactttaccc tgacaatcag ctctctgcag 240 cccgaggatt tcgccaccta ctattgtcag cagttctctg agtccacctg gacatttggc 300 cagggcacaa agctggagat caagcgtacg gtggccgctc ccagcgtgtt catctttccc 360 ccttctgacg agcagctgaa gtctggcacc gcttccgtgg tgtgcctgct gaacaatttc 420 taccccagag aggccaaggt gcagtggaag gtggataacg ctctgcagtc cggcaatagc 480 caggagtctg tgaccgagca ggactccaag gatagcacat attctctgtc ttccaccctg 540 acactgtcta aggccgacta cgagaagcac aaggtgtatg cttgcgaggt gacccatcag 600 ggcctgagct ctcctgtgac aaagtccttt aatcgcggcg agtgttga 648 <210> 117 <211> 444 <212> PRT <213> Artificial Sequence <400> 117 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Ile Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 118 <211> 1335 <212> DNA <213> Artificial Sequence <400> 118 caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggctccag cgtgaaggtg 60 tcttgcaagg cttccggcta caccttctct agatattgga tcgagtgggt gcgccaggct 120 ccaggacagg gactggagtg gatgggcgag ttcatccctg gctccgacac cacaaactac 180 gctcagaagt ttcagggcag ggtgaccatc acagccgata tctccaccag cacagcttat 240 atggagctgt cttccctgag gagcgaggac acagccgtgt actattgtgc tagaggcggc 300 ctgaggcgga tggattactg gggccagggc accctggtga cagtgagctc tgcctctaca 360 aagggccctt ccgtgttccc actggctccc tgctccagaa gcacatctga gtccaccgcc 420 gctctgggct gtctggtgaa ggactacttc cctgagccag tgaccgtgtc ctggaacagc 480 ggcgccctga catctggcgt gcacaccttt ccagctgtgc tgcagtccag cggcctgtac 540 tccctgtctt ccgtggtgac agtgcccagc tcttccctgg gcaccaagac atatacctgc 600 aacgtggacc ataagccttc caataccaag gtggataaga gggtggagag caagtacgga 660 ccaccttgcc caccatgtcc agctcctgag tttctgggag gaccatccgt gttcctgttt 720 cctccaaagc ctaaggacac cctgatgatc agccggacac ctgaggtgac ctgcgtggtg 780 gtggacgtgt ctcaggagga tccagaggtg cagttcaact ggtacgtgga tggcgtggag 840 gtgcacaatg ctaagaccaa gccaagagag gagcagttta attccacata ccgcgtggtg 900 agcgtgctga ccgtgctgca tcaggattgg ctgaacggca aggagtataa gtgcaaggtg 960 tccaataagg gcctgcccag ctctatcgag aagacaatca gcaaggctaa gggacagcct 1020 agggagccac aggtgtacac cctgccccct tctcaggagg agatgacaaa gaaccaggtg 1080 tccctgacct gtctggtgaa gggcttctat ccaagcgaca tcgctgtgga gtgggagtct 1140 aatggccagc ccgagaacaa ttacaagacc acaccacccg tgctggactc tgatggctcc 1200 ttctttctgt attctaggct gacagtggat aagtcccggt ggcaggaggg caacgtgttt 1260 agctgctctg tgatgcacga ggccctgcac aatcattata cccagaagtc cctgagcctg 1320 tctctgggca agtga 1335 <210> 119 <211> 215 <212> PRT <213> Artificial Sequence <400> 119 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Asp Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 120 <211> 648 <212> DNA <213> Artificial Sequence <400> 120 gacatccaga tgacacagag cccatccagc ctgtccgcca gcgtgggcga tagggtgacc 60 atcacatgcc gggcttcttc cagcgtgtct tccacctacc tgcactggta tcagcagaag 120 cccggcaagg cccctaagct gctgatctac accacaagca ccctggcttc tggcgtgcca 180 tccaggttct ctggctccgg cagcggcaca tcttataccc tgacaatcag ctctctgcag 240 cccgaggact ttgccaccta ctattgtcag cagttctctg attccacctg gacatttggc 300 cagggcacaa agctggagat caagcgtacg gtggccgctc ccagcgtgtt catctttccc 360 ccttctgacg agcagctgaa gtctggcacc gcttccgtgg tgtgcctgct gaacaatttc 420 taccccagag aggccaaggt gcagtggaag gtggataacg ctctgcagtc cggcaatagc 480 caggagtctg tgaccgagca ggactccaag gatagcacat attctctgtc ttccaccctg 540 acactgtcta aggccgacta cgagaagcac aaggtgtatg cttgcgaggt gacccatcag 600 ggcctgagct ctcctgtgac aaagtccttt aatcgcggcg agtgttga 648 <210> 121 <211> 444 <212> PRT <213> Artificial Sequence <400> 121 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Ile Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 122 <211> 1335 <212> DNA <213> Artificial Sequence <400> 122 caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggctccag cgtgaaggtg 60 tcttgcaagg cttccggcta caccttctct agatattgga tcgagtgggt gcgccaggct 120 ccaggacagg gactggagtg gatgggcgag ttcatccctg gctccgacac cacaaactac 180 gctcagaagt ttcagggcag ggtgaccatc acagccgata tctccaccag cacagcttat 240 atggagctgt cttccctgag gagcgaggac acagccgtgt actattgtgc tagaggcggc 300 ctgaggcgga tggattactg gggccagggc accctggtga cagtgagctc tgcctctaca 360 aagggccctt ccgtgttccc actggctccc tgctccagaa gcacatctga gtccaccgcc 420 gctctgggct gtctggtgaa ggactacttc cctgagccag tgaccgtgtc ctggaacagc 480 ggcgccctga catctggcgt gcacaccttt ccagctgtgc tgcagtccag cggcctgtac 540 tccctgtctt ccgtggtgac agtgcccagc tcttccctgg gcaccaagac atatacctgc 600 aacgtggacc ataagccttc caataccaag gtggataaga gggtggagag caagtacgga 660 ccaccttgcc caccatgtcc agctcctgag tttctgggag gaccatccgt gttcctgttt 720 cctccaaagc ctaaggacac cctgatgatc agccggacac ctgaggtgac ctgcgtggtg 780 gtggacgtgt ctcaggagga tccagaggtg cagttcaact ggtacgtgga tggcgtggag 840 gtgcacaatg ctaagaccaa gccaagagag gagcagttta attccacata ccgcgtggtg 900 agcgtgctga ccgtgctgca tcaggattgg ctgaacggca aggagtataa gtgcaaggtg 960 tccaataagg gcctgcccag ctctatcgag aagacaatca gcaaggctaa gggacagcct 1020 agggagccac aggtgtacac cctgccccct tctcaggagg agatgacaaa gaaccaggtg 1080 tccctgacct gtctggtgaa gggcttctat ccaagcgaca tcgctgtgga gtgggagtct 1140 aatggccagc ccgagaacaa ttacaagacc acaccacccg tgctggactc tgatggctcc 1200 ttctttctgt attctaggct gacagtggat aagtcccggt ggcaggaggg caacgtgttt 1260 agctgctctg tgatgcacga ggccctgcac aatcattata cccagaagtc cctgagcctg 1320 tctctgggca agtga 1335 <210> 123 <211> 215 <212> PRT <213> Artificial Sequence <400> 123 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Glu Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 124 <211> 648 <212> DNA <213> Artificial Sequence <400> 124 gacatccaga tgacacagag cccatccagc ctgtccgcca gcgtgggcga tagggtgacc 60 atcacatgcc gggcttcttc cagcgtgtct tccacctacc tgcactggta tcagcagaag 120 cccggcaagg cccctaagct gctgatctac accacaagca ccctggcttc tggcgtgcca 180 tccaggttct ctggctccgg cagcggcaca tcttataccc tgacaatcag ctctctgcag 240 cccgaggact ttgccaccta ctattgtcag cagttctctg agtccacctg gacatttggc 300 cagggcacaa agctggagat caagcgtacg gtggccgctc ccagcgtgtt catctttccc 360 ccttctgacg agcagctgaa gtctggcacc gcttccgtgg tgtgcctgct gaacaatttc 420 taccccagag aggccaaggt gcagtggaag gtggataacg ctctgcagtc cggcaatagc 480 caggagtctg tgaccgagca ggactccaag gatagcacat attctctgtc ttccaccctg 540 acactgtcta aggccgacta cgagaagcac aaggtgtatg cttgcgaggt gacccatcag 600 ggcctgagct ctcctgtgac aaagtccttt aatcgcggcg agtgttga 648 <210> 125 <211> 444 <212> PRT <213> Artificial Sequence <400> 125 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Ile Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 126 <211> 1335 <212> DNA <213> Artificial Sequence <400> 126 caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggctccag cgtgaaggtg 60 tcttgcaagg cttccggcta caccttctct agatattgga tcgagtgggt gcgccaggct 120 ccaggacagg gactggagtg gatgggcgag ttcatccctg gctccgacac cacaaactac 180 gctcagaagt ttcagggcag ggtgaccatc acagccgata tctccaccag cacagcttat 240 atggagctgt cttccctgag gagcgaggac acagccgtgt actattgtgc tagaggcggc 300 ctgaggcgga tggattactg gggccagggc accctggtga cagtgagctc tgcctctaca 360 aagggccctt ccgtgttccc actggctccc tgctccagaa gcacatctga gtccaccgcc 420 gctctgggct gtctggtgaa ggactacttc cctgagccag tgaccgtgtc ctggaacagc 480 ggcgccctga catctggcgt gcacaccttt ccagctgtgc tgcagtccag cggcctgtac 540 tccctgtctt ccgtggtgac agtgcccagc tcttccctgg gcaccaagac atatacctgc 600 aacgtggacc ataagccttc caataccaag gtggataaga gggtggagag caagtacgga 660 ccaccttgcc caccatgtcc agctcctgag tttctgggag gaccatccgt gttcctgttt 720 cctccaaagc ctaaggacac cctgatgatc agccggacac ctgaggtgac ctgcgtggtg 780 gtggacgtgt ctcaggagga tccagaggtg cagttcaact ggtacgtgga tggcgtggag 840 gtgcacaatg ctaagaccaa gccaagagag gagcagttta attccacata ccgcgtggtg 900 agcgtgctga ccgtgctgca tcaggattgg ctgaacggca aggagtataa gtgcaaggtg 960 tccaataagg gcctgcccag ctctatcgag aagacaatca gcaaggctaa gggacagcct 1020 agggagccac aggtgtacac cctgccccct tctcaggagg agatgacaaa gaaccaggtg 1080 tccctgacct gtctggtgaa gggcttctat ccaagcgaca tcgctgtgga gtgggagtct 1140 aatggccagc ccgagaacaa ttacaagacc acaccacccg tgctggactc tgatggctcc 1200 ttctttctgt attctaggct gacagtggat aagtcccggt ggcaggaggg caacgtgttt 1260 agctgctctg tgatgcacga ggccctgcac aatcattata cccagaagtc cctgagcctg 1320 tctctgggca agtga 1335 <210> 127 <211> 215 <212> PRT <213> Artificial Sequence <400> 127 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Asp Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 128 <211> 648 <212> DNA <213> Artificial Sequence <400> 128 gacatccaga tgacacagag cccatccagc ctgtccgcca gcgtgggcga tagggtgacc 60 atcacatgcc gggcttcttc cagcgtgtct tccacctacc tgcactggta tcagcagaag 120 cccggcaagg cccctaagct gtggatctac accacaagca ccctggcttc tggcgtgcca 180 tccaggttct ctggctccgg cagcggcaca tcttataccc tgacaatcag ctctctgcag 240 cccgaggact ttgccaccta ctattgtcag cagttctctg attccacctg gacatttggc 300 cagggcacaa agctggagat caagcgtacg gtggccgctc ccagcgtgtt catctttccc 360 ccttctgacg agcagctgaa gtctggcacc gcttccgtgg tgtgcctgct gaacaatttc 420 taccccagag aggccaaggt gcagtggaag gtggataacg ctctgcagtc cggcaatagc 480 caggagtctg tgaccgagca ggactccaag gatagcacat attctctgtc ttccaccctg 540 acactgtcta aggccgacta cgagaagcac aaggtgtatg cttgcgaggt gacccatcag 600 ggcctgagct ctcctgtgac aaagtccttt aatcgcggcg agtgttga 648 <210> 129 <211> 444 <212> PRT <213> Artificial Sequence <400> 129 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Ile Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 130 <211> 1335 <212> DNA <213> Artificial Sequence <400> 130 caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggctccag cgtgaaggtg 60 tcttgcaagg cttccggcta caccttctct agatattgga tcgagtgggt gcgccaggct 120 ccaggacagg gactggagtg gatgggcgag ttcatccctg gctccgacac cacaaactac 180 gctcagaagt ttcagggcag ggtgaccatc acagccgata tctccaccag cacagcttat 240 atggagctgt cttccctgag gagcgaggac acagccgtgt actattgtgc tagaggcggc 300 ctgaggcgga tggattactg gggccagggc accctggtga cagtgagctc tgcctctaca 360 aagggccctt ccgtgttccc actggctccc tgctccagaa gcacatctga gtccaccgcc 420 gctctgggct gtctggtgaa ggactacttc cctgagccag tgaccgtgtc ctggaacagc 480 ggcgccctga catctggcgt gcacaccttt ccagctgtgc tgcagtccag cggcctgtac 540 tccctgtctt ccgtggtgac agtgcccagc tcttccctgg gcaccaagac atatacctgc 600 aacgtggacc ataagccttc caataccaag gtggataaga gggtggagag caagtacgga 660 ccaccttgcc caccatgtcc agctcctgag tttctgggag gaccatccgt gttcctgttt 720 cctccaaagc ctaaggacac cctgatgatc agccggacac ctgaggtgac ctgcgtggtg 780 gtggacgtgt ctcaggagga tccagaggtg cagttcaact ggtacgtgga tggcgtggag 840 gtgcacaatg ctaagaccaa gccaagagag gagcagttta attccacata ccgcgtggtg 900 agcgtgctga ccgtgctgca tcaggattgg ctgaacggca aggagtataa gtgcaaggtg 960 tccaataagg gcctgcccag ctctatcgag aagacaatca gcaaggctaa gggacagcct 1020 agggagccac aggtgtacac cctgccccct tctcaggagg agatgacaaa gaaccaggtg 1080 tccctgacct gtctggtgaa gggcttctat ccaagcgaca tcgctgtgga gtgggagtct 1140 aatggccagc ccgagaacaa ttacaagacc acaccacccg tgctggactc tgatggctcc 1200 ttctttctgt attctaggct gacagtggat aagtcccggt ggcaggaggg caacgtgttt 1260 agctgctctg tgatgcacga ggccctgcac aatcattata cccagaagtc cctgagcctg 1320 tctctgggca agtga 1335 <210> 131 <211> 215 <212> PRT <213> Artificial Sequence <400> 131 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Glu Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 132 <211> 648 <212> DNA <213> Artificial Sequence <400> 132 gacatccaga tgacacagag cccatccagc ctgtccgcca gcgtgggcga tagggtgacc 60 atcacatgcc gggcttcttc cagcgtgtct tccacctacc tgcactggta tcagcagaag 120 cccggcaagg cccctaagct gtggatctac accacaagca ccctggcttc tggcgtgcca 180 tccaggttct ctggctccgg cagcggcaca tcttataccc tgacaatcag ctctctgcag 240 cccgaggact ttgccaccta ctattgtcag cagttctctg agtccacctg gacatttggc 300 cagggcacaa agctggagat caagcgtacg gtggccgctc ccagcgtgtt catctttccc 360 ccttctgacg agcagctgaa gtctggcacc gcttccgtgg tgtgcctgct gaacaatttc 420 taccccagag aggccaaggt gcagtggaag gtggataacg ctctgcagtc cggcaatagc 480 caggagtctg tgaccgagca ggactccaag gatagcacat attctctgtc ttccaccctg 540 acactgtcta aggccgacta cgagaagcac aaggtgtatg cttgcgaggt gacccatcag 600 ggcctgagct ctcctgtgac aaagtccttt aatcgcggcg agtgttga 648 <210> 133 <211> 444 <212> PRT <213> Artificial Sequence <400> 133 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Glu Ile Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 134 <211> 1335 <212> DNA <213> Artificial Sequence <400> 134 caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggctccag cgtgaaggtg 60 tcctgcaagg ctagcggcta caccttctct agatattgga tcgagtgggt gcgccaggct 120 ccaggacagg gactggagtg gatgggcgag ttcatccctg gctccgacac cacaaactac 180 gctcagaagt ttcagggcag ggtgaccatc acagccgaga tctctaccaa tacagcttat 240 atggagctgt cttccctgag gtccgaggac acagccgtgt actattgtgc tagaggcggc 300 ctgaggcgga tggattactg gggccagggc accctggtga cagtgagctc tgcctctaca 360 aagggccctt ccgtgttccc actggctccc tgctccagaa gcacatctga gtccaccgcc 420 gctctgggct gtctggtgaa ggactacttc cctgagccag tgaccgtgtc ctggaacagc 480 ggcgccctga catctggcgt gcacaccttt ccagctgtgc tgcagtccag cggcctgtac 540 tccctgtctt ccgtggtgac agtgcccagc tcttccctgg gcaccaagac atatacctgc 600 aacgtggacc ataagccttc caataccaag gtggataaga gggtggagag caagtacgga 660 ccaccttgcc caccatgtcc agctcctgag tttctgggag gaccatccgt gttcctgttt 720 cctccaaagc ctaaggacac cctgatgatc agccggacac ctgaggtgac ctgcgtggtg 780 gtggacgtgt ctcaggagga tccagaggtg cagttcaact ggtacgtgga tggcgtggag 840 gtgcacaatg ctaagaccaa gccaagagag gagcagttta attccacata ccgcgtggtg 900 agcgtgctga ccgtgctgca tcaggattgg ctgaacggca aggagtataa gtgcaaggtg 960 tccaataagg gcctgcccag ctctatcgag aagacaatca gcaaggctaa gggacagcct 1020 agggagccac aggtgtacac cctgccccct tctcaggagg agatgacaaa gaaccaggtg 1080 tccctgacct gtctggtgaa gggcttctat ccaagcgaca tcgctgtgga gtgggagtct 1140 aatggccagc ccgagaacaa ttacaagacc acaccacccg tgctggactc tgatggctcc 1200 ttctttctgt attctaggct gacagtggat aagtcccggt ggcaggaggg caacgtgttt 1260 agctgctctg tgatgcacga ggccctgcac aatcattata cccagaagtc cctgagcctg 1320 tctctgggca agtga 1335 <210> 135 <211> 215 <212> PRT <213> Artificial Sequence <400> 135 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Asp Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 136 <211> 648 <212> DNA <213> Artificial Sequence <400> 136 gacatccaga tgacacagag cccatccagc ctgtccgcca gcgtgggcga tagggtgacc 60 atcacatgcc gggcttcttc cagcgtgtct tccacctacc tgcactggta tcagcagaag 120 cccggcaagg cccctaagct gctgatctac accacaagca ccctggcttc tggcgtgcca 180 tccaggttct ctggctccgg cagcggcaca gactttaccc tgacaatcag ctctctgcag 240 cccgaggact tcgccaccta ctattgtcag cagttctctg attccacctg gacatttggc 300 cagggcacaa agctggagat caagcgtacg gtggccgctc ccagcgtgtt catctttccc 360 ccttctgacg agcagctgaa gtctggcacc gcttccgtgg tgtgcctgct gaacaatttc 420 taccccagag aggccaaggt gcagtggaag gtggataacg ctctgcagtc cggcaatagc 480 caggagtctg tgaccgagca ggactccaag gatagcacat attctctgtc ttccaccctg 540 acactgtcta aggccgacta cgagaagcac aaggtgtatg cttgcgaggt gacccatcag 600 ggcctgagct ctcctgtgac aaagtccttt aatcgcggcg agtgttga 648 <210> 137 <211> 444 <212> PRT <213> Artificial Sequence <400> 137 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Glu Ile Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 138 <211> 1335 <212> DNA <213> Artificial Sequence <400> 138 caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggctccag cgtgaaggtg 60 tcctgcaagg ctagcggcta caccttctct agatattgga tcgagtgggt gcgccaggct 120 ccaggacagg gactggagtg gatgggcgag ttcatccctg gctccgacac cacaaactac 180 gctcagaagt ttcagggcag ggtgaccatc acagccgaga tctctaccaa tacagcttat 240 atggagctgt cttccctgag gtccgaggac acagccgtgt actattgtgc tagaggcggc 300 ctgaggcgga tggattactg gggccagggc accctggtga cagtgagctc tgcctctaca 360 aagggccctt ccgtgttccc actggctccc tgctccagaa gcacatctga gtccaccgcc 420 gctctgggct gtctggtgaa ggactacttc cctgagccag tgaccgtgtc ctggaacagc 480 ggcgccctga catctggcgt gcacaccttt ccagctgtgc tgcagtccag cggcctgtac 540 tccctgtctt ccgtggtgac agtgcccagc tcttccctgg gcaccaagac atatacctgc 600 aacgtggacc ataagccttc caataccaag gtggataaga gggtggagag caagtacgga 660 ccaccttgcc caccatgtcc agctcctgag tttctgggag gaccatccgt gttcctgttt 720 cctccaaagc ctaaggacac cctgatgatc agccggacac ctgaggtgac ctgcgtggtg 780 gtggacgtgt ctcaggagga tccagaggtg cagttcaact ggtacgtgga tggcgtggag 840 gtgcacaatg ctaagaccaa gccaagagag gagcagttta attccacata ccgcgtggtg 900 agcgtgctga ccgtgctgca tcaggattgg ctgaacggca aggagtataa gtgcaaggtg 960 tccaataagg gcctgcccag ctctatcgag aagacaatca gcaaggctaa gggacagcct 1020 agggagccac aggtgtacac cctgccccct tctcaggagg agatgacaaa gaaccaggtg 1080 tccctgacct gtctggtgaa gggcttctat ccaagcgaca tcgctgtgga gtgggagtct 1140 aatggccagc ccgagaacaa ttacaagacc acaccacccg tgctggactc tgatggctcc 1200 ttctttctgt attctaggct gacagtggat aagtcccggt ggcaggaggg caacgtgttt 1260 agctgctctg tgatgcacga ggccctgcac aatcattata cccagaagtc cctgagcctg 1320 tctctgggca agtga 1335 <210> 139 <211> 215 <212> PRT <213> Artificial Sequence <400> 139 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Glu Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 140 <211> 648 <212> DNA <213> Artificial Sequence <400> 140 gacatccaga tgacacagag cccatccagc ctgtccgcca gcgtgggcga tagggtgacc 60 atcacatgcc gggcttcttc cagcgtgtct tccacctacc tgcactggta tcagcagaag 120 cccggcaagg cccctaagct gctgatctac accacaagca ccctggcttc tggcgtgcca 180 tccaggttct ctggctccgg cagcggcaca gactttaccc tgacaatcag ctctctgcag 240 cccgaggatt tcgccaccta ctattgtcag cagttctctg agtccacctg gacatttggc 300 cagggcacaa agctggagat caagcgtacg gtggccgctc ccagcgtgtt catctttccc 360 ccttctgacg agcagctgaa gtctggcacc gcttccgtgg tgtgcctgct gaacaatttc 420 taccccagag aggccaaggt gcagtggaag gtggataacg ctctgcagtc cggcaatagc 480 caggagtctg tgaccgagca ggactccaag gatagcacat attctctgtc ttccaccctg 540 acactgtcta aggccgacta cgagaagcac aaggtgtatg cttgcgaggt gacccatcag 600 ggcctgagct ctcctgtgac aaagtccttt aatcgcggcg agtgttga 648 <210> 141 <211> 444 <212> PRT <213> Artificial Sequence <400> 141 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Glu Ile Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 142 <211> 1335 <212> DNA <213> Artificial Sequence <400> 142 caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggctccag cgtgaaggtg 60 tcctgcaagg ctagcggcta caccttctct agatattgga tcgagtgggt gcgccaggct 120 ccaggacagg gactggagtg gatgggcgag ttcatccctg gctccgacac cacaaactac 180 gctcagaagt ttcagggcag ggtgaccatc acagccgaga tctctaccaa tacagcttat 240 atggagctgt cttccctgag gtccgaggac acagccgtgt actattgtgc tagaggcggc 300 ctgaggcgga tggattactg gggccagggc accctggtga cagtgagctc tgcctctaca 360 aagggccctt ccgtgttccc actggctccc tgctccagaa gcacatctga gtccaccgcc 420 gctctgggct gtctggtgaa ggactacttc cctgagccag tgaccgtgtc ctggaacagc 480 ggcgccctga catctggcgt gcacaccttt ccagctgtgc tgcagtccag cggcctgtac 540 tccctgtctt ccgtggtgac agtgcccagc tcttccctgg gcaccaagac atatacctgc 600 aacgtggacc ataagccttc caataccaag gtggataaga gggtggagag caagtacgga 660 ccaccttgcc caccatgtcc agctcctgag tttctgggag gaccatccgt gttcctgttt 720 cctccaaagc ctaaggacac cctgatgatc agccggacac ctgaggtgac ctgcgtggtg 780 gtggacgtgt ctcaggagga tccagaggtg cagttcaact ggtacgtgga tggcgtggag 840 gtgcacaatg ctaagaccaa gccaagagag gagcagttta attccacata ccgcgtggtg 900 agcgtgctga ccgtgctgca tcaggattgg ctgaacggca aggagtataa gtgcaaggtg 960 tccaataagg gcctgcccag ctctatcgag aagacaatca gcaaggctaa gggacagcct 1020 agggagccac aggtgtacac cctgccccct tctcaggagg agatgacaaa gaaccaggtg 1080 tccctgacct gtctggtgaa gggcttctat ccaagcgaca tcgctgtgga gtgggagtct 1140 aatggccagc ccgagaacaa ttacaagacc acaccacccg tgctggactc tgatggctcc 1200 ttctttctgt attctaggct gacagtggat aagtcccggt ggcaggaggg caacgtgttt 1260 agctgctctg tgatgcacga ggccctgcac aatcattata cccagaagtc cctgagcctg 1320 tctctgggca agtga 1335 <210> 143 <211> 215 <212> PRT <213> Artificial Sequence <400> 143 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Asp Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 144 <211> 648 <212> DNA <213> Artificial Sequence <400> 144 gacatccaga tgacacagag cccatccagc ctgtccgcca gcgtgggcga tagggtgacc 60 atcacatgcc gggcttcttc cagcgtgtct tccacctacc tgcactggta tcagcagaag 120 cccggcaagg cccctaagct gctgatctac accacaagca ccctggcttc tggcgtgcca 180 tccaggttct ctggctccgg cagcggcaca tcttataccc tgacaatcag ctctctgcag 240 cccgaggact ttgccaccta ctattgtcag cagttctctg attccacctg gacatttggc 300 cagggcacaa agctggagat caagcgtacg gtggccgctc ccagcgtgtt catctttccc 360 ccttctgacg agcagctgaa gtctggcacc gcttccgtgg tgtgcctgct gaacaatttc 420 taccccagag aggccaaggt gcagtggaag gtggataacg ctctgcagtc cggcaatagc 480 caggagtctg tgaccgagca ggactccaag gatagcacat attctctgtc ttccaccctg 540 acactgtcta aggccgacta cgagaagcac aaggtgtatg cttgcgaggt gacccatcag 600 ggcctgagct ctcctgtgac aaagtccttt aatcgcggcg agtgttga 648 <210> 145 <211> 444 <212> PRT <213> Artificial Sequence <400> 145 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Glu Ile Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 146 <211> 1335 <212> DNA <213> Artificial Sequence <400> 146 caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggctccag cgtgaaggtg 60 tcctgcaagg ctagcggcta caccttctct agatattgga tcgagtgggt gcgccaggct 120 ccaggacagg gactggagtg gatgggcgag ttcatccctg gctccgacac cacaaactac 180 gctcagaagt ttcagggcag ggtgaccatc acagccgaga tctctaccaa tacagcttat 240 atggagctgt cttccctgag gtccgaggac acagccgtgt actattgtgc tagaggcggc 300 ctgaggcgga tggattactg gggccagggc accctggtga cagtgagctc tgcctctaca 360 aagggccctt ccgtgttccc actggctccc tgctccagaa gcacatctga gtccaccgcc 420 gctctgggct gtctggtgaa ggactacttc cctgagccag tgaccgtgtc ctggaacagc 480 ggcgccctga catctggcgt gcacaccttt ccagctgtgc tgcagtccag cggcctgtac 540 tccctgtctt ccgtggtgac agtgcccagc tcttccctgg gcaccaagac atatacctgc 600 aacgtggacc ataagccttc caataccaag gtggataaga gggtggagag caagtacgga 660 ccaccttgcc caccatgtcc agctcctgag tttctgggag gaccatccgt gttcctgttt 720 cctccaaagc ctaaggacac cctgatgatc agccggacac ctgaggtgac ctgcgtggtg 780 gtggacgtgt ctcaggagga tccagaggtg cagttcaact ggtacgtgga tggcgtggag 840 gtgcacaatg ctaagaccaa gccaagagag gagcagttta attccacata ccgcgtggtg 900 agcgtgctga ccgtgctgca tcaggattgg ctgaacggca aggagtataa gtgcaaggtg 960 tccaataagg gcctgcccag ctctatcgag aagacaatca gcaaggctaa gggacagcct 1020 agggagccac aggtgtacac cctgccccct tctcaggagg agatgacaaa gaaccaggtg 1080 tccctgacct gtctggtgaa gggcttctat ccaagcgaca tcgctgtgga gtgggagtct 1140 aatggccagc ccgagaacaa ttacaagacc acaccacccg tgctggactc tgatggctcc 1200 ttctttctgt attctaggct gacagtggat aagtcccggt ggcaggaggg caacgtgttt 1260 agctgctctg tgatgcacga ggccctgcac aatcattata cccagaagtc cctgagcctg 1320 tctctgggca agtga 1335 <210> 147 <211> 215 <212> PRT <213> Artificial Sequence <400> 147 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Glu Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 148 <211> 648 <212> DNA <213> Artificial Sequence <400> 148 gacatccaga tgacacagag cccatccagc ctgtccgcca gcgtgggcga tagggtgacc 60 atcacatgcc gggcttcttc cagcgtgtct tccacctacc tgcactggta tcagcagaag 120 cccggcaagg cccctaagct gctgatctac accacaagca ccctggcttc tggcgtgcca 180 tccaggttct ctggctccgg cagcggcaca tcttataccc tgacaatcag ctctctgcag 240 cccgaggact ttgccaccta ctattgtcag cagttctctg agtccacctg gacatttggc 300 cagggcacaa agctggagat caagcgtacg gtggccgctc ccagcgtgtt catctttccc 360 ccttctgacg agcagctgaa gtctggcacc gcttccgtgg tgtgcctgct gaacaatttc 420 taccccagag aggccaaggt gcagtggaag gtggataacg ctctgcagtc cggcaatagc 480 caggagtctg tgaccgagca ggactccaag gatagcacat attctctgtc ttccaccctg 540 acactgtcta aggccgacta cgagaagcac aaggtgtatg cttgcgaggt gacccatcag 600 ggcctgagct ctcctgtgac aaagtccttt aatcgcggcg agtgttga 648 <210> 149 <211> 444 <212> PRT <213> Artificial Sequence <400> 149 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Ile Pro Gly Ser Asp Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Glu Ile Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 150 <211> 1335 <212> DNA <213> Artificial Sequence <400> 150 caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggctccag cgtgaaggtg 60 tcctgcaagg ctagcggcta caccttctct agatattgga tcgagtgggt gcgccaggct 120 ccaggacagg gactggagtg gatgggcgag ttcatccctg gctccgacac cacaaactac 180 gctcagaagt ttcagggcag ggtgaccatc acagccgaga tctctaccaa tacagcttat 240 atggagctgt cttccctgag gtccgaggac acagccgtgt actattgtgc tagaggcggc 300 ctgaggcgga tggattactg gggccagggc accctggtga cagtgagctc tgcctctaca 360 aagggccctt ccgtgttccc actggctccc tgctccagaa gcacatctga gtccaccgcc 420 gctctgggct gtctggtgaa ggactacttc cctgagccag tgaccgtgtc ctggaacagc 480 ggcgccctga catctggcgt gcacaccttt ccagctgtgc tgcagtccag cggcctgtac 540 tccctgtctt ccgtggtgac agtgcccagc tcttccctgg gcaccaagac atatacctgc 600 aacgtggacc ataagccttc caataccaag gtggataaga gggtggagag caagtacgga 660 ccaccttgcc caccatgtcc agctcctgag tttctgggag gaccatccgt gttcctgttt 720 cctccaaagc ctaaggacac cctgatgatc agccggacac ctgaggtgac ctgcgtggtg 780 gtggacgtgt ctcaggagga tccagaggtg cagttcaact ggtacgtgga tggcgtggag 840 gtgcacaatg ctaagaccaa gccaagagag gagcagttta attccacata ccgcgtggtg 900 agcgtgctga ccgtgctgca tcaggattgg ctgaacggca aggagtataa gtgcaaggtg 960 tccaataagg gcctgcccag ctctatcgag aagacaatca gcaaggctaa gggacagcct 1020 agggagccac aggtgtacac cctgccccct tctcaggagg agatgacaaa gaaccaggtg 1080 tccctgacct gtctggtgaa gggcttctat ccaagcgaca tcgctgtgga gtgggagtct 1140 aatggccagc ccgagaacaa ttacaagacc acaccacccg tgctggactc tgatggctcc 1200 ttctttctgt attctaggct gacagtggat aagtcccggt ggcaggaggg caacgtgttt 1260 agctgctctg tgatgcacga ggccctgcac aatcattata cccagaagtc cctgagcctg 1320 tctctgggca agtga 1335 <210> 151 <211> 215 <212> PRT <213> Artificial Sequence <400> 151 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp 35 40 45 Ile Tyr Thr Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Asp Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 152 <211> 648 <212> DNA <213> Artificial Sequence <400> 152 gacatccaga tgacacagag cccatccagc ctgtccgcca gcgtgggcga tagggtgacc 60 atcacatgcc gggcttcttc cagcgtgtct tccacctacc tgcactggta tcagcagaag 120 cccggcaagg cccctaagct gtggatctac accacaagca ccctggcttc tggcgtgcca 180 tccaggttct ctggctccgg cagcggcaca tcttataccc tgacaatcag ctctctgcag 240 cccgaggact ttgccaccta ctattgtcag cagttctctg attccacctg gacatttggc 300 cagggcacaa agctggagat caagcgtacg gtggccgctc ccagcgtgtt catctttccc 360 ccttctgacg agcagctgaa gtctggcacc gcttccgtgg tgtgcctgct gaacaatttc 420 taccccagag aggccaaggt gcagtggaag gtggataacg ctctgcagtc cggcaatagc 480 caggagtctg tgaccgagca ggactccaag gatagcacat attctctgtc ttccaccctg 540 acactgtcta aggccgacta cgagaagca...

Claims

1. An isolated antigen-binding polypeptide or antigen-binding portion thereof that binds CD47, wherein the antigen-binding polypeptide comprises the following complementarity determining regions: The heavy chain CDR1 as shown in the amino acid sequence of SEQ ID NO: 5; The heavy chain CDR2 as shown in the amino acid sequence of SEQ ID NO: 10; The heavy chain CDR3 as shown in the amino acid sequence of SEQ ID NO: 15; The light chain CDR1 as shown in the amino acid sequence of SEQ ID NO: 20; The light chain CDR2 as shown in the amino acid sequence of SEQ ID NO: 25; and The light chain CDR3 is shown in the amino acid sequence of SEQ ID NO:

30.

2. The isolated antigen-binding polypeptide or antigen-binding portion thereof according to claim 1, wherein the antigen-binding polypeptide is selected from the group consisting of: (a) comprises a heavy chain variable region comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 41 and a light chain variable region comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 42; or (b) comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 87, and the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:

88.

3. The antigen-binding polypeptide or antigen-binding portion thereof according to claim 2, wherein the antigen-binding polypeptide comprises a heavy chain variable region and a light chain variable region, wherein The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 41, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 42; The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 87, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 88; The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 82; or The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 79, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

80.

4. The antigen-binding polypeptide or antigen-binding portion thereof according to claim 1, wherein The antigen binding polypeptide comprises a heavy chain according to SEQ ID NO: 105 and a light chain according to SEQ ID NO: 107; The antigen binding polypeptide comprises a heavy chain according to SEQ ID NO: 181 and a light chain according to SEQ ID NO: 183; or The antigen binding polypeptide comprises a heavy chain according to SEQ ID NO: 185 and a light chain according to SEQ ID NO:

187.

5. The antigen-binding polypeptide or antigen-binding portion thereof according to claim 1, wherein the antigen-binding polypeptide is chimeric or humanized.

6. The antigen-binding polypeptide or antigen-binding portion thereof according to claim 1, wherein the antigen-binding polypeptide or antigen-binding portion thereof is selected from the group consisting of a monoclonal antibody, a fusion protein, a multispecific antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, an isolated CDR region and a scFv.

7. The antigen-binding polypeptide or antigen-binding portion thereof according to claim 1, wherein The antigen binding polypeptide is of IgG1, IgG2, IgG3 or IgG4 type.

8. The antigen-binding polypeptide or antigen-binding portion thereof according to claim 7, wherein the IgG4-type antigen-binding polypeptide has an S228P mutation according to the EU numbering index.

9. The antigen-binding polypeptide or antigen-binding portion thereof according to any one of claims 1 to 8, wherein the antigen-binding polypeptide or antigen-binding portion thereof exhibits one or a combination of the following properties: (a) binds to CD47 with a KD value of 1.89E-08 or less; (b) blocking the binding of CD47 to SIRPα; (c) promoting macrophage-mediated phagocytosis of cells expressing CD47; (d) no obvious induction of apoptosis of CD4 + T cells; (e) does not cause substantial erythropenia, anemia or hemagglutination of red cells; and (f) The antigen-binding polypeptide or the antigen-binding portion thereof has a melting temperature T≥62°C and an aggregation temperature Tagg≥61°C.

10. An immunoconjugate comprising an antigen-binding polypeptide or antigen-binding portion thereof according to any one of claims 1 to 9, and a therapeutic agent linked or conjugated to the antigen-binding polypeptide or antigen-binding portion thereof.

11. The immunoconjugate of claim 10, wherein the therapeutic agent is a cytotoxic drug, a radioisotope, an immunomodulator or an antibody.

12. A composition comprising component A and a pharmaceutically acceptable carrier, wherein component A is an antigen-binding polypeptide or antigen-binding portion thereof according to any one of claims 1-9, or an immunoconjugate according to claim 10 or 11.

13. An isolated nucleic acid encoding the antigen-binding polypeptide or antigen-binding portion thereof according to any one of claims 1 to 9.

14. A vector comprising the isolated nucleic acid according to claim 13.

15. A host cell comprising the vector according to claim 14 or having the isolated nucleic acid according to claim 13 integrated into its genome.

16. A method for preparing an antigen-binding polypeptide or antigen-binding portion thereof according to any one of claims 1 to 9, comprising: The host cell of claim 15 is cultured under conditions suitable for expressing a nucleic acid encoding an antigen-binding polypeptide or antigen-binding portion thereof according to any one of claims 1 to 9, and the expressed antigen-binding polypeptide or antigen-binding portion thereof is isolated.

17. Use of the isolated antigen-binding polypeptide or antigen-binding portion thereof according to any one of claims 1 to 9, the immunoconjugate according to claim 10 or 11, or the composition according to claim 12 in the preparation of a medicament for reducing tumors or inhibiting tumor cell growth in a subject.

18. Use of an antigen-binding polypeptide or antigen-binding portion thereof according to any one of claims 1 to 9, an immunoconjugate according to claim 10 or 11, or a composition according to claim 12 in the preparation of a medicament for treating cancer in a subject in need thereof.

19. The use according to claim 18, characterized in that The cancer is selected from the group consisting of leukemia, lymphoma, ovarian cancer, breast cancer, colon cancer, rectal cancer, bladder cancer, urothelial cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, esophageal cancer, renal cell carcinoma, thyroid cancer, head and neck cancer, testicular cancer, skin cancer, vascular cancer, brain cancer, eye cancer, oropharyngeal cancer, uterine cancer, neuroblastoma, melanoma, and myelodysplastic syndrome.

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