TW201915017A - BCMA (B cell maturation antigen)-targeting antibody and application thereof - Google Patents

Abstract

The present invention relates to a BCMA (B cell maturation antigen)-specific antibody and a BCMA-targeting immune effector cell. The BCMA-specific antibody has excellent binding properties to BCMA, and after being prepared into a chimeric antigen receptor-modified T cell, the BCMA-specific antibody can well exhibit a killing effect on cells expressing the BCMA. The invention also provides the chimeric antigen receptor-modified T cell prepared by use of the antibody and use thereof.

Description

Target BCMA antibody and its application

The present invention pertains to the field of tumor immunotherapy or diagnosis; more specifically, the present invention relates to antibodies targeting BCMA and uses thereof.

Multiple myeloma (MM) is a common hematological malignancy, accounting for 2% of all cancer deaths. MM is a heterogeneous disease and is mostly caused by chromosomal translocations of t (11;14), t (4;14), t (8;14), del (13), del (17) (among others) (Drach et al., (1998) Blood 92 (3): 802-809; Gertz et al., (2005) Blood 106 (8): 2837-2840; Facon et al., (2001) Blood 97 (6): 1566-1571) . The main condition of multiple myeloma (MM) is the infinite expansion and enrichment of plasma cells in the bone marrow, which leads to osteonecrosis. Patients with MM may experience a variety of disease-related symptoms due to the psychological burden of bone marrow infiltration, bone destruction, renal failure, immunodeficiency, and cancer diagnosis. At present, the main treatment options are chemotherapy and stem cell transplantation. The chemotherapy drugs are mainly steroid, thalidomide, lenalidomide, bortezomib or a combination of various cytotoxic agents. For younger patients, high-dose chemotherapy can be used. Autologous stem cell transplantation.

BCMA (B-cell maturation antigen) is a B-cell maturation antigen, a type III transmembrane protein consisting of 185 amino acid residues, belonging to the TNF receptor superfamily, whose ligand belongs to the TNF superfamily, such as proliferation-inducing ligands. (APRIL), B lymphocyte stimulating factor (BAFF), when BCMA binds to its ligand, initiates proliferation and survival of B cells. BCMA is highly expressed on the surface of plasma cells and multiple myeloma cells, but not in hematopoietic stem cells and other normal tissue cells. Therefore, BCMA can be an ideal target for targeted treatment of MM.

In view of this, there is an urgent need in the art for specific antibodies against BCMA as well as immune effector cells targeting BCMA.

The present invention aims to provide a specific antibody against BCMA and an immune effector cell targeting BCMA.

In a first aspect, the invention provides an antibody that targets BCMA, the antibody being selected from the group consisting of: (1) an antibody comprising a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO: HCDR1 as shown in 60 or 62, and/or comprising HCDR2 as shown in SEQ ID NO: 2, 61 or 63, and/or comprising any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. The HCDR3 is shown; (2) an antibody comprising a light chain variable region comprising the LCDR1 of SEQ ID NO: 6, and/or comprising the LCDR2 of SEQ ID NO: 7, And/or comprising the LCDR3 of any one of SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10; (3) an antibody comprising (1) a heavy chain variable region of the antibody and (2) And a variant of the antibody according to any one of (1) to (3), which is provided with any one of (1) to (3) The antibodies described have the same or similar activities.

In a specific embodiment, the antibody is selected from the group consisting of: (1) an antibody comprising the HCDR1 of SEQ ID NO: 1, the HCDR of SEQ ID NO: 2, and the HCDR3 of SEQ ID NO: And LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8; (2) antibody comprising HCDR1 and SEQ ID shown in SEQ ID NO: NO: HCDR2 shown in 2, HCDR3 shown in SEQ ID NO: 4, and LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 9; An antibody, HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 5, and LCDR1 and SEQ ID NO: 7 shown in SEQ ID NO: LCDR2, LCDR3 shown in SEQ ID NO: 10; (4) an antibody comprising the HCDR1 shown in SEQ ID NO: 60, the HCDR2 shown in SEQ ID NO: 61, and the HCDR3 shown in SEQ ID NO: LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, LCDR3 shown in SEQ ID NO: 10; (5) antibody comprising HCDR1, SEQ ID NO shown by SEQ ID NO: : HCDR2 shown in 63, HCDR3 shown in SEQ ID NO: 5, and LCDR1, SEQ ID NO: 7 shown in SEQ ID NO: 6. The LCDR3 shown in the above, and the LCDR3 shown in SEQ ID NO: 10; (6) The antibody of any one of (1) to (5), and having (1) to (5) The same or similar activity of any of the antibodies described.

In a specific embodiment, the antibody is selected from the group consisting of: (1) an antibody having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 13, and SEQ ID NO: 17 The amino acid sequence, the amino acid sequence shown in SEQ ID NO: 21, the amino acid sequence shown in SEQ ID NO: 56, or the amino acid sequence shown in SEQ ID NO: 58; (2) antibody The light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 11, the amino acid sequence shown in SEQ ID NO: 15, or the amino acid shown in SEQ ID NO: (3) an antibody comprising (1) a heavy chain variable region of the antibody and (2) a light chain variable region of the antibody; (4) an antibody, any one of (1) to (3) A variant of the antibody, which has the same or similar activity as the antibody of any one of (1) to (3).

In a specific embodiment, the antibody is selected from the group consisting of: (1) an antibody, the heavy chain variable region of the antibody having the amino acid sequence set forth in SEQ ID NO: 13 and the light chain of the antibody is The variable region has the amino acid sequence shown by SEQ ID NO: 11; (2) an antibody, wherein the heavy chain variable region of the antibody has the amino acid sequence shown by SEQ ID NO: 17 and the antibody is light The chain variable region has the amino acid sequence of SEQ ID NO: 15; (3) the antibody, the heavy chain variable region of the antibody having the amino acid sequence of SEQ ID NO: 21 and the antibody a light chain variable region having the amino acid sequence of SEQ ID NO: 19; (4) an antibody having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 56 and The light chain variable region of the antibody has the amino acid sequence of SEQ ID NO: 19; (5) the antibody, the heavy chain variable region of the antibody having the amino acid sequence of SEQ ID NO: 58 And the light chain variable region of the antibody has the amino acid sequence of SEQ ID NO: 19; (6) The antibody, which is a variant of the antibody according to any one of (1) to (5), And any of (1)~(5) The same or similar activity of the antibody described below.

In a second aspect, the invention provides an antibody which recognizes the same epitope as the antibody of the first aspect of the invention.

In a third aspect, the invention provides a nucleic acid encoding the antibody of the first or second aspect of the invention.

In a fourth aspect, the present invention provides a performance vector comprising the nucleic acid of the third aspect of the invention.

In a fifth aspect, the present invention provides a host cell comprising the expression vector of the fourth aspect of the invention or the nucleic acid according to the third aspect of the invention integrated in the genome.

In a sixth aspect, the present invention provides the use of the antibody of the first or second aspect of the present invention, a target drug, an antibody drug conjugate or a multifunctional antibody for preparing a tumor cell of a specific target BCMA; Or for preparing a reagent for diagnosing a tumor, the tumor exhibiting BCMA; or for preparing a chimeric antigen receptor-modified immune cell; preferably, the immune cell comprises: T lymphocytes, NK cells or NKT lymphocytes.

In a seventh aspect, the present invention provides a multifunctional immunoconjugate comprising: the antibody of the first or second aspect of the invention; and a functional molecule linked thereto; The functional molecule is selected from the group consisting of a molecule that targets a tumor surface marker, a molecule that inhibits tumors, a molecule that targets a surface marker of an immune cell, or a detectable label. In a specific embodiment, the tumor suppressing molecule is an antitumor cytokine or an antitumor toxin. Preferably, the cytokine comprises: IL-12, IL-15, type I interferon, TNF-alpha. In a specific embodiment, the molecule of the surface marker of the target immune cell is an antibody or a ligand that binds to an immunocyte surface marker; preferably, the immune cell surface marker comprises: CD3, CD16 Further, CD28, more preferably, the antibody that binds to an immunocyte surface marker is an anti-CD3 antibody. In a specific embodiment, the molecule of the surface marker of the target immune cell is an antibody that binds to a T cell surface marker.

In an eighth aspect, the invention provides a nucleic acid encoding the multifunctional immunoconjugate of the seventh aspect of the invention.

In a ninth aspect, the present invention provides the use of the multifunctional immunoconjugate of the seventh aspect of the present invention, for preparing an antitumor drug, or for preparing a reagent for diagnosing a tumor, the tumor exhibiting BCMA; or for preparing Chimeric antigen receptor-modified immune cells; preferably, the immune cells include: T lymphocytes, NK cells, or NKT lymphocytes.

In a tenth aspect, the present invention provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, the extracellular domain comprising the antibody of the first or second aspect of the invention, the antibody preferably Chain antibody or domain antibody.

In a specific embodiment, the intracellular signal domain includes one or more co-stimulation signal domains and/or primary signal domains.

In a specific embodiment, the chimeric antigen receptor further comprises a hinge domain.

In a specific embodiment, the transmembrane domain is selected from the group consisting of alpha, beta, zeta, TCD, CD3, CD3, CD4 CD64, CD80, CD86, CD134, CD137, CD152, CD154, and the transmembrane region of PD1; and/or the costimulatory signal domain is selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, Intracellular signal region of OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C SLP76, TRIM, FcεRIγ, MyD88, and 41BBL; and/or The primary signal domain is selected from the group consisting of TCRξ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as “ICOS”) and CD66d, and CD3ζ, more preferably, the transmembrane domain a transmembrane domain selected from the group consisting of CD8α, CD4, CD45, PD1, CD154 and CD28; and/or said costimulatory signal domain is selected from the group consisting of CD137, CD134, CD28 and OX40; and/or said primary signal domain is selected from the group consisting of CD3ζ, most Preferably, the transmembrane domain is selected from the group consisting of CD8α or CD28, and the costimulatory signal domain is selected from the intracellular signal domain of CD137 or CD28. Signal domain is selected from CD3ζ.

In a specific embodiment, the chimeric antigen receptor comprises the following sequentially linked antibodies, a transmembrane region and an intracellular signal region: the antibody of the first or second aspect of the invention, the transmembrane region of CD8 And the CD3ζ; the antibody according to the first or second aspect of the invention, the transmembrane region of CD8, the intracellular signal region of CD137, and the CD3ζ; the antibody according to the first or second aspect of the invention, the transmembrane region of CD28, The intracellular signal region of CD28 and CD3ζ; or the antibody of the first or second aspect of the invention, the transmembrane region of CD28, the intracellular signal region of CD28, CD137 and CD3ζ.

In an eleventh aspect, the invention provides a nucleic acid encoding the chimeric antigen receptor of the tenth aspect of the invention.

In a twelfth aspect, the present invention provides a performance vector comprising the nucleic acid of the eleventh aspect of the present invention.

In a thirteenth aspect, the present invention provides a virus comprising the vector of the twelfth aspect of the invention. In a preferred embodiment, the virus is a lentivirus.

In a fourteenth aspect, the present invention provides the chimeric antigen receptor according to the tenth aspect of the present invention, or the nucleic acid according to the eleventh aspect of the present invention, or the expression vector according to the twelfth aspect of the present invention, or the present invention Use of the virus of the thirteenth aspect of the invention for the preparation of genetically modified immune cells targeting tumor cells expressing BCMA. In a preferred embodiment, the tumor exhibiting BCMA is multiple myeloma.

In a fifteenth aspect, the present invention provides a genetically modified immune cell transduced with the nucleic acid of the eleventh aspect of the invention, or the expression vector of the twelfth aspect of the invention or the thirteenth aspect of the invention The virus; or the chimeric antigen receptor thereof according to the tenth aspect of the invention, wherein the immune cell is preferably a T lymphocyte, an NK cell or an NKT cell.

In a specific embodiment, the genetically modified immune cell further exhibits a sequence other than the chimeric antigen receptor of the tenth aspect of the invention, the other sequence comprising a cytokine, or another chimera An antigen receptor, or a chemokine receptor, or an siRNA that reduces PD-1 expression, or a protein that blocks PD-L1, or a TCR, or a safety switch; preferably, the cytokine includes IL-12, Preferably, the chemokine receptor comprises CCR2, CCR5, CXCR2, or CXCR4; preferably, the safety switch comprises iCaspase-9, Truncated EGFR Or RQR8.

In a sixteenth aspect, the present invention provides the use of the genetically modified immune cell of the fifteenth aspect of the present invention, which is characterized in that it is used for preparing a tumor suppressing drug, wherein the tumor is a tumor exhibiting BCMA, preferably In one embodiment, the tumor exhibiting BCMA is multiple myeloma.

In a seventeenth aspect, the present invention provides a pharmaceutical composition comprising: the antibody of the first or second aspect of the invention or the nucleic acid encoding the antibody; or the immunoconjugate of the seventh aspect of the invention or A nucleic acid encoding the conjugate; or a chimeric antigen receptor according to the tenth aspect of the invention, or a nucleic acid encoding the chimeric antigen receptor; or the genetically modified immune cell of the fifteenth aspect of the invention.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

Through extensive and intensive research, the inventors have unexpectedly discovered antibodies that specifically bind to BCMA, and these antibodies can be applied to the preparation of various targeted antitumor drugs and drugs for diagnosing tumors. The present invention has been completed on this basis.

The technical terms used herein have the same or similar meanings as commonly understood by those skilled in the art to which the invention pertains. To facilitate an understanding of the invention, some terms are defined as follows.

The term "BCMA" herein refers to a B cell maturation antigen consisting of a type III transmembrane protein consisting of 184 amino acid residues (NCBI Reference Sequence: NP_001183.2) and an amino acid sequence as set forth in SEQ ID No: 37. Show. In a specific embodiment, BCMA refers to human BCMA.

The term "APRIL" herein refers to an A proliferation-inducing ligand which is a proliferation inducing ligand consisting of 184 amino acid residues (NCBI Reference Sequence: NP_003799.1) and belongs to the TNF superfamily.

The term "antibody" as used herein refers to an antigen binding protein of the immune system. The term "antibody" as referred to herein includes intact full length antibody having an antigen binding region and any fragment thereof retained by an "antigen binding portion" or "antigen binding region", or a single strand thereof such as a single chain variable fragment (scFv) ). A native antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains or antigen-binding fragments thereof interconnected by a disulfide bond. The term "antibody" also includes all recombinant forms of antibodies, particularly the antibodies described herein, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and antibody fragments that bind to antigens and derivatives hereinafter. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. VH and VL can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FR). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The constant region of the antibody mediates binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (C1q) of the classical complement system.

Antibody fragments include, but are not limited to, (i) Fab fragments consisting of VL, VH, CL, and CH1 domains, including Fab' and Fab'-SH; (ii) Fd fragments consisting of VH and CH1 domains; (iii) An Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a single variable region (Ward et al., 1989, Nature 341: 544-546); (v) a F(ab')2 fragment , a bivalent fragment comprising two linked Fab fragments; (vi) a single-chain Fv molecule antigen binding site (Bird et al, 1988, Science 242: 423-426; Huston et al, 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883); (vii) bispecific single-chain Fv dimer (PCT/US92/09965); (viii) "dimer" or "trisomy", constructed by gene fusion for multivalent or multiple Specificity fragments (Tomlinson et al, 2000, Methods Enzymol. 326: 461-479; WO94/13804; Holliger et al, 1993, Proc. Natl. Acad. Sci. USA 90: 6444-6448); and (ix) identical or The scFv genetically fused to different antibodies (Coloma & Morrison, 1997, Nature Biotechnology 15, 159-163).

The term "Fc" or "Fc region" as used herein includes a polypeptide comprising an antibody constant region other than the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinges at the N-terminus of these domains. For IgA and IgM, Fc can include a J chain. For IgG, Fc includes the immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is generally defined as comprising residues C226 or P230 at its carboxy terminus, where numbering is according to the EU index of Kabat. For human IgGl, Fc is defined herein to include residue P232 to its carboxy terminus, where numbering is based on the EU index in Kabat. Fc may refer to this region of isolation, or to that region of an Fc polypeptide, such as an antibody, environment. The above "hinge" includes a flexible polypeptide comprising an amino acid between the first and second constant domains of the antibody. Structurally, the IgG CH1 domain ends at position EU220 and the IgG CH2 domain begins at residue EU237. Thus, for IgG, the antibody hinge herein is defined to include 221 (D221 of IgG1) to 231 (A231 of IgG1), where the numbering is according to the EU index of Kabat.

The term "parent antibody" or "parent immunoglobulin" as used herein includes unmodified antibodies which are then modified to produce variants. The parent antibody can be a naturally occurring antibody, or a variant or modified version of a naturally occurring antibody. A parent antibody can refer to the antibody itself, a composition comprising the parent antibody, or an amino acid sequence encoding the same. The term "parent antibody" or "parent immunoglobulin" as used herein includes murine or chimeric antibodies that are subsequently modified to produce a humanized antibody.

The term "variant antibody" or "antibody variant" as used herein includes an antibody sequence that differs from the parent antibody sequence due to at least one amino acid modification compared to the parent. The variant antibody sequences herein preferably have at least about 80%, most preferably at least about 90%, more preferably at least about 95% amino acid sequence identity to the parent antibody sequence. An antibody variant can refer to the antibody itself, comprising the composition of the parent antibody, or encoding other amino acid sequences.

The term "variant" as used herein includes antibody sequences that differ from the parent antibody sequence due to at least one amino acid modification compared to the parent. In a specific embodiment, a variant antibody sequence herein has at least about 80%, preferably at least about 90%, more preferably at least about 95%, more preferably at least about 97%, more preferably at least about 98 with the parent antibody sequence. %, most preferably at least about 99% amino acid sequence identity. An antibody variant can refer to the antibody itself, the composition comprising the parent antibody, or the amino acid sequence encoding the same. The term "amino acid modification" includes amino acid substitutions, additions and/or deletions, and "amino acid substitution" means replacement of an amino acid at a specific position in the parent polypeptide sequence with another amino acid. For example, substituting R94K means that the arginine at position 94 is replaced by an amine acid, and "amino acid insertion" as used herein means the addition of an amino acid at a specific position in the parent polypeptide sequence. As used herein, "amino acid deletion" or "deletion" means removal of an amino acid at a particular position in the parent polypeptide sequence.

The term "conservative modification" or "conservative sequence modification" as used herein means an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions, and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and mutagenesis of PCR media. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. A family of amino acid residues having similar side chains have been defined in the art. These families include amino acids containing basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, aspartame, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg, alanine, lysine, amine) Acid, isoleucine, valine, phenylalanine, methionine, beta branched side chains (eg, sulphate, valine, isoleucine) and aromatic side chains (eg, tyramine) Acid, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of the antibody of the invention or in the framework regions can be replaced with other amino acid residues of the same side chain family, and the altered antibody (variant antibody) can be tested. Reserved features.

All immunoglobulin heavy chain constant region positions discussed in the present invention are based on Kabat's EU index number (Kabat et al., 1991, sequences of proteins of immunological interest, 5th edition, United States Public Health Service, National Institutes of Health, Bethesda, integrated by reference to the full text). "EU index of Kabat" refers to the residue numbering of the human IgGl EU antibody as described by Edelman et al., 1969, Biochemistry 63: 78-85.

The term "antigenic epitope" as used herein, also referred to as an epitope, may consist of a contiguous sequence of BCMA protein sequences or a discontinuous three-dimensional structure of the BCMA protein sequence.

The term "chimeric antigen receptor" or "CAR" as used herein, refers to a polypeptide comprising an extracellular domain capable of binding an antigen, a transmembrane domain, and a cytoplasmic signal that is transmitted to a domain (ie, an intracellular signal domain), intracellularly A signal domain is a protein that transmits information to a cell via a defined signal transduction pathway to regulate cellular activity, or a protein that acts as an effector through a corresponding signal to the messenger, including the primary signal domain, and A functional signal transduction domain (ie, a costimulatory signal domain) derived from a stimulus molecule as defined below may be included. The intracellular signal domain produces a signal that promotes immune effector function of cells of the CAR (eg, CAR T cells), examples of immune effector functions, such as in CART cells, including cell lytic activity and helper activity, including cytokine secretion.

The term "primary signal domain" modulates the initial activation of the TCR complex in an irritating manner. In one aspect, the primary signal domain is initiated by, for example, binding of a TCR/CD3 complex to a peptide-loaded MHC molecule, thereby mediating T cell responses (including, but not limited to, proliferation, activation, differentiation, etc.). The primary signal domain that functions in an irritating manner may comprise an immunoreceptor tyrosine promoter motif or an ITAM signal transduction motif. Examples of primary signal domains comprising ITAM that are particularly useful in the present invention include, but are not limited to, those derived from TCR, FcR, FcR, CD3, CD3, CD3, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"). And the sequence of CD66d, in the particular CAR of the invention, in any one or more of the CARs of the invention, the intracellular signal transduction domain comprises an intracellular signalling sequence, such as the primary signal domain of CD3ξ.

The term "co-stimulatory signal domain" refers to a "co-stimulatory molecule" which is a related binding partner on a T cell that specifically binds to a costimulatory ligand, whereby a co-stimulatory response of a vector T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules or ligands of non-antigen receptors required for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137) ).

In the present invention, in one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the intracellular signal transduction domain is derived from A functional signal transduction domain that stimulates a molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain, the intracellular signal transduction domain comprising a function derived from a costimulatory molecule A sexual signaling domain and a functional signal transduction domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular conduction domain, the intracellular signal transduction domain comprising one or more costimulatory molecules At least two functional signal transduction domains and a functional signal transduction domain derived from a stimulatory molecule. In one aspect, the CAR comprises an optional leader sequence at the amino acid (ND end) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (eg, scFv) during processing and localization of the CAR cell to the cell membrane.

The term "CD3" is defined herein as a protein provided by GenBan Accession No. BAG36664.1, or an equivalent residue from a non-human species such as a mouse, rodent, monkey, donkey, and the like. "CD3ξ domain" is defined as an amino acid residue from the cytoplasmic domain of the ξ chain that is sufficient to functionally deliver the initial signal required for T cell activation. In one aspect, the cytoplasmic domain of sputum comprises residues 52 to 164 of GenBan Accession No. BAG36664.1, a functional ortholog thereof - from non-human species such as mice, rodents, monkeys, baboons, etc. Valence residue.

The term "4-1BB" herein refers to a member of the TNFR superfamily having an amino acid sequence of GenBank Acc. No. AAA62478.2, or from a non-human species such as a mouse, a rodent, a monkey, a donkey, etc. Valency residue; "4-1BB costimulatory domain" is defined as the amino acid sequence 214-255 of GenBank ACC. No. AAA62478.2, or from non-classified species such as mice, rodents, monkeys, baboons, etc. Equivalent residue. In one aspect, the "4-1BB costimulatory domain" is the sequence provided in SEQ ID NO: 35, or an equivalent residue from a non-human species such as a mouse, rodent, monkey, donkey, and the like.

The term "interferon" as used herein refers to a full-length interferon, or substantially retains the biological activity of a full-length wild-type interferon (eg, maintaining a full length of at least 80%, preferably at least 90%, more preferably at least 95%, 98%, or 99%) Interferon fragment (truncated interferon) or interferon mutant. Interferons include type I interferons (eg, interferon alpha and interferon beta) and type II interferons (eg, interferon gamma).

The antibody of the present invention or a variant thereof can be applied to the preparation of various target antitumor drugs as well as drugs for diagnosing tumors, in particular, for the preparation of immunological effector cells of the target BCMA.

anti- BCMA Antibody

In the present disclosure, antigen binding proteins having an antigen binding region based on scFv, including antibodies, are described. Where recombinant BCMA was used, scFv was selected from the human scFv phage display gene bank. These molecules show fine specificity. For example, the antibody only recognizes K562 cells that stably express BCMA, and does not recognize K562 cells.

In some embodiments, the invention encompasses an antibody having a scFv sequence fused to one or more heavy chain constant regions to form an antibody having a human immunoglobulin Fc region to produce a bivalent protein, thereby increasing antibody Overall affinity and stability. In addition, the Fc portion allows for the direct conjugation of other molecules (including but not limited to fluorescent dyes, cytotoxins, radioisotopes, etc.) to antibodies such as those used in antigen quantification studies in order to immobilize antibodies for affinity measurement, for targeted delivery. Therapeutic agents, immunocytokines are used to test the cytotoxicity of Fc vectors and many other applications.

The results presented herein highlight the specificity, sensitivity, and utility of the antibodies of the invention in targeting BCMA.

The molecules of the invention are based on the use of phage display to identify and select single-chain variable fragments (scFv), the amino acid sequence of which provides the specificity of the molecule for BCMA and forms the basis of all of the antigen-binding proteins disclosed herein. . Thus, the scFv can be used to design a range of different "antibody" molecules including, for example, full length antibodies, fragments thereof such as Fab and F(ab')2, fusion proteins (including scFv_Fc), multivalent antibodies, ie, having the same More than one specific antibody of an antigen or a different antigen, for example, a bispecific T cell-binding antibody (BiTE), a tri-antibody, etc. (Cuesta et al, Multivalent antibodies: when design surpasses evolution, Trends in Biotechnology 28: 355-362, 2010).

In one embodiment where the antigen binding protein is a full length antibody, the heavy and light chains of the antibody of the invention may be full length (eg, the antibody may comprise at least one and preferably two intact heavy chains, and at least one and preferably two The intact light chain) may alternatively comprise an antigen binding moiety (Fab, F(ab')2, Fv or scFv). In other embodiments, the antibody heavy chain constant region is selected, for example, from IgGl, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. The choice of antibody type will depend on the immune effector function that the designed antibody is intended to elicit. Suitable amino acid sequences for the constant regions of various immunoglobulin isotypes and methods for producing a wide variety of antibodies are known in the art to which the present invention pertains in the construction of recombinant immunoglobulins.

In a first aspect, the invention provides an antibody or fragment thereof that binds to BCMA, comprising a heavy chain CDR1 comprising an amino acid sequence of any one of SEQ ID NO: 1, 60, 62, and/or comprising SEQ ID NO: 2 , the heavy chain CDR2 of any of the amino acid sequences of 61, 63, and/or the heavy chain CDR3 comprising any of the amino acid sequences of SEQ ID NO: 3, 4, 5. In another aspect, the invention provides an antibody or fragment thereof that binds to BCMA, comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, and/or comprising an amino acid sequence of SEQ ID NO: Light chain CDR2, and/or light chain CDR3 comprising any of the amino acid sequences of SEQ ID NO: 8, 9, and 10. In another aspect, the invention provides an antibody or fragment thereof that binds to BCMA, comprising a heavy chain CDR1 comprising an amino acid sequence of any one of SEQ ID NO: 1, 60, 62, and/or comprising SEQ ID NO: 2, a heavy chain CDR2 of any amino acid sequence of 61,63, and/or a heavy chain CDR3 comprising an amino acid sequence of any one of SEQ ID NOs: 3, 4, 5, and/or an amine comprising SEQ ID NO: 6. The light chain CDR1 of the base acid sequence, and/or the light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and/or the light chain comprising the amino acid sequence of any of SEQ ID NO: 8, 9, 10 CDR3. Preferably, the BCMA-binding antibody or fragment thereof comprises a heavy chain CDR1 comprising an amino acid sequence of any one of SEQ ID NO: 1, 60, 62, and an amino acid comprising any one of SEQ ID NO: 2, 61, 63 a heavy chain CDR2 of the sequence, and a heavy chain CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 3, 4, 5, and/or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, and comprising The light chain CDR2 of the amino acid sequence of SEQ ID NO: 7, and the light chain CDR3 comprising the amino acid sequence of any of SEQ ID NO: 8, 9, and 10. More preferably, the BCMA-binding antibody or fragment thereof comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, 60, 62, and an amino acid sequence comprising SEQ ID NO: 2, 61, 63 a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 3, 4, 5, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, and comprising SEQ ID NO The light chain CDR2 of the amino acid sequence of 7 and the light chain CDR3 comprising the amino acid sequence of any of SEQ ID NO: 8, 9, and 10.

In another aspect, the invention provides an antibody or fragment thereof that binds to BCMA comprising a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 13, 17, 21, 56, 58.

In another aspect, the invention provides an antibody or fragment thereof that binds to BCMA comprising a light chain variable region sequence selected from the group consisting of SEQ ID NOs: 11, 15, 19 .

Given that each of the heavy and light chain variable region sequences can bind to BCMA, the heavy and light chain variable region sequences can be "mixed and matched" to produce the anti-BCMA binding molecules of the invention.

In another aspect, the invention provides variants of an antibody or fragment thereof that binds BCMA. Thus the invention provides an antibody or fragment thereof having a heavy chain and/or light chain variable region that is at least 80% identical to the variable region sequence of a heavy or light chain. Preferably, the amino acid sequence identity of the heavy and/or light chain variable regions is at least 85%, more preferably at least 90%, most preferably at least 95%, especially 96%, more particularly 97%, even more specific. 98%, most particularly 99%, including for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% and 100%. The variant can be obtained by using the antibody described in the present application as a parent antibody, by yeast library screening, phage library screening, point mutation or the like. As the method used in Example 10 of the present application, the antibody 23F10 was used as the parent antibody, and the phage library screening method was used for mutation modification.

In another aspect, the invention provides an antibody that recognizes the same epitope as the anti-BCMA antibody described above.

anti- BCMA Antibody characteristics

Standard assays for assessing the binding capacity of antibodies, such as antibodies against BCMA, are known in the art and include, for example, ELISA, biacore, Western blots, and flow cytometry analysis. Suitable assays are described in detail in the examples.

Nucleic acids, vectors and host cells

The invention also provides nucleic acids, vectors, and host cells comprising the nucleic acid or vector, which encode an antibody that binds to BCMA and fragments thereof. The nucleic acid can be located in intact cells, in cell lysates, or in partially purified or substantially purified form.

The nucleic acids of the invention can be obtained using standard molecular biology techniques, for example, by standard PCR amplification or cDNA transduction techniques, to obtain light and heavy chains encoding antibodies or cDNA encoding VH and VL segments. For antibodies obtained from immunoglobulin gene gene pools (eg, using phage display technology), one or more nucleic acids encoding the antibodies can be recovered from the gene bank. Methods for introducing foreign nucleic acids into host cells are generally known in the art and can vary with the host cell used.

Preferred nucleic acid molecules of the invention are those selected from the group consisting of SEQ ID NO: 12, 16, 20 which encode a light chain variable region, and/or which encode a heavy chain variable region selected from the group consisting of SEQ ID NO: 14, 18, 22, Those of 57,59. More preferred are nucleic acid molecules comprising a sequence of SEQ ID NO: 14 encoding a heavy chain, and a sequence comprising SEQ ID NO: 12 encoding a light chain or comprising a sequence encoding SEQ ID NO: 18, And a sequence comprising the SEQ ID NO: 16 encoding a light chain or comprising a sequence encoding the heavy chain of SEQ ID NO: 22, and comprising the sequence encoding the light chain SEQ ID NO: 20 or comprising the heavy chain SEQ ID NO: 57, And a sequence comprising the SEQ ID NO: 20 encoding a light chain or comprising a sequence encoding the heavy chain of SEQ ID NO: 59, and comprising the sequence encoding the light chain of SEQ ID NO: 20.

To express a protein, a nucleic acid encoding an antibody of the invention can be integrated into a performance vector. A variety of performance vectors are available for protein expression. The expression vector can include a self-replicating extra-chromosomal vector, or a vector integrated into the host genome. Expression vectors for use in the present invention include, but are not limited to, those which enable expression of proteins in mammalian cells, bacteria, insect cells, yeast, and in vitro systems. As is known in the art, a variety of performance vectors are commercially available or otherwise available. It can be used in the present invention to express antibodies.

Immunoconjugate

The invention also provides a multifunctional immunoconjugate comprising an antibody described herein and further comprising at least one other type of functional molecule. The functional molecule is selected from, but not limited to, a molecule that targets a tumor surface marker, a molecule that inhibits tumors, a molecule that targets a surface marker of an immune cell, or a detectable label. The antibody and the functional molecule can form a conjugate by covalent attachment, coupling, attachment, crosslinking, and the like.

In a preferred embodiment, the immunoconjugate may comprise: an antibody of the invention and at least one molecule that targets a tumor surface marker or a tumor suppressor molecule. The tumor suppressing molecule may be an anti-tumor cytokine or an anti-tumor toxin; preferably, the cytokine includes, but is not limited to, IL-2, IL-7, IL-12, IL. -15, type I IFN, TNF-alpha. In a specific embodiment, the molecule of the target tumor surface marker is a molecule that targets a surface marker of the same tumor to which the antibody of the invention targets. For example, the molecule of the target tumor surface marker can be an antibody or ligand that binds to a tumor surface marker, for example, can cooperate with the antibody of the present invention to more accurately target tumor cells.

In a preferred embodiment, the immunoconjugate can comprise: an antibody of the invention and a detectable label. The detectable label includes, but is not limited to, a fluorescent label, a chromogenic label; for example, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, a radioactive material, a positron emitting metal, and a non- Radioactive paramagnetic metal ions. More than one marker may also be included. Markers for labeling antibodies for detection and/or analysis and/or diagnostic purposes depend on the particular detection/analysis/diagnostic techniques and/or methods used, such as immunohistochemical staining (tissue) samples, flow cytometry, etc. . Suitable labels for detection/analysis/diagnostic techniques and/or methods known in the art are well known to those skilled in the art to which the present invention pertains.

In a preferred embodiment, the immunoconjugate can comprise: an antibody of the invention and a molecule that is a surface marker of the target immune cell. The molecule of the surface marker of the target immune cell may be an antibody or a ligand that binds to an immunocyte surface marker, and is capable of recognizing an immune cell, which carries the antibody of the present invention to the immune cell, and the antibody of the present invention can immunize the cell Targeting tumor cells, thereby inducing immune cells to specifically kill tumors. The immune cell surface marker may be selected from the group consisting of CD3, CD16, CD28, and more preferably, the antibody that binds to the immune cell surface marker is an anti-CD3 antibody. The immune cells can be selected from the group consisting of T cells, NK cells, and NKT cells.

As a means of chemically producing an immunoconjugate by direct or indirect (eg, via a conjugate) conjugation, the immunoconjugate can be produced as a fusion protein comprising an antibody of the invention and a suitable Other proteins. The fusion protein can be produced by methods known in the art, for example, by constructing a nucleic acid molecule comprising a nucleotide sequence encoding the antibody and encoding a suitable labeled nucleoside, and subsequently expressing the nucleic acid molecule. Acid sequence.

Another aspect of the invention provides a nucleic acid molecule encoding at least one antibody, functional variant or immunoconjugate thereof of the invention. Once the relevant sequences are obtained, the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually carried out by transferring it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by a conventional method.

The invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins. The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.

Containing resistance BCMA Chimeric antigen receptor

The invention also provides a plurality of chimeric antigen receptors (CARs) comprising an antibody or antibody fragment of the invention, the CAR-T cells exhibiting anti-tumor properties. In some embodiments, cells (e.g., T cells) are transduced with a viral vector encoding CAR. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the cells can stably express CAR.

In a preferred embodiment, the BCMA binding portion of CAR is a scFv antibody fragment that retains an equivalent affinity binding force, such as it binds to the same antigen with comparable efficacy, as compared to the IgG antibody from which it is derived. The antibody fragment is functional, thereby providing a biochemical reaction, such as initiating an immune response, inhibiting the initiation of signal transduction from its target antigen, inhibiting kinase activity, and the like. Accordingly, the present invention provides a BCMA-CAR comprising a WT1 binding domain engineered into a T cell, and a method for use in adoptive immunotherapy.

In one aspect, the anti-BCMA antigen binding domain of CAR is a scFv antibody fragment that is humanized relative to the murine sequence scFv from which it is derived.

In one aspect, the CAR of the invention combines the antigen binding domain of a particular antibody with an intracellular signal transduction molecule. For example, in some aspects, intracellular signal transduction molecules include, but are not limited to, CD3 ξ chain, 4-1BB and CD28 signal transduction modules, and combinations thereof.

In one aspect, the BCMA-CAR comprises at least one intracellular signal transduction domain that selects a CD137 (4-1BB) signal transduction domain, a CD28 signal transduction domain, a CD3 deuterium signal domain, and any combination thereof. In one aspect, the BCMA-CAR comprises at least one intracellular signal transduction domain derived from one or more costimulatory molecules that are not CD137 (4-1BB) or CD28.

By way of illustration, the sequence of BCMA-CAR can be 7A12-BBZ (SEQ ID NO: 75), 25C2-BBZ (SEQ ID NO: 76), 25D2-BBZ (SEQ ID NO: 77), 7G2-BBZ (SEQ ID NO: 78), 7A12-28Z (SEQ ID NO: 79), 7A12-28BBZ (SEQ ID NO: 80), 7G2-28Z (SEQ ID NO: 81), 7G2-28BBZ (SEQ ID NO: 82), 25C2-28Z (SEQ ID NO: 83), 25C2-28BBZ (SEQ ID NO: 84), 25D2-28Z (SEQ ID NO: 85), 25D2-28BBZ (SEQ ID NO: 86), SEQ ID NO: 75 above Transmembrane and intracellular domains of -86 Those skilled in the art to which the present invention pertains can select conventional transmembrane and intracellular domains for substitution, and all fall within the scope of this application.

Chimeric antigen receptor modified T cell

The invention also provides immune cells comprising a chimeric antigen receptor of the invention.

In another aspect, the present invention provides a chimeric antigen receptor-modified T cell which further carries a coding sequence for a foreign cytokine; preferably, the cytokine comprises: IL-12, IL-15 or IL -twenty one. The immune cells are preferably selected from T lymphocytes, NK cells or NKT cells.

In another aspect, the chimeric antigen receptor-modified T cells provided by the invention further comprise a PD-L1 blocker or a protein that blocks PD-L1, such as native PD-1, or a mutation capable of binding to PD-L1. PD-1, or a fragment of native or mutant PD-1 capable of binding to PD-L1, or an antibody against PD-L1. By way of illustration, the PD-L1 blocker may comprise the amino acid sequence encoded by SEQ ID NO:70.

Drug composition

The antibody of the present invention, the immunoconjugate comprising the antibody, and the genetically modified immune cell can be applied to the preparation of a pharmaceutical composition or a diagnostic reagent. The composition may comprise a pharmaceutically acceptable carrier in addition to an effective amount of the antibody, immunoconjugate or immune cell. The term "pharmaceutically acceptable" means that when the molecular body and composition are suitably administered to an animal or a human, they do not produce an adverse, allergic or other untoward reaction.

Specific examples of some substances which can be used as pharmaceutically acceptable carriers or components thereof are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof such as carboxymethyl cellulose Sodium, ethyl cellulose and methyl cellulose; western yellow gum powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, Sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; Coloring agents; flavoring agents; compressed tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers.

The composition of the present invention can be formulated into various dosage forms as needed, and can be administered by a physician in accordance with factors such as patient type, age, body weight, and general disease condition, mode of administration, and the like. The mode of administration can be, for example, injection or other treatment.

Advantages of the invention:

1. The invention provides specific antibodies against BCMA;

2. The invention provides immune effector cells that target BCMA;

3. The antibody of the present invention is capable of efficiently binding to tumor cells expressing BCMA, and the immune effector cells of the present invention exhibit significant cytotoxic ability against tumor cells expressing BCMA, and therefore, the antibody and immune effector cells of the present invention can be effectively and safely applied. The treatment of multiple myeloma has laid the material foundation for the treatment of multiple myeloma.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the manufacturer. Recommended conditions. Example 1. Preparation of BCMA recombinant protein a. BCMA_huFc, BCMA_muFc expression plastid construction

The extracellular segment of the human BCMA, Met1-Ala54 (SEQ ID NO: 38) gene (SEQ ID NO: 39), was inserted in vitro, and the gene was inserted into the eukaryotic expression plastid of the Fc segment Asp104-Lys330 containing the human IgG1 heavy chain constant region. In the middle, the middle is joined by "GS" to form a fusion expression protein BCMA_huFc (SEQ ID NO: 40), and the corresponding gene sequence is shown in SEQ ID NO: 41.

The BCMA extracellular domain gene (SEQ ID NO: 39) was inserted into the eukaryotic expression plastid of the Fc fragment Arg100-Lys324 containing the murine IgG1 heavy chain constant region, and ligated in the middle with "GS" to form a fusion expression protein BCMA_muFc (SEQ ID NO: 42), the corresponding gene sequence is shown in SEQ ID NO:43. b. Transient transfection performance BCMA_huFc, BCMA_muFc (1) One day before transfection, inoculate 6~7x10 ⁵ /mL 293F cells in 125 mL culture flask; (2) Adjust 3x10 ⁷ cells in 28 mL FreeStyle ^TM 293 on the day of transfection (3) Prepare the lipid-DNA complex according to the following procedure: Dilute 30 μg of DNA with Opti-MEM I, final volume 1 mL, mix thoroughly and dilute 60 μL 293fectin ^TM with Opti-MEM I, final volume 1 mL, mix well incubated at room temperature for 5 minutes, (4) DNA was mixed with the diluted 293fectin ^(TM), incubated at room temperature 20 minutes; (5) 2 mL DNA-293fectin ^TM complex was added to 28 mL cells, 37 Degree, 8% CO ₂ , 125 rpm for 3 to 4 days, and the supernatant was collected. c. Purification of BCMA_huFc and BCMA_muFc (1) Centrifugal culture at 13,000 rpm for 15 min; (2) Affinity purification using protein A filler, the specific steps are as follows: Equilibration: 10 column volume of equilibrium buffer balance protein A filler. Loading: All samples processed with 0.45 μm filter were loaded. Washing: 20 column volume balance buffer wash until no effluent from the effluent. Stripping: Add 10 column volumes of elution buffer to elute the target protein (pre-add 6% neutralization buffer in the collection tube). Solution formulation balance buffer: PBS pH 7.4 elution buffer: 0.1 M glycine pH 2.6 neutralization buffer: 1 M Tris (3) After elution with a 0.22 μm membrane, use a millipore ultrafiltration tube with a cutoff of 10 KD Concentration, concentration to a volume of 1 mL, desalting using a PD-Midi desalting column, and collecting 1.5 mL of the sample. SDS-PAGE was run through 2 μg of protein concentration by OD ₂₈₀ /1.47, and the results are shown in Fig. 1. Example 2. Construction of K562-BCMA stable cell line 1. Construction of pWPT-BCMA packaging plastid

The full-length (SEQ ID NO: 37) gene of human BCMA was synthesized in vitro and inserted into the restriction enzyme cleavage site MluI, SalI (SEQ ID NO: 44). Insert into the lentiviral packaging plastid pWPT by double restriction enzyme cleavage. 2. Packaging of lentivirus a) Lenti-x 293T was digested and plated to a 10 cm dish at 8 × 10 ⁶ cells and cultured at 37 ° C. b) The next morning: Prepare the plastid/PEI mixture pWPT-BCMA 5 μg psPAX .2 7.5 μg pMD2.G 2.5 μg was added to 800 μL of DMEM for incubation. The corresponding PEI volume was 45 μL, and incubated for 5 min in 800 μL OMEM. c) Add the plastid mixture dropwise to the PEI incubation solution, mix gently and incubate for 20 min at room temperature. d) Add the prepared plastid/PEI mixture dropwise to the cells and mix. Change the solution after 5 h. e) After 72 h, the virus was collected and filtered through a 0.45 μm filter and stored at 4 °C. 3. BCMA virus infects K562 cells a) Day 1: Afternoon: Well-grown K562 cells were plated at 1 × 10 ⁵ cells to a 6 cm dish. b) The next afternoon: K562 cells were discarded, 3 mL of fresh complete medium was added, 1 mL of virus stock was added, and a final concentration of 6 μg/mL of polybrene was added. c) On the third morning: Remove the supernatant and add 5 mL of fresh complete medium. d) Day 6 morning: Take some cells for flow detection. 4. Identification of K562-BCMA mixed transgenic plants a) K562-BCMA mixed transgenic plants and K562 negative cells were washed with 1% NCS (PBS containing 1% calf serum) for 2 times and then cultured with primary antibody: huBCMA antibody (abcam , #17323) diluted with 1% NCS 1:1000, each was added to 50 μL of 4 ° C for 45 min. b) Cells were washed twice with 1% NCS and then cultured with secondary antibody: DyLight488-labeled goat anti-rat IgG (abcam, #ab98420) was diluted 1% NCS 1:200, 50 μL each, and incubated at 4 °C for 45 min. . c) After the cells were washed 3 times in 1% NCS and resuspended in 1% NCS, using Guava easyCyte ^TM HT System instruments to detect, the results shown in FIG. 2-A. 5. K562-BCMA single transgenic plants were plated a) K562-BCMA mixed transgenic cells were counted and plated by single dilution plants by limiting dilution method. b) Observe the growth of the transgenic plants one week later and add additional medium. c) After two weeks, the cells in the wells grown by the single transgenic plants were expanded and cultured. 6. The K562-BCMA single transgenic plants were identified and identified as mixed transgenic plants. The experimental results are shown in Figure 2-B. Among them, 4 single transgenic plants were BCMA positive transgenic plants. Example 3. Screening for BCMA- specific scFv using a full-human phage display gene library

The phage display gene library used in the present invention is a whole human natural scFv phage gene library constructed by the company, and the storage capacity is 1E+11. Highly specific scFv fragments for BCMA are obtained using screening methods known to those skilled in the art to which the present invention pertains. Briefly, 10 μg/mL of antigen BCMA_huFc and human Fc fragment were coated on the immunotube, respectively. To reduce the effect of the Fc segment, the phage gene pool was added to the immunotube coated with the human Fc segment for 1 hr. The supernatant was added to the immunotube coated with BCMA_huFc for 1.5 hours, then the non-specific phage was washed away, the bound phage was eluted and infected with E. coli TG1 in logarithmic growth phase. The cultured phage was expanded and the expanded phage gene pool was purified using PEG/NaCl precipitation for the next round of screening. The panning was performed for 3-4 cycles to enrich for scFv phage transfectants that specifically bind to BCMA. Positive transgenic strains were determined by standard ELISA methods for BCMA_huFc. ELISA uses the human Fc fragment as an unrelated antigen to verify antibody specificity. A total of 2470 transgenic strains were screened, of which 160 transgenic strains were ELISA-specifically bound to BCMA_huFc and did not bind to human Fc segments. Among them, 76 transgenic plants with high signal values were picked to obtain 23 single sequences. The 23 transgenic plants were purified to obtain three specific binding K562-BCMA cells (Fig. 4), and the transgenic plants were named 7G2, 7A12, 23F10. By sequencing analysis, the heavy chain variable region of 7A12 is the amino acid sequence shown by SEQ ID NO: 13, the light chain variable region is the amino acid sequence shown by SEQ ID NO: 11; and the 7G2 heavy chain can be The variable region is the amino acid sequence shown by SEQ ID NO: 17, the light chain variable region is the amino acid sequence shown by SEQ ID NO: 15, and the heavy chain variable region of 23F10 is represented by SEQ ID NO: The amino acid sequence, the light chain variable region is the amino acid sequence shown in SEQ ID NO: 19.

The amino acid sequence of the heavy chain variable region of 7A12 (SEQ ID NO: 13):

EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR YPYLAFDY WGQGTLVTVSS (Bold letters underlined to show CDR sequences)

Nucleotide sequence of the heavy chain variable region of 7A12 (SEQ ID NO: 14):

GAGGTGCAATTGCTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGTTACCCATACCTGGCATTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT

Amino acid sequence of the light chain variable region of 7A12 (SEQ ID NO: 11):

EIVLTQSPGTLSLSPGERATLSC RASQSVSSSYLA WYQQKPGQAPRLLIY GASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGYPPSY TFGQGTKVEIK (Bold characters are underlined to show CDR sequences)

Nucleotide sequence of the light chain variable region of 7A12 (SEQ ID NO: 12):

GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTACGGTTACCCACCATCTTACACGTTCGGCCAGGGGACCAAAGTGGAAATCAAA

The amino acid sequence of the heavy chain variable region of 7G2 (SEQ ID NO: 17):

EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK LSGDAAMDY WGQGTLVTVSS (Bold characters are underlined to show CDR sequences)

Nucleotide sequence of the heavy chain variable region of 7G2 (SEQ ID NO: 17):

GAGGTGCAATTGCTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAACTGTCTGGTGATGCAGCAATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT

Amino acid sequence of the light chain variable region of 7G2 (SEQ ID NO: 15):

EIVLTQSPGTLSLSPGERATLSC RASQSVSSSYLA WYQQKPGQAPRLLIY GASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGYPPRY TFGQGTKVEIK (Bold characters are underlined to show CDR sequences)

Nucleotide sequence of the light chain variable region of 7G2 (SEQ ID NO: 16):

GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTACGGTTACCCACCAAGATACACGTTCGGCCAGGGGACCAAAGTGGAAATCAAA

Amino acid sequence of the heavy chain variable region of 23F10 (SEQ ID NO: 21):

EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK VRPFWGTFDY WGQGTLVTVSS (Bold characters are underlined to show CDR sequences)

The amino acid sequence of the heavy chain variable region of 23F10 (SEQ ID NO: 22):

GAGGTGCAATTGCTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGTTCGTCCATTCTGGGGTACTTTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT

The amino acid sequence of the heavy chain variable region of 23F10 (SEQ ID NO: 19):

EIVLTQSPGTLSLSPGERATLSC RASQSVSSSYLA WYQQKPGQAPRLLIY GASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYFNPPEY TFGQGTKVEIK (Bold letters underlined to show CDR sequences)

The amino acid sequence of the heavy chain variable region of 23F10 (SEQ ID NO: 20):

Example 4. GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTACTTCAACCCACCAGAATACACGTTCGGCCAGGGGACCAAAGTGGAAATCAAA fusion antibodies an anti BCMA scFv_Fc Construction and performance of transient transfected eukaryotic cells purified Activity Assay

Primers were designed for the VH and VL fragments of 7G2, 7A12, and 23F10, respectively, and a linker consisting of 15 flexible amino acids (GGGGSGGGGSGGGGS) was inserted to form a scFv; a restriction enzyme cleavage site and a protective base of NheI were inserted upstream of VH. Downstream of VL inserts the restriction enzyme cleavage site of BamHI and protects the base. The PCR product was analyzed by 1% agarose gel electrophoresis and purified and recovered. The restriction enzyme was cleaved and then ligated into V152 (purchased from Shanghai Ruijin Biotechnology Co., Ltd.) eukaryotic expression vector. Logarithmic growth phase 293F cells were transiently transfected with 293fectinTM Transfection reagent (Invitrogen, 12347-019) or polyethyleneimine (PEI) (Sigma-Aldrich, 408727). After 5-7 days of transfection, the culture supernatant was collected for affinity purification by Protein A. The obtained antibodies were quantitatively and qualitatively analyzed by SDS PAGE (Fig. 5).

The binding of the antibody to K562 stably expressing BCMA was tested by flow cytometry. The specific method for FACs detection is as follows: cells are collected, cells are washed once with growth medium, resuspended in PBS, and the cell concentration is adjusted to 4E+5 cells/mL. The gradient diluted scFv_Fc fusion antibody was incubated with the cells for 30 minutes on ice with an initial concentration of 500 nM, 5 fold dilutions, for a total of 7 gradients. Thereafter, it was incubated with a FITC-labeled anti-mouse IgG secondary antibody. After two washing steps, using the Guava easyCyte ^TM HT System instruments to detect. Figure 6 shows the binding of the antibody 7A12, 7G2, 23F10 scFv_Fc fusion form and K562-BCMA. All three antibodies had a concentration-dependent binding with an EC ₅₀ of 3.13 nM, 3.42 nM, and 5.61 nM, respectively. Example 5. Determination of the affinity of an antibody using surface plasmon resonance (SPR)

The affinity of different antibodies for BCMA was determined using biacore T200. The specific practices are as follows:

BCMA_huFc was coated on a CM5 wafer by means of amine coupling, coated to about 500 RU, and the gradient-diluted antibody was passed through the channel for coating the antigen as a mobile phase at a flow rate of 30 μL/min. The running buffer was HBS-N and the temperature was 25 degrees. The experimental data was analyzed by BIAevaluation 3.2 and the kinetic curves were fitted using a 1:1 langmuir model. The KD of 7A12 (scFv_Fc) was 663 pM, the KD of 7G2 (scFv_Fc) was 499 pM, and the KD of 23F10 (scFv_Fc) was 667 pM (see Figure 7). The specific parameters are shown in the following table: Example 6. Determination of binding of antibodies to tumor cell lines using FACs

RPMI8226 is a peripheral blood B lymphocyte in human multiple myeloma. The specific method for FACs detection is as follows: cells are collected, cells are washed once with growth medium, resuspended in PBS, and the cell concentration is adjusted to 4E+5 cells/mL. The gradient diluted scFv_Fc fusion antibody was incubated with the cells for 30 minutes on ice with an initial concentration of 500 nM, 5 fold dilutions, for a total of 7 gradients. Thereafter, it was incubated with a FITC-labeled anti-mouse IgG secondary antibody. After two washing steps, using the Guava easyCyte ^TM HT System instruments to detect. The antibody 7A12, 7G2, 23F10 scFv_Fc fusion format shown in Figure 8 has a concentration-dependent binding on the cell line RPMI8226. Example 7. Competitive binding of anti- BCMA antibody to BCMA ligand APRIL . Experiment 1. Expression of purified recombinant APRIL fusion protein

The fusion protein of the Fc fragment Asp104-Lys330 expressing human APRIL His115-Leu250 and human IgG1 heavy chain constant region was recombined, and the fusion protein APRIL_huFc (SEQ ID NO: 45) was ligated in the middle, and the corresponding gene sequence was SEQ ID NO: 46. Transient transfection performance purification was performed as described in Example 1. 2. Competitive ELISA

50 μg/mL 100 μL/empty BCMA_muFc was applied to the ELISA plate and coated overnight at 4 degrees. On the next day, the plate was washed 3 times with PBS, and PBS containing 2% skim milk powder was added thereto, and the mixture was blocked at room temperature for 1 hour. Simultaneously add 40 ng/mL APRIL_huFc and gradient diluted antibody 7A12, 7G2, or 23F10 (starting concentration 200 nM, 3-fold dilution, 7 gradients). The cells were incubated for 1 hour at room temperature, washed three times with PBST, washed three times with PBS, and added with a 1:1000 dilution of HRP-labeled mouse anti-human Fc antibody, incubated for 1 hour at room temperature, washed three times with PBST, and washed three times with PBS. Add TMB to color and read with a microplate reader.

The experimental results are shown in Fig. 9. 7A12, 7G2, and 23F10 all significantly inhibited the binding of APRIL and BCMA. This demonstrates that the antibodies of the invention inhibit the binding of BCMA to its natural ligand. Example 8. Construction of an Anti physical BCMA construct chimeric antigen by physical body (CAR) of a. An anti-BCMA antibody 7A12 chimeric antigen by

Lentiviral plastids expressing the second and third generation chimeric antigen receptors of antibody 7A12 were constructed using PRRLSIN-cPPT.EF-1α as a vector, including PRRLSIN-cPPT.EF-1α-7A12-28Z, PRRLSIN-cPPT.EF -1α-7A12-BBZ and PRRLSIN-cPPT.EF-1α-7A12-28BBZ. The 7A12-28Z sequence consists of CD8α signal peptide (SEQ ID NO: 23), 7A12 scFv (SEQ ID NO: 47), CD8 hinge (SEQ ID NO: 25), CD28 transmembrane region (SEQ ID NO: 27), and intracellular The signal transduction domain (SEQ ID NO: 29) and the intracellular domain CD3 (CD ID NO: 31) of CD3; the 7A12-BBZ sequence consists of the CD8α signal peptide (SEQ ID NO: 23), 7A12 scFv (SEQ ID NO: 47), CD8 hinge (SEQ ID NO: 25) and transmembrane region (SEQ ID NO: 33), CD137 intracellular signal transduction domain (SEQ ID NO: 35), and CD3ξ (SEQ ID NO: 31); 7A12 The -28BBZ sequence consists of CD8α signal peptide (SEQ ID NO: 23), 7A12-scFv (SEQ ID NO: 47), CD8 hinge (SEQ ID NO: 25), CD28 transmembrane region (SEQ ID NO: 27), and intracellular Segment (SEQ ID NO: 29), CD137 intracellular signal transduction domain (SEQ ID NO: 35), and CD3ξ composition (SEQ ID NO: 31). b. Construction of anti-BCMA antibody 7G2 chimeric antigen receptor plastid

Using PRRLSIN-cPPT.EF-1α as a vector, a lentiviral plastid representing the second and third generation chimeric antigen receptors of antibody 7G2 was constructed, including PRRLSIN-cPPT.EF-1α-7G2-28Z, PRRLSIN-cPPT.EF -1α-7G2-BBZ and PRRLSIN-cPPT.EF-1α-7G2-28BBZ. The 7G2-28Z sequence consists of CD8α signal peptide (SEQ ID NO: 23), 7G2 scFv (SEQ ID NO: 48), CD8 hinge (SEQ ID NO: 25), CD28 transmembrane region (SEQ ID NO: 27), and intracellular The signal transduction domain (SEQ ID NO: 29) and the intracellular domain CD3ξ (SEQ ID NO: 31) of CD3; the 7G2-BBZ sequence consists of the CD8α signal peptide (SEQ ID NO: 23), 7G2 scFV (SEQ ID NO: 48), CD8 hinge (SEQ ID NO: 25) and transmembrane region (SEQ ID NO: 33), CD137 intracellular signal transduction domain (SEQ ID NO: 35), and CD3ξ (SEQ ID NO: 31); 7G2 The -28BBZ sequence consists of CD8α signal peptide (SEQ ID NO: 23), 7G2-scFv (SEQ ID NO: 48), CD8 hinge (SEQ ID NO: 25), CD28 transmembrane region (SEQ ID NO: 27), and intracellular Segment (SEQ ID NO: 29), CD137 intracellular signal transduction domain (SEQ ID NO: 35), and CD3ξ composition (SEQ ID NO: 31). Example 9. Preparation of CAR-T cells 1. Lentiviral packaging, virus concentration and titer determination of the target BCMA CAR lentiviral vector a. Lentiviral packaging 1) Inoculate 293T cells in 10 cm cell culture dishes, Cultured at 37 ° C, 5% CO ₂ overnight for transfection, the medium is DMEM containing 10% fetal bovine serum (Gibco); 2) the target gene plastid PRRLSIN-cPPT.EF-1α-EGFP (Mock) or Related CAR plastids 5.4 μg and packaging plastid pRsv-REV 6.2 μg, RRE-PMDLg 6.2 μg, Vsvg 2.4 μg dissolved in 800 μL blank DMEM medium, and mix; 3) Dissolve 60 μg PEI in 800 μL of serum-free DMEM medium, gently mix (or vortex at 1000 rpm for 5 seconds), incubate for 5 min at room temperature; 4) Formation of transfection complex: Add the plastid mixture to the PEI mixture, immediately after the addition Mix by spin or gently mix and incubate for 20 min at room temperature; 5) Add 1.6 mL of the transfection complex into a 10 cm dish containing 11 mL of DMEM medium (no need to change); 4-5 h after, The transfected 293T cells were exchanged with 10% FBS DMEM medium; the cells were incubated at 37 ° C for 72 h, and the virus solution was collected. b. Lentivirus concentration 1) 5X PEG8000 NaCl preparation: weigh NaCl 8.766 g, PEG8000 50 g dissolved in 200 mL Milli-Q pure water; 121 ° C 30 min wet heat sterilization for 30 min; stored at 4 ° C; 2) use 0.45 The μm filter is used to filter the lentivirus solution. For each 30 mL of the filtered virus initial solution, add 7.5 mL of 5X PEG-8000 NaCl stock solution; mix once every 20~30 min for 3-5 times; overnight at 4 °C 4 ° C, 4000 g, centrifugation for 20 min; 3) Aspirate the supernatant, let stand the tube for 1-2 minutes, absorb the residual liquid; add the appropriate amount of lentivirus solution to dissolve the lentiviral precipitate; after packaging, store At -80 ° C. c. Lentiviral titer determination 1) Inoculate 293T cells in a 6 well plate with 1 × 10 ⁵ cell number, 1 mL/well; add 0.6 μL/mL polybrene solution with initial concentration of 10 μg/μL, final concentration 6 μg /mL; 37 ° C, 5% CO ₂ culture for 1 hour, the culture medium is DMEM containing 10% fetal bovine serum; 2) Add 10 μL / well of virus concentrate 5 times dilution, 3 gradients, 37 ° C, 5% CO ₂ culture; 3) 72 hours after infection, trypsin digest (30 s) 293T cells, add 1 mL DMEM (10% FBS) to terminate digestion, transfer the cell suspension into a 2 mL centrifuge tube (second aliquot), Centrifuge at 5000 rpm for 5 min, discard the broth; wash twice with PBS (2% NBS); 4) Add 50 μL of PE-SA (1:200 dilution) antibody to the control cells for 45 min on ice, PBS (2%) NBS) washed twice and resuspended as a control; 5) Detection group +50 μL 1:50 diluted biotin-Goat anti human IgG, F(ab')2 antibody, incubated on ice for 45 min; PBS (2 Wash twice with % NBS); add 50 μL of PE-SA (1:200 dilution) antibody for 45 min on ice; 6) Resuspend the cells by adding 2 mL PBS (2% NBS), centrifuge at 4 ° C, 5000 rpm / min. Discard the solution for 5 minutes; repeat twice; 7) Add 500 μL PB S (2% NBS), transferred to the flow tube. Flow cytometry was used to detect PE channels. The positive rate was 5-20%, and the titer (PFUs/mL) = cell number × positive rate / virus volume. 2. Lentiviral transduction of T lymphocytes ------ Preparation of CAR-positive T lymphocytes 1) Activation of T lymphocytes: cultured in a lymphocyte culture medium at a density of about 1 × 10 ⁶ /mL, and according to magnetic beads : The cell ratio was 2:1, and the magnetic beads (Invitrogen) coated with anti-CD3 and CD28 antibodies and recombinant human IL-2 (Shanghai Huaxin Biotech Co., Ltd.) with a final concentration of 500 U/mL were added for 48 h stimulation. 2) One day before the infection, Retronectin coated 24 well plates, the final concentration of retronectin was 5 μg/mL, and was cultured overnight at 4 degrees; 3) Discard the retronecectin solution (PBS) in the 24-well plate and wash in 1 mL PBS. 4) Add the concentrated lentivirus to the PBMCs cells according to MOI=10, 1000 g, centrifuge for 40 min, transfer to the cell culture incubator; 5) Expand the culture: the infected cells adopt 5× every other day. Passage was carried out at a density of 10 ⁵ /mL, and recombinant human IL-2 at a final concentration of 500 U/mL was supplemented in the lymphocyte culture solution. 3. Expression of T lymphocyte chimeric antigen receptor 1) Lentivirus-infected T lymphocytes On the 7th day of culture, 1×10 ⁶ T cells were taken in a centrifuge tube; 2) Centrifuge for 5 min at 4° C., 5000 rpm. Discard the broth and wash twice with PBS; 3) Add 50 μL of biotin-Goat anti human IgG, F(ab')2 antibody (1:50 dilution) to the cells to be tested, and incubate for 45 min on ice; PBS (2 % NBS) wash twice; add 50 μL PE-SA (1:200 dilution) antibody for 45 min on ice; 4) Resuspend the cells by adding 2 mL PBS (2% NBS), centrifuge at 4 ° C, 5000 rpm / min. Discard the clear solution for 5 minutes; repeat twice; 5) Add 500 μL PBS (2% NBS) and transfer to a flow tube. Flow cytometry detects PE channels and determines the proportion of CAR-positive T cells. The positive rates of Mock, 7A12-28Z, 7A12-BBZ, 7A12-28BBZ, 7G2-28Z, 7G2-BBZ and 7G2-28BBZ T cell infections in in vitro poisoning experiments are shown in Figure 10, which are 72.8%, 60.8%, respectively. 48.7%, 57.4%, 67.5%, 68.8%, 63.6%. 4. Determination of cytotoxicity of target BCMA CAR T cells

CytoTox 96 non-radioactive cytotoxicity detection kit (Promega) was used, with specific reference to the CytoTox 96 non-radioactive cytotoxicity detection kit instructions.

Target cells: 75 μL of 2×10 ⁵ /mL K562, K562-BCMA and RPMI-8226 cells were seeded in 96 well plates, respectively. Effector cells: CAR T cells with T-Mock and different chimeric antigen receptors at a target ratio of 3:1, 1:1 or 1:3. Each group has 4 rehabilitation wells and takes the average of 4 rehabilitation wells. The detection time was 18 h of cell culture. Each experimental group and each control group were as follows: Each experimental group: each target cell + CAR T showing different chimeric antigen receptors; control group 1: maximum release of LDH from target cells; control group 2: spontaneous release of target cells LDH; control group 3: effector cells spontaneously release LDH;

The cytotoxicity formula is: cytotoxicity % = [(experimental group - effector cell control - target cell control) / (target cell maximum lysis amount - target cell control)] × 100%

The results showed that CAR T cells with different chimeric antigen receptors had significant in vitro toxicity to BCMA-positive K562-BCMA and RPMI-8226 cells, especially to RPMI-8226 cells endogenously expressing BCMA. It was stronger, and there was almost no poisoning effect on BCMA-negative K562 cells (Fig. 11A). 5. NOD/SCID mouse subcutaneous xenograft model for treatment of multiple myeloma peripheral blood B lymphocyte RPMI-8226

RPMI-8226 cells were inoculated into 40 NOD/SCID mice, respectively, and the number of cells inoculated was 8×10 ⁶ /mouse. On the 12th day after subcutaneous inoculation of tumor cells, the tumor volume averaged 75 mm ³ and was randomly divided into 4 groups. The tail vein was injected with 1×10 ⁷ CAR T, and the intraperitoneal injection of cyclophosphamide was given at a dose of 100 mg/Kg. The residual T cells in the mice were pre-cleared, and on the 17th day after CAR T injection, the mice were sacrificed by neck.

The tumor size of the mice was analyzed. The results are shown in Fig. 11B. Compared with the UTD group, the antitumor effects of the 7A12-28Z, 7A12-BBZ and 7A12-28BBZ treatment groups were very significant, and the 17th day of CAR T injection 7A12- In the 28Z treatment group, there were 1 tumor regression in 7 mice, 7 tumors in 7A12-BBZ treatment group, and 7 tumors in 7A12-28BBZ treatment group. The tumor inhibition rates were 7A12-28Z. (84.6%), 7A12-BBZ (65.4%) and 7A12-28BBZ (100%). Example 10. Modification of Antibody 23F10

In this example, 23F10 was used as the parent antibody, and 23F10 was modified by phage display method. The construction of the 23F10-based phage gene library retained the CDR3 region of the light chain as well as the heavy chain, and two phage gene banks were constructed by randomizing the CDR1 and CDR2 of the light chain or the CDR1 and CDR2 of the heavy chain, respectively, by degenerate primers. The primer information is as follows: 2.1 Construction of 23F10 mutant

A vane body was first constructed based on antibody 23F10 (scFv) (SEQ ID NO: 55). For the phage gene pool randomized to the CDR1 and CDR2 of the light chain, the fragment 1 was amplified by PCR using the primers LMF and BL1R; the fragment 2 was amplified by PCR using the primers BL2F and FdR; and then fragment 1 and fragment 2 were ligated by bridge PCR. The full length of the scFv was sequenced, and then the full-length fragment was cleaved with NcoI and NotI restriction enzymes and ligated into the phylogenetic body of the same restriction enzyme cleavage by T4 ligase. And electroporation into the TG1 competent cells, the storage capacity is 1.50E+9. For the phage gene pool randomized to the heavy chain CDR1 and CDR2, the fragment 3 was amplified by PCR using the primers LMF and BH1R; the fragment 4 was amplified by PCR using the primers BH2F and FdR; and then fragment 3 and fragment 4 were ligated by bridge PCR. The full length of the scFv was sequenced, and then the full-length fragment was cleaved with NcoI and NotI restriction enzymes and ligated into the phylogenetic body of the same restriction enzyme cleavage by T4 ligase. And electroporation into TG1 competent cells, the storage capacity is 2.2E+9.

Screening of phage gene pools. Refer to the method in Example 3 of this patent. The initial concentration of the antigen BCMA_huFc was 20 nM and a 5-fold gradient dilution was performed for the next round of screening. Panning was performed for 2-3 cycles to enrich for scFv phage transfectants that specifically bind to BCMA_huFc. Positive transgenic strains were determined by standard ELISA methods for BCMA_huFc. ELISA uses the human Fc fragment as an unrelated antigen to verify antibody specificity. A total of 80 ELISA-positive transgenic plants were picked and re-induced and the dissociation constant Kd of the supernatant was induced by biacore. Among them, there are two transgenic strains, 25C2, which are 10 times lower than the parental transgenic strain 23F10, as shown in the following table:

After sequencing, the light chains of the transgenic plants 25C2 and 25D2 were identical to those of 23F10. Figure 12 compares the heavy chain amino acid sequences of the transgenic strains 25C2, 25D2 and 23F10, wherein the transgenic 25C2 has 5 point mutations on the heavy chain compared to the parent antibody 23F10 (SEQ ID NO: 56, 57 are 25C2, respectively). The amino acid sequence and the nucleotide sequence of the heavy chain variable region), wherein there are two on the CDR1, the thirty amino acid at position 31 becomes glycine, and the tyrosine at position 32 becomes asparagine. There are two on the CDR2, the 54th serine acid becomes asparagine, the 59th tyrosine becomes phenylalanine, and there is one in the framework region, and the 30th serine changes to glycine. Transgenic strain 25D2 has four point mutations on the heavy chain compared to the parental antibody 23F10 (SEQ ID NO: 58 and 59 are the amino acid sequence and nucleotide sequence of the heavy chain variable region of 25D2, respectively), 3 of which Located in the CDR2 region, the 54th serine acid becomes glycine, the 57th serine becomes asparagine, the 59th tyrosine becomes amphetamine, the framework region has one, and the 30th serine becomes fine. Amino acid.

The sequence of HC1 of 25C2 is set forth in SEQ ID NO: 60, and the sequence of HCDR2 of 25C2 is set forth in SEQ ID NO: 61. The sequence of HCDR1 of 25D2 is set forth in SEQ ID NO: 62, and the sequence of HCDR2 of 25D2 is set forth in SEQ ID NO: 63. The nucleotide sequence and the amino acid sequence of the 25C2 scFv are shown in SEQ ID NO: 64, 65, respectively, and the nucleotide sequence and the amino acid sequence of the 25D2 scFv are shown in SEQ ID NOS: 66, 67, respectively. 2.2 Purification of transgenic 25C2 and 25D2 (scFv_Fc)

As shown in Example 4, a suitable restriction enzyme cleavage site and a protecting base were inserted upstream of the VH, and a suitable restriction enzyme cleavage site and a protecting base were inserted downstream of the VL. The PCR product was analyzed by 1% agarose gel electrophoresis and purified and recovered. The restriction enzyme was cleaved and ligated into the eukaryotic expression vector V152 containing human Fc fragment (purchased from Shanghai Ruijin Biotechnology Co., Ltd.). Transiently transfected into 293F cells by 293Fectin and expressed.

The aggregation of 25C2 and 25D2 was analyzed by SEC. As shown in Figs. 13A and 13B, the monomeric form of the antibody was 91% and 97%, respectively. Compared with the parent antibody 23F10 (30% monomer rate), the increase was 61% and 67%, respectively, and the degree of aggregation was significantly reduced. After concentration by ultrafiltration, the obtained antibodies were quantitatively and qualitatively analyzed by SDS PAGE. The yields were 80 μg/mL and 60 μg/mL, respectively (yield = final product mass/transfection volume). 2.3 Binding characteristics of 25C2 and 25D2 of transgenic plants

Cells were harvested using K562 cells (K562-BCMA) and K562 stably expressing human BCMA, washed with complete growth medium, and plated into U-bottom microtiter plates at approximately 1 to 5 x 10 ⁵ cells/well. . The gradient diluted scFv_Fc fusion antibody was incubated with K562-BCMA/K562 for 30 minutes on ice, followed by incubation with FITC-labeled anti-human Fc as a secondary antibody. After two washing steps, using the Guava easyCyte ^TM HT System instrumental analysis, experimental data using GraphPad Prism, to obtain EC _50. Figure 14 shows the binding of 25C2, 25D2 to K562-BCMA and K562 cells. The results showed that the stability of the two types of transgenic plants, 25C2, 25D2 and K562-BCMA, had an EC ₅₀ of 2.594 nM and 1.891 nM, respectively. Compared with 23F10, it is increased by 3~4 times. 2.5 Specificity determination of 25C2 and 25D2 transgenic plants

The specificity of antibodies 23F10, 25C2, 25D2 was determined by ELISA.

2 μg/mL recombinant human BCMA_Fc, mouse BCMA_Fc, TACI_huFc (R&D, #174TC), BAFF R (R&D, #1162-BR) were applied to the immunoplate at 4 degrees overnight. The next day, 300 μL/well 2% MPBS was added for 2 hours, then 200 nM purified antibody (scFv format) was added, incubated at 37 degrees for 1 hour, washed three times with PBST (PBS containing 0.05% Tween-20), and washed three times with PBS. Then, a 1:4000 diluted HRP-labeled anti-Flag marker antibody (sigma, #A8592-1MG) was added, incubated at 37 degrees for 1 hour, washed three times with PBST (PBS containing 0.05% Tween-20), and washed three times with PBS. Add 100 μL/well of TMBS matrix, develop color for 10-15 minutes, and add 50 μL of 2 M sulfuric acid to stop the reaction.

As a result, as shown in Fig. 14B, antibodies 7A12, 23F10, 25C2, 25D2 specifically bind human BCMA, and do not bind human TACI and human BAFF R. Among them, antibodies 25C2, 25D2 and mouse BCMA have weaker binding. Preparation 11. 25C2, CAR-T cells embodiment 25D2

Referring to the procedure of Example 8, chimeric antigen receptor plastids of 25C2 and 25D2 were respectively constructed. a. Construction of chimeric antigen receptor plastid of 25C2

The lentiviral plastid PRRLSIN-cPPT.EF-1α-25C2-BBZ representing the second-generation chimeric antigen receptor of antibody 25C2 was constructed using PRRLSIN-cPPT.EF-1α as a vector. The lentiviral plastid PRRLSIN-cPPT.EF-1α-25D2-BBZ representing the second-generation chimeric antigen receptor of antibody 25D2 was constructed using PRRLSIN-cPPT.EF-1α as a vector.

The 25C2-BBZ sequence consists of CD8α signal peptide (SEQ ID NO: 23), 25C2 scFv (SEQ ID NO: 64), CD8 hinge (SEQ ID NO: 25) and transmembrane region (SEQ ID NO: 33), CD137 intracellular The signal transmission domain (SEQ ID NO: 35) and CD3ξ (SEQ ID NO: 31) are composed.

The 25D2-BBZ sequence consists of CD8α signal peptide (SEQ ID NO: 23), 25D2 scFV (SEQ ID NO: 66), CD8 hinge (SEQ ID NO: 25) and transmembrane region (SEQ ID NO: 33), CD137 intracellular The signal transmission domain (SEQ ID NO: 35) and CD3ξ (SEQ ID NO: 31) are composed.

Referring to the procedure of Example 9, the plastids PRRLSIN-cPPT.EF-1α-25C2-BBZ, PRRLSIN-cPPT.EF-1α-25D2-BBZ were subjected to lentiviral packaging, T cell infection, and amplification to obtain a chimeric antigen. Receptor-modified T cells 25C2-BBZ and 25D2-BBZ. Example 12. Preparation of CAR-T cells expressing soluble PD1

In this example, CAR-T cells expressing soluble PD1 were prepared using the scFv of antibody 7A12. The preparation method is as follows: 1. Fully synthesize the signal peptide sequence of PD-1 (SEQ ID NO: 68), the PD-1 extracellular domain sequence (SEQ ID NO: 69), and the sequence of CH3 (SEQ ID NO: 70). And transferred into the T vector to obtain the plastid T-sPD1-Fc.

Using T-sPD1-Fc plastid as a model, the upstream primer 5'-acgcgtcctagcgctaccggtcgccaccatgcagatcccacaggcgccc-3' (SEQ ID NO: 71) and the downstream primer 5'-ctctcggggctgcccaccatacaccagggtttggaactggc-3' (SEQ ID NO: 72) were amplified by PCR to obtain sPD1. Sequence; sPD1-CH3 fragment was obtained by amplification with the upstream primer 5'-tatggtgggcaccccccagagccacag-3' (SEQ ID NO: 73) and the downstream primer 5'-aaaattcaaagtctgtttcactttacccggagacagggag-3' (SEQ ID NO: 74).

The sPD1-CH3 fragment and the fragment of 7A12-BBZ (SEQ ID NO: 75) were PCR spliced and amplified to obtain sPD1-CH3-7A12-BBZ, and the sequence of 7A12-BBZ was composed of CD8α signal peptide (SEQ ID NO: 23), 7A12 scFv (SEQ ID NO: 47), CD8 hinge (SEQ ID NO: 25) and transmembrane region (SEQ ID NO: 33), CD137 intracellular signal transduction domain (SEQ ID NO: 35), and CD3ξ (SEQ ID) NO: 31) Composition.

The 5' end of the above constructed fragment sPD1-CH3-7A12-BBZ carries the MluI restriction enzyme cleavage site, and the 3' end carries the SalI restriction enzyme cleavage site. The cleavage of the chimeric antigen receptor of the target BCMA expressing the sPD-1-CH3 protein was obtained by cleavage with MluI and SalI double restriction enzymes and ligating into the PRRLSIN-cPPT.EF-1α vector which was cleaved by the same restriction enzyme.

Referring to the procedure of Example 9, T cells sPD-1-7A12-BBZ expressing sPD1 and 7A12-BBZ were obtained. Example 13. In vitro cell killing experiment

In vitro poisoning experiments were performed using 25C2-BBZ T cells, 25D2-BBZ T cells, 7A12-BBZ T cells, C11D5.3-BBZ T cells, and sPD-1-7A12-BBZ T cells as effector cells, among which C11D5.3 -BBZ (SEQ ID NO: 87) is a second generation CAR prepared using anti-BCMA mouse anti-C11D5.3 (see CN201580073309.6). The target cells were human myeloma cells NCI-H929 and multiple myeloma peripheral blood B lymphocytes RPMI-8226.

CytoTox 96 non-radioactive cytotoxicity detection kit (Promega) was used. For specific methods, refer to the CytoTox 96 non-radioactive cytotoxicity detection kit instructions.

The effector cells were inoculated in the 96 well plate according to the effective target ratio of 3:1, 1:1 or 1:3, and 50 μL of 2×10 ⁵ /mL NCI-H929 and RPMI-8226 cells were inoculated respectively to the corresponding 96 well culture. plate.

Five wells were set in each group, and the plates were placed in a cell culture incubator for 18 h.

The experimental groups and the control groups were set as follows: experimental group: each target cell + T lymphocytes showing different chimeric antigen receptors; control group 1: maximal release of LDH from target cells; control group 2: target cells spontaneously Release of LDH; control group 3: effector cells spontaneously release LDH. The calculation formula is: % cytotoxicity = [(experimental group - effector cell spontaneous group - target cell spontaneous group) / (target cell max - target cell spontaneous)] * 100.

The experimental results of cytotoxicity are shown in Figure 15. Example 14. Cell cytotoxicity test in mice

8×10 ⁶ RPMI-8226 cells were inoculated subcutaneously into the right axillary area of B-NDG mice, and on the 18th day after subcutaneous inoculation of tumor cells, the average tumor volume was about 243 mm ³ , and multiple myeloma peripheral blood B lymphocytes RPMI were obtained. -8226 B-NDG mouse subcutaneous xenograft model.

The mouse subcutaneous xenograft model was divided into 3 groups, 4 rats in each group, respectively, injected with 25C2-BBZ, 25D2-BBZ, and untransfected T cells (UTD) at a dose of 5×10 ⁶ . The results are shown in the following table. On the 32nd and 36th day after tumor cell inoculation, the tumors of all 4 mice in the 25C2-BBZ and 25D2-BBZ treatment groups all resolved.

The mouse subcutaneous xenograft model was divided into 3 groups, 4 in each group, injected with 25C2-BBZ, 25D2-BBZ, C11D5.3-BBZ, 7A12-BBZ and untransfected T cells (UTD), CAR T injection. The dose was 1 × 10 ⁶ and the tumor regression was as shown in the following table and Figure 16.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it is to be understood that various modifications and changes may be made to the present invention, which is to be understood by those skilled in the art.

Figure 1 shows an SDS electropherogram of BCMA_huFc, BCMA_muFc (reduction conditions);

Figure 2-A and Figure 2-B show the performance of BCMA in the stable cell line K562-BCMA detected by FACs.

Figure 3 shows the results of ELISA assays for antibodies 7A12, 7G2, 23F10.

Figure 4 shows the binding of antibodies 7A12, 7G2, 23F10 to K562-BCMA and K562 detected by FACs.

Figure 5 shows the purified anti-BCMA scFv_Fc antibody (reduction conditions) by SDS PAGE analysis.

Figure 6 shows the binding of purified scFv_Fc to K562-BCMA by gradient dilution of FACs assay.

Figure 7 shows the affinity of antibodies 7A12, 7G2, 23F10 and BCMA as determined by Biacore.

Figure 8 shows the binding of antibody 7A12, 7G2, 23F10 to RPMI8226 cell line determined by FACs.

Figure 9 shows the competitive binding of ELISA to antibodies and APRIL by ELISA.

Figure 10 shows the positive rate of BCMA-CAR T virus-infected T lymphocytes detected by FACS.

Figure 11A shows the results of an in vitro toxicity test of BCMA-CAR T positive for BCMA and negative cells, and Figure 11B shows the results of in vivo experiments of BCMA-CAR T mice.

Figure 12 compares the heavy chain amino acid sequences of the transgenic strains 25C2, 25D2 and 23F10.

Fig. 13A shows the aggregation of antibody 25C2, Fig. 13B shows the aggregation of antibody 25D2, and Fig. 13C shows the aggregation of antibodies 23F10 and 7A12.

Figure 14A shows the binding of 25C2, 25D2 to K562-BCMA and K562 cells, and Figure 14B shows the specificity of the ELISA assay for antibodies 23F10, 25C2, 25D2.

Figure 15 shows the results of experiments with cytotoxicity of 25C2-BBZ, 25D2-BBZ, 7A12-BBZ, C11D5.3-BBZ, and sPD-1-7A12-BBZ.

Figure 16 shows the results of subcutaneous xenografts of 25C2-BBZ, 25D2-BBZ, C11D5.3-BBZ, 7A12-BBZ.

<110> Koji Biomedical (Shanghai) Co., Ltd. <120> Target BCMA antibody and its application <130> P2018-0136 <150> CN201710058581. 8 <151> 2017-01-23 <150> CN201710920346. 7 <151> 2017-09-30 <160> 87 <170> PatentIn version 3. 5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <400> 1 Ser Tyr Ala Met Ser 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial sequence (Artificial sequence) <400> 2 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 8 <212> PRT <213> Artificial sequence <400> 3 Tyr Pro Tyr Leu Ala Phe Asp Tyr 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial sequence <400> 4 Leu Ser Gly Asp Ala Ala Met Asp Tyr 1 5 <210> 5 <211> 10 <212> PRT <213> Artificial sequence <400> 5 Val Arg Pro Phe Trp Gly Thr Phe Asp Tyr 1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial sequence <400> 6 Arg Ala Ser Gln Ser Val Ser Ser Serr Tyr Leu Ala 1 5 10 <210> 7 <211> 7 <212> PRT <213> Artificial sequence (Artificial sequence) < 400> 7 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 8 <211> 9 212> PRT <213> Artificial sequence <400> 8 Gln Gln Tyr Gly Tyr Pro Pro Ser Tyr 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial sequence <400> > 9 Gln Gln Tyr Gly Tyr Pro Pro Arg Tyr 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <400> 10 Gln Gln Tyr Phe Asn Pro Pro Glu Tyr 1 5 < 210> 11 <211> 109 <212> PRT <213> Artificial sequence <400> 11 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Tyr Pro Pro 85 90 95 Ser Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 12 <211 327 <212> DNA <213> Artificial sequence (Artificial sequence) <400> 12 gaaatcgtgt taacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcttgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggagcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg atccgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagtacggtt Acccaccatc ttacacgttc 300 ggccagggga ccaaagtgga aatcaaa 327 <210> 13 <211> 117 <212> PRT <213> Artificial sequence <400> 13 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Pro Tyr Leu Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 14 <211> 351 <212> DNA <213> Artificial Sequence <400> 14 gaggtgcaat tgctggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gcgttaccca 300 tacctggcat tcgactactg gggccaagga accctggtca ccgtctcgag t 351 <210> 15 <211> 109 <212> PRT <213> Artificial sequence <400> 15 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 G Ly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Tyr Pro Pro 85 90 95 Arg Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 16 <211> 327 <212> DNA <213> Artificial sequence <400> 16 gaaatcgtgt taacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcttgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggagcatcca gcagggccac tggcatccca 180 Gacaggttca gtggcagtgg atccgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagtacggtt acccaccaag atacacgttc 300 ggccagggga ccaaagtgga aatcaaa 327 <210> 17 <211> 118 <212> PRT <213> Artificial sequence <400> 17 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Ser Gly Asp Ala Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 18 <211> 354 <212> DNA <213 > artificial sequence (artificial sequence) <400> 18 gaggtgcaat tgctggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaactgtct 300 ggtgatgcag caatggacta ctggggccaa ggaaccctgg Tcaccgtctc gagt 354 <210> 19 <211> 109 <212> PRT <213> Artificial sequence <400> 19 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pr o Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Phe Asn Pro Pro 85 90 95 Glu Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 20 <211> 327 <212> DNA <213> Artificial sequence <400> 20 gaaatcgtgt taacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcttgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggagcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg atccgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagtacttca acccaccaga atacacgttc 300 ggccagggga ccaaagtgga aatcaaa 327 <210> 21 <211> 119 <212> PRT <213> artificial sequence (artificialSequence) <400> 21 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Arg Pro Phe Trp Gly Thr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 < 210> 22 <211> 357 <212> DNA <213> artificial sequence (artificial sequence) <400> 22 gaggtgcaat tgctggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa Cacgctgtat 240 ctgcagatga acagcctgag agccgaggac acggccgtat attact Gtgc gaaagttcgt 300 ccattctggg gtactttcga ctactggggc caaggaaccc tggtcaccgt ctcgagt 357 <210> 23 <211> 21 <212> PRT <213> Artificial sequence <400> 23 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 24 <211> 63 <212> DNA <213> Artificial sequence <400> 24 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccg 63 <210> 25 < 211> 45 <212> PRT <213> Artificial Sequence <400> 25 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 26 <211> 135 <212> DNA <213> Artificial Sequence <400> 26 Accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gact Tcgcct gtgat 135 <210> 27 <211> 27 <212> PRT <213> Artificial sequence <400> 27 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 28 <211> 81 <212> DNA <213> Artificial sequence <400> 28 ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60 gcctttatta ttttctgggt g 81 <210> 29 <211> 41 <212> PRT <213> Artificial sequence <400> 29 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 30 <211> 123 <212> DNA <213> Artificial Sequence <400> 30 aggagtaaga ggagcaggct cctgcacagt Gactacatga acatgactcc ccgccgcccc 60 gggccaaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120 tcc 123 <210> 31 <211> 113 <212> P RT <213> Artificial sequence <400> 31 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 50 55 60 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 65 70 75 80 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 85 90 95 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 100 105 110 Arg <210> 32 <211> 339 <212> DNA <213> artificial sequence (artificial sequence) <400> 32 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgcag agaaggaaga accctcagga aggcctgtac 180 aatgaactgc agaaagataa gatggcggag gcctacagtg Agattgggat gaaaggcgag 240 cgccggaggg gcaaggggca cgatggcctt taccaggg Tc tcagtacagc caccaaggac 300 acctacgacg cccttcacat gcaggccctg ccccctcgc 339 <210> 33 <211> 21 <212> PRT <213> Artificial sequence <400> 33 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr 20 <210> 34 <211> 63 <212> DNA <213> Artificial sequence <400> 34 atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60 acc 63 <210> 35 <211 > 42 <212> PRT <213> Artificial sequence <400> 35 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 36 <211> 126 <212> DNA <213> Artificial sequence <400> 36 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag Accagtacaa 60 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120 gaactg 126 <210> 37 <211 > 184 <212> PRT <213> Artificial sequence <400> 37 Met Leu Gln Met Ala Gly Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser 1 5 10 15 Leu Leu His Ala Cys Ile Pro Cys Gln Leu Arg Cys Ser Ser Asn Thr 20 25 30 Pro Pro Leu Thr Cys Gln Arg Tyr Cys Asn Ala Ser Val Thr Asn Ser 35 40 45 Val Lys Gly Thr Asn Ala Ile Leu Trp Thr Cys Leu Gly Leu Ser Leu 50 55 60 Ile Ile Ser Leu Ala Val Phe Val Leu Met Phe Leu Leu Arg Lys Ile 65 70 75 80 Asn Ser Glu Pro Leu Lys Asp Glu Phe Lys Asn Thr Gly Ser Gly Leu 85 90 95 Leu Gly Met Ala Asn Ile Asp Leu Glu Lys Ser Arg Thr Gly Asp Glu 100 105 110 Ile Ile Leu Pro Arg Gly Leu Glu Tyr Thr Val Glu Glu Cys Thr Cys 115 120 125 Glu Asp Cys Ile Lys Ser Lys Pro Lys Val Asp Ser Asp His Cys Phe 130 135 140 Pro Leu Pro Ala Met Glu Glu Gly Ala Thr Ile Leu Val Thr Thr Lys 145 150 155 160 Thr Asn Asp Tyr Cys Lys Ser Leu Pro Ala Ala Leu Ser Ala Thr Glu 165 170 175 Ile Glu Lys Ser Ile Ser Ala Arg 180 <210> 38 <211> 54 <212 > PRT <213> Human Process Artificial sequence <400> 38 Met Leu Gln Met Ala Gly Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser 1 5 10 15 Leu Leu His Ala Cys Ile Pro Cys Gln Leu Arg Cys Ser Ser Asn Thr 20 25 30 Pro Pro Leu Thr Cys Gln Arg Tyr Cys Asn Ala Ser Val Thr Asn Ser 35 40 45 Val Lys Gly Thr Asn Ala 50 <210> 39 <211> 162 <212> DNA <213> Artificial Sequence <400> 39 atgctgcaga Tggccggcca gtgcagccag aacgagtact tcgacagcct gctgcacgcc 60 tgcatcccct gccagctgcg gtgcagcagc aacacccccc ccctgacctg ccagcggtac 120 tgcaacgcca gcgtgaccaa cagcgtgaag ggcaccaacg cc 162 <210> 40 <211> 283 <212> PRT <213> Artificial sequence <400> 40 Met Leu Gln Met Ala Gly Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser 1 5 10 15 Leu Leu His Ala Cys Ile Pro Cys Gln Leu Arg Cys Ser Ser Asn Thr 20 25 30 Pro Pro Leu Thr Cys Gln Arg Tyr Cys Asn Ala Ser Val Thr Asn Ser 35 40 45 Val Lys Gly Thr Asn Ala Gly Ser Asp Lys Thr His Thr Cys Pro Pro 50 55 60 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 65 70 75 80 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 85 90 95 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 100 105 110 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 115 120 125 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 130 135 140 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 145 150 155 160 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 165 170 175 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 180 185 190 Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe 195 200 205 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 210 215 220 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 225 230 235 240 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 245 250 255 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 260 265 270 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 275 280 <210> 41 <211> 849 <212> DNA <213> Artificial sequence (Artificial sequence) <400> 41 atgctgcaga tggccggcca gtgcagccag aacgagtact tcgacagcct gctgcacgcc 60 tgcatcccct gccagctgcg gtgcagcagc aacacccccc ccctgacctg ccagcggtac 120 tgcaacgcca gcgtgaccaa cagcgtgaag ggcaccaacg ccggatccga caaaactcac 180 acatgcccac cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc 240 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 300 gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 360 cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 420 gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 480 aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga 540 gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 600 ctgtggtgcc tggtcaaagg cttctatccc Agcgacatcg ccgtggagtg ggagagcaat 660 gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 720 ttcctctata gcaagctcac cgtggacaag agcaggtggc agcaggggaa Cgtcttctca 780 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 840 ccgggtaaa 849 <210> 42 <211> 281 <212> PRT <213> Artificial sequence <400> 42 Met Leu Gln Met Ala Gly Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser 1 5 10 15 Leu Leu His Ala Cys Ile Pro Cys Gln Leu Arg Cys Ser Ser Asn Thr 20 25 30 Pro Pro Leu Thr Cys Gln Arg Tyr Cys Asn Ala Ser Val Thr Asn Ser 35 40 45 Val Lys Gly Thr Asn Ala Gly Ser Arg Asp Cys Gly Cys Lys Pro Cys 50 55 60 Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys 65 70 75 80 Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val 85 90 95 Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe 100 105 110 Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu 115 120 125 Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His 130 135 140 Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala 145 150 155 160 Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg 165 170 175 Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met 180 185 190 Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro 195 200 205 Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn 210 215 220 Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val 225 230 235 240 Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr 245 250 255 Phe Thr Cys Ser Val Leu His Glu Gly Leu His Ass His His Thr Glu 260 265 270 Lys Ser Leu Ser His Ser Pro Gly Lys 275 280 <210> 43 <211> 843 <212> DNA <213> Artificial Sequence ( Artificial sequence) <400> 43 atgctgcaga tggccggcca gtgcagccag aacgagtact tcgacagcct gctgcacgcc 60 tgcatcccct gccagctgcg gtgcagcagc aacacccccc ccctgacctg ccagcggtac 120 tgcaacgcca gcgtgaccaa cagcgtgaag ggcaccaacg ccggatccag ggattgtggt 180 tgtaagcctt gcatatgtac agtcccagaa gtatcatctg tcttcatctt ccccccaaag 240 cccaaggatg tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc 300 agcaaggatg atcccgaggt cca gttcagc tggtttgtag atgatgtgga ggtgcacaca 360 gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt cagtgaactt 420 cccatcatgc accaggactg gctcaatggc aaggagttca aatgcagggt caacagtgca 480 gctttccctg cccccatcga gaaaaccatc tccaaaacca aaggcagacc gaaggctcca 540 caggtgtaca ccattccacc tcccaaggag cagatggcca aggataaagt cagtctgacc 600 tgcatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag 660 ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc ttacttcgtc 720 tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt cacctgctct 780 gtgttacatg agggcctgca caaccaccat actgagaaga gcctctccca ctctcctggt 840 aaa 843 <210> 44 <211> 589 <212> DNA <213> artificial sequence (artificial sequence) <400> 44 acgcgtccta gcgctaccgg tcgccaccat gttgcagatg gctgggcagt gctcccaaaa 60 tgaatatttt gacagtttgt tgcatgcttg cataccttgt caacttcgat Gttcttctaa 120 tactcctcct ctaacatgtc agcgttattg taatgcaagt gtgaccaatt cagtgaaagg 180 aacgaatgcg attctctgga cctgtttggg actgagctta ataatttctt tggcagtttt 240 cgtgctaatg tttttgctaa ggaagataaa ctctgaacca ttaaaggacg agtttaaaaa 300 cacaggatca ggtctcctgg gcatggctaa cattgacctg gaaaagagca ggactggtga 360 tgaaattatt cttccgagag gcctcgagta cacggtggaa gaatgcacct gtgaagactg 420 catcaagagc aaaccgaagg tcgactctga ccattgcttt ccactcccag ctatggagga 480 aggcgcaacc attcttgtca ccacgaaaac gaatgactat tgcaagagcc tgccagctgc 540 tttgagtgct acggagatag agaaatcaat ttctgctagg taagtcgac 589 <210> 45 <211> 365 <212 > PRT <213> Artificial sequence <400> 45 His Ser Val Leu His Leu Val Pro Ile Asn Ala Thr Ser Lys Asp Asp 1 5 10 15 Ser Asp Val Thr Glu Val Met Trp Gln Pro Ala Leu Arg Arg Gly Arg 20 25 30 Gly Leu Gln Ala Gln Gly Tyr Gly Val Arg Ile Gln Asp Ala Gly Val 35 40 45 Tyr Leu Leu Tyr Ser Gln Val Leu Phe Gln Asp Val Thr Phe Thr Met 50 55 60 Gly Gln Val Val Ser Arg Glu Gly Gln Gly Arg Gln Glu Thr Leu Phe 65 70 75 80 Arg Cys Ile Arg Ser Met Pro Ser His Pro Asp Arg Ala Tyr Asn Ser 85 90 95 Cys Tyr Ser Ala Gly Val Phe His Leu His Gln Gly Asp Ile Leu Se r 100 105 110 Val Ile Ile Pro Arg Ala Arg Ala Lys Leu Asn Leu Ser Pro His Gly 115 120 125 Thr Phe Leu Gly Phe Val Lys Leu Gly Ser Asp Lys Thr His Thr Cys 130 135 140 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 145 150 155 160 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 165 170 175 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 180 185 190 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 195 200 205 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 210 215 220 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 225 230 235 240 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 245 250 255 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 260 265 270 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys 275 280 285 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 290 295 300 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 30 5 310 315 320 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 325 330 335 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 340 345 350 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 355 360 365 <210> 46 <211> 1095 <212> DNA <213> Artificial sequence <400> 46 cacagcgtgc tgcacctggt gcccatcaac gccaccagca aggacgacag cgacgtgacc 60 gaggtgatgt ggcagcccgc cctgcggcgg ggccggggcc tgcaggccca gggctacggc 120 gtgcggatcc aggacgccgg cgtgtacctg ctgtacagcc aggtgctgtt ccaggacgtg 180 accttcacca tgggccaggt ggtgagccgg gagggccagg gccggcagga gaccctgttc 240 cggtgcatcc ggagcatgcc cagccacccc gaccgggcct acaacagctg ctacagcgcc 300 ggcgtgttcc acctgcacca gggcgacatc ctgagcgtga tcatcccccg ggcccgggcc 360 aagctgaacc tgagccccca cggcaccttc ctgggcttcg tgaagctggg atccgacaaa 420 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 480 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 540 gtggtggacg tgagccacga agaccctgag gtcaagttc a actggtacgt ggacggcgtg 600 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 660 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 720 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 780 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 840 gtcagcctgt ggtgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 900 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 960 tccttcttcc tctatagcaa gctcaccgtg gacaagagca Ggtggcagca ggggaacgtc 1020 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1080 ctgtctccgg gtaaa 1095 <210> 47 <211> 242 <212> PRT <213> Artificial sequence <400> 47 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Pro Tyr Leu Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser 130 135 140 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 145 150 155 160 Val Ser Ser Serrr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala 165 170 175 Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro 180 185 190 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 195 200 205 Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr 210 215 220 Gly Tyr Pro Pro Ser Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 225 230 235 240 Lys Arg <210> 48 <211> 243 <212> PRT <213> Artificial Sequence <400> 48 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gl n Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Ser Gly Asp Ala Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu 130 135 140 Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln 145 150 155 160 Ser Val Ser Ser Serrr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 165 170 175 Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile 180 185 190 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 210 215 220 Tyr Gly Tyr Pro Pro Arg Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu 225 230 235 240 Ile Lys Arg <210> 49 <211> 26 <212> DNA <213> Artificial Sequence <400> 49 caggaaacag Ctatgaccat gattac 26 <210> 50 <211> 80 <212> DNA <213> Artificial sequence <220> <221> misc_feature <222> (44). . (45) <223> n is a, c, g, or t <220> <221> misc_feature <222> (47). . (48) <223> n is a, c, g, or t <220> <221> misc_feature <222> (50). . (51) <223> n is a, c, g, or t <220> <221> misc_feature <222> (53). . (54) <223> n is a, c, g, or t <220> <221> misc_feature <222> (56). . (57) <223> n is a, c, g, or t <220> <221> misc_feature <222> (59). . (60) <223> n is a, c, g, or t <400> 50 tgagacccac tccagcccct tccctggagc ctggcggacc camnnmnnmn nmnnmnnmnn 60 aaaggtgaat ccggaggctg 80 <210> 51 <211> 67 <212> DNA <213> Artificial sequence (Artificial sequence ) <220> <221> misc_feature <222> (18). . (19) <223> n is a, c, g, or t <220> <221> misc_feature <222> (24). . (25) <223> n is a, c, g, or t <220> <221> misc_feature <222> (27). . (28) <223> n is a, c, g, or t <220> <221> misc_feature <222> (30). . (31) <223> n is a, c, g, or t <220> <221> misc_feature <222> (33). . (34) <223> n is a, c, g, or t <220> <221> misc_feature <222> (39). . (40) <223> n is a, c, g, or t <220> <221> misc_feature <222> (45). . (46) <223> n is a, c, g, or t <400> 51 ggctggagtg ggtctcannk attnnknnkn nknnkggtnn kacannktac gcagactccg 60 tgaaggg 67 <210> 52 <211> 30 <212> DNA <213> Artificial sequence <400> 52 gacgttagta aatgaatttt ctgtatgagg 30 <210> 53 <211> 85 <212> DNA <213> Artificial sequence <220> <221> misc_feature <222> (44). . (45) <223> n is a, c, g, or t <220> <221> misc_feature <222> (50). . (51) <223> n is a, c, g, or t <220> <221> misc_feature <222> (53). . (54) <223> n is a, c, g, or t <220> <221> misc_feature <222> (56). . (57) <223> n is a, c, g, or t <220> <221> misc_feature <222> (59). . (60) <223> n is a, c, g, or t <220> <221> misc_feature <222> (62). . (63) <223> n is a, c, g, or t <220> <221> misc_feature <222> (65). . (66) <223> n is a, c, g, or t <400> 53 gatgaggagc ctgggagcct ggccaggttt ctgctggtac camnntaamn nmnnmnnmnn 60 mnnmnnctga ctggccctgc aagag 85 <210> 54 <211> 58 <212> DNA <213> Artificial sequence (Artificial Sequence) <220> <221> misc_feature <222> (23). . (24) <223> n is a, c, g, or t <220> <221> misc_feature <222> (26). . (27) <223> n is a, c, g, or t <220> <221> misc_feature <222> (29). . (30) <223> n is a, c, g, or t <220> <221> misc_feature <222> (32). . (33) <223> n is a, c, g, or t <220> <221> misc_feature <222> (35). . (36) <223> n is a,  c,  g,   Or   t   <400>   54   Ccaggctccc   Aggctcctca   Tcnnknnknn   Knnknnkagg   Gccactggca   Tcccagac   58   <210>   55   <211>   244   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   55   Glu   Val   Gln   Leu   Leu   Glu   Ser   Gly   Gly   Gly   Leu   Val   Gln   Pro   Gly   Gly   1   5   10   15   Ser   Leu   Arg   Leu   Ser   Cys   Ala   Ala   Ser   Gly   Phe   Thr   Phe   Ser   Ser   Tyr   20   25   30   Ala   Met   Ser   Trp   Val   Arg   Gln   Ala   Pro   Gly   Lys   Gly   Leu   Glu   Trp   Val   35   40   45   Ser   Ala   Ile   Ser   Gly   Ser   Gly   Gly   Ser   Thr   Tyr   Tyr   Ala   Asp   Ser   Val   50   55   60   Lys   Gly   Arg   Phe   Thr   Ile   Ser   Arg   Asp   Asn   Ser   Lys   Asn   Thr   Leu   Tyr   65   70   75   80   Leu   Gln   Met   Asn   Ser   Leu   Arg   Ala   Glu   Asp   Thr   Ala   Val   Tyr   Tyr   Cys   85   90   95   Ala   Lys   Val   Arg   Pro   Phe   Trp   Gly   Thr   Phe   Asp   Tyr   Trp   Gly   Gln   Gly   100   105   110   Thr   Leu   Val   Thr   Val   Ser   Ser   Gly   Gly   Gly   Gly   Ser   Gly   Gly   Gly   Gly   115   120   125   Ser   Gly   Gly   Gly   Gly   Ser   Glu   Ile   Val   Leu   Thr   Gln   Ser   Pro   Gly   Thr   130   135   140   Leu   Ser   Leu   Ser   Pro   Gly   Glu   Arg   Ala   Thr   Leu   Ser   Cys   Arg   Ala   Ser   145   150   155   160   Gln   Ser   Val   Ser   Ser   Ser   Tyr   Leu   Ala   Trp   Tyr   Gln   Gln   Lys   Pro   Gly   165   170   175   Gln   Ala   Pro   Arg   Leu   Leu   Ile   Tyr   Gly   Ala   Ser   Ser   Arg   Ala   Thr   Gly   180   185   190   Ile   Pro   Asp   Arg   Phe   Ser   Gly   Ser   Gly   Ser   Gly   Thr   Asp   Phe   Thr   Leu   195   200   205   Thr   Ile   Ser   Arg   Leu   Glu   Pro   Glu   Asp   Phe   Ala   Val   Tyr   Tyr   Cys   Gln   210   215   220   Gln   Tyr   Phe   Asn   Pro   Pro   Glu   Tyr   Thr   Phe   Gly   Gln   Gly   Thr   Lys   Val   225   230   235   240   Glu   Ile   Lys   Arg   <210>   56   <211>   119   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   56   Glu   Val   Gln   Leu   Leu   Glu   Ser   Gly   Gly   Gly   Leu   Val   Gln   Pro   Gly   Gly   1   5   10   15   Ser   Leu   Arg   Leu   Ser   Cys   Ala   Ala   Ser   Gly   Phe   Thr   Phe   Gly   Gly   Asn   20   25   30   Ala   Met   Ser   Trp   Val   Arg   Gln   Ala   Pro   Gly   Lys   Gly   Leu   Glu   Trp   Val   35   40   45   Ser   Ala   Ile   Ser   Gly   Asn   Gly   Gly   Ser   Thr   Phe   Tyr   Ala   Asp   Ser   Val   50   55   60   Lys   Gly   Arg   Phe   Thr   Ile   Ser   Arg   Asp   Asn   Ser   Lys   Asn   Thr   Leu   Tyr   65   70   75   80   Leu   Gln   Met   Asn   Ser   Leu   Arg   Ala   Glu   Asp   Thr   Ala   Val   Tyr   Tyr   Cys   85   90   95   Ala   Lys   Val   Arg   Pro   Phe   Trp   Gly   Thr   Phe   Asp   Tyr   Trp   Gly   Gln   Gly   100   105   110   Thr   Leu   Val   Thr   Val   Ser   Ser   115   <210>   57   <211>   357   <212>   DNA   <213>   Artificial sequence   Sequence)   <400>   57   Gaggtgcaat   Tgctggagtc   Tgggggaggc   Ttggtacagc   Ctggggggtc   Cctgagactc   60   Tcctgtgcag   Cctccggatt   Cacctttggc   Ggtaatgcca   Tgtcctgggt   Ccgccaggct   120   Ccagggaagg   Ggctggagtg   Ggtctcagca   Attagtggta   Atggtggtag   Tacattctac   180   Gcagactccg   Tgaagggccg   Gttcaccatc   Tccagagaca   Attccaagaa   Cacgctgtat   240   Ctgcagatga   Acagcctgag   Agccgaggac   Acggccgtat   Attactgtgc   Gaaagttcgt   300   Ccattctggg   Gtactttcga   Ctactggggc   Caaggaaccc   Tggtcaccgt   Ctcgagt   357   <210>   58   <211>   119   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   58   Glu   Val   Gln   Leu   Leu   Glu   Ser   Gly   Gly   Gly   Leu   Val   Gln   Pro   Gly   Gly   1   5   10   15   Ser   Leu   Arg   Leu   Ser   Cys   Ala   Ala   Ser   Gly   Phe   Thr   Phe   Arg   Ser   Tyr   20   25   30   Ala   Met   Ser   Trp   Val   Arg   Gln   Ala   Pro   Gly   Lys   Gly   Leu   Glu   Trp   Val   35   40   45   Ser   Ala   Ile   Ser   Gly   Gly   Gly   Gly   Asn   Thr   Phe   Tyr   Ala   Asp   Ser   Val   50   55   60   Lys   Gly   Arg   Phe   Thr   Ile   Ser   Arg   Asp   Asn   Ser   Lys   Asn   Thr   Leu   Tyr   65   70   75   80   Leu   Gln   Met   Asn   Ser   Leu   Arg   Ala   Glu   Asp   Thr   Ala   Val   Tyr   Tyr   Cys   85   90   95   Ala   Lys   Val   Arg   Pro   Phe   Trp   Gly   Thr   Phe   Asp   Tyr   Trp   Gly   Gln   Gly   100   105   110   Thr   Leu   Val   Thr   Val   Ser   Ser   115   <210>   59   <211>   357   <212>   DNA   <213>   Artificial sequence   Sequence)   <400>   59   Gaggtgcaat   Tgctggagtc   Tgggggaggc   Ttggtacagc   Ctggggggtc   Cctgagactc   60   Tcctgtgcag   Cctccggatt   Cacctttagg   Agctatgcca   Tgagctgggt   Ccgccaggct   120   Ccagggaagg   Ggctggagtg   Ggtctcagct   Attagtggcg   Gtggtggtaa   Cacattctac   180   Gcagactccg   Tgaagggccg   Gttcaccatc   Tccagagaca   Attccaagaa   Cacgctgtat   240   Ctgcagatga   Acagcctgag   Agccgaggac   Acggccgtat   Attactgtgc   Gaaagttcgt   300   Ccattctggg   Gtactttcga   Ctactggggc   Caaggaaccc   Tggtcaccgt   Ctcgagt   357   <210>   60   <211>   5   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   60   Gly   Asn   Ala   Met   Ser   1   5   <210>   61   <211>   17   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   61   Ala   Ile   Ser   Gly   Asn   Gly   Gly   Ser   Thr   Phe   Tyr   Ala   Asp   Ser   Val   Lys   1   5   10   15   Gly   <210>   62   <211>   5   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   62   Ser   Tyr   Ala   Met   Ser   1   5   <210>   63   <211>   17   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   63   Ala   Ile   Ser   Gly   Gly   Gly   Gly   Asn   Thr   Phe   Tyr   Ala   Asp   Ser   Val   Lys   1   5   10   15   Gly   <210>   64   <211>   732   <212>   DNA   <213>   Artificial sequence   Sequence)   <400>   64   Gaggtgcaat   Tgctggagtc   Tgggggaggc   Ttggtacagc   Ctggggggtc   Cctgagactc   60   Tcctgtgcag   Cctccggatt   Cacctttggc   Ggtaatgcca   Tgtcctgggt   Ccgccaggct   120   Ccagggaagg   Ggctggagtg   Ggtctcagca   Attagtggta   Atggtggtag   Tacattctac   180   Gcagactccg   Tgaagggccg   Gttcaccatc   Tccagagaca   Attccaagaa   Cacgctgtat   240   Ctgcagatga   Acagcctgag   Agccgaggac   Acggccgtat   Attactgtgc   Gaaagttcgt   300   Ccattctggg   Gtactttcga   Ctactggggc   Caaggaaccc   Tggtcaccgt   Ctcgagtggt   360   Ggaggcggtt   Caggcggagg   Tggttctggc   Ggtggcggat   Cggaaatcgt   Gttaacgcag   420   Tctccaggca   Ccctgtcttt   Gtctccaggg   Gaaagagcca   Ccctctcttg   Cagggccagt   480   Cagagtgtta   Gcagcagcta   Cttagcctgg   Taccagcaga   Aacctggcca   Ggctcccagg   540   Ctcctcatct   Atggagcatc   Cagcagggcc   Actggcatcc   Cagacaggtt   Cagtggcagt   600   Ggatccggga   Cagacttcac   Tctcaccatc   Agcagactgg   Agcctgaaga   Ttttgcagtg   660   Tattactgtc   Agcagtactt   Caacccacca   Gaatacacgt   Tcggccaggg   Gaccaaagtg   720   Gaaatcaaac   Gt   732   <210>   65   <211>   244   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   65   Glu   Val   Gln   Leu   Leu   Glu   Ser   Gly   Gly   Gly   Leu   Val   Gln   Pro   Gly   Gly   1   5   10   15   Ser   Leu   Arg   Leu   Ser   Cys   Ala   Ala   Ser   Gly   Phe   Thr   Phe   Gly   Gly   Asn   20   25   30   Ala   Met   Ser   Trp   Val   Arg   Gln   Ala   Pro   Gly   Lys   Gly   Leu   Glu   Trp   Val   35   40   45   Ser   Ala   Ile   Ser   Gly   Asn   Gly   Gly   Ser   Thr   Phe   Tyr   Ala   Asp   Ser   Val   50   55   60   Lys   Gly   Arg   Phe   Thr   Ile   Ser   Arg   Asp   Asn   Ser   Lys   Asn   Thr   Leu   Tyr   65   70   75   80   Leu   Gln   Met   Asn   Ser   Leu   Arg   Ala   Glu   Asp   Thr   Ala   Val   Tyr   Tyr   Cys   85   90   95   Ala   Lys   Val   Arg   Pro   Phe   Trp   Gly   Thr   Phe   Asp   Tyr   Trp   Gly   Gln   Gly   100   105   110   Thr   Leu   Val   Thr   Val   Ser   Ser   Gly   Gly   Gly   Gly   Ser   Gly   Gly   Gly   Gly   115   120   125   Ser   Gly   Gly   Gly   Gly   Ser   Glu   Ile   Val   Leu   Thr   Gln   Ser   Pro   Gly   Thr   130   135   140   Leu   Ser   Leu   Ser   Pro   Gly   Glu   Arg   Ala   Thr   Leu   Ser   Cys   Arg   Ala   Ser   145   150   155   160   Gln   Ser   Val   Ser   Ser   Ser   Tyr   Leu   Ala   Trp   Tyr   Gln   Gln   Lys   Pro   Gly   165   170   175   Gln   Ala   Pro   Arg   Leu   Leu   Ile   Tyr   Gly   Ala   Ser   Ser   Arg   Ala   Thr   Gly   180   185   190   Ile   Pro   Asp   Arg   Phe   Ser   Gly   Ser   Gly   Ser   Gly   Thr   Asp   Phe   Thr   Leu   195   200   205   Thr   Ile   Ser   Arg   Leu   Glu   Pro   Glu   Asp   Phe   Ala   Val   Tyr   Tyr   Cys   Gln   210   215   220   Gln   Tyr   Phe   Asn   Pro   Pro   Glu   Tyr   Thr   Phe   Gly   Gln   Gly   Thr   Lys   Val   225   230   235   240   Glu   Ile   Lys   Arg   <210>   66   <211>   732   <212>   DNA   <213>   Artificial sequence   Sequence)   <400>   66   Gaggtgcaat   Tgctggagtc   Tgggggaggc   Ttggtacagc   Ctggggggtc   Cctgagactc   60   Tcctgtgcag   Cctccggatt   Cacctttagg   Agctatgcca   Tgagctgggt   Ccgccaggct   120   Ccagggaagg   Ggctggagtg   Ggtctcagct   Attagtggcg   Gtggtggtaa   Cacattctac   180   Gcagactccg   Tgaagggccg   Gttcaccatc   Tccagagaca   Attccaagaa   Cacgctgtat   240   Ctgcagatga   Acagcctgag   Agccgaggac   Acggccgtat   Attactgtgc   Gaaagttcgt   300   Ccattctggg   Gtactttcga   Ctactggggc   Caaggaaccc   Tggtcaccgt   Ctcgagtggt   360   Ggaggcggtt   Caggcggagg   Tggttctggc   Ggtggcggat   Cggaaatcgt   Gttaacgcag   420   Tctccaggca   Ccctgtcttt   Gtctccaggg   Gaaagagcca   Ccctctcttg   Cagggccagt   480   Cagagtgtta   Gcagcagcta   Cttagcctgg   Taccagcaga   Aacctggcca   Ggctcccagg   540   Ctcctcatct   Atggagcatc   Cagcagggcc   Actggcatcc   Cagacaggtt   Cagtggcagt   600   Ggatccggga   Cagacttcac   Tctcaccatc   Agcagactgg   Agcctgaaga   Ttttgcagtg   660   Tattactgtc   Agcagtactt   Caacccacca   Gaatacacgt   Tcggccaggg   Gaccaaagtg   720   Gaaatcaaac   Gt   732   <210>   67   <211>   244   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   67   Glu   Val   Gln   Leu   Leu   Glu   Ser   Gly   Gly   Gly   Leu   Val   Gln   Pro   Gly   Gly   1   5   10   15   Ser   Leu   Arg   Leu   Ser   Cys   Ala   Ala   Ser   Gly   Phe   Thr   Phe   Arg   Ser   Tyr   20   25   30   Ala   Met   Ser   Trp   Val   Arg   Gln   Ala   Pro   Gly   Lys   Gly   Leu   Glu   Trp   Val   35   40   45   Ser   Ala   Ile   Ser   Gly   Gly   Gly   Gly   Asn   Thr   Phe   Tyr   Ala   Asp   Ser   Val   50   55   60   Lys   Gly   Arg   Phe   Thr   Ile   Ser   Arg   Asp   Asn   Ser   Lys   Asn   Thr   Leu   Tyr   65   70   75   80   Leu   Gln   Met   Asn   Ser   Leu   Arg   Ala   Glu   Asp   Thr   Ala   Val   Tyr   Tyr   Cys   85   90   95   Ala   Lys   Val   Arg   Pro   Phe   Trp   Gly   Thr   Phe   Asp   Tyr   Trp   Gly   Gln   Gly   100   105   110   Thr   Leu   Val   Thr   Val   Ser   Ser   Gly   Gly   Gly   Gly   Ser   Gly   Gly   Gly   Gly   115   120   125   Ser   Gly   Gly   Gly   Gly   Ser   Glu   Ile   Val   Leu   Thr   Gln   Ser   Pro   Gly   Thr   130   135   140   Leu   Ser   Leu   Ser   Pro   Gly   Glu   Arg   Ala   Thr   Leu   Ser   Cys   Arg   Ala   Ser   145   150   155   160   Gln   Ser   Val   Ser   Ser   Ser   Tyr   Leu   Ala   Trp   Tyr   Gln   Gln   Lys   Pro   Gly   165   170   175   Gln   Ala   Pro   Arg   Leu   Leu   Ile   Tyr   Gly   Ala   Ser   Ser   Arg   Ala   Thr   Gly   180   185   190   Ile   Pro   Asp   Arg   Phe   Ser   Gly   Ser   Gly   Ser   Gly   Thr   Asp   Phe   Thr   Leu   195   200   205   Thr   Ile   Ser   Arg   Leu   Glu   Pro   Glu   Asp   Phe   Ala   Val   Tyr   Tyr   Cys   Gln   210   215   220   Gln   Tyr   Phe   Asn   Pro   Pro   Glu   Tyr   Thr   Phe   Gly   Gln   Gly   Thr   Lys   Val   225   230   235   240   Glu   Ile   Lys   Arg   <210>   68   <211>   60   <212>   DNA   <213>   Artificial sequence   Sequence)   <400>   68   Atgcagatcc   Cacaggcgcc   Ctggccagtc   Gtctgggcgg   Tgctacaact   Gggctggcgg   60   <210>   69   <211>   450   <212>   DNA   <213>   Artificial sequence   Sequence)   <400>   69   Ccaggatggt   Tcttagactc   Cccagacagg   Ccctggaacc   Cccccacctt   Ctccccagcc   60   Ctgctcgtgg   Tgaccgaagg   Ggacaacgcc   Accttcacct   Gcagcttctc   Caacacatcg   120   Gagagcttcg   Tgctaaactg   Gtaccgcatg   Agccccagca   Accagacgga   Caagctggcc   180   Gccttccccg   Aggaccgcag   Ccaggccggc   Caggactgcc   Gcttccgtgt   Cacacaactg   240   Cccaacgggc   Gtgacttcca   Catgagcgtg   Gtcagggccc   Ggcgcaatga   Cagcggcacc   300   Tacctctgtg   Gggccatctc   Cctggccccc   Aaggcgcaga   Tcaaagagag   Cctgcgggca   360   Gagctcaggg   Tgacagagag   Aagggcagaa   Gtgcccacag   Cccaccccag   Cccctcaccc   420   Aggccagccg   Gccagttcca   Aaccctggtg   450   <210>   70   <211>   321   <212>   DNA   <213>   Artificial sequence   Sequence)   <400>   70   Cccccatgcc   Caccatgccc   Agcacctgag   Ttcctggggg   Gaccatcagt   Cttcctgttc   60   Cccccaaaac   Ccaaggacac   Tctcatgatc   Tcccggaccc   Ctgaggtcac   Gtgcgtggtg   120   Gtggacgtga   Gccaggaaga   Ccccgaggtc   Cagttcaact   Ggtacgtgga   Tggcgtggag   180   Gtgcataatg   Ccaagacaaa   Gccgcgggag   Gagcagttca   Acagcacgta   Ccgtgtggtc   240   Agcgtcctca   Ccgtcctgca   Ccaggactgg   Ctgaacggca   Aggagtacaca   Gtgcaaggtc   300   Tccaacaaag   Gcctcccgtc   c   321   <210>   71   <211>   49   <212>   DNA   <213>   Artificial sequence   Sequence)   <400>   71   Acgcgtccta   Gcgctaccgg   Tcgccaccat   Gcagatccca   Caggcgccc   49   <210>   72   <211>   41   <212>   DNA   <213>   Artificial sequence   Sequence)   <400>   72   Ctctcggggc   Tgcccaccat   Acaccagggt   Ttggaactgg   c   41   <210>   73   <211>   27   <212>   DNA   <213>   Artificial sequence   Sequence)   <400>   73   Tatggtgggc   Agccccgaga   Gccacag   27   <210>   74   <211>   40   <212>   DNA   <213>   Artificial sequence   Sequence)   <400>   74   Aaaattcaaa   Gtctgtttca   Ctttacccgg   Agacagggag   40   <210>   75   <211>   465   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   75   Glu   Val   Gln   Leu   Leu   Glu   Ser   Gly   Gly   Gly   Leu   Val   Gln   Pro   Gly   Gly   1   5   10   15   Ser   Leu   Arg   Leu   Ser   Cys   Ala   Ala   Ser   Gly   Phe   Thr   Phe   Ser   Ser   Tyr   20   25   30   Ala   Met   Ser   Trp   Val   Arg   Gln   Ala   Pro   Gly   Lys   Gly   Leu   Glu   Trp   Val   35   40   45   Ser   Ala   Ile   Ser   Gly   Ser   Gly   Gly   Ser   Thr   Tyr   Tyr   Ala   Asp   Ser   Val   50   55   60   Lys   Gly   Arg   Phe   Thr   Ile   Ser   Arg   Asp   Asn   Ser   Lys   Asn   Thr   Leu   Tyr   65   70   75   80   Leu   Gln   Met   Asn   Ser   Leu   Arg   Ala   Glu   Asp   Thr   Ala   Val   Tyr   Tyr   Cys   85   90   95   Ala   Arg   Tyr   Pro   Tyr   Leu   Ala   Phe   Asp   Tyr   Trp   Gly   Gln   Gly   Thr   Leu   100   105   110   Val   Thr   Val   Ser   Ser   Gly   Gly   Gly   Gly   Ser   Gly   Gly   Gly   Gly   Ser   Gly   115   120   125   Gly   Gly   Gly   Ser   Glu   Ile   Val   Leu   Thr   Gln   Ser   Pro   Gly   Thr   Leu   Ser   130   135   140   Leu   Ser   Pro   Gly   Glu   Arg   Ala   Thr   Leu   Ser   Cys   Arg   Ala   Ser   Gln   Ser   145   150   155   160   Val   Ser   Ser   Ser   Tyr   Leu   Ala   Trp   Tyr   Gln   Gln   Lys   Pro   Gly   Gln   Ala   165   170   175   Pro   Arg   Leu   Leu   Ile   Tyr   Gly   Ala   Ser   Ser   Arg   Ala   Thr   Gly   Ile   Pro   180   185   190   Asp   Arg   Phe   Ser   Gly   Ser   Gly   Ser   Gly   Thr   Asp   Phe   Thr   Leu   Thr   Ile   195   200   205   Ser   Arg   Leu   Glu   Pro   Glu   Asp   Phe   Ala   Val   Tyr   Tyr   Cys   Gln   Gln   Tyr   210   215   220   Gly   Tyr   Pro   Pro   Ser   Tyr   Thr   Phe   Gly   Gln   Gly   Thr   Lys   Val   Glu   Ile   225   230   235   240   Lys   Arg   Thr   Thr   Thr   Pro   Ala   Pro   Arg   Pro   Pro   Thr   Pro   Ala   Pro   Thr   245   250   255   Ile   Ala   Ser   Gln   Pro   Leu   Ser   Leu   Arg   Pro   Glu   Ala   Cys   Arg   Pro   Ala   260   265   270   Ala   Gly   Gly   Ala   Val   His   Thr   Arg   Gly   Leu   Asp   Phe   Ala   Cys   Asp   Ile   275   280   285   Tyr   Ile   Trp   Ala   Pro   Leu   Ala   Gly   Thr   Cys   Gly   Val   Leu   Leu   Leu   Ser   290   295   300   Leu   Val   Ile   Thr   Leu   Tyr   Cys   Lys   Arg   Gly   Arg   Lys   Lys   Leu   Leu   Tyr   305   310   315   320   Ile   Phe   Lys   Gln   Pro   Phe   Met   Arg   Pro   Val   Gln   Thr   Thr   Gln   Glu   Glu   325   330   335   Asp   Gly   Cys   Ser   Cys   Arg   Phe   Pro   Glu   Glu   Glu   Glu   Gly   Gly   Cys   Glu   340   345   350   Leu   Arg   Val   Lys   Phe   Ser   Arg   Ser   Ala   Asp   Ala   Pro   Ala   Tyr   Lys   Gln   355   360   365   Gly   Gln   Asn   Gln   Leu   Tyr   Asn   Glu   Leu   Asn   Leu   Gly   Arg   Arg   Glu   Glu   370   375   380   Tyr   Asp   Val   Leu   Asp   Lys   Arg   Arg   Gly   Arg   Asp   Pro   Glu   Met   Gly   Gly   385   390   395   400   Lys   Pro   Arg   Arg   Lys   Asn   Pro   Gln   Glu   Gly   Leu   Tyr   Asn   Glu   Leu   Gln   405   410   415   Lys   Asp   Lys   Met   Ala   Glu   Ala   Tyr   Ser   Glu   Ile   Gly   Met   Lys   Gly   Glu   420   425   430   Arg   Arg   Arg   Gly   Lys   Gly   His   Asp   Gly   Leu   Tyr   Gln   Gly   Leu   Ser   Thr   435   440   445   Ala   Thr   Lys   Asp   Thr   Tyr   Asp   Ala   Leu   His   Met   Gln   Ala   Leu   Pro   Pro   450   455   460   Arg   465   <210>   76   <211>   467   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   76   Glu   Val   Gln   Leu   Leu   Glu   Ser   Gly   Gly   Gly   Leu   Val   Gln   Pro   Gly   Gly   1   5   10   15   Ser   Leu   Arg   Leu   Ser   Cys   Ala   Ala   Ser   Gly   Phe   Thr   Phe   Gly   Gly   Asn   20   25   30   Ala   Met   Ser   Trp   Val   Arg   Gln   Ala   Pro   Gly   Lys   Gly   Leu   Glu   Trp   Val   35   40   45   Ser   Ala   Ile   Ser   Gly   Asn   Gly   Gly   Ser   Thr   Phe   Tyr   Ala   Asp   Ser   Val   50   55   60   Lys   Gly   Arg   Phe   Thr   Ile   Ser   Arg   Asp   Asn   Ser   Lys   Asn   Thr   Leu   Tyr   65   70   75   80   Leu   Gln   Met   Asn   Ser   Leu   Arg   Ala   Glu   Asp   Thr   Ala   Val   Tyr   Tyr   Cys   85   90   95   Ala   Lys   Val   Arg   Pro   Phe   Trp   Gly   Thr   Phe   Asp   Tyr   Trp   Gly   Gln   Gly   100   105   110   Thr   Leu   Val   Thr   Val   Ser   Ser   Gly   Gly   Gly   Gly   Ser   Gly   Gly   Gly   Gly   115   120   125   Ser   Gly   Gly   Gly   Gly   Ser   Glu   Ile   Val   Leu   Thr   Gln   Ser   Pro   Gly   Thr   130   135   140   Leu   Ser   Leu   Ser   Pro   Gly   Glu   Arg   Ala   Thr   Leu   Ser   Cys   Arg   Ala   Ser   145   150   155   160   Gln   Ser   Val   Ser   Ser   Ser   Tyr   Leu   Ala   Trp   Tyr   Gln   Gln   Lys   Pro   Gly   165   170   175   Gln   Ala   Pro   Arg   Leu   Leu   Ile   Tyr   Gly   Ala   Ser   Ser   Arg   Ala   Thr   Gly   180   185   190   Ile   Pro   Asp   Arg   Phe   Ser   Gly   Ser   Gly   Ser   Gly   Thr   Asp   Phe   Thr   Leu   195   200   205   Thr   Ile   Ser   Arg   Leu   Glu   Pro   Glu   Asp   Phe   Ala   Val   Tyr   Tyr   Cys   Gln   210   215   220   Gln   Tyr   Phe   Asn   Pro   Pro   Glu   Tyr   Thr   Phe   Gly   Gln   Gly   Thr   Lys   Val   225   230   235   240   Glu   Ile   Lys   Arg   Thr   Thr   Thr   Pro   Ala   Pro   Arg   Pro   Pro   Thr   Pro   Ala   245   250   255   Pro   Thr   Ile   Ala   Ser   Gln   Pro   Leu   Ser   Leu   Arg   Pro   Glu   Ala   Cys   Arg   260   265   270   Pro   Ala   Ala   Gly   Gly   Ala   Val   His   Thr   Arg   Gly   Leu   Asp   Phe   Ala   Cys   275   280   285   Asp   Ile   Tyr   Ile   Trp   Ala   Pro   Leu   Ala   Gly   Thr   Cys   Gly   Val   Leu   Leu   290   295   300   Leu   Ser   Leu   Val   Ile   Thr   Leu   Tyr   Cys   Lys   Arg   Gly   Arg   Lys   Lys   Leu   305   310   315   320   Leu   Tyr   Ile   Phe   Lys   Gln   Pro   Phe   Met   Arg   Pro   Val   Gln   Thr   Thr   Gln   325   330   335   Glu   Glu   Asp   Gly   Cys   Ser   Cys   Arg   Phe   Pro   Glu   Glu   Glu   Glu   Gly   Gly   340   345   350   Cys   Glu   Leu   Arg   Val   Lys   Phe   Ser   Arg   Ser   Ala   Asp   Ala   Pro   Ala   Tyr   355   360   365   Lys   Gln   Gly   Gln   Asn   Gln   Leu   Tyr   Asn   Glu   Leu   Asn   Leu   Gly   Arg   Arg   370   375   380   Glu   Glu   Tyr   Asp   Val   Leu   Asp   Lys   Arg   Arg   Gly   Arg   Asp   Pro   Glu   Met   385   390   395   400   Gly   Gly   Lys   Pro   Arg   Arg   Lys   Asn   Pro   Gln   Glu   Gly   Leu   Tyr   Asn   Glu   405   410   415   Leu   Gln   Lys   Asp   Lys   Met   Ala   Glu   Ala   Tyr   Ser   Glu   Ile   Gly   Met   Lys   420   425   430   Gly   Glu   Arg   Arg   Arg   Gly   Lys   Gly   His   Asp   Gly   Leu   Tyr   Gln   Gly   Leu   435   440   445   Ser   Thr   Ala   Thr   Lys   Asp   Thr   Tyr   Asp   Ala   Leu   His   Met   Gln   Ala   Leu   450   455   460   Pro   Pro   Arg   465   <210>   77   <211>   467   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   77   Glu   Val   Gln   Leu   Leu   Glu   Ser   Gly   Gly   Gly   Leu   Val   Gln   Pro   Gly   Gly   1   5   10   15   Ser   Leu   Arg   Leu   Ser   Cys   Ala   Ala   Ser   Gly   Phe   Thr   Phe   Arg   Ser   Tyr   20   25   30   Ala   Met   Ser   Trp   Val   Arg   Gln   Ala   Pro   Gly   Lys   Gly   Leu   Glu   Trp   Val   35   40   45   Ser   Ala   Ile   Ser   Gly   Gly   Gly   Gly   Asn   Thr   Phe   Tyr   Ala   Asp   Ser   Val   50   55   60   Lys   Gly   Arg   Phe   Thr   Ile   Ser   Arg   Asp   Asn   Ser   Lys   Asn   Thr   Leu   Tyr   65   70   75   80   Leu   Gln   Met   Asn   Ser   Leu   Arg   Ala   Glu   Asp   Thr   Ala   Val   Tyr   Tyr   Cys   85   90   95   Ala   Lys   Val   Arg   Pro   Phe   Trp   Gly   Thr   Phe   Asp   Tyr   Trp   Gly   Gln   Gly   100   105   110   Thr   Leu   Val   Thr   Val   Ser   Ser   Gly   Gly   Gly   Gly   Ser   Gly   Gly   Gly   Gly   115   120   125   Ser   Gly   Gly   Gly   Gly   Ser   Glu   Ile   Val   Leu   Thr   Gln   Ser   Pro   Gly   Thr   130   135   140   Leu   Ser   Leu   Ser   Pro   Gly   Glu   Arg   Ala   Thr   Leu   Ser   Cys   Arg   Ala   Ser   145   150   155   160   Gln   Ser   Val   Ser   Ser   Ser   Tyr   Leu   Ala   Trp   Tyr   Gln   Gln   Lys   Pro   Gly   165   170   175   Gln   Ala   Pro   Arg   Leu   Leu   Ile   Tyr   Gly   Ala   Ser   Ser   Arg   Ala   Thr   Gly   180   185   190   Ile   Pro   Asp   Arg   Phe   Ser   Gly   Ser   Gly   Ser   Gly   Thr   Asp   Phe   Thr   Leu   195   200   205   Thr   Ile   Ser   Arg   Leu   Glu   Pro   Glu   Asp   Phe   Ala   Val   Tyr   Tyr   Cys   Gln   210   215   220   Gln   Tyr   Phe   Asn   Pro   Pro   Glu   Tyr   Thr   Phe   Gly   Gln   Gly   Thr   Lys   Val   225   230   235   240   Glu   Ile   Lys   Arg   Thr   Thr   Thr   Pro   Ala   Pro   Arg   Pro   Pro   Thr   Pro   Ala   245   250   255   Pro   Thr   Ile   Ala   Ser   Gln   Pro   Leu   Ser   Leu   Arg   Pro   Glu   Ala   Cys   Arg   260   265   270   Pro   Ala   Ala   Gly   Gly   Ala   Val   His   Thr   Arg   Gly   Leu   Asp   Phe   Ala   Cys   275   280   285   Asp   Ile   Tyr   Ile   Trp   Ala   Pro   Leu   Ala   Gly   Thr   Cys   Gly   Val   Leu   Leu   290   295   300   Leu   Ser   Leu   Val   Ile   Thr   Leu   Tyr   Cys   Lys   Arg   Gly   Arg   Lys   Lys   Leu   305   310   315   320   Leu   Tyr   Ile   Phe   Lys   Gln   Pro   Phe   Met   Arg   Pro   Val   Gln   Thr   Thr   Gln   325   330   335   Glu   Glu   Asp   Gly   Cys   Ser   Cys   Arg   Phe   Pro   Glu   Glu   Glu   Glu   Gly   Gly   340   345   350   Cys   Glu   Leu   Arg   Val   Lys   Phe   Ser   Arg   Ser   Ala   Asp   Ala   Pro   Ala   Tyr   355   360   365   Lys   Gln   Gly   Gln   Asn   Gln   Leu   Tyr   Asn   Glu   Leu   Asn   Leu   Gly   Arg   Arg   370   375   380   Glu   Glu   Tyr   Asp   Val   Leu   Asp   Lys   Arg   Arg   Gly   Arg   Asp   Pro   Glu   Met   385   390   395   400   Gly   Gly   Lys   Pro   Arg   Arg   Lys   Asn   Pro   Gln   Glu   Gly   Leu   Tyr   Asn   Glu   405   410   415   Leu   Gln   Lys   Asp   Lys   Met   Ala   Glu   Ala   Tyr   Ser   Glu   Ile   Gly   Met   Lys   420   425   430   Gly   Glu   Arg   Arg   Arg   Gly   Lys   Gly   His   Asp   Gly   Leu   Tyr   Gln   Gly   Leu   435   440   445   Ser   Thr   Ala   Thr   Lys   Asp   Thr   Tyr   Asp   Ala   Leu   His   Met   Gln   Ala   Leu   450   455   460   Pro   Pro   Arg   465   <210>   78   <211>   466   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   78   Glu   Val   Gln   Leu   Leu   Glu   Ser   Gly   Gly   Gly   Leu   Val   Gln   Pro   Gly   Gly   1   5   10   15   Ser   Leu   Arg   Leu   Ser   Cys   Ala   Ala   Ser   Gly   Phe   Thr   Phe   Ser   Ser   Tyr   20   25   30   Ala   Met   Ser   Trp   Val   Arg   Gln   Ala   Pro   Gly   Lys   Gly   Leu   Glu   Trp   Val   35   40   45   Ser   Ala   Ile   Ser   Gly   Ser   Gly   Gly   Ser   Thr   Tyr   Tyr   Ala   Asp   Ser   Val   50   55   60   Lys   Gly   Arg   Phe   Thr   Ile   Ser   Arg   Asp   Asn   Ser   Lys   Asn   Thr   Leu   Tyr   65   70   75   80   Leu   Gln   Met   Asn   Ser   Leu   Arg   Ala   Glu   Asp   Thr   Ala   Val   Tyr   Tyr   Cys   85   90   95   Ala   Lys   Leu   Ser   Gly   Asp   Ala   Ala   Met   Asp   Tyr   Trp   Gly   Gln   Gly   Thr   100   105   110   Leu   Val   Thr   Val   Ser   Ser   Gly   Gly   Gly   Gly   Ser   Gly   Gly   Gly   Gly   Ser   115   120   125   Gly   Gly   Gly   Gly   Ser   Glu   Ile   Val   Leu   Thr   Gln   Ser   Pro   Gly   Thr   Leu   130   135   140   Ser   Leu   Ser   Pro   Gly   Glu   Arg   Ala   Thr   Leu   Ser   Cys   Arg   Ala   Ser   Gln   145   150   155   160   Ser   Val   Ser   Ser   Ser   Tyr   Leu   Ala   Trp   Tyr   Gln   Gln   Lys   Pro   Gly   Gln   165   170   175   Ala   Pro   Arg   Leu   Leu   Ile   Tyr   Gly   Ala   Ser   Ser   Arg   Ala   Thr   Gly   Ile   180   185   190   Pro   Asp   Arg   Phe   Ser   Gly   Ser   Gly   Ser   Gly   Thr   Asp   Phe   Thr   Leu   Thr   195   200   205   Ile   Ser   Arg   Leu   Glu   Pro   Glu   Asp   Phe   Ala   Val   Tyr   Tyr   Cys   Gln   Gln   210   215   220   Tyr   Gly   Tyr   Pro   Pro   Arg   Tyr   Thr   Phe   Gly   Gln   Gly   Thr   Lys   Val   Glu   225   230   235   240   Ile   Lys   Arg   Thr   Thr   Thr   Pro   Ala   Pro   Arg   Pro   Pro   Thr   Pro   Ala   Pro   245   250   255   Thr   Ile   Ala   Ser   Gln   Pro   Leu   Ser   Leu   Arg   Pro   Glu   Ala   Cys   Arg   Pro   260   265   270   Ala   Ala   Gly   Gly   Ala   Val   His   Thr   Arg   Gly   Leu   Asp   Phe   Ala   Cys   Asp   275   280   285   Ile   Tyr   Ile   Trp   Ala   Pro   Leu   Ala   Gly   Thr   Cys   Gly   Val   Leu   Leu   Leu   290   295   300   Ser   Leu   Val   Ile   Thr   Leu   Tyr   Cys   Lys   Arg   Gly   Arg   Lys   Lys   Leu   Leu   305   310   315   320   Tyr   Ile   Phe   Lys   Gln   Pro   Phe   Met   Arg   Pro   Val   Gln   Thr   Thr   Gln   Glu   325   330   335   Glu   Asp   Gly   Cys   Ser   Cys   Arg   Phe   Pro   Glu   Glu   Glu   Glu   Gly   Gly   Cys   340   345   350   Glu   Leu   Arg   Val   Lys   Phe   Ser   Arg   Ser   Ala   Asp   Ala   Pro   Ala   Tyr   Lys   355   360   365   Gln   Gly   Gln   Asn   Gln   Leu   Tyr   Asn   Glu   Leu   Asn   Leu   Gly   Arg   Arg   Glu   370   375   380   Glu   Tyr   Asp   Val   Leu   Asp   Lys   Arg   Arg   Gly   Arg   Asp   Pro   Glu   Met   Gly   385   390   395   400   Gly   Lys   Pro   Arg   Arg   Lys   Asn   Pro   Gln   Glu   Gly   Leu   Tyr   Asn   Glu   Leu   405   410   415   Gln   Lys   Asp   Lys   Met   Ala   Glu   Ala   Tyr   Ser   Glu   Ile   Gly   Met   Lys   Gly   420   425   430   Glu   Arg   Arg   Arg   Gly   Lys   Gly   His   Asp   Gly   Leu   Tyr   Gln   Gly   Leu   Ser   435   440   445   Thr   Ala   Thr   Lys   Asp   Thr   Tyr   Asp   Ala   Leu   His   Met   Gln   Ala   Leu   Pro   450   455   460   Pro   Arg   465   <210>   79   <211>   468   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   79   Glu   Val   Gln   Leu   Leu   Glu   Ser   Gly   Gly   Gly   Leu   Val   Gln   Pro   Gly   Gly   1   5   10   15   Ser   Leu   Arg   Leu   Ser   Cys   Ala   Ala   Ser   Gly   Phe   Thr   Phe   Ser   Ser   Tyr   20   25   30   Ala   Met   Ser   Trp   Val   Arg   Gln   Ala   Pro   Gly   Lys   Gly   Leu   Glu   Trp   Val   35   40   45   Ser   Ala   Ile   Ser   Gly   Ser   Gly   Gly   Ser   Thr   Tyr   Tyr   Ala   Asp   Ser   Val   50   55   60   Lys   Gly   Arg   Phe   Thr   Ile   Ser   Arg   Asp   Asn   Ser   Lys   Asn   Thr   Leu   Tyr   65   70   75   80   Leu   Gln   Met   Asn   Ser   Leu   Arg   Ala   Glu   Asp   Thr   Ala   Val   Tyr   Tyr   Cys   85   90   95   Ala   Arg   Tyr   Pro   Tyr   Leu   Ala   Phe   Asp   Tyr   Trp   Gly   Gln   Gly   Thr   Leu   100   105   110   Val   Thr   Val   Ser   Ser   Gly   Gly   Gly   Gly   Ser   Gly   Gly   Gly   Gly   Ser   Gly   115   120   125   Gly   Gly   Gly   Ser   Glu   Ile   Val   Leu   Thr   Gln   Ser   Pro   Gly   Thr   Leu   Ser   130   135   140   Leu   Ser   Pro   Gly   Glu   Arg   Ala   Thr   Leu   Ser   Cys   Arg   Ala   Ser   Gln   Ser   145   150   155   160   Val   Ser   Ser   Ser   Tyr   Leu   Ala   Trp   Tyr   Gln   Gln   Lys   Pro   Gly   Gln   Ala   165   170   175   Pro   Arg   Leu   Leu   Ile   Tyr   Gly   Ala   Ser   Ser   Arg   Ala   Thr   Gly   Ile   Pro   180   185   190   Asp   Arg   Phe   Ser   Gly   Ser   Gly   Ser   Gly   Thr   Asp   Phe   Thr   Leu   Thr   Ile   195   200   205   Ser   Arg   Leu   Glu   Pro   Glu   Asp   Phe   Ala   Val   Tyr   Tyr   Cys   Gln   Gln   Tyr   210   215   220   Gly   Tyr   Pro   Pro   Ser   Tyr   Thr   Phe   Gly   Gln   Gly   Thr   Lys   Val   Glu   Ile   225   230   235   240   Lys   Arg   Thr   Thr   Thr   Pro   Ala   Pro   Arg   Pro   Pro   Thr   Pro   Ala   Pro   Thr   245   250   255   Ile   Ala   Ser   Gln   Pro   Leu   Ser   Leu   Arg   Pro   Glu   Ala   Cys   Arg   Pro   Ala   260   265   270   Ala   Gly   Gly   Ala   Val   His   Thr   Arg   Gly   Leu   Asp   Phe   Ala   Cys   Asp   Phe   275   280   285   Trp   Val   Leu   Val   Val   Val   Gly   Gly   Val   Leu   Ala   Cys   Tyr   Ser   Leu   Leu   290   295   300   Val   Thr   Val   Ala   Phe   Ile   Ile   Phe   Trp   Val   Arg   Ser   Lys   Arg   Ser   Arg   305   310   315   320   Leu   Leu   His   Ser   Asp   Tyr   Met   Asn   Met   Thr   Pro   Arg   Arg   Pro   Gly   Pro   325   330   335   Thr   Arg   Lys   His   Tyr   Gln   Pro   Tyr   Ala   Pro   Pro   Arg   Asp   Phe   Ala   Ala   340   345   350   Tyr   Arg   Ser   Arg   Val   Lys   Phe   Ser   Arg   Ser   Ala   Asp   Ala   Pro   Ala   Tyr   355   360   365   Gln   Gln   Gly   Gln   Asn   Gln   Leu   Tyr   Asn   Glu   Leu   Asn   Leu   Gly   Arg   Arg   370   375   380   Glu   Glu   Tyr   Asp   Val   Leu   Asp   Lys   Arg   Arg   Gly   Arg   Asp   Pro   Glu   Met   385   390   395   400   Gly   Gly   Lys   Pro   Gln   Arg   Arg   Lys   Asn   Pro   Gln   Glu   Gly   Leu   Tyr   Asn   405   410   415   Glu   Leu   Gln   Lys   Asp   Lys   Met   Ala   Glu   Ala   Tyr   Ser   Glu   Ile   Gly   Met   420   425   430   Lys   Gly   Glu   Arg   Arg   Arg   Gly   Lys   Gly   His   Asp   Gly   Leu   Tyr   Gln   Gly   435   440   445   Leu   Ser   Thr   Ala   Thr   Lys   Asp   Thr   Tyr   Asp   Ala   Leu   His   Met   Gln   Ala   450   455   460   Leu   Pro   Pro   Arg   465   <210>   80   <211>   510   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   80   Glu   Val   Gln   Leu   Leu   Glu   Ser   Gly   Gly   Gly   Leu   Val   Gln   Pro   Gly   Gly   1   5   10   15   Ser   Leu   Arg   Leu   Ser   Cys   Ala   Ala   Ser   Gly   Phe   Thr   Phe   Ser   Ser   Tyr   20   25   30   Ala   Met   Ser   Trp   Val   Arg   Gln   Ala   Pro   Gly   Lys   Gly   Leu   Glu   Trp   Val   35   40   45   Ser   Ala   Ile   Ser   Gly   Ser   Gly   Gly   Ser   Thr   Tyr   Tyr   Ala   Asp   Ser   Val   50   55   60   Lys   Gly   Arg   Phe   Thr   Ile   Ser   Arg   Asp   Asn   Ser   Lys   Asn   Thr   Leu   Tyr   65   70   75   80   Leu   Gln   Met   Asn   Ser   Leu   Arg   Ala   Glu   Asp   Thr   Ala   Val   Tyr   Tyr   Cys   85   90   95   Ala   Arg   Tyr   Pro   Tyr   Leu   Ala   Phe   Asp   Tyr   Trp   Gly   Gln   Gly   Thr   Leu   100   105   110   Val   Thr   Val   Ser   Ser   Gly   Gly   Gly   Gly   Ser   Gly   Gly   Gly   Gly   Ser   Gly   115   120   125   Gly   Gly   Gly   Ser   Glu   Ile   Val   Leu   Thr   Gln   Ser   Pro   Gly   Thr   Leu   Ser   130   135   140   Leu   Ser   Pro   Gly   Glu   Arg   Ala   Thr   Leu   Ser   Cys   Arg   Ala   Ser   Gln   Ser   145   150   155   160   Val   Ser   Ser   Ser   Tyr   Leu   Ala   Trp   Tyr   Gln   Gln   Lys   Pro   Gly   Gln   Ala   165   170   175   Pro   Arg   Leu   Leu   Ile   Tyr   Gly   Ala   Ser   Ser   Arg   Ala   Thr   Gly   Ile   Pro   180   185   190   Asp   Arg   Phe   Ser   Gly   Ser   Gly   Ser   Gly   Thr   Asp   Phe   Thr   Leu   Thr   Ile   195   200   205   Ser   Arg   Leu   Glu   Pro   Glu   Asp   Phe   Ala   Val   Tyr   Tyr   Cys   Gln   Gln   Tyr   210   215   220   Gly   Tyr   Pro   Pro   Ser   Tyr   Thr   Phe   Gly   Gln   Gly   Thr   Lys   Val   Glu   Ile   225   230   235   240   Lys   Arg   Thr   Thr   Thr   Pro   Ala   Pro   Arg   Pro   Pro   Thr   Pro   Ala   Pro   Thr   245   250   255   Ile   Ala   Ser   Gln   Pro   Leu   Ser   Leu   Arg   Pro   Glu   Ala   Cys   Arg   Pro   Ala   260   265   270   Ala   Gly   Gly   Ala   Val   His   Thr   Arg   Gly   Leu   Asp   Phe   Ala   Cys   Asp   Phe   275   280   285   Trp   Val   Leu   Val   Val   Val   Gly   Gly   Val   Leu   Ala   Cys   Tyr   Ser   Leu   Leu   290   295   300   Val   Thr   Val   Ala   Phe   Ile   Ile   Phe   Trp   Val   Arg   Ser   Lys   Arg   Ser   Arg   305   310   315   320   Leu   Leu   His   Ser   Asp   Tyr   Met   Asn   Met   Thr   Pro   Arg   Arg   Pro   Gly   Pro   325   330   335   Thr   Arg   Lys   His   Tyr   Gln   Pro   Tyr   Ala   Pro   Pro   Arg   Asp   Phe   Ala   Ala   340   345   350   Tyr   Arg   Ser   Lys   Arg   Gly   Arg   Lys   Lys   Leu   Leu   Tyr   Ile   Phe   Lys   Gln   355   360   365   Pro   Phe   Met   Arg   Pro   Val   Gln   Thr   Thr   Gln   Glu   Glu   Asp   Gly   Cys   Ser   370   375   380   Cys   Arg   Phe   Pro   Glu   Glu   Glu   Glu   Gly   Gly   Cys   Glu   Leu   Arg   Val   Lys   385   390   395   400   Phe   Ser   Arg   Ser   Ala   Asp   Ala   Pro   Ala   Tyr   Gln   Gln   Gly   Gln   Asn   Gln   405   410   415   Leu   Tyr   Asn   Glu   Leu   Asn   Leu   Gly   Arg   Arg   Glu   Glu   Tyr   Asp   Val   Leu   420   425   430   Asp   Lys   Arg   Arg   Gly   Arg   Asp   Pro   Glu   Met   Gly   Gly   Lys   Pro   Gln   Arg   435   440   445   Arg   Lys   Asn   Pro   Gln   Glu   Gly   Leu   Tyr   Asn   Glu   Leu   Gln   Lys   Asp   Lys   450   455   460   Met   Ala   Glu   Ala   Tyr   Ser   Glu   Ile   Gly   Met   Lys   Gly   Glu   Arg   Arg   Arg   465   470   475   480   Gly   Lys   Gly   His   Asp   Gly   Leu   Tyr   Gln   Gly   Leu   Ser   Thr   Ala   Thr   Lys   485   490   495   Asp   Thr   Tyr   Asp   Ala   Leu   His   Met   Gln   Ala   Leu   Pro   Pro   Arg   500   505   510   <210>   81   <211>   469   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   81   Glu   Val   Gln   Leu   Leu   Glu   Ser   Gly   Gly   Gly   Leu   Val   Gln   Pro   Gly   Gly   1   5   10   15   Ser   Leu   Arg   Leu   Ser   Cys   Ala   Ala   Ser   Gly   Phe   Thr   Phe   Ser   Ser   Tyr   20   25   30   Ala   Met   Ser   Trp   Val   Arg   Gln   Ala   Pro   Gly   Lys   Gly   Leu   Glu   Trp   Val   35   40   45   Ser   Ala   Ile   Ser   Gly   Ser   Gly   Gly   Ser   Thr   Tyr   Tyr   Ala   Asp   Ser   Val   50   55   60   Lys   Gly   Arg   Phe   Thr   Ile   Ser   Arg   Asp   Asn   Ser   Lys   Asn   Thr   Leu   Tyr   65   70   75   80   Leu   Gln   Met   Asn   Ser   Leu   Arg   Ala   Glu   Asp   Thr   Ala   Val   Tyr   Tyr   Cys   85   90   95   Ala   Lys   Leu   Ser   Gly   Asp   Ala   Ala   Met   Asp   Tyr   Trp   Gly   Gln   Gly   Thr   100   105   110   Leu   Val   Thr   Val   Ser   Ser   Gly   Gly   Gly   Gly   Ser   Gly   Gly   Gly   Gly   Ser   115   120   125   Gly   Gly   Gly   Gly   Ser   Glu   Ile   Val   Leu   Thr   Gln   Ser   Pro   Gly   Thr   Leu   130   135   140   Ser   Leu   Ser   Pro   Gly   Glu   Arg   Ala   Thr   Leu   Ser   Cys   Arg   Ala   Ser   Gln   145   150   155   160   Ser   Val   Ser   Ser   Ser   Tyr   Leu   Ala   Trp   Tyr   Gln   Gln   Lys   Pro   Gly   Gln   165   170   175   Ala   Pro   Arg   Leu   Leu   Ile   Tyr   Gly   Ala   Ser   Ser   Arg   Ala   Thr   Gly   Ile   180   185   190   Pro   Asp   Arg   Phe   Ser   Gly   Ser   Gly   Ser   Gly   Thr   Asp   Phe   Thr   Leu   Thr   195   200   205   Ile   Ser   Arg   Leu   Glu   Pro   Glu   Asp   Phe   Ala   Val   Tyr   Tyr   Cys   Gln   Gln   210   215   220   Tyr   Gly   Tyr   Pro   Pro   Arg   Tyr   Thr   Phe   Gly   Gln   Gly   Thr   Lys   Val   Glu   225   230   235   240   Ile   Lys   Arg   Thr   Thr   Thr   Pro   Ala   Pro   Arg   Pro   Pro   Thr   Pro   Ala   Pro   245   250   255   Thr   Ile   Ala   Ser   Gln   Pro   Leu   Ser   Leu   Arg   Pro   Glu   Ala   Cys   Arg   Pro   260   265   270   Ala   Ala   Gly   Gly   Ala   Val   His   Thr   Arg   Gly   Leu   Asp   Phe   Ala   Cys   Asp   275   280   285   Phe   Trp   Val   Leu   Val   Val   Val   Gly   Gly   Val   Leu   Ala   Cys   Tyr   Ser   Leu   290   295   300   Leu   Val   Thr   Val   Ala   Phe   Ile   Ile   Phe   Trp   Val   Arg   Ser   Lys   Arg   Ser   305   310   315   320   Arg   Leu   Leu   His   Ser   Asp   Tyr   Met   Asn   Met   Thr   Pro   Arg   Arg   Pro   Gly   325   330   335   Pro   Thr   Arg   Lys   His   Tyr   Gln   Pro   Tyr   Ala   Pro   Pro   Arg   Asp   Phe   Ala   340   345   350   Ala   Tyr   Arg   Ser   Arg   Val   Lys   Phe   Ser   Arg   Ser   Ala   Asp   Ala   Pro   Ala   355   360   365   Tyr   Gln   Gln   Gly   Gln   Asn   Gln   Leu   Tyr   Asn   Glu   Leu   Asn   Leu   Gly   Arg   370   375   380   Arg   Glu   Glu   Tyr   Asp   Val   Leu   Asp   Lys   Arg   Arg   Gly   Arg   Asp   Pro   Glu   385   390   395   400   Met   Gly   Gly   Lys   Pro   Gln   Arg   Arg   Lys   Asn   Pro   Gln   Glu   Gly   Leu   Tyr   405   410   415   Asn   Glu   Leu   Gln   Lys   Asp   Lys   Met   Ala   Glu   Ala   Tyr   Ser   Glu   Ile   Gly   420   425   430   Met   Lys   Gly   Glu   Arg   Arg   Arg   Gly   Lys   Gly   His   Asp   Gly   Leu   Tyr   Gln   435   440   445   Gly   Leu   Ser   Thr   Ala   Thr   Lys   Asp   Thr   Tyr   Asp   Ala   Leu   His   Met   Gln   450   455   460   Ala   Leu   Pro   Pro   Arg   465   <210>   82   <211>   511   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   82   Glu   Val   Gln   Leu   Leu   Glu   Ser   Gly   Gly   Gly   Leu   Val   Gln   Pro   Gly   Gly   1   5   10   15   Ser   Leu   Arg   Leu   Ser   Cys   Ala   Ala   Ser   Gly   Phe   Thr   Phe   Ser   Ser   Tyr   20   25   30   Ala   Met   Ser   Trp   Val   Arg   Gln   Ala   Pro   Gly   Lys   Gly   Leu   Glu   Trp   Val   35   40   45   Ser   Ala   Ile   Ser   Gly   Ser   Gly   Gly   Ser   Thr   Tyr   Tyr   Ala   Asp   Ser   Val   50   55   60   Lys   Gly   Arg   Phe   Thr   Ile   Ser   Arg   Asp   Asn   Ser   Lys   Asn   Thr   Leu   Tyr   65   70   75   80   Leu   Gln   Met   Asn   Ser   Leu   Arg   Ala   Glu   Asp   Thr   Ala   Val   Tyr   Tyr   Cys   85   90   95   Ala   Lys   Leu   Ser   Gly   Asp   Ala   Ala   Met   Asp   Tyr   Trp   Gly   Gln   Gly   Thr   100   105   110   Leu   Val   Thr   Val   Ser   Ser   Gly   Gly   Gly   Gly   Ser   Gly   Gly   Gly   Gly   Ser   115   120   125   Gly   Gly   Gly   Gly   Ser   Glu   Ile   Val   Leu   Thr   Gln   Ser   Pro   Gly   Thr   Leu   130   135   140   Ser   Leu   Ser   Pro   Gly   Glu   Arg   Ala   Thr   Leu   Ser   Cys   Arg   Ala   Ser   Gln   145   150   155   160   Ser   Val   Ser   Ser   Ser   Tyr   Leu   Ala   Trp   Tyr   Gln   Gln   Lys   Pro   Gly   Gln   165   170   175   Ala   Pro   Arg   Leu   Leu   Ile   Tyr   Gly   Ala   Ser   Ser   Arg   Ala   Thr   Gly   Ile   180   185   190   Pro   Asp   Arg   Phe   Ser   Gly   Ser   Gly   Ser   Gly   Thr   Asp   Phe   Thr   Leu   Thr   195   200   205   Ile   Ser   Arg   Leu   Glu   Pro   Glu   Asp   Phe   Ala   Val   Tyr   Tyr   Cys   Gln   Gln   210   215   220   Tyr   Gly   Tyr   Pro   Pro   Arg   Tyr   Thr   Phe   Gly   Gln   Gly   Thr   Lys   Val   Glu   225   230   235   240   Ile   Lys   Arg   Thr   Thr   Thr   Pro   Ala   Pro   Arg   Pro   Pro   Thr   Pro   Ala   Pro   245   250   255   Thr   Ile   Ala   Ser   Gln   Pro   Leu   Ser   Leu   Arg   Pro   Glu   Ala   Cys   Arg   Pro   260   265   270   Ala   Ala   Gly   Gly   Ala   Val   His   Thr   Arg   Gly   Leu   Asp   Phe   Ala   Cys   Asp   275   280   285   Phe   Trp   Val   Leu   Val   Val   Val   Gly   Gly   Val   Leu   Ala   Cys   Tyr   Ser   Leu   290   295   300   Leu   Val   Thr   Val   Ala   Phe   Ile   Ile   Phe   Trp   Val   Arg   Ser   Lys   Arg   Ser   305   310   315   320   Arg   Leu   Leu   His   Ser   Asp   Tyr   Met   Asn   Met   Thr   Pro   Arg   Arg   Pro   Gly   325   330   335   Pro   Thr   Arg   Lys   His   Tyr   Gln   Pro   Tyr   Ala   Pro   Pro   Arg   Asp   Phe   Ala   340   345   350   Ala   Tyr   Arg   Ser   Lys   Arg   Gly   Arg   Lys   Lys   Leu   Leu   Tyr   Ile   Phe   Lys   355   360   365   Gln   Pro   Phe   Met   Arg   Pro   Val   Gln   Thr   Thr   Gln   Glu   Glu   Asp   Gly   Cys   370   375   380   Ser   Cys   Arg   Phe   Pro   Glu   Glu   Glu   Glu   Gly   Gly   Cys   Glu   Leu   Arg   Val   385   390   395   400   Lys   Phe   Ser   Arg   Ser   Ala   Asp   Ala   Pro   Ala   Tyr   Gln   Gln   Gly   Gln   Asn   405   410   415   Gln   Leu   Tyr   Asn   Glu   Leu   Asn   Leu   Gly   Arg   Arg   Glu   Glu   Tyr   Asp   Val   420   425   430   Leu   Asp   Lys   Arg   Arg   Gly   Arg   Asp   Pro   Glu   Met   Gly   Gly   Lys   Pro   Gln   435   440   445   Arg   Arg   Lys   Asn   Pro   Gln   Glu   Gly   Leu   Tyr   Asn   Glu   Leu   Gln   Lys   Asp   450   455   460   Lys   Met   Ala   Glu   Ala   Tyr   Ser   Glu   Ile   Gly   Met   Lys   Gly   Glu   Arg   Arg   465   470   475   480   Arg   Gly   Lys   Gly   His   Asp   Gly   Leu   Tyr   Gln   Gly   Leu   Ser   Thr   Ala   Thr   485   490   495   Lys   Asp   Thr   Tyr   Asp   Ala   Leu   His   Met   Gln   Ala   Leu   Pro   Pro   Arg   500   505   510   <210>   83   <211>   470   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   83   Glu   Val   Gln   Leu   Leu   Glu   Ser   Gly   Gly   Gly   Leu   Val   Gln   Pro   Gly   Gly   1   5   10   15   Ser   Leu   Arg   Leu   Ser   Cys   Ala   Ala   Ser   Gly   Phe   Thr   Phe   Ser   Ser   Tyr   20   25   30   Ala   Met   Ser   Trp   Val   Arg   Gln   Ala   Pro   Gly   Lys   Gly   Leu   Glu   Trp   Val   35   40   45   Ser   Ala   Ile   Ser   Gly   Ser   Gly   Gly   Ser   Thr   Tyr   Tyr   Ala   Asp   Ser   Val   50   55   60   Lys   Gly   Arg   Phe   Thr   Ile   Ser   Arg   Asp   Asn   Ser   Lys   Asn   Thr   Leu   Tyr   65   70   75   80   Leu   Gln   Met   Asn   Ser   Leu   Arg   Ala   Glu   Asp   Thr   Ala   Val   Tyr   Tyr   Cys   85   90   95   Ala   Lys   Val   Arg   Pro   Phe   Trp   Gly   Thr   Phe   Asp   Tyr   Trp   Gly   Gln   Gly   100   105   110   Thr   Leu   Val   Thr   Val   Ser   Ser   Gly   Gly   Gly   Gly   Ser   Gly   Gly   Gly   Gly   115   120   125   Ser   Gly   Gly   Gly   Gly   Ser   Glu   Ile   Val   Leu   Thr   Gln   Ser   Pro   Gly   Thr   130   135   140   Leu   Ser   Leu   Ser   Pro   Gly   Glu   Arg   Ala   Thr   Leu   Ser   Cys   Arg   Ala   Ser   145   150   155   160   Gln   Ser   Val   Ser   Ser   Ser   Tyr   Leu   Ala   Trp   Tyr   Gln   Gln   Lys   Pro   Gly   165   170   175   Gln   Ala   Pro   Arg   Leu   Leu   Ile   Tyr   Gly   Ala   Ser   Ser   Arg   Ala   Thr   Gly   180   185   190   Ile   Pro   Asp   Arg   Phe   Ser   Gly   Ser   Gly   Ser   Gly   Thr   Asp   Phe   Thr   Leu   195   200   205   Thr   Ile   Ser   Arg   Leu   Glu   Pro   Glu   Asp   Phe   Ala   Val   Tyr   Tyr   Cys   Gln   210   215   220   Gln   Tyr   Phe   Asn   Pro   Pro   Glu   Tyr   Thr   Phe   Gly   Gln   Gly   Thr   Lys   Val   225   230   235   240   Glu   Ile   Lys   Arg   Thr   Thr   Thr   Pro   Ala   Pro   Arg   Pro   Pro   Thr   Pro   Ala   245   250   255   Pro   Thr   Ile   Ala   Ser   Gln   Pro   Leu   Ser   Leu   Arg   Pro   Glu   Ala   Cys   Arg   260   265   270   Pro   Ala   Ala   Gly   Gly   Ala   Val   His   Thr   Arg   Gly   Leu   Asp   Phe   Ala   Cys   275   280   285   Asp   Phe   Trp   Val   Leu   Val   Val   Val   Gly   Gly   Val   Leu   Ala   Cys   Tyr   Ser   290   295   300   Leu   Leu   Val   Thr   Val   Ala   Phe   Ile   Ile   Phe   Trp   Val   Arg   Ser   Lys   Arg   305   310   315   320   Ser   Arg   Leu   Leu   His   Ser   Asp   Tyr   Met   Asn   Met   Thr   Pro   Arg   Arg   Pro   325   330   335   Gly   Pro   Thr   Arg   Lys   His   Tyr   Gln   Pro   Tyr   Ala   Pro   Pro   Arg   Asp   Phe   340   345   350   Ala   Ala   Tyr   Arg   Ser   Arg   Val   Lys   Phe   Ser   Arg   Ser   Ala   Asp   Ala   Pro   355   360   365   Ala   Tyr   Gln   Gln   Gly   Gln   Asn   Gln   Leu   Tyr   Asn   Glu   Leu   Asn   Leu   Gly   370   375   380   Arg   Arg   Glu   Glu   Tyr   Asp   Val   Leu   Asp   Lys   Arg   Arg   Gly   Arg   Asp   Pro   385   390   395   400   Glu   Met   Gly   Gly   Lys   Pro   Gln   Arg   Arg   Lys   Asn   Pro   Gln   Glu   Gly   Leu   405   410   415   Tyr   Asn   Glu   Leu   Gln   Lys   Asp   Lys   Met   Ala   Glu   Ala   Tyr   Ser   Glu   Ile   420   425   430   Gly   Met   Lys   Gly   Glu   Arg   Arg   Arg   Gly   Lys   Gly   His   Asp   Gly   Leu   Tyr   435   440   445   Gln   Gly   Leu   Ser   Thr   Ala   Thr   Lys   Asp   Thr   Tyr   Asp   Ala   Leu   His   Met   450   455   460   Gln   Ala   Leu   Pro   Pro   Arg   465   470   <210>   84   <211>   512   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   84   Glu   Val   Gln   Leu   Leu   Glu   Ser   Gly   Gly   Gly   Leu   Val   Gln   Pro   Gly   Gly   1   5   10   15   Ser   Leu   Arg   Leu   Ser   Cys   Ala   Ala   Ser   Gly   Phe   Thr   Phe   Ser   Ser   Tyr   20   25   30   Ala   Met   Ser   Trp   Val   Arg   Gln   Ala   Pro   Gly   Lys   Gly   Leu   Glu   Trp   Val   35   40   45   Ser   Ala   Ile   Ser   Gly   Ser   Gly   Gly   Ser   Thr   Tyr   Tyr   Ala   Asp   Ser   Val   50   55   60   Lys   Gly   Arg   Phe   Thr   Ile   Ser   Arg   Asp   Asn   Ser   Lys   Asn   Thr   Leu   Tyr   65   70   75   80   Leu   Gln   Met   Asn   Ser   Leu   Arg   Ala   Glu   Asp   Thr   Ala   Val   Tyr   Tyr   Cys   85   90   95   Ala   Lys   Val   Arg   Pro   Phe   Trp   Gly   Thr   Phe   Asp   Tyr   Trp   Gly   Gln   Gly   100   105   110   Thr   Leu   Val   Thr   Val   Ser   Ser   Gly   Gly   Gly   Gly   Ser   Gly   Gly   Gly   Gly   115   120   125   Ser   Gly   Gly   Gly   Gly   Ser   Glu   Ile   Val   Leu   Thr   Gln   Ser   Pro   Gly   Thr   130   135   140   Leu   Ser   Leu   Ser   Pro   Gly   Glu   Arg   Ala   Thr   Leu   Ser   Cys   Arg   Ala   Ser   145   150   155   160   Gln   Ser   Val   Ser   Ser   Ser   Tyr   Leu   Ala   Trp   Tyr   Gln   Gln   Lys   Pro   Gly   165   170   175   Gln   Ala   Pro   Arg   Leu   Leu   Ile   Tyr   Gly   Ala   Ser   Ser   Arg   Ala   Thr   Gly   180   185   190   Ile   Pro   Asp   Arg   Phe   Ser   Gly   Ser   Gly   Ser   Gly   Thr   Asp   Phe   Thr   Leu   195   200   205   Thr   Ile   Ser   Arg   Leu   Glu   Pro   Glu   Asp   Phe   Ala   Val   Tyr   Tyr   Cys   Gln   210   215   220   Gln   Tyr   Phe   Asn   Pro   Pro   Glu   Tyr   Thr   Phe   Gly   Gln   Gly   Thr   Lys   Val   225   230   235   240   Glu   Ile   Lys   Arg   Thr   Thr   Thr   Pro   Ala   Pro   Arg   Pro   Pro   Thr   Pro   Ala   245   250   255   Pro   Thr   Ile   Ala   Ser   Gln   Pro   Leu   Ser   Leu   Arg   Pro   Glu   Ala   Cys   Arg   260   265   270   Pro   Ala   Ala   Gly   Gly   Ala   Val   His   Thr   Arg   Gly   Leu   Asp   Phe   Ala   Cys   275   280   285   Asp   Phe   Trp   Val   Leu   Val   Val   Val   Gly   Gly   Val   Leu   Ala   Cys   Tyr   Ser   290   295   300   Leu   Leu   Val   Thr   Val   Ala   Phe   Ile   Ile   Phe   Trp   Val   Arg   Ser   Lys   Arg   305   310   315   320   Ser   Arg   Leu   Leu   His   Ser   Asp   Tyr   Met   Asn   Met   Thr   Pro   Arg   Arg   Pro   325   330   335   Gly   Pro   Thr   Arg   Lys   His   Tyr   Gln   Pro   Tyr   Ala   Pro   Pro   Arg   Asp   Phe   340   345   350   Ala   Ala   Tyr   Arg   Ser   Lys   Arg   Gly   Arg   Lys   Lys   Leu   Leu   Tyr   Ile   Phe   355   360   365   Lys   Gln   Pro   Phe   Met   Arg   Pro   Val   Gln   Thr   Thr   Gln   Glu   Glu   Asp   Gly   370   375   380   Cys   Ser   Cys   Arg   Phe   Pro   Glu   Glu   Glu   Glu   Gly   Gly   Cys   Glu   Leu   Arg   385   390   395   400   Val   Lys   Phe   Ser   Arg   Ser   Ala   Asp   Ala   Pro   Ala   Tyr   Gln   Gln   Gly   Gln   405   410   415   Asn   Gln   Leu   Tyr   Asn   Glu   Leu   Asn   Leu   Gly   Arg   Arg   Glu   Glu   Tyr   Asp   420   425   430   Val   Leu   Asp   Lys   Arg   Arg   Gly   Arg   Asp   Pro   Glu   Met   Gly   Gly   Lys   Pro   435   440   445   Gln   Arg   Arg   Lys   Asn   Pro   Gln   Glu   Gly   Leu   Tyr   Asn   Glu   Leu   Gln   Lys   450   455   460   Asp   Lys   Met   Ala   Glu   Ala   Tyr   Ser   Glu   Ile   Gly   Met   Lys   Gly   Glu   Arg   465   470   475   480   Arg   Arg   Gly   Lys   Gly   His   Asp   Gly   Leu   Tyr   Gln   Gly   Leu   Ser   Thr   Ala   485   490   495   Thr   Lys   Asp   Thr   Tyr   Asp   Ala   Leu   His   Met   Gln   Ala   Leu   Pro   Pro   Arg   500   505   510   <210>   85   <211>   470   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   85   Glu   Val   Gln   Leu   Leu   Glu   Ser   Gly   Gly   Gly   Leu   Val   Gln   Pro   Gly   Gly   1   5   10   15   Ser   Leu   Arg   Leu   Ser   Cys   Ala   Ala   Ser   Gly   Phe   Thr   Phe   Arg   Ser   Tyr   20   25   30   Ala   Met   Ser   Trp   Val   Arg   Gln   Ala   Pro   Gly   Lys   Gly   Leu   Glu   Trp   Val   35   40   45   Ser   Ala   Ile   Ser   Gly   Gly   Gly   Gly   Asn   Thr   Phe   Tyr   Ala   Asp   Ser   Val   50   55   60   Lys   Gly   Arg   Phe   Thr   Ile   Ser   Arg   Asp   Asn   Ser   Lys   Asn   Thr   Leu   Tyr   65   70   75   80   Leu   Gln   Met   Asn   Ser   Leu   Arg   Ala   Glu   Asp   Thr   Ala   Val   Tyr   Tyr   Cys   85   90   95   Ala   Lys   Val   Arg   Pro   Phe   Trp   Gly   Thr   Phe   Asp   Tyr   Trp   Gly   Gln   Gly   100   105   110   Thr   Leu   Val   Thr   Val   Ser   Ser   Gly   Gly   Gly   Gly   Ser   Gly   Gly   Gly   Gly   115   120   125   Ser   Gly   Gly   Gly   Gly   Ser   Glu   Ile   Val   Leu   Thr   Gln   Ser   Pro   Gly   Thr   130   135   140   Leu   Ser   Leu   Ser   Pro   Gly   Glu   Arg   Ala   Thr   Leu   Ser   Cys   Arg   Ala   Ser   145   150   155   160   Gln   Ser   Val   Ser   Ser   Ser   Tyr   Leu   Ala   Trp   Tyr   Gln   Gln   Lys   Pro   Gly   165   170   175   Gln   Ala   Pro   Arg   Leu   Leu   Ile   Tyr   Gly   Ala   Ser   Ser   Arg   Ala   Thr   Gly   180   185   190   Ile   Pro   Asp   Arg   Phe   Ser   Gly   Ser   Gly   Ser   Gly   Thr   Asp   Phe   Thr   Leu   195   200   205   Thr   Ile   Ser   Arg   Leu   Glu   Pro   Glu   Asp   Phe   Ala   Val   Tyr   Tyr   Cys   Gln   210   215   220   Gln   Tyr   Phe   Asn   Pro   Pro   Glu   Tyr   Thr   Phe   Gly   Gln   Gly   Thr   Lys   Val   225   230   235   240   Glu   Ile   Lys   Arg   Thr   Thr   Thr   Pro   Ala   Pro   Arg   Pro   Pro   Thr   Pro   Ala   245   250   255   Pro   Thr   Ile   Ala   Ser   Gln   Pro   Leu   Ser   Leu   Arg   Pro   Glu   Ala   Cys   Arg   260   265   270   Pro   Ala   Ala   Gly   Gly   Ala   Val   His   Thr   Arg   Gly   Leu   Asp   Phe   Ala   Cys   275   280   285   Asp   Phe   Trp   Val   Leu   Val   Val   Val   Gly   Gly   Val   Leu   Ala   Cys   Tyr   Ser   290   295   300   Leu   Leu   Val   Thr   Val   Ala   Phe   Ile   Ile   Phe   Trp   Val   Arg   Ser   Lys   Arg   305   310   315   320   Ser   Arg   Leu   Leu   His   Ser   Asp   Tyr   Met   Asn   Met   Thr   Pro   Arg   Arg   Pro   325   330   335   Gly   Pro   Thr   Arg   Lys   His   Tyr   Gln   Pro   Tyr   Ala   Pro   Pro   Arg   Asp   Phe   340   345   350   Ala   Ala   Tyr   Arg   Ser   Arg   Val   Lys   Phe   Ser   Arg   Ser   Ala   Asp   Ala   Pro   355   360   365   Ala   Tyr   Gln   Gln   Gly   Gln   Asn   Gln   Leu   Tyr   Asn   Glu   Leu   Asn   Leu   Gly   370   375   380   Arg   Arg   Glu   Glu   Tyr   Asp   Val   Leu   Asp   Lys   Arg   Arg   Gly   Arg   Asp   Pro   385   390   395   400   Glu   Met   Gly   Gly   Lys   Pro   Gln   Arg   Arg   Lys   Asn   Pro   Gln   Glu   Gly   Leu   405   410   415   Tyr   Asn   Glu   Leu   Gln   Lys   Asp   Lys   Met   Ala   Glu   Ala   Tyr   Ser   Glu   Ile   420   425   430   Gly   Met   Lys   Gly   Glu   Arg   Arg   Arg   Gly   Lys   Gly   His   Asp   Gly   Leu   Tyr   435   440   445   Gln   Gly   Leu   Ser   Thr   Ala   Thr   Lys   Asp   Thr   Tyr   Asp   Ala   Leu   His   Met   450   455   460   Gln   Ala   Leu   Pro   Pro   Arg   465   470   <210>   86   <211>   512   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   86   Glu   Val   Gln   Leu   Leu   Glu   Ser   Gly   Gly   Gly   Leu   Val   Gln   Pro   Gly   Gly   1   5   10   15   Ser   Leu   Arg   Leu   Ser   Cys   Ala   Ala   Ser   Gly   Phe   Thr   Phe   Arg   Ser   Tyr   20   25   30   Ala   Met   Ser   Trp   Val   Arg   Gln   Ala   Pro   Gly   Lys   Gly   Leu   Glu   Trp   Val   35   40   45   Ser   Ala   Ile   Ser   Gly   Gly   Gly   Gly   Asn   Thr   Phe   Tyr   Ala   Asp   Ser   Val   50   55   60   Lys   Gly   Arg   Phe   Thr   Ile   Ser   Arg   Asp   Asn   Ser   Lys   Asn   Thr   Leu   Tyr   65   70   75   80   Leu   Gln   Met   Asn   Ser   Leu   Arg   Ala   Glu   Asp   Thr   Ala   Val   Tyr   Tyr   Cys   85   90   95   Ala   Lys   Val   Arg   Pro   Phe   Trp   Gly   Thr   Phe   Asp   Tyr   Trp   Gly   Gln   Gly   100   105   110   Thr   Leu   Val   Thr   Val   Ser   Ser   Gly   Gly   Gly   Gly   Ser   Gly   Gly   Gly   Gly   115   120   125   Ser   Gly   Gly   Gly   Gly   Ser   Glu   Ile   Val   Leu   Thr   Gln   Ser   Pro   Gly   Thr   130   135   140   Leu   Ser   Leu   Ser   Pro   Gly   Glu   Arg   Ala   Thr   Leu   Ser   Cys   Arg   Ala   Ser   145   150   155   160   Gln   Ser   Val   Ser   Ser   Ser   Tyr   Leu   Ala   Trp   Tyr   Gln   Gln   Lys   Pro   Gly   165   170   175   Gln   Ala   Pro   Arg   Leu   Leu   Ile   Tyr   Gly   Ala   Ser   Ser   Arg   Ala   Thr   Gly   180   185   190   Ile   Pro   Asp   Arg   Phe   Ser   Gly   Ser   Gly   Ser   Gly   Thr   Asp   Phe   Thr   Leu   195   200   205   Thr   Ile   Ser   Arg   Leu   Glu   Pro   Glu   Asp   Phe   Ala   Val   Tyr   Tyr   Cys   Gln   210   215   220   Gln   Tyr   Phe   Asn   Pro   Pro   Glu   Tyr   Thr   Phe   Gly   Gln   Gly   Thr   Lys   Val   225   230   235   240   Glu   Ile   Lys   Arg   Thr   Thr   Thr   Pro   Ala   Pro   Arg   Pro   Pro   Thr   Pro   Ala   245   250   255   Pro   Thr   Ile   Ala   Ser   Gln   Pro   Leu   Ser   Leu   Arg   Pro   Glu   Ala   Cys   Arg   260   265   270   Pro   Ala   Ala   Gly   Gly   Ala   Val   His   Thr   Arg   Gly   Leu   Asp   Phe   Ala   Cys   275   280   285   Asp   Phe   Trp   Val   Leu   Val   Val   Val   Gly   Gly   Val   Leu   Ala   Cys   Tyr   Ser   290   295   300   Leu   Leu   Val   Thr   Val   Ala   Phe   Ile   Ile   Phe   Trp   Val   Arg   Ser   Lys   Arg   305   310   315   320   Ser   Arg   Leu   Leu   His   Ser   Asp   Tyr   Met   Asn   Met   Thr   Pro   Arg   Arg   Pro   325   330   335   Gly   Pro   Thr   Arg   Lys   His   Tyr   Gln   Pro   Tyr   Ala   Pro   Pro   Arg   Asp   Phe   340   345   350   Ala   Ala   Tyr   Arg   Ser   Lys   Arg   Gly   Arg   Lys   Lys   Leu   Leu   Tyr   Ile   Phe   355   360   365   Lys   Gln   Pro   Phe   Met   Arg   Pro   Val   Gln   Thr   Thr   Gln   Glu   Glu   Asp   Gly   370   375   380   Cys   Ser   Cys   Arg   Phe   Pro   Glu   Glu   Glu   Glu   Gly   Gly   Cys   Glu   Leu   Arg   385   390   395   400   Val   Lys   Phe   Ser   Arg   Ser   Ala   Asp   Ala   Pro   Ala   Tyr   Gln   Gln   Gly   Gln   405   410   415   Asn   Gln   Leu   Tyr   Asn   Glu   Leu   Asn   Leu   Gly   Arg   Arg   Glu   Glu   Tyr   Asp   420   425   430   Val   Leu   Asp   Lys   Arg   Arg   Gly   Arg   Asp   Pro   Glu   Met   Gly   Gly   Lys   Pro   435   440   445   Gln   Arg   Arg   Lys   Asn   Pro   Gln   Glu   Gly   Leu   Tyr   Asn   Glu   Leu   Gln   Lys   450   455   460   Asp   Lys   Met   Ala   Glu   Ala   Tyr   Ser   Glu   Ile   Gly   Met   Lys   Gly   Glu   Arg   465   470   475   480   Arg   Arg   Gly   Lys   Gly   His   Asp   Gly   Leu   Tyr   Gln   Gly   Leu   Ser   Thr   Ala   485   490   495   Thr   Lys   Asp   Thr   Tyr   Asp   Ala   Leu   His   Met   Gln   Ala   Leu   Pro   Pro   Arg   500   505   510   <210>   87   <211>   412   <212>   PRT   <213>   Artificial sequence   Sequence)   <400>   87   Gln   Ile   Gln   Leu   Val   Gln   Ser   Gly   Pro   Glu   Leu   Lys   Lys   Pro   Gly   Glu   1   5   10   15   Thr   Val   Lys   Ile   Ser   Cys   Lys   Ala   Ser   Gly   Tyr   Thr   Phe   Thr   Asp   Tyr   20   25   30   Ser   Ile   Asn   Trp   Val   Lys   Arg   Ala   Pro   Gly   Lys   Gly   Leu   Lys   Trp   Met   35   40   45   Gly   Trp   Ile   Asn   Thr   Glu   Thr   Arg   Glu   Pro   Ala   Tyr   Ala   Tyr   Asp   Phe   50   55   60   Arg   Gly   Arg   Phe   Ala   Phe   Ser   Leu   Glu   Thr   Ser   Ala   Ser   Thr   Ala   Tyr   65   70   75   80   Leu   Gln   Ile   Asn   Asn   Leu   Lys   Tyr   Glu   Asp   Thr   Ala   Thr   Tyr   Phe   Cys   85   90   95   Ala   Leu   Asp   Tyr   Ser   Tyr   Ala   Met   Asp   Tyr   Trp   Gly   Gln   Gly   Thr   Ser   100   105   110   Val   Thr   Val   Ser   Ser   Gly   Gly   Gly   Gly   Ser   Gly   Gly   Gly   Gly   Ser   Gly   115   120   125   Gly   Gly   Gly   Ser   Asp   Ile   Val   Leu   Thr   Gln   Ser   Pro   Pro   Ser   Leu   Ala   130   135   140   Met   Ser   Leu   Gly   Lys   Arg   Ala   Thr   Ile   Ser   Cys   Arg   Ala   Ser   Glu   Ser   145   150   155   160   Val   Thr   Ile   Leu   Gly   Ser   His   Leu   Ile   His   Trp   Tyr   Gln   Gln   Lys   Pro   165   170   175   Gly   Gln   Pro   Pro   Thr   Leu   Leu   Ile   Gln   Leu   Ala   Ser   Asn   Thr   Thr   Thr   180   185   190   Pro   Ala   Pro   Arg   Pro   Pro   Thr   Pro   Ala   Pro   Thr   Ile   Ala   Ser   Gln   Pro   195   200   205   Leu   Ser   Leu   Arg   Pro   Glu   Ala   Cys   Arg   Pro   Ala   Ala   Gly   Gly   Ala   Val   210   215   220   His   Thr   Arg   Gly   Leu   Asp   Phe   Ala   Cys   Asp   Ile   Tyr   Ile   Trp   Ala   Pro   225   230   235   240   Leu   Ala   Gly   Thr   Cys   Gly   Val   Leu   Leu   Leu   Ser   Leu   Val   Ile   Thr   Leu   245   250   255   Tyr   Cys   Lys   Arg   Gly   Arg   Lys   Lys   Leu   Leu   Tyr   Ile   Phe   Lys   Gln   Pro   260   265   270   Phe   Met   Arg   Pro   Val   Gln   Thr   Thr   Gln   Glu   Glu   Asp   Gly   Cys   Ser   Cys   275   280   285   Arg   Phe   Pro   Glu   Glu   Glu   Glu   Gly   Gly   Cys   Glu   Leu   Arg   Val   Lys   Phe   290   295   300   Ser   Arg   Ser   Ala   Asp   Ala   Pro   Ala   Tyr   Lys   Gln   Gly   Gln   Asn   Gln   Leu   305   310   315   320   Tyr   Asn   Glu   Leu   Asn   Leu   Gly   Arg   Arg   Glu   Glu   Tyr   Asp   Val   Leu   Asp   325   330   335   Lys   Arg   Arg   Gly   Arg   Asp   Pro   Glu   Met   Gly   Gly   Lys   Pro   Arg   Arg   Lys   340   345   350   Asn   Pro   Gln   Glu   Gly   Leu   Tyr   Asn   Glu   Leu   Gln   Lys   Asp   Lys   Met   Ala   355   360   365   Glu   Ala   Tyr   Ser   Glu   Ile   Gly   Met   Lys   Gly   Glu   Arg   Arg   Arg   Gly   Lys   370   375   380   Gly   His   Asp   Gly   Leu   Tyr   Gln   Gly   Leu   Ser   Thr   Ala   Thr   Lys   Asp   Thr   385   390   395   400   Tyr   Asp   Ala   Leu   His   Met   Gln   Ala   Leu   Pro   Pro   Arg   405   410

Claims (28)

An antibody against BCMA, characterized in that the antibody is selected from the group consisting of: (1) an antibody comprising a heavy chain variable region comprising SEQ ID NO: 1, 60 or 62 The HCDR1 is shown, and/or comprises the HCDR2 of SEQ ID NO: 2, 61 or 63, and/or comprises the HCDR3 of any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. (2) an antibody comprising a light chain variable region comprising the LCDR1 of SEQ ID NO: 6, and/or comprising the LCDR2 of SEQ ID NO: 7, and/or comprising SEQ ID NO: 8, LCDR3 of any one of SEQ ID NO: 9 or SEQ ID NO: 10; (3) an antibody comprising (1) a heavy chain variable region of the antibody and (2) the antibody The variant of the antibody according to any one of (1) to (3), which is the same as the antibody of any one of (1) to (3). Or similar activity. The antibody according to claim 1, wherein the antibody is selected from the group consisting of: (1) an antibody comprising the HCDR1 represented by SEQ ID NO: 1, the HCDR2 represented by SEQ ID NO: 2, and SEQ ID NO: : HCDR3 shown in 3, LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, LCDR3 shown in SEQ ID NO: 8, and (2) antibody comprising SEQ ID NO: 1. HCDR1 shown in HCDR1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 4, and LCDR2 shown in SEQ ID NO: 6, LCDR2 and SEQ ID NO: 9 shown in SEQ ID NO: LCDR3; (3) antibody, HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 5, and LCDR1, SEQ shown in SEQ ID NO: 6. ID NO: LCDR2 shown in 7 and LCDR3 shown in SEQ ID NO: 10; (4) Antibody comprising HCDR1 shown in SEQ ID NO: 60, HCDR2 shown in SEQ ID NO: 61, and SEQ ID NO: HCDR3 shown in 5 and LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, LCDR3 shown in SEQ ID NO: 10; (5) antibody comprising SEQ ID NO: 62 HCDR1, HCDR2 shown in SEQ ID NO: 63, HCDR3 shown in SEQ ID NO: 5, and SEQ ID NO: 6 The LCDR1, SEQ ID NO: LCDR2, shown in FIG. 7 SEQ ID NO: LCDR3 10 shown. (6) The antibody of any one of (1) to (5), which has the same or similar activity as the antibody according to any one of (1) to (5). The antibody according to claim 1, wherein the antibody is selected from the group consisting of: (1) an antibody, wherein the heavy chain variable region of the antibody has the amino acid sequence of SEQ ID NO: 13, SEQ. ID NO: the amino acid sequence shown by 17, the amino acid sequence shown by SEQ ID NO: 21, the amino acid sequence shown by SEQ ID NO: 56, or the amino acid shown by SEQ ID NO: (2) an antibody, wherein the light chain variable region of the antibody has the amino acid sequence of SEQ ID NO: 11, the amino acid sequence of SEQ ID NO: 15, or SEQ ID NO: 19 The amino acid sequence shown; (3) an antibody comprising (1) a heavy chain variable region of the antibody and (2) a light chain variable region of the antibody; (4) an antibody, (1)~( The variant of the antibody according to any one of (1) to (3), which has the same or similar activity as the antibody according to any one of (1) to (3). The antibody according to claim 3, wherein the antibody is selected from the group consisting of: (1) an antibody, wherein the heavy chain variable region of the antibody has the amino acid sequence of SEQ ID NO: 13 and The light chain variable region of the antibody has the amino acid sequence of SEQ ID NO: 11; (2) the antibody, the heavy chain variable region of the antibody having the amino acid sequence of SEQ ID NO: And the light chain variable region of the antibody has the amino acid sequence shown by SEQ ID NO: 15; (3) the antibody, the heavy chain variable region of the antibody having the amino group shown by SEQ ID NO: An acid sequence and the light chain variable region of the antibody has the amino acid sequence set forth in SEQ ID NO: 19; (4) an antibody, the heavy chain variable region of the antibody having the SEQ ID NO: 56 An amino acid sequence and the light chain variable region of the antibody has the amino acid sequence set forth in SEQ ID NO: 19; (5) an antibody having a heavy chain variable region of SEQ ID NO: 58 The amino acid sequence of the antibody and the light chain variable region of the antibody has the amino acid sequence of SEQ ID NO: 19; (6) The antibody of any one of (1) to (5) Variant The same or similar (1) to (5) as claimed in any one antibody activity. The antibody, which recognizes the same epitope as the antibody of any one of claims 1-4. A nucleic acid encoding the antibody of any one of claims 1 to 5. A performance vector comprising the nucleic acid of claim 6. A host cell comprising the expression vector of claim 7 or a nucleic acid as defined in claim 6 integrated in the genome. Use of the antibody according to any one of claims 1 to 5, for the preparation of a target drug, an antibody drug conjugate or a multifunctional antibody for a tumor cell of a specific target BCMA; or a reagent for preparing a tumor for diagnosis, The tumor exhibits BCMA; or is used to prepare chimeric antigen receptor-modified immune cells; preferably, the immune cells include: T lymphocytes, NK cells, or NKT lymphocytes. A multifunctional immunoconjugate comprising: the antibody of any one of claims 1 to 5; and a functional molecule linked thereto; the functional molecule Selected from: a molecule that targets a tumor surface marker, a molecule that inhibits tumors, a molecule that targets a surface marker of an immune cell, or a detectable label. The multifunctional immunoconjugate according to claim 10, wherein the tumor suppressing molecule is an antitumor cytokine or an antitumor toxin. Preferably, the cytokine comprises: IL-12, IL-15, type I interferon, TNF-alpha. The multifunctional immunoconjugate according to claim 10, wherein the molecule of the surface marker of the target immune cell is an antibody or a ligand that binds to an immunocyte surface marker; preferably, the Immunocyte surface markers include: CD3, CD16, CD28, and more preferably, the antibody that binds to an immunocyte surface marker is an anti-CD3 antibody. The multifunctional immunoconjugate of claim 12, wherein the molecule of the surface marker of the target immune cell is an antibody that binds to a T cell surface marker. The nucleic acid encoding the multifunctional immunoconjugate of any of claims 10-13. Use of the multifunctional immunoconjugate of any of claims 10-13 for the preparation of an anti-tumor drug, or for the preparation of a medicament for diagnosing a tumor, the tumor exhibiting BCMA; or for preparing a chimeric antigen receptor modification The immune cells; preferably, the immune cells include: T lymphocytes, NK cells or NKT lymphocytes. A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises the antibody of any one of claims 1 to 5, and the antibody is preferably a single chain antibody or a domain antibody. . The chimeric antigen receptor according to claim 16, wherein the intracellular signal domain comprises one or more costimulatory signal domains and/or a primary signal domain. The chimeric antigen receptor according to claim 16, wherein the chimeric antigen receptor further comprises a hinge domain. The chimeric antigen receptor according to claim 17, wherein the transmembrane domain is selected from the group consisting of an alpha, beta, zeta chain of TCR, CD3 epsilon, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and the transmembrane region of PD1; and/or the costimulatory signal domain is selected from CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C SLP76, TRIM, FcεRIγ, MyD88, and 41BBL The intracellular signal region; and/or the primary signal domain is selected from the group consisting of TCRξ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as “ICOS”) and CD66d, and CD3ζ, More preferably, the transmembrane domain is selected from the transmembrane domain of CD8α, CD4, CD45, PD1, CD154 and CD28; and/or the costimulatory signal domain is selected from the group consisting of CD137, CD134, CD28 and OX40; The primary signal domain is selected from the group consisting of CD3ζ; optimally, the transmembrane domain is selected from the group consisting of CD8α or CD28, and the costimulatory signal domain is selected from the group consisting of CD137 CD28 intracellular domain of the signal, the primary signal domain is selected from CD3ζ. The chimeric antigen receptor according to claim 16, wherein the chimeric antigen receptor comprises the following sequentially linked antibodies, a transmembrane region and an intracellular signal region: any one of claims 1-5 The antibody, the transmembrane region of CD8, and the CD3ζ; the antibody according to any one of claims 1 to 5, the transmembrane region of CD8, the intracellular signal region of CD137, and CD3ζ; the antibody according to any one of claims 1 to 5; The transmembrane region of CD28, the intracellular signal region of CD28, and CD3ζ; or the antibody of any of claims 1-5, the transmembrane region of CD28, the intracellular signal region of CD28, CD137 and CD3ζ. A nucleic acid encoding the chimeric antigen receptor of any of claims 16-20. A performance vector comprising the nucleic acid of claim 21. A virus, characterized in that the virus comprises the vector of claim 22. The use of the chimeric antigen receptor of any one of claims 16 to 20, or the nucleic acid of claim 21, or the expression vector of claim 22, or the virus of claim 23, for use in preparing a standard The target expresses genetically modified immune cells of tumor cells of BCMA. A genetically modified immune cell characterized by transducing the nucleic acid of claim 21, or the expression vector of claim 22 or the virus of claim 23; or it is represented by claim 16-20 The chimeric antigen receptor according to any one of the above, wherein the immune cell is preferably a T lymphocyte, an NK cell or an NKT cell. The genetically modified immune cell of claim 25, which further comprises a sequence other than the chimeric antigen receptor of any one of claims 16-20, the other sequence comprising a cytokine Or another chimeric antigen receptor, or a chemokine receptor, or an siRNA that reduces PD-1 expression, or a protein that blocks PD-L1, or a TCR, or a safety switch; preferably, the cell The factor comprises IL-12, IL-15, IL-21, or type I interferon; preferably, the chemokine receptor comprises CCR2, CCR5, CXCR2, or CXCR4; preferably, the safety switch comprises iCaspase-9, Truncated EGFR or RQR8. Use of the genetically modified immune cell of any one of claims 25-27, characterized in that it is used for the preparation of a tumor suppressing drug, the tumor being a tumor exhibiting BCMA. A pharmaceutical composition, comprising: the antibody of any one of claims 1 to 5, or the nucleic acid encoding the antibody; or the immunoconjugate of any one of claims 10-15 or encoding the suffix The nucleic acid of any one of claims 16 to 20, or the nucleic acid encoding the chimeric antigen receptor, or the genetically modified immune cell of any one of claims 25-26.