CN115340610A

CN115340610A - Bispecific CAR T cells and uses thereof

Info

Publication number: CN115340610A
Application number: CN202110516661.XA
Authority: CN
Inventors: 朱保国; 职慧军; 罗翀; 张长风; 王影; 陈怡�; 柯军; 舒望云; 于洋; 钟凯伦
Original assignee: Livzon Mabpharm Inc
Current assignee: Livzon Mabpharm Inc
Priority date: 2021-05-12
Filing date: 2021-05-12
Publication date: 2022-11-15

Abstract

The present disclosure relates to a bispecific CAR T cell and uses thereof. The Chimeric Antigen Receptor (CAR) comprises a first polypeptide and a second polypeptide, wherein: the first polypeptide comprises: (ii) a first binding protein, (ii) a first spacer, (iii) a first transmembrane domain, (iv) a costimulatory signaling domain of 4-1BB, and (v) a TCR CD3 zeta signaling domain; and the second polypeptide comprises: (ii) a second binding protein, (ii) a second spacer, (iii) a second transmembrane domain, (iv) a costimulatory signaling domain of 4-1BB, and (v) a TCR CD3 zeta signaling domain. The CAR-T cells prepared from the chimeric antigen receptor of the present disclosure can specifically kill tumor cells, and are more advantageous in inhibiting tumor recurrence than CD19 or CD20 single-target CAR-T cells.

Description

Bispecific CAR T cells and uses thereof

Technical Field

The present disclosure relates to a bispecific CAR T cell and uses thereof, in particular to a bispecific CAR T cell targeting CD19 and CD20 and uses thereof.

Background

T cells modified with Chimeric Antigen Receptors (CARs) specifically recognize and kill tumor cells, an emerging fourth method of tumor treatment following surgery, radiation therapy and chemotherapy. Many studies have shown that adoptive cellular immunotherapy, typified by CAR-T cells, achieves a very good therapeutic effect in the immunotherapy of tumors, in particular in the treatment of hematological tumors.

The principle of the CAR-T cell therapy method is to express CAR on T cells by genetic modification, and then to return the modified T cells to the patient after in vitro expansion, so that they can be activated and proliferated under the stimulation of tumor cells carrying specific target antigens, thereby exerting immune effect, releasing cytokines and lysing tumor cells. A typical CAR molecule generally comprises an intracellular domain of the CD3 zeta chain, one or more costimulatory signals, a transmembrane region and a spacer domain, and an extracellular domain responsible for recognition and specific binding to a tumor antigen, usually a single chain antibody sequence (scFv). The intracellular domain of the CD3 zeta chain contains Immunoreceptor Tyrosine Activation Motifs (ITAMs) which are capable of providing a first signal (also referred to as primary cytoplasmic signal) required for T cell activation; commonly used costimulatory molecules are the intracellular domains of CD28, CD137 (4-1 BB), icos, OX40, etc., which provide a secondary signal for T cell activation.

Two CAR T cell therapy products targeting the B cell surface antigen CD19 are currently approved by the FDA in the united states for the treatment of relapsed or refractory B-lymphocyte leukemia and non-hodgkin lymphoma (NHL).

CD19 is a glycoprotein on the surface of B cells and is expressed from the early stages of B cell development until it differentiates into plasma cells. CD19 is one of the members of the immunoglobulin (Ig) superfamily, and is one of the components of the B cell surface signaling complex, involved in the process of signal transduction that regulates B cell receptors. It is generally accepted that expression of CD19 is restricted to B cell lines (B-cell lines) and not expressed on the surface of pluripotent hematopoietic stem cells. This feature allows CD19 to be a safe therapeutic target without developing autoimmune diseases or irreversible bone marrow toxic damage. CD19 is also expressed on the surface of most B-cell lymphomas, mantle cell lymphomas, acute Lymphocytic Leukemia (ALL), chronic Lymphocytic Leukemia (CLL), hairy cell leukemia and a portion of acute myeloid leukemia cells. Thus, CD19 is a very valuable immunotherapeutic target in the treatment of leukemia/lymphoma.

CD20 is another B cell surface antigen that is expressed primarily in the developmental process from pre-B cells to memory B cells, but not in hematopoietic stem cells, pro-B cells, mature plasma cells, and other normal tissues. Expressed in more than 90% of B cell lymphomas and 1/3 of acute B lymphocytic leukemia (B-ALL). Thus, like CD19, CD20 is a tumor target antigen for the treatment of B-cell lymphomas and B-lymphocyte leukemias. Monoclonal antibody medicines aiming at CD20 targets are available on the market, and the medicine has obvious curative effect on B cell lymphoma/leukemia. Meanwhile, CAR-T cell therapy targeting CD20 antigen is also under development, and preliminary clinical experimental data show that it has significant efficacy in treating relapsed or refractory B cell lymphoma/leukemia.

Current treatment data for two CD19 CAR-T cell therapeutic products that have been approved by the FDA in the united states indicate that the response rate to B cell lymphoma is as high as 50-80% in the early stages of treatment, but the recurrence rate after one year is close to 50%. There are several causes of recurrence, one of which is important is tumor recurrence resulting from loss of target antigen. This increase in the number of targets will reduce the likelihood of recurrence due to loss of a single target.

At present, CD19 and CD20 bispecific CAR-T cells are prepared and applied, but the reports are that CD19 and CD20 single-chain antibodies (ScFv) are connected through a linker, and a set of CAR structures such as a hinge region, a transmembrane region, a costimulatory domain and an activation domain are shared, so that the CAR structures are called Tandem CAR structures (Tandem CARs and TanCARs) (figure 1). Due to the structural design, the two antibodies are close to each other in space, so that the possibility of mutual interference exists. And one of the antibodies, after binding to the corresponding antigen, is unable to bind to the second antigen due to steric hindrance.

Disclosure of Invention

To solve the problems of the prior art, the inventors designed and prepared a double cis chimeric antigen receptor (Bicistronic CARs) in a parallel structure, and designed CD19 CAR and CD20CAR as two independent structural units, which have the same primary cytoplasmic signaling sequence CD3 ζ and costimulatory molecule 4-1BB, and can independently recognize and bind to their respective target antigens, respectively (fig. 2). In vitro and in vivo experimental results show that the CAR-T cells prepared by the CAR element with double targets of CD19 and CD20 can kill tumor cells specifically, and have more advantages than the CAR-T cells with single targets of CD19 or CD20 in the aspect of inhibiting tumor recurrence.

The present disclosure provides compositions and methods for treating cancer and other diseases. The cancer may be a hematologic malignancy, a solid tumor, a primary or metastatic tumor.

In one aspect, the present disclosure provides a chimeric antigen receptor comprising a first polypeptide and a second polypeptide, wherein:

the first polypeptide comprises: (ii) a first binding protein, (ii) a first spacer, (iii) a first transmembrane domain, (iv) a costimulatory signaling domain of 4-1BB, and (v) a TCR CD3 zeta signaling domain; and is

The second polypeptide comprises: (ii) a second binding protein, (ii) a second spacer, (iii) a second transmembrane domain, (iv) a costimulatory signaling domain of 4-1BB, and (v) a TCR CD3 zeta signaling domain.

In one aspect, the disclosure provides polynucleotides encoding the aforementioned chimeric antigen receptors.

In one aspect, the disclosure provides a vector comprising the foregoing polynucleotide.

In one aspect, the present disclosure provides an engineered cell comprising the aforementioned vector.

In one aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned chimeric antigen receptor, polynucleotide, vector and/or engineered cell.

In one aspect, the present disclosure provides the use of the aforementioned chimeric antigen receptors, polynucleotides, vectors, engineered cells and/or pharmaceutical compositions in the manufacture of a medicament for the treatment or prevention of diseases, conditions and disorders, including tumors.

In one aspect, the present disclosure provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue in a subject, the method comprising administering to the subject an effective amount of the aforementioned chimeric antigen receptor, polynucleotide, vector, engineered cell, and/or pharmaceutical composition.

In one aspect, the present disclosure provides methods of administering the aforementioned chimeric antigen receptors, polynucleotides, vectors, engineered cells and/or pharmaceutical compositions for the treatment or prevention of diseases, conditions and disorders, including cancer, and uses of such aforementioned chimeric antigen receptors, polynucleotides, vectors, engineered cells and/or pharmaceutical compositions for treatment or prevention. In some embodiments, the cells, populations, and compositions are administered to a subject or patient to be treated having a particular disease or disorder, e.g., by adoptive cell therapy (e.g., adoptive T cell therapy). In some embodiments, cells and compositions (e.g., engineered compositions and end-of-production products after incubation and/or other processing steps) prepared by the provided methods are administered to a subject, such as a subject having or at risk of a disease or disorder. In some aspects, the methods thereby treat, for example, one or more symptoms of a disease or disorder, e.g., by reducing tumor burden in a cancer expressing an antigen recognized by an engineered T cell.

Drawings

Figure 1 shows tandem CAR structures.

Figure 2 shows a parallel CAR structure.

Figure 3 shows a schematic diagram of the dual CAR domains of the present disclosure.

FIG. 4 shows a map of plasmid pAC 001L.

Figure 5 shows the results of flow-testing CAR-T cell CD19 CAR, CD20CAR positivity rates.

FIG. 6 shows the results of Western-blot detection of the expression of CD19 CAR and CD20CAR in human T cells.

Figure 7 shows the long-term tumor killing effect of single CAR-T cells in vitro.

FIG. 8 shows a comparison of the long-term killing activity of different CD19/CD20CAR T cells against the target cell K562-CD 19.

Figure 9 shows the cumulative fold expansion of different CD19/CD20CAR T cells when co-cultured with the target cell K562-CD 19.

FIG. 10 shows a comparison of the long-term killing activity of different CD19/CD20CAR T cells against the target cell K562-CD20.

Figure 11 shows the cumulative fold expansion of different CD19/CD20CAR T cells when co-cultured with the target cell K562-CD20.

Figure 12 shows a comparison of long term killing activity of different CD19/CD20CAR T cells against the target cells K562-CD19+ K562-CD20.

Figure 13 shows the cumulative fold expansion of different CD19/CD20CAR T cells when co-cultured with the target cell K562-CD19+ K562-CD20.

Figure 14 shows LDH assay to detect killing activity of CD19 or CD20CAR and CD19/CD20CAR T cells against target cells.

Figure 15 shows cytokine levels in supernatants after 48h of CAR #10 vector transduced and untransduced T cells co-cultured with different target cells.

Figure 16 is an image of tumor imaging in NSG mice with or without infused CAR T cells.

Figure 17 shows tumor fluorescence model intensity in NSG mice with or without infused CAR T cells.

Detailed Description

In the present disclosure, unless defined otherwise, scientific and technical terms used herein have the meanings that are commonly understood by those of skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and conventional procedures used extensively in the relevant art. Meanwhile, for better understanding of the present disclosure, definitions and explanations of related terms are provided below.

The term "comprising" as used herein refers to compositions, methods, and one or more components thereof useful with the embodiments, as well as open to the inclusion of unspecified elements, whether or not useful. In general, those skilled in the art will appreciate that the terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.).

The use of the terms "a" and "an" and "the" and similar referents in the context of describing particular embodiments of the application (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided with respect to certain embodiments herein, is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. The abbreviation "for example" is derived from latin exemplification and is used herein to indicate a non-limiting example. Thus, the abbreviation "such as" is synonymous with the term "such as". No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

The term "about" as used herein refers to a measurable value, such as an amount, duration, etc., and includes variations from the specified value by 20%, 10%, 5%, 1%, 0.5%, or 0.1%.

The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. The antibody may be an intact immunoglobulin derived from a natural source or a recombinant source and may be an immunoreactive part of an intact immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies of the present disclosure may exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, fv, fab and F (ab), as well as single chain antibodies and humanized antibodies.

The term "antigen" or "Ag" as used herein is defined as a molecule that elicits an immune response. The immune response may involve antibody production, or activation of specific immunocompetent cells, or both. The skilled person will appreciate that any macromolecule, including almost any protein or peptide, may be used as an antigen. Furthermore, the antigen may be derived from recombinant DNA or genomic DNA. The skilled person will understand that any DNA comprising a nucleotide sequence or part of a nucleotide sequence encoding a protein that elicits an immune response thus encodes an "antigen" as the term is used herein. Furthermore, one skilled in the art will appreciate that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Furthermore, the skilled person will understand that an antigen need not be encoded by a "gene" at all. It will be apparent that the antigen may be produced synthetically or may be derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

The term "anti-tumor effect" as used herein refers to a biological effect that can be manifested by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancer condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the disclosure to prevent the initial development of a tumor.

The term "autologous" as used herein refers to any substance that is derived from the same individual and subsequently reintroduced into the individual.

"allogeneic" refers to grafts derived from different animals of the same species.

"xenogeneic" refers to grafts derived from animals of different species.

The term "cancer" as used herein is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the blood and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.

"encoding" refers to the inherent nature of a particular sequence of nucleotides in a polynucleotide. Such as genes, cDNAs or mRNAs, are used as templates for the synthesis of other polymers and macromolecules in biological processes, and have defined nucleotide sequences (i.e., rRNA, tRNA and mRNA) or defined amino acid sequences, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand (whose nucleotide sequence is identical to the mRAN sequence and is typically provided in the sequence listing) and the non-coding strand (which serves as a transcription template for a gene or cDNA) may be referred to as encoding the protein or other product of the gene or cDNA.

"homologous" refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of two compared sequences is occupied by the same base or amino acid monomer subunit, for example if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, two sequences are 60% homologous if 6 of 10 positions in the two sequences are matching or homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Typically, the comparison is made when the two sequences are aligned to give maximum homology.

The term "co-stimulatory ligand" as used herein includes molecules on antigen presenting cells (e.g., APCs, dendritic cells, B cells, and other immune cells) that specifically bind to cognate co-stimulatory molecules on T cells, thereby providing a signal that mediates T cell responses, including but not limited to proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, binding of the TCR/CD3 complex to peptide-loaded MHC molecules. Costimulatory ligands can include, but are not limited to, CD7, B7-1 (CD 80), B7-2 (CD 86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands also include antibodies that specifically bind to, inter alia, costimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD 83.

"costimulatory molecule" or "costimulatory receptor" refers to an associated binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, toll ligand receptors. Costimulatory molecules also include non-naturally engineered proteins.

As used herein, "co-stimulatory signals" refer to signals that, in combination with a primary signal, e.g., TCR/CD3 linkage, result in up-or down-regulation of T cell proliferation and/or key molecules.

The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., the TCR/CD3 complex) to its cognate ligand, thereby mediating a signaling event, such as, but not limited to, signaling through the TCR/CD3 complex. Stimulation may mediate altered expression of certain molecules, such as down-regulation of TGF-B, and/or recombination of cytoskeletal structures, among others.

The term "stimulatory molecule" as used herein refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell.

As used herein, a "stimulatory ligand" refers to a ligand that, when present on an antigen presenting cell (e.g., APC, dendritic cell, B cell, etc.), can specifically bind to an associated binding partner (referred to herein as a "stimulatory molecule") on the T cell, thereby mediating a primary response of the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, inter alia, peptide-loaded mhc class i molecules, anti-CD 3 antibodies, superagonist anti-CD 28 antibodies, and superagonist anti-CD 2 antibodies.

A "vector" is a composition of matter that comprises an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphoteric compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. The term should also be construed to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, lentiviruses, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like. Examples of non-viral vectors include, but are not limited to, CRISPR vector systems, sleeping Beauty transposon systems, and the like. As used herein, "activation" refers to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cell" especially refers to a T cell undergoing cell division.

The terms "peptide," "polypeptide," and "protein" as used herein are used interchangeably and refer to a compound comprising amino acid residues covalently linked by peptide bonds. The protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute the sequence of the protein or peptide. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers) and longer chains (which are commonly referred to in the art as proteins, of which there are many types), "polypeptides" including, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably throughout and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. The nucleic acid molecule may be single-stranded or double-stranded. The term "gene" refers to a sequence of DNA or RNA that encodes a molecule that has a function.

The term "binding protein" includes native protein binding domains (e.g., receptors, ligands, cytokines, cytokine receptors), antibodies or fragments thereof (e.g., fab, scFv, diabodies, variable region-derived binders, VHH nanobodies), alternative scaffold-derived protein binding domains (e.g., fn3 variants, ankyrin repeat variants, centyrin variants, avimer, affibodies), or any protein that recognizes a particular antigen.

"Signal peptide". The co-stimulatory molecules or CARs of the present disclosure may comprise a signal peptide such that when the co-stimulatory molecule or CAR is expressed within a cell, such as a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface where it is expressed. The core of the signal peptide may comprise a long stretch of hydrophobic amino acids that have a tendency to form a single alpha-helix. The signal peptide may start with a small fraction of positively charged amino acids, which helps to enforce the proper topology of the polypeptide during transport. At the end of the signal peptide, there is usually a stretch of amino acids recognized and cleaved by the signal peptidase. The signal peptidase may cleave during or after completion of the translocation to generate free signal peptide and mature protein. The free signal peptide is then digested by a specific protease. The signal peptide may be at the amino terminus of the molecule.

The present disclosure provides compositions and methods for treating cancer and other diseases. The cancer may be a hematological malignancy, a solid tumor, a primary or metastatic tumor.

In some embodiments, the binding protein is selected from a native protein binding domain (e.g., cytokine receptor), an antibody fragment (e.g., fab, scFv, diabody, variable region-derived binder, VHH nanobody), a surrogate scaffold-derived protein binding domain (e.g., fn3 variant, ankyrin repeat variant, centyrin variant, avimer, affibody), or any protein that recognizes a particular antigen.

In some embodiments, the first binding protein of the first polypeptide comprises a first antigen-binding domain and the second binding protein of the second polypeptide comprises a second antigen-binding domain, wherein at least one of the first antigen-binding domain and the second antigen-binding domain binds an antigen on a tumor cell.

In some embodiments, the first antigen-binding domain of the first polypeptide portion and the second antigen-binding domain of the second polypeptide portion bind to different antigens or different epitopes of the same antigen on the tumor cell.

In some embodiments, the first antigen binding domain of the first polypeptide binds to antigen CD19; more preferably, the second antigen-binding domain of the second polypeptide binds the antigen CD20; preferably, the first antigen-binding domain of the first polypeptide comprises an anti-CD 19 scFv, preferably, the anti-CD 19 scFv is a humanized anti-CD 19 scFv; preferably, the first antigen-binding domain of the first polypeptide comprises an amino acid sequence that is 80% or more identical, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the light chain variable region sequence and the heavy chain variable region sequence in the humanized anti-CD 19 scFv represented by SEQ ID NO: 20; more preferably, the first antigen-binding domain of the first polypeptide comprises the light chain variable region sequence and the heavy chain variable region sequence of the humanized anti-CD 19 scFv represented by SEQ ID NO. 20; more preferably, the first binding protein comprises the humanized anti-CD 19 scFv amino acid sequence shown in SEQ ID NO 20; preferably, the second antigen-binding domain of the second polypeptide comprises an anti-CD 20 scFv, preferably, the anti-CD 20 scFv is a humanized anti-CD 20 scFv; preferably, the second antigen-binding domain of the second polypeptide comprises an amino acid sequence that is 80% or more identical to the light chain variable region sequence and the heavy chain variable region sequence of the humanized anti-CD 20 scFv represented by SEQ ID NO 21 or SEQ ID NO 22, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical; more preferably, the second antigen-binding domain of the second polypeptide comprises the light chain variable region sequence and the heavy chain variable region sequence of the humanized anti-CD 20 scFv shown in SEQ ID NO:21 or SEQ ID NO: 22; more preferably, the second binding protein comprises the humanized anti-CD 20 scFv amino acid sequence shown in SEQ ID NO 21 or SEQ ID NO 22.

In some embodiments, the first antigen-binding domain of the first polypeptide binds antigen CD20, more preferably, the second antigen-binding domain of the second polypeptide binds antigen CD19; preferably, the first antigen-binding domain of the first polypeptide comprises an anti-CD 20 scFv, preferably the anti-CD 20 scFv is a humanized anti-CD 20 scFv; preferably, the first antigen binding domain of the first polypeptide comprises an amino acid sequence that is 80% or more identical to the light chain variable region sequence and the heavy chain variable region sequence of the humanized anti-CD 20 scFv shown in SEQ ID NO:21 or SEQ ID NO:22, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical; more preferably, the first antigen-binding domain of the first polypeptide comprises the light chain variable region sequence and the heavy chain variable region sequence of the humanized anti-CD 20 scFv shown in SEQ ID NO:21 or SEQ ID NO: 22; more preferably, the first binding protein comprises the humanized anti-CD 20 scFv amino acid sequence shown in SEQ ID NO 21 or SEQ ID NO 22; preferably, the second antigen-binding domain of the second polypeptide comprises an anti-CD 19 scFv, preferably, the anti-CD 19 scFv is a humanized anti-CD 19 scFv; preferably, the second antigen-binding domain of the second polypeptide comprises an amino acid sequence that is 80% or more identical, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the light chain variable region sequence and the heavy chain variable region sequence of the humanized anti-CD 19 scFv of SEQ ID No. 20; more preferably, the second antigen-binding domain of the second polypeptide comprises the light chain variable region sequence and the heavy chain variable region sequence of the humanized anti-CD 19 scFv represented by SEQ ID NO: 20; more preferably, the second binding protein comprises the humanized anti-CD 19 scFv amino acid sequence shown in SEQ ID NO 20.

In some embodiments, a linker is included between the light chain variable region sequence and the heavy chain variable region sequence in the anti-CD 19 scFv and/or between the light chain variable region sequence and the heavy chain variable region sequence in the anti-CD 20 scFv, preferably the linker is a flexible linker, more preferably the amino acid sequence of the flexible linker is set forth in SEQ ID NO: 23.

In some embodiments, the chimeric antigen receptor further comprises a peptide cleavage site linking the first polypeptide portion and the second polypeptide portion.

In some embodiments, the peptide cleavage site is a self-cleavage site.

In some embodiments, the self-cleavage site is selected from the group consisting of a T2A, P2A, E2A, or F2A site. Preferably, the amino acid sequence of the T2A is shown as SEQ ID NO. 15. Preferably, the amino acid sequence of the P2A is shown as SEQ ID NO 16. Preferably, the amino acid sequence of the E2A is shown as SEQ ID NO 17. Preferably, the amino acid sequence of the F2A is shown as SEQ ID NO. 18;

in some embodiments, a linker is added to the N-terminus of the self-cleavage site, and the amino acid sequence of the linker is shown in SEQ ID NO. 19. The purpose of adding the joint is to increase the cutting efficiency.

In some embodiments, the first binding polypeptide further comprises a first signal peptide. Preferably, the amino acid sequence of the first signal peptide is shown as SEQ ID NO. 1.

In some embodiments, the second binding polypeptide further comprises a second signal peptide. Preferably, the amino acid sequence of the second signal peptide is shown in SEQ ID NO 2.

In some embodiments, the first and second spacers comprise amino acid sequences having 80% or more identity to the amino acid sequence set forth in SEQ ID No. 3, preferably amino acid sequences having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity; more preferably, the amino acid sequences of the first and second spacers are as shown in SEQ ID NO 3.

In some embodiments, the amino acid sequences of the first and second transmembrane domains comprise amino acid sequences having 80% or more identity to the amino acid sequence set forth in SEQ ID No. 4, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity; more preferably, the amino acid sequences of the first and second transmembrane domains are as shown in SEQ ID NO 4.

In some embodiments, the amino acid sequence of the co-stimulatory signaling domain of 4-1BB comprises an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID No. 7, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical; more preferably, the amino acid sequence of the co-stimulatory signaling domain of 4-1BB is as shown in SEQ ID NO 7.

In some embodiments, the amino acid sequence of the TCR CD3 zeta signaling domain comprises an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID No. 13 or SEQ ID No. 14, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical; more preferably, the amino acid sequence of the TCR CD3 zeta signaling domain is as set forth in SEQ ID NO 13 or SEQ ID NO 14; more preferably, the amino acid sequence of the TCR CD3 zeta signaling domain is set forth in SEQ ID NO 13.

In some embodiments, the chimeric antigen receptor comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID No. 42, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity; more preferably, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 42.

In another aspect, the present disclosure provides a polynucleotide encoding the aforementioned chimeric antigen receptor.

In one embodiment, a polynucleotide of the present disclosure comprises a first gene encoding a first polypeptide and a second gene encoding a second polypeptide, wherein: the first polypeptide comprises a first antigen binding domain, a spacer, a transmembrane domain, a 4-1BB costimulatory signaling domain, and a CD 3-zeta signaling domain, and the second polypeptide comprises a second antigen binding domain, a spacer, a transmembrane domain, a costimulatory signaling domain of 4-1BB, and a CD 3-epsilon signaling domain; wherein the first and second antigen-binding domains bind to different antigens on the cancer cell.

In some embodiments, the polynucleotide further comprises a third nucleic acid sequence encoding a peptide cleavage site to join the first gene and the second gene.

In some embodiments, the first and second genes further comprise a nucleic acid sequence encoding a signal peptide.

In some embodiments, the polynucleotide is linked to an inducible suicide gene.

In some embodiments, the inducible suicide gene is linked to the polynucleotide described above through a nucleic acid sequence encoding a 2A peptide.

In some embodiments, the first polypeptide comprises an antigen binding domain that targets the target CD 19. In addition, the second polypeptide comprises an antigen binding domain that targets the target CD20.

In some embodiments, the first polypeptide comprises an antigen binding domain that targets the target CD20. In addition, the second polypeptide comprises an antigen binding domain that targets the target CD 19.

In a preferred embodiment, the CD 19-targeted CAR and the CD 20-targeted CAR are linked by a self-cleaving 2A peptide (T2A) (as in figure 3), and can be automatically cleaved into two CAR molecules upon intracellular synthesis. Wherein the CD 19-targeting CAR comprises a humanized CD19 single chain antibody (ScFv) humanized from the murine CD19 monoclonal antibody FMC63, a Spacer (Spacer), a transmembrane region (TM), a 4-1BB co-stimulatory domain, and a T cell activation domain from CD3 ζ. CD 20-targeting CARs comprise a humanized CD20 single chain antibody (ScFv) from a humanized ofatumumab (ofatumumab) of CD20, a spacer, a transmembrane region sequence, a 4-1BB costimulatory domain, and a T cell activation domain from CD3 ζ.

In some embodiments, the polynucleotide encoding the aforementioned chimeric antigen receptor comprises a nucleic acid sequence having 80% or more identity to the nucleic acid sequence set forth in SEQ ID No. 43, preferably a nucleic acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity; preferably, the polynucleotide is as set forth in SEQ ID NO 43.

In some embodiments, the engineered cells are T cells (CD 4 and CD8T cells) or NK cells (NKT and NK92 cells).

In some embodiments, the antigen binding domain is a scFv or VHH nanobody.

In some embodiments, the engineered cell comprises an inactivated gene for PD-1, TIM3, or LAG3 by a gene knockout method.

In some embodiments, the engineered cell is an engineered T cell or an engineered NK cell.

In some embodiments, the engineered T cell is a CD 4T cell or a CD8T cell.

In some embodiments, the engineered NK cell is an NKT cell or an NK-92 cell.

In another aspect, the present disclosure provides a vector comprising the foregoing polynucleotide.

In some embodiments, a T cell can be transduced with a lentiviral vector to express a multiple signaling Chimeric Antigen Receptor (CAR) system with or without a membrane-bound fusion protein, wherein the lentiviral vector comprises an isolated polynucleotide encoding a plurality of polypeptides selected from a first polypeptide and a second polypeptide, wherein:

the first polypeptide portion comprises: (ii) a first binding protein, (ii) a first spacer, (iii) a first transmembrane domain, (iv) a costimulatory signaling domain of 4-1BB, and (v) a TCR CD3 zeta signaling domain; and is

The second polypeptide portion comprises: (ii) a second binding protein, (ii) a second spacer, (iii) a second transmembrane domain, (iv) a costimulatory signaling domain of 4-1BB, and (v) a TCR CD3 zeta signaling domain.

In some embodiments, the spacer is optional.

In some embodiments, the vector is a viral vector selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, and a retroviral vector. In some other embodiments, the vector is a non-viral vector selected from the group consisting of a CRISPR vector system and a Sleeping Beauty transposon system.

In some embodiments, the genes encoding the multiple polypeptide subunits may be linked into a single vector construct using a 2A peptide gene comprising T2A, P2A, E2A, or F2A.

In another embodiment of the present disclosure, an engineered immune cell comprises an isolated polynucleotide molecule encoding an engineered membrane-bound single-chain variable fragment (scFv) against a B lymphocyte antigen, wherein the engineered membrane-bound B cell antigen comprises (i) a signal peptide, (ii) a single-chain variable fragment (scFv), (iii) a spacer, (iv) a transmembrane domain, (v) a 41BB co-stimulatory domain, and (vi) a CD 3-zeta signaling domain.

In some embodiments, the single-chain variable fragment (scFv) antigen is directed against CD19, wherein the engineered T cells produce a membrane-bound fusion protein capable of recognizing the CD19 protein expressed on the surface of B cells, but not aberrant T cell proliferation. T cells expressing scFv against CD19 with CAR retain the memory potential of the TSCM-like phenotype.

In some embodiments, the single-chain variable fragment (scFv) antigen is directed against CD20, wherein the engineered T cells produce a membrane-bound fusion protein capable of recognizing the CD20 protein expressed on the surface of B cells, but not aberrant T cell proliferation. T cells expressing scFv against CD20 with CARs retained the memory potential of the TSCM-like phenotype.

In some embodiments, the single-chain variable fragment (scFv) antigen is directed against both CD19 and CD20, wherein the engineered T cells produce a membrane-bound fusion protein capable of recognizing CD19 and CD20 proteins expressed on the surface of B cells, but not aberrant T cell proliferation. T cells expressing scFv against CD19 and CD20 with CARs retained the memory potential of TSCM-like phenotypes.

In another embodiment, an engineered immune cell comprises a polynucleotide comprising a first gene encoding a first polypeptide and a second gene encoding a second polypeptide, wherein the first polypeptide comprises (i) a signal peptide, (ii) a first binding protein, (iii) a spacer, (iv) a transmembrane domain, (v) a 41BB co-stimulatory domain, and (vi) a CD3 zeta signaling domain; and the second polypeptide comprises (i) a signal peptide, (ii) an extracellular domain of an immunomodulatory cytokine or cytokine receptor, (iii) a spacer, (iv) a transmembrane domain, (v) a costimulatory signaling domain of 4-1BB, and (vi) a CD3 zeta signaling domain; wherein the binding protein binds to an antigen on the cancer cell; wherein the first gene and the second gene are linked by a gene encoding a 2A peptide.

In preferred embodiments, the present disclosure provides a single vector expressing two Chimeric Antigen Receptors (CARs) each comprising an extracellular and an intracellular domain. The extracellular domain of the CAR comprises a target-specific binding element or otherwise referred to as an antigen-binding portion. The intracellular domain, or alternatively the cytoplasmic domain, comprises a costimulatory signaling region and a zeta or epsilon chain moiety. A spacer (spacer) may be introduced between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. The term "spacer domain" as used herein generally refers to any oligo-or polypeptide that functions to connect a transmembrane domain to an extracellular domain or a cytoplasmic domain in a polypeptide chain. The spacer may comprise up to 300 amino acids, preferably 10 to 100 amino acids, most preferably 25 to 50 amino acids. In some alternatives, the spacer is derived from a CD8a or CD28 extracellular domain. In some alternatives, the spacer comprises an Ig hinge region. In some preferred forms, the Ig hinge region is an IgG4 hinge region.

The transmembrane domain may be derived from natural or synthetic sources. Where the source is native, the domain may be derived from any membrane-binding or transmembrane protein (i.e. comprising at least the transmembrane region thereof), for example the α, β, ε or ζ chain of a T cell receptor, CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will contain predominantly hydrophobic residues, such as leucine and valine. Optionally, a short oligo or polypeptide linker, preferably between 2 and 10 amino acids in length, may form a link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. Glycine-serine diads provide particularly suitable linkers.

The cytoplasmic domain or otherwise intracellular signaling domain of the CAR of the present disclosure is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR has been placed. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including secretion of cytokines. The term "intracellular signaling domain" thus refers to a portion of a protein that transduces effector function signals and directs a cell to perform a specialized function. Although the entire intracellular signaling domain may generally be used, in many cases, the entire strand need not be used. To the extent that truncated portions of intracellular signaling domains are used, such truncated portions may be used in place of the entire chain, so long as they transduce effector function signals. The term intracellular signaling domain is therefore intended to include any truncated portion of an intracellular signaling domain sufficient to transduce an effector function signal.

The primary cytoplasmic signaling sequence modulates primary activation of the TCR complex in either a stimulatory or inhibitory manner. The primary cytoplasmic signaling sequence that functions in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.

Examples of ITAMs comprising primary cytoplasmic signaling sequences particularly useful in the present disclosure include those derived from CD3 ζ, fcR γ, fcR β, CD3 γ, CD3 δ, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66 d. Particularly preferably, the cytoplasmic signaling molecule in the CAR of the present disclosure comprises a cytoplasmic signaling sequence derived from CD3 ζ or CD3 epsilon.

In preferred embodiments, the cytoplasmic domain of the CAR can be designed to comprise a CD3 zeta chain (CD 3-zeta) or CD3 epsilon chain (CD 3-epsilon) signaling domain that binds to any other desired cytoplasmic domain useful in the context of the CARs of the present disclosure. For example, the cytoplasmic domain of the CAR or any other costimulatory molecule can include a CD3 zeta chain portion and a costimulatory signaling region. A costimulatory signaling region refers to a portion of a CAR that comprises the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules required for an effective response of lymphocytes to antigens in addition to the primary antigen receptor or its ligand. Examples of such molecules include CD27, 4-1BB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, among others. Preferably, the co-stimulatory signaling element or co-stimulatory molecule in the CARs of the present disclosure is 4-1BB and/or ICOS. And it is particularly preferred that the cytoplasmic domain comprises a combination of ICOS/CD 3-zeta or 4-1BB/CD 3-epsilon or 4-1BB/CD 3-zeta.

In another embodiment, the multi-costimulatory signal CAR T cell further comprises an inducible suicide gene.

Strategies for multiple gene co-expression with a single vector include multiple promoters, fusion proteins, proteolytic cleavage sites between genes, internal ribosome entry sites, and the use of "self-cleaving" 2A peptides. The 2A peptide is a viral oligopeptide 18-22 amino acids (aa) long, which mediates "cleavage" of the polypeptide during translation in eukaryotic cells. The designation "2A" refers to a specific region of the viral genome, and different viruses 2A are often named according to the virus from which they are derived. The first 2A was found to be F2A (foot and mouth disease virus), after which E2A (equine rhinitis virus), P2A (porcine teschovirus-12A) and T2A (thosa asigna virus 2A) were also identified. It has recently been found that the mechanism of 2A mediated "self-cleavage" is that the ribosome skips the formation of a glycosyl-prolyl peptide bond at the C-terminus of 2A.

DNA encoding the novel CAR system was synthesized and cloned into a lentiviral vector. These vector plasmids will be produced under quality control into mature lentiviral particles in 293T cells, and T cells or other immune cells will be transduced with lentiviruses comprising the novel CAR structures of the present disclosure, e.g., NK or NKT cells isolated from PBMCs of patients. The transduced immune cells will grow and expand in the bioreactor for about 10 days to reach therapeutic numbers. After quality control is released, these CAR-expressing immune cells will be infused back into the patient for medical use.

The disclosure also provides cells, cell populations, and compositions (including pharmaceutical and therapeutic compositions) comprising the cells and populations, e.g., cells and populations produced by the provided methods, and methods, e.g., therapeutic methods for administering the cells and compositions to a subject, e.g., a patient.

Also provided are compositions, including pharmaceutical compositions and formulations, comprising cells for administration, e.g., unit dosage form compositions comprising a number of cells for administration in a given dose or fraction thereof. Pharmaceutical compositions and formulations typically comprise one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition comprises at least one additional therapeutic agent.

The term "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional components that would have unacceptable toxicity to the subject to which the formulation is to be administered.

"pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than the active ingredient, which is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

In some aspects, the choice of vector is determined in part by the particular cell and/or method of administration. Thus, there are a number of suitable formulations. For example, the pharmaceutical composition may comprise a preservative. Suitable preservatives may include, for example, methyl paraben, propyl paraben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixture thereof is typically present in an amount of about 0.0001% to about 2% by weight of the total weight of the composition. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, for example methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG).

In some aspects, a buffer is included in the composition. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffers is used. The buffering agent or mixture thereof is typically present in an amount of about 0.001 weight% to about 4 weight% of the total weight of the composition. Methods of preparing administrable pharmaceutical compositions are known.

The formulation may comprise an aqueous solution. The formulation or composition may also comprise more than one active ingredient useful for the particular indication, disease or condition being treated with the cells, preferably those having activities complementary to the cells, wherein the respective activities do not adversely affect each other. Such active ingredients are suitably present in combination in an amount effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.

In some embodiments, the pharmaceutical composition comprises an amount of cells effective to treat or prevent a disease or disorder, e.g., a therapeutically effective amount or a prophylactically effective amount. In some embodiments, treatment or prevention efficacy is monitored by periodic assessment of the treated subject. The desired dose can be delivered by administering the cells as a single bolus, multiple boluses, or continuous infusion.

The cells and compositions can be administered using standard administration techniques, formulations, and/or devices. Administration of the cells may be autologous or heterologous. For example, immunoresponsive cells or progenitor cells can be obtained from one subject and administered to the same subject or to a different, compatible subject. Peripheral blood-derived immunoresponsive cells or progeny thereof (e.g., derived in vivo, ex vivo, or in vitro) can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When a therapeutic composition (e.g., a pharmaceutical composition comprising genetically modified immunoresponsive cells) is administered, it is typically formulated in a unit dose injectable form (solution, suspension, emulsion).

Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal and intraperitoneal administration. In some embodiments, the cells are administered to the subject by intravenous, intraperitoneal, or subcutaneous injection administration using peripheral systemic delivery.

In some embodiments, the compositions are provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered in some aspects to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide longer contact times with specific tissues. The liquid or viscous composition can comprise a carrier, which can be a solvent or dispersion medium, comprising, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

Sterile injectable solutions can be prepared by introducing the cells into a solvent, for example, by mixing with a suitable carrier, diluent or excipient, such as sterile water, physiological saline, glucose, dextrose, and the like. The compositions may contain auxiliary substances such as wetting, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-increasing additives, preservatives, flavoring agents, and/or colors, depending on the route of administration and the desired preparation. In certain aspects, reference may be made to standard text to prepare suitable preparations.

Various additives may be added that enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Formulations for in vivo administration are generally sterile. Sterility can be readily achieved by, for example, filtration through sterile filtration membranes.

Also provided are methods of administering the cells, populations and compositions to treat or prevent diseases, conditions and disorders, including cancer, and uses of such cells, populations and compositions to treat or prevent diseases, conditions and disorders, including cancer. In some embodiments, the cells, populations, and compositions are administered to a subject or patient to be treated having a particular disease or disorder, for example, by adoptive cell therapy (e.g., adoptive T cell therapy). In some embodiments, cells and compositions prepared by the provided methods (e.g., engineered compositions and production end products after incubation and/or other processing steps) are administered to a subject, e.g., a subject having or at risk of a disease or disorder. In some aspects, the methods thereby treat, for example, one or more symptoms of a disease or disorder, e.g., by reducing tumor burden in a cancer expressing an antigen recognized by an engineered T cell.

Methods of cell administration for adoptive cell therapy are known and can be used in conjunction with the methods and compositions provided. For example, methods of adoptive T cell therapy are described in, e.g., U.S. patent application publication Nos. 2003/0170238 to Gruenberg et al; U.S. Pat. nos. 4,690,915 to Rosenberg; rosenberg (2011) Nat Rev Clin Oncol.8 (10): 577-85). See, e.g., themeli et al (2013) Nat Biotechnol.31 (10): 928-933; tsukahara et al (2013) Biochem Biophys Res Commun 438 (1): 84-9; davila et al (2013) PLoS ONE 8 (4): e61338.

As used herein, a "subject" is a mammal, e.g., a human or other animal, and typically a human. In some embodiments, the subject, e.g., patient, to whom the cells, cell populations, or compositions are administered is a mammal, typically a primate, e.g., a human. In some embodiments, the primate is a monkey or ape. The subject may be male or female and may be of any suitable age, including infant, juvenile, adolescent, adult and geriatric subjects. In some embodiments, the subject is a non-primate mammal, e.g., a rodent.

As used herein, "treating" (and grammatical variations thereof, such as "treating" or "treatment") refers to ameliorating or alleviating, in whole or in part, a disease or condition or disorder, or a symptom, adverse effect or outcome or phenotype associated therewith. Desirable therapeutic effects include, but are not limited to, prevention of occurrence or recurrence of disease, alleviation of symptoms, alleviation of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, alleviation or palliation of the disease state, and remission or improved prognosis. These terms do not imply a complete cure for the disease or complete elimination of any symptoms or impact on all symptoms or consequences.

As used herein, "delaying the progression of a disease" refers to delaying, hindering, slowing, arresting, stabilizing, inhibiting and/or delaying the progression of a disease (e.g., cancer). The delay may be of varying lengths of time depending on the history of the disease and/or the individual being treated. It will be apparent to those skilled in the art that a sufficient or significant delay may actually include prevention, since the individual does not develop the disease. For example, the development of advanced cancers, such as metastases, may be delayed.

As used herein, "preventing" includes providing prevention against the occurrence or recurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the cells and compositions provided are used to delay the progression of a disease or slow the progression of a disease.

As used herein, "inhibiting" a function or activity refers to decreasing the function or activity when compared to the same condition (except for the condition or parameter of interest), or when compared to another condition. For example, a cell that inhibits tumor growth decreases the growth rate of the tumor as compared to the growth rate of the tumor in the absence of the cell.

In the context of administration, an "effective amount" of an agent (e.g., a chimeric antigen receptor, polynucleotide, vector, pharmaceutical preparation, cell, or composition) refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result, e.g., a therapeutic or prophylactic result.

A "therapeutically effective amount" of an agent (e.g., a pharmaceutical agent or cell) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic effect (e.g., for the treatment of a disease, condition, or disorder, and/or the pharmacokinetic or pharmacodynamic effect of the treatment). The therapeutically effective amount may vary depending on factors such as the disease state, the age, sex, and weight of the subject, and the cell population administered. In some embodiments, provided methods comprise administering the cells and/or compositions in an effective amount, e.g., a therapeutically effective amount.

A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, because a prophylactic dose is used in a subject prior to or early in the disease, the prophylactically effective amount will be less than the therapeutically effective amount. In the case of a lower tumor burden, the prophylactically effective amount will be higher than the therapeutically effective amount in certain aspects.

In some embodiments, cell therapy, such as adoptive T cell therapy, is performed by autologous transfer, wherein cells are isolated and/or otherwise prepared from a subject to receive the cell therapy or a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., a patient, in need of treatment, and the cells are administered to the same subject after isolation and processing.

In some embodiments, cell therapy, such as adoptive T cell therapy, is performed by allogeneic transfer, wherein cells are isolated and/or otherwise prepared from a subject other than the subject to receive or ultimately receive the cell therapy, e.g., the first subject. In such embodiments, the cells are then administered to a different subject of the same species, e.g., a second subject. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype (supertype) as the first subject.

In some embodiments, the subject has been treated with a therapeutic agent that targets a disease or disorder, such as a tumor, prior to administration of the cells or cell-containing composition. In some aspects, the subject is refractory or non-responsive to other therapeutic agents. In some embodiments, the subject has a persistent or recurrent disease, e.g., after treatment with another therapeutic intervention, including chemotherapy, radiation, and/or Hematopoietic Stem Cell Transplantation (HSCT), e.g., allogeneic HSCT. In some embodiments, the administration is effective to treat the subject despite the subject having become resistant to the additional therapy.

In some embodiments, the subject is responsive to an additional therapeutic agent, and treatment with the therapeutic agent reduces the burden of the disease. In some aspects, the subject initially responds to the therapeutic agent, but over time exhibits a recurrence of the disease or disorder. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk of relapse, e.g., at high risk of relapse, and the cells are therefore administered prophylactically, e.g., to reduce the likelihood of relapse or prevent relapse.

In some aspects, the subject has not received pre-treatment with an additional therapeutic agent. Among the diseases, conditions, and disorders that are treated with the provided compositions, cells, methods, and uses are tumors, including solid tumors, hematologic malignancies, and melanoma, and infectious diseases, such as viral or other pathogen infections, e.g., HIV, HCV, HBV, CMV, and parasitic diseases. In some embodiments, the disease or disorder is a tumor, cancer, malignancy, neoplasm, or other proliferative disease or disorder. Such diseases include, but are not limited to, leukemia, lymphoma, e.g., chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), non-hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B-cell lymphoma, B-cell malignancy, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone and brain cancer, ovarian cancer, epithelial cancer, renal cell cancer, pancreatic adenocarcinoma, hodgkin's lymphoma, systemic anaplastic large cell lymphoma or primary cutaneous anaplastic large cell lymphoma (pcALCL), cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma and/or mesothelioma.

In some embodiments, the cells are administered at a desired dose, which in certain aspects comprises a desired dose or number of cells or cell types and/or a desired ratio of cell types. Thus, in some embodiments, the dosage of cells is based on the total number of cells (or number per kilogram of body weight) and a desired ratio of individual populations or subtypes, e.g., a ratio of CD4+ to CD8 +. In some embodiments, the dose of cells is based on the desired total number of cells or individual cell types (or number per kilogram body weight) in an individual population. In some embodiments, the dose is based on a combination of such characteristics, e.g., a desired number of total cells, a desired ratio, and a desired total number of cells in an individual population.

In some embodiments, a population or subset of cells, e.g., CD8+ and CD4+ T cells, is administered at or within an allowable difference of the desired dose of total cells, e.g., the desired dose of T cells. In some aspects, the desired dose is a desired number of cells or cells per unit weight of the subject to which the cells are administered, e.g., cells/kg. In some aspects, the desired dose is at or above the minimum cell number or the minimum cell number per unit body weight. In some aspects, in total cells, administered at a desired dose, a population or subtype of individuals is present at or near a desired output ratio (e.g., a ratio of CD4+ to CD8 +), e.g., within some allowable difference or error of such ratio.

In some embodiments, the cells are administered at or within a permissive for difference in a desired dose of one or more individual populations or subtypes of cells, e.g., a desired dose of CD4+ cells and/or a desired dose of CD8+ cells. In some aspects, the desired dose is the number of cells of a desired subtype or population, or the desired number of such cells per unit body weight of the subject to which the cells are administered, e.g., cells/kg. In some aspects, the desired dose is at or above the minimum number of cells of the population or subtype, or the minimum number of cells of the population or subtype per unit weight.

Thus, in some embodiments, the dose is based on a desired fixed dose and a desired ratio of total cells, and/or is based on a desired fixed dose of one or more, e.g., individual subtypes or subpopulations. Thus, in some embodiments, the dose is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4+ to CD8+ cells, and/or on a desired fixed or minimum dose of CD4+ and/or CD8+ cells.

In certain embodiments, the cells or individual populations or subsets of cells are administered to the subject in a range of about one million to about one billion cells, such as 100 to about 500 million cells (e.g., about 500 million cells, about 2500 million cells, about 5 million cells, about 10 million cells, about 50 million cells, about 200 million cells, about 300 million cells, about 400 million cells, or a range defined by any of the above values), such as about 1000 to about 1000 million cells (e.g., about 2000 million cells, about 3000 million cells, about 4000 million cells, about 6000 million cells, about 7000 million cells, about 8000 million cells, about 9000 million cells, about 100 million cells, about 250 million cells, about 500 million cells, about 750 million cells, about 900 million cells, or a range defined by any of the above values), and in some cases, about 1 to about 500 million cells (e.g., about 1.2 hundred million cells, about 5 hundred million cells, about 5.5 million cells, about 5.8 million cells, or a range defined by any of the above values).

In some embodiments, the dose of total cells and/or the dose of an individual subpopulation of cells is at or about 10 ⁴ Individual cells per kilogram (kg) body weight to at or about 10 ⁹ In the range between individual cells per kilogram (kg) of body weight, e.g. 10 ⁵ And 10 ⁶ Between individual cells/kg body weight, e.g., at least or at least about or at or about 1X 10 ⁵ 1.5X 10 cells/kg ⁵ Individual cells/kg, 2X 10 ⁵ Individual cell/kg or 1X 10 ⁶ Individual cells/kg body weight. For example, in some embodiments, the cell is at or about 10 ⁴ And at or about 10 ⁹ Between T cells per kilogram (kg) of body weight or within some margin of error thereof, e.g. 10 ⁵ And 10 ⁶ Between T cells/kg body weight, e.g., at least or at least about or at or about 1X 10 ⁵ 1.5X 10T cells/kg ⁵ Individual T cells/kg, 2X 10 ⁵ T cells/kg, or 1X 10 ⁶ Individual T cells/kg body weight.

In some embodiments, the cell is at or about 10 ⁴ And at or about 10 ⁹ Between individual CD4+ and/or CD8+ cells/kilogram (kg) body weight or within a certain margin of error thereof, e.g. 10 ⁵ And 10 ⁶ Between CD4+ and/or CD8+ cells/kg body weight, e.g., at least or at least about or at or about 1X 10 ⁵ 1.5X 10 CD4+ and/or CD8+ cells/kg ⁵ 2X 10 CD4+ and/or CD8+ cells/kg ⁵ CD4+ and/or CD8+ cells/kg, or 1X 10 ⁶ Individual CD4+ and/or CD8+ cells/kg body weight.

In some embodiments, the cells are in the range of greater than and/or at least about 1 × 10 ⁶ About 2.5X 10 ⁶ About, an5×10 ⁶ About 7.5X 10 ⁶ Or about 9X 10 ⁶ CD4+ cells, and/or at least about 1X 10 ⁶ About 2.5X 10 ⁶ About 5X 10 ⁶ About 7.5X 10 ⁶ Or about 9X 10 ⁶ CD8+ cells, and/or at least about 1X 10 ⁶ About 2.5X 10 ⁶ About 5X 10 ⁶ About 7.5X 10 ⁶ Or about 9X 10 ⁶ Individual T cells or a range of errors thereof. In some embodiments, the cell is at about 10 ⁸ To 10 ¹² Between or about 10 ¹⁰ To 10 ¹¹ Between T cells, about 10 ⁸ To 10 ¹² Between or about 10 ¹⁰ To 10 ¹¹ Between CD4+ cells, and/or about 10 ⁸ To 10 ¹² Between or about 10 ¹⁰ To 10 ¹¹ Between individual CD8+ cells, or within certain tolerances thereof.

In some embodiments, the cells are administered at a desired output ratio or within a tolerance range thereof for a plurality of cell populations or subtypes, e.g., CD4+ and CD8+ cells or subtypes. In some aspects, the desired ratio may be a particular ratio or may be a range of ratios. For example, in some embodiments, a desired ratio (e.g., CD4+ to CD8+ cell) is between or about 5. In some aspects, the difference is allowed to be within about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value between these ranges.

For the prevention or treatment of a disease, the appropriate dosage may depend on the type of disease to be treated, the type of cell or recombinant receptor, the severity and course of the disease, whether the cells are administered for prophylactic or therapeutic purposes, previous therapy, the clinical history and response to the cells of the subject, and the judgment of the attending physician. In some embodiments, the composition and cells are suitably administered to the subject at once or through a series of treatments.

The cells can be administered by any suitable means, for example by bolus injection, by injection, for example, intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subperiodic injection, subconjunctival injection, sub-Tenon injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered parenterally, intrapulmonary, and intranasally, and, if topical treatment is desired, intralesionally. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration of cells. In some embodiments, it is administered by administering the cells by multiple bolus injections, e.g., over a period of no more than 3 days, or by continuous infusion.

In some embodiments, the cells are administered as part of a combination therapy, e.g., simultaneously or sequentially in any order with additional therapeutic intervention, e.g., an antibody or engineered cell or receptor or agent, e.g., a cytotoxic or therapeutic agent. In some embodiments, the cells are co-administered simultaneously or sequentially in any order, in association with one or more additional therapeutic agents or with additional therapeutic interventions. In certain instances, the cells are co-administered in sufficient temporal proximity with the additional therapy such that the population of cells enhances the effect of the one or more additional therapeutic agents, and vice versa. In some embodiments, the cell is administered prior to the one or more additional therapeutic agents. In some embodiments, the cell is administered after the one or more additional therapeutic agents. In some embodiments, one or more additional agents include a cytokine, such as IL-2, to enhance persistence. In some embodiments, the method comprises administration of a chemotherapeutic agent.

After administration of the cells, in some embodiments, the biological activity of the engineered cell population is measured, for example, by any of a number of known methods. Parameters evaluated include the specific binding of engineered or native T cells or other immune cells to an antigen, evaluated in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cell to destroy a target cell can be measured using any suitable method known in the art, for example, as described in, e.g., kochenderfer et al, j.immunotherapy,32 (7): 689-702 (2009), and Herman et al, j.immunological Methods,285 (1): 25-40 (2004). In certain embodiments, the biological activity of a cell is measured by determining the expression and/or secretion of one or more cytokines, such as CD107a, IFN γ, IL-2, and TNF. In some aspects, biological activity is measured by assessing clinical outcome, such as tumor burden or reduction in burden.

In certain embodiments, the engineered cell is further modified in a variety of ways to increase its therapeutic or prophylactic efficacy. For example, a population-expressed engineered CAR or TCR may be coupled directly or indirectly to a targeting moiety through a linker. The practice of coupling compounds such as CARs or TCRs to targeting moieties is known in the art. See, e.g., wadwa et al, j.drug Targeting 3 (1995) and U.S. Pat. No.5,087,616.

In some embodiments, a repeat dose method is provided, wherein a first dose of cells is administered followed by one or more second consecutive doses. When administered to a subject in an adoptive therapy approach, the timing and size of multiple doses of the cells are typically designed to increase the efficacy and/or activity and/or function of antigen-expressing T cells (e.g., CAR-expressing T cells). In some embodiments, repeated administration reduces the downregulation or inhibitory activity that can occur when an inhibitory immune molecule, e.g., PD-1 and/or PD-L1, is upregulated on antigen-expressing, e.g., CAR-expressing, T cells. The methods comprise administering a first dose, typically followed by one or more consecutive doses, and having a specific time frame between different doses.

In the context of adoptive cell therapy, administration of a given "dose" includes administration of a given amount or number of cells as a single composition and/or a single uninterrupted administration (e.g., as a single injection or continuous infusion), and also includes administration of a given amount or number of cells provided in multiple individual compositions or infusions as a fractionated dose over a specified period of time (no more than 3 days). Thus, in certain instances, a first or continuous dose is a single or continuous administration of a specified number of cells given or initiated at a single time point. However, in certain instances, the first or continuous dose is administered as multiple injections or infusions over a period of no more than three days, e.g., three or two days, once per day, or multiple infusions over a period of one day.

Thus, in some aspects, the first dose of cells is administered as a single pharmaceutical composition. In some embodiments, successive doses of cells are administered in a single pharmaceutical composition.

In some embodiments, the first dose of cells is administered in a plurality of compositions that collectively comprise the first dose of cells. In some embodiments, successive doses of cells are administered in multiple compositions that collectively comprise the successive doses of cells. In some aspects, additional consecutive doses may be administered in multiple compositions over a period of no more than 3 days.

The term "fractionated dose" refers to a dose that is divided such that it is administered over more than one day. This type of administration is included in the present method and is considered a single dose.

Thus, in some aspects, the first dose and/or the continuous dose may be administered as a divided dose. For example, in some embodiments, a dose may be administered to a subject within 2 days or 3 days. An exemplary method of divided dosing includes administering 25% of the dose on the first day and administering the remaining 75% of the dose on the second day. In other embodiments, 33% of the first dose may be administered on the first day and the remaining 67% may be administered on the second day. In some aspects, 10% of the dose is administered on the first day, 30% of the dose is administered on the second day, and 60% of the dose is administered on the third day. In some embodiments, the split dose is not distributed over more than 3 days.

With respect to a prior dose, e.g., a first dose, the term "consecutive dose" refers to a dose administered to the same subject after a prior (e.g., first) dose without administering any intermediate dose to the subject in between. However, the term does not include the second, third, etc. injection or infusion in a series of infusions or injections contained within a single fractionated dose. Thus, unless otherwise specified, a second infusion over a period of one, two or three days is not considered a "continuous" dose as used herein. Likewise, the second, third, etc. of a series of multiple doses within a divided dose are also not considered "intermediate" doses in the sense of "continuous" doses. Thus, unless otherwise indicated, a dose administered over a period of more than three days after the start of a first or previous dose is considered a "continuous" dose, even if the subject received a second or subsequent injection or infusion of cells after the start of the first dose, so long as the second or subsequent injection or infusion occurred within three days after the start of the first or previous dose.

Thus, unless otherwise indicated, multiple administrations of the same cells over a period of up to 3 days are considered a single dose, and administration of cells within 3 days of the initial administration is not considered a continuous dose, nor is it considered an intermediate dose for the purpose of determining whether the second dose is "continuous" with the first dose.

In some embodiments, multiple consecutive doses are administered in some aspects using the same timing guidelines as for timing between the first dose and the first consecutive dose, e.g., by administering the first and multiple consecutive doses, each consecutive dose being administered over a period of time, wherein the inhibitory immune molecule, e.g., CD19 and/or CD20, has been upregulated in the cells of the subject as a result of the administered first dose. Empirically determining when to provide a continuous dose is within the level of the skilled artisan, for example by assessing CD19 and/or CD20 expression levels in CAR-expressing cells from peripheral blood or other bodily fluids.

In some embodiments, the timing between the first dose and the first consecutive dose, or between the first and multiple consecutive doses, is such that each consecutive dose is administered over a period of greater than about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or more. In some embodiments, consecutive doses are administered over a period of less than about 28 days after the first or immediately preceding dose. The further plurality of further consecutive doses or doses is also referred to as subsequent doses or subsequent consecutive doses.

The size of the first and/or one or more successive doses of cells is typically designed to provide improved efficacy and/or reduced risk of toxicity. In some aspects, the dose or size of the first dose or any consecutive dose is any dose or amount as described above. In some embodiments, the number of cells in the first dose or any consecutive dose is about 0.5 x 10 ⁶ Cells/kg subject body weight and 5X 10 ⁶ Between about 0.75X 10 cells/kg subject body weight ⁶ Cells/kg subject weight and 3X 10 ⁶ Between one cell/kg subject body weight, or about 1X 10 ⁶ Individual cells/kg subject body weight and 2X 10 ⁶ Between individual cells/kg subject body weight, both values included.

As used herein, "first dose" is used to describe the timing of a given dose before administration of successive or subsequent doses. The term does not necessarily imply that the subject has never previously received a dose of cell therapy, or even that the subject has never previously received a dose of the same cells or cells expressing the same recombinant receptor or targeting the same antigen.

In some embodiments, the receptor, e.g., CAR, expressed by the cells in the consecutive dose comprises at least one immunoreactive epitope as the receptor, e.g., CAR, expressed by the cells of the first dose. In some aspects, the receptor, e.g., CAR, expressed by the cells administered by the successive doses is the same as, or substantially the same as, the receptor, e.g., CAR, expressed by the cells administered in the first dose.

Recombinant receptors, e.g., CARs, expressed by cells administered to a subject at various doses typically recognize or specifically bind to a molecule expressed in, associated with, and/or specific to the disease or disorder to be treated or cells thereof. Upon specific binding to a molecule, e.g., an antigen, the receptor typically delivers an immunostimulatory signal, such as an ITAM transduction signal, into the cell, thereby promoting an immune response that targets the disease or disorder. For example, in some embodiments, the first dose of cells expresses a CAR that specifically binds to an antigen expressed by cells or tissues of the disease or disorder or associated with the disease or disorder.

In the present disclosure, the DNA sequence encoding the above CAR structure is optimized, including using codons commonly used in human cells, removing repetitive sequences and RNA unstable sequences, etc., and then artificially synthesized in vitro. The humanized single-chain antibody ScFv sequence is adopted by the antibodies of CD19 and CD20, so that the immune rejection reaction induced in a human body by a murine prototype antibody sequence can be reduced, and the duration of the infused CAR-T cells in the body is prolonged. The synthetic CAR gene fragment was cloned into self-inactivating (self-inactivated) lentiviral vectors, resulting in CAR vector plasmids (CAR transfer plasmids). After co-transfecting HEK293T cells together with third generation lentivirus packaging plasmids, lentivirus particles in culture solution are harvested and purified to obtain the recombinant lentivirus capable of expressing CD19 and CD20 double CAR genes. Human T cells are transduced in vitro by the recombinant lentivirus, and the CD19CD20 double-targeted CAR-T cells are prepared after amplification. Furthermore, the present disclosure uses one vector to express two CARs simultaneously, which is more efficient in obtaining cells that simultaneously express two CARs than if the two vectors were expressed separately.

The experimental results of this disclosure demonstrate that the effect of chimeric antigen receptors with 4-1BB and CD3 ζ and 4-1BB and CD3 ζ in combination (CAR # 10) as an shunt structure of intracellular signaling domains is superior to the effect of chimeric antigen receptors with Icos and CD3 ζ and 4-1BB and CD3 ζ combinations (CAR # 7), icos, 4-1BB and CD3 ζ and 4-1BB and CD3 ζ combinations (CAR #8 and CAR # 9), CD28G and CD3 ζ and 4-1BB and CD3 ζ combinations (CAR # 11), OX40 and CD3 ζ and 4-1BB and CD3 ζ combinations (CAR # 12) as shunt structures of intracellular signaling domains for cells expressing dual CARs.

TABLE 1 amino acid sequence numbering of CAR components

TABLE 2 amino acid numbering corresponding to CAR and nucleic acid encoding same

CAR numbering	Amino acid numbering	Nucleic acid numbering
			CAR#1	SEQ ID NO:24	SEQ ID NO.25
CAR#2	SEQ ID NO:26	SEQ ID NO.27
			CAR#3	SEQ ID NO:28	SEQ ID NO.29
CAR#4	SEQ ID NO:30	SEQ ID NO.31
			CAR#5	SEQ ID NO:32	SEQ ID NO.33
CAR#6	SEQ ID NO:34	SEQ ID NO.35
			CAR#7	SEQ ID NO:36	SEQ ID NO.37
CAR#8	SEQ ID NO:38	SEQ ID NO.39
			CAR#9	SEQ ID NO:40	SEQ ID NO.41
CAR#10	SEQ ID NO:42	SEQ ID NO.43
			CAR#11	SEQ ID NO:44	SEQ ID NO.45
CAR#12	SEQ ID NO:46	SEQ ID NO.47
			CAR#13	SEQ ID NO:48	SEQ ID NO:49

For purposes of clarity and brevity, features may be described herein as part of the same or separate embodiments, however, it is to be understood that the scope of the present disclosure may include some embodiments having combinations of all or some of the features described.

Examples

Example 1 structural design of CAR and construction of vector plasmid

To obtain the best active dual CAR structure by comparison, this example designed the following three classes of CAR candidate structures:

the first type: CD19 targeting CAR, a total of 6 were designed, numbered #1, #2, #3, #4, #5, #6.

The second type: CD19/CD20 dual CAR targeting CD19 and CD20, 6 in total, numbered #7, #8, #9, #10, #11, #12, respectively, were designed.

In the third category: a CD 20-targeting CAR, numbered #13.

The three CAR classes are shown in table 3.

TABLE 3 constituent elements of CAR

The DNA sequence encoding the CAR with the above numbering was optimized, synthesized by Kisrey Biotech, and cloned between EcoRI and BamHI sites of a self-inactivating lentiviral vector pAC001L (from Abcyte Therapeutics, USA, see FIG. 4 for structural diagram) to obtain a corresponding lentiviral vector plasmid, and the cloned CAR gene was transcribed and expressed by the human EF-1a promoter. The synthetic CD19 CAR and CD20CAR gene fragments have about 30 bases of homologous sequence to each other and at the same time they have about 25-30bp of homologous sequence to the EcoRI and BamHI sides of pAC001L, respectively, the pAC001L vector is double cut with restriction enzymes EcoRI and BamHI, a notch is cut out from the pAC001L vector, the vector fragments are separated by agarose gel electrophoresis, the vector fragments are recovered by cutting gel, the synthetic CAR gene fragments are cloned onto a vector backbone plasmid by In-Fusion homologous recombination technique (the target fragment and linearized vector are mixed with premixed enzyme reagents and incubated at 50 ℃ for 15 minutes), the constructed lentiviral vector plasmid is transformed into e.coli DH5 α cell competent bacteria (purchased from Takara, cat # 5-1600-020), single cloned by coating a resistance plate, after enzyme digestion and sequencing identification, engineered bacteria are preserved.

Example 2 preparation of lentivirus

The lentiviral vector plasmids constructed in example 1 were purified and co-transfected with HEK293T cells along with three packaging plasmids of a third generation lentiviral packaging system (expressing HIV-gag-pol, rev and VSV-G, respectively, from Abcyte Therapeutics, USA, with original numbering pKC3A, pKC3B, pKC3C, respectively) to obtain lentiviruses encoding CD19 mono CAR, CD20 mono CAR or CD19/CD20 bis CAR, respectively. Comprises the following steps:

1. 293T was digested with trypsin, and then inoculated at 182cm using DMEM medium containing 10% Fetal Bovine Serum (FBS) ² Cell flasks, 1.6X 10 per flask ⁷ And (4) putting the total cells into a carbon dioxide incubator for overnight culture.

2. And (3) transfecting the cells with 80-90% of confluence at the next day, and replacing the cells with serum-free DMEM medium (30 mL per bottle) 1-2 hours before transfection.

3. Transfection: setting a tube A and a tube B, wherein the tube A is 2.8mL of Opti-MEM culture medium and 240 mu L of 1mg/mL Polyetherimide (PEI) solution; tube B was 2.8mL Opti-MEM medium + 36. Mu.g gag-pol packaging plasmid + 24. Mu.g Rev packaging plasmid + 12. Mu.g VSV-G packaging plasmid + 48. Mu.g of the gene of interest plasmid. And after the tube A and the tube B are respectively mixed uniformly, sucking the solution in the tube B and slowly dripping the solution into the tube A, after mixing uniformly, standing at room temperature for 15 minutes, and then adding the solution into a cell bottle for mixing.

4. 6 hours after transfection, the medium was replaced with 60mL of DMEM medium containing 2% FBS for 48 hours.

5. And (3) harvesting viruses: filtering the culture medium supernatant by a 0.45 mu m filter, adding 25-30 mL of a centrifuge tube into each tube, adding 3mL of 20% sucrose solution into the bottom, carrying out vacuum centrifugation at 25000rpm and 4 ℃ for 2 hours after trimming, removing the supernatant, and adding 200 mu L of OpTsizer T cell culture medium into each centrifuge tube to resuspend the virus overnight at 4 ℃.

6. The next day of harvesting, the virus solution in the centrifuge tubes was removed, the bottoms of the two centrifuge tubes were washed with 200 μ L of OpTsizer T cell medium and mixed with the virus solution, and the virus solution was stored at-80 ℃ after packaging and sent to the sample to test for virus titer.

Example 3 preparation of CAR-T cells

Peripheral Blood Mononuclear Cells (PBMC) are separated from the collected blood by using a cell separation system (Sepax C-Pro) to remove unwanted impurities such as red blood cells, granulocytes, platelets, plasma and the like. After completion of the isolation, the PBMC were transferred to a cell culture bag through a tube adapter, a sampling bag was connected to the culture bag through the tube adapter, and a small amount of PBMC was subjected to cell counting (AO/PI method) and flow assay for CD3, CD14, CD19, and CD56. And (4) calculating the total number of T cells in the PBMC according to the positive proportion of the flow-detection CD3, taking the PBMC according to the total number of the T cells required by the next operation, and freezing and storing redundant PBMC.

Taking fresh or revived PBMC, according to the theoretical total number of T cells: CD3/CD28 beads =1, beads were added, T cells were positive selected and activated from PBMCs, and the Day was counted as Day0 (Day 0), and the cells were divided into several portions for preparing different CAR-T cells. The next Day (i.e., day 1) lentiviruses encoding CD19 mono CAR, CD20 mono CAR or CD19/CD20 dual CAR, prepared in example 2, were used to transduce, respectively, to prepare corresponding CAR-T cells. The culture medium is supplemented after counting by using AO/PI every Day, the culture medium is maintained at 5E +05 cells/ml after liquid supplementation, the culture is carried out till the 5 th Day (Day 5), and the positive rate of CAR-T cells is detected by using Protein-L, CD19, CD20 antigen or anti-antibody respectively through a flow cytometer. And continuously expanding the T cells to the number of the cells meeting the requirements of feedback and detection, and harvesting the CAR-T cells after the magnetic beads are removed for subsequent experiments.

Example 4 expression of CD19 CAR and CD20CAR on the surface of Dual-Targeted CAR-T cells

1) Flow cytometry detection of CD19 CAR and CD20CAR expression on the surface of transduced T cells

The CAR positive rate of single-target and double-target CAR-T cells is detected in a flow mode, the detection and analysis results are shown in figure 5, the double-target CAR viral vectors constructed by the method can effectively transduce human primary T cells and express CD19 CAR and CD20CAR molecules, and the efficiency is slightly lower or equivalent to that of T cells transduced by respective single CAR viral vectors. The results shown in figure 5 are the expression of CD19 (CAR # 2), CD20 (CAR # 13) and CD19/CD20CAR (CAR # 10) on the surface of T cells. Compared to non-transduced negative T cells, single-target CAR-T cells (CAR # 2) had a CD19 CAR positive rate of about 81%, and double-target CAR-T cells (CAR # 10) had a CD19 CAR positive rate of about 78% (fig. 5A-C). Compared to non-transduced negative T cells, single-targeted CAR-T cells (CAR # 13) had a CD20CAR positive rate of about 59%, and dual-targeted CAR-T cells (CAR # 10) had a CD20CAR positive rate of about 48% (fig. 5D-F).

2) Immunoblotting (Western-blot) to examine the expression of CAR and the cleavage efficiency of 2A peptide

CD19 CAR, CD20CAR, or CD19/CD20CAR vector transduced T cells and non-transduced negative T cells were treated with cell lysate and total protein was extracted to obtain protein samples. Adding a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer solution into a protein sample, boiling, carrying out electrophoresis by using 12% protein prefabricated gel, transferring the protein in the gel onto a polyvinylidene fluoride (PVDF) membrane by using an electric transfer method after the electrophoresis is finished, cleaning and sealing the PVDF membrane, and carrying out Western-blot detection by using anti-human CD3 as a primary antibody. Figure 6 shows the results of the detection of CAR negative T cells (NT), CD19 (CAR # 2), CD20 (CAR # 13) and CD19/CD20 (CAR # 10) CAR vector transduced T cells in sequence. As shown in figure 6, in CAR #10 vector transduced T cells, both CD19 and CD20 CARs were predominantly present as a single CAR with a small amount of fusion protein unbroken, demonstrating the high efficiency of T2A-mediated self-cleavage. In fact, the signal at the fusion protein position is mainly from insufficiently reduced single CAR dimers, as compared to two single CAR-T samples. The expression of dual CAR T cell CARs in lower amounts than single CAR T cells may be a result of its slightly lower positive rate. In this experiment, the positive rates of CD19, CD20 and CD19/CD20CAR T detected by Protein-L flow cytometry were 87%, 89% and 73%, respectively.

Example 5 Single CAR-T cell in vitro Long-term killing experiments

Constructing a human chronic myelogenous leukemia cell K562 expressing CD19 or CD20 for an in-vitro killing experiment, wherein the method comprises the steps of cloning a synthesized gene fragment containing a CMV promoter and a human CD19 or CD20 DNA sequence into a lentiviral vector pLL-CMV-GFP-T2A-Puro (the cargo number is SBI # LL100 PA-1), and replacing a GFP coding sequence between a vector SnaBI and a BsrGI enzyme cutting site to obtain 2 corresponding lentiviral vector plasmids; two lentivirus vector plasmids and three packaging plasmids (pMDLg/pRRE, pRSV-Rev, pMD2.G, addgene cargo numbers are #12251, #12253 and # 12259) of a third generation lentivirus packaging system are co-transfected into HEK293T cells respectively, a culture solution containing virus particles is obtained, the culture solution is purified by steps of ultracentrifugation or ion exchange chromatography, molecular sieve and the like and then transduced into K562 cells, a monoclonal is screened by a limiting dilution method, the expression condition of CD19 or CD20 on the screened cell strains is detected by a flow cytometer, cell strains with high expression of CD19 or CD20 are selected for establishing a library and preserving the seeds, and the cell strains are named as K562-CD19 and K562-CD20 respectively.

A series of CD19 mono CAR-T cells (effector cells) constructed were co-cultured in vitro with the target cells K562-CD19, respectively, at a ratio of effector cells to target cells of 1 (E/T = 1. Counting by using AO/PI every two days, detecting the proportion of CAR-T cells and target cells by using a flow cytometer, calculating the number of effector cells and target cells, taking a certain amount of effector cells and target cells which are mixed and cultured, and supplementing the target cells to readjust the ratio of E: T to 1 for continuous culture. CAR-T cytotoxic activity was calculated by the percentage of target cells after co-culture,% killing = (initial tumor cell proportion on day X-tumor cell proportion on day X +2 flow assay)/initial tumor cell proportion on day X × 100%. FIG. 7 shows the results of in vitro long-term killing experiments on CD19 CAR-T (CAR # 1-6) cells, and it can be seen from FIG. 7 that the long-term multiple killing effect of CAR #3 (costimulatory molecules ICOS and 4-1 BB) on tumors can last for 13 days, and the long-term tumor killing ability of CAR #2 (costimulatory molecules 4-1 BB) is inferior to CAR #3.

Example 6 Dual CAR-T cells Long-term killing in vitro

The constructed series of CD19/CD20 dual CAR-T cells (CAR # 7-12) were co-cultured in vitro with the target cells K562-CD19, K562-CD20 or K562-CD19+ K562-CD20, respectively, at a ratio of effector cells to target cells of 1 (E/T = 1. Effector cells were stimulated multiple times every two days with the detection and addition of target cells as in example 5. The fold expansion of CAR-T cells during the co-culture phase was estimated by integrating the results of dilution fold and cell count, with the number of CAR-T cells = AO/PI count density X CAR-T cell proportion by volume by flow assay two days after each co-culture, and the fold expansion of CAR-T cells = CAR-T cell number calculated day X + 2/total number of starting CAR-T cells day X. FIG. 8 is a comparison of the long-term killing activity of different CD19/CD20CAR T cells against K562-CD19 target cells. Figure 9 is the cumulative fold expansion of different CD19/CD20CAR T cells when co-cultured with the target cell K562-CD 19. The experimental results of figures 8 and 9 show that the killing effect of dual CAR-expressing T cells on CD19 target cells K562-CD19 is optimal for CAR #9, followed by CAR #10; the in vitro amplification effect is similar to that of the in vitro amplification effect. FIG. 10 is a comparison of long-term killing of target cells K562-CD20 by different CD19/CD20CAR T cells. Figure 11 is the cumulative fold expansion of different CD19/CD20CAR T cells when co-cultured with target cells K562-CD20. The experimental results of figures 10 and 11 show that the killing effect of the different CAR T cells on CD20 target cells K562-CD20 was optimized for CAR #10, CAR #12, followed by CAR #9; the in vitro amplification effect is similar to the three. FIG. 12 is a comparison of long-term killing activity of different CD19/CD20CAR T cells against K562-CD19+ K562-CD20 target cells. FIG. 13 is the cumulative fold expansion of different CD19/CD20CAR T cells when co-cultured with target cells K562-CD19+ K562-CD20. The experimental results of fig. 12 and 13 show that the killing and expansion effect of the different prepared dual CAR T cells on K562-CD19+ K562-CD20 was optimized for CAR #10 under simultaneous stimulation of the dual target cells. Combining the experimental results, CAR #10 was selected as the subject for subsequent experiments.

Example 7 detection of specific killing by Lactate Dehydrogenase (LDH) method

This example investigates the specific killing of tumor cells by CD19/CD20 CAR-T cells in vitro and in comparison to CD19 or CD20 CAR-T cells. CD19 CAR T cells (CAR # 2), CD20CAR T cells (CAR # 13) and CD19/CD20CAR T cells (CAR # 10) were co-cultured in vitro at different ratios with target cells expressing CD19 or CD20 antigen (K562-CD 19 or K562-CD 20), respectively, and cytotoxic activity was detected by LDH release assay after 18 hours (fig. 14). The experimental results in figure 14 show that CAR # 10-expressing dual CAR T cells can specifically kill both CD 19-and CD 20-positive target cells with comparable killing efficiency to corresponding single CAR-T cells.

Example 8 assay of specific Release of cytokines

CD19/CD20 double CAR-T cells (CAR # 10) were mixed with target cells K562-CD19, K562-CD20, K562-CD19+ K562-CD20 at a ratio of 1, and cytokines were detected in the supernatant after 48h of culture, as shown in FIG. 15. As can be seen in FIG. 15, the CAR # 10-expressing T cells, when co-cultured with single-target or double-target positive cells, produced significant amounts of cytokines such as IFN-. Gamma.TNF-. Alpha.and IL-2, as compared to CAR negative T cells (NTs).

Example 9 animal drug efficacy test

In vivo efficacy experiments, mice transplanted with CD19+ CD20+ human lymphoma Raji cells were used as models. Raji cells (1E + 06/mouse) expressing luciferase are delivered into female NSG mice of 6-8 weeks of age through tail veins, after 5 days, small animal imaging is carried out to observe the signal intensity of tumor cells, after the proliferation of the tumor cells in most animals is confirmed to be visible, 56 mice are randomly selected into 7 groups according to the tumor signal intensity, 8 mice in each group are delivered through tail veins, phosphate Buffered Saline (PBS), untransduced human T cells and T cells expressing single CAR or double CAR are delivered.

The experiments were divided into the following 7 groups:

model group: inputting PSB buffer solution only;

NT group: transfusing 1.5e8 cells/kg untransduced human T cells;

CD19 (CAR # 2) single CAR group: transfusing 1.5e8 cells/kg CD19 CAR-T cells;

CD20 (CAR # 13) single CAR group: input 1.5E8 cells/kgCD 20 CAR-T cells;

dual CAR (CAR # 10) low dose group: input 0.5E8 cells/kg (low) CAR-T cells;

dual CAR (CAR # 10) dose groups: input 1.5E8 cells/kg (medium) CAR-T cells;

dual CAR (CAR # 10) high dose group: 5E8 cells/kg (high) CAR-T cells were input.

FIG. 16 is an image of tumors in NSG mice. Figure 17 shows tumor fluorescence model intensity in NSG mice. Fluorescence signals in the animal were collected in vivo according to the times shown in fig. 16. The experimental results of fig. 16 and 17 show that CD19/CD20 dual CAR-T cells, CD19 single CAR-T cells, and CD20 single CAR-T cells were equally effective at inhibiting tumor growth early in the experiment, but the recurrence rate of tumors in the late dual CAR-T cell experimental group was significantly lower than that of the two single CAR cell experimental groups, suggesting that CD19/CD20 dual CAR T cells can effectively prevent tumor recurrence.

Sequence listing

<110> Lizhuzumab Biotechnology Co., ltd, zhuhai City

<120> bispecific CAR T cells and uses thereof

<130> MTI20197

<160> 49

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 1

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

1 5 10 15

Gly Ser Thr Gly

20

<210> 2

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 2

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> 3

<211> 45

<212> PRT

<213> Artificial Sequence

<400> 3

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

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

20 25 30

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

35 40 45

<210> 4

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 4

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> 5

<211> 27

<212> PRT

<213> Artificial Sequence

<400> 5

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> 6

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 6

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

1 5 10 15

Gly Cys Ile Leu Ile

20

<210> 7

<211> 42

<212> PRT

<213> Artificial Sequence

<400> 7

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> 8

<211> 41

<212> PRT

<213> Artificial Sequence

<400> 8

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> 9

<211> 41

<212> PRT

<213> Artificial Sequence

<400> 9

Arg Ser Lys Arg Ser Arg Gly Gly 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> 10

<211> 38

<212> PRT

<213> Artificial Sequence

<400> 10

Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn

1 5 10 15

Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg

20 25 30

Leu Thr Asp Val Thr Leu

35

<210> 11

<211> 38

<212> PRT

<213> Artificial Sequence

<400> 11

Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn

1 5 10 15

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

20 25 30

Leu Thr Asp Val Thr Leu

35

<210> 12

<211> 42

<212> PRT

<213> Artificial Sequence

<400> 12

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

1 5 10 15

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

20 25 30

Ala Asp Ala His Ser Thr Leu Ala Lys Ile

35 40

<210> 13

<211> 112

<212> PRT

<213> Artificial Sequence

<400> 13

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 14

<211> 112

<212> PRT

<213> Artificial Sequence

<400> 14

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys 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 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 15

<211> 18

<212> PRT

<213> Artificial Sequence

<400> 15

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

Gly Pro

<210> 16

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 16

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

1 5 10 15

Pro Gly Pro

<210> 17

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 17

Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

1 5 10 15

Asn Pro Gly Pro

20

<210> 18

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 18

Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val

1 5 10 15

Glu Ser Asn Pro Gly Pro

20

<210> 19

<211> 3

<212> PRT

<213> Artificial Sequence

<400> 19

Gly Ser Gly

1

<210> 20

<211> 245

<212> PRT

<213> Artificial Sequence

<400> 20

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

1 5 10 15

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

20 25 30

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn

195 200 205

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

210 215 220

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

225 230 235 240

Val Thr Val Ser Ser

245

<210> 21

<211> 246

<212> PRT

<213> Artificial Sequence

<400> 21

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

1 5 10 15

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

20 25 30

Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 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 Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Tyr Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp Gly Gln Gly Thr

225 230 235 240

Thr Val Thr Val Ser Ser

245

<210> 22

<211> 247

<212> PRT

<213> Artificial Sequence

<400> 22

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 Gly Ser Thr Ser Gly

100 105 110

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

115 120 125

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

130 135 140

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr Ala Met His

145 150 155 160

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

165 170 175

Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val Lys Gly Arg

180 185 190

Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr Leu Gln Met

195 200 205

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

210 215 220

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

225 230 235 240

Thr Thr Val Thr Val Ser Ser

245

<210> 23

<211> 18

<212> PRT

<213> Artificial Sequence

<400> 23

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

1 5 10 15

Ser Ser

<210> 24

<211> 492

<212> PRT

<213> Artificial Sequence

<400> 24

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 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

210 215 220

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

225 230 235 240

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

245 250 255

Gln Gly Thr Thr Val Thr Val Ser Ser 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 Lys Pro Phe Trp Val Leu Val Val Val Gly

305 310 315 320

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

325 330 335

Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met

340 345 350

Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro

355 360 365

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

370 375 380

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys 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> 1476

<212> DNA

<213> Artificial Sequence

<400> 25

atggagaccg ataccctcct gctctgggtc ctgctcctgt gggtccccgg aagcacaggc 60

gacattcaga tgacacagtc cccctccagc ctcagcgcta gcgtcggcga cagggtgaca 120

atcacatgca gggcctccca ggacattagc aagtacctca actggtatca gcagaagcct 180

ggcaaggctc ccaagctcct catctaccac acaagcaggc tgcactccgg cgtcccctcc 240

agattcagcg gctccggctc cggcacagac tacacactga caatctccag cctccagcct 300

gaggacttcg ctacctacta ctgccagcag ggaaacacac tgccctacac attcggaggc 360

ggaaccaagg tcgagattaa gggaagcacc tccggcggcg gcagcggagg cggaagcggc 420

ggaggaggct ccagccaggt ccagctccag gagtccggcc ctggcctcgt gaagcctagc 480

cagacactgt ccctgacatg cacagtgtcc ggcgtcagcc tccccgacta cggagtgtcc 540

tggattagac agcctcccgg aaagggactg gagtggctcg gcgtcatctg gggaagcgag 600

acaacctact acaactccgc cctcaagtcc agactcacca ttagcaggga caactccaag 660

aacacactgt acctccagat gaactccctg agggccgagg acacagccgt ctactactgc 720

gctaagcact actactacgg aggctcctac gctatggact actggggaca gggaaccaca 780

gtgacagtgt ccagcaccac aacccctgcc cctagacctc ccacacccgc tcccacaatc 840

gctagccagc ctctgtccct gaggcccgag gcctgcagac ctgccgctgg cggagccgtc 900

cacacaagag gactggactt cgcttgcgac aagccctttt gggtgctggt ggtggttggt 960

ggagtcctgg cttgctatag cttgctagta acagtggcct ttattatttt ctgggtgagg 1020

agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactccccg ccgcccaggg 1080

cctacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 1140

agagtcaagt tctccagatc cgccgacgct cccgcttaca agcagggaca gaaccagctc 1200

tacaacgagc tcaacctcgg caggagagag gaatacgacg tcctggacaa gaggagagga 1260

agagaccctg agatgggagg caagcccaga aggaagaacc ctcaggaggg actgtacaac 1320

gagctccaga aggacaaaat ggctgaggct tactccgaga ttggcatgaa gggagagagg 1380

agaaggggca agggacacga cggactgtac cagggactgt ccaccgctac caaggacaca 1440

tacgacgctc tgcacatgca ggctctgcct cccagg 1476

<210> 26

<211> 488

<212> PRT

<213> Artificial Sequence

<400> 26

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 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

210 215 220

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

225 230 235 240

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

245 250 255

Gln Gly Thr Thr Val Thr Val Ser Ser 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 Lys Arg

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

His Met Gln Ala Leu Pro Pro Arg

485

<210> 27

<211> 1464

<212> DNA

<213> Artificial Sequence

<400> 27

atggagaccg ataccctcct gctctgggtc ctgctcctgt gggtccccgg aagcacaggc 60

gacattcaga tgacacagtc cccctccagc ctcagcgcta gcgtcggcga cagggtgaca 120

atcacatgca gggcctccca ggacattagc aagtacctca actggtatca gcagaagcct 180

ggcaaggctc ccaagctcct catctaccac acaagcaggc tgcactccgg cgtcccctcc 240

agattcagcg gctccggctc cggcacagac tacacactga caatctccag cctccagcct 300

gaggacttcg ctacctacta ctgccagcag ggaaacacac tgccctacac attcggaggc 360

ggaaccaagg tcgagattaa gggaagcacc tccggcggcg gcagcggagg cggaagcggc 420

ggaggaggct ccagccaggt ccagctccag gagtccggcc ctggcctcgt gaagcctagc 480

cagacactgt ccctgacatg cacagtgtcc ggcgtcagcc tccccgacta cggagtgtcc 540

tggattagac agcctcccgg aaagggactg gagtggctcg gcgtcatctg gggaagcgag 600

acaacctact acaactccgc cctcaagtcc agactcacca ttagcaggga caactccaag 660

aacacactgt acctccagat gaactccctg agggccgagg acacagccgt ctactactgc 720

gctaagcact actactacgg aggctcctac gctatggact actggggaca gggaaccaca 780

gtgacagtgt ccagcaccac aacccctgcc cctagacctc ccacacccgc tcccacaatc 840

gctagccagc ctctgtccct gaggcccgag gcctgcagac ctgccgctgg cggagccgtc 900

cacacaagag gactggactt cgcttgcgat atctacattt gggctcccct cgccggaacc 960

tgcggagtgc tcctgctcag cctcgtgatt accctctact gcaagagggg caggaagaag 1020

ctcctgtaca ttttcaaaca gcctttcatg aggcccgtcc agacaaccca ggaggaagac 1080

ggatgctcct gcaggttccc tgaggaagag gaaggcggat gcgagctcag agtcaagttc 1140

tccagatccg ccgacgctcc cgcttaccag cagggacaga accagctcta caacgagctc 1200

aacctcggca ggagagagga atacgacgtc ctggacaaga ggagaggaag agaccctgag 1260

atgggaggca agcccagaag gaagaaccct caggagggac tgtacaacga gctccagaag 1320

gacaaaatgg ctgaggctta ctccgagatt ggcatgaagg gagagaggag aaggggcaag 1380

ggacacgacg gactgtacca gggactgtcc accgctacca aggacacata cgacgctctg 1440

cacatgcagg ctctgcctcc cagg 1464

<210> 28

<211> 524

<212> PRT

<213> Artificial Sequence

<400> 28

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 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

210 215 220

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

225 230 235 240

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

245 250 255

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

305 310 315 320

Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu Thr

325 330 335

Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr Met

340 345 350

Asn Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp Val

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520

<210> 29

<211> 1572

<212> DNA

<213> Artificial Sequence

<400> 29

atggagaccg ataccctcct gctctgggtc ctgctcctgt gggtccccgg aagcacaggc 60

gacattcaga tgacacagtc cccctccagc ctcagcgcta gcgtcggcga cagggtgaca 120

atcacatgca gggcctccca ggacattagc aagtacctca actggtatca gcagaagcct 180

ggcaaggctc ccaagctcct catctaccac acaagcaggc tgcactccgg cgtcccctcc 240

agattcagcg gctccggctc cggcacagac tacacactga caatctccag cctccagcct 300

gaggacttcg ctacctacta ctgccagcag ggaaacacac tgccctacac attcggaggc 360

ggaaccaagg tcgagattaa gggaagcacc tccggcggcg gcagcggagg cggaagcggc 420

ggaggaggct ccagccaggt ccagctccag gagtccggcc ctggcctcgt gaagcctagc 480

cagacactgt ccctgacatg cacagtgtcc ggcgtcagcc tccccgacta cggagtgtcc 540

tggattagac agcctcccgg aaagggactg gagtggctcg gcgtcatctg gggaagcgag 600

acaacctact acaactccgc cctcaagtcc agactcacca ttagcaggga caactccaag 660

aacacactgt acctccagat gaactccctg agggccgagg acacagccgt ctactactgc 720

gctaagcact actactacgg aggctcctac gctatggact actggggaca gggaaccaca 780

gtgacagtgt ccagcaccac aacccctgcc cctagacctc ccacacccgc tcccacaatc 840

gctagccagc ctctgtccct gaggcccgag gcctgcagac ctgccgctgg cggagccgtc 900

cacacaagag gactggactt cgcttgcgat atcttctggc tccccattgg ctgcgctgcc 960

ttcgtcgtgg tctgcattct gggatgcatt ctgatttgct ggctgacaaa gaaaaagtac 1020

tccagctccg tgcacgaccc taacggagag tacatgaaca tgagggccgt caacacagcc 1080

aagaaaagca ggctgacaga cgtcaccctc aagaggggca ggaagaagct cctgtacatt 1140

ttcaaacagc ctttcatgag gcccgtccag acaacccagg aggaagacgg atgctcctgc 1200

aggttccctg aggaagagga aggcggatgc gagctcagag tcaagttctc cagatccgcc 1260

gacgctcccg cttaccagca gggacagaac cagctctaca acgagctcaa cctcggcagg 1320

agagaggaat acgacgtcct ggacaagagg agaggaagag accctgagat gggaggcaag 1380

cccagaagga agaaccctca ggagggactg tacaacgagc tccagaagga caaaatggct 1440

gaggcttact ccgagattgg catgaaggga gagaggagaa ggggcaaggg acacgacgga 1500

ctgtaccagg gactgtccac cgctaccaag gacacatacg acgctctgca catgcaggct 1560

ctgcctccca gg 1572

<210> 30

<211> 529

<212> PRT

<213> Artificial Sequence

<400> 30

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 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

210 215 220

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

225 230 235 240

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

245 250 255

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

340 345 350

Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg

355 360 365

Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

Arg

<210> 31

<211> 1587

<212> DNA

<213> Artificial Sequence

<400> 31

atggagaccg ataccctcct gctctgggtc ctgctcctgt gggtccccgg aagcacaggc 60

gacattcaga tgacacagtc cccctccagc ctcagcgcta gcgtcggcga cagggtgaca 120

atcacatgca gggcctccca ggacattagc aagtacctca actggtatca gcagaagcct 180

ggcaaggctc ccaagctcct catctaccac acaagcaggc tgcactccgg cgtcccctcc 240

agattcagcg gctccggctc cggcacagac tacacactga caatctccag cctccagcct 300

gaggacttcg ctacctacta ctgccagcag ggaaacacac tgccctacac attcggaggc 360

ggaaccaagg tcgagattaa gggaagcacc tccggcggcg gcagcggagg cggaagcggc 420

ggaggaggct ccagccaggt ccagctccag gagtccggcc ctggcctcgt gaagcctagc 480

cagacactgt ccctgacatg cacagtgtcc ggcgtcagcc tccccgacta cggagtgtcc 540

tggattagac agcctcccgg aaagggactg gagtggctcg gcgtcatctg gggaagcgag 600

acaacctact acaactccgc cctcaagtcc agactcacca ttagcaggga caactccaag 660

aacacactgt acctccagat gaactccctg agggccgagg acacagccgt ctactactgc 720

gctaagcact actactacgg aggctcctac gctatggact actggggaca gggaaccaca 780

gtgacagtgt ccagcaccac aacccctgcc cctagacctc ccacacccgc tcccacaatc 840

gctagccagc ctctgtccct gaggcccgag gcctgcagac ctgccgctgg cggagccgtc 900

cacacaagag gactggactt cgcttgcgat atctacattt gggctcccct cgccggaacc 960

tgcggagtgc tcctgctcag cctcgtgatt accctctact gcaggagtaa gaggagcagg 1020

ggaggccaca gtgactacat gaacatgact ccccgccgcc ccgggcccac ccgcaagcat 1080

taccagccct atgccccacc acgcgacttc gcagcctatc gctccaagag gggcaggaag 1140

aagctcctgt acattttcaa acagcctttc atgaggcccg tccagacaac ccaggaggaa 1200

gacggatgct cctgcaggtt ccctgaggaa gaggaaggcg gatgcgagct cagagtcaag 1260

ttctccagat ccgccgacgc tcccgcttac cagcagggac agaaccagct ctacaacgag 1320

ctcaacctcg gcaggagaga ggaatacgac gtcctggaca agaggagagg aagagaccct 1380

gagatgggag gcaagcccag aaggaagaac cctcaggagg gactgtacaa cgagctccag 1440

aaggacaaaa tggctgaggc ttactccgag attggcatga agggagagag gagaaggggc 1500

aagggacacg acggactgta ccagggactg tccaccgcta ccaaggacac atacgacgct 1560

ctgcacatgc aggctctgcc tcccagg 1587

<210> 32

<211> 491

<212> PRT

<213> Artificial Sequence

<400> 32

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 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

210 215 220

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

225 230 235 240

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

245 250 255

Gln Gly Thr Thr Val Thr Val Ser Ser 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 Phe Trp Val Leu Val Val Val Gly Gly

305 310 315 320

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

325 330 335

Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn

340 345 350

Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 33

<211> 1473

<212> DNA

<213> Artificial Sequence

<400> 33

atggagaccg ataccctcct gctctgggtc ctgctcctgt gggtccccgg aagcacaggc 60

gacattcaga tgacacagtc cccctccagc ctcagcgcta gcgtcggcga cagggtgaca 120

atcacatgca gggcctccca ggacattagc aagtacctca actggtatca gcagaagcct 180

ggcaaggctc ccaagctcct catctaccac acaagcaggc tgcactccgg cgtcccctcc 240

agattcagcg gctccggctc cggcacagac tacacactga caatctccag cctccagcct 300

gaggacttcg ctacctacta ctgccagcag ggaaacacac tgccctacac attcggaggc 360

ggaaccaagg tcgagattaa gggaagcacc tccggcggcg gcagcggagg cggaagcggc 420

ggaggaggct ccagccaggt ccagctccag gagtccggcc ctggcctcgt gaagcctagc 480

cagacactgt ccctgacatg cacagtgtcc ggcgtcagcc tccccgacta cggagtgtcc 540

tggattagac agcctcccgg aaagggactg gagtggctcg gcgtcatctg gggaagcgag 600

acaacctact acaactccgc cctcaagtcc agactcacca ttagcaggga caactccaag 660

aacacactgt acctccagat gaactccctg agggccgagg acacagccgt ctactactgc 720

gctaagcact actactacgg aggctcctac gctatggact actggggaca gggaaccaca 780

gtgacagtgt ccagcaccac aacccctgcc cctagacctc ccacacccgc tcccacaatc 840

gctagccagc ctctgtccct gaggcccgag gcctgcagac ctgccgctgg cggagccgtc 900

cacacaagag gactggactt cgcttgcgat atcttttggg tgctggtggt ggttggtgga 960

gtcctggctt gctatagctt gctagtaaca gtggccttta ttattttctg ggtgaggagt 1020

aagaggagca ggggaggcca cagtgactac atgaacatga ctccccgccg ccccgggccc 1080

acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaga 1140

gtcaagttct ccagatccgc cgacgctccc gcttaccagc agggacagaa ccagctctac 1200

aacgagctca acctcggcag gagagaggaa tacgacgtcc tggacaagag gagaggaaga 1260

gaccctgaga tgggaggcaa gcccagaagg aagaaccctc aggagggact gtacaacgag 1320

ctccagaagg acaaaatggc tgaggcttac tccgagattg gcatgaaggg agagaggaga 1380

aggggcaagg gacacgacgg actgtaccag ggactgtcca ccgctaccaa ggacacatac 1440

gacgctctgc acatgcaggc tctgcctccc agg 1473

<210> 34

<211> 488

<212> PRT

<213> Artificial Sequence

<400> 34

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 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

210 215 220

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

225 230 235 240

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

245 250 255

Gln Gly Thr Thr Val Thr Val Ser Ser 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 Ala Leu

325 330 335

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

340 345 350

Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp

355 360 365

Ala His Ser Thr Leu Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

His Met Gln Ala Leu Pro Pro Arg

485

<210> 35

<211> 1464

<212> DNA

<213> Artificial Sequence

<400> 35

atggagaccg ataccctcct gctctgggtc ctgctcctgt gggtccccgg aagcacaggc 60

gacattcaga tgacacagtc cccctccagc ctcagcgcta gcgtcggcga cagggtgaca 120

atcacatgca gggcctccca ggacattagc aagtacctca actggtatca gcagaagcct 180

ggcaaggctc ccaagctcct catctaccac acaagcaggc tgcactccgg cgtcccctcc 240

agattcagcg gctccggctc cggcacagac tacacactga caatctccag cctccagcct 300

gaggacttcg ctacctacta ctgccagcag ggaaacacac tgccctacac attcggaggc 360

ggaaccaagg tcgagattaa gggaagcacc tccggcggcg gcagcggagg cggaagcggc 420

ggaggaggct ccagccaggt ccagctccag gagtccggcc ctggcctcgt gaagcctagc 480

cagacactgt ccctgacatg cacagtgtcc ggcgtcagcc tccccgacta cggagtgtcc 540

tggattagac agcctcccgg aaagggactg gagtggctcg gcgtcatctg gggaagcgag 600

acaacctact acaactccgc cctcaagtcc agactcacca ttagcaggga caactccaag 660

aacacactgt acctccagat gaactccctg agggccgagg acacagccgt ctactactgc 720

gctaagcact actactacgg aggctcctac gctatggact actggggaca gggaaccaca 780

gtgacagtgt ccagcaccac aacccctgcc cctagacctc ccacacccgc tcccacaatc 840

gctagccagc ctctgtccct gaggcccgag gcctgcagac ctgccgctgg cggagccgtc 900

cacacaagag gactggactt cgcttgcgat atctacattt gggctcccct cgccggaacc 960

tgcggagtgc tcctgctcag cctcgtgatt accctctact gcgccctcta cctgttgaga 1020

agagatcaaa gattgcctcc agacgctcac aaaccgccag gaggtgggtc ttttcggact 1080

ccgattcagg aggaacaggc cgatgcacat tccactctgg ccaaaatcag agtcaagttc 1140

tccagatccg ccgacgctcc cgcttaccag cagggacaga accagctcta caacgagctc 1200

aacctcggca ggagagagga atacgacgtc ctggacaaga ggagaggaag agaccctgag 1260

atgggaggca agcccagaag gaagaaccct caggagggac tgtacaacga gctccagaag 1320

gacaaaatgg ctgaggctta ctccgagatt ggcatgaagg gagagaggag aaggggcaag 1380

ggacacgacg gactgtacca gggactgtcc accgctacca aggacacata cgacgctctg 1440

cacatgcagg ctctgcctcc cagg 1464

<210> 36

<211> 992

<212> PRT

<213> Artificial Sequence

<400> 36

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 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

210 215 220

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

225 230 235 240

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

245 250 255

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

305 310 315 320

Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu Thr Lys

325 330 335

Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr Met Phe

340 345 350

Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp Val Thr

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val

485 490 495

Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu

500 505 510

Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Glu Ile Val Leu Thr

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Lys Val Glu Ile Lys Gly Ser Thr Ser Gly Gly Gly Ser Gly Gly Gly

625 630 635 640

Ser Gly Gly Gly Gly Ser Ser Gln Val Gln Leu Val Gln Ser Gly Ala

645 650 655

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

725 730 735

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

740 745 750

Tyr Trp Phe Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

755 760 765

Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

770 775 780

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

785 790 795 800

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

<210> 37

<211> 2976

<212> DNA

<213> Artificial Sequence

<400> 37

atggagaccg ataccctcct gctctgggtc ctgctcctgt gggtccccgg aagcacaggc 60

gacattcaga tgacacagtc cccctccagc ctcagcgcta gcgtcggcga cagggtgaca 120

atcacatgca gggcctccca ggacattagc aagtacctca actggtatca gcagaagcct 180

ggcaaggctc ccaagctcct catctaccac acaagcaggc tgcactccgg cgtcccctcc 240

agattcagcg gctccggctc cggcacagac tacacactga caatctccag cctccagcct 300

gaggacttcg ctacctacta ctgccagcag ggaaacacac tgccctacac attcggaggc 360

ggaaccaagg tcgagattaa gggaagcacc tccggcggcg gcagcggagg cggaagcggc 420

ggaggaggct ccagccaggt ccagctccag gagtccggcc ctggcctcgt gaagcctagc 480

cagacactgt ccctgacatg cacagtgtcc ggcgtcagcc tccccgacta cggagtgtcc 540

tggattagac agcctcccgg aaagggactg gagtggctcg gcgtcatctg gggaagcgag 600

acaacctact acaactccgc cctcaagtcc agactcacca ttagcaggga caactccaag 660

aacacactgt acctccagat gaactccctg agggccgagg acacagccgt ctactactgc 720

gctaagcact actactacgg aggctcctac gctatggact actggggaca gggaaccaca 780

gtgacagtgt ccagcaccac aacccctgcc cctagacctc ccacacccgc tcccacaatc 840

gctagccagc ctctgtccct gaggcccgag gcctgcagac ctgccgctgg cggagccgtc 900

cacacaagag gactggactt cgcttgcgac ttctggctcc ccattggctg cgctgccttc 960

gtcgtggtct gcattctggg atgcattctg atttgctggc tgacaaagaa aaagtactcc 1020

agctccgtgc acgaccctaa cggagagtac atgttcatga gggccgtcaa cacagccaag 1080

aaaagcaggc tgacagacgt caccctcaga gtcaagttct ccagatccgc cgacgctccc 1140

gcttacaagc agggacagaa ccagctctac aacgagctca acctcggcag gagagaggaa 1200

tacgacgtcc tggacaagag gagaggaaga gaccctgaga tgggaggcaa gcccagaagg 1260

aagaaccctc aggagggact gtacaacgag ctccagaagg acaaaatggc tgaggcttac 1320

tccgagattg gcatgaaggg agagaggaga aggggcaagg gacacgacgg actgtaccag 1380

ggactgtcca ccgctaccaa ggacacatac gacgctctgc acatgcaggc tctgcctccc 1440

aggggctccg gcgagggcag gggaagtctt ctaacatgcg gggacgtgga ggaaaatccc 1500

ggcccaatgg ctctccctgt gacagctctt cttctgcccc tggcccttct gcttcacgct 1560

gccaggccag aaatcgtgct cactcaatcc cctgcaaccc tttcccttag ccctggagaa 1620

cgggccacaa tgacttgtag agcttctagc tccgtgaact atatggactg gtatcaacag 1680

aaacctggac aggctcctag accttggatc tacgccactt ctaatcttgc ttctggcgtg 1740

cctgccagat tctctggaag cggatctggc acagacttca cactgaccat cagctctctt 1800

gagcctgagg actttgccgt gtattactgc cagcaatgga gctttaatcc tcctaccttc 1860

ggcggaggaa ccaaagtgga gatcaagggc tccacaagcg gaggaggctc cggcggaggc 1920

tccggaggcg gcggaagctc ccaagtgcaa ctggttcagt ctggcgctga ggtgaagaaa 1980

cctggagcta gcgtgaaagt gtcctgtaaa gctagcggct acacattcac atcttataat 2040

atgcattggg tgaggcaggc tcctggccag ggactggaat ggataggagc tatctatcct 2100

gggaacggag ataccagcta taaccagaaa ttcaagggaa aggctacact cacaagagat 2160

acctctacca gcacagtgta tatggagctg tctagcctta gatccgagga cacagctgtt 2220

tattactgtg ccagaagcaa ttactacggc tccagctatt ggtttttcga tgtgtgggga 2280

caaggcacta ccgtcacagt ttctagcaca accacacccg ctcccaggcc ccctacccct 2340

gcccctacca ttgcctccca acccctcagc ctcagacctg aagcctgtag gcccgctgcc 2400

ggaggcgctg tgcataccag gggcctcgat tttgcctgtg atatctacat ctgggcgccc 2460

ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaaacgg 2520

ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 2580

caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 2640

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 2700

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 2760

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 2820

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 2880

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 2940

tacgacgccc ttcacatgca ggccctgccc cctcgc 2976

<210> 38

<211> 1035

<212> PRT

<213> Artificial Sequence

<400> 38

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 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

210 215 220

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

225 230 235 240

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

245 250 255

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

305 310 315 320

Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu Thr

325 330 335

Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr Met

340 345 350

Asn Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp Val

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Glu

515 520 525

Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly

530 535 540

Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

725 730 735

Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

1025 1030 1035

<210> 39

<211> 3105

<212> DNA

<213> Artificial Sequence

<400> 39

atggagaccg ataccctcct gctctgggtc ctgctcctgt gggtccccgg aagcacaggc 60

gacattcaga tgacacagtc cccctccagc ctcagcgcta gcgtcggcga cagggtgaca 120

atcacatgca gggcctccca ggacattagc aagtacctca actggtatca gcagaagcct 180

ggcaaggctc ccaagctcct catctaccac acaagcaggc tgcactccgg cgtcccctcc 240

agattcagcg gctccggctc cggcacagac tacacactga caatctccag cctccagcct 300

gaggacttcg ctacctacta ctgccagcag ggaaacacac tgccctacac attcggaggc 360

ggaaccaagg tcgagattaa gggaagcacc tccggcggcg gcagcggagg cggaagcggc 420

ggaggaggct ccagccaggt ccagctccag gagtccggcc ctggcctcgt gaagcctagc 480

cagacactgt ccctgacatg cacagtgtcc ggcgtcagcc tccccgacta cggagtgtcc 540

tggattagac agcctcccgg aaagggactg gagtggctcg gcgtcatctg gggaagcgag 600

acaacctact acaactccgc cctcaagtcc agactcacca ttagcaggga caactccaag 660

aacacactgt acctccagat gaactccctg agggccgagg acacagccgt ctactactgc 720

gctaagcact actactacgg aggctcctac gctatggact actggggaca gggaaccaca 780

gtgacagtgt ccagcaccac aacccctgcc cctagacctc ccacacccgc tcccacaatc 840

gctagccagc ctctgtccct gaggcccgag gcctgcagac ctgccgctgg cggagccgtc 900

cacacaagag gactggactt cgcttgcgat atcttctggc tccccattgg ctgcgctgcc 960

ttcgtcgtgg tctgcattct gggatgcatt ctgatttgct ggctgacaaa gaaaaagtac 1020

tccagctccg tgcacgaccc taacggagag tacatgaaca tgagggccgt caacacagcc 1080

aagaaaagca ggctgacaga cgtcaccctc aagaggggca ggaagaagct cctgtacatt 1140

ttcaaacagc ctttcatgag gcccgtccag acaacccagg aggaagacgg atgctcctgc 1200

aggttccctg aggaagagga aggcggatgc gagctcagag tcaagttctc cagatccgcc 1260

gacgctcccg cttaccagca gggacagaac cagctctaca acgagctcaa cctcggcagg 1320

agagaggaat acgacgtcct ggacaagagg agaggaagag accctgagat gggaggcaag 1380

cccagaagga agaaccctca ggagggactg tacaacgagc tccagaagga caaaatggct 1440

gaggcttact ccgagattgg catgaaggga gagaggagaa ggggcaaggg acacgacgga 1500

ctgtaccagg gactgtccac cgctaccaag gacacatacg acgctctgca catgcaggct 1560

ctgcctccca ggggctccgg cgaaggcagg ggaagtcttc taacatgcgg ggacgtggag 1620

gaaaatcccg gccccatggc tctccctgtg acagctcttc ttctgcccct ggcccttctg 1680

cttcacgctg ccaggccaga aatcgtgctc actcaatccc ctgcaaccct ttcccttagc 1740

cctggagaac gggccacaat gacttgtaga gcttctagct ccgtgaacta tatggactgg 1800

tatcaacaga aacctggaca ggctcctaga ccttggatct acgccacttc taatcttgct 1860

tctggcgtgc ctgccagatt ctctggaagc ggatctggca cagacttcac actgaccatc 1920

agctctcttg agcctgagga ctttgccgtg tattactgcc agcaatggag ctttaatcct 1980

cctaccttcg gcggaggaac caaagtggag atcaagggct ccacaagcgg aggaggctcc 2040

ggcggaggct ccggaggcgg cggaagctcc caagtgcaac tggttcagtc tggcgctgag 2100

gtgaagaaac ctggagctag cgtgaaagtg tcctgtaaag ctagcggcta cacattcaca 2160

tcttataata tgcattgggt gaggcaggct cctggccagg gactggaatg gataggagct 2220

atctatcctg ggaacggaga taccagctat aaccagaaat tcaagggaaa ggctacactc 2280

acaagagata cctctaccag cacagtgtat atggagctgt ctagccttag atccgaggac 2340

acagctgttt attactgtgc cagaagcaat tactacggct ccagctattg gtttttcgat 2400

gtgtggggac aaggcactac cgtcacagtt tctagcacaa ccacccccgc ccccaggccc 2460

cctacccctg ctcctaccat tgccagccaa cccctcagcc tcagacctga agcctgtagg 2520

cccgctgccg gaggcgctgt gcataccagg ggcctcgatt ttgcctgtga tatctatatc 2580

tgggcccctc tggccggcac ctgtggcgtc ttgctccttt cattggtgat tactctttac 2640

tgtaagagag gtcgcaaaaa gctgctgtac atattcaaac aaccctttat gagacccgtc 2700

caaaccaccc aagaagagga tggatgtagc tgtaggtttc ccgaagagga ggagggagga 2760

tgtgaactta gagtgaaatt cagcaggtcc gcagatgccc ccgcctatca gcagggtcaa 2820

aaccagctgt acaatgaact taatctcgga agaagggagg aatatgacgt gctggacaaa 2880

agaaggggaa gagatccaga gatgggcggc aaacccagaa gaaaaaaccc acaggagggg 2940

ctctataatg aactgcagaa agacaagatg gccgaggcat attccgaaat cggaatgaag 3000

ggcgaaagga gaagaggcaa aggacacgat gggctgtatc agggcctgag caccgccacc 3060

aaagacacct atgacgccct ccatatgcag gccctccctc ccaga 3105

<210> 40

<211> 1036

<212> PRT

<213> Artificial Sequence

<400> 40

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 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

210 215 220

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

225 230 235 240

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

245 250 255

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

305 310 315 320

Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu Thr

325 330 335

Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr Met

340 345 350

Asn Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp Val

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Glu

515 520 525

Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly

530 535 540

Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

Lys Gly Ser Thr Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035

<210> 41

<211> 3108

<212> DNA

<213> Artificial Sequence

<400> 41

atggagaccg ataccctcct gctctgggtc ctgctcctgt gggtccccgg aagcacaggc 60

gacattcaga tgacacagtc cccctccagc ctcagcgcta gcgtcggcga cagggtgaca 120

atcacatgca gggcctccca ggacattagc aagtacctca actggtatca gcagaagcct 180

ggcaaggctc ccaagctcct catctaccac acaagcaggc tgcactccgg cgtcccctcc 240

agattcagcg gctccggctc cggcacagac tacacactga caatctccag cctccagcct 300

gaggacttcg ctacctacta ctgccagcag ggaaacacac tgccctacac attcggaggc 360

ggaaccaagg tcgagattaa gggaagcacc tccggcggcg gcagcggagg cggaagcggc 420

ggaggaggct ccagccaggt ccagctccag gagtccggcc ctggcctcgt gaagcctagc 480

cagacactgt ccctgacatg cacagtgtcc ggcgtcagcc tccccgacta cggagtgtcc 540

tggattagac agcctcccgg aaagggactg gagtggctcg gcgtcatctg gggaagcgag 600

acaacctact acaactccgc cctcaagtcc agactcacca ttagcaggga caactccaag 660

aacacactgt acctccagat gaactccctg agggccgagg acacagccgt ctactactgc 720

gctaagcact actactacgg aggctcctac gctatggact actggggaca gggaaccaca 780

gtgacagtgt ccagcaccac aacccctgcc cctagacctc ccacacccgc tcccacaatc 840

gctagccagc ctctgtccct gaggcccgag gcctgcagac ctgccgctgg cggagccgtc 900

cacacaagag gactggactt cgcttgcgat atcttctggc tccccattgg ctgcgctgcc 960

ttcgtcgtgg tctgcattct gggatgcatt ctgatttgct ggctgacaaa gaaaaagtac 1020

tccagctccg tgcacgaccc taacggagag tacatgaaca tgagggccgt caacacagcc 1080

aagaaaagca ggctgacaga cgtcaccctc aagaggggca ggaagaagct cctgtacatt 1140

ttcaaacagc ctttcatgag gcccgtccag acaacccagg aggaagacgg atgctcctgc 1200

aggttccctg aggaagagga aggcggatgc gagctcagag tcaagttctc cagatccgcc 1260

gacgctcccg cttaccagca gggacagaac cagctctaca acgagctcaa cctcggcagg 1320

agagaggaat acgacgtcct ggacaagagg agaggaagag accctgagat gggaggcaag 1380

cccagaagga agaaccctca ggagggactg tacaacgagc tccagaagga caaaatggct 1440

gaggcttact ccgagattgg catgaaggga gagaggagaa ggggcaaggg acacgacgga 1500

ctgtaccagg gactgtccac cgctaccaag gacacatacg acgctctgca catgcaggct 1560

ctgcctccca ggggctccgg cgaaggcagg ggaagtcttc taacatgcgg ggacgtggag 1620

gaaaatcccg gccccatggc tctccctgtg acagctcttc ttctgcccct ggcccttctg 1680

cttcacgctg ccaggccaga gattgtgctc acccaaagcc ctgccaccct gtccctgtcc 1740

cccggagaga gggccacact gtcctgcaga gccagccagt ccgtgtccag ctatctggct 1800

tggtatcagc aaaagcctgg ccaagcccct agactcctga tctacgatgc ctccaacagg 1860

gccacaggca ttcccgctag attctccgga agcggaagcg gaaccgattt caccctgacc 1920

atcagcagcc tggagcctga ggatttcgct gtgtattact gtcagcaaag atccaactgg 1980

cctatcacat tcggccaggg aaccaggctg gaaatcaaag gctccacatc cggaggaggc 2040

agcggcggcg gctccggagg cggcggaagc tccgaggtcc agctcgtgga aagcggaggc 2100

ggcctggtcc agcctggcag gagcctcaga ctcagctgtg ccgctagcgg attcacattc 2160

aatgactatg ccatgcattg ggtcagacaa gcccctggca aaggcctcga gtgggtcagc 2220

acaatctcct ggaatagcgg aagcattggc tatgccgata gcgtcaaggg aagattcacc 2280

atcagcagag acaacgccaa aaagtccctg tatctgcaaa tgaatagcct cagagctgag 2340

gataccgctc tgtattactg tgccaaagac attcagtatg gcaattacta ttacggaatg 2400

gacgtgtggg gccagggcac caccgtgacc gtgagcagca caaccacccc cgcccccagg 2460

ccccctaccc ctgctcctac cattgccagc caacccctca gcctcagacc tgaagcctgt 2520

aggcccgctg ccggaggcgc tgtgcatacc aggggcctcg attttgcctg tgatatctat 2580

atctgggccc ctctggccgg cacctgtggc gtcttgctcc tttcattggt gattactctt 2640

tactgtaaga gaggtcgcaa aaagctgctg tacatattca aacaaccctt tatgagaccc 2700

gtccaaacca cccaagaaga ggatggatgt agctgtaggt ttcccgaaga ggaggaggga 2760

ggatgtgaac ttagagtgaa attcagcagg tccgcagatg cccccgccta tcagcagggt 2820

caaaaccagc tgtacaatga acttaatctc ggaagaaggg aggaatatga cgtgctggac 2880

aaaagaaggg gaagagatcc agagatgggc ggcaaaccca gaagaaaaaa cccacaggag 2940

gggctctata atgaactgca gaaagacaag atggccgagg catattccga aatcggaatg 3000

aagggcgaaa ggagaagagg caaaggacac gatgggctgt atcagggcct gagcaccgcc 3060

accaaagaca cctatgacgc cctccatatg caggccctcc ctcccaga 3108

<210> 42

<211> 1000

<212> PRT

<213> Artificial Sequence

<400> 42

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 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

210 215 220

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

225 230 235 240

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

245 250 255

Gln Gly Thr Thr Val Thr Val Ser Ser 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 Lys Arg

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Glu Gly Arg Gly Ser

485 490 495

Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala Leu

500 505 510

Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

595 600 605

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

610 615 620

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

625 630 635 640

Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

His Met Gln Ala Leu Pro Pro Arg

995 1000

<210> 43

<211> 3000

<212> DNA

<213> Artificial Sequence

<400> 43

atggagaccg ataccctcct gctctgggtc ctgctcctgt gggtccccgg aagcacaggc 60

gacattcaga tgacacagtc cccctccagc ctcagcgcta gcgtcggcga cagggtgaca 120

atcacatgca gggcctccca ggacattagc aagtacctca actggtatca gcagaagcct 180

ggcaaggctc ccaagctcct catctaccac acaagcaggc tgcactccgg cgtcccctcc 240

agattcagcg gctccggctc cggcacagac tacacactga caatctccag cctccagcct 300

gaggacttcg ctacctacta ctgccagcag ggaaacacac tgccctacac attcggaggc 360

ggaaccaagg tcgagattaa gggaagcacc tccggcggcg gcagcggagg cggaagcggc 420

ggaggaggct ccagccaggt ccagctccag gagtccggcc ctggcctcgt gaagcctagc 480

cagacactgt ccctgacatg cacagtgtcc ggcgtcagcc tccccgacta cggagtgtcc 540

tggattagac agcctcccgg aaagggactg gagtggctcg gcgtcatctg gggaagcgag 600

acaacctact acaactccgc cctcaagtcc agactcacca ttagcaggga caactccaag 660

aacacactgt acctccagat gaactccctg agggccgagg acacagccgt ctactactgc 720

gctaagcact actactacgg aggctcctac gctatggact actggggaca gggaaccaca 780

gtgacagtgt ccagcaccac aacccctgcc cctagacctc ccacacccgc tcccacaatc 840

gctagccagc ctctgtccct gaggcccgag gcctgcagac ctgccgctgg cggagccgtc 900

cacacaagag gactggactt cgcttgcgat atctacattt gggctcccct cgccggaacc 960

tgcggagtgc tcctgctcag cctcgtgatt accctctact gcaagagggg caggaagaag 1020

ctcctgtaca ttttcaaaca gcctttcatg aggcccgtcc agacaaccca ggaggaagac 1080

ggatgctcct gcaggttccc tgaggaagag gaaggcggat gcgagctcag agtcaagttc 1140

tccagatccg ccgacgctcc cgcttaccag cagggacaga accagctcta caacgagctc 1200

aacctcggca ggagagagga atacgacgtc ctggacaaga ggagaggaag agaccctgag 1260

atgggaggca agcccagaag gaagaaccct caggagggac tgtacaacga gctccagaag 1320

gacaaaatgg ctgaggctta ctccgagatt ggcatgaagg gagagaggag aaggggcaag 1380

ggacacgacg gactgtacca gggactgtcc accgctacca aggacacata cgacgctctg 1440

cacatgcagg ctctgcctcc caggggctcc ggcgaaggca ggggaagtct tctaacatgc 1500

ggggacgtgg aggaaaatcc cggccccatg gctctccctg tgacagctct tcttctgccc 1560

ctggcccttc tgcttcacgc tgccaggcca gagattgtgc tcacccaaag ccctgccacc 1620

ctgtccctgt cccccggaga gagggccaca ctgtcctgca gagccagcca gtccgtgtcc 1680

agctatctgg cttggtatca gcaaaagcct ggccaagccc ctagactcct gatctacgat 1740

gcctccaaca gggccacagg cattcccgct agattctccg gaagcggaag cggaaccgat 1800

ttcaccctga ccatcagcag cctggagcct gaggatttcg ctgtgtatta ctgtcagcaa 1860

agatccaact ggcctatcac attcggccag ggaaccaggc tggaaatcaa aggctccaca 1920

tccggaggag gcagcggcgg cggctccgga ggcggcggaa gctccgaggt ccagctcgtg 1980

gaaagcggag gcggcctggt ccagcctggc aggagcctca gactcagctg tgccgctagc 2040

ggattcacat tcaatgacta tgccatgcat tgggtcagac aagcccctgg caaaggcctc 2100

gagtgggtca gcacaatctc ctggaatagc ggaagcattg gctatgccga tagcgtcaag 2160

ggaagattca ccatcagcag agacaacgcc aaaaagtccc tgtatctgca aatgaatagc 2220

ctcagagctg aggataccgc tctgtattac tgtgccaaag acattcagta tggcaattac 2280

tattacggaa tggacgtgtg gggccagggc accaccgtga ccgtgagcag cacaaccacc 2340

cccgccccca ggccccctac ccctgctcct accattgcca gccaacccct cagcctcaga 2400

cctgaagcct gtaggcccgc tgccggaggc gctgtgcata ccaggggcct cgattttgcc 2460

tgtgatatct atatctgggc ccctctggcc ggcacctgtg gcgtcttgct cctttcattg 2520

gtgattactc tttactgtaa gagaggtcgc aaaaagctgc tgtacatatt caaacaaccc 2580

tttatgagac ccgtccaaac cacccaagaa gaggatggat gtagctgtag gtttcccgaa 2640

gaggaggagg gaggatgtga acttagagtg aaattcagca ggtccgcaga tgcccccgcc 2700

tatcagcagg gtcaaaacca gctgtacaat gaacttaatc tcggaagaag ggaggaatat 2760

gacgtgctgg acaaaagaag gggaagagat ccagagatgg gcggcaaacc cagaagaaaa 2820

aacccacagg aggggctcta taatgaactg cagaaagaca agatggccga ggcatattcc 2880

gaaatcggaa tgaagggcga aaggagaaga ggcaaaggac acgatgggct gtatcagggc 2940

ctgagcaccg ccaccaaaga cacctatgac gccctccata tgcaggccct ccctcccaga 3000

<210> 44

<211> 1003

<212> PRT

<213> Artificial Sequence

<400> 44

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 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

210 215 220

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

225 230 235 240

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

245 250 255

Gln Gly Thr Thr Val Thr Val Ser Ser 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 Phe Trp Val Leu Val Val Val Gly Gly

305 310 315 320

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

325 330 335

Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn

340 345 350

Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Glu Gly

485 490 495

Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro

500 505 510

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

995 1000

<210> 45

<211> 3009

<212> DNA

<213> Artificial Sequence

<400> 45

atggagaccg ataccctcct gctctgggtc ctgctcctgt gggtccccgg aagcacaggc 60

gacattcaga tgacacagtc cccctccagc ctcagcgcta gcgtcggcga cagggtgaca 120

atcacatgca gggcctccca ggacattagc aagtacctca actggtatca gcagaagcct 180

ggcaaggctc ccaagctcct catctaccac acaagcaggc tgcactccgg cgtcccctcc 240

agattcagcg gctccggctc cggcacagac tacacactga caatctccag cctccagcct 300

gaggacttcg ctacctacta ctgccagcag ggaaacacac tgccctacac attcggaggc 360

ggaaccaagg tcgagattaa gggaagcacc tccggcggcg gcagcggagg cggaagcggc 420

ggaggaggct ccagccaggt ccagctccag gagtccggcc ctggcctcgt gaagcctagc 480

cagacactgt ccctgacatg cacagtgtcc ggcgtcagcc tccccgacta cggagtgtcc 540

tggattagac agcctcccgg aaagggactg gagtggctcg gcgtcatctg gggaagcgag 600

acaacctact acaactccgc cctcaagtcc agactcacca ttagcaggga caactccaag 660

aacacactgt acctccagat gaactccctg agggccgagg acacagccgt ctactactgc 720

gctaagcact actactacgg aggctcctac gctatggact actggggaca gggaaccaca 780

gtgacagtgt ccagcaccac aacccctgcc cctagacctc ccacacccgc tcccacaatc 840

gctagccagc ctctgtccct gaggcccgag gcctgcagac ctgccgctgg cggagccgtc 900

cacacaagag gactggactt cgcttgcgat atcttttggg tgctggtggt ggttggtgga 960

gtcctggctt gctatagctt gctagtaaca gtggccttta ttattttctg ggtgaggagt 1020

aagaggagca ggggaggcca cagtgactac atgaacatga ctccccgccg ccccgggccc 1080

acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaga 1140

gtcaagttct ccagatccgc cgacgctccc gcttaccagc agggacagaa ccagctctac 1200

aacgagctca acctcggcag gagagaggaa tacgacgtcc tggacaagag gagaggaaga 1260

gaccctgaga tgggaggcaa gcccagaagg aagaaccctc aggagggact gtacaacgag 1320

ctccagaagg acaaaatggc tgaggcttac tccgagattg gcatgaaggg agagaggaga 1380

aggggcaagg gacacgacgg actgtaccag ggactgtcca ccgctaccaa ggacacatac 1440

gacgctctgc acatgcaggc tctgcctccc aggggctccg gcgaaggcag gggaagtctt 1500

ctaacatgcg gggacgtgga ggaaaatccc ggccccatgg ctctccctgt gacagctctt 1560

cttctgcccc tggcccttct gcttcacgct gccaggccag agattgtgct cacccaaagc 1620

cctgccaccc tgtccctgtc ccccggagag agggccacac tgtcctgcag agccagccag 1680

tccgtgtcca gctatctggc ttggtatcag caaaagcctg gccaagcccc tagactcctg 1740

atctacgatg cctccaacag ggccacaggc attcccgcta gattctccgg aagcggaagc 1800

ggaaccgatt tcaccctgac catcagcagc ctggagcctg aggatttcgc tgtgtattac 1860

tgtcagcaaa gatccaactg gcctatcaca ttcggccagg gaaccaggct ggaaatcaaa 1920

ggctccacat ccggaggagg cagcggcggc ggctccggag gcggcggaag ctccgaggtc 1980

cagctcgtgg aaagcggagg cggcctggtc cagcctggca ggagcctcag actcagctgt 2040

gccgctagcg gattcacatt caatgactat gccatgcatt gggtcagaca agcccctggc 2100

aaaggcctcg agtgggtcag cacaatctcc tggaatagcg gaagcattgg ctatgccgat 2160

agcgtcaagg gaagattcac catcagcaga gacaacgcca aaaagtccct gtatctgcaa 2220

atgaatagcc tcagagctga ggataccgct ctgtattact gtgccaaaga cattcagtat 2280

ggcaattact attacggaat ggacgtgtgg ggccagggca ccaccgtgac cgtgagcagc 2340

acaaccaccc ccgcccccag gccccctacc cctgctccta ccattgccag ccaacccctc 2400

agcctcagac ctgaagcctg taggcccgct gccggaggcg ctgtgcatac caggggcctc 2460

gattttgcct gtgatatcta tatctgggcc cctctggccg gcacctgtgg cgtcttgctc 2520

ctttcattgg tgattactct ttactgtaag agaggtcgca aaaagctgct gtacatattc 2580

aaacaaccct ttatgagacc cgtccaaacc acccaagaag aggatggatg tagctgtagg 2640

tttcccgaag aggaggaggg aggatgtgaa cttagagtga aattcagcag gtccgcagat 2700

gcccccgcct atcagcaggg tcaaaaccag ctgtacaatg aacttaatct cggaagaagg 2760

gaggaatatg acgtgctgga caaaagaagg ggaagagatc cagagatggg cggcaaaccc 2820

agaagaaaaa acccacagga ggggctctat aatgaactgc agaaagacaa gatggccgag 2880

gcatattccg aaatcggaat gaagggcgaa aggagaagag gcaaaggaca cgatgggctg 2940

tatcagggcc tgagcaccgc caccaaagac acctatgacg ccctccatat gcaggccctc 3000

cctcccaga 3009

<210> 46

<211> 1000

<212> PRT

<213> Artificial Sequence

<400> 46

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 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

210 215 220

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

225 230 235 240

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

245 250 255

Gln Gly Thr Thr Val Thr Val Ser Ser 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 Ala Leu

325 330 335

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

340 345 350

Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp

355 360 365

Ala His Ser Thr Leu Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Glu Gly Arg Gly Ser

485 490 495

Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala Leu

500 505 510

Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

595 600 605

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

610 615 620

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

625 630 635 640

Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

His Met Gln Ala Leu Pro Pro Arg

995 1000

<210> 47

<211> 3000

<212> DNA

<213> Artificial Sequence

<400> 47

atggagaccg ataccctcct gctctgggtc ctgctcctgt gggtccccgg aagcacaggc 60

gacattcaga tgacacagtc cccctccagc ctcagcgcta gcgtcggcga cagggtgaca 120

atcacatgca gggcctccca ggacattagc aagtacctca actggtatca gcagaagcct 180

ggcaaggctc ccaagctcct catctaccac acaagcaggc tgcactccgg cgtcccctcc 240

agattcagcg gctccggctc cggcacagac tacacactga caatctccag cctccagcct 300

gaggacttcg ctacctacta ctgccagcag ggaaacacac tgccctacac attcggaggc 360

ggaaccaagg tcgagattaa gggaagcacc tccggcggcg gcagcggagg cggaagcggc 420

ggaggaggct ccagccaggt ccagctccag gagtccggcc ctggcctcgt gaagcctagc 480

cagacactgt ccctgacatg cacagtgtcc ggcgtcagcc tccccgacta cggagtgtcc 540

tggattagac agcctcccgg aaagggactg gagtggctcg gcgtcatctg gggaagcgag 600

acaacctact acaactccgc cctcaagtcc agactcacca ttagcaggga caactccaag 660

aacacactgt acctccagat gaactccctg agggccgagg acacagccgt ctactactgc 720

gctaagcact actactacgg aggctcctac gctatggact actggggaca gggaaccaca 780

gtgacagtgt ccagcaccac aacccctgcc cctagacctc ccacacccgc tcccacaatc 840

gctagccagc ctctgtccct gaggcccgag gcctgcagac ctgccgctgg cggagccgtc 900

cacacaagag gactggactt cgcttgcgat atctacattt gggctcccct cgccggaacc 960

tgcggagtgc tcctgctcag cctcgtgatt accctctact gcgccctcta cctgttgaga 1020

agagatcaaa gattgcctcc agacgctcac aaaccgccag gaggtgggtc ttttcggact 1080

ccgattcagg aggaacaggc cgatgcacat tccactctgg ccaaaatcag agtcaagttc 1140

tccagatccg ccgacgctcc cgcttaccag cagggacaga accagctcta caacgagctc 1200

aacctcggca ggagagagga atacgacgtc ctggacaaga ggagaggaag agaccctgag 1260

atgggaggca agcccagaag gaagaaccct caggagggac tgtacaacga gctccagaag 1320

gacaaaatgg ctgaggctta ctccgagatt ggcatgaagg gagagaggag aaggggcaag 1380

ggacacgacg gactgtacca gggactgtcc accgctacca aggacacata cgacgctctg 1440

cacatgcagg ctctgcctcc caggggctcc ggcgaaggca ggggaagtct tctaacatgc 1500

ggggacgtgg aggaaaatcc cggccccatg gctctccctg tgacagctct tcttctgccc 1560

ctggcccttc tgcttcacgc tgccaggcca gagattgtgc tcacccaaag ccctgccacc 1620

ctgtccctgt cccccggaga gagggccaca ctgtcctgca gagccagcca gtccgtgtcc 1680

agctatctgg cttggtatca gcaaaagcct ggccaagccc ctagactcct gatctacgat 1740

gcctccaaca gggccacagg cattcccgct agattctccg gaagcggaag cggaaccgat 1800

ttcaccctga ccatcagcag cctggagcct gaggatttcg ctgtgtatta ctgtcagcaa 1860

agatccaact ggcctatcac attcggccag ggaaccaggc tggaaatcaa aggctccaca 1920

tccggaggag gcagcggcgg cggctccgga ggcggcggaa gctccgaggt ccagctcgtg 1980

gaaagcggag gcggcctggt ccagcctggc aggagcctca gactcagctg tgccgctagc 2040

ggattcacat tcaatgacta tgccatgcat tgggtcagac aagcccctgg caaaggcctc 2100

gagtgggtca gcacaatctc ctggaatagc ggaagcattg gctatgccga tagcgtcaag 2160

ggaagattca ccatcagcag agacaacgcc aaaaagtccc tgtatctgca aatgaatagc 2220

ctcagagctg aggataccgc tctgtattac tgtgccaaag acattcagta tggcaattac 2280

tattacggaa tggacgtgtg gggccagggc accaccgtga ccgtgagcag cacaaccacc 2340

cccgccccca ggccccctac ccctgctcct accattgcca gccaacccct cagcctcaga 2400

cctgaagcct gtaggcccgc tgccggaggc gctgtgcata ccaggggcct cgattttgcc 2460

tgtgatatct atatctgggc ccctctggcc ggcacctgtg gcgtcttgct cctttcattg 2520

gtgattactc tttactgtaa gagaggtcgc aaaaagctgc tgtacatatt caaacaaccc 2580

tttatgagac ccgtccaaac cacccaagaa gaggatggat gtagctgtag gtttcccgaa 2640

gaggaggagg gaggatgtga acttagagtg aaattcagca ggtccgcaga tgcccccgcc 2700

tatcagcagg gtcaaaacca gctgtacaat gaacttaatc tcggaagaag ggaggaatat 2760

gacgtgctgg acaaaagaag gggaagagat ccagagatgg gcggcaaacc cagaagaaaa 2820

aacccacagg aggggctcta taatgaactg cagaaagaca agatggccga ggcatattcc 2880

gaaatcggaa tgaagggcga aaggagaaga ggcaaaggac acgatgggct gtatcagggc 2940

ctgagcaccg ccaccaaaga cacctatgac gccctccata tgcaggccct ccctcccaga 3000

<210> 48

<211> 491

<212> PRT

<213> Artificial Sequence

<400> 48

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 Thr Thr Thr Pro

260 265 270

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

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 49

<211> 1473

<212> DNA

<213> Artificial Sequence

<400> 49

atggctctcc ctgtgacagc tcttcttctg cccctggccc ttctgcttca cgctgccagg 60

ccagagattg tgctcaccca aagccctgcc accctgtccc tgtcccccgg agagagggcc 120

acactgtcct gcagagccag ccagtccgtg tccagctatc tggcttggta tcagcaaaag 180

cctggccaag cccctagact cctgatctac gatgcctcca acagggccac aggcattccc 240

gctagattct ccggaagcgg aagcggaacc gatttcaccc tgaccatcag cagcctggag 300

cctgaggatt tcgctgtgta ttactgtcag caaagatcca actggcctat cacattcggc 360

cagggaacca ggctggaaat caaaggctcc acatccggag gaggcagcgg cggcggctcc 420

ggaggcggcg gaagctccga ggtccagctc gtggaaagcg gaggcggcct ggtccagcct 480

ggcaggagcc tcagactcag ctgtgccgct agcggattca cattcaatga ctatgccatg 540

cattgggtca gacaagcccc tggcaaaggc ctcgagtggg tcagcacaat ctcctggaat 600

agcggaagca ttggctatgc cgatagcgtc aagggaagat tcaccatcag cagagacaac 660

gccaaaaagt ccctgtatct gcaaatgaat agcctcagag ctgaggatac cgctctgtat 720

tactgtgcca aagacattca gtatggcaat tactattacg gaatggacgt gtggggccag 780

ggcaccaccg tgaccgtgag cagcacaacc acccccgccc ccaggccccc tacccctgct 840

cctaccattg ccagccaacc cctcagcctc agacctgaag cctgtaggcc cgctgccgga 900

ggcgctgtgc ataccagggg cctcgatttt gcctgtgata tctatatctg ggcccctctg 960

gccggcacct gtggcgtctt gctcctttca ttggtgatta ctctttactg taagagaggt 1020

cgcaaaaagc tgctgtacat attcaaacaa ccctttatga gacccgtcca aaccacccaa 1080

gaagaggatg gatgtagctg taggtttccc gaagaggagg agggaggatg tgaacttaga 1140

gtgaaattca gcaggtccgc agatgccccc gcctatcagc agggtcaaaa ccagctgtac 1200

aatgaactta atctcggaag aagggaggaa tatgacgtgc tggacaaaag aaggggaaga 1260

gatccagaga tgggcggcaa acccagaaga aaaaacccac aggaggggct ctataatgaa 1320

ctgcagaaag acaagatggc cgaggcatat tccgaaatcg gaatgaaggg cgaaaggaga 1380

agaggcaaag gacacgatgg gctgtatcag ggcctgagca ccgccaccaa agacacctat 1440

gacgccctcc atatgcaggc cctccctccc aga 1473

Claims

1. A chimeric antigen receptor comprising a first polypeptide and a second polypeptide, wherein:

The second polypeptide comprises: (ii) a second binding protein, (ii) a second spacer, (iii) a second transmembrane domain, (iv) a costimulatory signaling domain of 4-1BB, and (v) a TCR CD3 zeta signaling domain;

preferably, the binding protein is selected from the group consisting of native protein binding domains (e.g. cytokines, cytokine receptors), antibody fragments (e.g. Fab, scFv, diabodies, variable region derived binders, VHH nanobodies), surrogate scaffold derived protein binding domains (e.g. Fn3 variants, ankyrin repeat variants, centyrin variants, avimers, affibodies) or any protein that recognizes a specific antigen.

2. The chimeric antigen receptor according to claim 1, wherein a first binding protein of the first polypeptide comprises a first antigen-binding domain and a second binding protein of the second polypeptide comprises a second antigen-binding domain, wherein at least one of the first antigen-binding domain and the second antigen-binding domain binds an antigen on a tumor cell;

preferably, the first antigen-binding domain of the first polypeptide and the second antigen-binding domain of the second polypeptide bind to different antigens or different epitopes of the same antigen on a tumor cell;

preferably, the first antigen binding domain of the first polypeptide binds to antigen CD19; more preferably, the second antigen-binding domain of the second polypeptide binds to the antigen CD20; preferably, the first antigen-binding domain of the first polypeptide comprises an anti-CD 19 scFv, preferably the anti-CD 19 scFv is a humanized anti-CD 19 scFv; preferably, the first antigen binding domain of the first polypeptide comprises an amino acid sequence that is 80% or more identical to the light chain variable region sequence and the heavy chain variable region sequence of the humanized anti-CD 19 scFv shown in SEQ ID NO:20, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical; more preferably, the first antigen-binding domain of the first polypeptide comprises the light chain variable region sequence and the heavy chain variable region sequence of the humanized anti-CD 19 scFv shown in SEQ ID NO: 20; more preferably, the first binding protein comprises the humanized anti-CD 19 scFv amino acid sequence shown in SEQ ID NO 20; preferably, the second antigen-binding domain of the second polypeptide comprises an anti-CD 20 scFv, preferably, the anti-CD 20 scFv is a humanized anti-CD 20 scFv; preferably, the second antigen-binding domain of the second polypeptide comprises an amino acid sequence that is 80% or more identical to the light chain variable region sequence and the heavy chain variable region sequence of the humanized anti-CD 20 scFv represented by SEQ ID NO 21 or SEQ ID NO 22, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical; more preferably, the second antigen-binding domain of the second polypeptide comprises the light chain variable region sequence and the heavy chain variable region sequence of the humanized anti-CD 20 scFv shown in SEQ ID NO 21 or SEQ ID NO 22; more preferably, the second binding protein comprises the humanized anti-CD 20 scFv amino acid sequence shown in SEQ ID NO 21 or SEQ ID NO 22;

preferably, the first antigen-binding domain of the first polypeptide binds antigen CD20, more preferably, the second antigen-binding domain of the second polypeptide binds antigen CD19; preferably, the first antigen-binding domain of the first polypeptide comprises an anti-CD 20 scFv, preferably, the anti-CD 20 scFv is a humanized anti-CD 20 scFv; preferably, the first antigen-binding domain of the first polypeptide comprises an amino acid sequence that is 80% or more identical to the light chain variable region sequence and the heavy chain variable region sequence of the humanized anti-CD 20 scFv represented by SEQ ID NO:21 or SEQ ID NO:22, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical; more preferably, the first antigen-binding domain of the first polypeptide comprises the light chain variable region sequence and the heavy chain variable region sequence of the humanized anti-CD 20 scFv shown in SEQ ID NO:21 or SEQ ID NO: 22; more preferably, the first binding protein comprises the humanized anti-CD 20 scFv amino acid sequence shown in SEQ ID NO 21 or SEQ ID NO 22; preferably, the second antigen-binding domain of the second polypeptide comprises an anti-CD 19 scFv, preferably, the anti-CD 19 scFv is a humanized anti-CD 19 scFv; preferably, the second antigen-binding domain of the second polypeptide comprises an amino acid sequence that is 80% or more identical, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the light chain variable region sequence and the heavy chain variable region sequence in the humanized anti-CD 19 scFv represented by SEQ ID NO 20; more preferably, the second antigen-binding domain of the second polypeptide comprises the light chain variable region sequence and the heavy chain variable region sequence of the humanized anti-CD 19 scFv represented by SEQ ID NO: 20; more preferably, the second binding protein comprises the humanized anti-CD 19 scFv amino acid sequence shown in SEQ ID NO 20;

preferably, a linker is included between the light chain variable region sequence and the heavy chain variable region sequence in the anti-CD 19 scFv and/or between the light chain variable region sequence and the heavy chain variable region sequence in the anti-CD 20 scFv, preferably the linker is a flexible linker, more preferably the amino acid sequence of the flexible linker is shown in SEQ ID NO: 23.

3. The chimeric antigen receptor according to claim 1 or 2, wherein the chimeric antigen receptor further comprises a peptide cleavage site linking the first polypeptide and the second polypeptide; preferably, the first binding polypeptide further comprises a first signal peptide; more preferably, the second binding polypeptide further comprises a second signal peptide;

preferably, the amino acid sequence of the first signal peptide is shown as SEQ ID NO. 1;

preferably, the amino acid sequence of the second signal peptide is shown as SEQ ID NO. 2;

preferably, the first and second spacers comprise amino acid sequences having 80% or more identity to the amino acid sequence set forth in SEQ ID No. 3, preferably amino acid sequences having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity; more preferably, the amino acid sequences of the first spacer and the second spacer are shown in SEQ ID NO. 3;

preferably, the amino acid sequences of the first and second transmembrane domains comprise amino acid sequences having 80% or more identity, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, to the amino acid sequence set forth in SEQ ID No. 4; more preferably, the amino acid sequences of the first transmembrane domain and the second transmembrane domain are shown in SEQ ID NO 4;

preferably, the amino acid sequence of the co-stimulatory signaling domain of 4-1BB comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO. 7, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity; more preferably, the amino acid sequence of the co-stimulatory signaling domain of 4-1BB is as shown in SEQ ID NO. 7;

preferably, the amino acid sequence of the TCR CD3 zeta signaling domain comprises an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID No. 13 or SEQ ID No. 14, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical; more preferably, the amino acid sequence of the TCR CD3 zeta signaling domain is as set forth in SEQ ID NO 13 or SEQ ID NO 14; more preferably, the amino acid sequence of the TCR CD3 zeta signaling domain is set forth in SEQ ID No. 13;

preferably, the peptide cleavage site is a self-cleavage site; more preferably, the self-cleavage site is selected from the group consisting of a T2A, P2A, E2A or F2A site; preferably, the amino acid sequence of the T2A is shown as SEQ ID NO. 15; preferably, the amino acid sequence of the P2A is shown as SEQ ID NO 16; preferably, the amino acid sequence of the E2A is shown as SEQ ID NO: 17; preferably, the amino acid sequence of the F2A is shown as SEQ ID NO. 18;

preferably, a linker is added at the N end of the peptide cutting site, and the amino acid sequence of the linker is shown as SEQ ID NO. 19;

preferably, the chimeric antigen receptor comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO. 42, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity; more preferably, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 42.

4. A polynucleotide encoding the chimeric antigen receptor of any one of claims 1-3;

preferably, the polynucleotide comprises a first gene encoding a first polypeptide comprising a first antigen binding domain, a spacer, a transmembrane domain, a 4-1BB costimulatory signaling domain, and a CD 3-zeta signaling domain, and a second gene encoding a second polypeptide comprising a second antigen binding domain, a spacer, a transmembrane domain, a 4-1BB costimulatory signaling domain, and a CD 3-epsilon signaling domain; wherein the first and second antigen-binding domains bind to different antigens on the cancer cell;

preferably, the polynucleotide further comprises a third nucleic acid sequence encoding a peptide cleavage site to link the first gene and the second gene;

preferably, the first and second genes further comprise a nucleic acid sequence encoding a signal peptide;

preferably, the polynucleotide comprises a nucleic acid sequence having 80% or more identity to the nucleic acid sequence as set forth in SEQ ID NO. 43, preferably a nucleic acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity; more preferably, the polynucleotide is as set forth in SEQ ID NO 43.

5. A vector comprising the polynucleotide of claim 4.

6. An engineered cell comprising the polynucleotide of claim 4 or the vector of claim 5;

preferably, the engineered cell is a T cell or NK cell;

preferably, the T cell is a CD 4T cell or a CD8T cell;

preferably, the NK cells are NKT cells or NK-92 cells;

preferably, the engineered cell comprises an inactivated gene for PD-1, TIM3 or LAG 3; more preferably, the inactivation is accomplished by a gene knock-out method.

7. A pharmaceutical composition comprising the chimeric antigen receptor of any one of claims 1-3, the polynucleotide of claim 4, the vector of claim 5 and/or the engineered cell of claim 6, and a pharmaceutically acceptable carrier.

8. Use of the chimeric antigen receptor of any one of claims 1-3, the polynucleotide of claim 4, the vector of claim 5, the engineered cell of claim 6, and/or the pharmaceutical composition of claim 7 in the manufacture of a medicament for the treatment or prevention of diseases, conditions, and disorders, including tumors;

preferably, the tumor is a cancer;

preferably, the cancer is selected from a hematologic malignancy, a solid tumor, a primary or metastatic tumor; more preferably, the cancer is a hematological cancer or myeloma;

preferably, the cancer is selected from leukemia, lymphoma, e.g. Chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), non-hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B-cell lymphoma, B-cell malignancy, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone and brain cancer, ovarian cancer, epithelial cancer, renal cell cancer, pancreatic adenocarcinoma, hodgkin's lymphoma, systemic or primary cutaneous anaplastic large cell lymphoma (pcALCL), cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma and/or mesothelioma.

9. A method of stimulating a T cell-mediated immune response to a target cell population or tissue in a subject, the method comprising administering to the subject an effective amount of the chimeric antigen receptor of any one of claims 1-3, the polynucleotide of claim 4, the vector of claim 5, the engineered cell of claim 6, and/or the pharmaceutical composition of claim 7.

10. A method of treating a tumor, the method comprising administering to a subject in need thereof an effective amount of the chimeric antigen receptor of any one of claims 1-3, the polynucleotide of claim 4, the vector of claim 5, the engineered cell of claim 6, and/or the pharmaceutical composition of claim 7.