CN108395481A - Construction of a CAR targeting CD20 and activity identification of engineered T cells - Google Patents

Abstract

The invention discloses construction of a targeted CD20 antigen chimeric antigen receptor and activity identification of an engineered T cell thereof, and particularly provides a CD20 targeted chimeric antigen receptor and a preparation method and application thereof, wherein an extracellular antigen binding domain of the chimeric antigen receptor comprises an antibody heavy chain variable region shown in SEQ ID No.12 and an antibody light chain variable region shown in SEQ ID No. 14. Experimental results show that the chimeric antigen receptor provided by the invention has extremely high killing capacity on tumor cells.

Description

Construction of a CAR targeting CD20 and activity identification of engineered T cells

technical field

The present invention provides a sequence component targeting CD20 antigen chimeric antigen receptor, and the preparation method and activity identification of modified T cells (CART20); the present invention identifies a CD20-positive B-cell lymphoma Chimeric antigen receptor structure.

Background technique

Hematological malignancies account for about 10% of human malignancies, and 95% of hematological malignancies are derived from B lymphocytes. Traditional chemotherapy and radiotherapy play an important role in the treatment of malignant tumors of the blood system. Some patients also have a significant effect, but most of them are difficult to cure. New and effective treatment methods have always been a hot spot for exploration in this field.

Adoptive T cell therapy has shown its strong curative effect and bright prospect in the clinical treatment of malignant tumors. Among them, the use of chimeric antigen receptor (Chimeric Antigen Receptor, CAR)-modified T cells to target CD19-expressing B-cell relapsed and refractory malignant tumors independently carried out by multiple centers has achieved unprecedented success. In particular, in a clinical trial using CART19 in the treatment of relapsed and refractory acute B-cell lymphoma (R/R B-ALL) conducted by the University of Pennsylvania School of Medicine, up to 94% of patients achieved complete remission. Despite the high initial response rate in this clinical trial, nearly 40% of patients relapsed after achieving complete remission at 1 month of treatment, and more than 60% of relapsed patients developed CD19-negative tumor cells escape. Therefore, there is an urgent need to screen out CART structures targeting B-cell lymphoma-associated antigens other than CD19 to treat patients with malignant lymphoma.

CD20 is a glycosylated protein, the first identified B cell membrane marker, also known as B1, encoded by the MS4A gene. The CD20 molecule has four transmembrane hydrophobic regions, and its N segment and C terminal are located on the cytoplasmic side, thus forming two closed loops outside the cell, which are called macrocycles and small loops. CD20 is specifically expressed in more than 95% of normal and cancerous B cells. These cells are in the developmental stages of pre-B cells and later, and CD20 does not stop expressing until they differentiate into plasma cells. Therefore, CD20 is an ideal target for immunotherapy of B cell malignancies.

Cellular immunotherapy is an emerging tumor treatment mode with significant curative effect, and it is a new type of autoimmune anti-cancer treatment. It is a method of using biotechnology and biological agents to culture and expand immune cells collected from patients in vitro and then infuse them back into the patient's body to stimulate and enhance the body's autoimmune function, so as to achieve the purpose of treating tumors. Those skilled in the art have been devoting themselves to developing new cellular immunotherapy in order to improve the effect of cellular immunotherapy and reduce its side effects.

Contents of the invention

The object of the present invention is to provide a CD20 targeting chimeric antigen receptor and its preparation method and application.

The invention relates to the construction of a CD20-targeting chimeric antigen receptor structure, a preparation method of CD20-targeting chimeric antigen receptor engineered T cells and activity identification thereof.

The first aspect of the present invention provides a chimeric antigen receptor (CAR) (sequence), the antigen binding domain (ie, scFv) of the chimeric antigen receptor includes the anti-CAR shown in SEQ ID NO.12 The heavy chain variable region, and the antibody light chain variable region shown in SEQ ID NO.14.

In another preferred example, the antigen-binding domain (scFv) of the chimeric antigen receptor is shown in the following formula I or formula II:

V _H -V _L ,(I); V _L -V _H ,(II)

Wherein, V _H is the variable region of the heavy chain of the antibody; V _L is the variable region of the light chain of the antibody; "-" is the connecting peptide or a peptide bond.

In another preferred example, the amino acid sequence of the connecting peptide is shown in SEQ ID NO.16.

In another preferred example, the structure of the chimeric antigen receptor is shown in the following formula:

L-scFv-H-TM-C-CD3ζ

in,

L is an optional leader sequence (Leader sequence, ie signal peptide sequence);

scFv is an antigen-binding domain;

H is the hinge region;

TM is the transmembrane domain;

C is costimulatory signal receptor tyrosine activation motif (costimulatory molecule);

CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

The antigen-binding domain and "-" are as above, respectively.

In another preferred example, the co-stimulatory signal receptor tyrosine activation motif includes a costimulatory signal receptor tyrosine activation motif derived from 4-1BB, and/or a costimulatory signal receptor tyrosine derived from CD28 amino acid activation motif.

In another preferred example, the sequence of L is shown in SEQ ID NO.32.

In another preferred example, the sequence of H is shown in SEQ ID NO.18 or 20.

In another preferred example, the sequence of TM includes the transmembrane region derived from CD8, preferably the sequence of TM is shown in SEQ ID NO.22.

In another preferred example, the sequence of TM includes the transmembrane region derived from CD28, preferably the sequence of TM is shown in SEQ ID NO.24.

In another preferred example, the amino acid sequence of the costimulatory signal receptor tyrosine activation motif derived from 4-1BB is shown in SEQ ID NO.26.

In another preferred example, the amino acid sequence of the CD28-derived co-stimulatory signal receptor tyrosine activation motif is shown in SEQ ID NO.28.

In another preferred example, the sequence of CD3ζ is shown in SEQ ID NO.30.

In another preferred example, the sequence of the chimeric antigen receptor is shown in SEQ ID NO.1, 4, 6, 8 or 10.

The second aspect of the present invention provides a nucleic acid molecule encoding the chimeric antigen receptor (CAR) described in the first aspect of the present invention.

In another preferred example, the nucleic acid molecule comprises a nucleic acid sequence encoding the hinge region selected from the following group:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.18 or 20;

(b) a polynucleotide whose sequence is shown in SEQ ID NO.19 or 21;

(c) The homology of the nucleotide sequence and the sequence shown in SEQ ID NO.19 or 21 ≥ 90% (preferably ≥ 95%), and the polynucleoside encoding the amino acid sequence shown in SEQ ID NO.18 or 20 acid;

(d) A polynucleotide complementary to the polynucleotide described in any one of (a)-(c).

In another preferred example, the nucleic acid molecule comprises a nucleic acid sequence encoding the transmembrane region of CD8 selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.22;

(b) a polynucleotide whose sequence is as shown in SEQ ID NO.23;

(c) a polynucleotide whose nucleotide sequence is ≥90% (preferably ≥95%) homologous to the sequence shown in SEQ ID NO.23 and encodes the amino acid sequence shown in SEQ ID NO.22;

(d) A polynucleotide complementary to the polynucleotide described in any one of (a)-(c).

In another preferred example, the nucleic acid molecule comprises a nucleic acid sequence encoding the transmembrane region of CD28 selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.24;

(b) a polynucleotide whose sequence is shown in SEQ ID NO.25;

(c) a polynucleotide whose nucleotide sequence is ≥90% (preferably ≥95%) homologous to the sequence shown in SEQ ID NO.25 and encodes the amino acid sequence shown in SEQ ID NO.24;

(d) A polynucleotide complementary to the polynucleotide described in any one of (a)-(c).

In another preferred example, the nucleic acid molecule comprises a co-stimulatory signal receptor tyrosine activation motif coding sequence, and the co-stimulatory signal receptor tyrosine activation motif coding sequence includes a co-stimulatory signal derived from 4-1BB Receptor tyrosine activation motif coding sequence, and/or CD28-derived co-stimulatory signal receptor tyrosine activation motif coding sequence, wherein

The co-stimulatory signal receptor tyrosine activation motif coding sequence derived from 4-1BB is selected from the following group:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.26;

(b) a polynucleotide whose sequence is as shown in SEQ ID NO.27;

(c) a polynucleotide whose nucleotide sequence is ≥90% (preferably ≥95%) homologous to the sequence shown in SEQ ID NO.27 and encodes the amino acid sequence shown in SEQ ID NO.26;

(d) a polynucleotide complementary to any of the polynucleotides described in (a)-(c);

The co-stimulatory signal receptor tyrosine activation motif coding sequence derived from CD28 is selected from the following group:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.28;

(b) a polynucleotide whose sequence is shown in SEQ ID NO.29;

(c) a polynucleotide whose nucleotide sequence is ≥90% (preferably ≥95%) homologous to the sequence shown in SEQ ID NO.29 and encodes the amino acid sequence shown in SEQ ID NO.28;

(d) A polynucleotide complementary to the polynucleotide described in any one of (a)-(c).

In another preferred example, the nucleic acid molecule comprises a nucleic acid sequence encoding the intracellular signaling domain of CD3ζ selected from the following group:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.30;

(b) a polynucleotide whose sequence is shown in SEQ ID NO.31;

(c) a polynucleotide whose nucleotide sequence is ≥90% (preferably ≥95%) homologous to the sequence shown in SEQ ID NO.30 and encodes the amino acid sequence shown in SEQ ID NO.31;

(d) A polynucleotide complementary to the polynucleotide described in any one of (a)-(c).

In another preference, the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.1, 4, 6, 8 or 10;

(b) a polynucleotide whose sequence is as shown in SEQ ID NO.2, 3, 5, 7, 9 or 11;

(c) The homology of the nucleotide sequence to the sequence shown in SEQ ID NO.2, 3, 5, 7, 9 or 11 is ≥95% (preferably ≥98%), and encodes SEQ ID NO.1, A polynucleotide of the amino acid sequence shown in 4, 6, 8 or 10;

(d) A polynucleotide complementary to the polynucleotide described in any one of (a)-(c).

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

In another preferred example, the nucleic acid molecule further includes a polynucleotide encoding a leader sequence (guide sequence, signal peptide), the amino acid sequence of the leader sequence is shown in SEQ ID NO.32; preferably, the encoding leader The polynucleotide of the sequence (signal peptide) is shown in SEQ ID NO.33.

In another preferred example, the sequence of the nucleic acid molecule is shown in SEQ ID NO.2, 3, 5, 7, 9 or 11.

The third aspect of the present invention provides a vector containing the nucleic acid molecule described in the second aspect of the present invention.

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

The fourth aspect of the present invention provides a host cell containing the vector of the third aspect of the present invention or the exogenous nucleic acid of the second aspect of the present invention integrated in the chromosome molecular.

In another preferred embodiment, the cells are isolated cells, and/or the cells are genetically engineered cells.

In another preferred embodiment, the cells are mammalian cells.

In another preferred example, the cells are T cells.

The fifth aspect of the present invention provides a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid described in the second aspect of the present invention The molecule, the vector of the third aspect of the present invention, or the cell of the fourth aspect of the present invention.

The sixth aspect of the present invention provides the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, or the fourth aspect of the present invention The use of the cells is for preparing medicines or preparations for treating tumors.

In another preferred example, the tumors include CD20 positive tumors.

The seventh aspect of the present invention provides a method for treating diseases, comprising administering an appropriate amount of the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, The carrier of the third aspect of the present invention, or the cell of the fourth aspect of the present invention, or the pharmaceutical composition of the fifth aspect of the present invention.

In another preferred example, the disease is a tumor.

The eighth aspect of the present invention provides a method for preparing CAR-T cells (CAR-modified T cells), the CAR-T cells expressing the chimeric antigen receptor described in the first aspect of the present invention,

The method includes the step of: transducing the nucleic acid molecule according to the second aspect of the present invention or the vector according to the third aspect of the present invention into T cells, thereby obtaining the CAR-T cells.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1. Structural diagram of chimeric antigen receptor targeting CD20. The elements of the designed CAR structure are shown in the figure, and the listed elements include: leader sequence, antigen recognition sequence (Leu16), hinge region, transmembrane region, co-stimulatory factor signal region and CD3zeta signal transduction region. Among them, CAR-T20.17 and CAR-T18 are the L235E-N297Q mutant forms of CAR-T20.9 and CAR-T20.12 in the IgG4Hinge-CH2-CH3 junction region, respectively.

Figure 2. Detection of transfection efficiency of CD20-targeted chimeric antigen receptor-engineered T cells. The protein L method was used to identify the expression level of the protein encoded by the CAR gene on the surface of the T cell membrane in CAR-T20s cells cultured to day 7 (A) and day 11 (B).

Figure 3. Take 1*10 ⁵ CART-20 cells cultured to the 6th day in sequence, and respectively mix with CD20 positive RAJI and RAMOS tumor cell lines, and CD20 negative MOLT-4 tumor cell line or no tumor cells, in 200μl The expression level of CD137 on the surface of the T cell membrane (A) and the secretion level of IFNγ in the culture supernatant (B) were detected after co-culture with the CAR-T20 cells shown in the figure at a ratio of 1:1 in GT-551 medium for 18 hours.

Figure 4. Take 1*10 ⁵ CART-20 cells cultured to the 13th day, respectively, with CD20-positive RAJI and RAMOS tumor cell lines, and CD20-negative MOLT-4 tumor cell lines or without adding tumor cells, in 200 μl The expression level of CD137 on the surface of the T cell membrane (A) and the secretion level of IFNγ in the culture supernatant (B) were detected after co-culture with the CAR-T20 cells shown in the figure at a ratio of 1:1 in GT-551 medium for 18 hours.

Figure 5. Detection of early apoptosis level of tumor cells induced by CART-20. Take 1*10 ⁴ CFSE-labeled CD20-negative (MOLT-4) or CD20-positive (RAJI, RAMOS) tumor cell lines, respectively, in 200 μl GT-551 medium according to the proportion shown in the figure, and cultured to the 11th day. The CAR-T20 cells were co-cultured for 4 hours, and the cell pellet was collected by centrifugation. The cells were washed twice with PBS and then stained with Annexin V-APC dye at a ratio of 1:50 in 100 μl of the staining solution for 30 minutes. After washing with PBS once, they were placed in a flow cytometer. The ratio of Annexin V positive cells in CFSE positive cells was analyzed above. The graphed results show the statistical analysis results of Annexin V positive cells in the corresponding co-culture samples.

Figure 6. Detection of late apoptosis level of tumor cells induced by CART-20. Figure A shows the ratio of CART-positive cells in the sample analyzed; take 1*10 ⁴ CFSE-labeled CD20-negative (MOLT-4) or CD20-positive (RAJI, RAMOS) tumor cell lines respectively, and mix them in 200 μl GT-551 medium according to The ratio shown in the figure was co-cultured with the corresponding T cells for 4 hours, and the cell pellet was collected by centrifugation. The cells were washed twice with PBS and then stained with PI dye (PI/RNase Staining Buffer) in 100 μl of staining solution for 30 minutes. After washing once with PBS, the cells were lost The ratio of Annexin V positive cells in CFSE positive cells was analyzed on a cytometer. Panel B shows the results of statistical analysis of PI-positive cells in the corresponding co-culture samples.

Detailed ways

Through extensive and in-depth research, the present inventors constructed various types of chimeric antigen receptors targeting CD20 antigen based on the sequence of CD20 murine monoclonal antibody leu16, and analyzed these chimeric antigen receptors in the original Analysis and identification of the expression level in generation T cells, in vitro activation ability and tumor cell killing efficiency. Finally, the discovery of a chimeric antigen receptor with better anti-tumor activity provides a new effective method and preparation for the clinical application of CAR-T in the treatment of CD20-positive leukemia and lymphoma.

In view of the differences in the affinity and killing mechanism of therapeutic antibodies targeting CD20, as well as the significant impact of different transmembrane domains and intracellular domains on the activity of chimeric antigen receptors, in the present invention, multiple anti-CD20 antibodies are used The amino acid sequence structure of the variable region combined with different transmembrane and intracellular parts, established a series of chimeric antigen receptors targeting CD20, and completed the expression of such chimeric antigen receptors in primary T cells, established The method for detecting the intensity of receptor expression was identified, and the ability of these CAR-T cells to recognize the CD20 antigen in vitro and in vivo was identified, as well as the differences in their activity in killing malignant tumors carrying the CD20 antigen in vitro and in vivo, providing a basis for the clinical application of CAR-T in the treatment of CD20 Positive leukemia and lymphoma provide new effective methods and agents.

chimeric antigen receptor

The present invention provides chimeric antigen receptors (CARs) comprising an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes target-specific binding elements (also known as antigen binding domains). The intracellular domain includes the co-stimulatory signaling region and the zeta chain portion. A co-stimulatory signaling region refers to a portion of an intracellular domain that includes co-stimulatory molecules. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes to antigens.

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

In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen-binding domain targeting CD20. The CAR of the present invention, when expressed in T cells, is capable of antigen recognition based on antigen binding specificity. When it binds its cognate antigen, it affects tumor cells, causing them not to grow, being induced to die, or otherwise affected, and resulting in a reduction or elimination of the patient's tumor burden. The antigen binding domain is preferably fused to an intracellular domain from one or more of the co-stimulatory molecule and the zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of a combination of the 4-1BB signaling domain, and the CD3ζ signaling domain.

In one embodiment, the CD20-targeted CAR of the present invention includes the specific signaling domain of the present invention (the transmembrane region of CD8, the intracellular signaling domains of CD137 and CD3ζ formed in series). Compared with CD20-targeted CARs in other ways, the signaling domain of the present invention significantly increases the anti-tumor activity and the in vivo persistence of CAR-T cells.

In a preferred embodiment of the present invention, the amino acid sequence of the chimeric antigen receptor (CAR) provided by the present invention is as follows:

(CAR-T20.9 and CAR-T20.10, SEQ ID NO.1):

The coding DNA sequence of CAR-T20.9 is as follows:

The coding DNA sequence of CAR-T20.10:

In another preferred embodiment of the present invention, the amino acid sequence of the chimeric antigen receptor is as follows:

(CAR-T20.11)

The coding DNA sequence of CAR-T20.11 is as follows:

In another preferred embodiment of the present invention, the amino acid sequence of the chimeric antigen receptor is as follows:

(CAR-T20.12)

The coding DNA sequence of CAR-T20.12 is:

In another preferred embodiment of the present invention, the amino acid sequence of the chimeric antigen receptor is as follows:

(CAR-T20.17)

The coding DNA sequence of CAR-T20.17 is as follows:

In another preferred embodiment of the present invention, the amino acid sequence of the chimeric antigen receptor is as follows:

(CAR-T20.18)

The coding DNA sequence of CAR-T20.18 is as follows:

antigen binding domain

In one embodiment, the CAR of the invention includes a target-specific binding element known as an antigen binding domain. The antigen binding domain of the CAR of the present invention is a specific binding element targeting CD20.

In a preferred embodiment of the present invention, the antigen-binding domain includes the heavy chain variable region and the light chain variable region of an anti-CD20 antibody.

In another preferred example, the amino acid sequence of the antibody heavy chain variable region is as follows:

Its coding DNA sequence is as follows:

In another preferred example, the amino acid sequence of the antibody light chain variable region is as follows:

Its coding DNA sequence is as follows:

In a preferred embodiment of the present invention, the amino acid linking sequence between the heavy chain variable region and the light chain variable region is as follows:

GGGGSGGGGS GGGGS 15 (SEQ ID NO. 16)

Its coding DNA sequence is as follows:

GGTGGCGGTG GCTCGGGCGG TGGTGGGTCG GGTGGCGGCG GATCT 45 (SEQ ID NO. 17)

hinge region and transmembrane region

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

In a preferred embodiment of the present invention, the hinge region includes the following amino acid sequence (IgG4 Hinge-CH2-CH3 hinge region):

Its coding DNA sequence is as follows:

Alternatively, the hinge region comprises the following amino acid sequence (IgG4 Hinge-CH2-CH3(L235E, N297Q)):

Its coding DNA sequence is as follows:

In a preferred embodiment of the present invention, the transmembrane region in the CAR of the present invention is a transmembrane region derived from CD28 (CD28TM) or a transmembrane region derived from CD8 (CD8TM).

In a preferred embodiment of the present invention, the transmembrane region (CD8TM) amino acid sequence derived from CD8 is as follows:

IYIWAPLAGT CGVLLLSLVI TLYC 24 (SEQ ID NO. 22)

Its coding DNA sequence is as follows:

In a preferred embodiment of the present invention, the transmembrane region (CD28TM) amino acid sequence derived from CD28 is as follows:

FWVLVVVGGV LACYSLLVTV AFIIFWV 27 (SEQ ID NO. 24);

CD28-derived transmembrane region (CD28TM) coding DNA sequence:

intracellular domain

The intracellular domain in the CAR of the present invention includes the signaling domain of 4-1BB and the signaling domain of CD3ζ.

In a preferred embodiment of the present invention, the intracellular domain of the CAR further includes a signaling domain of CD28.

Preferably, the intracellular signaling domain of 4-1BB comprises the following amino acid sequence:

KRGRKKLLYI FKQPFMRPVQ TTQEEDGCSC RFPEEEEGGC EL 42 (SEQ ID NO. 26)

Its coding DNA sequence is as follows:

Preferably, the intracellular signaling domain derived from CD28 comprises the following amino acid sequence:

RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR S 41 (SEQ ID NO. 28)

Its coding DNA sequence is as follows:

Preferably, the intracellular signaling domain of CD3ζ comprises the following amino acid sequence:

Its coding DNA sequence is as follows:

carrier

The present invention also provides a DNA construct encoding the CAR sequence of the present invention.

A nucleic acid sequence encoding a desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by using standard technology, directly isolated from cells and tissues containing the gene. Alternatively, the gene of interest can be produced synthetically.

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

Briefly summarized, expression of a natural or synthetic nucleic acid encoding a CAR is generally achieved by operably linking a nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter, and incorporating the construct into an expression vector. This vector is suitable for replication and integration in eukaryotic cells. A typical cloning vector contains transcriptional and translational terminators, an initial sequence and a promoter useful for regulating the expression of the desired nucleic acid sequence.

The expression constructs of the invention can also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, eg, US Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety. In another embodiment, the present invention provides gene therapy vectors.

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

Furthermore, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO01/96584; WO01/29058; and US Patent No. 6,326,193).

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

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

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

In order to assess the expression of the CAR polypeptide or a part thereof, the expression vector introduced into the cell may also contain either or both of a selectable marker gene or a reporter gene, so as to seek transfected or infected cell populations from viral vectors. Identification and selection of expressing cells. In other aspects, selectable markers can be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. Typically, a reporter gene is a gene that is absent from or expressed by a recipient organism or tissue and that encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. Expression of the reporter gene is measured at an appropriate time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are known and can be prepared using known techniques or obtained commercially. Typically, the construct with the minimum of 5 flanking regions showing the highest level of reporter gene expression was identified as a promoter. Such a promoter region can be linked to a reporter gene and used to assess the ability of the agent to regulate promoter-driven transcription.

Methods of introducing genes into cells and expressing genes into cells are known in the art. In the context of an expression vector, the vector can be easily introduced into host cells, eg, mammalian, bacterial, yeast or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

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

Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used method of inserting genes into mammalian, eg human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, eg, US Patent Nos. 5,350,674 and 5,585,362.

Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipid-based systems. plastid. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (eg, an artificial membrane vesicle).

Where a non-viral delivery system is used, an exemplary delivery vehicle is liposomes. The use of lipid formulations is contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with a lipid. Lipid-associated nucleic acids can be encapsulated into the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, attached via linker molecules associated with both liposomes and oligonucleotides To liposomes, entrapped in liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, associated with lipids, contained in lipids as a suspension, contained in micelles or Complexes with micelles, or otherwise associated with lipids. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. For example, they may exist in a bilayer structure, as micelles or have a "collapsed" structure. They may also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which occur naturally in the cytoplasm as well as compounds comprising long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

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

In a preferred embodiment of the present invention, the DNA construct further includes a signal peptide coding sequence. Preferably, the signal peptide sequence is linked upstream of the nucleic acid sequence of the antigen tuberculosis domain. Preferably, the signal peptide is a human CD8a signal peptide.

Preferably, the amino acid sequence of the signal peptide is as follows:

Amino acid sequence of CD8 Leader sequence:

MALPVTALLL PLALLLHAAR P 21 (SEQ ID NO. 32)

CD8 leader sequence (CD8 Leader sequence) coding DNA sequence sequence:

ATGGCCTTAC CAGTGACCGC CTTGCTCCTG CCGCTGGCCT TGCTGCTCCA CGCCGCCAGG 60

CCG 63 (SEQ ID NO. 33).

therapeutic application

The invention includes cells (eg, T cells) transduced with a lentiviral vector (LV) encoding a CAR of the invention. Transduced T cells can elicit a CAR-mediated T-cell response.

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

In one embodiment, the invention includes a type of cell therapy in which T cells are genetically modified to express a CAR of the invention, and the CAR-T cells are infused into a recipient in need thereof. The injected cells are able to kill the recipient's tumor cells. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

In one embodiment, the CAR-T cells of the invention can undergo robust in vivo T cell expansion for extended amounts of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-modified T cells induce an immune response specific for the antigen-binding domain in the CAR. For example, anti-CD20 CAR-T cells elicit a specific immune response against CD20-expressing cells.

Although the data disclosed herein specifically disclose lentiviral vectors comprising an anti-CD20 scFv, hinge and transmembrane regions, and 4-1BB and CD3ζ signaling domains, the invention should be construed to include reference to each of the construct components. Any number of variations of one.

Treatable cancers include tumors that are not or substantially not vascularized, as well as vascularized tumors. Cancer can include non-solid tumors (such as hematological tumors, eg, leukemias and lymphomas) or can include solid tumors. Cancer types treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignancies, such as sarcomas, carcinomas, and melanomas. Also includes adult tumors/cancers and childhood tumors/cancers.

Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic , monocytic and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. The different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcomas, myxosarcomas, liposarcomas, mesotheliomas, lymphoid malignancies, pancreatic cancer, ovarian cancer, .

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

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

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

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

In general, cells activated and expanded as described herein are useful in the treatment and prevention of disease arising in immunocompromised individuals. In particular, the CAR-modified T cells of the present invention are used to treat CCL. In certain embodiments, cells of the invention are used to treat patients at risk of developing CCL. Accordingly, the present invention provides a method of treating or preventing CCL comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the present invention.

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

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

When an "immunologically effective amount", "antitumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the precise amount of a composition of the invention to be administered can be determined by a physician, taking into account the patient (subject ) with individual differences in age, weight, tumor size, degree of infection or metastasis, and disease. It may generally be stated that a pharmaceutical composition comprising T cells as described herein may be dosed at a dose of 10 ⁴ to 10 ⁹ cells/kg body weight, preferably at a dose of 10 ⁵ to 10 ⁶ cells/kg body weight (including all integers within those ranges value) applied. T cell compositions can also be administered multiple times at these doses. Cells can be administered using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical art by monitoring the patient for signs of disease and adjusting treatment accordingly.

Administration of the compositions to a subject may be by any convenient means, including by spraying, injection, swallowing, infusion, implantation or implantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by i.v. injection. Compositions of T cells can be injected directly into tumors, lymph nodes or sites of infection.

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

Dosages administered to a patient for the above treatments will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be implemented according to practice accepted in the art. Usually, 1×10 ⁶ to 1×10 ¹⁰ modified T cells (such as CAR-T20 cells) of the present invention can be administered to the patient for each treatment or each course of treatment, for example, through intravenous infusion .

Advantages of the present invention include:

(1) In the chimeric antigen receptor of the present invention, its extracellular antigen-binding domain is a specific anti-CD20scFv, and the CAR formed by the specific anti-CD20scFv in combination with a specific hinge region and an intracellular domain shows a great It has the ability to kill tumor cells, and has less cytotoxicity and low side effects.

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

(3) The CAR-modified T cells of the present invention have a longer survival time in vivo and stronger anti-tumor efficacy; the CAR after optimizing the IgG4Hinge-CH2-CH3 junction region can avoid the binding of Fc receptors and the subsequent ADCC effect (antibody-dependent cytotoxicity).

The construction of embodiment 1 lentiviral expression vector

The coding plasmid was entrusted to Shanghai Boyi Biotechnology Co., Ltd. for full-length DNA synthesis and cloning construction. The cloning vector is pWPT lentiviral vector, and the cloning sites are BamH I and Sal I sites. The specific sequence structure is shown in Figure 1. The amino acid and nucleotide sequences of each element are as described above.

Example 2 Preparation of CAR-T cells

(1) Take healthy human venous blood, and obtain mononuclear cells (PBMCs) by means of density gradient centrifugation.

(2) On day 0, PBMCs were cultured in GT-T551 cell culture medium containing 2% human serum albumin, and the final cell concentration was adjusted to 2×10 ⁶ cell/mL. Cells were inoculated into cell culture flasks previously coated with a final concentration of 5 μg/mL LCD3 monoclonal antibody (OKT3) and a final concentration of 10 μg/mL of Retronectin (purchased from TAKARA). Recombinant human interleukin 2 (IL-2) with a final concentration of 1000 U/mL was added to the medium, and cultured in an incubator at 37° C. with a saturated humidity of 5% CO ₂ .

(3) On the second day, add fresh culture medium, concentrate and purify CAR20s lentivirus solution, protamine sulfate (12ug/ml), and a final concentration of 1000U/mL IL-2. Place in a 37°C, 5% CO ₂ incubator for 12 hours after infection, discard the culture medium, add fresh medium, and continue culturing in a 37°C, 5% CO ₂ incubator.

(4) From day 6, CART20s cells can be used for corresponding activity detection tests.

Example 3 Detection of the integration rate of the CAR gene in the T cell genome and the expression level of the encoded protein on the membrane surface.

(1) Take 0.5×10 ⁶ CART-20s cell samples cultured to the 7th day (Fig. 2A) and 11th day (Fig. 2B) in Example 2, respectively, and analyze them on the flow cytometer after Protein L staining The expression level of CAR20 protein on the surface of T cell membrane. The results showed that the CAR structures designed in this study could be expressed in the corresponding modified T cells and localized on the cell membrane surface.

Example 4 Detection of CAR-T20s activation ability in vitro

(1) The CAR-T20s cells cultured to day 6 in Example 2 were used to detect the activation level of the indicator proteins CD137 and IFNγ. Take 1*10 ⁵ CART-20 cells cultured to the 6th day in turn, and respectively mix with CD20-positive RAJI and RAMOS tumor cell lines, and CD20-negative MOLT-4 tumor cell line or no tumor cells, in 200μl GT-551 The expression level of CD137 on the surface of the T cell membrane was detected by the elapsed time method after being cultured in the medium at a ratio of 1:1 for 18 hours (Fig. 3A), and the secretion level of IFNγ in the culture supernatant was detected by ELISA method (Fig. 3B).

(2) The CAR-T20s cells cultured to the 13th day in Example 2 were used to detect the cell activation level indicator proteins CD137 and IFNγ. Figure 4, 1*10 ⁵ CART-20 cells cultured to the 6th day were taken in turn, respectively mixed with CD20-positive RAJI and RAMOS tumor cell lines, and CD20-negative MOLT-4 tumor cell line or without adding tumor cells, in 200 μl After cultured in GT-551 medium at a ratio of 1:1 for 18 hours, the expression level of CD137 on the surface of T cells was detected by the elapsed method (Fig. 4A), and the secretion level of IFNγ in the culture supernatant was detected by ELISA (Fig. 4B).

(3) From the results in Figure 3 and Figure 4, we can draw the following conclusions: the third-generation CAR based on the Leu-16 sequence has better CD137 activation level and IFNγ release level than the second-generation CAR; Leu16VH-VL Or the sequence of VL-VH has no obvious difference in IFNgamma release and CD137 expression for the in vitro activity of CART20 constructed based on the leu-16 antibody sequence.

Example 5 Detection of Early Apoptotic Activity of Tumor Cells Induced by CAR-T20s Cells

(1) The CAR-T20s cells cultured to the 11th day in Example 2 were used with 1*10 ⁴ CFSE-labeled CD20-negative (MOLT-4) or CD20-positive (RAJI, RAMOS) tumor cell lines according to the ratio shown in the figure , co-cultured in 200 μl GT-551 medium for 4 hours, collected the cell pellet by centrifugation, washed the cells twice with PBS, stained with Annexin V-APC dye in 100 μl dyeing solution at a ratio of 1:50 for 30 minutes, washed once with PBS, and then lost The ratio of Annexin V positive cells in CFSE positive cells was analyzed on a cytometer.

(2) The result of Fig. 5 shows that the third-generation CAR constructed based on the Leu16 sequence can better induce the early apoptosis of CD20-positive tumor cells than the second-generation CAR.

Example 6 Detection of late apoptosis activity of tumor cells induced by CAR-T20s cells

(1) Another batch of CAR-T20 cells prepared by the method in Example 2 (Fig. 6A), were mixed with 1*10 ⁴ CFSE-labeled CD20-negative (MOLT-4) or CD20-positive cells according to the ratio shown in Fig. 6B. The (RAJI, RAMOS) tumor cell line was co-cultured in 200 μl GT-551 medium for 4 hours, and the cell pellet was collected by centrifugation. After the cells were washed twice with PBS, they were stained with PI dye in 100 μl dyeing solution at a ratio of 1:50 for 30 minutes, and washed with PBS. After 1 pass, the ratio of PI-positive cells among CFSE-positive cells was analyzed on a flow cytometer.

(2) The results shown in Fig. 6B show that the third-generation CAR (CAR-T20.9, CAR-T20.17) based on the Leu16 sequence is compared with the second-generation CAR (CAR-T20.12, CAR-T20.18) Can better induce late apoptosis of CD20 positive tumor cells.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately.

In addition, various combinations of different embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the idea of the present invention, they should also be regarded as the disclosed content of the present invention.

sequence listing

<110> CBM Biotechnology (Shanghai) Co., Ltd.

CBM Biotechnology (Wuxi) Co., Ltd.

<120> Construction of a CAR targeting CD20 and activity identification of engineered T cells

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

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

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agcgacatcg ccgtggagtg ggagagcaac ggccagcccg agaacaacta caagaccacc 1320

ccccccgtgc tggacagcga cggcagcttc ttcctgtaca gccgcctgac cgtggacaag 1380

agccgctggc aggagggcaa cgtgttcagc tgcagcgtga tgcacgaggc cctgcacaac 1440

cactacaccc agaagagcct gagcctgagc ctgggcaagt tctgggtgct ggtggtggtg 1500

ggcggcgtgc tggcctgcta cagcctgctg gtgaccgtgg ccttcatcat cttctgggtg 1560

cgcagcaagc gcagccgcct gctgcacagc gactacatga acatgacccc ccgccgcccc 1620

ggccccaccc gcaagcacta ccagccctac gccccccccc gcgacttcgc cgcctaccgc 1680

agcaagcgcg gccgcaagaa gctgctgtac atcttcaagc agcccttcat gcgccccgtg 1740

cagaccaccc aggagggagga cggctgcagc tgccgcttcc ccgaggagga ggagggcggc 1800

tgcgagctgc gcgtgaagtt cagccgcagc gccgacgccc ccgcctacaa gcagggccag 1860

aaccagctgt acaacgagct gaacctgggc cgccgcgagg agtacgacgt gctggacaag 1920

cgccgcggcc gcgaccccga gatgggcggc aagccccgcc gcaagaaccc ccaggagggc 1980

ctgtacaacg agctgcagaa ggacaagatg gccgaggcct acagcgagat cggcatgaag 2040

ggcgagcgcc gccgcggcaa gggccacgac ggcctgtacc agggcctgag caccgccacc 2100

aaggacacct acgacgccct gcacatgcag gccctgcccc cccgctaa 2148

<210> 4

<211> 673

<212> PRT

<213> Artificial sequence

<400> 4

Gly Ser Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

Arg

<210> 5

<211> 2022

<212>DNA

<213> Artificial sequence

<400> 5

ggatccatgg ccttaccagt gaccgccttg ctcctgccgc tggccttgct gctccacgcc 60

gccaggccgg acatcgttct gacccagtct ccagcaatct tgtctgcatc tccaggggag 120

aaggtcacca tgacctgcag agccagctca agtgtaaatt acatggactg gtaccagaag 180

aagccaggct cctcccccaa accttggatt tatgccacat ccaacctggc ttctggagtc 240

cctgctcgct tctctggcag tgggtctggg acctcttact ctctcacaat cagcagagtc 300

gaggctgaag atgctgccac ttattactgc cagcagtgga gcttcaaccc accacacgttc 360

ggtggtggga ccaagctgga gatcaaaggt ggcggtggct cgggcggtgg tgggtcgggt 420

ggcggcggat ctgaggtcca gctccagcag tctggagctg agctggtgaa gcctggggct 480

tcagtgaaga tgtcctgcaa ggcttctgga tacacattca ctagttataa tatgcactgg 540

gtaaagcaga cacctggaca gggcctggaa tggattggag ctatttatcc aggaaatggt 600

gatacttcct acaatcagaa gttcaagggc aaggccaacac tgactgcaga caaatcctcc 660

agcacagcct acatgcagct cagcagcctg acatctgaag actctgctga ctattactgt 720

gcgaggagta actactacgg tagtagctac tggttcttcg atgtctgggg cgcagggacc 780

acggtcaccg tctcttcaga gagcaagtac ggaccgccct gccccccttg ccctgccccc 840

gagttcctgg gcggacccag cgtgttcctg ttccccccca agcccaagga caccctgatg 900

atcagccgga cccccgaggt gacctgcgtg gtggtggacg tgagccagga agatcccgag 960

gtccagttca attggtacgt ggacggcgtg gaagtgcaca acgccaagac caagcccaga 1020

gaggaacagt tcaacagcac ctaccgggtg gtgtctgtgc tgaccgtgct gcaccaggac 1080

tggctgaacg gcaaagaata caagtgcaag gtgtccaaca agggcctgcc cagcagcatc 1140

gaaaagacca tcagcaaggc caagggccag cctcgcgagc cccaggtgta caccctgcct 1200

ccctcccagg aagagatgac caagaaccag gtgtccctga cctgcctggt gaagggcttc 1260

taccccagcg acatcgccgt ggagtggggag agcaacggcc agcctgagaa caactacaag 1320

accacccctc ccgtgctgga cagcgacggc agcttcttcc tgtacagccg gctgaccgtg 1380

gacaagagcc ggtggcagga aggcaacgtc tttagctgca gcgtgatgca cgaggccctg 1440

cacaaccact acacccagaa gagcctgagc ctgtccctgg gcaagatcta catctgggcg 1500

cccttggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccct ttactgcaaa 1560

cggggcagaa agaaactcct gtatatattc aaacaaccat ttatgagacc agtacaaact 1620

actcaagagg aagatggctg tagctgccga tttccagaag aagaagaagg aggatgtgaa 1680

ctgagagtga agttcagcag gagcgcagac gcccccgcgt acaagcaggg ccagaaccag 1740

ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 1800

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1860

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1920

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1980

acctacgacg cccttcacat gcaggccctg ccccctcgct ag 2022

<210> 6

<211> 671

<212> PRT

<213> Artificial sequence

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

<210> 7

<211> 2016

<212> DNA

<213> Artificial sequence

<400> 7

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaggtcc agctccagca gtctggagct gagctggtga agcctggggc ttcagtgaag 120

atgtcctgca aggcttctgg atacacattc actagttata atatgcactg ggtaaagcag 180

acacctggac agggcctgga atggattgga gctatttatc caggaaatgg tgatacttcc 240

tacaatcaga agttcaaggg caaggccaca ctgactgcag acaaatcctc cagcacagcc 300

tacatgcagc tcagcagcct gacatctgaa gactctgctg actattactg tgcgaggagt 360

aactactacg gtagtagcta ctggttcttc gatgtctggg gcgcagggac cacggtcacc 420

gtctcttcag gtggcggtgg ctcgggcggt ggtgggtcgg gtggcggcgg atctgacatc 480

gttctgaccc agtctccagc aatcttgtct gcatctccag gggagaaggt caccatgacc 540

tgcagagcca gctcaagtgt aaattacatg gactggtacc agaagaagcc aggctcctcc 600

cccaaacctt ggatttatgc cacatccaac ctggcttctg gagtccctgc tcgcttctct 660

ggcagtgggt ctgggacctc ttactctctc acaatcagca gagtcgaggc tgaagatgct 720

gccacttatt actgccagca gtggagcttc aacccaccca cgttcggtgg tgggaccaag 780

ctggagatca aagagagcaa gtacggaccg ccctgccccc cttgccctgc ccccgagttc 840

ctgggcggac ccagcgtgtt cctgttcccc cccaagccca aggacaccct gatgatcagc 900

cggacccccg aggtgacctg cgtggtggtg gacgtgagcc aggaagatcc cgaggtccag 960

ttcaattggt acgtggacgg cgtggaagtg cacaacgcca agaccaagcc cagagaggaa 1020

cagttcaaca gcacctaccg ggtggtgtct gtgctgaccg tgctgcacca ggactggctg 1080

aacggcaaag aatacaagtg caaggtgtcc aacaagggcc tgcccagcag catcgaaaag 1140

accatcagca aggccaaggg ccagcctcgc gagccccagg tgtacaccct gcctccctcc 1200

caggaagaga tgaccaagaa ccaggtgtcc ctgacctgcc tggtgaaggg cttctacccc 1260

agcgacatcg ccgtggagtg ggagagcaac ggccagcctg agaacaacta caagaccacc 1320

cctcccgtgc tggacagcga cggcagcttc ttcctgtaca gccggctgac cgtggacaag 1380

agccggtggc aggaaggcaa cgtctttagc tgcagcgtga tgcacgaggc cctgcacaac 1440

cactacaccc agaagagcct gagcctgtcc ctgggcaaga tctacatctg ggcgcccttg 1500

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg caaacggggc 1560

agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1620

gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1680

gtgaagttca gcaggagcgc agacgccccc gcgtacaagc agggccagaa ccagctctat 1740

aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1800

gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1860

ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1920

aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1980

gacgcccttc acatgcaggc cctgccccct cgctag 2016

<210> 8

<211> 715

<212> PRT

<213> Artificial sequence

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

705 710 715

<210> 9

<211> 2148

<212>DNA

<213> Artificial sequence

<400> 9

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaggtcc agctccagca gtctggagct gagctggtga agcctggggc ttcagtgaag 120

atgtcctgca aggcttctgg atacacattc actagttata atatgcactg ggtaaagcag 180

acacctggac agggcctgga atggattgga gctatttatc caggaaatgg tgatacttcc 240

tacaatcaga agttcaaggg caaggccaca ctgactgcag acaaatcctc cagcacagcc 300

tacatgcagc tcagcagcct gacatctgaa gactctgctg actattactg tgcgaggagt 360

aactactacg gtagtagcta ctggttcttc gatgtctggg gcgcagggac cacggtcacc 420

gtctcttcag gtggcggtgg ctcgggcggt ggtgggtcgg gtggcggcgg atctgacatc 480

gttctgaccc agtctccagc aatcttgtct gcatctccag gggagaaggt caccatgacc 540

tgcagagcca gctcaagtgt aaattacatg gactggtacc agaagaagcc aggctcctcc 600

cccaaacctt ggatttatgc cacatccaac ctggcttctg gagtccctgc tcgcttctct 660

ggcagtgggt ctgggacctc ttactctctc acaatcagca gagtcgaggc tgaagatgct 720

gccacttatt actgccagca gtggagcttc aacccaccca cgttcggtgg tgggaccaag 780

ctggagatca aagagagcaa gtacggaccg ccctgccccc cttgccctgc ccccgagttc 840

gagggcggac ccagcgtgtt cctgttcccc cccaagccca aggacaccct gatgatcagc 900

cggacccccg aggtgacctg cgtggtggtg gacgtgagcc aggaagatcc cgaggtccag 960

ttcaattggt acgtggacgg cgtggaagtg cacaacgcca agaccaagcc cagagaggaa 1020

cagttccaaa gcacctaccg ggtggtgtct gtgctgaccg tgctgcacca ggactggctg 1080

aacggcaaag aatacaagtg caaggtgtcc aacaagggcc tgcccagcag catcgaaaag 1140

accatcagca aggccaaggg ccagcctcgc gagccccagg tgtacaccct gcctccctcc 1200

caggaagaga tgaccaagaa ccaggtgtcc ctgacctgcc tggtgaaggg cttctacccc 1260

agcgacatcg ccgtggagtg ggagagcaac ggccagcctg agaacaacta caagaccacc 1320

cctcccgtgc tggacagcga cggcagcttc ttcctgtaca gccggctgac cgtggacaag 1380

agccggtggc aggaaggcaa cgtctttagc tgcagcgtga tgcacgaggc cctgcacaac 1440

cactacaccc agaagagcct gagcctgtcc ctgggcaagt tttgggtgct ggtggtggtt 1500

ggtggagtcc tggcttgcta tagcttgcta gtaacagtgg cctttattat tttctgggtg 1560

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 1620

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 1680

tccaaacggg gcagaaagaa actcctgtat atattcaaac aaccatttat gagaccagta 1740

caaactactc aagaggaaga tggctgtagc tgccgatttc cagaagaaga agaaggagga 1800

tgtgaactga gagtgaagtt cagcaggagc gcagacgccc ccgcgtacaa gcagggccag 1860

aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag 1920

agacgtggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc 1980

ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 2040

ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 2100

aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgctaa 2148

<210> 10

<211> 671

<212> PRT

<213> Artificial sequence

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

<210> 11

<211> 2016

<212> DNA

<213> Artificial sequence

<400> 11

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaggtcc agctccagca gtctggagct gagctggtga agcctggggc ttcagtgaag 120

atgtcctgca aggcttctgg atacacattc actagttata atatgcactg ggtaaagcag 180

acacctggac agggcctgga atggattgga gctatttatc caggaaatgg tgatacttcc 240

tacaatcaga agttcaaggg caaggccaca ctgactgcag acaaatcctc cagcacagcc 300

tacatgcagc tcagcagcct gacatctgaa gactctgctg actattactg tgcgaggagt 360

aactactacg gtagtagcta ctggttcttc gatgtctggg gcgcagggac cacggtcacc 420

gtctcttcag gtggcggtgg ctcgggcggt ggtgggtcgg gtggcggcgg atctgacatc 480

gttctgaccc agtctccagc aatcttgtct gcatctccag gggagaaggt caccatgacc 540

tgcagagcca gctcaagtgt aaattacatg gactggtacc agaagaagcc aggctcctcc 600

cccaaacctt ggatttatgc cacatccaac ctggcttctg gagtccctgc tcgcttctct 660

ggcagtgggt ctgggacctc ttactctctc acaatcagca gagtcgaggc tgaagatgct 720

gccacttatt actgccagca gtggagcttc aacccaccca cgttcggtgg tgggaccaag 780

ctggagatca aagagagcaa gtacggaccg ccctgccccc cttgccctgc ccccgagttc 840

gagggcggac ccagcgtgtt cctgttcccc cccaagccca aggacaccct gatgatcagc 900

cggacccccg aggtgacctg cgtggtggtg gacgtgagcc aggaagatcc cgaggtccag 960

ttcaattggt acgtggacgg cgtggaagtg cacaacgcca agaccaagcc cagagaggaa 1020

cagttccaaa gcacctaccg ggtggtgtct gtgctgaccg tgctgcacca ggactggctg 1080

aacggcaaag aatacaagtg caaggtgtcc aacaagggcc tgcccagcag catcgaaaag 1140

accatcagca aggccaaggg ccagcctcgc gagccccagg tgtacaccct gcctccctcc 1200

caggaagaga tgaccaagaa ccaggtgtcc ctgacctgcc tggtgaaggg cttctacccc 1260

agcgacatcg ccgtggagtg ggagagcaac ggccagcctg agaacaacta caagaccacc 1320

cctcccgtgc tggacagcga cggcagcttc ttcctgtaca gccggctgac cgtggacaag 1380

agccggtggc aggaaggcaa cgtctttagc tgcagcgtga tgcacgaggc cctgcacaac 1440

cactacaccc agaagagcct gagcctgtcc ctgggcaaga tctacatctg ggcgcccttg 1500

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg caaacggggc 1560

agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1620

gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1680

gtgaagttca gcaggagcgc agacgccccc gcgtacaagc agggccagaa ccagctctat 1740

aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1800

gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1860

ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1920

aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1980

gacgcccttc acatgcaggc cctgccccct cgctag 2016

<210> 12

<211> 122

<212> PRT

<213> Artificial sequence

<400> 12

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Ala Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 13

<211> 366

<212>DNA

<213> Artificial sequence

<400> 13

gaggtccagc tccagcagtc tggagctgag ctggtgaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact agttataata tgcactgggt aaagcagaca 120

cctggacagg gcctggaatg gattggagct atttatccag gaaatggtga tacttcctac 180

aatcagaagt tcaagggcaa ggccaacactg actgcagaca aatcctccag cacagcctac 240

atgcagctca gcagcctgac atctgaagac tctgctgact attackgtgc gaggagtaac 300

tactacggta gtagctactg gttcttcgat gtctggggcg cagggaccac ggtcaccgtc 360

tcttca 366

<210> 14

<211> 106

<212> PRT

<213> Artificial sequence

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 15

<211> 318

<212>DNA

<213> Artificial sequence

<400> 15

gacatcgttc tgacccagtc tccagcaatc ttgtctgcat ctccaggggga gaaggtcacc 60

atgacctgca gagccagctc aagtgtaaat tacatggact ggtaccagaa gaagccaggc 120

tcctccccca aaccttggat ttatgccaca tccaacctgg cttctggagt ccctgctcgc 180

ttctctggca gtgggtctgg gacctcttac tctctcacaa tcagcagagt cgaggctgaa 240

gatgctgcca cttattactg ccagcagtgg agcttcaacc cacccacgtt cggtggtggg 300

accaagctgg agatcaaa 318

<210> 16

<211> 15

<212> PRT

<213> Artificial sequence

<400> 16

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

1 5 10 15

<210> 17

<211> 45

<212> DNA

<213> Artificial sequence

<400> 17

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatct 45

<210> 18

<211> 229

<212> PRT

<213> Artificial sequence

<400> 18

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Leu Gly Lys

225

<210> 19

<211> 687

<212>DNA

<213> Artificial sequence

<400> 19

gagagcaagt acggaccgcc ctgcccccct tgccctgccc ccgagttcct gggcggaccc 60

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag 120

gtgacctgcg tggtggtgga cgtgagccag gaagatcccg aggtccagtt caattggtac 180

gtggacggcg tggaagtgca caacgccaag accaagccca gagaggaaca gttcaacagc 240

acctaccggg tggtgtctgt gctgaccgtg ctgcaccagg actggctgaa cggcaaagaa 300

tacaagtgca aggtgtccaa caagggcctg cccagcagca tcgaaaagac catcagcaag 360

gccaagggcc agcctcgcga gccccaggtg tacaccctgc ctccctccca ggaagagatg 420

accaagaacc aggtgtccct gacctgcctg gtgaagggct tctaccccag cgacatcgcc 480

gtggagtggg agagcaacgg ccagcctgag aacaactaca agaccacccc tcccgtgctg 540

gacagcgacg gcagcttctt cctgtacagc cggctgaccg tggacaagag ccggtggcag 600

gaaggcaacg tctttagctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagagcctga gcctgtccct gggcaag 687

<210> 20

<211> 229

<212> PRT

<213> Artificial sequence

<400> 20

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Leu Gly Lys

225

<210> 21

<211> 687

<212>DNA

<213> Artificial sequence

<400> 21

gagagcaagt acggaccgcc ctgcccccct tgccctgccc ccgagttcga gggcggaccc 60

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag 120

gtgacctgcg tggtggtgga cgtgagccag gaagatcccg aggtccagtt caattggtac 180

gtggacggcg tggaagtgca caacgccaag accaagccca gagaggaaca gttccaaagc 240

acctaccggg tggtgtctgt gctgaccgtg ctgcaccagg actggctgaa cggcaaagaa 300

tacaagtgca aggtgtccaa caagggcctg cccagcagca tcgaaaagac catcagcaag 360

gccaagggcc agcctcgcga gccccaggtg tacaccctgc ctccctccca ggaagagatg 420

accaagaacc aggtgtccct gacctgcctg gtgaagggct tctaccccag cgacatcgcc 480

gtggagtggg agagcaacgg ccagcctgag aacaactaca agaccacccc tcccgtgctg 540

gacagcgacg gcagcttctt cctgtacagc cggctgaccg tggacaagag ccggtggcag 600

gaaggcaacg tctttagctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagagcctga gcctgtccct gggcaag 687

<210> 22

<211> 24

<212> PRT

<213> Artificial sequence

<400> 22

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> 23

<211> 72

<212>DNA

<213> Artificial sequence

<400> 23

atctacatct gggcgccctt ggccggggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 24

<211> 27

<212> PRT

<213> Artificial sequence

<400> 24

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> 25

<211> 81

<212>DNA

<213> Artificial sequence

<400> 25

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt g 81

<210> 26

<211> 42

<212> PRT

<213> Artificial sequence

<400> 26

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> 27

<211> 126

<212>DNA

<213> Artificial sequence

<400> 27

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 28

<211> 41

<212> PRT

<213> Artificial sequence

<400> 28

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> 29

<211> 123

<212>DNA

<213> Artificial sequence

<400> 29

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 30

<211> 113

<212> PRT

<213> Artificial sequence

<400> 30

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Arg

<210> 31

<211> 336

<212>DNA

<213> Artificial sequence

<400> 31

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 32

<211> 21

<212> PRT

<213> Artificial sequence

<400> 32

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

1 5 10 15

His Ala Ala Arg Pro

20

<210> 33

<211> 63

<212>DNA

<213> Artificial sequence

<400> 33

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

Claims (10)

1. A chimeric antigen receptor (CAR), characterized in that the antigen binding domain of the chimeric antigen receptor comprises the antibody heavy chain variable region shown in SEQ ID NO.12, and SEQ ID NO. The antibody light chain variable region shown in 14. 2. The chimeric antigen receptor according to claim 1, wherein the antigen binding domain (scFv) of the chimeric antigen receptor is as shown in formula I or formula II: V _H -V _L ,(I); V _L -V _H ,(II) Wherein, V _H is the variable region of the heavy chain of the antibody; V _L is the variable region of the light chain of the antibody; "-" is the connecting peptide or a peptide bond. 3. The chimeric antigen receptor according to claim 2, wherein the structure of the chimeric antigen receptor is as follows: L-scFv-H-TM-C-CD3ζ in, L is an optional leader sequence (Leader sequence, ie signal peptide sequence); scFv is an antigen-binding domain; H is the hinge region; TM is the transmembrane domain; C is a co-stimulatory signal receptor tyrosine activation motif; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ; The antigen-binding domain and "-" are as above, respectively. 4. A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the chimeric antigen receptor (CAR) according to claim 1. 5. nucleic acid molecule as claimed in claim 4, is characterized in that, described nucleic acid molecule comprises the nucleic acid sequence of encoding described hinge region selected from the group: (a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.18 or 20; (b) a polynucleotide whose sequence is shown in SEQ ID NO.19 or 21; (c) The homology of the nucleotide sequence and the sequence shown in SEQ ID NO.19 or 21 ≥ 90% (preferably ≥ 95%), and the polynucleoside encoding the amino acid sequence shown in SEQ ID NO.18 or 20 acid; (d) a polynucleotide complementary to any of the polynucleotides described in (a)-(c); and / or The nucleic acid molecule comprises a nucleic acid sequence selected from the transmembrane region of the encoding CD8 of the following group: (a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.22; (b) a polynucleotide whose sequence is as shown in SEQ ID NO.23; (c) a polynucleotide whose nucleotide sequence is ≥90% (preferably ≥95%) homologous to the sequence shown in SEQ ID NO.23 and encodes the amino acid sequence shown in SEQ ID NO.22; (d) a polynucleotide complementary to any of the polynucleotides described in (a)-(c); and / or The nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of the transmembrane region of the CD28 encoded: (a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.24; (b) a polynucleotide whose sequence is shown in SEQ ID NO.25; (c) a polynucleotide whose nucleotide sequence is ≥90% (preferably ≥95%) homologous to the sequence shown in SEQ ID NO.25 and encodes the amino acid sequence shown in SEQ ID NO.24; (d) a polynucleotide complementary to any of the polynucleotides described in (a)-(c); and / or The nucleic acid molecule comprises a costimulatory signal receptor tyrosine activation motif coding sequence, and the costimulatory signal receptor tyrosine activation motif coding sequence includes a costimulatory signal receptor tyrosine activation motif derived from 4-1BB sequence coding sequence, and/or CD28-derived costimulatory signal receptor tyrosine activation motif coding sequence, wherein the costimulatory signal receptor tyrosine activation motif coding sequence derived from 4-1BB is selected from the following group: (a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.26; (b) a polynucleotide whose sequence is as shown in SEQ ID NO.27; (c) a polynucleotide whose nucleotide sequence is ≥90% (preferably ≥95%) homologous to the sequence shown in SEQ ID NO.27 and encodes the amino acid sequence shown in SEQ ID NO.26; (d) a polynucleotide complementary to any of the polynucleotides described in (a)-(c); The co-stimulatory signal receptor tyrosine activation motif coding sequence derived from CD28 is selected from the following group: (a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.28; (b) a polynucleotide whose sequence is shown in SEQ ID NO.29; (c) a polynucleotide whose nucleotide sequence is ≥90% (preferably ≥95%) homologous to the sequence shown in SEQ ID NO.29 and encodes the amino acid sequence shown in SEQ ID NO.28; (d) a polynucleotide complementary to any of the polynucleotides described in (a)-(c); and / or The nucleic acid molecule comprises the nucleic acid sequence of the intracellular signaling domain of the CD3ζ encoding selected from the following group: (a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.30; (b) a polynucleotide whose sequence is shown in SEQ ID NO.31; (c) a polynucleotide whose nucleotide sequence is ≥90% (preferably ≥95%) homologous to the sequence shown in SEQ ID NO.30 and encodes the amino acid sequence shown in SEQ ID NO.31; (d) A polynucleotide complementary to the polynucleotide described in any one of (a)-(c). 6. A carrier, characterized in that the carrier contains the nucleic acid molecule of claim 4. 7. A host cell, characterized in that the host cell contains the vector according to claim 6 or the nucleic acid molecule of claim 5 is integrated in the chromosome. 8. A pharmaceutical composition, characterized in that the composition contains a pharmaceutically acceptable carrier and the chimeric antigen receptor as claimed in claim 1, the nucleic acid molecule as claimed in claim 4, and the nucleic acid molecule as claimed in claim 6. The carrier, or the cell described in claim 7. 9. The use of the chimeric antigen receptor according to claim 1, the nucleic acid molecule according to claim 4, the carrier according to claim 6, or the cell according to claim 7, for the preparation of a drug for treating tumors or preparation. 10. A method for preparing CAR-T cells (CAR-modified T cells), characterized in that the CAR-T cells express the chimeric antigen receptor according to claim 1, The method comprises the step of: transducing the nucleic acid molecule according to claim 4 or the vector according to claim 6 into T cells, thereby obtaining the CAR-T cells.