CN113402620A - Fusion protein of cytokine combined chimeric antigen receptor and application thereof - Google Patents

Abstract

The invention relates to the field of biomedicine, in particular to a fusion protein of a cytokine combined chimeric antigen receptor and application thereof. The fusion protein comprises a chimeric antigen receptor, 2A peptide, IL-7, 2A peptide and CCL3 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. According to the invention, two cytokines IL-7 and CCL-3 are added during CAR vector construction, and the two cytokines can enhance the anti-apoptosis effect of CAR T cells and reduce the immunosuppressive effect of the CAR T cells, thereby helping the CAR T cells to enhance the anti-tumor effect.

Description

Fusion protein of cytokine combined chimeric antigen receptor and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a fusion protein of a cytokine combined 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 found in human embryonic trophoblast and choriocarcinoma cellsHigh expression, no expression or low expression in normal tissue and cancer tissue, and high expression in epithelial cancer tissue. 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, IL-7, a 2A peptide and CCL3 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 IL-7 is shown as SEQ ID NO. 2;

the amino acid sequence of the CCL3 is shown as 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:

compared with the conventional CAR T cell technology, the two cytokines IL-7 and CCL-3 are added during CAR vector construction, and the two cytokines can enhance the anti-apoptosis effect of CAR T cells and reduce the immunosuppressive effect of the CAR T cells, so that the CAR T cells are helped to enhance the anti-tumor effect. Through comparison experiments, the CAR T cells prepared by the invention have better anti-tumor effect in various tumors, and the treatment effect of the CAR T cells is superior to that of a normal treatment scheme applied clinically at present and another similar application cytokine treatment scheme published, so the CAR T cells have better clinical application potential.

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 structural diagram of a CAR in one embodiment of the invention; shows a schematic diagram of the construction of a reverse transcription vector of a mouse secondary CAR targeting Trop2 (T2-m 28z) and a cytokine fusion expression mCAR (T2-m 28z IL-7 CCL 3);

FIG. 2 is a preparation of mouse CAR T cells in one embodiment of the invention; infection of mouse CD3 with retrovirus⁺T cells, transduction efficiency of mouse T cells detected by flow cytometry (a) negative control no treated cells; (B) normal control mCAR T cells; (C) the mCAR T cell is designed by the scheme;

FIG. 3 is a graph showing the in vitro lysis of tumor cells by T2-mCR T in one embodiment of the present invention; 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, ns:p > 0.05；

FIG. 4 shows an embodiment of the present invention in which T2-mDAR T cells have intracellular IFN-The level of expression of γ; detecting with MC38-Trop2 by adopting flow cytometry⁺The proportion of cells positive for IFN- γ expression in CAR-positive CD 8-positive cells and CD 4-positive cells after cell co-culture; results of experimental data are expressed as mean ± SEM, p values calculated by one-way analysisp< 0.05，***p< 0.001；

FIG. 5 shows the change in expression of the apoptotic protein FAS level of T2-mCR T cells in one embodiment of the present invention; CAR T cells and MC38-Trop2⁺After 48 hours of co-culture, FAS expression on different T cells was determined by flow cytometry; a, normal comparison, B, design of the scheme, and C, counting the three experimental data and then drawing an analysis result; results of experimental data are expressed as mean + -SEM calculated by one-way anovapValue ofp< 0.0001；

FIG. 6 shows the change in expression of the immunosuppressive factor PD1 levels of T2-mCR T cells in one embodiment of the invention; detection of PD-1 in vitro and MC38-Trop2 by flow cytometry⁺Expression of PD-1 of mCAR T cells after 12 hours of co-culture; a, normal comparison, B, design of the scheme, and C, counting the three experimental data and then drawing an analysis result; results of experimental data are expressed as mean + -SEM calculated by one-way anovapValuep< 0.01；

FIG. 7 is a schematic flow chart of the experimental procedure of the anti-tumor effect in C57B6 mouse according to an embodiment of the present invention;

FIG. 8 is an in vivo anti-tumor effect of CAR T cells on mouse colon cancer in one embodiment of the invention; c57BL/6 mice were inoculated subcutaneously in the back with MC38-Trop2⁺Injecting 80 mg/kg Cyclophosphamide (CY) into abdominal cavity 11 days after inoculation of the tumor cells of the mice, and infusing different T cells back through tail veins 14 days after inoculation, wherein the graph is a survival curve of the mice; results of experimental data are expressed as mean ± SEM, and the number of mice is indicated in different groupsp< 0.05，***p< 0.001，**** p< 0.0001；

FIG. 9 is an in vivo anti-tumor effect of CAR T cells on mouse colon cancer following replacement of a costimulatory signal in one embodiment of the invention; c57BL/6 mice were inoculated subcutaneously in the back with MC38-Trop2⁺MouseTumor cells, namely injecting 80 mg/kg of Cyclophosphamide (CY) into the abdominal cavity 11 days after inoculation, returning different T cells through tail veins 14 days after inoculation, and periodically measuring the size of the tumor and the survival condition of the mouse; results of experimental data are expressed as mean ± SEM, and the number of mice is indicated in different groupsp< 0.05；

FIG. 10 is a graph of the in vivo anti-tumor effect of different cytokine combinations on CAR T cells on mouse colon cancer in one embodiment of the invention; c57BL/6 mice were inoculated subcutaneously in the back with MC38-Trop2⁺Injecting 80 mg/kg of Cyclophosphamide (CY) into the abdominal cavity 11 days after inoculation of the tumor cells of the mice, returning different T cells through tail veins 14 days after inoculation, and periodically measuring the size of the tumor and the survival condition of the mice; results of experimental data are expressed as mean ± SEM, statistically labeledp< 0.05，**p< 0.01；

FIG. 11 is a graph showing the in vivo anti-tumor activity of the present design using different tumor models in one embodiment of the present invention; BALB/C mice dorsal subcutaneous inoculation CT26-Trop2⁺Injecting 80 mg/kg of Cyclophosphamide (CY) into the abdominal cavity 7 days after inoculation of the tumor cells of the mice, returning different T cells through tail veins 11 days after inoculation, and periodically measuring the size of the tumor and the survival condition of the mice; results of experimental data are expressed as mean ± SEM, number of mice n =5, statistically labeled p-value: nsp> 0.05, **p< 0.01；

FIG. 12 is a graph demonstrating the in vivo anti-tumor treatment protocol designed in the present case in different tumor models in one embodiment of the present invention; c57BL/6 mice were inoculated subcutaneously on the back at 2X 10⁶LLC-Trop2⁺Injecting 80 mg/kg of Cyclophosphamide (CY) into the abdominal cavity 7 days after inoculation of the tumor cells of the mice, returning different T cells through tail veins 11 days after inoculation, and periodically measuring the size of the tumor and the survival condition of the mice; results of experimental data are expressed as mean ± SEM, number of mice n =6, statistically labeled p-value: **p< 0.01；

FIG. 13 shows an example of the in vivo anti-tumor therapy of the present invention in a human tumor model; NCG mice were inoculated subcutaneously on the back at 5X 10⁵Colo-205, infusing different T cells back through tail veins 7 days after inoculation, and periodically measuring the size of tumors and the survival condition of mice; animals were sacrificed on day 24 to take material and compare tumor size; the negative control is human T cells which are not transduced, the normal control is CAR T cells of T2-28z, and the design is CAR T cells which are transduced with 7P 3; results of experimental data are expressed as mean ± SEM, number of mice n =6, statistically labeled p-value: *p< 0.05， **p< 0.01。

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 invention relates to a fusion protein, which comprises a chimeric antigen receptor, 2A peptide, IL-7, 2A peptide and CCL3 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 IL-7 is shown as SEQ ID NO. 2;

the amino acid sequence of the CCL3 is shown as SEQ ID NO. 3.

According to the invention, two cytokines IL-7 and CCL3 are connected in series on the same CAR receptor vector through connecting peptide 2A and CAR receptor, and the CAR T cell prepared by using the vector can normally recognize corresponding tumor antigen to activate anti-tumor effect, and can also express the two cytokines, activate and enhance CAR T cells and other effector immune cells, so that the treatment effect of the CAR T cell is enhanced. From the results the inventors demonstrated that this design had a stronger anti-tumor effect than the normal control CAR T cell treatment.

The polypeptides of the invention (e.g., signal peptide, portions of chimeric antigen receptor) may be independently selected from the same or different species, such as murine (mouse, rat), rabbit, sheep, goat, horse, chicken, bovine, canine, and 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. 5.

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

In some embodiments, the amino acid sequence of the hinge region is set forth in SEQ ID NO 6.

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 region, and more preferably has the amino acid sequence shown in SEQ ID NO 7.

In some embodiments, the transmembrane domain is the CD27 transmembrane domain, and more preferably has the amino acid sequence shown in SEQ ID NO. 8.

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;

in some embodiments, the intracellular signaling region is selected from the group consisting of CD27 and CD3 intracellular domains;

preferably, the amino acid sequence of the intracellular domain of CD28 is shown in SEQ ID NO 9.

Preferably, the amino acid sequence of the intracellular domain of CD27 is shown in SEQ ID NO 10.

Preferably, the amino acid sequence of the intracellular domain of CD3 is shown in SEQ ID NO. 11.

In some embodiments, the N-terminus of the fusion protein further has a signal peptide.

In some embodiments, the amino acid sequence of the signal peptide is set forth in SEQ ID NO 4.

In some embodiments, the amino acid sequence of the fusion protein is set forth in SEQ ID NO 12 or SEQ ID NO 13.

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, lung cancer.

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

Examples

1 construction of T2-mCER molecules co-expressing IL-7 and CCL3

A mouse CAR gene constructed based on a retroviral vector framework can be efficiently transduced into a mouse T cell, and the mCER fusion protein co-expressing mouse protein IL-7 and CCL3 is constructed on the basis of the original mouse secondary CAR molecule T2-m28 z. The IL-7 and CCL3 proteins of the mouse are connected to the 3' end of the CAR gene without a stop codon by utilizing a cleavable fusion protein linker adapter sequence (2A peptide, P2A), and because P2A can be cut by itself independent of protease, two domains of the CAR gene are broken to achieve the result of co-expression of three molecules. So far, a murine CAR structure of T2-m28z IL7 CCL3 (7P 3) is constructed, the amino acid sequence of the murine CAR structure is shown as SEQ ID NO: 12, and the structure is shown as figure 1.

2 preparation of CAR T cells Co-expressing IL-7 and CCL3

The invention prepares mCER T cells according to the prior literature experimental procedures, and detects the transduction efficiency of different mCER by flow cytometry. The experimental result shows that (figure 2), the preparation efficiencies of T2-m28z and the CAR T designed by the scheme are relatively close to each other and are all about 65%, and the successful preparation is indicated.

3. (7P 3) comparison with Normal control (T2-m 28z) CAR-T cells in vitro tumor killing Activity

The primary function of the CAR T cell is to recognize and kill tumor cells, and the detection method of LDH release is used for comparing T2-m28z with that of the CAR T cell lysis tumor cell MC38-Trop2 designed in the scheme (7P 3)⁺And 4T1-Trop2⁺The ability of the cell to perform. The experimental results show (fig. 3) that both CAR T cells of 7P3 and 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, indicating that 7P3 CAR T cells have tumor killing activity. But there was no difference in tumor lysis ability between comparison 7P3 and T2-m28 z.

4. The CAR T cells designed by the invention have higher IFN-gamma expression level

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 show (fig. 4) that the CAR T cells of the 7P3 group designed in this case express a high proportion of IFN- γ positive cells, both in CD8 positive and CD4 positive CAR T cells, compared to the normal control T2-m28z group. The above experimental results demonstrate that secreted expressed IL-7 and CCL3 can enhance the expression of CAR T cell IFN- γ.

5. IL-7 and CCL3 proteins have inhibitory effects on FAS protein expression induced by CAR T cell activation

After the CAR T cells recognize the related antigens on the surface of tumor cells, CAR intracellular signals are triggered, CAR T cells are activated, the activated T cells induce the high expression of an apoptosis protein FAS, and further induce AICD effect, and the sustained activation capacity of the T cells is inhibited, the effect is beneficial to controlling autoimmune diseases caused by the over-activation of the T cells, but the tumor immune response requiring the sustained T cell activation is inhibited, and the anti-tumor activity of the CAR T cells is reduced. To validate the effect of IL-7 and CCL3 proteins on FAS protein expression induced by CAR T cell activation, we examined the FAS expression level of CAR T cells after 48 hours of co-culture with tumor cells. The experimental result shows (fig. 5), compared with the normal control T2-m28z CAR T cell, the proportion of expressing the FAS protein after the co-culture of 7P3 and the tumor cell is reduced, which indicates that the IL-7 and CCL3 proteins can inhibit the expression of the FAS protein, and further can inhibit the apoptosis of the activated CAR T cell, and enhance the sustained activation capability and the anti-tumor activity of the CAR T.

6. IL-7 and CCL3 proteins are capable of reducing the proportion of PD-1 positive cells in 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 (FIG. 6), increased proportion of PD-1 positive cells was observed in CAR T cells co-cultured with tumor cells. However, compared with the normal control T2-m28z CAR T cell group, the proportion of PD-1 positive cells in the 7P3 CAR T cells is obviously reduced, and the result shows that IL-7 and CCL3 proteins can reduce the proportion of PD-1 positive cells in the CAR T cells, so that the anti-tumor activity of the CAR T cells is improved.

7. In vivo tumor killing experiment

The procedure of the experiment on the in vivo antitumor effect of all CAR T cells of the present invention on mouse colon cancer is shown in fig. 7; c57BL/6 mice were inoculated subcutaneously in the back with MC38-Trop2⁺Tumor cells of mice were inoculated with 80 mg/kg Cyclophosphamide (CY) intraperitoneally after day 11 after inoculation, different T cells were returned via tail vein after day 14 after inoculation, and the size of tumor and survival of mice were periodically measured twice a week.

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 7P3 CAR T cells requires further validation in mice to determine their actual therapeutic effect on solid tumors. In vitro experiments, the function of 7P3 and T2-m28z in killing tumor cells is not different, 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 7P3 CAR T cells, we chose to verify whether 7P3 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.

First, we chose the subcutaneous tumor-bearing MC38-Trop2 in the right side of immunocompromised C57BL/6 mice⁺After the tumor cells, 80 mg/kg Cyclophosphamide (CY) was intraperitoneally injected after day 11, and 1X 10 of different groups were returned via tail vein on day fourteen⁶Tumor size measurements of tumor-bearing mice were performed twice a week on individual mCAR T cells. The experimental results show (fig. 8) that the tumor growth of the mice of the T cell group of the reinfused 7P3 CAR of this case was significantly inhibited, and no animal died after 100 days of tumor bearing, which was statistically different from the other groups. Meanwhile, compared with the sizes of tumors at different time points, the 7P3 CAR T cell group of the present application also showed significantly slower tumor growth than the other groups. The above experimental results demonstrate that in the mouse colorectal model in vivo, the 7P3 CAR T cells have stronger anti-tumor function than the normal control T2-m28z CAR T cells.

8. Intracellular costimulatory signal was changed from CD28 to CD27

CD27 is also an important T cell activating costimulatory signaling molecule, and prior studies have shown that its intracellular signaling segment can be interchanged with the corresponding segment of CD28, and also has the function of activating CAR T cells. The amino acid sequence of the fusion protein with the replacement intracellular costimulatory signal of CD27 is shown in SEQ ID NO. 13. After the different CAR T cells were returned following the above experimental protocol, the results showed (figure 9) that CCL3 and IL-7 protein expression had a significant gain in anti-tumor activity on both CAR T cells containing intracellular signaling of CD27, as well as CD 28. It was shown that CCL3 and IL-7 proteins had similar effects on CAR T cells containing different species of costimulatory signals.

9. Comparison of the design of the Combined CCL3 and IL-7 with cytokines alone

CCL3 and IL-7 have different effects on T cell function, in order to observe the synergistic effect of two factors in CAR T cells, we constructed CAR T cells containing only one factor (3 CAR is a CAR T cell co-expressing CCL3, and 7 CAR is a CAR T cell co-expressing IL-7), designed CAR T cells by reinfusion of CAR T cells only expressing a single factor or the present case co-expressing a dual factor, and compared different combinations by reinfusion of two single-factor CAR T cellsThe antitumor effect of (1). C57BL/6 mice were inoculated subcutaneously in the back with MC38-Trop2⁺Tumor cells of mice were inoculated with 80 mg/kg Cyclophosphamide (CY) intraperitoneally 11 days after inoculation, different T cells were returned via tail vein 14 days after inoculation, and the size of tumor and survival of mice were measured periodically. The experimental results show (fig. 10) that compared with other groups, the design (7P 3) has obvious advantage of antitumor activity, and the combined application of the CCL3 and the IL-7 is superior to the application of any single factor and is also superior to the common application of two single-factor CAR T cells.

10. CT-26 tumor cell line experiments for colorectal cancer

To verify that the design is equally effective in different colorectal cancer tumors, we replaced the CT-26 tumor model of another tumor cell to test the anti-tumor effect of the design, and synchronously compared the anti-tumor activity difference of the combined application scheme (co-expressing cytokines IL-7 and CCL19, 7P 19) with another reported cytokine. BALB/C mice dorsal subcutaneous inoculation CT26-Trop2⁺Tumor cells of mice were inoculated with 80 mg/kg Cyclophosphamide (CY) intraperitoneally 7 days after inoculation, different T cells were returned via tail vein 11 days after inoculation, and the size of tumor and survival of mice were measured periodically. The experimental results show that (fig. 11), 7P19 and 7P3 can effectively inhibit the growth of the CT26 tumor model. Although there was no statistical difference between the combination of the present design (7P 3) and the control regimen 7P19, the present design was superior to the control regimen 7P19 compared to the end-stage survival, indicating that the present design is still the optimal treatment regimen.

11. Lung cancer cell LLC tumor cell line experiment

To verify that the design is equally effective in different types of tumors, we replaced the use of an LLC tumor-forming model of another lung cancer tumor cell to test the anti-tumor effect of the design. C57BL/6 mice were inoculated subcutaneously on the back at 2X 10⁶LLC-Trop2⁺Tumor cells of mice were inoculated with 80 mg/kg Cyclophosphamide (CY) intraperitoneally 7 days after inoculation, different T cells were returned via tail vein 11 days after inoculation, and the size of tumor and mice were measured periodicallySurvival situation. The experimental results show (fig. 12) that the design (7P 3) has a significant advantage of antitumor activity compared with the normal control (T2-m 28z) and the untreated group.

12. The NCG mouse is used for verifying the antitumor activity of the scheme design on human tumor cells

To verify the antitumor activity of the present design in a human tumor model, a colo-205 human colorectal cancer model was established using immunodeficient NCG mice, followed by reinfusion of different CAR T cells and observation of their effect on tumor growth. NCG mice were inoculated subcutaneously on the back at 5X 10⁵Individual colo-205 human colorectal cancer cells were reinfused with different T cells via tail vein after 7 days post inoculation, and tumor size and mouse survival were measured periodically. Animals were sacrificed at day 24 to take material and compare tumor sizes. The negative control was human T cells that were not transduced, the normal control was CAR T cells from T2-28z, and the present case was designed to be CAR T cells transduced with 7P 3. The experimental results show (fig. 13), the design of the scheme has better anti-tumor activity in the human tumor model, and is superior to the treatment effect of other control groups.

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

<110> Zhongshan university

Fusion protein of <120> cytokine combined chimeric antigen receptor and application thereof

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Val Ser Ser

<210> 2

<211> 154

<212> PRT

<213> artificial sequence

<400> 2

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

1 5 10 15

Leu Val Leu Leu Pro Val Thr Ser Ser Glu Cys His Ile Lys Asp Lys

20 25 30

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

35 40 45

Asp Lys Met Thr Gly Thr Asp Ser Asn Cys Pro Asn Asn Glu Pro Asn

50 55 60

Phe Phe Arg Lys His Val Cys Asp Asp Thr Lys Glu Ala Ala Phe Leu

65 70 75 80

Asn Arg Ala Ala Arg Lys Leu Lys Gln Phe Leu Lys Met Asn Ile Ser

85 90 95

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

100 105 110

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

115 120 125

Lys Asn Asp Ala Cys Phe Leu Lys Arg Leu Leu Arg Glu Ile Lys Thr

130 135 140

Cys Trp Asn Lys Ile Leu Lys Gly Ser Ile

145 150

<210> 3

<211> 92

<212> PRT

<213> artificial sequence

<400> 3

Met Lys Val Ser Thr Thr Ala Leu Ala Val Leu Leu Cys Thr Met Thr

1 5 10 15

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

20 25 30

Ala Cys Cys Phe Ser Tyr Ser Arg Lys Ile Pro Arg Gln Phe Ile Val

35 40 45

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

50 55 60

Leu Thr Lys Arg Asn Arg Gln Ile Cys Ala Asp Ser Lys Glu Thr Trp

65 70 75 80

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

85 90

<210> 4

<211> 20

<212> PRT

<213> artificial sequence

<400> 4

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

1 5 10 15

Gly Ala Tyr Gly

20

<210> 5

<211> 19

<212> PRT

<213> artificial sequence

<400> 5

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

1 5 10 15

Pro Gly Pro

<210> 6

<211> 45

<212> PRT

<213> artificial sequence

<400> 6

Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val His Pro Thr

1 5 10 15

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

20 25 30

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

35 40 45

<210> 7

<211> 28

<212> PRT

<213> artificial sequence

<400> 7

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

1 5 10 15

Leu Leu Val Thr Val Ala Leu Cys Val Ile Trp Thr

20 25

<210> 8

<211> 21

<212> PRT

<213> artificial sequence

<400> 8

Ile Phe Val Thr Phe Ser Ser Met Phe Leu Ile Phe Val Leu Gly Ala

1 5 10 15

Ile Leu Phe Phe His

20

<210> 9

<211> 41

<212> PRT

<213> artificial sequence

<400> 9

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

1 5 10 15

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

20 25 30

Ala Arg Asp Phe Ala Ala Tyr Arg Pro

35 40

<210> 10

<211> 47

<212> PRT

<213> artificial sequence

<400> 10

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

1 5 10 15

Glu Pro Cys Pro Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Ala Ile

20 25 30

Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Phe Tyr Pro

35 40 45

<210> 11

<211> 113

<212> PRT

<213> artificial sequence

<400> 11

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Arg

<210> 12

<211> 797

<212> PRT

<213> artificial sequence

<400> 12

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 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 Phe His Val Ser Phe Arg Tyr Ile

515 520 525

Phe Gly Ile Pro Pro Leu Ile Leu Val Leu Leu Pro Val Thr Ser Ser

530 535 540

Glu Cys His Ile Lys Asp Lys Glu Gly Lys Ala Tyr Glu Ser Val Leu

545 550 555 560

Met Ile Ser Ile Asp Glu Leu Asp Lys Met Thr Gly Thr Asp Ser Asn

565 570 575

Cys Pro Asn Asn Glu Pro Asn Phe Phe Arg Lys His Val Cys Asp Asp

580 585 590

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

595 600 605

Phe Leu Lys Met Asn Ile Ser Glu Glu Phe Asn Val His Leu Leu Thr

610 615 620

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

625 630 635 640

Lys Asn Val Lys Glu Gln Lys Lys Asn Asp Ala Cys Phe Leu Lys Arg

645 650 655

Leu Leu Arg Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Lys Gly Ser

660 665 670

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

675 680 685

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

690 695 700

Pro Met Lys Val Ser Thr Thr Ala Leu Ala Val Leu Leu Cys Thr Met

705 710 715 720

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

725 730 735

Thr Ala Cys Cys Phe Ser Tyr Ser Arg Lys Ile Pro Arg Gln Phe Ile

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795

<210> 13

<211> 798

<212> PRT

<213> artificial sequence

<400> 13

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 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 Ile Phe Val Thr Phe

305 310 315 320

Ser Ser Met Phe Leu Ile Phe Val Leu Gly Ala Ile Leu Phe Phe His

325 330 335

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

340 345 350

Glu Pro Cys Pro Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Ala Ile

355 360 365

Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Phe Tyr Pro Lys

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

Ile Phe Gly Ile Pro Pro Leu Ile Leu Val Leu Leu Pro Val Thr Ser

530 535 540

Ser Glu Cys His Ile Lys Asp Lys Glu Gly Lys Ala Tyr Glu Ser Val

545 550 555 560

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

565 570 575

Asn Cys Pro Asn Asn Glu Pro Asn Phe Phe Arg Lys His Val Cys Asp

580 585 590

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

595 600 605

Gln Phe Leu Lys Met Asn Ile Ser Glu Glu Phe Asn Val His Leu Leu

610 615 620

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

625 630 635 640

Glu Lys Asn Val Lys Glu Gln Lys Lys Asn Asp Ala Cys Phe Leu Lys

645 650 655

Arg Leu Leu Arg Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Lys Gly

660 665 670

Ser Ile Asp Tyr Lys Asp Asp Asp Asp Lys Val Asp Gly Ser Gly Ala

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

Pro Thr Ala Cys Cys Phe Ser Tyr Ser Arg Lys Ile Pro Arg Gln Phe

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795

Claims (11)

1. The fusion protein is characterized by comprising a chimeric antigen receptor, a 2A peptide, IL-7, a 2A peptide and CCL3 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 IL-7 is shown as SEQ ID NO. 2;

the amino acid sequence of the CCL3 is shown as SEQ ID NO. 3.

2. The fusion protein of claim 1, wherein the 2A peptide is a P2A peptide.

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

4. The fusion protein of claim 1, wherein the transmembrane domain is selected from the group consisting of an alpha, beta, or zeta chain of a T cell receptor, CD epsilon, CD134, CD137, CD154, KIRDS, OX, CD, LFA-1(CD11, CD), ICOS (CD278), 4-1BB (CD137), GITR, CD, BAFFR, HVEM (LITR), SLAMF, NKp (KLRF), CD160, CD, IL2 beta, IL2 gamma, IL7 alpha, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, LFA-1, ITGAM, CD11, ITGAX, CD11, ITGB, CD160, ITGALC 160, ACAG-229, ITGAD (TAMGB), CD100, TAMG, CD-100, TAMGW, CD-CD, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG 2C.

5. The fusion protein of claim 1, wherein the intracellular signaling region is selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-related antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B C-H C, a ligand that specifically binds to CD C, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF C, NKp C (KLRF C), CD160, CD C alpha, CD C beta, IL 2C gamma, IL 7C alpha, ITGA C, VLA C, CD 49C, CD C, ITGA C, CD C-ITGA C, CD C, GAITGB 11, GAITGB, GAITGA C, CD C, GAITGB 11, GAITGA C, CD C, GAITGB C, CD C, GAITGB 11, CD C, GAITCD C, CD C, GAITGB C, GAITCD C, GAITGB C, CD C, GAITCD C, GAITB C, GAITCD C, GAITGB 11-C, GAITGB C, GAITB C, GAITCD C, CD C, GAITGB, GAITCD C, GAITGB C, GAITCD C, GAITB C, GAITCD C, GAITB C, GAITCD C, GAITB C, GAITCD C, GAIT, CD160(BY55), PSGL1, CD100(SEMA4D), 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 endodomain.

6. The fusion protein of any one of claims 1 to 5, wherein the N-terminus of the fusion protein further comprises a signal peptide.

7. The fusion protein of claim 6, wherein the signal peptide has the amino acid sequence shown in SEQ ID NO. 4.

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

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

A T cell comprising the nucleic acid of claim 8 or transformed with the vector of claim 9.

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

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