CN111575241A

CN111575241A - T lymphocyte of chimeric chondroitin sulfate proteoglycan 4 receptor and preparation method and application thereof

Info

Publication number: CN111575241A
Application number: CN202010477197.3A
Authority: CN
Inventors: 陈健; 吴海涛; 邱士真; 周霆; 陈敏; 方一
Original assignee: Eye and ENT Hospital of Fudan University
Current assignee: Eye and ENT Hospital of Fudan University
Priority date: 2020-05-29
Filing date: 2020-05-29
Publication date: 2020-08-25
Anticipated expiration: 2040-05-29
Also published as: CN111575241B

Abstract

The invention provides a T lymphocyte of a chimeric chondroitin sulfate proteoglycan 4 receptor, which comprises an extracellular segment structure, a human CD28 transmembrane sequence and an intracellular segment structure; the extracellular domain structure is formed by connecting a signal peptide sequence, an optimized CSPG4 single-chain antibody sequence and a sequence taking a human immunoglobulin G2 mutant sequence as a hinge region in series in sequence; the gene sequence of the optimized CSPG4 single-chain antibody is shown in any one of SEQ ID NO. 33-42; the gene sequence of the hinge region is shown as SEQ ID NO. 31; the intracellular segment structure is formed by connecting a human CD28 or 41BB intracellular segment co-stimulatory domain sequence and a human CD3 zeta intracellular segment sequence in series. The invention also provides a preparation method of the T lymphocyte of the chimeric chondroitin sulfate proteoglycan 4 receptor. Simultaneously provides the application in preparing the medicine for treating the head and neck tumor. The optimized CSPG4.CAR-T cells can effectively kill CSPG4 positive tumor cells in vitro and in vivo.

Description

T lymphocyte of chimeric chondroitin sulfate proteoglycan 4 receptor and preparation method and application thereof

Technical Field

The invention belongs to the field of bioengineering, relates to a drug for treating tumors, and particularly relates to T lymphocyte (CSPG4.CAR-T) of a chimeric chondroitin sulfate proteoglycan 4 receptor, a preparation method and application thereof.

Background

The head and neck cancer refers to epithelial malignant tumor of upper respiratory digestive tract including nasal cavity, paranasal sinus, oral cavity, pharyngeal cavity (including nasopharynx, oropharynx, laryngopharynx) and larynx. Head and neck cancer is one of the common malignant tumors of human, the incidence rate of the head and neck cancer is 6 th of all malignant tumors, the head and neck cancer is frequently seen in men, and the head and neck cancer is related to age and better seen in people over 60 years old. Recent tumor statistics in the united states found about 6.8 million new head and neck cancers in the last 1 year, of which 70.4% were males. In our country, about 13 ten thousand new head and neck cancers occur every year, and about 7 ten thousand patients die of head and neck cancer. The pathological type of the head and neck cancer is mainly squamous carcinoma, accounting for more than 95%, so most head and neck cancers are Head and Neck Squamous Cell Carcinoma (HNSCC), and the HNSCC also becomes a main research object of the head and neck cancer. Surgery, radiotherapy and chemotherapy are still the main methods for the treatment of HNSCC at present. However, the important blood vessels of the head and the neck are dense in nerves and difficult to operate, and radiotherapy and chemotherapy have great damage to normal tissues and have the difficult problems of drug resistance and relapse, so the overall treatment effect is still not ideal at present, and the 5-year survival rate is only about 50%. Meanwhile, the requirements of patients on the postoperative life quality are continuously improved, and a treatment means with obvious curative effect and low side effect is urgently needed to be developed.

Immunotherapy is the fourth major therapy of tumor, which means that the specific recognition and killing effects of immune effector cells such as natural killer cells, CD8+ T cells and the like on tumor cells are improved by corresponding immunological means, and finally the purpose of treating tumor is achieved. The tumor immunotherapy does not generate drug resistance similar to that after radiotherapy and chemotherapy, has lasting curative effect and even immunological memory function, and can also design specific targeted therapy according to the specific self condition of a tumor patient so as to fit the idea of precise therapy. The tumor immunotherapy mainly comprises monoclonal antibody therapy, T cell therapy, tumor vaccine therapy, cytokine therapy and the like, wherein the superiority of the monoclonal antibody and the T cell therapy in the tumor therapy makes the monoclonal antibody and the T cell therapy increasingly stressed and have the potential of becoming first-line tumor therapy.

Chimeric Antigen Receptor (CAR) T cell therapy is currently the hottest of tumor immunotherapy research. The CAR is mainly formed by coupling 2 parts, one part is a single chain antibody (scFv) which can specifically bind to a tumor-associated antigen, and the other part is an activating receptor which can activate T cells to play immune effector functions. CAR-T therapy against the CD19 antigen on the surface of B cells has met with great success clinically in the treatment of B cell-derived hematological tumors, but has fallen into struggle in the study of the treatment of solid tumors, one of the most prominent reasons being the high heterogeneity of solid tumors and the lack of a stably expressed tumor-specific antigen. Therefore, the excavation of tumor-specific antigens to explore novel effective therapeutic targets is a difficult point and key to the current research on immunotherapy of all solid tumors, including HNSCC.

Chondroitin sulfate proteoglycan 4(CSPG4) is a cell surface proteoglycan, is strictly regulated in the distribution of normal tissue cells, is mainly expressed in normal tissues such as cartilage and the like, and has main functions possibly related to the growth, migration, adhesion and interaction with extracellular matrix of cells. CSPG4 was originally found in melanoma and later studies found that the neuro-glial antigen 2(NG2) present in the central nervous system was its cognate protein. The research detects postoperative specimens of various solid tumors including malignant melanoma, breast cancer, mesothelioma, pancreatic cancer, head and neck squamous cell carcinoma and the like, and finds that the expression of CSPG4 in tumors of many patients is obviously higher than that of normal tissues, but the specific action and mechanism of the CSPG4 in tumor development are not clear. The tumor stem cells are cells which have self-renewal capacity and can generate heterogeneous tumor cells in the tumor, have important action on the occurrence and development of the tumor, are insensitive to the conventional chemoradiotherapy means, and are one of the key factors of tumor recurrence after chemoradiotherapy. Research shows that CSPG4 expression is often found in tumor stem cells of malignant melanoma, brain glioma and colon cancer, and the research suggests that the targeted CSPG4 treatment may become a novel effective weapon for killing tumor stem cells and radically treating tumor recurrence.

The T lymphocyte of the chimeric chondroitin sulfate proteoglycan 4 receptor has an extracellular section structure which is formed by connecting a signal peptide sequence, a 763.74 or optimized scFv sequence and human immunoglobulin G1(IgG1) or optimized hinge sequence in series; the transmembrane region sequence is the transmembrane region sequence of CD 28; the intracellular segment structure is formed by connecting a human 41BB intracellular segment co-stimulatory domain sequence or a CD28 intracellular segment co-stimulatory domain sequence with a human CD3 zeta intracellular segment sequence in series. The structure schematic diagram is shown in FIG. 12:

signal peptide sequence: MEFGLSWLFLVAILKGVQCSR (SEQ ID NO. 43).

CD28 transmembrane sequence: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO. 44).

CD3 ζ sequence: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO. 45).

Firstly, the expression level of CSPG4 and the relation with a head and neck tumor stem cell marker CD44 are detected in head and neck tumor tissues and cell lines, and the feasibility and the value of CSPG4 as an immunotherapy target of head and neck cancer are explored. Meanwhile, current CSPG4 target-targeted cspg4.car T cells have been preliminarily demonstrated to be effective on cell lines of various tumors. However, the therapeutic effect of cspg4.car T reported at present is not significant enough, the structure has yet to be optimized, and no further study on the specific therapeutic effect of HNSCC is available. Based on the above research reports, we further designed and optimized a series of CARs targeting the CSPG4 protein and observed its therapeutic effect on HNSCC cell lines in vitro and in vivo.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides a T lymphocyte (CSPG4.CAR-T) of a chimeric chondroitin sulfate proteoglycan 4 receptor and a preparation method and application thereof, and the T lymphocyte (CSPG4.CAR-T) of the chimeric chondroitin sulfate proteoglycan 4 receptor and the preparation method and application thereof aim to solve the technical problem that the effect of the CSPG4.CAR T cell designed aiming at a CSPG4 target spot in the prior art on treating tumors is not obvious.

The invention provides a T lymphocyte of a chimeric chondroitin sulfate proteoglycan 4 receptor, which comprises an extracellular segment structure, a human CD28 transmembrane sequence and an intracellular segment structure; the extracellular domain structure is formed by connecting a signal peptide sequence, an optimized CSPG4 single-chain antibody sequence and a sequence taking a human immunoglobulin G2 mutant sequence as a hinge region in series in sequence; the gene sequence of the optimized CSPG4 single-chain antibody is shown in any one of SEQ ID NO. 33-42; the gene sequence of the hinge region is shown as SEQ ID NO. 31; the intracellular segment structure is formed by connecting a human CD28 or 41BB intracellular segment co-stimulatory domain sequence and a human CD3 zeta intracellular segment sequence in series.

Further, the sequence of the signal peptide is shown as SEQ ID NO. 43.

Further, the human CD28 transmembrane sequence is shown as SEQ ID NO. 44.

Further, the human CD3 zeta intracellular segment sequence is shown in SEQ ID NO. 45.

Furthermore, the human CD28 intracellular segment co-stimulatory domain sequence is shown in SEQ ID NO. 26.

Furthermore, the sequence of the human 41BB intracellular segment co-stimulatory domain is shown as SEQ ID NO. 27.

The invention also provides a preparation method of the T lymphocyte of the chimeric chondroitin sulfate proteoglycan 4 receptor, which comprises the following steps:

1) respectively synthesizing optimized CSPG4 single-chain antibody sequence and human immunoglobulin G2 mutant sequence; the gene sequence of the optimized CSPG4 single-chain antibody is shown in any one of SEQ ID NO. 33-42; the gene sequence of the hinge region is shown as SEQ ID NO. 31;

2) the optimized CSPG4 single-chain antibody sequence, the human immunoglobulin G2 mutant sequence, the human CD28 or 41BB intracellular segment co-stimulatory domain sequence and the human CD3 zeta intracellular segment sequence are connected and cloned into a vector;

3) resuspending the human kidney epithelial cell line in a DMEM medium containing 10% fetal calf serum by mass percentage, adding the lentivirus packaging plasmid and the vector in the step 2) into the human kidney epithelial cell line medium by using a liposome transfection system, collecting cell supernatant after 48 hours, and freezing and storing the cell supernatant in a refrigerator at-80 ℃ for later use;

4) culturing primary T cells in RPMI-1640 medium containing 10% by mass of fetal bovine serum, 100U/ml of penicillin and 100 mu g/ml of streptomycin sulfate; activating T cells by using magnetic beads coated with anti-CD 3 and anti-CD 28 antibodies, adding the lentivirus prepared in the step 3) after co-incubation for infection, removing cell supernatant after infection and replacing the cell supernatant with a fresh T cell complete culture medium; and culturing to obtain T lymphocyte of chimeric chondroitin sulfate proteoglycan 4 receptor.

The invention also provides application of the T lymphocyte of the chimeric chondroitin sulfate proteoglycan 4 receptor in preparing a medicament for treating head and neck tumors.

The invention also provides a vector which contains an extracellular segment structure, a human CD28 transmembrane sequence and an intracellular segment structure; the extracellular domain structure is formed by connecting a signal peptide sequence, an optimized CSPG4 single-chain antibody sequence and a sequence taking a human immunoglobulin G2 mutant sequence as a hinge region in series in sequence; the gene sequence of the optimized CSPG4 single-chain antibody is shown in any one of SEQ ID NO. 33-42; the gene sequence of the hinge region is shown as SEQ ID NO. 31; the intracellular segment structure is formed by connecting a human CD28 or 41BB intracellular segment co-stimulatory domain sequence and a human CD3 zeta intracellular segment sequence in series.

The invention also provides a lentivirus containing the vector.

We first found that there is high expression of CSPG4 protein in many head and neck malignant tumor specimens and cell lines, and that targeting CSPG4 has feasibility for treating head and neck squamous cell carcinoma. Meanwhile, the co-expression condition of CSPG4 and a head and neck tumor stem cell marker CD44 is found, and the fact that the target CSPG4 has important significance in treating head and neck squamous cell carcinoma is suggested.

We found that CSPG4 monoclonal antibody 763.74, which is currently studied, is used as a single chain antibody (scFv) sequence, human immunoglobulin G1(hIgG1) is used as a hinge region, and a CD28 or 41BB intracellular segment sequence is used for forming CSPG4.CAR-T cells, which can kill CSPG4 positive target cells in vitro well, but have poor capacity of killing targeted tumor cells in vivo.

In one aspect, the invention provides for reducing the affinity of cspg4. CARs for IgGFc receptors (Fc γ R) by optimizing the hinge region IgG1 sequence in the CAR structure. We attempted to truncate the CH2CH3 sequence of IgG1 (CHC), truncate (CHs), replace it with mutated IgG2(CHM) or replace it with a mutated truncated low affinity nerve growth factor receptor (NMS) sequence applied in cd44.CAR, and found that only cspg4.CHM. CAR efficiently expresses CAR on the cell membrane surface and recognizes CSPG4 protein. Further in vitro and in vivo studies demonstrated a significant improvement in the in vivo antitumor activity of cspg4.chm. car-T.

On the other hand, the scFv sequence of CSPG4 monoclonal antibody 763.74 is optimized, and the optimized CSPG4.CAR-T is also capable of specifically recognizing CSPG4 protein. This optimized CAR-T had significantly reduced self-activation and depletion signals and exhibited greater killing activity in vitro assays compared to CAR-T previously at 763.74 scfv. Optimized cspg4.car-T cells were better able to control tumor proliferation in tumor mouse models and were found to have more infiltration into the tumor microenvironment in tumor sections.

Compared with the prior art, the invention has remarkable technical progress. The invention designs and optimizes the CSPG4.CAR-T cell, and proves that the optimized CSPG4.CAR-T cell can effectively kill CSPG4 positive tumor cells in vitro and in vivo, thereby providing a new method and thought for the clinical treatment of CSPG4 positive solid tumors.

Drawings

FIG. 1.A: Q-PCR detects the transcription level of CSPG4 mRNA in head and neck tumors and paracancerous control tissues. B: the immunization group tests the CSPG4 protein expression level in the head and neck tumor and the paracancer control tissue. C: immunohistochemical detection of CSPG4 protein expression level in head and neck tumor and head and neck inflammatory polyp tissues.

FIG. 2. A: Q-PCR detects the transcription level of CSPG4 mRNA in multiple cell lines of head and neck tumors and a nasopharyngeal normal squamous epithelial cell line NP 69. B: western Blot was used to measure the expression level of total protein of CSPG4 in multiple cell lines of head and neck tumors and in NP69, a normal squamous epithelial cell line of nasopharynx. C: the flow-type technology detects the expression level of CSPG4 protein on the surface of cell membrane in a plurality of cell lines of head and neck tumor and a nasopharyngeal normal squamous epithelial cell line NP 69.

FIG. 3. A: the TCGA big database analyzed the correlation of the transcript levels of CSPG4 mRNA and CD44 mRNA in head and neck squamous carcinoma specimens. B: immunofluorescence detects the coexpression condition of CSPG4 protein and CD44 protein in the head and neck tumor specimen. C: flow-based technology was used to detect co-expression of CSPG4 and CD44 proteins in head and neck tumor cell lines.

FIG. 4. A: in vitro killing assay of target cells by cspg4.igg1.cd28.car and cspg4.igg1.41bb. car. B: cspg4.igg1.cd28.car and cspg4.igg1.41bb. car killing assay of target cells in vivo.

FIG. 5. A. flow assay for hinge region engineered CSPG4.CAR expression. And B, detecting the recognition capability of the hinge region modified CSPG4.CAR on the CSPG4 protein by using a flow technology.

FIG. 6.A in vitro killing assay of CSPG4.CAR. T with CHM as the hinge region. In vivo killing assay of cspg4.car. t cells with CHM as the hinge region.

Figure 7 flow-based assays detect expression of cspg4.cd28.car and cspg4.41bb.car T cells in the absence of antigen stimulation to activate the relevant receptor CD69 and ICOS and the T cell depletion-related receptor PD-1.

FIG. 8.A flow-based assay 763.74ScFv sequence optimized CSPG4.CAR expression. B: the flow technology detects the specific recognition capability of CSPG4.CAR on CSPG4 antigen after ScFv sequence optimization. And C, detecting the expression of an activation related receptor CD69 and a T cell exhaustion related receptor PD-1 of CSPG4.CAR after ScFv sequence optimization by using a flow technology.

Figure 9. a. 2 optimized cspg4.car infected human primary T cells were selected and the expression of the corresponding primary T cell activation-related receptor CD69 and T cell depletion-related receptor PD-1 was detected by flow-based techniques. B: optimized in vitro killing test of target cells by CSPG4.CAR. C: ELISA test the ability of optimized cspg4.car to secrete relevant cellular inflammatory factors when killing tumors.

Figure 10.a in vivo killing assay of target cells by cspg4.car after optimization. B: survival curves of tumor bearing mice after optimized cspg4.car. t cell treatment.

FIG. 11.A Western Blot assay optimized 4G10 protein expression levels of CSPG4.CAR at rest. B: immunofluorescence assay for T cell infiltration in tumor tissue after optimized cspg4.car. T cell treatment.

FIG. 12 is a schematic diagram showing the structure of T lymphocytes of the chimeric chondroitin sulfate proteoglycan 4 receptor.

Detailed Description

Example 1: expression of CSPG4 in head and neck squamous cell carcinoma

Detection of CSPG4 mRNA in tumors: fresh or frozen tumor tissue at-80 deg.C is cut into pieces, 1ml TRIzon reagent is added into about 50mg tissue, and then homogenized on a homogenizer, and mRNA is extracted by using the CW0581S kit of the Kangwei century according to the instructions. The extracted mRNA was assayed for concentration and then reverse transcribed into cDNA using Takara RR036A kit (Prov.) as per the protocol. The expression level of CSPG4 in the cDNA was then determined using a real-time fluorescence quantification kit (Takara RR420A) following the protocol, using the CSPG4 primer:

Former：ctggagaatggtggaagag(SEQ ID NO.1)

Reverse：ggacagtgacagtgaagg(SEQ ID NO.2)

GAPDH primer:

Former：caaggtcatccatgacaactttg(SEQ ID NO.3)

Reverse：gtccaccaccctgttgctgtag(SEQ ID NO.4)。

staining of tumor tissue sections: firstly, tumor tissues are immersed in 4% paraformaldehyde for fixation at 4 ℃, and white tablets are obtained after embedding and slicing (the thickness of the tablet is 4 microns). Then, the procedure was followed according to the instructions of the Ready-to-use immunohistochemistry kit (Shanghai Biotech, C506333-0020). CSPG4 antibody: atlas Antibodies HPA 002951. After staining, the tissue sections were recorded under a microscope at bright field. The immunohistochemical result is subjected to histochemical scoring (H-Score) by a pathologist with abundant experience, and the specific method comprises the steps of classifying the staining intensity of cells into 4 grades of no, weak, medium and strong, and recording 0 Score if no yellow particle-like precipitate exists; weak light yellow, 1 point is marked; brown yellow is middle, 2 points are marked; the dark brown-yellow color is strong, and 3 points are recorded. And multiplying the fraction by the percentage of the corresponding cell, wherein the total value is the score value in the range of 0-300.

As shown in fig. 1, a: the CSPG4 mRNA transcript levels in head and neck tumors were significantly higher than in paracancerous control tissues. B: the expression level of CSPG4 protein in the head and neck tumor is obviously higher than that of the paracancer control tissue. C: the expression level of CSPG4 protein in head and neck tumors was significantly higher than that of polyp tissue.

And (4) conclusion: the CSPG4 transcription and protein expression levels were abnormally increased in clinical specimens of head and neck squamous cell carcinoma.

Example 2: transcription and expression of CSPG4 in human head and neck squamous carcinoma cell line

CSPG4 mRNA detection in cell lines: the test cells were washed with PBS and the pellet was collected, and 1ml of TRIzon was added per 1,000,000 cells for lysis, followed by the same detection method as that of tumor mRNA in example 1.

Detecting the expression of CSPG4 protein in a cell line by Western blot: collecting cells, extracting total protein by a protein extraction kit (Biyun, P0013) according to the instruction, determining the protein concentration by a BCA method, adding a sample loading buffer solution to prepare an electrophoresis sample, performing polyacrylamide gel electrophoresis, performing membrane transfer, sealing, primary antibody and secondary antibody incubation, and then dripping an ECL developing reagent into a gel image processing system for developing and analyzing. The CSPG4 antibody used: abcam ab 139408.

Flow cytometry analysis of CSPG4 protein expression in cell lines: the test cells were washed with FACS buffer (phosphate buffered saline PBS + 2% fetal bovine serum), incubated with primary and secondary antibodies, and stained with DAPI, then tested on a BD LSR Fortessa flow cytometer (BD bioscience) and then analyzed for data using flowjosoftware (Tree Star). The CSPG4 antibody used: santa Cruz sc 53389.

As shown in fig. 2, a: the transcription level of CSPG4 mRNA in multiple cell lines of head and neck tumors is significantly higher than that of the normal squamous epithelial cell line NP 69. B: the total protein expression level of CSPG4 in multiple cell lines of head and neck tumors is significantly higher than that of the normal squamous epithelial cell line NP 69. C: the CSPG4 protein is mainly expressed on the surface of cell membrane in multiple cell lines of head and neck tumor.

The conclusion is that the transcription of CSPG4 in the head and neck squamous carcinoma cell line is obviously higher than that of normal nasopharyngeal epithelial cell NP69, the expression level of the total protein of CSPG4 is also obviously increased, and the CSPG4 is mainly positioned outside the cell membrane, thus providing the possibility of CAR-T targeted therapy.

Example 3: CSPG4 has co-expression characteristics with tumor stem cell marker CD44 in head and neck squamous carcinoma

Cancer and genetic tumor map (TCGA) big data analysis: the gene transcript information of all head and neck squamous carcinoma specimens is downloaded from TCGA big database (website: https:// cancer. nih. gov), and correlation of CSPG4 and CD44 gene transcription levels is analyzed by Pearson test.

Tumor tissue sections CSPG4 co-stained with CD 44: the procedure for obtaining white tumor tissue slices was the same as in example 1. The white slices are sealed after baking, dewaxing, hydrating, antigen repairing, enzyme inactivation, sealing, primary and secondary antibody incubation and DAPI counterstaining, and finally observed and analyzed under a fluorescence microscope. The antibodies used were: CSPG4 antibody: atlas Antibodies HPA 002951; CD44 antibody: biolegend 103006.

Flow cytometry analysis of CSPG4 and CD44 protein co-expression in head and neck squamous carcinoma cell lines: the specific test procedure is shown in example 2. The CSPG4 antibody used: santa Cruz sc 53389; CD44 antibody: miltenyi Biotec 130-.

As shown in fig. 3, a: analysis of a TCGA big database finds that the transcription level of CSPG4 mRNA and the transcription level of CD44 mRNA in a head and neck squamous carcinoma specimen are positively correlated. B: immunofluorescence shows that the CSPG4 protein and CD44 protein in the head and neck tumor specimen are widely co-expressed. C: the cell line expressing the CD44 protein in the head and neck tumor also expresses the CSPG4 protein.

And (4) conclusion: tumor cells expressing a tumor stem cell marker CD44 in head and neck squamous cell carcinoma often express CSPG4 protein at the same time, and targeted therapy of CSPG4 positive cells is expected to kill tumor stem cells of head and neck tumors.

Example 4: preliminary construction and killing effect of CAR

Primary construction of car plasmid and lentivirus: the single-chain antibody (scFv) sequence of CSPG4 monoclonal antibody 763.74, human immunoglobulin G1(hIgG1) as a hinge sequence, a transmembrane sequence of human CD28 as a transmembrane region, an intracellular segment sequence of human CD28 or 41BB as a co-stimulatory molecule, and finally a tandem human CD3 zeta intracellular segment sequence form a primary CSPG4.CAR, namely CSPG4.hIgG1.CD28 and CSPG4.hIgG1.41BB CAR. The amino acid sequence was codon-optimized, converted to a base sequence and synthesized by the company (Qinglan Biotech). The amino acid sequence is shown in the following table:

the base sequences of all CARs were finally cloned into the pHR-SFFV-IRES-EGFP vector by means of Gibson ligation (NEB # E2611L). 293T cells (TaKaRa #632180) were resuspended in DMEM medium (Gibco #11995-065) containing 10% fetal bovine serum and 500ng of the lentiviral packaging plasmids pCMVdR8.92(Addge #8455) and pMD2.G (Addge #12259) and 500ng of the CSPG4.CAR plasmid were added to 293T cell medium using the Lipofectin system (Mirus #2300) following the protocol of the Lipofectin. After 48 hours, cell supernatants were collected and stored frozen in a freezer at-80 ℃ for further use.

Human primary T cell culture and lentivirus infection: human primary T cells were collected from healthy, informed volunteers. Primary T cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100U/ml penicillin and 100. mu.g/ml streptomycin sulfate (all reagents were purchased from Gibco). To maintain proliferation of T cells, 100U/ml hIL-2(Sigma-Aldrich) was added to the medium. T cells were activated using magnetic beads (Life Technologies #11132D) coated with anti-CD 3 and anti-CD 28 antibodies. T cells were incubated with magnetic beads at 1: 3 for 24 hours, and then adding the prepared lentivirus for infection. After 18 hours the cell supernatant was removed and replaced with fresh T cell complete medium. After 5 days of T cell stimulation with magnetic beads, the beads were removed and fresh complete T cell culture medium was added every 2-3 days.

CAR-T in vitro killing assay: and mixing K562 target cells expressing CSPG4 and mCherry fluorescence double-positive with K562 non-target cells not expressing CSPG4 and mCherry fluorescence according to a cell ratio of 1:1, and incubating the mixture with CAR-T cells according to a cell ratio of a specific experimental arrangement for 3 days before detecting by an up-flow cytometer. Analyzing the proportion (CK%) of the target cells in the K562 mixed cell group without the T cells as a control group; t cells were differentiated from the K562 cell population by CD3 staining in the T cell-containing panel, and then the proportion (EX%) of K562 target cells in the K562 cell population was analyzed, and the killing efficiency of T cells was (1-EX%/CK%) × 100%)

CAR-T in vivo killing assay 5 to 8 week old immunodeficient (NSG) mice were inoculated subcutaneously on the back with 1 × 10⁶K562-CSPG4 target cells expressing firefly luciferase (Luc) gene, and NSG mouse tail vein injection of 5 × 10 after the target cells are stable in vivo for 4 days⁶Individual CAR-T cells were treated. Tumor proliferation in mice was next monitored weekly by animal in vivo imaging techniques. The specific implementation operation is as follows: injecting firefly luciferin substrate into the abdominal cavity of the tumor-bearing mouse, wherein the substrate dosage is 1.5mg/g of the weight of the mouse; after 10 minutes the substrate was fully circulated to the whole body of the mouse, the mouse was anesthetized with 2.5% -3.5% isoflurane gas for imaging. Bioluminescence imaging was performed by IVIS spectral imaging system (Perkin Elmer) and fluorescence quantification data was obtained by in vivo imaging software (Perkin Elmer).

As shown in fig. 4, a: cspg4.igg1.cd28.car and cspg4.igg1.41bb. car are effective in killing tumor cells in vitro. B: cspg4.igg1.cd28.car and cspg4.igg1.41bb. car are not effective in controlling tumor proliferation in vivo.

And (4) conclusion: cspg4.higg1.cd28 and cspg4.higg1.41bb CAR T killed target cells well in vitro but hardly controlled tumor proliferation at all in vivo.

Example 5: optimization of CAR hinge region Structure

Optimization of car hinge region structure: since it has been reported that the crystallizable fragment (Fc) function threshold CH2CH3 sequence of hIgG1 antibody has a high affinity for the Fc γ receptor of other cells, which may thus cause non-specific activation of the corresponding CAR T cells, leading to a reduced tumor suppression effect in the complex cellular environment in vivo, we attempted to optimize the hIgG1 hinge region CH2CH3 sequence of the cspg4.CAR structure. We tried to remove the CH2CH3 sequence (CH2CH3 Cut, CHC), truncate (CH2CH3 Short, CHS), mutate (CH2CH3 Mutant, CHM) and replace it with a Mutant truncated low affinity nerve growth factor receptor (NMS), using unmodified CH2CH3(CH2CH3 Wide type, CHW) as a control, and carrying out subsequent detection verification, and the specific plasmid construction method is shown in example 4. The amino acid sequences used are shown in the following table:

the expression and antigen recognition ability detection of CSPG4.CAR after hinge region modification was verified by first we detected the expression level of CSPG4.CAR by flow technique using antibodies recognizing hIgG or NMS. We subsequently constructed a CSPG4 knock-out cell line (CSPG4-KO) using CRISPR/Cas9 technology, and further treated with CSPG4 wild type (CSPG4-WT) and CSPG4-KO and CSPG4.car T cells at 3: 1 for 24 hours, the expression level of the T cell surface activating receptor CD69 was measured by flow technique, and the specific recognition ability of CSPG4.car T on CSPG4 antigen was measured. Flow assay method referring to example 2, the hIgG antibody used: jackson ImmunoResearch 109-; NMS antibody: BD 560326; CD69 antibody: biolegend 310906.

As shown in FIG. 5, A-the expression of CSPG4.CAR after engineering the sequence of hinge region IgG1. And B, the recognition capability of the CSPG4 protein of the CSPG4.CAR with the sequence of the hinge region IgG1 modified, and only the CSPG4 antigen can be specifically recognized by the CSPG4.CAR with the mutated CHM as the hinge region.

And (4) conclusion: only the CSPG4.CAR with CHM as the hinge region can normally express the CSPG4.CAR and can specifically recognize CSPG4 antigen.

Example 6: in vitro and in vivo killing effect of CSPG4.CHM. CAR

We constructed cspg4.chm.cd28.car and cspg4.chm.41bb. car T, respectively, to test their killing effect on target cells in vitro and in vivo, and the specific procedure was as in example 4.

As shown in fig. 6, a cspg4.car. t cells with CHM as the hinge region can effectively kill tumor cells in vitro. Cspg4.car. t cells with CHM as the hinge region can significantly inhibit but fail to completely control tumor proliferation in vivo.

And (4) conclusion: cspg4.chm.cd28 and cspg4.chm.41bb CAR T killed target cells well in vitro and inhibited but did not completely control tumor proliferation in vivo.

Example 7: CAR has strong activation and depletion signals in resting state

In order to find out the reason for poor tumor killing effect of cspg4.car T in vivo, we detected the expression levels of the activation-related proteins CD69, ICOS and the depletion-related protein PD-1 of cspg4.car T in the resting state by using a flow-type technique, and the specific operation method refers to example 2. The CD69 antibody used was Biolegend 310906; ICOS antibody: biolegend 313520; PD-1 antibody: eBioscience 12-2799-42.

As shown in figure 7, cspg4.cd28.car and cspg4.41bb.car T cells also expressed activation-related receptors CD69 and ICOS without antigen stimulation, while also expressing the T cell depletion-related receptor PD-1.

And (4) conclusion: cspg4.cd28.car and cspg4.41bb. car T express strong activation and depletion signals in the resting state, which may be related to their poor tumor killing effect in vivo.

Example 8: optimization of car scFv sequence

We optimized the framework structure of 763.74scFv sequence and examined and evaluated the expression ability, specific antigen recognition ability and self-activation and depletion signals in resting state of optimized cspg4.car, as described in examples 5 and 7. Optimized cspg4.car scFv amino acid sequence is shown in the table below.

As shown in FIG. 8, the expression of CSPG4.CAR was optimized by the ScFv sequence 763.74, and it was found that CSPG4.CAR was expressed well and at an increased level in each optimized version. B: CSPG4.CAR can specifically recognize CSPG4 antigen after optimizing ScFv sequence 763.74. After optimizing the ScFv sequence of 763.74, the expression of the self-activation signal CD69 and the depletion signal PD-1 of CSPG4.CAR are both reduced obviously.

And (4) conclusion: the optimized CSPG4.CAR can normally express CSPG4.CAR on T cell membranes and can specifically recognize CSPG4 antigen, and the self-activation and depletion signals in a resting state are remarkably reduced.

Example 9: in vitro killing effect of scFv optimized CSPG4.CAR

We selected 2 representative optimized scfvs, h-CSPG4 and m-CSPG4-5, and then prepared lentiviruses to infect human primary T cells, and flow-technology examined their self-activation and depletion signals, as well as killing effect on target cells in vitro (see example 4). Meanwhile, the inflammatory cytokine secreted by the T cell in the killing process is detected by an ELISA method, cell supernatant is taken and processed according to the instruction of a kit, and then the absorbance is detected at 450nm under an enzyme-linked immunosorbent assay, and analysis data is recorded. The ELISA kit used: interferon gamma (IFN- γ): BD 555142; tumor necrosis factor α (TNF- α): BD 555212; interleukin 2 (IL-2): BD 555190.

As shown in fig. 9, a 2 optimized cspg4.car infected human primary T cells were selected and found to have significantly reduced expression of both the self-activation signal CD69 and the depletion signal PD-1. B: the optimized CSPG4.CAR has stronger effect of killing the tumor in vitro. C: the optimized CSPG4.CAR has better capability of secreting cell factors and inflammatory factors when killing tumors.

And (4) conclusion: H-CSPG4.CHM.41BB.CAR and m-CSPG4-5.CHM.41BB.CAR T cells are remarkably reduced in the self-activating signal CD69 and the exhaustion signal PD-1 in the resting state, and the in vitro killing capacity and the secretion capacity of the cell inflammatory factors are remarkably enhanced.

Example 10: in vivo killing effect of scFv optimized CSPG4.CAR

We further tested the in vivo killing effect of h-cspg4.chm.41bb. car and m-CSPG4-5.chm.41bb. cart cells on NSG mice, quantitated the growth curve of the tumor and the survival time of the mice, and specifically conducted reference example 4.

As shown in fig. 10, the ability of optimized cspg4.car to kill tumors in vivo was significantly enhanced. B: the optimized CSPG4.CAR. T cells can obviously prolong the survival time of tumor-bearing mice.

And (4) conclusion: h-CSPG4.CHM.41BB. CAR and m-CSPG4-5.CHM.41BB. CAR T cells can significantly control the proliferation of tumors and prolong the survival time of tumor-bearing mice in vivo experiments.

Example 11: mechanism analysis of killing effect improvement of scFv optimized CSPG4.CAR

We further analyzed the level of autophosphorylation of h-cspg4.chm.41bb. car and m-CSPG4-5.chm.41bb. car T cells at rest using the anti-phosphotyrosine antibody clone 4G10 by Western Blot assay, working with reference to example 2. Meanwhile, tumors in mice in vivo experiments are taken out for immunofluorescence analysis of infiltration conditions of T cells, Ki67 reflects proliferation conditions of the tumors, GFP and CD3 suggest infiltration conditions of the T cells, and the specific operation method refers to example 3.

As shown in fig. 11, the phosphorylation level of cspg4.car in resting state after optimization was significantly reduced. B: optimized cspg4.car. t cells were able to better infiltrate in the tumor microenvironment.

And (4) conclusion: the autophosphorylation level of h-CSPG4.CHM.41BB. CAR and m-CSPG4-5.CHM.41BB. CAR T cells in an unstimulated state is obviously lower than that before optimization, the cells can be more widely proliferated and infiltrated in tumors, and the cells are favorable for better exerting tumor inhibition effect in the tumors in the bodies of the cells.

Sequence listing

<110> eye, ear, nose and throat department hospital affiliated to the university of Compound Dan

<120> T lymphocyte of chimeric chondroitin sulfate proteoglycan 4 receptor, preparation method and application thereof

<160>45

<170>SIPOSequenceListing 1.0

<210>1

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

ctggagaatg gtggaagag 19

<210>2

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

ggacagtgac agtgaagg 18

<210>3

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

caaggtcatc catgacaact ttg 23

<210>4

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

gtccaccacc ctgttgctgt ag 22

<210>5

<211>238

<212>PRT

<213>Homo sapiens

<400>5

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

1 5 10 15

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

20 25 30

Asp Tyr Ser Met His Trp Val Lys Lys Thr Pro Gly Lys Gly Leu Lys

35 40 45

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

50 55 60

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

65 70 75 80

Val Tyr Leu Gln Ile Asn Asn Leu Arg Asn Glu Asp Thr Ala Thr Tyr

85 90 95

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

100 105 110

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

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

130 135 140

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

145 150 155 160

Lys Asn Leu His Trp Tyr Gln Gln Lys Ser His Arg Ser Pro Arg Leu

165 170 175

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

180 185 190

Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Asn Ile Asn Ser Val

195 200 205

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

210 215 220

Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

225 230 235

<210>6

<211>231

<212>PRT

<213>Homo sapiens

<400>6

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

1 5 10 15

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

20 25 30

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

35 40 45

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

50 55 60

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

65 70 75 80

Asn Ser Thr Tyr Arg Val Val SerVal Leu Thr Val Leu His Gln Asp

85 90 95

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

100 105 110

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

115 120 125

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

130 135 140

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

145 150 155 160

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

165 170 175

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

180 185 190

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

195 200 205

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

210 215 220

Leu Ser Leu Ser Pro Gly Lys

225 230

<210>7

<211>41

<212>PRT

<213>Homo sapiens

<400>7

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>8

<211>42

<212>PRT

<213>Homo sapiens

<400>8

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>9

<211>231

<212>PRT

<213>Homo sapiens

<400>9

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

1 5 1015

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

20 25 30

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

35 40 45

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

50 55 60

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

65 70 75 80

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

85 90 95

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

100 105 110

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

115 120 125

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

130 135 140

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

145 150 155 160

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

165 170 175

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

180 185 190

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

195 200 205

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

210 215 220

Leu Ser Leu Ser Pro Gly Lys

225 230

<210>10

<211>14

<212>PRT

<213>Homo sapiens

<400>10

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10

<210>11

<211>124

<212>PRT

<213>Homo sapiens

<400>11

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

1 5 10 15

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

20 25 30

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

35 40 45

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

50 55 60

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

65 70 75 80

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

85 90 95

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

100 105 110

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

115 120

<210>12

<211>228

<212>PRT

<213>Homo sapiens

<400>12

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Ser Pro Gly Lys

225

<210>13

<211>140

<212>PRT

<213>Homo sapiens

<400>13

Lys Glu Ala Cys Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys

1 5 10 15

Lys Ala Cys Asn Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn

20 25 30

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

35 40 45

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

50 55 60

Gln Ser Met Ser Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg

65 70 75 80

Cys Ala Tyr Gly Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala

85 90 95

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

100 105 110

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

115 120 125

Glu Ala Ala Arg Ala Ala Asp Ala Glu Cys Glu Glu

130 135 140

<210>14

<211>234

<212>PRT

<213>Homo sapiens

<400>14

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Trp Ile Asn Thr Ala Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

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

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

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

115 120 125

Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys Glu

130 135 140

Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Tyr Lys Asn Leu

145 150 155 160

His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys

165 170 175

Tyr Gly Ser Asp Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser

180 185 190

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu

195 200 205

Asp Ala Ala Thr Tyr Tyr Cys Leu Gln Gly Tyr Ser Thr Pro Trp Thr

210 215 220

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

225 230

<210>15

<211>234

<212>PRT

<213>Homo sapiens

<400>15

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

1 5 10 15

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

20 25 30

Ser Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Ala Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

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

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp

115 120 125

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

130 135 140

Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Tyr Lys Asn Leu

145 150 155 160

His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys

165 170 175

Tyr Gly Ser Asp Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser

180 185 190

Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr Glu

195 200 205

Asp Phe Gly Met Tyr Phe Cys Leu Gln Gly Tyr Ser Thr Pro Trp Thr

210 215 220

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

225 230

<210>16

<211>234

<212>PRT

<213>Homo sapiens

<400>16

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Trp Ile Asn Thr Ala Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

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

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp

115 120 125

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

130 135 140

Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Tyr Lys Asn Leu

145 150 155 160

His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys

165 170 175

Tyr Gly Ser Asp Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser

180 185 190

Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr Glu

195 200 205

Asp Phe Gly Met Tyr Phe Cys Leu Gln Gly Tyr Ser Thr Pro Trp Thr

210 215 220

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

225 230

<210>17

<211>234

<212>PRT

<213>Homo sapiens

<400>17

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

1 5 10 15

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

20 25 30

Ser Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Ala Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe Cys

85 90 95

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

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp

115 120 125

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

130 135 140

Arg Val SerLeu Ser Cys Arg Ala Ser Gln Thr Ile Tyr Lys Asn Leu

145 150 155 160

His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys

165 170 175

Tyr Gly Ser Asp Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser

180 185 190

Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr Glu

195 200 205

Asp Phe Gly Met Tyr Phe Cys Leu Gln Gly Tyr Ser Thr Pro Trp Thr

210 215 220

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

225 230

<210>18

<211>234

<212>PRT

<213>Homo sapiens

<400>18

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

1 5 10 15

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

20 25 30

Ser Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Ala Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

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

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp

115 120 125

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

130 135 140

Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Tyr Lys Asn Leu

145 150 155 160

His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys

165 170 175

Tyr Gly Ser Asp Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser

180 185 190

Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr Glu

195 200 205

Asp Phe Gly Met Tyr Phe Cys Leu Gln Gly Tyr Ser Thr Pro Trp Thr

210 215 220

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

225 230

<210>19

<211>234

<212>PRT

<213>Homo sapiens

<400>19

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

1 5 10 15

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

20 25 30

Ser Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Ala Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

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

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp

115 120 125

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

130 135 140

Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Tyr Lys Asn Leu

145 150 155 160

His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys

165 170 175

Tyr Gly Ser Asp Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser

180 185 190

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

195 200 205

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

210 215 220

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

225 230

<210>20

<211>234

<212>PRT

<213>Homo sapiens

<400>20

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Trp Ile Asn Thr Ala Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

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

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp

115 120 125

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

130 135 140

Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Tyr Lys Asn Leu

145 150 155 160

His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys

165 170 175

Tyr Gly Ser Asp Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser

180 185 190

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

195 200 205

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

210 215 220

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

225 230

<210>21

<211>234

<212>PRT

<213>Homo sapiens

<400>21

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

1 5 10 15

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

20 25 30

Ser Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Ala Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe Cys

85 90 95

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

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp

115 120 125

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

130 135 140

Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Tyr Lys Asn Leu

145 150 155 160

His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys

165 170 175

Tyr Gly Ser Asp Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser

180 185 190

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

195 200 205

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

210 215 220

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

225 230

<210>22

<211>234

<212>PRT

<213>Homo sapiens

<400>22

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Trp Ile Asn Thr Ala Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

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

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp

115 120 125

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

130 135 140

Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Tyr Lys Asn Leu

145 150 155 160

His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys

165 170 175

Tyr Gly Ser Asp Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser

180 185 190

Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr Glu

195 200 205

Asp Phe Gly Met Tyr Phe Cys Leu Gln Gly Tyr Ser Thr Pro Trp Thr

210 215 220

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

225 230

<210>23

<211>234

<212>PRT

<213>Homo sapiens

<400>23

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

1 5 10 15

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

20 25 30

Ser Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

3540 45

Gly Trp Ile Asn Thr Ala Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe Cys

85 90 95

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

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp

115 120 125

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

130 135 140

Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Tyr Lys Asn Leu

145 150 155 160

His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys

165 170 175

Tyr Gly Ser Asp Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser

180 185 190

Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr Glu

195 200205

Asp Phe Gly Met Tyr Phe Cys Leu Gln Gly Tyr Ser Thr Pro Trp Thr

210 215 220

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

225 230

<210>24

<211>714

<212>DNA

<213>Homo sapiens

<400>24

atggcccagg tcaaactgaa ggagtctgga cctgagctga agaagcctgg agagacagtc 60

aagatctcct gcaaggcttc tggttatacc ttcacagact attcaatgca ctgggtgaag 120

aagactccag gaaagggttt aaagtggctg ggctggataa acactgcgac tggtgagcca 180

acatatgcag atgacttcaa gggacggttt gccatctctt tggaaacctc tgccaggact 240

gtctatttgc agatcaataa tctcagaaat gaggacacgg ctacatattt ctgttttagt 300

tactacgact actggggcca aggcaccacg gtcaccgtct cctcaggtgg gggcggttca 360

ggcggaggtg gctctggcgg tggcggattg gacatcaagc tcagtcagtc tccatccatc 420

ctgtctgtga ctccaggtga aacagtcagt ctttcctgta gggccagcca gactatttac 480

aagaacctac actggtatca acagaaatca catcggtctc caaggcttct catcaagtat 540

ggttctgatt ccatctctgg catcccctcc aggttcactg gcagtggatc agggacagat 600

tacactctca atatcaacag tgtgaagccc gaagatgaag gaatatatta ctgtcttcaa 660

ggttacagta caccttggac gttcggtgga gggaccaagc tggaaataaa acgg 714

<210>25

<211>684

<212>DNA

<213>Homo sapiens

<400>25

gagcgcaaat gttgtgtcga gtgcccaccg tgcccagcac cacctgtggc aggaccgtca 60

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 120

acgtgcgtgg tggtggacgt gagccacgaa gaccccgagg tccagttcaa ctggtacgtg 180

gacggcgtgg aggtgcataa tgccaagaca aagccacggg aggagcagtt ccaaagcacg 240

ttccgtgtgg tcagcgtcct caccgttgtg caccaggact ggctgaacgg caaggagtac 300

aagtgcaagg tctccaacaa aggcctccca gcccccatcg agaaaaccat ctccaaaacc 360

aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 420

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 480

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 540

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 600

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660

agcctctccc tgtctccggg taaa 684

<210>26

<211>123

<212>DNA

<213>Homo sapiens

<400>26

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210>27

<211>158

<212>DNA

<213>Homo sapiens

<400>27

cagatccagc tggtgcagag cggccccgag ctaaacgggg cagaaagaaa ctcctgtata 60

tattcaaaca accatttatg agaccagtac aaactactca agaggaagat ggctgtagct 120

gccgatttcc agaagaagaa gaaggaggat gtgaactg 158

<210>28

<211>684

<212>DNA

<213>Homo sapiens

<400>28

gagcgcaaat gttgtgtcga gtgcccaccg tgcccagcac cacctgtggc aggaccgtca 60

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 120

acgtgcgtgg tggtggacgt gagccacgaa gaccccgagg tccagttcaa ctggtacgtg 180

gacggcgtgg aggtgcataa tgccaagaca aagccacggg aggagcagtt ccaaagcacg 240

ttccgtgtgg tcagcgtcct caccgttgtg caccaggact ggctgaacgg caaggagtac 300

aagtgcaagg tctccaacaa aggcctccca gcccccatcg agaaaaccat ctccaaaacc 360

aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 420

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 480

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 540

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 600

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660

agcctctccc tgtctccggg taaa 684

<210>29

<211>42

<212>DNA

<213>Homo sapiens

<400>29

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc ca 42

<210>30

<211>372

<212>DNA

<213>Homo sapiens

<400>30

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 60

ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 120

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 180

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 240

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 300

aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 360

tccaaagcca aa 372

<210>31

<211>684

<212>DNA

<213>Homo sapiens

<400>31

gagcgcaaat gttgtgtcga gtgcccaccg tgcccagcac cacctgtggc aggaccgtca 60

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 120

acgtgcgtgg tggtggacgt gagccacgaa gaccccgagg tccagttcaa ctggtacgtg 180

gacggcgtgg aggtgcataa tgccaagaca aagccacggg aggagcagtt ccaaagcacg 240

ttccgtgtgg tcagcgtcct caccgttgtg caccaggact ggctgaacgg caaggagtac 300

aagtgcaagg tctccaacaa aggcctccca gcccccatcg agaaaaccat ctccaaaacc 360

aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 420

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 480

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 540

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 600

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660

agcctctccc tgtctccggg taaa 684

<210>32

<211>420

<212>DNA

<213>Homo sapiens

<400>32

aaagaggcct gccccaccgg cctgtacacc cacagcggag agtgctgcaa ggcctgcaac 60

ctgggagagg gcgtggccca gccttgcggc gccaatcaga ccgtgtgcga gccctgcctg 120

gacagcgtga ccttcagcga cgtggtgtcc gccaccgagc cctgcaagcc ttgcaccgag 180

tgtgtgggcc tgcagagcat gagcgccccc tgcgtggaag ccgacgacgc cgtgtgtaga 240

tgcgcctacg gctactacca ggacgagaca accggcagat gcgaggcctg tagagtgtgc 300

gaggccggca gcggcctggt gttcagttgt caggacaagc agaacaccgt gtgtgaagag 360

tgccccgacg gcacctacag cgacgaggcc gcccgggccg ccgacgccga gtgcgaggaa 420

<210>33

<211>702

<212>DNA

<213>Homo sapiens

<400>33

caggtgcagc tggtgcagag cggcagcgag ctgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc gactacagca tgcactgggt gaggcaggcc 120

cccggccagg gcctggagtg gatgggctgg atcaacaccg ccaccggcga gcccacctac 180

gccgacgact tcaagggcag gttcgtgttc agcctggaca ccagcgtgag caccgcctac 240

ctgcagatca gcagcctgaa ggccgaggac accgccgtgt actactgctt cagctactac 300

gactactggg gccagggcac caccgtgacc gtgagcagcg gtgggggcgg ttcaggcgga 360

ggtggctctg gcggtggcgg agagatcgtg ctgacccaga gccccgactt ccagagcgtg 420

acccccaagg agaaggtgac catcacctgc agggccagcc agaccatcta caagaacctg 480

cactggtacc agcagaagcc cgaccagagc cccaagctgc tgatcaagta cggcagcgac 540

agcatcagcg gcatccccag caggttcagc ggcagcggca gcggcaccga cttcaccctg 600

accatcaaca gcctggaggc cgaggacgcc gccacctact actgcctgca gggctacagc 660

accccctgga ccttcggcgg cggcaccaag gtggagatca ag 702

<210>34

<211>702

<212>DNA

<213>Homo sapiens

<400>34

cagatccagc tggtgcagag cggccccgag ctgaagaagc ccggcgagac cgtgaagatc 60

agctgcaagg ccagcggcta caccttcacc gactacagca tgaactgggt gaagcaggcc 120

cccggcaagg gcctgaagtg gatgggctgg atcaacaccg ccaccggcga gcccacctac 180

gccgacgact tcaagggcag gttcgccttc agcctggaga ccagcgccag caccgcctac 240

ctgcagatca acaacctgaa gaacgaggac accgccacct acttctgctt cagctactac 300

gactactggg gccagggcac caccctgacc gtgagcagcg gtgggggcgg ttcaggcgga 360

ggtggctctg gcggtggcgg agacatcgtg ctgacccaga gccccgccac cctgagcgtg 420

acccccggcg acagggtgag cctgagctgc agggccagcc agaccatcta caagaacctg 480

cactggtacc agcagaagag ccacgagagc cccaggctgc tgatcaagta cggcagcgac 540

agcatcagcg gcatccccag caggttcagc ggcagcggca gcggcaccga cttcaccctg 600

agcatcaaca gcgtggagac cgaggacttc ggcatgtact tctgcctgca gggctacagc 660

accccctgga ccttcggcgg cggcaccaag ctggagatca ag 702

<210>35

<211>702

<212>DNA

<213>Homo sapiens

<400>35

cagatccagc tggtgcagag cggccccgag ctgaagaagc ccggcgagac cgtgaagatc 60

agctgcaagg ccagcggcta caccttcacc gactacagca tgcactgggt gaagcaggcc 120

cccggcaagg gcctgaagtg gatgggctgg atcaacaccg ccaccggcga gcccacctac 180

gccgacgact tcaagggcag gttcgccttc agcctggaga ccagcgccag caccgcctac 240

ctgcagatca acaacctgaa gaacgaggac accgccacct acttctgctt cagctactac 300

gactactggg gccagggcac caccctgacc gtgagcagcg gtgggggcgg ttcaggcgga 360

ggtggctctg gcggtggcgg agacatcgtg ctgacccaga gccccgccac cctgagcgtg 420

acccccggcg acagggtgag cctgagctgc agggccagcc agaccatcta caagaacctg 480

cactggtacc agcagaagag ccacgagagc cccaggctgc tgatcaagta cggcagcgac 540

agcatcagcg gcatccccag caggttcagc ggcagcggca gcggcaccga cttcaccctg 600

agcatcaaca gcgtggagac cgaggacttc ggcatgtact tctgcctgca gggctacagc 660

accccctgga ccttcggcgg cggcaccaag ctggagatca ag 702

<210>36

<211>702

<212>DNA

<213>Homo sapiens

<400>36

cagatccagc tggtgcagag cggccccgag ctgaagaagc ccggcgagac cgtgaagatc 60

agctgcaagg ccagcggcta caccttcacc gactacagca tgaactgggt gaagcaggcc 120

cccggcaagg gcctgaagtg gatgggctgg atcaacaccg ccaccggcga gcccacctac 180

gccgacgact tcaagggcag gttcgccttc agcctggaga ccagcgccag caccgcctac 240

ctgcagatca acaacctgaa gaacgaggac atggccacct acttctgctt cagctactac 300

gactactggg gccagggcac caccctgacc gtgagcagcg gtgggggcgg ttcaggcgga 360

ggtggctctg gcggtggcgg agacatcgtg ctgacccaga gccccgccac cctgagcgtg 420

acccccggcg acagggtgag cctgagctgc agggccagcc agaccatcta caagaacctg 480

cactggtacc agcagaagag ccacgagagc cccaggctgc tgatcaagta cggcagcgac 540

agcatcagcg gcatccccag caggttcagc ggcagcggca gcggcaccga cttcaccctg 600

agcatcaaca gcgtggagac cgaggacttc ggcatgtact tctgcctgca gggctacagc 660

accccctgga ccttcggcgg cggcaccaag ctggagatca ag 702

<210>37

<211>702

<212>DNA

<213>Homo sapiens

<400>37

cagatccagc tggtgcagag cggccccgag ctgaagaagc ccggcgagac cgtgaagatc 60

agctgcaagg ccagcggcta caccttcacc gactacagca tgaactgggt gaagcaggcc 120

cccggcaagg gcctgaagtg gatgggctgg atcaacaccg ccaccggcga gcccacctac 180

gccgacgact tcaagggcag gttcgccttc agcctggaga ccagcgccag caccgcctac 240

ctgcagatca acaacctgaa gaacgaggac accgccacct acttctgctt cagctactac 300

gactactggg gccagggcac caccctgacc gtgagcagcg gtgggggcgg ttcaggcgga 360

ggtggctctg gcggtggcgg agacatcgtg ctgacccaga gccccgccac cctgagcgtg 420

acccccggcg acagcgtgag cctgagctgc agggccagcc agaccatcta caagaacctg 480

cactggtacc agcagaagag ccacgagagc cccaggctgc tgatcaagta cggcagcgac 540

agcatcagcg gcatccccag caggttcagc ggcagcggca gcggcaccga cttcaccctg 600

agcatcaaca gcgtggagac cgaggacttc ggcatgtact tctgcctgca gggctacagc 660

accccctgga ccttcggcgg cggcaccaag ctggagatca ag 702

<210>38

<211>702

<212>DNA

<213>Homo sapiens

<400>38

cagatccagc tggtgcagag cggccccgag ctgaagaagc ccggcgagac cgtgaagatc 60

agctgcaagg ccagcggcta caccttcacc gactacagca tgaactgggt gaagcaggcc 120

cccggcaagg gcctgaagtg gatgggctgg atcaacaccg ccaccggcga gcccacctac 180

gccgacgact tcaagggcag gttcgccttc agcctggaga ccagcgccag caccgcctac 240

ctgcagatca acaacctgaa gaacgaggac accgccacct acttctgctt cagctactac 300

gactactggg gccagggcac caccctgacc gtgagcagcg gtgggggcgg ttcaggcgga 360

ggtggctctg gcggtggcgg agacatcgtg atgacccaga gccccgccac cctgagcgtg 420

acccccggcg acagggtgag cctgagctgc agggccagcc agaccatcta caagaacctg 480

cactggtacc agcagaagag ccacgagagc cccaggctgc tgatcaagta cggcagcgac 540

agcatcagcg gcatccccag caggttcagc ggcagcggca gcggcagcga cttcaccctg 600

agcatcaaca gcgtggagcc cgaggacgtc ggcgtgtact actgcctgca gggctacagc 660

accccctgga ccttcggcgg cggcaccaag ctggagatca ag 702

<210>39

<211>702

<212>DNA

<213>Homo sapiens

<400>39

cagatccagc tggtgcagag cggccccgag ctgaagaagc ccggcgagac cgtgaagatc 60

agctgcaagg ccagcggcta caccttcacc gactacagca tgcactgggt gaagcaggcc 120

cccggcaagg gcctgaagtg gatgggctgg atcaacaccg ccaccggcga gcccacctac 180

gccgacgact tcaagggcag gttcgccttc agcctggaga ccagcgccag caccgcctac 240

ctgcagatca acaacctgaa gaacgaggac accgccacct acttctgctt cagctactac 300

gactactggg gccagggcac caccctgacc gtgagcagcg gtgggggcgg ttcaggcgga 360

ggtggctctg gcggtggcgg agacatcgtg atgacccaga gccccgccac cctgagcgtg 420

acccccggcg acagggtgag cctgagctgc agggccagcc agaccatcta caagaacctg 480

cactggtacc agcagaagag ccacgagagc cccaggctgc tgatcaagta cggcagcgac 540

agcatcagcg gcatccccag caggttcagc ggcagcggca gcggcagcga cttcaccctg 600

agcatcaaca gcgtggagcc cgaggacgtc ggcgtgtact actgcctgca gggctacagc 660

accccctgga ccttcggcgg cggcaccaag ctggagatca ag 702

<210>40

<211>702

<212>DNA

<213>Homo sapiens

<400>40

cagatccagc tggtgcagag cggccccgag ctgaagaagc ccggcgagac cgtgaagatc 60

agctgcaagg ccagcggcta caccttcacc gactacagca tgaactgggt gaagcaggcc 120

cccggcaagg gcctgaagtg gatgggctgg atcaacaccg ccaccggcga gcccacctac 180

gccgacgact tcaagggcag gttcgccttc agcctggaga ccagcgccag caccgcctac 240

ctgcagatca acaacctgaa gaacgaggac atggccacct acttctgctt cagctactac 300

gactactggg gccagggcac caccctgacc gtgagcagcg gtgggggcgg ttcaggcgga 360

ggtggctctg gcggtggcgg agacatcgtg atgacccaga gccccgccac cctgagcgtg 420

acccccggcg acagggtgag cctgagctgc agggccagcc agaccatcta caagaacctg 480

cactggtacc agcagaagag ccacgagagc cccaggctgc tgatcaagta cggcagcgac 540

agcatcagcg gcatccccag caggttcagc ggcagcggca gcggcagcga cttcaccctg 600

agcatcaaca gcgtggagcc cgaggacgtc ggcgtgtact actgcctgca gggctacagc 660

accccctgga ccttcggcgg cggcaccaag ctggagatca ag 702

<210>41

<211>702

<212>DNA

<213>Homo sapiens

<400>41

cagatccagc tggtgcagag cggccccgag ctgaagaagc ccggcgagac cgtgaagatc 60

agctgcaagg ccagcggcta caccttcacc gactacagca tgcactgggt gaagcaggcc 120

cccggcaagg gcctgaagtg gatgggctgg atcaacaccg ccaccggcga gcccacctac 180

gccgacgact tcaagggcag gttcgccttc agcctggaga ccagcgccag caccgcctac 240

ctgcagatca acaacctgaa gaacgaggac accgccacct acttctgctt cagctactac 300

gactactggg gccagggcac caccctgacc gtgagcagcg gtgggggcgg ttcaggcgga 360

ggtggctctg gcggtggcgg agacatcgtg ctgacccaga gccccgccac cctgagcgtg 420

acccccggcg acagcgtgag cctgagctgc agggccagcc agaccatcta caagaacctg 480

cactggtacc agcagaagag ccacgagagc cccaggctgc tgatcaagta cggcagcgac 540

agcatcagcg gcatccccag caggttcagc ggcagcggca gcggcaccga cttcaccctg 600

agcatcaaca gcgtggagac cgaggacttc ggcatgtact tctgcctgca gggctacagc 660

accccctgga ccttcggcgg cggcaccaag ctggagatca ag 702

<210>42

<211>702

<212>DNA

<213>Homo sapiens

<400>42

cagatccagc tggtgcagag cggccccgag ctgaagaagc ccggcgagac cgtgaagatc 60

agctgcaagg ccagcggcta caccttcacc gactacagca tgaactgggt gaagcaggcc 120

cccggcaagg gcctgaagtg gatgggctgg atcaacaccg ccaccggcga gcccacctac 180

gccgacgact tcaagggcag gttcgccttc agcctggaga ccagcgccag caccgcctac 240

ctgcagatca acaacctgaa gaacgaggac atggccacct acttctgctt cagctactac 300

gactactggg gccagggcac caccctgacc gtgagcagcg gtgggggcgg ttcaggcgga 360

ggtggctctg gcggtggcgg agacatcgtg ctgacccaga gccccgccac cctgagcgtg 420

acccccggcg acagcgtgag cctgagctgc agggccagcc agaccatcta caagaacctg 480

cactggtacc agcagaagag ccacgagagc cccaggctgc tgatcaagta cggcagcgac 540

agcatcagcg gcatccccag caggttcagc ggcagcggca gcggcaccga cttcaccctg 600

agcatcaaca gcgtggagac cgaggacttc ggcatgtact tctgcctgca gggctacagc 660

accccctgga ccttcggcgg cggcaccaag ctggagatca ag 702

<210>43

<211>21

<212>PRT

<213>Homo sapiens

<400>43

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Ser Arg

20

<210>44

<211>27

<212>PRT

<213>Homo sapiens

<400>44

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>45

<211>112

<212>PRT

<213>Homo sapiens

<400>45

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 2530

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Claims

1.A T lymphocyte of a chimeric chondroitin sulfate proteoglycan 4 receptor, comprising: comprises an extracellular segment structure, a human CD28 transmembrane sequence and an intracellular segment structure; the extracellular domain structure is formed by connecting a signal peptide sequence, an optimized CSPG4 single-chain antibody sequence and a sequence taking a human immunoglobulin G2 mutant sequence as a hinge region in series in sequence; the gene sequence of the optimized CSPG4 single-chain antibody is shown in any one of SEQ ID NO. 33-42; the gene sequence of the hinge region is shown as SEQ ID NO. 31; the intracellular segment structure is formed by connecting a human CD28 or 41BB intracellular segment co-stimulatory domain sequence and a human CD3 zeta intracellular segment sequence in series.

2. The T lymphocyte of a chimeric chondroitin sulfate proteoglycan 4 receptor of claim 1, wherein: the sequence of the signal peptide is shown in SEQ ID NO. 43.

3. The T lymphocyte of a chimeric chondroitin sulfate proteoglycan 4 receptor of claim 1, wherein: the transmembrane sequence of the human CD28 is shown as SEQ ID NO. 44.

4. The T lymphocyte of a chimeric chondroitin sulfate proteoglycan 4 receptor of claim 1, wherein: the human CD3 zeta intracellular segment sequence is shown in SEQ ID NO. 45.

5. The T lymphocyte of a chimeric chondroitin sulfate proteoglycan 4 receptor of claim 1, wherein: the human CD28 intracellular segment co-stimulatory domain sequence is shown in SEQ ID NO. 26.

6. The T lymphocyte of a chimeric chondroitin sulfate proteoglycan 4 receptor of claim 1, wherein: the sequence of the human 41BB intracellular segment co-stimulatory domain is shown as SEQ ID NO. 27.

7. The method for producing a T lymphocyte of a chimeric chondroitin sulfate proteoglycan 4 receptor of claim 1, comprising the steps of:

1) respectively synthesizing optimized CSPG4 single-chain antibody sequence and hinge region formed by human immunoglobulin G2 mutant sequence; the gene sequence of the optimized CSPG4 single-chain antibody is shown in any one of SEQ ID NO. 33-42; the gene of the hinge region is shown as SEQ ID NO. 31;

2) the optimized CSPG4 single-chain antibody sequence, the human immunoglobulin G2 mutation sequence, the human CD28 transmembrane sequence, the human CD28 or 41BB intracellular segment co-stimulatory domain sequence and the human CD3 zeta intracellular segment sequence are cloned into a vector after being connected;

4) culturing primary T cells in RPMI-1640 medium containing 10% by mass of fetal bovine serum, 100U/ml of penicillin and 100 mu g/ml of streptomycin sulfate; activating T cells by using magnetic beads coated with anti-CD 3 and anti-CD 28 antibodies, adding the lentivirus prepared in the step 3) after co-incubation for infection, removing cell supernatant after infection, and replacing the cell supernatant with a fresh complete T cell culture medium; and culturing to obtain T lymphocyte of chimeric chondroitin sulfate proteoglycan 4 receptor.

8. Use of a chimeric chondroitin sulfate proteoglycan 4 receptor T lymphocyte of claim 1 in the preparation of a medicament for treating a head and neck tumor.

9.A carrier, characterized by: contains extracellular segment structure, human CD28 transmembrane sequence and intracellular segment structure; the extracellular domain structure is formed by connecting a signal peptide sequence, an optimized CSPG4 single-chain antibody sequence and a sequence taking a human immunoglobulin G2 mutant sequence as a hinge region in series in sequence; the gene sequence of the optimized CSPG4 single-chain antibody is shown in any one of SEQ ID NO. 33-42; the gene sequence of the hinge region is shown as SEQ ID NO. 31; the intracellular segment structure is formed by connecting a human CD28 or 41BB intracellular segment co-stimulatory domain sequence and a human CD3 zeta intracellular segment sequence in series.

10.A lentivirus, characterized by: comprising the vector of claim 9.