1. A Chimeric Antigen Receptor (CAR), wherein the structure of said chimeric antigen receptor is represented by formula I:

L-scFv1-I-scFv2-H-TM-C-CD3ζ (I)

in the formula (I), the compound is shown in the specification,

each "-" is independently a linker peptide or a peptide bond;

l is an optional signal peptide sequence;

i is a flexible joint;

h is an optional hinge region;

TM is a transmembrane domain;

c is a costimulatory signal molecule;

CD3 ζ is the cytoplasmic signaling sequence derived from CD3 ζ;

both scFv1 and scFv2 are antigen binding domains that target CD19, one and CD 20.

2. The CAR of claim 1, wherein the scFv1 is an antigen binding domain targeting CD20 and the scFv2 is an antigen binding domain targeting CD 19.

3. The CAR of claim 1, wherein the chimeric antigen receptor has the structure of formula II:

L-V_L1-V_H1-I-V_H2-V_L2-H-TM-C-CD3ζ (II)

in the formula, V_H1Is the heavy chain variable region of the anti-CD 20 antibody; v_L1Is the light chain variable region of the anti-CD 20 antibody; v_L2Is the light chain variable region of the anti-CD 19 antibody; v_H2Is the heavy chain variable region of anti-CD 19 antibody(ii) a "-" is a linker peptide or peptide bond;

elements L, I, H, TM, C, CD3 ζ are as recited in claim 1.

4. The CAR of claim 1, wherein the amino acid sequence of the CAR is as set forth in SEQ ID NO 15 or 16.

5. A nucleic acid molecule encoding the chimeric antigen receptor of claim 1.

6. A vector comprising the nucleic acid molecule of claim 2.

7. A host cell comprising the vector or chromosome of claim 3 integrated with an exogenous nucleic acid molecule of claim 2 or expressing the chimeric antigen receptor of claim 1.

8. A method of making a CAR-T cell that expresses the chimeric antigen receptor of claim 1, comprising the steps of: transforming the nucleic acid molecule of claim 2 or the vector of claim 3 into a T cell, thereby obtaining the CAR-T cell.

9. A formulation comprising the chimeric antigen receptor of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, or the host cell of claim 4, and a pharmaceutically acceptable carrier, diluent, or excipient.

10. Use of the chimeric antigen receptor of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, or the host cell of claim 4 for the preparation of a medicament or formulation for the prevention and/or treatment of cancer or tumor.

Background

Hematological malignancies account for about 10% of human malignancies, and 95% of hematological malignancies are of B lymphocyte origin. Traditional chemotherapy and radiotherapy play an important role in treating malignant tumors of the blood system, and some patients have obvious curative effect but most of the patients are difficult to cure. New, effective treatments have been the focus of this field.

Adoptive T cell therapy has shown its powerful therapeutic effect and promising prospects in clinical treatment of malignant tumors, and is currently known as one of the most promising methods for treating hematological tumors. The surface of most B cell malignant tumors highly expresses CD19, and a plurality of T cells which are independently developed and modified by a Chimeric Antigen Receptor (CAR) and target B cells expressing CD19 to relapse and refractory malignant tumors have not been successful before. Currently, two CAR-T products approved by the FDA for marketing, both directed against CD19 antigen, have expanded indications as well, such as chronic lymphocytic leukemia. Despite the outstanding efficacy of anti-CD 19CAR-T, many studies have shown that CD19 Chimeric Antigen Receptor (CAR) T cell therapy also has a number of problems, with some patients having poor therapeutic efficacy and being prone to relapse, including susceptibility of tumor cells to antigen escape. For example, a recent CD19 CAR-cell therapy trial showed that complete remission was achieved in 90% of patients, but 11% of these patients eventually relapsed, primarily CD 19-negative tumors. In particular, complete remission was achieved in up to 94% of patients in a clinical trial using CART19 for relapsed, refractory acute B-cell lymphoma (R/R B-ALL) conducted at the university of pennsylvania medical school. Despite the high initial response rate of this clinical trial, approximately 40% of patients had relapsed after 1 month of treatment to complete remission, and a proportion of patients with relapses that were greater than 60% had escape of CD 19-negative tumor cells. Antigen escape expression of the NY-ESO1 specific T cell receptor has been found in adoptive transfer, and cancer vaccines for the treatment of melanoma. Spontaneous mutation and selective amplification are the main reasons for antigen escape.

Therefore, there is an urgent need in the art to develop methods for effectively treating tumors and preventing antigen escape.

Disclosure of Invention

The present invention aims to provide a method for effectively treating tumors and preventing antigen escape.

The invention aims to provide a combined chimeric antigen receptor targeting CD19 and CD20, and a preparation method and application thereof.

Specifically, the invention aims to provide a sequence targeting CD19 and CD20 combined antigen chimeric antigen receptor, and a preparation method and activity identification of modified T cell (CAR-T19/20) thereof. The present invention provides a chimeric antigen receptor structure for the treatment of CD19CD20 positive B cell lymphoma.

In a first aspect of the invention, there is provided a Chimeric Antigen Receptor (CAR) having the structure shown in formula I below:

L-scFv1-I-scFv2-H-TM-C-CD3ζ (I)

in the formula (I), the compound is shown in the specification,

each "-" is independently a linker peptide or a peptide bond;

l is an optional signal peptide sequence;

i is a flexible joint;

h is an optional hinge region;

TM is a transmembrane domain;

c is a costimulatory signal molecule;

CD3 ζ is the cytoplasmic signaling sequence derived from CD3 ζ;

both scFv1 and scFv2 are antigen binding domains that target CD19, one and CD 20.

In another preferred example, the scFv1 is an antigen binding domain targeting CD20 and the scFv2 is an antigen binding domain targeting CD 19.

In another preferred embodiment, the antigen binding domain targeting CD20 has the structure shown in formula a or formula B below:

V_H1-V_L1 (A)；V_L1-V_H1 (B)

in the formula, V_H1Is the heavy chain variable region of the anti-CD 20 antibody; v_L1Is the light chain variable region of the anti-CD 20 antibody; "-" is a linker peptide or peptide bond.

In another preferred embodiment, the antigen binding domain targeting CD20 has the structure shown in formula B.

In another preferred embodiment, the V_H1The amino acid sequence of (A) is shown as SEQ ID NO. 1, the V_L1The amino acid sequence of (A) is shown as SEQ ID NO. 2; or

The V is_H1The amino acid sequence of (A) is shown as SEQ ID NO. 3, the V_L1The amino acid sequence of (A) is shown in SEQ ID NO. 4.

In another preferred embodiment, V is_H1And V_L1Are connected by a flexible linker (or connecting peptide) which is 1-4 consecutive sequences shown in SEQ ID NO:7(GGGGS), preferably 2-4, more preferably 3-4.

In another preferred embodiment, the antigen binding domain targeting CD19 has the structure shown in formula C or formula D below:

V_L2-V_H2 (C)；V_H2-V_L2 (D)

in the formula, V_L2Is the light chain variable region of the anti-CD 19 antibody; v_H2Is the heavy chain variable region of the anti-CD 19 antibody; "-" is a linker peptide or peptide bond.

In another preferred embodiment, the antigen binding domain targeting CD19 has the structure shown in formula D.

In another preferred embodiment, the V_L2The amino acid sequence of (A) is shown as SEQ ID NO. 5, V_H2The amino acid sequence of (A) is shown in SEQ ID NO. 6.

In another preferred embodiment, V is_H2And V_L2Are connected by a flexible linker (or connecting peptide) which is 1-4 consecutive sequences shown in SEQ ID NO:7(GGGGS), preferably 2-4, more preferably 3-4.

In another preferred embodiment, the scFv1 and/or scFv2 is a single chain antibody variable region fragment of murine, human, chimeric of human and murine, or fully humanized.

In another preferred embodiment, the chimeric antigen receptor has the structure shown in formula II below:

L-V_L1-V_H1-I-V_H2-V_L2-H-TM-C-CD3ζ (II)

wherein each element is as described above.

In another preferred embodiment, the sequence of the flexible linker I comprises 2 to 6, preferably 3 to 4 consecutive sequences of SEQ ID NO 7 (GGGGS).

In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of: CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

In another preferred embodiment, L is a signal peptide derived from CD 8.

In another preferred embodiment, the amino acid sequence of L is shown in SEQ ID NO 8.

In another preferred embodiment, the H is a hinge region of a protein selected from the group consisting of: CD8, CD28, CD137, Ig4, or a combination thereof.

In another preferred embodiment, the H is a hinge region from Ig 4.

In another preferred embodiment, the amino acid sequence of H is shown in SEQ ID NO 9.

In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

In another preferred embodiment, the TM is a CD8 or CD28 derived transmembrane region.

In another preferred embodiment, the TM has the sequence shown in SEQ ID NO 10 or 11.

In another preferred embodiment, C is a costimulatory signal molecule for a protein selected from the group consisting of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

In another preferred embodiment, the C is a co-stimulatory signaling molecule from 4-1BB or CD 28.

In another preferred embodiment, the amino acid sequence of C is shown in SEQ ID NO 12 or 13.

In another preferred embodiment, the amino acid sequence of CD3 ζ is set forth as SEQ ID NO. 14.

In another preferred embodiment, the amino acid sequence of the CAR is as shown in SEQ ID NO 15 or 16.

In a second aspect of the invention, there is provided a nucleic acid molecule encoding the chimeric antigen receptor of the first aspect of the invention.

In another preferred embodiment, the nucleic acid molecule is isolated.

In another preferred embodiment, the nucleotide sequence of said nucleic acid molecule is as shown in SEQ ID NO 17 or 18.

In a third aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the second aspect of the invention.

In another preferred embodiment, the vector comprises DNA and RNA.

In another preferred embodiment, the carrier is selected from the group consisting of: a plasmid, a viral vector, a transposon, or a combination thereof.

In another preferred embodiment, the vector comprises a DNA virus or a retroviral vector.

In another preferred embodiment, the carrier is selected from the group consisting of: a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a combination thereof.

In another preferred embodiment, the vector is a lentiviral vector.

In a fourth aspect of the invention, there is provided a host cell comprising a vector or chromosome of the third aspect of the invention into which has been integrated an exogenous nucleic acid molecule of the second aspect of the invention or which expresses a chimeric antigen receptor of the first aspect of the invention.

In another preferred embodiment, the cell is an isolated cell.

In another preferred embodiment, the cell is a genetically engineered cell.

In another preferred embodiment, the cell is a mammalian cell.

In another preferred embodiment, the cell is a CAR-T cell and/or a CAR-NK cell.

In another preferred example, the cell targets both CD19 and CD 20.

In a fifth aspect of the invention there is provided a method of making a CAR-T cell expressing a chimeric antigen receptor according to the first aspect of the invention, comprising the steps of: (ii) transferring the nucleic acid molecule of the second aspect of the invention or the vector of the third aspect of the invention into a T cell, thereby obtaining the CAR-T cell.

In another preferred embodiment, the method further comprises the step of performing a functional and efficacy test on the obtained CAR-T cells.

In a sixth aspect of the invention, there is provided a formulation comprising a chimeric antigen receptor according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, or a host cell according to the fourth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

In another preferred embodiment, the formulation is a liquid formulation.

In another preferred embodiment, the formulation is in the form of an injection.

In another preferred embodiment, the preparation contains the host cell of the fourth aspect of the invention at a concentration of 1X 10³-1×10⁸Individual cells/ml, preferably 1X 10⁴-1×10⁷Individual cells/ml.

In a seventh aspect of the present invention, there is provided a use of the chimeric antigen receptor of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the vector of the third aspect of the present invention, or the host cell of the fourth aspect of the present invention, for the preparation of a medicament or formulation for the prevention and/or treatment of cancer or tumor.

In another preferred embodiment, the tumor is selected from the group consisting of: a hematologic tumor, a solid tumor, or a combination thereof; preferably, the tumor is a hematological tumor.

In another preferred embodiment, the hematological tumor is selected from the group consisting of: acute Myeloid Leukemia (AML), Multiple Myeloma (MM), Chronic Lymphocytic Leukemia (CLL), Acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

In another preferred embodiment, the solid tumor is selected from the group consisting of: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymph cancer, nasopharyngeal cancer, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, or a combination thereof.

In an eighth aspect of the invention, there is provided a kit for preparing a cell according to the fourth aspect of the invention, the kit comprising a container, and a nucleic acid molecule according to the second aspect of the invention, or a vector according to the third aspect of the invention, in the container.

In a ninth aspect of the invention there is provided a cell according to the fourth aspect of the invention, or a formulation according to the sixth aspect of the invention, for use in the prevention and/or treatment of cancer or a tumour.

In a tenth aspect of the invention, there is provided a method of treating a disease comprising administering to a subject in need thereof an amount of a cell according to the fourth aspect of the invention, or a formulation according to the sixth aspect of the invention.

In another preferred embodiment, the disease is cancer or a tumor.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a schematic structure of a combined chimeric antigen receptor targeting CD19CD 20. The structure of the CAR includes a leader sequence, an antigen recognition sequence, a connecting region, a transmembrane region, a costimulatory factor signaling region, and a CD3zeta signaling region.

FIG. 2 shows the measurement of transfection efficiency of targeting CD19CD20 combined with chimeric antigen receptor engineered T cells. The Protein L method identified the expression level of the Protein encoded by the CAR gene on the surface of the T cell membrane in CAR-T19/20s cells cultured up to day 7.

FIG. 3 shows the expression level of CD137 on the surface of T cell membrane (FIG. 3A) and the secretion level of IFN γ in the culture supernatant (FIG. 3B). Specifically, 1 × 10 is taken in order⁵The CAR-T19/20s cells cultured up to day 7, K562-CD19+ tumor cell line positive to CD19, K562-CD20+ tumor cell line positive to CD20, K562-CD19+ CD20+ tumor cell line double positive to CD19CD20, RAJI tumor cell line naturally expressing CD19 and CD20, and K562 tumor cell line double negative to CD19CD20 or without addition of tumor cells were cultured in 200. mu.l GT-551 medium at a ratio of 1:1 for 18h, and then the expression level of CD137 on the surface of T cell membrane and the secretion level of IFN γ in the culture supernatant were measured, respectively.

FIG. 4 shows the CAR-T19/20s cell tumor killing activity assay, mainly by measuring the level of secretion of LDH in the supernatant after co-culture. Specifically, 1 × 10 is taken respectively⁴The secretion levels of LDH were measured after 8h coculture with the corresponding T cells in 100. mu.l GT-551 medium in the ratios indicated in the figure, for CD19 positive K562-CD19+ tumor cell lines, CD20 positive K562-CD20+ tumor cell lines, CD19CD20 double positive K562-CD19+ CD20+ tumor cell lines, RAJI naturally expressing CD19 and CD20, RAMOS tumor cell lines or CD19CD20 double negative K562 tumor cell lines, respectively, and the percentage LDH release was statistically analyzed in the corresponding cocultured samples.

FIG. 5 shows the CAR-T19/20s cell tumor killing activity assay, mainly by measuring the expression level of CD107a on the surface of T cell membrane after co-culture. Specifically, 1 × 10 is taken respectively⁵The CAR-T19/20s cells were cultured in 200. mu.l GT-551 medium at a ratio of 1:2 for 4h without addition of tumor cells and then tested for the expression level of CD107a on the surface of T cell membrane, respectively, in a K562-CD19+ tumor cell line positive to CD19, a K562-CD20+ tumor cell line positive to CD20, a K562-CD19+ CD20+ tumor cell line double positive to CD19CD20, RAJI naturally expressing CD19 and CD20, a RAMOS tumor cell line or a K562 tumor cell line double negative to CD19CD 20.

FIG. 6 shows the results of killing of tumor cells by CAR-T19/20s cells in RAJI-Luc/NSG leukemia model mice. FIG. 6A shows the detection of transfection efficiency of CAR-T19/20s cells, and the Protein L method identifies the expression level of the CAR gene-encoding Protein on the surface of T cell membrane in CAR-T19/20s cells cultured up to day 9. Sequentially taking 1 × 10⁵CAR-T19/20s cells cultured up to day 10 (FIG. 6B), day 18 (FIG. 6C), K562-CD19+ tumor cell line positive for CD19, K562-CD20+ tumor cell line positive for CD20, K562-CD19+ CD20+ tumor cell line double positive for CD19CD20, RAJI and RAMOS tumor cell lines naturally expressing CD19 and CD20, and K562 and MOLT4 tumor cell lines double negative for CD19CD20 or without addition of tumor cells were cultured in 200. mu.l GT-551 medium at a 1:1 ratio for 18h, respectively, and the expression level of CD137 on the surface of T cell membrane was examined. Figure 6D shows the culture supernatant IFN gamma secretion level. FIG. 6E shows the mean body weight change and mean fluorescence intensity change over 21 days, recorded every 7 days, for mice injected with CAR-T19/20s cells. FIG. 6F shows live imaging of CAR-T19/20s cell injected mice on days 0 (D0), 7 (D7), 14 (D14) and 21 (D21) CAR T cell injection.

FIG. 7 shows preliminary functional validation results for CAR-T20.1, CAR-T20.2, CAR-T20.4.

FIG. 7A shows the results of the T cell transfection efficiency assay, identifying the expression level of the CAR gene-encoding protein on the surface of the T cell membrane in CAR-T20s cells cultured up to day 7 by the DNA copy number method. FIG. 7B shows the level of secretion of TFN γ in the supernatant after coculture of CAR-T20s cells with target cells. FIG. 7C shows the expression level of CD137 on the surface of T cell membrane after co-culture.

FIG. 8 shows the functional validation results of CAR-T20.5, CAR-T20.6, CAR-T20.7, CAR-T20.8, CAR-T20.9, CAR-T20.10

FIG. 8A shows the results of the examination of the transfection efficiency of T cells, and the expression level of the Protein encoded by the CAR gene on the surface of the T cell membrane in CAR-T20s cells cultured up to day 7 was identified by the Protein L method. FIG. 8B shows the level of secretion of IFN γ in the supernatant after coculture of CAR-T20s cells with target cells. FIG. 8C shows the expression level of CD137 on the surface of T cell membrane after co-culture.

FIG. 9 shows functional validation results for CAR-T20.11, CAR-T20.12, CAR-T20.13, CAR-T20.14, CAR-T20.15, CAR-T20.16. FIG. 9A shows the results of the examination of the transfection efficiency of T cells, and the expression level of the Protein encoded by the CAR gene on the surface of the T cell membrane in CAR-T20s cells cultured up to day 7 was identified by the Protein L method. FIG. 9B shows the level of secretion of IFN γ in the supernatant after coculture of CAR-T20s cells with target cells. Fig. 9C shows the expression level of CD137 on the surface of T cell membrane after co-culture.

FIG. 10 shows the functional validation results of CAR-T20.17, CAR-T20.18, CAR-T20.19.

FIG. 10A shows the results of the measurement of the transfection efficiency of T cells. Figure 10B shows the secretion level of IFN γ in the supernatant after co-culture. FIG. 10C shows the detection of the killing ability of CAR-T20s cells against tumor cells, mainly by measuring the level of secretion of LDH in the supernatant after co-culture. Fig. 10D shows the expression level of CD137 on the surface of T cell membrane.

FIG. 11 shows the inhibitory effect of CAR-T19/20s cells on transplanted tumor cells in mice.

FIG. 12 shows the experimental procedure for the CART-20TN-OF-19 phase I clinical trial.

FIG. 13 shows the results of the test for the copy number of the CAR.

FIG. 14 shows the tumor size change in one subject.

In the figure, TN-OF-19 and TN-20OF-19 have the same meaning, and TN-LEU-19 and TN-20LEU-19 have the same meaning, and represent CART cells having a corresponding CAR structure.

Detailed Description

The present inventors have extensively and intensively studied and, for the first time, unexpectedly found a CAR-T cell that targets both CD19 and CD 20. Specifically, the invention provides a chimeric antigen receptor targeting both CD19 and CD20, which comprises a signal peptide, an scFv against CD20, an scFv against CD19, a hinge region, a transmembrane region and an intracellular T cell signal region, wherein the anti-CD 20scFv and the CD19scFv are obtained by mass screening and are connected through a peptide segment with a plurality of repetitive structures (G4S). The CAR-T cell can simultaneously recognize two antigens of CD19 and CD20, so that the risk of immune escape caused by down-regulation or deletion of antigen expression in the process of recognizing single-target CAR-T cell therapy is reduced. Compared with CAR-T cells which target single antigen and CAR-T cells which target double targets (targeting CD19 and CD20), the CAR-T cells which simultaneously recognize two targets have stronger killing capacity on tumor cells, and have the advantages of lower cytotoxicity, low side effect, wider treatment range, lower recurrence rate and better curative effect. The present invention has been completed based on this finding.

Term(s) for

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

The term "administering" refers to the physical introduction of the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal cord or other parenteral routes of administration, e.g., by injection or infusion.

The term "antibody" (Ab) shall include, but is not limited to, an immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises a constant domain CL. The VH and VL regions may be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

CD20

Despite the outstanding efficacy of anti-CD 19CAR-T, many studies have shown that CD19 Chimeric Antigen Receptor (CAR) T cell therapy also has a number of problems, with some patients having poor therapeutic efficacy and being prone to relapse, including susceptibility of tumor cells to antigen escape.

To prevent CD19CAR-T antigen escape, the present invention designs a combination bispecific CAR (i.e., BICAR) that targets both CD19 and CD20, such that when the CD19 antigen escapes and is not expressed in tumor cells, tumor cells can be cleared in vivo by recognizing CD 20.

CD20 is expressed in most B cell acute lymphoblastic leukemia patients, including some CD19 negative patients after anti-CD 19CAR-T treatment, and CD20 is a glycosylated protein that is the first established B cell membrane marker, also known as B1, encoded by the MS4A gene. The CD20 molecule is a hydrophobic region with four transmembrane domains, with the N and C termini located on the cytosolic side, thus forming two closed loops outside the cell, called the macrocycle and the minicycle, respectively. CD20 is specifically expressed in more than 95% of normal and cancerous B cells, which are at a pre-B cell stage and later in development, and do not stop expression until differentiation into plasma cells, CD 20. The present invention employs CD20 as another target for B cell malignancy immunotherapy.

Bispecific chimeric antigen receptors targeting CD19 and CD20

The cellular immunotherapy is a new tumor treatment mode with obvious curative effect, and is a novel autoimmune anticancer treatment method. It uses biological technology and biological preparation to culture and expand immune cells collected from patient in vitro and then to return them to patient, to excite and enhance body's self-immune function, so as to achieve the goal of curing tumor. Those skilled in the art have been working on the development of new cellular immunotherapy to improve the effect of cellular immunotherapy and reduce its side effects.

The present invention proposes a reasonably optimized single-chain design and system, i.e. in combination with a bispecific CAR, which can efficiently integrate into primary human T cells, targeting both CD19 and CD20 when the T cells are activated. The CAR-T cells of the invention recognize two antigens (CD19 and CD20) and are a very effective potential means of preventing antigen escape.

The invention uses CARs of CD19 and CD20 that are targeted bi-directionally, with enhanced affinity, increased activity of T cells, with additive or synergistic effects compared to CARs targeted to a single antigen. In addition, dual-targeted CAR-T therapy is broader due to the heterogeneity of expression levels of CD19 and CD20 in tumor cells. CAR-T targeting both CD19 and CD20 on the surface of tumor cells can reduce the likelihood of antigen escape due to down-regulation or deletion of a single surface antigen.

Bispecific means that the same CAR can specifically bind and immunologically recognize two different antigens, and the CAR can generate an immune response when binding any one antigen.

The CD19 and CD20 bispecific CARs of the invention are single structures comprising scFv against CD19 and CD 20. Where the CAR comprises CD19scFv and CD20scFv, the amino acid sequence, order and hinge of CD19scFv and CD20scFv are the major contributors to their function.

Specifically, the Chimeric Antigen Receptors (CARs) of the invention include an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding member (also referred to as an antigen-binding domain). The intracellular domain includes a costimulatory signaling region and a zeta chain moiety. The costimulatory signaling region refers to a portion of the intracellular domain that includes the costimulatory molecule. Costimulatory molecules are cell surface molecules required for efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.

A linker may be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular domain or a cytoplasmic domain of a polypeptide chain. The linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

In a preferred embodiment of the invention, the extracellular domain of the CAR provided by the invention comprises an antigen binding domain that targets CD19CD20 association. The CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds its associated antigen, it affects the tumor cells, causing the tumor cells to not grow, to be driven to death, or to otherwise be affected, and causing the patient's tumor burden to shrink or be eliminated. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and the zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the 4-1BB signaling domain, and the CD3zeta signaling domain in combination.

As used herein, "antigen binding domain" and "single chain antibody fragment" each refers to a Fab fragment, Fab 'fragment, F (ab')₂A fragment, or a single Fv fragment. Fv antibodies contain the variable regions of the antibody heavy chain, the variable regions of the light chain, but no constant regions, and have the smallest antibody fragment of the entire antigen binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding. The antigen binding domain is typically a scFv (single-chain variable fragment). The size of the scFv is typically 1/6 for a whole antibody. Single chain antibodies are preferably a sequence of amino acids encoded by a single nucleotide chain. In a preferred embodiment of the invention, the scFv comprises an antibody that specifically recognizes CD19CD 20.

For the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domains may be selected, or modified by amino acid substitutions, to avoid binding such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex.

The intracellular domains in the CAR of the invention include the signaling domain of 4-1BB and the signaling domain of CD3 ζ.

Preferably, the structure of the CAR of the invention comprises, in order, a signal peptide sequence (also known as a leader), an antigen recognition sequence (antigen binding domain), a hinge region, a transmembrane region, a costimulatory factor signal region, and a CD3zeta signaling region (zeta chain moiety), the order of attachment being shown in figure 1.

In another preferred embodiment, the CAR of the invention is TN-LEU-19. Wherein the antigen binding domain targeting CD20 comprises the Leu16 antibody derived heavy chain single variable region sequence (SEQ ID NO:1) and light chain single variable region (VL) sequence (SEQ ID NO: 2).

Leu16 antibody-derived single chain Variable Heavy (VH) sequence:

EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSS(SEQ ID NO:1)

leu16 antibody-derived single chain variable light chain (VL) sequence:

DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIK(SEQ ID NO:2)

in another preferred embodiment, the CAR OF the invention is TN-OF-19. Wherein the antigen binding domain targeting CD20 comprises the Ofatumumab antibody-derived single chain variable heavy chain sequence (SEQ ID NO:3) and a single chain variable light chain sequence (SEQ ID NO: 4).

The sequence of the single-chain variable heavy chain (VH) derived from Ofatumumab antibody:

EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSS(SEQ ID NO:3)

single chain variable region light chain (VL) sequence from Ofatumumab antibody:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK(SEQ ID NO:4)

in another preferred embodiment, the antigen binding domain targeting CD19 in a CAR of the invention comprises the FMC63 antibody derived single chain variable light chain (VL) sequence (SEQ ID NO:5) and single chain variable heavy chain sequence (SEQ ID NO: 6).

FMC63 antibody-derived single chain variable light chain (VL) amino acid sequence:

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT(SEQ ID NO:5)

FMC63 antibody-derived single chain variable region light chain (VL) nucleotide sequence:

FMC63 antibody-derived single chain variable heavy chain (VH) amino acid sequence:

EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(SEQ ID NO:6)

FMC63 antibody-derived single chain variable heavy chain (VH) nucleotide sequence:

in particular, the sequence of the other elements in the CAR of the invention is as follows:

leader sequence is the leader sequence of CD8 antigen:

MALPVTALLLPLALLLHAARP(SEQ ID NO:8)

the connecting sequence between the heavy chain and the light chain of the single-chain variable region (namely, the flexible joint I) is as follows:

amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO:19)

Nucleic acid sequence: ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatct (SEQ ID NO:20)

The Hinge region is selected from the sequences of IgG4 Hinge-CH2-CH 3:

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:9)

the transmembrane region is the sequence of the transmembrane region of CD8(CD8TM) or CD28(CD28TM) antigen:

CD8TM:IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:10)

CD28TM:FWVLVVVGGVLACYSLLVTVAFIIFWV(SEQ ID NO:11)

the costimulatory factor signaling region is derived from the sequence of the intracellular signaling motif of 4-1BB or CD 28:

4-1BB:KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:12)

CD28:RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:13)

the signaling region for CD3 ζ is derived from the sequence of the tyrosine-centered immunoreceptor activation motif (ITAM) of CD3 ζ in the TCR complex:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:14)

in a preferred embodiment, the complete nucleic acid and amino acid sequences of two CARs constructed in accordance with the invention are as follows:

complete nucleic acid sequence OF TN-OF-19

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAAGGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGCGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGAGGTGGTGGATCCGAGGTGAAGCTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCCGGCAGCCCCCCAGGAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCGGCAGCACCTCCGGCAGCGGCAAGCCTGGCAGCGGCGAGGGCAGCACCAAGGGCGACATCCAGATGACCCAGACCACCTCCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCCGACGGCACCGTCAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTTAGCGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCCACCTACTTTTGCCAGCAGGGCAACACACTGCCCTACACCTTTGGCGGCGGAACAAAGCTGGAAATCACCGAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCTATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG(SEQ ID NO:18)

Complete amino acid sequence OF TN-OF-19

MALPVTALLLPLALLLHAARPEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:16)

Complete nucleic acid sequence of TN-LEU-19

ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCACAGGTGACATTGTGCTGACCCAATCTCCAGCTATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAATTACATGGACTGGTACCAGAAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTTTTAATCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAGGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGCGAGGTGCAGCTGCAGCAGTCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAGCAGACACCTGGACAGGGCCTGGAATGGATTGGAGCTATTTATCCAGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGACTATTACTGTGCAAGATCTAATTATTACGGTAGTAGCTACTGGTTCTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGGAGGTGGTGGATCCGAGGTGAAGCTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCCGGCAGCCCCCCAGGAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCGGCAGCACCTCCGGCAGCGGCAAGCCTGGCAGCGGCGAGGGCAGCACCAAGGGCGACATCCAGATGACCCAGACCACCTCCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCCGACGGCACCGTCAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTTAGCGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCCACCTACTTTTGCCAGCAGGGCAACACACTGCCCTACACCTTTGGCGGCGGAACAAAGCTGGAAATCACCGAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCTATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG(SEQ ID NO:17)

Complete amino acid sequence of TN-LEU-19

METDTLLLWVLLLWVPGSTGDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSS STAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSEVKLQESGPGLVAPSQSLSVTCTVS GVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYY GGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:15)

The design of the BICAR of the present invention has the following advantages:

first, CD19, CD20 are expressed in the vast majority of malignant B cell tumors. Secondly, in general, expansion of CAR structures to increase T cell recognition capacity is often encountered with increased poor targeting, cytotoxicity and increased side effects, however, CD19, CD20 do not as such, since both are expressed only in B cells, with the same tumor toxicity profile. Finally, expression of CD19, CD20 in B cells can promote survival of B cells. And loss of both antigens during treatment is a very low probability event. Therefore, the CD19 and CD20 are expected to provide effective guarantee for antigen escape in malignant B cells.

BICAR has the following advantages compared with single CAR of CD19 and CD 20:

first, when BICAR is expressed in a single T cell, the DNA footprint is significantly reduced (40% reduction in DNA length) compared to two independent CARs, and the size of the structure can significantly affect the packaging and transduction efficiency of the viral vector, which directly affects clinical efficacy. Second, BICAR can significantly reduce treatment costs (BICAR is fully compatible with current T cell production processes and does not add additional burden) and increase clinical cure rates compared to a mix of two different single CARs. Finally, the CD19 and the CD20 are clearly and safely verified in a large number of clinical studies.

In the invention, 2 types OF chimeric antigen receptor structures targeting CD19CD20 antigen, namely TN-LEU-19 and TH-OF-19, are constructed on the basis OF the sequence OF a murine monoclonal antibody FMC63 OF CD19 and the sequences OF CD20 murine monoclonal antibodies LEU-16 and Ofatumumab, and the analysis and identification OF the expression level, in vitro activation capacity, tumor cell killing efficiency and the like OF the 2 types OF chimeric antigen receptors in primary T cells are completed. Finally, the T cells modified by TN-LEU-19, TH-OF-19 chimeric antigen receptors are found to have strong capacities OF killing in vitro and eliminating in vivo malignant tumors carrying CD19 and CD20 positive antigens, and Ofatumumab is better than LEU16, so that a new effective method and a preparation are provided for clinical application OF CAR-T to treatment OF CD19 and CD20 positive leukemia and lymphoma.

The present invention is designed and optimized for single-chain, bispecific CARs that have a strong killing capacity against B-cell malignancies expressing CD19 or CD 20. BICAR can target a single T-cell product to two clinically validated antigens associated with B-cell leukemia and lymphoma, ultimately reducing the risk of tumor recurrence due to loss or escape of a single antigen. The invention can be further used for novel BICAR design, increasing antigen pair to expand applicability and improve curative effect cancer of T cell therapy.

Chimeric antigen receptor T cells (CAR-T cells)

As used herein, the terms "CAR-T cell", "CAR-T", "CART", "CAR-T cell of the invention" all refer to a CAR-T cell of the fourth aspect of the invention that targets both CD19 and CD 20. The CAR structure of the CAR-T cell specifically includes, in order, an scFv against CD19, an scFv against CD20, a hinge region, a transmembrane region, and an intracellular T cell signal region, and the anti-CD 20scFv and the CD19scFv are linked by a multiple repeat (G4S) peptide segment. Compared with the CAR-T targeting a single antigen, the CAR-T cell which can simultaneously recognize two targets has stronger killing performance and wider treatment range.

Carrier

Nucleic acid sequences encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.

The present invention also provides a vector into which the expression cassette of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, since they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.

In brief summary, an expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence.

The expression constructs of the invention may also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the invention provides a gene therapy vector.

The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and Molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors comprise an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

Additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50bp apart, and activity begins to decline. Depending on the promoter, it appears that the individual elements may function cooperatively or independently to initiate transcription.

An example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1 α (EF-1 α). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40(SV40) early promoter, the mouse mammary cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr (Epstein-Barr) virus immediate early promoter, the rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

To assess the expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cells can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in a host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, the reporter gene is the following: which is not present in or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al, 2000FEBS Letters479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, the construct with the minimum of 5 flanking regions that showed the highest level of reporter gene expression was identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate the ability of an agent to modulate promoter-driven transcription.

Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vector may be readily introduced into a host cell by any method known in the art, e.g., mammalian, bacterial, yeast or insect cells. For example, the expression vector may be transferred into a host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing the polynucleotide into a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.

Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).

In the case of non-viral delivery systems, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for use to introduce nucleic acids into host cells (ex vivo or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution comprising the lipid, mixed with the lipid, associated with the lipid, contained as a suspension in the lipid, contained in or complexed with a micelle, or otherwise associated with the lipid. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. For example, they may be present in bilayer structures, either as micelles or with a "collapsed" structure. They may also simply be dispersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include fatty droplets that occur naturally in the cytoplasm as well as such compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

In a preferred embodiment of the invention, the vector is a lentiviral vector.

Preparation

The invention provides a composition comprising the CAR-T cell of the fourth aspect of the invention and a pharmaceutically acceptable carrierA body, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the CAR-T cells are present in the formulation at a concentration of 1X 10³-1×10⁸Individual cells/ml, more preferably 1X 10⁴-1×10⁷Individual cells/ml.

In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The formulations of the present invention are preferably formulated for intravenous administration.

Therapeutic applications

The invention includes therapeutic applications of cells (e.g., T cells) transduced with Lentiviral Vectors (LV) encoding expression cassettes of the invention. The transduced T cells can target markers CD19 and CD20 of tumor cells, and synergistically activate the T cells to cause T cell immune response, so that the killing efficiency of the T cells on the tumor cells is remarkably improved.

Accordingly, the present invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal comprising the steps of: administering to the mammal the CAR-T cells of the invention.

In one embodiment, the invention includes a class of cell therapy in which autologous T cells (or allogeneic donors) from a patient are isolated, activated, genetically engineered to produce CAR-T cells, and subsequently injected into the same patient. In this way, the probability of graft versus host disease is very low and antigens are recognized by T cells in an MHC-unrestricted manner. Furthermore, one CAR-T can treat all cancers expressing this antigen. Unlike antibody therapy, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

In one embodiment, the CAR-T cells of the invention can undergo robust in vivo T cell expansion and can last for an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-modified T cell induces an immune response specific to the antigen binding domain in the CAR. For example, CAR-T cells against CD19CD20 elicit specific immune responses against cells expressing CD19 and CD 20.

Although the data disclosed herein specifically disclose lentiviral vectors comprising an anti-CD 19CD20scFv, hinge and transmembrane regions, and 4-1BB and CD3zeta signaling domains, the invention should be construed to include any number of variations on each of the construct components.

Treatable cancers include tumors that are not vascularized or have not substantially vascularized, as well as vascularized tumors. The cancer may comprise a non-solid tumor (such as a hematological tumor, e.g., leukemia and lymphoma) or may comprise a solid tumor. The types of cancer treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, such as sarcomas, carcinomas and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematological) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, granulo-monocytic, monocytic and erythrocytic leukemias), chronic leukemias (such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, hodgkin's disease, non-hodgkin's lymphoma (indolent and higher forms), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

A solid tumor is an abnormal mass of tissue that generally does not contain cysts or fluid regions. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.

The CAR-modified T cells of the invention may also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals. Preferably, the mammal is a human.

For ex vivo immunization, at least one of the following occurs in vitro prior to administration of the cells into a mammal: i) expanding the cell, ii) introducing a nucleic acid encoding the CAR into the cell, and/or iii) cryopreserving the cell.

Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cells can be autologous with respect to the recipient. Alternatively, the cells may be allogeneic, syngeneic (syngeneic), or xenogeneic with respect to the recipient.

In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

The invention provides a method of treating a tumor comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the invention.

The CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, a pharmaceutical composition of the invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.

The pharmaceutical compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease-although the appropriate dosage may be determined by clinical trials.

When referring to an "immunologically effective amount", "an anti-tumor effective amount", "a tumor-inhibiting effective amount", or a "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, extent of infection or metastasis, and individual differences in the condition of the patient (subject). It can be generally pointed out that: pharmaceutical compositions comprising T cells described herein can be in the range of 10⁴To 10⁹Dosage of individual cells/kg body weight, preferably 10⁵To 10⁶Doses of individual cells per kg body weight (including all integer values within those ranges) are administered. The T cell composition may also be administered multiple times at these doses. Cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al, New Eng.J.of Med.319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject composition may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by i.v. injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.

In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) any number of relevant treatment modalities, including but not limited to treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or efavirenz therapy for psoriasis patients or other therapy for PML patients. In further embodiments, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In a further embodiment, the cell composition of the invention is administered to the patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) bone marrow transplantation with a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an injection of the expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered pre-or post-surgery.

The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The proportion of doses administered to a patient may be in accordance with accepted practice in the art. Typically, 1X 10 may be administered per treatment or per course of treatment⁶1 to 10¹⁰The modified T cells of the invention (e.g., CAR-T-19/20 cells) are administered to a patient, for example, by intravenous infusion.

The advantages of the invention include:

(1) the extracellular antigen binding domain of the chimeric antigen receptor is specific anti-CD 20scFv and anti-CD 19scFv, and the specific anti-CD 20scFv and the specific anti-CD 19scFv are combined with a specific hinge region and an intracellular domain to form a CAR, so that the CAR shows great killing capacity on tumor cells, and has the advantages of low cytotoxicity and low side effect.

(2) The chimeric antigen receptor provided by the invention can realize stable expression and membrane localization of CAR protein after a lentivirus carrying the CAR gene infects T cells.

(3) The CAR-modified T cells have long survival time in vivo and strong anti-tumor efficacy; the optimized CAR for the IgG4 Hinge-CH2-CH3 junction region was able to avoid Fc receptor binding and subsequent ADCC (antibody-dependent cellular cytotoxicity).

(4) Compared with two independent CAR, the bispecific chimeric antigen receptor of the invention simultaneously contains anti-CD 20scFv and anti-CD 19scFv, the DNA footprint is obviously reduced (the DNA length is reduced by 40%), the structure size is short, and the packaging and transduction efficiency of a virus vector are facilitated, so that the clinical curative effect is directly improved. And the bispecific CAR is lower in cost, higher in cure rate and safer.

(5) The TN-LEU-19, TH-OF-19 chimeric antigen receptor modified T cells have very strong capacities OF killing in vitro and eliminating malignant tumors carrying CD19 and CD20 positive antigens in vivo, and have stronger Ofatumumab, so that a novel effective method and a preparation are provided for clinical application OF CAR-T to treatment OF CD19 and CD20 positive leukemia and lymphoma.

(6) The CAR-T cell disclosed by the invention has a killing effect on most malignant B cell tumors, is wider in treatment range and larger in coverage rate, and can effectively prevent tumor cells from escaping.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

EXAMPLE 1 construction of lentiviral expression vectors

Synthesizing and cloning full-length DNA to realize the construction of coding plasmid. The pWPT lentiviral vector is selected as a cloning vector, and the cloning sites are BamH I and Sal I sites. The structures of the 2 CARs designed in the present invention are shown in fig. 1. The nucleic acid sequence OF TN-LEU-19 is shown as SEQ ID NO. 17, and the nucleic acid sequence OF TN-OF-19 is shown as SEQ ID NO. 18.

Example 2 preparation of CAR-T cells

(1) Healthy human venous blood is taken and separated by a density gradient centrifugation method to obtain mononuclear cells (PBMCs).

(2) On day 0, PBMCs were inoculated into cell culture flasks previously coated with 5. mu.g/mL CD3 monoclonal antibody (OKT3) and 10. mu.g/mL Retronectin (available from TAKARA), GT-551 medium containing 1% human albumin, to which recombinant human interleukin 2(IL-2) was added at a final concentration of 1000U/mL, and saturated humidity of 5% CO at 37 ℃₂Culturing in an incubator.

(3) On day 1, the supernatant from the cultured PBMCs was slowly aspirated and discarded, and fresh GT-551 cell culture medium containing 1% human albumin was added to which recombinant human interleukin 2(IL-2) was added to a final concentration of 1000U/mL at 37 ℃ with a saturation humidity of 5% CO₂The incubator continues to culture.

(4) On day 3, fresh medium was added, purified TN-LEU-19 or TN-OF-19 lentivirus solution, protamine sulfate (12. mu.g/mL), and IL-2 at a final concentration OF 1000U/mL were concentrated. Standing at 37 deg.C for 5% CO₂After 12 hours of infection in the incubator, the culture medium was discarded and fresh medium was added at 37 ℃ with 5% CO₂The incubator continues to perform the cultivation.

(5) From day 6 onwards, CAR-T19/20s (CART-TN-OF-19 and CART-TN-LEU-19) cells were taken for the corresponding activity assay.

Example 3 detection of integration Rate of CAR Gene in T cell genome and expression level of its encoded protein on Membrane surface

Respectively taking 0.5 × 10⁶The CAR-T19/20s cell samples cultured to day 7 in example 2 were tested for transfection efficiency of targeting CD19CD20 combined chimeric antigen receptor engineered T cells. The Protein L method identified the expression level of the Protein encoded by the CAR gene on the surface of the T cell membrane in CAR-T19/20s cells cultured up to day 7.

Results as shown in figure 2,2 CAR structures designed in the present invention (figure 1) can be expressed in their respective modified T cells and complete the cell membrane surface localization.

Example 4 detection of CAR-T19/20s in vitro activation Capacity

The CAR-T19/20s cells cultured up to day 7 in example 2 were used for the detection of the cell activation level indicators CD137 and IFN γ. Sequentially taking 1 × 10⁵CAR-T19/20 cells cultured up to day 7, K562-CD19+, K562-CD20+, K562-CD19+ CD20+ which are positive to CD19, CD20, respectively, and Raji (naturally expressing CD19CD20) tumor cell lines, and K562 tumor cell lines which are negative to CD19CD20, or without addition of tumor cells, were cultured in 200. mu.l GT-551 medium at a ratio of 1:1 for 18h, and then the expression level of CD137 on the surface of T cell membranes and the secretion level of IFN γ in the culture supernatant were measured, respectively.

As a result, as shown in fig. 3A and 3B, the expression of CD137 was detected on the surface of all 2 CART cells, and the expression of IFN γ was detected in the culture supernatant. Wherein the CD137 activation level and IFN gamma release level OF TN-OF-19 are higher than those OF TN-LEU-19.

Example 5 detection of the level of LDH released by CAR-T19/20s cells killing target tumors in vitro

The CAR-T19/20s cells prepared in example 2 were tested as follows:

an experimental well, an effector cell control well, a target cell maximum release well, a medium control well, and a volume control well (target cells have CD19, CD20 positive K562-CD19, K562-CD20, K562-CD19-CD20, and Raji cells; effector cells have NT, CART-TN-LEU-19, CART-TN-OF-19) were provided, respectively.

Setting the effective target ratio, namely the effective cell: target cells 5: 1,10: 1,20: 1,40: 1. cell number: target cell 1X 10⁴50 ul/well. The effector cells were 5X 10 cells, respectively⁴1X 10 per hole⁵Hole, 2X 10⁵Hole, 5X 10⁵A hole. Experimental wells, adding effector cells at different dilution ratios: target cells 5: 1,10: 1,20: 1,40: 1, a total of 100ul (50 ul effector cells +50ul target cells) of the two cells are plated on a cell culture plate, and 3 replicates are set; effector cell control wells, i.e. effector cells: target cells 5: 0,10: 0,20: 0,40: 0, adding 5X 10 effector cells⁴Hole, 10⁵Hole, 2X 10⁵Hole/hole，5×10⁵Per well cells +50ul medium, set up 2 replicates. Control well of target cells, target cells 1X 10 were added⁴Well, 50ul, +50ul medium. Maximum release hole for target cell, i.e. 1X 10 target cell is added⁴50ul and 50ul of culture medium, and 10ul of lysate is added after incubation for 3h and 15 min; adding 100ul of culture medium into the culture medium control hole; volume control wells, 100ul of medium was added.

After incubation for 3h15min, 10ul of lysate was added to the maximum release wells of the target cells, followed by incubation at 37 ℃. Centrifugation at 250g for 4min transferred 50 ul/well cell supernatant to a new microplate. Add 50 ul/well substrate mix (protected from light, 12ml detection buffer added to one vial of substrate mix and mixed). Incubate for 30min at room temperature in the dark. Finally 50 ul/well stop solution was added and the plate was read at 490nm within 1h and the data was analyzed.

The results are shown in fig. 4, 2 CART cells can well induce apoptosis of BCMA positive tumor cells and release LDH. Wherein, the CART-TN-LEU-19 can kill the tumor cells positive to the CD19CD20 better than the CART-TN-OF-19, and release higher LDH or the like.

Example 6 detection of the level of CD107a Release during the Induction of tumor cell killing by CAR-T19/20s cells

Respectively taking 1 × 10⁵Effector cells CAR-T19/20s (CART-TN-OF-19 and CART-TN-LEU-19) and target cells tumor cells 2X 10⁵The cells were co-cultured. The target cells are K562-CD19+, K562-CD20+, K562-CD19+ CD20+, K562 cells, Raji cells and Romas cells respectively. At the same time, 3. mu.l of Anti-Human CD107Ape was added to each well for staining, and the cells were incubated at 37 ℃ in 5% CO₂Incubate for 1 hour, then add 3. mu.l of 1% Golgistop per well and incubate at 37 ℃ with 5% CO₂The culture was carried out for 3.5 hours. Then 2. mu.l of CD8FITC and 1.5. mu.l of CD3APC were added to each well at 37 ℃ in 5% CO₂Culturing for 30 min. Add 200uL FACS Buffer into each well, centrifuge for 5min at 300g, pour off the liquid in the enzyme label plate quickly, remove the residual liquid with absorbent paper, and wash with FACS Buffer once. 7-AAD staining, using FACS Buffer (1:300) dilution, each hole with 200uL heavy suspension cells, 10min later using flow cytometry detection, care and light protection, statistical analysis results.

The results are shown in fig. 5, 2 CART cells can better induce the release of CD107a in the process of killing tumor cells. Wherein, the release OF CD107a in the killing process is slightly higher in the CART-TN-OF-19 compared with the CART-TN-LEU-19, and the killing effect is stronger.

Example 7 inhibitory Effect of CAR-T19/20s on transplanted tumor cells in mice

The tumor cells injected in the animal body are Raji, and the tumor cells Raji carry a luciferase reporter gene (Raji expressing luciferase). The experiment adopts that firstly tumor cells Raji are injected into a mouse body to grow for one week, and then effector T cells are injected, wherein the effector T cells are divided into three groups, namely NT, CART-TN-LEU-19 and CART-TN-OF-19.

FIG. 6A shows that the expression level OF CD137 and the secretion level OF IFN gamma in the culture supernatant are the same as before in the Protein L method for detecting the transfection efficiency OF the targeting CD19CD20 combined chimeric antigen receptor engineered T cells, and the results in FIG. 6A show that TN-LEU-19 and TN-OF-19 have higher transfection efficiency. FIGS. 6B and 6C show that the expression level OF CD137 on the surface OF T cell membranes at day 10 and day 18 is high, and TN-LEU-19 and TN-OF-19 can be expressed. FIG. 6D shows the IFN-. gamma.secretion level in the culture supernatant on day 10, indicating that TN-OF-19 is significantly higher than TN-LEU-19.

Expanded effector T cells (sent to the animal testing center of medical university of Nanjing) were injected into mice via tail vein at day 21, then the fluorescence intensity in mice (imaged by IVIS fluorescence) and the weight of mice were recorded every seven days, and the experiment was stopped at day 21 to analyze the statistics.

The results are shown in FIG. 6E, and both CART-TN-OF-19 and CART-TN-LEU-19 cells can inhibit the growth OF tumor cells in mice better than NT cells. FIG. 6E (left) shows the body weight change OF mice after infusion OF effector T cells in three groups OF mice, with significant weight loss OF NT as compared to both CART-TN-OF-19 and CART-TN-LEU-19, and slight weight gain in both CART cell groups. Fig. 6E (right) shows the mean of the fluorescence intensity in three groups of mice, and the results show that the mean of the fluorescence intensity in NT group mice is significantly increased, while the mean of the fluorescence intensity in two CART cell group mice is significantly decreased, even difficult to detect.

FIG. 6F is a photograph of in vivo fluorescence intensity IVIS imaging in mice. The results showed that after tail vein injection of effector T cells, the fluorescence intensity in mice of both CART cell groups was weak after the seventh day, while the NT group was strong, as was the case at day 14, and the NT group mice had all died at day 21, while the mice of both CART cell groups were still growing normally.

Example 8 screening and functional validation of CAR-T20.1, CAR-T20.2, CAR-T20.4

The construction and detection of CART cells were carried out in reference to examples 2, 3, 4, and 5.

Full-length DNA synthesis and cloning are firstly carried out to realize the construction of coding plasmids. CAR-T20.1,20.2,20.4 (structure shown in Table 1 and sequence shown in Table 2) was designed, followed by functional validation.

Resuscitating PBMC, infecting to obtain CAR-T20s cells, and starting on day 6, taking CAR-T20s cells for corresponding activity detection assay

Respectively taking 0.5 × 10⁶The transfection efficiency of the cells was measured by DNA copy number of the CAR-T20s cell samples cultured up to day 7.

As a result, as shown in FIG. 7A, the DNA copy number was not very high as compared with the positive control. Then, co-culture was performed, and the activation level of cells was measured using CAR-T20s cells cultured up to day 7 for the detection of the markers CD137 and IFN γ.

Sequentially taking 1 × 10⁵And (3) culturing the CAR-T20 cells cultured to the 7 th day with a CD20 positive Raji and Ramos and a negative cell K562 and a Karpas tumor cell line for 18 hours according to a ratio of 1:1, and then respectively detecting the expression level of CD137 on the surface of the T cell membrane and the secretion level of IFN gamma in culture supernatant.

Results As shown in FIGS. 7B and 7C, CAR-T20.1, CAR-T20.2, and CAR-T20.4 constructed in this example were not effective.

Example 9 screening and functional validation of CAR-T20.5, CAR-T20.6, CAR-T20.7, CAR-T20.8, CAR-T20.9, CAR-T20.10

The construction and detection of CART cells were carried out in reference to examples 2, 3, 4, and 5.

Full-length DNA synthesis and cloning are firstly carried out to realize the construction of coding plasmids. CAR-T20.5, CAR-T20.6, CAR-T20.7, CAR-T20.8, CAR-T20.9, CAR-T20.10 (structure shown in Table 1, sequence shown in Table 2) were designed, followed by functional validation.

Resuscitating PBMC, infecting to obtain CAR-T20s cells, and starting on day 6, taking CAR-T20s cells for corresponding activity detection assay

Respectively taking 0.5 × 10⁶A0.5X 10 sample of CAR-T20s cells cultured up to day 7 was obtained⁶Samples of CAR-T20s cells cultured up to day 7 were tested for T cell transfection efficiency. The expression level of the Protein encoded by the CAR gene on the surface of the T cell membrane in CAR-T20s cells cultured up to day 7 was identified by the Protein L method.

The results are shown in FIG. 8A, and compared with CAR-T20.1, the positive rate of CAR-T20.10 is higher in CAR-T20.5, CAR-T20.6, CAR-T20.7, CAR-T20.8, CAR-T20.9 and CAR-T20.10, which are only CAR-T20.9.

Then, co-culture was performed, and the activation level of cells was measured using CAR-T20s cells cultured up to day 7 for the detection of the markers CD137 and IFN γ. Sequentially taking 1 × 10⁶And (3) culturing the CAR-T20s cells cultured to the 7 th day with a CD20 positive Raji and Ramos cell and a negative cell K562 and a Karpas tumor cell line for 18 hours according to a ratio of 1:1, and then respectively detecting the expression level of CD137 on the surface of the T cell membrane and the secretion level of IFN gamma in culture supernatant.

The results are shown in FIGS. 8B and 8C, and indicate that only CAR-T20.9, CAR-T20.10 among CAR-T20.5, CAR-T20.6, CAR-T20.7, CAR-T20.8, CAR-T20.9, CAR-T20.10, expression level of CD137 on the cell membrane surface of CAR-T20.10 is activated and IFN γ is released from the culture supernatant, i.e., CAR-T20.9, CAR-T20.10 is effective.

Example 10 screening and functional validation of CAR-T20.11, CAR-T20.12, CAR-T20.13, CAR-T20.14, CAR-T20.15, CAR-T20.16

The construction and detection of CART cells were carried out in reference to examples 2, 3, 4, and 5.

Full-length DNA synthesis and cloning are firstly carried out to realize the construction of coding plasmids. CAR-T20.11, CAR-T20.12, CAR-T20.13, CAR-T20.14, CAR-T20.15, CAR-T20.16 (structure shown in Table 1, sequence shown in Table 2) were designed, followed by functional validation.

Resuscitating PBMC, infecting to obtain CAR-T20s cells, and starting on day 6, taking CAR-T20s cells for corresponding activity detection assay

Respectively taking 0.5 × 10⁶A0.5X 10 sample of CAR-T20s cells cultured up to day 7 was obtained⁶Samples of CAR-T20s cells cultured up to day 7 were tested for T cell transfection efficiency. The expression level of the Protein encoded by the CAR gene on the surface of the T cell membrane in CAR-T20s cells cultured up to day 7 was identified by the Protein L method.

The results are shown in FIG. 9A, and compared with CAR-T20.1, CAR-T20.9, CAR-T20.10, the positive rate was higher in CAR-T20.11, CAR-T20.12, CAR-T20.14, CAR-T20.16, and very low in CAR-T20.13, CAR-T20.15.

Then, co-culture was performed, and the activation level of cells was measured using CAR-T20s cells cultured up to day 7 for the detection of the markers CD137 and IFN γ. Sequentially taking 1 × 10⁶And respectively culturing the CAR-T20s cells cultured to the 7 th day with Raji and Ramos which are positive for CD20 and K562 and negative cells, and respectively detecting the expression level of CD137 on the surface of the T cell membrane and the secretion level of IFN gamma in culture supernatant after a Molt-4 tumor cell line is cultured for 18 hours according to the ratio of 1: 1.

Results as shown in fig. 9B and 9C, the results indicated that after co-culture with CD20 positive Raji, Ramos, CAR-T20.9, CAR-T20.10, CAR-T20.11, CAR-T20.12, CAR-T20.13, CAR-T20.14, expression level of T cell membrane surface CD137 in CAR-T20.16 was activated and released very high, CAR-T20.10, CAR-T20.14 was the highest, and for the remaining times, only CAR-T20.15 did not respond. Accordingly, the same applies to IFN γ release from the culture supernatant. The results show that CAR-T20.10 and CAR-T20.14 are the best in this screen.

Example 11 screening and functional validation of CAR-T20.17, CAR-T20.18, CAR-T20.19

The construction and detection methods of CART cells were performed with reference to examples 2, 3, 4, and 5.

Full-length DNA synthesis and cloning are firstly carried out to realize the construction of coding plasmids. Functional validation was performed after designing CAR-T20.17, CAR-T20.18, CAR-T20.19 (structure shown in Table 1, sequence shown in Table 2).

Resuscitating PBMC, infecting to obtain CAR-T20s cells, and starting on day 6, taking CAR-T20s cells for corresponding activity detection assay

Respectively taking 0.5 × 10⁶A0.5X 10 sample of CAR-T20s cells cultured up to day 7 was obtained⁶Samples of CAR-T20s cells cultured up to day 7 were tested for T cell transfection efficiency. The expression level of the Protein encoded by the CAR gene on the surface of the T cell membrane in CAR-T20s cells cultured up to day 7 was identified by the Protein L method.

The results are shown in FIG. 10A, and compared with CAR-T20.9, CAR-T20.12 and CAR-T20.14, the positive rate of CAR-T20.17, CAR-T20.18 and CAR-T20.19 is higher and the expression is better.

Then, co-culture was performed, and the activation level of cells was measured using CAR-T20s cells cultured up to day 7 for the detection of the markers CD137 and IFN γ. Sequentially taking 1 × 10⁶And after the CAR-T20s cells cultured to the 7 th day are respectively cultured with Raji and Ramos positive CD20 and Karpas-620 negative cells and Molt-4 tumor cell lines for 18 hours according to the ratio of 1:1, the expression level of CD137 on the surface of the T cell membrane and the secretion level of IFN gamma in culture supernatant are respectively detected.

Results the expression levels of CD137 on the cell membrane surface of T cell in CAR-T20.17, CAR-T20.18, CAR-T20.19, after co-culture with Raji, Ramos, positive for CD20, were activated and released very high as shown in FIGS. 10B, 10D. Correspondingly, the IFN gamma in the culture supernatant is released similarly to CAR-T20.17, CAR-T20.18 and CAR-T20.19. The CAR-T20s cells were subsequently tested for killing in vitro the levels of LDH released from the target tumor, the specific test method referred to in example 5

Target cells had Raji, Romas positive for CD20, Molt4 negative for CD 20. The final killing result shows that the CAR-T20.17, CAR-T20.18 and CAR-T20.19 have strong killing effect on Raji and Romas which are positive to the target cell CD 20.

The results of examples 8-11 are summarized below:

the applicant of the present invention has conducted a number of experiments to screen out a number of CARs with better effect, and by comparison, 20.1,20.2,20.4, 20.5, 20.6, 20.7, 20.8, 20.15 were found to be substantially ineffective, 20.11, 20.12,20.13 had some effect, but not as effective as 20.9,20.10, 20.14, 20.16,20.17,20.18,20.19, of which 20.18,20.19 were the best. In addition to the above structure, CD20Scfv was connected in tandem with scFv of CD19 (FMC63) to form a novel bispecific chimeric antigen receptor.

The CART cells involved in examples 8-11 comprise chimeric antigen receptor structures as shown in table 1 below and sequences as shown in table 2 below.

TABLE 1 chimeric antigen receptors and structures thereof

TABLE 2 chimeric antigen receptors and sequences thereof

Example 12 in vivo experiments in mice OF CART-TN-OF-19 and CART-TN-LEU-19

The inhibitory effect OF CART-TN-OF-19 and CART-TN-LEU-19 cells on transplanted tumor cells in mice was examined by referring to example 7, and NT was used as a control group.

The results are shown in fig. 11, 2 CART cells can better inhibit tumor cell expansion, and the fluorescence intensity in mice of the CART cell group is very weak, while the fluorescence intensity in the NT group is very strong. Wherein, the CART-TN-OF-19 can better inhibit or kill tumor cells than the CART-TN-LEU-19 in-vivo experiments.

Example 13 phase I clinical trial OF CART-TN-OF-19

A total of 3 volunteers (nos. C001, C002 and C003) were subjected to phase I clinical trials by the ethical committee, and the key criteria for volunteer selection were as follows: age 18-75 years, having received DLBCL therapy for more than 2 times, PD after or ineligible for auto-SCT, not treated with anti-CD 19, inactive CNS, with adequate liver, kidney, heart and hematopoietic functions.

The experimental procedure is shown in FIG. 12.

Clinical responses of the subjects are shown in table 3. The results showed that three patients achieved Partial Remission (PR) after 4 weeks of treatment and one patient achieved Complete Remission (CR) after 12 weeks of treatment. Since two additional patient experiments are still in progress, part of the data is not yet available.

TABLE 3 clinical response of subjects

Adverse events after treatment were as shown in table 4, with 1 of 3 patients experiencing grade 2 cytokine release syndrome, 2 experiencing grade 1 cytokine release syndrome, no neurotoxic events, no Dose Limiting Toxicities (DLTs) were observed in dose escalations, and cytopenia was primarily associated with Cy/Flu lymphocyte clearance. It should be noted that the development OF a certain degree OF cytokine release syndrome after treatment also laterally illustrates the effectiveness OF CART treatment, no particularly severe cytokine cytokines were developed in 3 patients, and CART-TN-OF-19 has a better safety profile.

TABLE 4 post-treatment adverse reactions of subjects

Detection of CAR copy numberAs shown in FIG. 13, it was shown that at 14 days, the CAR copy number peaked (above 10)⁵Copies/microgram gDNA) and still have a very high copy number after 56 days.

The change in tumor size in one subject is shown in figure 14 and shows that the tumor volume is significantly reduced and almost disappeared after 4 weeks of treatment.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai Seebiman Biotech Co., Ltd

WUXI CELLULAR BIOPHARMACEUTICAL Group Ltd.

<120> combined chimeric antigen receptor targeting CD19 and CD20 and application thereof

<130> P2018-1802

<140> CN 202010188038.1

<141> 2020-03-17

<160> 39

<170> PatentIn version 3.5

<210> 1

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

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Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

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Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

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Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

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Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Asp Tyr Tyr Cys

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Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp

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Gly Ala Gly Thr Thr Val Thr Val Ser Ser

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<210> 2

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met

20 25 30

Asp Trp Tyr Gln Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 3

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 4

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Glu Ile Val Leu Thr Gln Ser Pro Ala 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 Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 5

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

100 105

<210> 6

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 7

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Gly Gly Gly Gly Ser

1 5

<210> 8

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

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> 9

<211> 229

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 10

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

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 Leu Tyr Cys

20

<210> 11

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

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> 12

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

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> 13

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

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> 14

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

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> 15

<211> 710

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Leu Ser

20 25 30

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

35 40 45

Val Asn Tyr Met Asp Trp Tyr Gln Lys Lys Pro Gly Ser Ser Pro Lys

50 55 60

Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg

85 90 95

Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe

100 105 110

Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser

115 120 125

Thr Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser

130 135 140

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

145 150 155 160

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

165 170 175

Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile

180 185 190

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

195 200 205

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

210 215 220

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Asp Tyr Tyr Cys

225 230 235 240

Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp

245 250 255

Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu

260 265 270

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

275 280 285

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

290 295 300

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

305 310 315 320

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

325 330 335

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

340 345 350

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

355 360 365

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

370 375 380

Thr Ser Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro

385 390 395 400

Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Met Thr Gln Thr

405 410 415

Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys

420 425 430

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys

435 440 445

Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His

450 455 460

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr

465 470 475 480

Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe

485 490 495

Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys

500 505 510

Leu Glu Ile Thr Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

515 520 525

Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser

530 535 540

Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg

545 550 555 560

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

565 570 575

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

580 585 590

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

595 600 605

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

610 615 620

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

625 630 635 640

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

645 650 655

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

660 665 670

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

675 680 685

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

690 695 700

Gln Ala Leu Pro Pro Arg

705 710

<210> 16

<211> 712

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

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 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

85 90 95

Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg

100 105 110

Ser Asn Trp Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

115 120 125

Gly Ser Thr Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

145 150 155 160

Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn

165 170 175

Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

180 185 190

Trp Val Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp

195 200 205

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser

210 215 220

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr

225 230 235 240

Tyr Cys Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp

245 250 255

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly

260 265 270

Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser

275 280 285

Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp

290 295 300

Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp

305 310 315 320

Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu

325 330 335

Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe

340 345 350

Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys

355 360 365

Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly

370 375 380

Gln Gly Thr Ser Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly

385 390 395 400

Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Met Thr

405 410 415

Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile

420 425 430

Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln

435 440 445

Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg

450 455 460

Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

465 470 475 480

Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr

485 490 495

Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly

500 505 510

Thr Lys Leu Glu Ile Thr Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

515 520 525

Cys Pro Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys

530 535 540

Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg

545 550 555 560

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

565 570 575

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

580 585 590

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

595 600 605

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

610 615 620

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

625 630 635 640

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

645 650 655

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

660 665 670

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

675 680 685

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

690 695 700

His Met Gln Ala Leu Pro Pro Arg

705 710

<210> 17

<211> 2130

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atggagacag acacactcct gctatgggtg ctgctgctct gggttccagg ttccacaggt 60

gacattgtgc tgacccaatc tccagctatc ctgtctgcat ctccagggga gaaggtcaca 120

atgacttgca gggccagctc aagtgtaaat tacatggact ggtaccagaa gaagccagga 180

tcctccccca aaccctggat ttatgccaca tccaacctgg cttctggagt ccctgctcgc 240

ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagagt ggaggctgaa 300

gatgctgcca cttattactg ccagcagtgg agttttaatc cacccacgtt cggagggggg 360

accaagctgg aaataaaagg cagtactagc ggtggtggct ccgggggcgg ttccggtggg 420

ggcggcagca gcgaggtgca gctgcagcag tctggggctg agctggtgaa gcctggggcc 480

tcagtgaaga tgtcctgcaa ggcttctggc tacacattta ccagttacaa tatgcactgg 540

gtaaagcaga cacctggaca gggcctggaa tggattggag ctatttatcc aggaaatggt 600

gatacttcct acaatcagaa gttcaaaggc aaggccacat tgactgcaga caaatcctcc 660

agcacagcct acatgcagct cagcagcctg acatctgagg actctgcgga ctattactgt 720

gcaagatcta attattacgg tagtagctac tggttcttcg atgtctgggg cgcagggacc 780

acggtcaccg tctcctcagg aggtggtgga tccgaggtga agctgcagga aagcggccct 840

ggcctggtgg cccccagcca gagcctgagc gtgacctgca ccgtgagcgg cgtgagcctg 900

cccgactacg gcgtgagctg gatccggcag ccccccagga agggcctgga atggctgggc 960

gtgatctggg gcagcgagac cacctactac aacagcgccc tgaagagccg gctgaccatc 1020

atcaaggaca acagcaagag ccaggtgttc ctgaagatga acagcctgca gaccgacgac 1080

accgccatct actactgcgc caagcactac tactacggcg gcagctacgc catggactac 1140

tggggccagg gcaccagcgt gaccgtgagc agcggcagca cctccggcag cggcaagcct 1200

ggcagcggcg agggcagcac caagggcgac atccagatga cccagaccac ctccagcctg 1260

agcgccagcc tgggcgaccg ggtgaccatc agctgccggg ccagccagga catcagcaag 1320

tacctgaact ggtatcagca gaagcccgac ggcaccgtca agctgctgat ctaccacacc 1380

agccggctgc acagcggcgt gcccagccgg tttagcggca gcggctccgg caccgactac 1440

agcctgacca tctccaacct ggaacaggaa gatatcgcca cctacttttg ccagcagggc 1500

aacacactgc cctacacctt tggcggcgga acaaagctgg aaatcaccga gagcaagtac 1560

ggaccgccct gccccccttg ccctatgttc tgggtgctgg tggtggtcgg aggcgtgctg 1620

gcctgctaca gcctgctggt caccgtggcc ttcatcatct tttgggtgaa acggggcaga 1680

aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 1740

gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgcgggtg 1800

aagttcagca gaagcgccga cgcccctgcc taccagcagg gccagaatca gctgtacaac 1860

gagctgaacc tgggcagaag ggaagagtac gacgtcctgg ataagcggag aggccgggac 1920

cctgagatgg gcggcaagcc tcggcggaag aacccccagg aaggcctgta taacgaactg 1980

cagaaagaca agatggccga ggcctacagc gagatcggca tgaagggcga gcggaggcgg 2040

ggcaagggcc acgacggcct gtatcagggc ctgtccaccg ccaccaagga tacctacgac 2100

gccctgcaca tgcaggccct gcccccaagg 2130

<210> 18

<211> 2136

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaaattg tgttgacaca gtctccagcc accctgtctt tgtctccagg ggaaagagcc 120

accctctcct gcagggccag tcagagtgtt agcagctact tagcctggta ccaacagaaa 180

cctggccagg ctcccaggct cctcatctat gatgcatcca acagggccac tggcatccca 240

gccaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagcctagag 300

cctgaagatt ttgcagttta ttactgtcag cagcgtagca actggccgat caccttcggc 360

caagggacac gactggagat taaaggcagt actagcggtg gtggctccgg gggcggttcc 420

ggtgggggcg gcagcagcga agtgcagctg gtggagtctg ggggaggctt ggtacagcct 480

ggcaggtccc tgagactctc ctgtgcagcc tctggattca cctttaatga ttatgccatg 540

cactgggtcc ggcaagctcc agggaagggc ctggagtggg tctcaactat tagttggaat 600

agtggttcca taggctatgc ggactctgtg aagggccgat tcaccatctc cagagacaac 660

gccaagaagt ccctgtatct gcaaatgaac agtctgagag ctgaggacac ggccttgtat 720

tactgtgcaa aagatataca gtacggcaac tactactacg gtatggacgt ctggggccaa 780

gggaccacgg tcaccgtctc ctcaggaggt ggtggatccg aggtgaagct gcaggaaagc 840

ggccctggcc tggtggcccc cagccagagc ctgagcgtga cctgcaccgt gagcggcgtg 900

agcctgcccg actacggcgt gagctggatc cggcagcccc ccaggaaggg cctggaatgg 960

ctgggcgtga tctggggcag cgagaccacc tactacaaca gcgccctgaa gagccggctg 1020

accatcatca aggacaacag caagagccag gtgttcctga agatgaacag cctgcagacc 1080

gacgacaccg ccatctacta ctgcgccaag cactactact acggcggcag ctacgccatg 1140

gactactggg gccagggcac cagcgtgacc gtgagcagcg gcagcacctc cggcagcggc 1200

aagcctggca gcggcgaggg cagcaccaag ggcgacatcc agatgaccca gaccacctcc 1260

agcctgagcg ccagcctggg cgaccgggtg accatcagct gccgggccag ccaggacatc 1320

agcaagtacc tgaactggta tcagcagaag cccgacggca ccgtcaagct gctgatctac 1380

cacaccagcc ggctgcacag cggcgtgccc agccggttta gcggcagcgg ctccggcacc 1440

gactacagcc tgaccatctc caacctggaa caggaagata tcgccaccta cttttgccag 1500

cagggcaaca cactgcccta cacctttggc ggcggaacaa agctggaaat caccgagagc 1560

aagtacggac cgccctgccc cccttgccct atgttctggg tgctggtggt ggtcggaggc 1620

gtgctggcct gctacagcct gctggtcacc gtggccttca tcatcttttg ggtgaaacgg 1680

ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 1740

caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 1800

cgggtgaagt tcagcagaag cgccgacgcc cctgcctacc agcagggcca gaatcagctg 1860

tacaacgagc tgaacctggg cagaagggaa gagtacgacg tcctggataa gcggagaggc 1920

cgggaccctg agatgggcgg caagcctcgg cggaagaacc cccaggaagg cctgtataac 1980

gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg 2040

aggcggggca agggccacga cggcctgtat cagggcctgt ccaccgccac caaggatacc 2100

tacgacgccc tgcacatgca ggccctgccc ccaagg 2136

<210> 19

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 20

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatct 45

<210> 21

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300

gggaccaagc tggagatcac a 321

<210> 22

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gaggtgaaac tgcaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccgtc 60

acatgcactg tctcaggggt ctcattaccc gactatggtg taagctggat tcgccagcct 120

ccacgaaagg gtctggagtg gctgggagta atatggggta gtgaaaccac atactataat 180

tcagctctca aatccagact gaccatcatc aaggacaact ccaagagcca agttttctta 240

aaaatgaaca gtctgcaaac tgatgacaca gccatttact actgtgccaa acattattac 300

tacggtggta gctatgctat ggactactgg ggccaaggaa cctcagtcac cgtctcctca 360

<210> 23

<211> 472

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Met Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro

1 5 10 15

Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Leu Ser Phe

20 25 30

Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile

35 40 45

Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Thr Val Glu Ala

65 70 75 80

Glu Asp Ala Ala Ser Tyr Phe Cys His Gln Trp Ser Ser Asn Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Ser Ser Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Asp Val Met Gly Val Asp Ser Gly Gly

115 120 125

Gly Leu Val Gln Pro Gly Gly Ser Arg Lys Leu Ser Cys Ala Ala Pro

130 135 140

Gly Phe Thr Phe Ser Ser Phe Gly Met His Trp Val Arg Gln Ala Pro

145 150 155 160

Glu Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser Pro Ser Ser Thr

165 170 175

Leu His Tyr Ala Asp Arg Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

180 185 190

Asn Pro Lys Asn Thr Leu Phe Leu Gln Met Lys Leu Pro Ser Leu Cys

195 200 205

Tyr Gly Leu Leu Gly Pro Arg Asp His Val His Arg Leu Leu Lys Thr

210 215 220

Arg Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val Pro Val Phe

225 230 235 240

Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

245 250 255

Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys

260 265 270

Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala

275 280 285

Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu

290 295 300

Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser Lys Arg Gly Arg

305 310 315 320

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

325 330 335

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

340 345 350

Glu Gly Gly Cys Glu Leu Glu Phe Arg Val Lys Phe Ser Arg Ser Ala

355 360 365

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

370 375 380

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

385 390 395 400

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

405 410 415

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

420 425 430

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

435 440 445

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

450 455 460

His Met Gln Ala Leu Pro Pro Arg

465 470

<210> 24

<211> 492

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

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 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Leu

20 25 30

Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser

35 40 45

Ser Leu Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro

50 55 60

Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser

85 90 95

Thr Val Glu Ala Glu Asp Ala Ala Ser Tyr Phe Cys His Gln Trp Ser

100 105 110

Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Ser Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asp Val Met Gly Val

130 135 140

Asp Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Arg Lys Leu Ser

145 150 155 160

Cys Ala Ala Pro Gly Phe Thr Phe Ser Ser Phe Gly Met His Trp Val

165 170 175

Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser

180 185 190

Pro Ser Ser Thr Leu His Tyr Ala Asp Arg Val Lys Gly Arg Phe Thr

195 200 205

Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe Leu Gln Met Lys Leu

210 215 220

Pro Ser Leu Cys Tyr Gly Leu Leu Gly Pro Arg Asp His Val His Arg

225 230 235 240

Leu Leu Lys Thr Arg Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe

245 250 255

Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg

260 265 270

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

275 280 285

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

290 295 300

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

305 310 315 320

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser

325 330 335

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

340 345 350

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

355 360 365

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Glu Phe Arg Val Lys Phe

370 375 380

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

385 390 395 400

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

405 410 415

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

420 425 430

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

435 440 445

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

450 455 460

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

465 470 475 480

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 25

<211> 377

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

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 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Leu

20 25 30

Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser

35 40 45

Ser Leu Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro

50 55 60

Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser

85 90 95

Thr Val Glu Ala Glu Asp Ala Ala Ser Tyr Phe Cys His Gln Trp Ser

100 105 110

Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Met

130 135 140

Gly Val Asp Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Arg Lys

145 150 155 160

Leu Ser Cys Ala Ala Pro Gly Phe Thr Phe Ser Ser Phe Gly Met His

165 170 175

Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val Ala Tyr Ile

180 185 190

Ser Ser Pro Ser Ser Thr Leu His Tyr Ala Asp Arg Val Lys Gly Arg

195 200 205

Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe Leu Gln Met

210 215 220

Lys Leu Pro Ser Leu Cys Tyr Gly Leu Leu Gly Pro Arg Asp His Val

225 230 235 240

His Arg Leu Leu Thr Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

245 250 255

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

260 265 270

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

275 280 285

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

290 295 300

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser Lys Arg Gly

305 310 315 320

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

325 330 335

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

340 345 350

Glu Glu Gly Gly Cys Glu Leu Glu Phe Glu Leu Gly Thr Phe Lys Thr

355 360 365

Asn Asp Leu Gln Gly Ser Cys Arg Ser

370 375

<210> 26

<211> 377

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

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 Asp Val Met Gly Val Asp Ser Gly Gly Gly Leu

20 25 30

Val Gln Pro Gly Gly Ser Arg Lys Leu Ser Cys Ala Ala Pro Gly Phe

35 40 45

Thr Phe Ser Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Glu Lys

50 55 60

Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser Pro Ser Ser Thr Leu His

65 70 75 80

Tyr Ala Asp Arg Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro

85 90 95

Lys Asn Thr Leu Phe Leu Gln Met Lys Leu Pro Ser Leu Cys Tyr Gly

100 105 110

Leu Leu Gly Pro Arg Asp His Val His Arg Leu Leu Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr

130 135 140

Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met

145 150 155 160

Thr Cys Arg Ala Ser Ser Ser Leu Ser Phe Met His Trp Tyr Gln Gln

165 170 175

Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu

180 185 190

Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser

195 200 205

Tyr Ser Leu Thr Ile Ser Thr Val Glu Ala Glu Asp Ala Ala Ser Tyr

210 215 220

Phe Cys His Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr

225 230 235 240

Lys Leu Glu Ile Thr Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

245 250 255

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

260 265 270

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

275 280 285

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

290 295 300

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser Lys Arg Gly

305 310 315 320

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

325 330 335

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

340 345 350

Glu Glu Gly Gly Cys Glu Leu Glu Phe Glu Leu Gly Thr Phe Lys Thr

355 360 365

Asn Asp Leu Gln Gly Ser Cys Arg Ser

370 375

<210> 27

<211> 396

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

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 Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu

20 25 30

Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser

35 40 45

Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro

50 55 60

Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser

85 90 95

Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr

100 105 110

Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

130 135 140

Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val

145 150 155 160

Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met

165 170 175

His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile Gly Ala

180 185 190

Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly

195 200 205

Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln

210 215 220

Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg

225 230 235 240

Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly Ala Gly

245 250 255

Thr Thr Val Thr Val Ser Ala Thr Arg Thr Thr Thr Pro Ala Pro Arg

260 265 270

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

275 280 285

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

290 295 300

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

305 310 315 320

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser

325 330 335

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

340 345 350

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

355 360 365

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Glu Phe Glu Leu Gly Thr

370 375 380

Phe Lys Thr Asn Asp Leu Gln Gly Ser Cys Arg Ser

385 390 395

<210> 28

<211> 396

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

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 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu

20 25 30

Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

35 40 45

Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Arg

50 55 60

Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser

65 70 75 80

Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser

85 90 95

Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr

115 120 125

Phe Asn Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Val

145 150 155 160

Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val

165 170 175

Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile His Trp Phe

180 185 190

Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser

195 200 205

Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala

225 230 235 240

Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr Phe Gly Gly

245 250 255

Gly Thr Lys Leu Glu Ile Lys Thr Arg Thr Thr Thr Pro Ala Pro Arg

260 265 270

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

275 280 285

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

290 295 300

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

305 310 315 320

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser

325 330 335

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

340 345 350

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

355 360 365

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Glu Phe Glu Leu Gly Thr

370 375 380

Phe Lys Thr Asn Asp Leu Gln Gly Ser Cys Arg Ser

385 390 395

<210> 29

<211> 715

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

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 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu

20 25 30

Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

35 40 45

Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Gln

50 55 60

Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser

65 70 75 80

Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser

85 90 95

Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser

100 105 110

Ala Asp Tyr Tyr Cys Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp

115 120 125

Phe Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile

145 150 155 160

Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys

165 170 175

Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Asp Trp

180 185 190

Tyr Gln Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr

195 200 205

Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser

210 215 220

Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala

225 230 235 240

Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly

245 250 255

Gly Gly Thr Lys Leu Glu Ile Lys Glu Ser Lys Tyr Gly Pro Pro Cys

260 265 270

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

275 280 285

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

290 295 300

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

305 310 315 320

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

325 330 335

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

340 345 350

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

355 360 365

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

370 375 380

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

385 390 395 400

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

405 410 415

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

420 425 430

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

435 440 445

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

450 455 460

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

465 470 475 480

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Phe Trp Val

485 490 495

Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr

500 505 510

Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu

515 520 525

His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg

530 535 540

Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg

545 550 555 560

Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

565 570 575

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

580 585 590

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

595 600 605

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

610 615 620

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

625 630 635 640

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

645 650 655

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

660 665 670

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

675 680 685

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

690 695 700

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

705 710 715

<210> 30

<211> 715

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

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 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu

20 25 30

Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

35 40 45

Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Gln

50 55 60

Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser

65 70 75 80

Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser

85 90 95

Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser

100 105 110

Ala Asp Tyr Tyr Cys Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp

115 120 125

Phe Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile

145 150 155 160

Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys

165 170 175

Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Asp Trp

180 185 190

Tyr Gln Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr

195 200 205

Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser

210 215 220

Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala

225 230 235 240

Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly

245 250 255

Gly Gly Thr Lys Leu Glu Ile Lys Glu Ser Lys Tyr Gly Pro Pro Cys

260 265 270

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

275 280 285

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

290 295 300

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

305 310 315 320

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

325 330 335

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

340 345 350

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

355 360 365

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

370 375 380

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

385 390 395 400

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

405 410 415

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

420 425 430

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

435 440 445

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

450 455 460

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

465 470 475 480

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Phe Trp Val

485 490 495

Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr

500 505 510

Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu

515 520 525

His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg

530 535 540

Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg

545 550 555 560

Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

565 570 575

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

580 585 590

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

595 600 605

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

610 615 620

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

625 630 635 640

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

645 650 655

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

660 665 670

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

675 680 685

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

690 695 700

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

705 710 715

<210> 31

<211> 671

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

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 Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Leu

20 25 30

Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser

35 40 45

Ser Val Asn Tyr Met Asp Trp Tyr Gln Lys Lys Pro Gly Ser Ser Pro

50 55 60

Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser

85 90 95

Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser

100 105 110

Phe Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

130 135 140

Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val

145 150 155 160

Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met

165 170 175

His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile Gly Ala

180 185 190

Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly

195 200 205

Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln

210 215 220

Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Asp Tyr Tyr Cys Ala Arg

225 230 235 240

Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp Gly Ala

245 250 255

Gly Thr Thr Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys

260 265 270

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

275 280 285

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

290 295 300

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

305 310 315 320

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

325 330 335

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

340 345 350

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

355 360 365

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

370 375 380

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

385 390 395 400

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

405 410 415

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

420 425 430

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

435 440 445

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

450 455 460

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

465 470 475 480

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ile Tyr Ile

485 490 495

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

500 505 510

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

515 520 525

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

530 535 540

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

545 550 555 560

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln

565 570 575

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

580 585 590

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

595 600 605

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

610 615 620

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

625 630 635 640

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

645 650 655

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

660 665 670

<210> 32

<211> 671

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

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 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu

20 25 30

Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

35 40 45

Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Gln

50 55 60

Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser

65 70 75 80

Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser

85 90 95

Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser

100 105 110

Ala Asp Tyr Tyr Cys Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp

115 120 125

Phe Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile

145 150 155 160

Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys

165 170 175

Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Asp Trp

180 185 190

Tyr Gln Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr

195 200 205

Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser

210 215 220

Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala

225 230 235 240

Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly

245 250 255

Gly Gly Thr Lys Leu Glu Ile Lys Glu Ser Lys Tyr Gly Pro Pro Cys

260 265 270

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

275 280 285

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

290 295 300

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

305 310 315 320

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

325 330 335

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

340 345 350

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

355 360 365

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

370 375 380

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

385 390 395 400

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

405 410 415

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

420 425 430

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

435 440 445

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

450 455 460

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

465 470 475 480

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ile Tyr Ile

485 490 495

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

500 505 510

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

515 520 525

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

530 535 540

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

545 550 555 560

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln

565 570 575

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

580 585 590

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

595 600 605

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

610 615 620

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

625 630 635 640

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

645 650 655

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

660 665 670

<210> 33

<211> 677

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

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 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr

35 40 45

Ala Phe Ser Tyr Ser Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln

50 55 60

Gly Leu Glu Trp Met Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp

65 70 75 80

Tyr Asn Gly Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser

85 90 95

Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val

115 120 125

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr

145 150 155 160

Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile

165 170 175

Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr

180 185 190

Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile

195 200 205

Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro Asp Arg Phe Ser Gly

210 215 220

Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

225 230 235 240

Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn Leu Glu Leu Pro Tyr

245 250 255

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Glu Ser

260 265 270

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly

275 280 285

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

290 295 300

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

305 310 315 320

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

325 330 335

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

340 345 350

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

355 360 365

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

370 375 380

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

385 390 395 400

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

405 410 415

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

420 425 430

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

435 440 445

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

450 455 460

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

465 470 475 480

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

485 490 495

Leu Gly Lys Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

500 505 510

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

515 520 525

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

530 535 540

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

545 550 555 560

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

565 570 575

Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

580 585 590

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

595 600 605

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

610 615 620

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

625 630 635 640

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

645 650 655

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

660 665 670

Ala Leu Pro Pro Arg

675

<210> 34

<211> 672

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

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 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

20 25 30

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Asn Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

85 90 95

Lys Lys Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Leu Tyr Tyr Cys Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr

115 120 125

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile

145 150 155 160

Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg

165 170 175

Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

180 185 190

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp

195 200 205

Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly

210 215 220

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp

225 230 235 240

Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile Thr Phe

245 250 255

Gly Gln Gly Thr Arg Leu Glu Ile Lys Glu Ser Lys Tyr Gly Pro Pro

260 265 270

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

275 280 285

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

290 295 300

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

305 310 315 320

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

325 330 335

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

340 345 350

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

355 360 365

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

370 375 380

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

385 390 395 400

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

405 410 415

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

420 425 430

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

435 440 445

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

450 455 460

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

465 470 475 480

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ile Tyr

485 490 495

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

500 505 510

Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile

515 520 525

Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp

530 535 540

Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

545 550 555 560

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

565 570 575

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

580 585 590

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

595 600 605

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

610 615 620

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

625 630 635 640

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

645 650 655

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

660 665 670

<210> 35

<211> 653

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

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 Gly Asp Val Met Gly Val Asp Ser Gly Gly Gly

20 25 30

Leu Val Gln Pro Gly Gly Ser Arg Lys Leu Ser Cys Ala Ala Pro Gly

35 40 45

Phe Thr Phe Ser Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Glu

50 55 60

Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser Pro Ser Ser Thr Leu

65 70 75 80

His Tyr Ala Asp Arg Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

85 90 95

Pro Lys Asn Thr Leu Phe Leu Gln Met Lys Leu Pro Ser Leu Cys Tyr

100 105 110

Gly Leu Leu Gly Pro Arg Asp His Val His Arg Leu Leu Lys Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val

145 150 155 160

Thr Met Thr Cys Arg Ala Ser Ser Ser Leu Ser Phe Met His Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser

180 185 190

Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Ser Tyr Ser Leu Thr Ile Ser Thr Val Glu Ala Glu Asp Ala Ala

210 215 220

Ser Tyr Phe Cys His Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala

225 230 235 240

Gly Thr Lys Leu Glu Ile Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

245 250 255

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

260 265 270

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

275 280 285

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

290 295 300

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

305 310 315 320

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

325 330 335

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

340 345 350

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

355 360 365

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

370 375 380

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

385 390 395 400

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

405 410 415

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

420 425 430

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

435 440 445

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

450 455 460

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ile Tyr Ile Trp Ala

465 470 475 480

Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

485 490 495

Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

500 505 510

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

515 520 525

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

530 535 540

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln

545 550 555 560

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

565 570 575

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

580 585 590

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

595 600 605

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

610 615 620

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

625 630 635 640

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

645 650

<210> 36

<211> 670

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

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 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu

20 25 30

Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

35 40 45

Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Arg

50 55 60

Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser

65 70 75 80

Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser

85 90 95

Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr

115 120 125

Phe Asn Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Val

145 150 155 160

Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val

165 170 175

Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile His Trp Phe

180 185 190

Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser

195 200 205

Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala

225 230 235 240

Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr Phe Gly Gly

245 250 255

Gly Thr Lys Leu Glu Ile Lys Glu Ser Lys Tyr Gly Pro Pro Cys Pro

260 265 270

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

275 280 285

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

290 295 300

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

305 310 315 320

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

325 330 335

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

340 345 350

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

355 360 365

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

370 375 380

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

385 390 395 400

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

405 410 415

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

420 425 430

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

435 440 445

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

450 455 460

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

465 470 475 480

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ile Tyr Ile Trp

485 490 495

Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile

500 505 510

Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys

515 520 525

Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys

530 535 540

Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val

545 550 555 560

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn

565 570 575

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

580 585 590

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

595 600 605

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

610 615 620

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

625 630 635 640

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

645 650 655

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

660 665 670

<210> 37

<211> 715

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 37

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 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu

20 25 30

Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

35 40 45

Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Gln

50 55 60

Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser

65 70 75 80

Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser

85 90 95

Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser

100 105 110

Ala Asp Tyr Tyr Cys Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp

115 120 125

Phe Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile

145 150 155 160

Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys

165 170 175

Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Asp Trp

180 185 190

Tyr Gln Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr

195 200 205

Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser

210 215 220

Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala

225 230 235 240

Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly

245 250 255

Gly Gly Thr Lys Leu Glu Ile Lys Glu Ser Lys Tyr Gly Pro Pro Cys

260 265 270

Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu

275 280 285

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

290 295 300

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

305 310 315 320

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

325 330 335

Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser Val Leu

340 345 350

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

355 360 365

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

370 375 380

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

385 390 395 400

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

405 410 415

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

420 425 430

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

435 440 445

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

450 455 460

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

465 470 475 480

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Phe Trp Val

485 490 495

Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr

500 505 510

Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu

515 520 525

His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg

530 535 540

Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg

545 550 555 560

Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

565 570 575

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

580 585 590

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

595 600 605

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

610 615 620

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

625 630 635 640

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

645 650 655

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

660 665 670

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

675 680 685

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

690 695 700

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

705 710 715

<210> 38

<211> 660

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 38

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 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu

20 25 30

Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

35 40 45

Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Gln

50 55 60

Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser

65 70 75 80

Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser

85 90 95

Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser

100 105 110

Ala Asp Tyr Tyr Cys Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp

115 120 125

Phe Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile

145 150 155 160

Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys

165 170 175

Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Asp Trp

180 185 190

Tyr Gln Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr

195 200 205

Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser

210 215 220

Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala

225 230 235 240

Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly

245 250 255

Gly Gly Thr Lys Leu Glu Ile Lys Glu Ser Lys Tyr Gly Pro Pro Cys

260 265 270

Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu

275 280 285

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

290 295 300

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

305 310 315 320

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

325 330 335

Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser Val Leu

340 345 350

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

355 360 365

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

370 375 380

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

385 390 395 400

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

405 410 415

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

420 425 430

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

435 440 445

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

450 455 460

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

465 470 475 480

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ile Tyr Ile

485 490 495

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

500 505 510

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

515 520 525

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

530 535 540

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

545 550 555 560

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln

565 570 575

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

580 585 590

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

595 600 605

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

610 615 620

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

625 630 635 640

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

645 650 655

Lys Asp Thr Tyr

660

<210> 39

<211> 672

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 39

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 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

20 25 30

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Asn Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

85 90 95

Lys Lys Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Leu Tyr Tyr Cys Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr

115 120 125

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile

145 150 155 160

Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg

165 170 175

Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

180 185 190

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp

195 200 205

Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly

210 215 220

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp

225 230 235 240

Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile Thr Phe

245 250 255

Gly Gln Gly Thr Arg Leu Glu Ile Lys Glu Ser Lys Tyr Gly Pro Pro

260 265 270

Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe

275 280 285

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

290 295 300

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

305 310 315 320

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

325 330 335

Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser Val

340 345 350

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

355 360 365

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

370 375 380

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

385 390 395 400

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

405 410 415

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

420 425 430

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

435 440 445

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

450 455 460

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

465 470 475 480

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ile Tyr

485 490 495

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

500 505 510

Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile

515 520 525

Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp

530 535 540

Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

545 550 555 560

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

565 570 575

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

580 585 590

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

595 600 605

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

610 615 620

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

625 630 635 640

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

645 650 655

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

660 665 670