CN113402621B

CN113402621B - Fusion protein containing chimeric antigen receptor and application thereof

Info

Publication number: CN113402621B
Application number: CN202110878307.1A
Authority: CN
Inventors: 黄朝峰; 陈焕鹏
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2021-08-02
Filing date: 2021-08-02
Publication date: 2021-12-10
Anticipated expiration: 2041-08-02
Also published as: CN113402621A

Abstract

The invention relates to the field of biomedicine, in particular to a fusion protein containing a chimeric antigen receptor and application thereof. The fusion protein comprises a chimeric antigen receptor, a 2A peptide, a first signal peptide, a Sirp alpha structure domain and IgG1 Fc which are sequentially connected in series; the chimeric antigen receptor comprises a scFv region, a hinge region, a transmembrane domain, and an intracellular signaling region. The Sirf CAR T cells can reduce tumor load and improve the survival rate of tumor-bearing mice, have no obvious blood toxicity and side effects, and have good clinical application potential. The sirpa-Fc fusion protein can enhance the anti-apoptotic and memory phenotype of CAR T cells and alter the immune microenvironment of tumor tissues.

Description

Fusion protein containing chimeric antigen receptor and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a fusion protein containing a chimeric antigen receptor and application thereof.

Background

Malignant tumors have become one of the leading causes of death worldwide. With the continuous development of science and technology, the early diagnosis and treatment of cancer can greatly improve the cure rate of cancer and improve the life quality of patients. However, most malignant tumors still have the problems of poor prognosis, easy relapse and the like. Among all tumor treatment means, immunotherapy is receiving attention and expectations from researchers. Immunotherapy is characterized by the use of the immune system itself to combat cancer, including antibody therapy, cell therapy and immunomodulator therapy. Of particular interest is cell therapy, which involves the return of a cell preparation to a patient, thereby activating the patient's own immune response to achieve anti-tumor effects. In cellular immunotherapy, Chimeric Antigen Receptor (CAR) modified T cell (CAR T) therapy has been proven to be effective in treating hematological malignancies, and has become a hotspot in the field of cancer treatment. With the official approval by the U.S. Food and Drug Administration (FDA) of two CAR T cell products (kymeriah and yescata) from norway and gilid in 2017 to market, CAR T therapy was initiated for the treatment of acute B-lymphoblastic leukemia as well as diffuse large B-cell lymphoma. Based on the breakthrough of CAR T in the treatment of hematological tumors, CAR T therapy is further expanded to the treatment of solid tumors, and is explored in various solid tumors including breast cancer, liver cancer, colorectal cancer, lung cancer and the like, but the solid tumors are different from the hematological tumors, lack safe and specific targets, and have complex tumor immune microenvironment, so that the treatment effect is poor. Therefore, there is an urgent need to find specific and safe therapeutic targets for solid tumors, optimize the structure of CAR, and improve the tumor microenvironment, thereby enhancing the anti-tumor effect of CAR T cells.

The Trophoblast cell-surface antigen 2 (Trop 2) is prepared fromTACSTD2The gene encodes an expressed type I transmembrane glycoprotein, isTACSTDOne member of the Gene family, humanTASTD2The gene is located in zone 3, zone 2 of the short arm of chromosome 1 (1 p 32). Trop2 is highly expressed in human embryonic trophoblast cells and choriocarcinoma cells, is not expressed or is low expressed in normal tissues and tissues beside cancer, and is highly expressed in epithelial cell cancer tissues. The related research shows that Trop2 is positively correlated with the metastasis and invasion degree of the tumor, and the prognosis of the tumor patient is influenced. Monoclonal-drug conjugates (ADCs) targeting Trop2 have excellent safety and efficacy in clinical therapy, leading to encouraging results in the treatment of refractory relapsed triple-negative breast cancer, and Trop2 has received increasing attention as a target for tumor-targeted therapy.

Disclosure of Invention

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the invention relates to a fusion protein, which comprises a chimeric antigen receptor, a 2A peptide, a first signal peptide, a Sirp alpha structure domain and IgG1 Fc which are connected in series in sequence;

the chimeric antigen receptor comprises a scFv region, a hinge region, a transmembrane domain, and an intracellular signaling region;

the amino acid sequence of the scFv is shown as SEQ ID NO. 1;

the amino acid sequence of the Sirp alpha domain is shown in SEQ ID NO. 2 or SEQ ID NO. 3.

According to a second aspect of the invention, it also relates to an isolated nucleic acid, the expression of which results in a fusion protein as described above.

According to a third aspect of the invention, it also relates to a vector comprising a nucleic acid as described above.

According to a fourth aspect of the invention, it also relates to a T cell containing a nucleic acid as described above, or transformed with a vector as described above.

According to a fifth aspect of the invention, it also relates to a composition comprising a pharmaceutically acceptable carrier and T cells as described above.

According to a sixth aspect of the present invention, it also relates to the use of a T cell as described above or a composition as described above for the preparation of a medicament for the prevention and/or treatment of solid tumors.

Compared with the prior art, the invention has the beneficial effects that:

1) the Sirf CAR T cells can reduce tumor load and improve the survival rate of tumor-bearing mice, have no obvious blood toxicity and side effects, and have good clinical application potential.

2) The sirpa-Fc fusion protein can enhance the anti-apoptotic and memory phenotype of CAR T cells and alter the immune microenvironment of tumor tissues.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic diagram of the principles of the present invention;

FIG. 2 is a schematic structural diagram of a CAR in one embodiment of the invention; shows a schematic diagram of retroviral vector construction for the mouse secondary CAR (T2-m 28 z) and Sirf mCAR (T2-m 28z sirpa-Fc) targeting Trop 2;

FIG. 3 is a preparation of mouse CAR T cells in one embodiment of the invention; infection of mouse CD3 with retrovirus⁺T cells, (a) the transduction efficiency of mCAR T cells was detected using flow cytometry; (B) flow detection of CD4 in T cell subsets of mice of each group⁺T and CD8⁺The proportion of T cells;

FIG. 4 is an embodiment of the invention in which a Sirf CAR T cell expresses a SIRPa-Fc fusion protein; the immunoblotting method detects the expression of SIRPa-Fc fusion protein in the supernatant of CAR T cell culture of T2-m28z and Sirf;

FIG. 5 shows the expression level of CD47 on the surface of tumor cells according to an embodiment of the present invention; flow detection of tumor cells MC38-Trop⁺(A) And 4T1-Trop2⁺(B) Expression of surface CD 47; respectively incubating the supernatants of the T2-m28z after amplification culture and the mCER T cells of the Sirf with the tumor cells for 1 h, and then competitively detecting the level of the CD47 on the surfaces of the tumor cells of the control and the tumor cells after the incubation of the supernatants by using an anti-CD 47 flow type antibody;

FIG. 6 shows the tumor phagocytic function of macrophages in an embodiment of the invention; the phagocytosis is detected by labeling CFSE with tumor cell MC38-Trop⁺BMDM cells grown in vitro for 6 days were grown according to 4: 1, co-culturing in different supernatants, and adding different groups of T cell supernatants (A) after in vitro culture; or a different set of T cell supernatants (B) after co-culture with tumor cells; collecting cells after 4 h, and detecting by flow detection F4/80⁺In macrophages of⁺Determining phagocytic tumor effect of mouse macrophages; results of experimental data are expressed as mean + -SEM calculated by one-way anovapValue, ns no statistical differencep< 0.05，** p< 0.01。；

FIG. 7 shows an embodiment of the present inventionIn the examples, T2-mCAR T has in vitro lysis effect on tumor cells; evaluating the cytotoxic effect of T2-mCAR T cells on Trop2 positive mouse tumor cells by measuring LDH release; (A) MC38-Trop2⁺；（B） 4T1-Trop2⁺(ii) a Results of experimental data are expressed as mean + -SEM calculated by one-way anovapValue of p< 0.05，** p< 0.01，*** p< 0.001，**** p< 0.0001；

FIG. 8 shows the intracellular IFN- γ expression level of T2-mCR T cells in one embodiment of the present invention. Detection of CD3 by flow cytometry⁺The proportion of cells in which IFN- γ expression is positive; and MC38-Trop2⁺After cell co-culture (A-B); and 4T1-Trop2⁺After cell co-culture (C-D); results of experimental data are expressed as mean ± SEM, p values calculated by one-way anovap< 0.05，**p< 0.01，***p< 0.001，**** p< 0.0001；

FIG. 9 shows the level of activation of T2-mCR T cells in one embodiment of the invention; (A) t cells and MC38-Trop2⁺After 48 hours of co-culture, CD69 expression on different T cells was determined by flow cytometry; (B) CD69 positive cells account for CD3⁺Proportion in T cells, UTD group served as negative control. Results of experimental data are expressed as mean + -SEM calculated by one-way anovapValue ofp< 0.0001；

FIG. 10 is a graph of the in vivo anti-tumor effect of Sirf CAR T cells on mouse colon cancer in one embodiment of the invention; (A) c57BL/6 mice were inoculated subcutaneously in the back with MC38-Trop2⁺The tumor cells of the mice are infused back with different T cells through tail veins on the 7 th day after inoculation, and the sizes of the tumors are measured regularly; (B) tumor size growth curve of tumor-bearing mice; (C) body weight change in tumor-bearing mice; (D) tumor tissues of mice were taken for comparison on day 31 after tumor formation; (E) comparing tumor weight between different groups of tumor-bearing mice; results of experimental data are expressed as mean ± SEM, number of mice N = 5/groupp< 0.05，**p< 0.01，***p< 0.001，**** p< 0.0001；

FIG. 11 is an in vivo anti-tumor effect of Sirf CAR T cells on mouse breast cancer in one embodiment of the invention; (A) BALB/c mice dorsal subcutaneous inoculation of 4T1-Trop2⁺Mouse tumor cells, different T cells are returned through tail veins 2 days and 7 days after inoculation, and the sizes of the tumors are measured periodically; (B) tumor size growth curve of tumor-bearing mice; results of experimental data are expressed as mean ± SEM, number of mice N = 5/groupp< 0.05，**p< 0.01，***p< 0.001，**** p< 0.0001；

FIG. 12 is a graph of Sirf CAR T increasing survival of tumor-bearing mice after CY pretreatment in one embodiment of the invention; (A) c57BL/6 mice were inoculated subcutaneously in the back with MC38-Trop2⁺Mouse tumor cells, a single injection of CY 80 mg/kg into abdominal cavity on 14 days after inoculation, and 1X 10 times of infusion by tail vein on the third day after CY treatment⁶Individual T cell (B) tumor-bearing mouse survival curves; (C) tumor growth curves of mice after neoplasia; (group N =5, CY; group N =5, T2-m28 z; group N =7, Sirf); the results of the experimental data are expressed as mean + -SEM and the comparison of the survival rates is calculated using the Log-rank (Mantel-Cox) testpValue ofp< 0.05，**p< 0.01，***p< 0.001，**** p< 0.0001；

FIG. 13 is a graph showing the ratio of immune cells in the spleen of a mouse according to an embodiment of the present invention; detecting the positive rate of CAR in spleen cells of tumor-bearing mice by flow cytometry; CD3⁺Positive rate of CAR in T cells (a); CD4⁺Positive rate of CAR in T cells (B); CD8⁺Positive rate of CAR in T cells (C); mouse spleen CD3⁺ CAR⁺Flow assay (D) of surface markers CD44 and CD62L of cells; results of experimental data are expressed as mean ± SEM, number of mice N = 3/groupp< 0.05，**p< 0.01，***p< 0.001，**** p< 0.0001；

Figure 14 is apoptosis of activated CAR T cells in one embodiment of the invention; detecting with MC38-Trop2 by flow cytometry⁺After the tumor cells are co-cultured for 48 h under the condition of no cytokine addition in vitro, the surface of the CAR T cells is markedAnnals CD44, CD62L and apoptotic events; (A) CD44 and CD62L of CAR T cells; (B) detecting the level of apoptosis in CAR-positive T cells with Annexin V/7-AAD; (C) level of apoptosis in CAR-negative T cells. Results of experimental data are expressed as mean ± SEM, N = 3/groupp< 0.05，**p< 0.01，***p< 0.001，**** p< 0.0001；

FIG. 15 shows the distribution of immune cells in tumor tissue of tumor-bearing mice according to an embodiment of the present invention; detecting the proportion of immune cells in tumor tissues of tumor-bearing mice by adopting flow cytometry; (A) CD4⁺ CAR⁺The ratio of T cells to total cell number; (B) CD8⁺ CAR⁺The ratio of T cells to total cell number; (C) CD45⁺ CD11c⁺The proportion of total number of cells of (a); (D) CD11b⁺Ly6G⁺The ratio of cells to total cell number; results of experimental data are expressed as mean ± SEM, number of mice N = 3/groupp< 0.05，**p< 0.01，***p< 0.001；

FIG. 16 is the PD-1 expression level following activation of CAR T cells in one embodiment of the invention; detecting with MC38-Trop2 by flow cytometry⁺After the tumor cells are co-cultured for 48 hours in vitro without any cytokine addition condition, PD-1 in the T cells⁺The results of the experimental data are expressed as mean + -SEM, bytTest analysis calculation p Value, N = 3; *p< 0.05，**p< 0.01；

FIG. 17 is a graphical representation of the hematological changes in tumor-bearing mice in accordance with an embodiment of the present invention; c57BL/6 mice were inoculated subcutaneously in the back with MC38-Trop2⁺The mouse tumor cells are back-transfused with T2-m28z and Sirf CAR T cells through tail vein at 14 days after inoculation, and routine changes of mouse peripheral blood are detected after 3 days; (A) white blood cell count, (B) red blood cell count, (C) platelet count, (D) hemoglobin concentration; results of experimental data are expressed as mean ± SEM, N = 4/group, ns means no statistical difference —, xp< 0.05；

FIG. 18 is a histological change of tumor-bearing mice in an embodiment of the present invention; c57BL/6 mice were inoculated subcutaneously in the back with MC38-Trop2⁺Tumor cells of mice, different CAR T cells were reinfused via tail vein on day 7 after inoculation, H31 days after tumor bearing&E staining to detect pathological changes in the heart, liver, lung and kidney of the mice;

FIG. 19 is the result of in vitro killing of human CAR T cells in one embodiment of the invention; RPMI 1640 complete Medium resuspension of tumor cells to 1X 10⁵each/mL, the corresponding CAR T cells were resuspended to 1X 10 with RPMI 1640⁵The ratio of the cells/mL to the tumor cells (E: T) is 5:1, after the tumor cells and the CAR T cells are cultured together in a 96-well round bottom plate for 8 hours, the content of Lactate Dehydrogenase (LDH) in supernatant is detected, and the efficiency of killing target cells of the CAR T cells is calculated; results of the experimental data are expressed as mean + -SEM, bytTest analysis calculation p Value, N = 4; ****p< 0.0001。

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

the amino acid sequence of the scFv is shown as SEQ ID NO. 1;

the amino acid sequence of the Sirp alpha domain is shown in SEQ ID NO 2 (murine) or SEQ ID NO 3 (human).

According to the invention, the CAR T cell technology is combined with a CD47 blocker SIRP alpha-Fc fusion protein, the CAR T cell becomes a brick-kick for destroying solid tumors, the function of the body autoimmune system is mobilized by expressing a blocker of a CD47 molecule on the surface of a tumor cell, the tumor tissue microenvironment is improved, and the anti-tumor function of the CAR T cell is enhanced, so that the purpose of inhibiting the solid tumors is achieved, and a reference and an idea are provided for the subsequent treatment of the solid tumors.

The polypeptides of the invention (e.g., IgG1 Fc, signal peptide, portions of chimeric antigen receptor) can be independently selected from the same or different species sources, e.g., mouse (mouse, rat), rabbit, sheep, goat, horse, chicken, cow, dog, and human.

Wherein the amino acid sequence of IgG1 Fc is shown as SEQ ID NO. 7 (murine) or SEQ ID NO. 8 (human).

In some embodiments, the 2A peptide is a P2A peptide.

In some embodiments, the amino acid sequence of the P2A peptide is set forth in SEQ ID NO 9.

In some embodiments, the hinge region is selected from the hinge regions of CD8 α.

In some embodiments, the hinge region has an amino acid sequence as set forth in SEQ ID NO 10 (murine) or SEQ ID NO 11 (human).

In some embodiments, the transmembrane domain is selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD epsilon, CD, OX, CD134, CD137, CD154, KIRDS, OX, CD, LFA-1(CD11, CD), ICOS (CD278), 4-1BB (CD137), GITR, CD, BAFFR, HVEM (LIGHTR), SLAMF, NKp (KLRF), CD160, CD, IL2 beta, IL2 gamma, IL7 alpha, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, LFA-1, ITGAM, CD11, ITGAX, CD11, ITGB, LFCD, ITGB, CD160, ITGAD-1, ACAT 160, ITGAE, CD103, ITGAM (TAAMGB), TAAMB), SLAM-100, CD-150, TAAMB, CD-100, TAAMGL, TAAMB, CD-CD (CD-CD, BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG 2C.

In some embodiments, the transmembrane domain is the CD28 transmembrane domain, and more preferably has the amino acid sequence shown in SEQ ID NO 12 or SEQ ID NO 13.

In some embodiments, the intracellular signaling domain is selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX 28, CD28, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD28, LIGHT, NKG2 28, B28-H28, ligands that specifically bind CD28, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF 28, NKp 28 (KLRF 28), CD160, CD28 alpha, CD28 beta, IL2 28 gamma, IL7 28 alpha, ITGA 28, VLA 28, CD49 28, ITGA 28, CD49 ITGA 28, CD28, GAITGL 28, CD28, GAITGL 28, CD28, GAITGB, CD28, GAITGL 28, GAITGB, CD28, GAITGB, GAITGL 28, CD28, GAITGL 28, CD28, GAITGL 28, CD28, GAITGL 28, CD28, GAITGL 28, GAITCD 28, CD28, GAITCD 28, GAITGL 28, GAITCD 28, CD28, GAITCD 28, GAITGL 28, CD28, GAITGL 28, At least one of CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, PKC θ, Fc ε RI γ, ZAP70, and CD3 endodomains.

In some embodiments, the intracellular signaling region is selected from the group consisting of CD28 and CD3 intracellular domains, preferably the amino acid sequence of CD28 is as set forth in SEQ ID NO: 14 (murine) or SEQ ID NO: 15 (human); the amino acid sequence of the intracellular domain of CD3 is shown in SEQ ID NO 16 (murine) or SEQ ID NO 17 (human).

In some embodiments, the amino acid sequence of the first signal peptide is as set forth in SEQ ID NO 4 (murine) or SEQ ID NO 5 (human).

In some embodiments, the N segment of the fusion protein further comprises a second signal peptide, the amino acid sequence of which is shown in SEQ ID NO 6.

In some embodiments, the amino acid sequence of the fusion protein is as set forth in SEQ ID NO 18 (murine) or SEQ ID NO 19 (human).

The invention also relates to an isolated nucleic acid, the expression of which results in a fusion protein as described above.

The nucleic acid may be DNA or RNA.

The invention also relates to a vector containing a nucleic acid as described above.

In some specific embodiments of the present disclosure, the vector is selected from a retroviral vector, a lentiviral vector, an adenovirus, an adeno-associated virus, or a CRISPR/CAS plasmid.

The invention also relates to T cells containing a nucleic acid as described above or transformed with a vector as described above.

In some specific embodiments of the present disclosure, the T cell is any one of a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a MAIT cell, a γ δ T cell.

In some embodiments, the T cell is CD3⁺A T cell;

in some embodiments, the T cell is CD3⁺CD4⁺T cells and/or CD3⁺CD8⁺T cells.

According to a further aspect of the invention, it also relates to a composition comprising a pharmaceutically acceptable carrier and T cells as described above.

The invention also relates to the use of a T cell as described above or a composition as described above for the preparation of a medicament for the prevention and/or treatment of a solid tumor.

In the present invention, "solid tumor" includes: bone, bone junction, muscle, lung, trachea, heart, spleen, artery, vein, capillary vessel, lymph node, lymphatic vessel, lymph fluid, oral cavity, pharynx, esophagus, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum, testis, vas deferens, penis, eye, ear, nose, tongue, skin, brain, brainstem, medulla oblongata, spinal cord, cerebrospinal fluid, nerve, thyroid, parathyroid, adrenal gland, pituitary, pineal gland, pancreatic islet, thymus, gonad gland, sublingual gland and parotid. In particular, it is preferred that contemplated tumors may be targeted, such as bile duct cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-hodgkin's lymphoma, multiple myeloma, kidney cancer, ovarian cancer, pancreatic cancer, glioma, melanoma, liver cancer, prostate cancer, and urinary bladder cancer.

In some embodiments, the solid tumor comprises colorectal cancer, breast cancer.

Embodiments of the present invention will be described in detail with reference to examples.

Examples

1. Construction of Sirf CAR T cells Co-expressing SIRP α -Fc fusion proteins

1.1 construction of T2-mCAR molecules Co-expressing SIRP α -Fc

Among the numerous molecules blocked by the CD47 antibody or receptor, the drug SIRPa-Fc fusion protein (TTI-621) was selected, and compared with the 150 kDa size of the antibody molecule, the SIRPa-Fc has the molecular weight of only 90kDa, and has better penetrability and tissue distribution. And the human SIRP alpha-Fc fusion protein has been proved to be capable of combining with CD47 molecules on the surface of tumor cells in vitro, thereby enhancing the phagocytic function of macrophages and activating the anti-tumor function of the whole immune system. Based on the fact that a mouse CAR gene constructed based on a retroviral vector framework can be efficiently transduced into a mouse T cell, the mCER structure of co-expression SIRPa-Fc is constructed on the basis of an original mouse secondary CAR molecule T2-m28z (A in figure 2). The mouse sirpa extracellular segment and the Fc segment of the murine IgG1 structure were fused and the mouse sirpa-Fc was ligated to the 3' end of the CAR gene without stop codon using a cleavable fusion protein linker adaptor sequence (2A peptide, P2A), which, due to the ability of P2A to cleave itself independently of proteases, allows the two domains to be cleaved and the co-expression of both molecules to be achieved. To this end, we constructed a murine CAR structure of T2-m28z SIRP α -Fc (Sirf) (B in FIG. 2) whose amino acid sequence is shown in SEQ ID NO: 18.

1.2 preparation of CAR T cells Co-expressing a SIRP α -Fc protein

Not only CD8 is reported in the literature⁺The T cells can be used for preparing CAR T cells, using CD4⁺T cell-produced CAR T cells can also cause direct cytotoxicity. At the same time, the CD4 is found in the clinical treatment of tumors⁺And CD8⁺The proportion of cells was in the range of 1: at 1, the therapeutic effect of CAR T will be more durable. Therefore, in subsequent experiments in this study, we selected to include both CD4⁺T cells also contain CD8⁺Mouse CD3 of T cells⁺T cells were used as cells for the subsequent preparation of mCAR T. mCAR T cells were prepared according to a previous experimental protocol and the transduction efficiency of different mcars was examined by flow cytometry. The experimental results show that the CAR T preparation efficiencies of T2-m28z and Sirf are both high, and are both around 90% (A in FIG. 3). And CD4 in the prepared mCAR T cells⁺And CD8⁺The ratios of (A) and (B) in FIG. 3 were also similar between the three groups, which also flanked that the secreted SIRPa-Fc fusion protein was directed against CD4⁺T cells and CD8⁺T cell clustering had no effect.

1.3 Sirf CAR T cells are able to secrete SIRP alpha-Fc fusion proteins

To further confirm that Sirf CAR T cells successfully expressed the sirpa-Fc protein in mice, we indicated the expression of secreted proteins in the cell supernatant by detection of HIS tag protein on the target protein. Experimental results found that, at the target band around 50kDa, expression of the target protein was found in the cell supernatant of Sirf CAR T, whereas no expression of the corresponding target protein was detected in the T2-m28z CAR T cell supernatant (fig. 4). Experimental results show that the constructed Sirf CAR T cells can express the SIRPa-Fc fusion protein and secrete the SIRPa-Fc fusion protein into cell culture supernatant.

1.4 SIRP alpha-Fc fusion protein capable of binding to the CD47 molecule on the surface of tumor cells

To verify whether sirpa-Fc fusion proteins in the Sirf CAR T cell supernatant were able to bind to CD47 on the surface of tumor cells, we tested the expression of the tumor cell CD47 molecule using an antibody competition binding method. The experimental result shows that the colorectal cancer cell MC38-Trop of the mouse⁺The surface of the tumor cell is highly expressed with CD47, and further the flow antibody of CD47 is used for detecting the expression of CD47 molecules on the surface of the tumor cell after being incubated with the supernatant, and the fluorescence intensity of CD47 molecules on the surface of the tumor cell after being incubated with the supernatant of T2-m28z is not changed, while the fluorescence intensity of CD47 molecules on the surface of the tumor cell after being incubated with the supernatant of the Sirf CAR T cell is obviously reduced (A in figure 5), which indicates that the SIRPa-Fc fusion protein secreted and expressed by the Sirf CAR T cell can be combined with CD47 on the surface of the tumor cell, competitively inhibits the recombination of the CD47 flow antibody, so that the flow detection value of CD47 is reduced. Another mouse breast cancer cell line expressing human Trop2, 4T1-Trop2⁺The same experimental results were also observed (B in fig. 5). The above experimental results show that the SIRP alpha-Fc fusion protein can be combined with CD47 indicated by mouse tumor cells.

1.5 SIRP alpha-Fc fusion protein enhances tumor phagocytosis in vitro by macrophages

The main approach of the CD47 antibody in tumor treatment is to block the 'eat me' signal of CD47-SIRP alpha, so that macrophages can play a role in phagocytosis of tumors, relieve immunosuppression and restore the anti-tumor capability of the immune system. sirpa-Fc expressed by Sirf CAR T cells has been shown to bind CD47 on the surface of tumor cells, blocking the rebinding of CD47 antibodies (figure 5). In order to verify that the SIRP alpha-Fc fusion protein secreted and expressed enhances the tumor phagocytosis of macrophages, culture supernatants of different CAR T cells are added into a co-culture system of mouse Bone marrow-derived macrophages (BMDM) and tumor cells, and the functions of the macrophages are detected. As a result, it was found that the phagocytosis of macrophages was enhanced after the addition of the supernatant of the Sirf CAR T cells, and was higher than that of the control T2-m28z group and the blank UTD group. WhileThere was no difference in macrophage phagocytosis between the UTD group and the traditional second generation CAR T2-m28z group (A in FIG. 6). It has been shown that IFN-gamma can strongly and advantageously activate macrophages, and CAR T cells highly express IFN-gamma after recognizing tumor cells, whether Sirf CAR T cells secreting SIRPa-Fc protein can more strongly activate macrophages in tumor therapy

We first collected the culture supernatant after co-culturing different T cells and tumor cells, and used it as the co-culturing medium of BMDM and tumor cells, and examined the phagocytic ability of macrophages. The results of the experiments showed that both mCAR T groups phagocytosed macrophages more than the control UTD group, whereas the Sirf group engulfed macrophages more strongly than the conventional T2-m28z group (B in fig. 6). This suggests that cytokines secreted by activated CAR T cells can also enhance tumor phagocytosis by macrophages. The above results demonstrate that Sirf CAR T cell culture supernatant containing sirpa-Fc fusion protein promotes tumor phagocytosis of macrophages in vitro.

2. The SIRP alpha-Fc does not influence the in vitro killing function of the Sirf CAR T cells

The surface of the T cell expresses CD47, and the CD47 antibody can be combined with the T cell to regulate the function of the T cell. Therefore, there is a need to further validate the effect of sirpa-Fc fusion proteins on CAR T cell killing tumor cell function in vitro.

2.1 the SIRP alpha-Fc fusion protein does not affect the tumor killing effect of CAR T cells

The primary function of the CAR T cells is to recognize and kill tumor cells, and the lysis of tumor cells MC38-Trop2 by T2-m28z and Sirf CAR T cells is compared by LDH release detection method⁺The ability of (c). Experimental results show that both Sirf and CAR T cells of T2-m28z and target cells were able to lyse tumor cells efficiently under co-culture conditions, and that the killing ability of CAR T cells to tumors was enhanced with increasing effector cell ratio (a in fig. 7), indicating that Sirf CAR T cells have tumor killing activity. However, there was no difference in tumor lysis ability between Sirf compared to T2-m28 z. In additionIn addition, in 4T1-Trop2⁺The same experiment was also performed on the tumor cell line (B in FIG. 7). The above experimental results demonstrate that sirpa-Fc fusion proteins do not affect the tumor cell killing function of CAR T cells.

2.2 SIRP alpha-Fc fusion proteins do not affect IFN-gamma expression of CAR T cells

IFN-gamma is an important anti-tumor factor, so that the CAR T cells and MC38-Trop2 are further detected by a flow cytometry method⁺IFN-gamma expression after co-culture of tumor cells. The experimental results showed that both Sirf and T2-m28z groups of CAR T cells highly expressed IFN- γ, both around 20% (a, B in fig. 8), compared to the control UTD group, and there was no difference in the expression amount of IFN- γ between the two. Detection and 4T1-Trop2⁺The same experiment was also performed with co-cultured CAR T cells, with T2-m28z expressing IFN- γ positive cells at a ratio of around 30% to Sirf CAR T cells (C, D in fig. 8). The above experimental results demonstrate that secretion of expressed sirpa-Fc fusion protein does not affect the expression of CAR T cell IFN- γ.

2.3 SIRP alpha-Fc fusion protein does not affect the level of activation of mCAR T cells

CAR T cells, upon recognizing the associated antigen on the surface of tumor cells, trigger CAR intracellular signaling, thereby activating CAR T cells, and CD47 has been reported to be associated with T cell activation. CD69 is an early activating molecule for T cells, is not expressed on resting T cells, and is highly expressed by CD69 under antigen stimulation and activation, suggesting a level of activation of T cells. To validate the effect of sirpa-Fc fusion proteins on CAR T cell activation, we examined the level of CD69 expression by T cells after 48 h of co-culture with tumor cells and the experimental results found that both Sirf and T2-m28z CAR T cells highly expressed CD69 after co-culture with tumor cells (a in fig. 9), indicating that both CAR T cells were activated and that there was no difference in CD69 expression between Sirf and T2-m28z (B in fig. 9). The above results demonstrate that expression of sirpa-Fc fusion protein does not affect activation of CAR T cells.

SIRP alpha-Fc enhances the antitumor function of Sirf CAR T cells in vivo

Important indicators for assessing the clinical efficacy of a tumor include the size of the tumor in a patient and the survival rate of the patient. Therefore, the anti-tumor function of Sirf CAR T cells requires further validation in mice to determine their actual therapeutic effect on solid tumors. In vitro experiments, Sirf and T2-m28z have no difference in the function of killing tumor cells, but CAR T cells are influenced by immune microenvironment and immune system in vivo, and various immune cells interact with each other, so that different curative effects can be displayed. Therefore, to better assess the in vivo anti-tumor function of Sirf CAR T cells, we still chose to verify whether Sirf CAR T cells could exert better anti-tumor effects under the conditions of the intact immune system and tumor immune microenvironment in an immunocompromised allograft tumor mouse model.

3.1 more effective Sirf CAR T in inhibiting growth of colorectal cancer in mice

First, we chose the subcutaneous tumor-bearing MC38-Trop2 in the right side of immunocompromised C57BL/6 mice⁺1X 10 by caudal vein infusion on the seventh day after tumor cells⁶Tumor size and body weight measurements of tumor-bearing mice were performed twice a week on individual mCAR T cells (a in fig. 10). Tumor volumes of control mice had reached the ethical anthropogenic end-point of the animals at day 31, and we performed tumor tissue sampling. The experimental results showed that the tumor growth was significantly inhibited in mice of the T-cell group of the reinfused Sirf CAR, statistically significantly different from that of the T2-m28z group (B in fig. 10), while there was no difference in the change in body weight of the mice of the three groups (C in fig. 10). After 31 days of material selection, photographs were taken comparing tumor size and the tumor volume and weight were found to be significantly less for the Sirf group than for the T2-m28z and UTD groups (D, E in fig. 10). The above experimental results demonstrate that Sirf CAR T cells have stronger anti-tumor function than T2-m28z CAR T cells in a mouse colorectal model in vivo experiments.

3.2 Sirf CAR T cells inhibit growth of mouse breast cancer

4T1 is a breast cancer cell line from BALB/c mice, and the literature reports that the growth characteristics of 4T1 cells in BALB/c tumor-bearing mice are very similar to the growth characteristics of human breast cancer, so that the cell is a very important animal model in the IV stage of human breast cancer and is commonly used for the treatment of breast cancerThe drug or treatment regimen is evaluated preclinically. In this study, we used BALB/c mice to inoculate 4T1-Trop2 subcutaneously⁺Tumor cells, and two reinfuses of 1X 10⁶Individual mCAR T cells (a in fig. 11) observed the anti-tumor effect of Sirf CAR T cells. The experimental results show that despite two mCAR T cell transfusions, the growth of UTD was only inhibited to some extent in mice in the T2-m28z group compared to the control group, and the tumor size gradually increased with time, and the tumor growth rates of UTD and T2-m28z were still fast. However, tumor growth was significantly slower in mice of the Sirf group than in both T2-m28z and UTD, and tumor volume was smaller (B in fig. 11). The above experimental results demonstrate that Sirf CAR T cells are more effective in inhibiting breast cancer growth.

3.3 Sirf CAR T cells increase survival of chemotherapy-pretreated tumor-bearing mice

Sirf CAR T cells have been shown to be effective in inhibiting tumor growth in colorectal cancer in mice, but this is a therapeutic effect after early cell reinfusion in mice bearing tumors. In clinical treatment, most of the patients find the tumors at an advanced stage, so whether the mCAR T cells infused back later have different anti-tumor effects after the tumor-bearing mice form tumors or not

Also, in current clinical CAR T cell reinfusion regimens, low doses of fludarabine and Cyclophosphamide (CY) are typically first selected for conditioning by intravenous drip in order to clear lymphocytes from the peripheral blood and reduce tumor burden. We also pretreated mice with cyclophosphamide prior to reinfusion therapy to investigate whether Sirf CAR T cells could prolong the survival time of tumor bearing mice. As shown in the in vivo experimental procedure (a in fig. 12), survival of the mice was observed for as long as 61 days, and it was found that there was no difference in survival rate between the mice of the T cell group into which T2-m28z CAR was reinfused and the mice of the CY group alone was injected. Whereas mice with returned Sirf CAR T cells had higher survival rates than both T2-m28z and UTD (B in fig. 12), the differences were statistically significant. Meanwhile, the change of the tumor size within 31 days after tumor formation is analyzed, and the tumors of all mice treated by cyclophosphamide are slightly reduced, but the tumor load of tumor-bearing mice is obviously reduced after the Sirf CAR T cells are further reinfused. The results show that the Sirf CAR T cells can effectively prolong the survival time of tumor-bearing mice pretreated by chemotherapeutic drugs and improve the survival rate.

4. Sirf CAR T cells improve tumor microenvironment and enhance self anti-apoptotic ability

In vivo experimental results show that the antitumor effect of the Sirf CAR T cells is significantly better than that of T2-m28z CAR T cells, and the Sirf CAR T cells reduce tumor burden and improve the survival rate of mice. In order to further explore the reasons, a series of in vitro experiments are carried out to carry out related mechanism research, and the changes of an immune system and a tumor immune microenvironment in tumor-bearing mice are synchronously compared.

4.1 Sirf CAR T cells persist in spleen of tumor-bearing mice to increase, T_CMIncrease of the ratio

At MC38-Trop2⁺The tail vein in the tumor-bearing mouse tumor model of (a) was reinfused with CAR T cells, and then the proportion of CAR T cells in the mouse spleen was examined. The results showed that CAR T cell retention was detectable in both spleen of tumor bearing mice of the T2-m28z and Sirf groups (a in figure 13). Wherein the CD3 of spleen of the Sirf group mice⁺The proportion of CAR T cells in T cells is higher than that in T2-m28z group. Analysis of CD3 subpopulations we found no difference in the proportion of CD4 positive CAR T cells between the Sirf and T2-m28z groups (B in fig. 13), but a higher proportion of CD8 positive CAR T cells in the Sirf group than in T2-m28z (C in fig. 13). Further analysis of the level of differentiation of this fraction of CAR T cells in the spleen revealed T cells in CAR T cells of the Sirf group_CM（CD44⁺ CD62⁺) The cell proportion of (a) is much higher than that of the T2-m28z group_CMHas been proved to be capable of exerting an antitumor function with high efficiency and durability. The above results indicate that more T with high antitumor activity is retained in spleen of tumor-bearing mice infused with Sirf CAR T_CMCAR T cells.

4.2 the Sirf CAR T cells have stronger anti-apoptosis ability, and the memory phenotype of the T cells is obvious

The above studies found that T in spleen of tumor-bearing mice_CMThe ratio of Sirf CAR T cells is higher, and the existing literature reports T_CMThe anti-apoptosis ability is strong, the survival time is long, and the in-vivo long-acting anti-tumor function is maintained, so that the in-vitro experiment further verifies that the Sirf CAR T cells have stronger anti-apoptosis ability. The experimental results show that CD44 in Sirf CAR T cells after co-culture with tumor cells⁺CD62L⁺T of_CMThe cell proportion was significantly higher than that of T2-m28z CAR T cells (A in FIG. 14). After the CAR T cells recognize and kill the tumor, the CAR T cells can also generate spontaneous apoptosis, and the apoptosis level of the T cells after the CAR T cells are co-cultured with the tumor cells is detected to find the apoptosis proportion (Annexin V) in the CAR-positive T cells in the Sirf group⁺/ 7-AAD⁺) Significantly lower than the T2-m28z group (B in fig. 14), but there was no difference in the level of apoptosis of this portion of T cells that were CAR negative between the two groups (C in fig. 14), suggesting that secretion of sirpa-Fc fusion protein expression may only be on activated CARs⁺T cells have a role. The above results demonstrate that expression of a sirpa-Fc fusion protein enhances the anti-apoptotic ability of Sirf CAR T cells.

4.3 Sirf CAR T improves the immune microenvironment of mouse tumor tissue regions

Immunosuppressive effects in the tumor microenvironment are one of the bottlenecks in CAR T treatment of solid tumors, and CAR T cell retention was detected in both Sirf and T2-m28z groups of tumors as detected in tumor tissue of tumor-bearing mice, where Sirf CAR T cells were returned to tumor tissue of either CD4⁺Or CD8⁺The proportion of CAR T cells was higher than that of T2-m28z group (a, B in fig. 15). Further examination of other immune cells in tumor tissue revealed CD11c in the Sirf group tumors⁺The DC cell ratio of (A) was higher than that of T2-m28z and UTD group (C in FIG. 15), while CD11b in tumor tissue of Sirf group⁺ Ly6G⁺The cellular proportion of MDSCs was significantly lower than the T2-m28z group (D in fig. 15). The above results demonstrate that Sirf CAR T cells are able to improve the tumor immune microenvironment of tumor-bearing mice.

4.4 decrease in PD-1 Positive cell proportion in Sirf CAR T cells

PD-1 is a star molecule in cancer immunotherapy, and blocking and down-regulating PD-1 molecules can significantly enhance the anti-tumor function of CAR T cells. In vitro results found that the proportion of PD-1 positive cells in CAR T cells after co-culture with tumor cells was significantly increased, and that the proportion of PD-1 positive cells in Sirf CAR T cells was lower than that of the T2-m28z CAR T cell group, with no difference between the PD-1 positive rates in non-CAR T cells (fig. 16). The above results demonstrate that the secretion of the expressed sirpa-Fc fusion protein is not only able to reduce the proportion of PD-1 positive cells in CAR T cells, but also has no additional effect on non-CAR T cells.

5. Safety of Sirf CAR T cell therapy

Since CD47 is expressed in normal tissues and cells, whether a CD47 blocker sirpa-Fc fusion protein will affect the function of normal tissues and organs during Sirf CAR T cell therapy is also a key to its successful application to clinical practice. Based on this consideration, we performed an initial assessment of therapeutic safety of Sirf CAR T cells.

The existing clinical data show that anemia and thrombocytopenia become dose-limiting toxicity of the CD47 antibody, so that the project also detects related indexes such as hemoglobin content, platelet count and erythrocyte number in model animals. The results show that there was no difference in the total number of white blood cells (a in fig. 17), total number of red blood cells (B in fig. 17), and hemoglobin (D in fig. 17) in the blood of the mice after transfusion of Sirf T2-m28z CAR T cells in comparison with the model group and the T2-m28z CAR T cell group. The content of platelets (C in FIG. 17) was significantly increased after transfusion back of Sirf T2-m28z CAR T cells compared to the model group, but was not significantly different from the T2-m28z CAR T cell group. The experimental results show that the transfusion of the Sirf CAR T cells can not cause anemia, thrombocytopenia and other blood toxicity caused by application of the CD47 antibody in the existing clinical practice, and the safety is better.

Further to assess the potential toxicity of Sirf CAR T cells to normal tissues, we examined pathological changes in important organs of mice, including heart, liver, lung and kidney, 31 days after reinfusion treatment, with no apparent significant morphological changes in the organs (fig. 18).

6. In vitro killing effect verification of human CAR T cells

The murine sequence in the fusion protein shown by SEQ ID NO. 18 was replaced with the humanized sequence shown by SEQ ID NO. 19 and the in vitro killing effect was verified.

The experimental results showed that the control model group, the T2-m28z CAR T cell group and the Sirf CAR T cells were both able to kill Trop2 positive cells MDA-MB-231 and BGC-823 tumor cell lines (FIG. 19). The above results indicate that the human T2-CAR T cells have specific target killing effect on Trop2 positive tumor cells.

In conclusion, the CAR T cell technology is combined with a CD47 blocker SIRP alpha-Fc fusion protein, the CAR T cell becomes a brick-kick for destroying solid tumors, the function of the body autoimmune system is mobilized by expressing a blocker of a CD47 molecule on the surface of a tumor cell, the microenvironment of tumor tissues is improved, and the anti-tumor function of the CAR T cell is enhanced, so that the aim of inhibiting the solid tumors is fulfilled, and a reference and an idea are provided for the subsequent treatment of the solid tumors.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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

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

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

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Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu

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

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

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

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Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys

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

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Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe Lys Gly Arg Phe Ala Phe

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

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

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

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

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

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Gly Pro Ile Arg Trp Tyr Arg Gly Val Gly Pro Ser Arg Leu Leu Ile

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Tyr Ser Phe Ala Gly Glu Tyr Val Pro Arg Ile Arg Asn Val Ser Asp

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Thr Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Asn Val

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Thr Pro Ala Asp Ala Gly Ile Tyr Tyr Cys Val Lys Phe Gln Lys Gly

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Ser Ser Glu Pro Asp Thr Glu Ile Gln Ser Gly Gly Gly Thr Glu Val

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

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Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile Tyr Asn Gln

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Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn Ile Thr Pro Ala

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

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

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Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

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Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile

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Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val

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Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val

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Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp

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Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln

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

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

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Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr

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Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu

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Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr

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

180 185 190

Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr

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Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys

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Ser Phe Ser Arg Thr Pro Gly Lys

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

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

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Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

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Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala Ala Asn Leu Gln Asp

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

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

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

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

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

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

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Arg

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

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

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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

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

100 105 110

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Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe Lys Gly

195 200 205

Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr Leu Gln

210 215 220

Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys Ala Arg

225 230 235 240

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

245 250 255

Ser Leu Val Thr Val Ser Ser Gln Ala Ser Asn Ser Thr Thr Thr Lys

260 265 270

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

275 280 285

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

290 295 300

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Leu Glu Leu Phe Trp Ala Leu

305 310 315 320

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

325 330 335

Ala Leu Cys Val Ile Trp Thr Asn Ser Arg Arg Asn Arg Leu Leu Gln

340 345 350

Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Leu Thr Arg Lys

355 360 365

Pro Tyr Gln Pro Tyr Ala Pro Ala Arg Asp Phe Ala Ala Tyr Arg Pro

370 375 380

Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala Ala Asn Leu Gln Asp

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Val Glu Glu Asn Pro Gly Pro Met Glu Pro Ala Gly Pro Ala Pro Gly

515 520 525

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

530 535 540

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

545 550 555 560

Ser Val Ser Val Ala Ala Gly Asp Ser Thr Val Leu Asn Cys Thr Leu

565 570 575

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

580 585 590

Pro Ser Arg Leu Leu Ile Tyr Ser Phe Ala Gly Glu Tyr Val Pro Arg

595 600 605

Ile Arg Asn Val Ser Asp Thr Thr Lys Arg Asn Asn Met Asp Phe Ser

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu

675 680 685

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

690 695 700

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

705 710 715 720

Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val

725 730 735

Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr

820 825 830

Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr

835 840 845

Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu

850 855 860

Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser

865 870 875 880

Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser

885 890 895

Arg Thr Pro Gly Lys

900

<210> 19

<211> 900

<212> PRT

<213> artificial sequence

<400> 19

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe Lys Gly

195 200 205

Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr Leu Gln

210 215 220

Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys Ala Arg

225 230 235 240

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

245 250 255

Ser Leu Val Thr Val Ser Ser Gln Ala Ser Asn Ser Thr Thr Thr Pro

260 265 270

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

275 280 285

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

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Leu Glu Phe Trp Val Leu Val

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Val Glu Glu Asn Pro Gly Pro Met Glu Pro Ala Gly Pro Ala Pro Gly

515 520 525

Arg Leu Gly Pro Leu Leu Cys Leu Leu Leu Ala Ala Ser Cys Ala Trp

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Pro Ala Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg

595 600 605

Val Thr Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser

610 615 620

Ile Ser Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys

625 630 635 640

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

645 650 655

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

660 665 670

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

Ser Pro Gly Lys

900

Claims

1. The fusion protein is characterized by comprising a chimeric antigen receptor, a 2A peptide, a first signal peptide, a Sirp alpha structure domain and IgG1 Fc which are sequentially connected in series;

the amino acid sequence of the scFv is shown as SEQ ID NO. 1;

the amino acid sequence of the Sirp alpha structure domain is shown as SEQ ID NO. 2 or SEQ ID NO. 3;

the 2A peptide is a P2A peptide.

2. The fusion protein of claim 1, wherein the hinge region is selected from the hinge region of CD8 α.

3. The fusion protein of claim 1, wherein the transmembrane domain is selected from the group consisting of the α, β, or ζ chain of the T cell receptor, CD epsilon, CD134, CD137, CD154, KIRDS, OX, CD, ICOS (CD278), GITR, CD, BAFFR, HVEM (LIGHT TR), SLAMF, NKp (KLRF), CD160, CD, IL2 β, IL2 γ, IL7 α, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGB, LFA-1, ITGAM, CD11, ITGAX, CD11, ITGB, CD, ITGB, TNFR (CD226, SLAMF 2, ACAG), TAAMB (TAAMGL), SLAMBR-100, SLAMBR (SLAMG), SLAMB-100, SLAMBR), SLAMBR, SLAMB (CD-150, SLAMBR), SLAMB, SLAMBR, SLAMB (CD-100, SLAMBR), SLAMB, SLAMBR, SLAMB, SLAMBR, SLE, CD49, CD-6, CD-CD 49, CD-6, CD-6, CD49, CD-6, and SLE, One of NKp30, NKp46, NKG2D and NKG 2C.

4. The fusion protein of claim 1, wherein the intracellular signaling region is selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX 28, CD28, PD-1, ICOS, CD28, LIGHT, NKG2 28, B28-H28, a ligand that specifically binds CD28, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHT TR), SLAMF 28, NKp 28 (KLRF 28), CD160, CD28 a, CD28 beta, IL2 28 gamma, IL7 28 alpha, ITGA 28, VLA 28, CD49 28, CD 3649 28, ITGA 28, VLA-6, CD 3649, ITGAITGAD, CD11 ITE 28, GAITGA 103, GAACA 28, GAMMA-28, CD28, GAITGL 28, CD28, GAITGA 28, CD28, GAITGL 28, CD28, GAITGL 28, CD-SLITGL 28, CD-SLITGL 28, CD-SLITGL 28, CD-III, CD28, CD-III, CD-SLITGL 28, CD28, BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, PKC θ, Fc ε RI γ, ZAP70, and CD3 endodomain.

5. The fusion protein of any one of claims 1-4, wherein the amino acid sequence of the first signal peptide is as set forth in SEQ ID NO. 4 or SEQ ID NO. 5.

6. The fusion protein of any one of claims 1 to 4, wherein the N segment of the fusion protein further comprises a second signal peptide, the amino acid sequence of which is shown in SEQ ID NO. 6.

7. An isolated nucleic acid, wherein the fusion protein of any one of claims 1 to 6 is expressed.

8. A vector comprising the nucleic acid of claim 7.

A T cell comprising the nucleic acid of claim 7 or transformed with the vector of claim 8.

10. A composition comprising a pharmaceutically acceptable carrier and the T cell of claim 9.