CN113481168A

CN113481168A - Agent for enhancing homing ability of CAR-T cells to solid tumor tissue and use thereof

Info

Publication number: CN113481168A
Application number: CN202110901011.7A
Authority: CN
Inventors: 张巍; 陈运帆; 赵永春; 徐艳敏; 赵文旭; 黄霞; 单娟娟; 陈军; 张茜真
Original assignee: Chongqing Precision Biotech Co ltd
Current assignee: Chongqing Precision Biotech Co ltd
Priority date: 2018-12-26
Filing date: 2018-12-26
Publication date: 2021-10-08
Also published as: CN109593726A

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a CAR-T cell, a reagent for enhancing the ability of the CAR-T cell to home to solid tumor tissues and application thereof. The agent is a DPP4 inhibitor, including but not limited to sitagliptin, vildagliptin, saxagliptin, alogliptin and linagliptin. The CAR-T cell expresses a Chimeric Antigen Receptor (CAR) comprising an ScFv, a hinge structure and a transmembrane structure and an intracellular activation signal domain, the amino acid sequence of the hinge structure being shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4 or SEQ ID No. 5. The invention provides a new application of the DPP4 inhibitor in tumor treatment, and the agent can enhance the tumor homing and tumor enrichment capacity of the CAR-T cells and improve the treatment effect of CAR-T therapy on solid tumors.

Description

Agent for enhancing homing ability of CAR-T cells to solid tumor tissue and use thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a CAR-T cell, a reagent for enhancing the ability of the CAR-T cell to home to solid tumor tissues and application thereof.

Background

CAR-T is called chimeric antigen receptor T cell immunotherapy, and achieves better performance in hematological tumors, but the CAR-T has lower effect in solid tumors than the hematological tumors, and one of the main reasons is that CAR-T is difficult to enter the solid tumors, the heterogeneity of the solid tumors is high, solid tumor targets are often expressed in normal tissues, and if CAR-T cells cannot be precisely targeted to tumor tissues, the safety problems of off-target risks and the like exist, which all affect the curative effect of CAR-T cells in solid tumor therapy. Therefore, the temperature of the molten metal is controlled,it is necessary to improve the tumor homing and infiltration capacity of CAR-T cells.

DPP4, also known as CD26, is a transmembrane serine protease with multiple biological functions. Film bonding typeCD26 Is one of the markers of T cell activation, and the expression quantity on the activated T cells is obviously increased. As a co-stimulatory molecule, it is,CD26 capable of interacting with a variety of cell surface or intracellular moleculesThe effects of CD26 on T cells, which are involved in the T cell signaling process, are still under investigation, and only at presentThe anti-CD 26 antibody has been studied for activation of T cells, but requires a higher antibody concentration than conventional T cell activating antibodies such as anti-CD 3 antibody or anti-TCR antibody.

The CD26 inhibitor, namely the DPP4 inhibitor can inhibit the shearing of DPP4 to incretins such as glucagon-like peptide (GLP) and glucose-dependent insulinotropic polypeptide (GIP) and the like, effectively controls blood sugar, and is an important antidiabetic medicament. However, the biological function of DPP4 in different tumors and the mechanism of action are still not well defined.

Aiming at the characteristic that DPP4 can interact with various molecules on the cell surface or in the cell,to increase CAR-T The homing capability and infiltration capability of cell tumor, the reduction of the miss risk and other safety problemsThe application utilizes a DPP4 inhibitor in combination with CAR-T cell immunotherapy technology to improve the effect of CAR-T cells in treating solid tumors.

Disclosure of Invention

It is an object of the invention to provide a CAR-T cell.

In order to achieve the purpose, the following scheme is adopted by the invention:

a CAR-T cell expressing a chimeric antigen receptor (CAR structure) comprising an extracellular antigen-recognition region and an intracellular activation signal domain, the extracellular antigen-recognition region comprising an ScFv, a hinge structure and a transmembrane structure, the amino acid sequence of the hinge structure being as set forth in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4 or SEQ ID No. 5.

In certain embodiments, the hinge region sequence in the CAR structure may be derived from: IgG, IgD, CD8, CD7, CD 4.

Further, the amino acid sequence of the transmembrane region of the CAR structure is shown as SEQ ID NO.6 or SEQ ID NO. 7.

In certain embodiments, the transmembrane region in the CAR structure may be derived from: CD8, CD28, CD3 epsilon, CD4, CD16, CD137, CD80, and CD 86.

Further, the amino acid sequence of the extracellular antigen recognition region of the CAR structure is shown as SEQ ID NO.11 or SEQ ID NO. 13.

Further, the intracellular activation signal domain comprises CD28 and/or CD137 and CD 3.

Further, the amino acid sequence of the intracellular activation signal domain is a tandem of SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10.

In certain embodiments, the intracellular signaling region in the CAR structure may be derived from: CD3, CD137, CD28, CD27, OX40, ICOS, GITR, CD2, CD40, PD-1, PD1L, B7-H3, lymphocyte function-associated antigen-1 (LFA-1), ICAM-1, CD7, NKG2C, CD83, CD86 and CD 127.

In certain embodiments, the VH of the ScFv is shown in SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21 and the VL of the ScFv is shown in SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO. 26.

Further, the amino acid sequence of the CAR structure is shown as SEQ ID NO.12 or SEQ ID NO. 14.

Further, the CAR-T cells target CEA or PSCA.

Carcinoembryonic antigen (CEA) is a tumor-associated antigen first extracted from colon cancer and embryonic tissue by Gold and Freedman in 1965, is an acidic glycoprotein having the characteristics of a human embryonic antigen, exists on the surface of cancer cells differentiated from endoderm cells, and is a structural protein of cell membranes. Formed in the cytosol, secreted outside the cell through the cell membrane and then into the surrounding body fluids. Therefore, the reagent can be detected from various body fluids and excretions such as serum, cerebrospinal fluid, milk, gastric juice, hydrothorax, ascites, urine, feces and the like.

According to a large amount of clinical practices, CEA is used as a specific marker for early diagnosis of colon cancer and rectal cancer, and the CEA value of malignant tumors of the gastrointestinal tract can be increased, and the CEA value is also increased in serum of breast cancer, lung cancer and other malignant tumors. Therefore, carcinoembryonic antigen is a broad-spectrum tumor marker, and although the carcinoembryonic antigen cannot be used as a specific index for diagnosing a certain malignant tumor, the carcinoembryonic antigen still has important clinical value in the aspects of differential diagnosis, disease condition monitoring, curative effect evaluation and the like of the malignant tumor.

The Prostate Stem Cell Antigen (PSCA) is a newly discovered cell surface antigen, and the research in recent years finds that the Prostate Stem Cell Antigen (PSCA) has a certain relation with the occurrence and development of the urinary tumor and the infiltration, metastasis and recurrence of the tumor.

In certain embodiments, the CAR-T targeted target may be CD19, CD20, CD123, CD22, CD16, NKG2D, BCMA, ROR1, CEA, Mesothelin (Mesothelin), PSCA, PSMA, PSA, MUC1, DLL3, FAP, c-Met, GPC-3, Her2, EpCAM, IL13R α 2, EGFR, EGFRvIII, GD-2, CD133, NY-ESO-1, MAGE A3, WT1, or any combination thereof.

In view of the above, it is also an object of the present invention to provide an agent that enhances the ability of said CAR-T cells to home to solid tumor tissue, which agent is capable of chemotactic migration and homing and infiltration of said CAR-T cells to tumor tissue.

an agent that enhances the ability of the CAR-T cell to home to solid tumor tissue the agent is a DPP4 inhibitor, the DPP4 inhibitor includes but is not limited to sitagliptin, vildagliptin, saxagliptin, alogliptin and linagliptin.

Preferably, the DPP4 inhibitor is sitagliptin.

DPP-4 inhibitor, i.e. dipeptidyl peptidase 4 inhibitor, is a kind of medicine for treating type 2 diabetes, and the medicine can inhibit the inactivation of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), improve the level of endogenous GLP-1 and GIP, promote the release of insulin by islet beta cells, and inhibit the secretion of glucagon by islet alpha cells, thereby improving insulin level, reducing blood sugar, and being difficult to induce hypoglycemia and increase body weight. The research of the applicant of the invention finds that the DPP-4 inhibitor can enhance the homing of the CAR-T cells to the solid tumor tissue and the infiltration of the CAR-T cells to the tumor tissue.

The invention also aims to provide an application of the reagent, in particular an application of a DPP4 inhibitor in the CAR-T cells.

In order to achieve the purpose, the invention adopts the following scheme:

the application of the DPP4 inhibitor in enhancing the tumor homing and tumor enrichment of the CAR-T cells and improving the treatment effect of CAR-T therapy on solid tumors.

Further, the tumor is a malignant tumor, including but not limited to lymphoma, prostate cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, lung cancer, kidney cancer, liver cancer, brain cancer, and skin cancer.

The DPP4 inhibitor enhances tumor homing and tumor enrichment capacity of the CAR-T cells, improving the therapeutic effect of CAR-T therapy on solid tumors. The normal tissue distribution of the CAR-T cells is reduced, the off-target risk of normal tissues is weakened, and the safety is higher.

The fourth object of the invention is to provide the use of said agent and said CAR-T cells, in particular in the treatment of CEA and/or PSCA highly expressed solid tumors.

The fifth object of the present invention is to provide a pharmaceutical composition, in particular a pharmaceutical composition comprising the agent, which comprises CAR-T cells and the DPP4 inhibitor.

The sixth purpose of the invention is to provide an application of the reagent, in particular to an application of the reagent in enhancing the tumor homing and tumor enrichment of CAR-T cells and improving the treatment effect of CAR-T therapy on solid tumors.

The invention has the beneficial effects that:

1) the DPP4 inhibitor provided by the invention can enhance the tumor homing and tumor enrichment capacity of CAR-T cells and improve the treatment effect of CAR-T therapy on solid tumors;

2) the DPP4 inhibitor provided by the invention can stimulate the infiltration of the CAR-T cells into tumor tissues and enhance the curative effect of the CAR-T cells on solid tumors;

3) the invention provides a new application of the DPP4 inhibitor, namely, the DPP4 inhibitor can be applied to the aspect of treating diabetes, and can also be combined with CAR-T technology to participate in the treatment of tumors.

Drawings

FIG. 1A shows CEA-CAR positivity, and FIG. 1B shows PSCA-CAR positivity.

FIG. 2 is a graph showing the results of the DPP4 inhibitor's ability to enhance chemotaxis of anti-CEA-CAR towards colorectal cancer cell lines.

FIG. 3 is a graph showing the results of DPP4 inhibitor enhancing the chemotactic ability of anti-PSCA-CAR towards CAR-T cervical cancer cell line Hela.

FIG. 4 is a graph of the effect of CEA-CAR-T in combination with DPPi inhibitor treatment on tumor proliferation in LoVo tumor-bearing mice.

FIG. 5A is the result of the detection of the CAR-T cell ratio in the tumor tissue of LoVo tumor-bearing mice, and FIG. 5B is the result of the detection of the copy number of the CAR gene in the tumor tissue of LoVo tumor-bearing mice.

FIG. 6 is a graph of the effect of CEA-CAR-T in combination with DPPi inhibitor treatment on tumor proliferation in Hela tumor-bearing mice.

FIG. 7A is the result of the detection of the CAR-T cell ratio in the tumor tissue of Hela tumor-bearing mice, and FIG. 7B is the result of the detection of the copy number of the CAR gene in the tumor tissue of Hela tumor-bearing mice.

Detailed Description

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental procedures, in the preferred embodiments, which do not specify specific conditions, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (third edition, sambrook et al), or according to the conditions recommended by the manufacturers. The examples are provided for better illustration of the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.

Example 1 an agent that enhances the ability of CAR-T cells to home to solid tumor tissue

An agent that enhances the ability of CAR-T cells to home to solid tumor tissue, wherein the DPP4 inhibitor is sitagliptin.

Example 2A CAR-T cell and preparation thereof

(1) Isolation of PBMC (peripheral blood mononuclear cells)

Hydroxyethyl starch (Shandong Qidu pharmaceutical Co., Ltd.) in an amount of 20 vol% was added to peripheral blood of healthy donors, and the mixture was mixed well and allowed to stand at room temperature for 30 min. PBMC was isolated using human lymphocyte isolate (Tianjin tertiary Biochemical Co.). The isolated cells were resuspended in fresh 1640 medium (Gibco) containing 10% FBS and placed in a 37 ℃ CO2 incubator for 2h to remove monocytes.

(2) Activation of T cells, viral infection and expansion

CD3(Miltenyi Biotec) and CD28(Miltenyi Biotec) coat PBMCs obtained in activation (1). The activated cells are added with CAR virus to infect the activated T cells, the CAR-T cells infected with the virus are subjected to amplification culture by using a 1640 culture solution containing 10% FBS (Gibco company), and meanwhile, 100IU/mL IL-2 (Liaoning satellite biologicals company) is added to stimulate proliferation.

The CAR structure is: ScFv-hinge-CD28TM-CD28-CD137-CD3Z

(3) Flow-based detection of CAR positivity

The CAR-T infected with virus 8d and the corresponding T cells not infected with virus were collected, washed 2 times with PBS, added with anti-human protein L antibody (Santa Cruz Co.), gently mixed, incubated at 4 ℃ for 30min, washed free of unbound antibody, and tested on the machine. As shown in fig. 1, fig. 1A is CEA-CAR positive rate; FIG. 1B shows the PSCA-CAR positivity.

Example 3 the ability of DPP4 inhibitors to enhance chemotaxis of CAR-T cells towards tumor cells

Since in vitro chemotaxis experiments are difficult to perform without the presence of chemokines, CXCL10 (PeproTech) was added exogenously to the in vitro experiments in order to enhance the sensitivity of the in vitro experiments, but not to affect the overall experimental trend.

After the LoVo (colorectal cancer cell line) and Hela (cervical cancer cell line) cells with good growth state are digested and collected, the cells are paved on a 6-hole plate at the constant temperature of 37 ℃ and CO₂The incubator is used for 2 days. The experimental group is added with 0.5mg/mL DPP4 inhibitor sitagliptin before collecting cells and reacts for 30 min. The cell supernatant was collected, centrifuged to remove cell debris, 600uL of the cell supernatant was placed in a 24-well plate, 100. mu.g/mL CXCL10 factor was added, and the plate was placed in a transwell chamber with a 5 μm pore size. CAR-T cells were collected and counted, taking 5X 10⁵Resuspending fresh cell culture medium, placing in a chamber, and standing at 37 deg.CConstant temperature CO₂Culturing in an incubator for 2 h. After 2h the chamber was removed and the cells in the lower chamber were collected and counted. The results are shown in FIG. 2 and FIG. 3, and FIG. 2 shows that the DPP4 inhibitor enhances the chemotactic ability of the anti-CEA-CAR for colorectal cancer cell lines, and the results show that the CEA-CAR-T has significantly enhanced migration ability to tumors after the addition of the DPP4 inhibitor, as shown by the CXCL10+ LoVo group and the DPPi + CXCL10+ LoVo group; FIG. 3 shows that the DPP4 inhibitor enhances the chemotactic ability of anti-PSCA-CAR towards CAR-T to cervical cancer cell line Hela, and as a result, the CEA-CAR-T migration ability to tumors is significantly enhanced after the addition of DPP4 inhibitor, as shown by CXCL10+ Hela group and DPPi + CXCL10+ Hela group. Taken together, the experimental results demonstrate the ability of DPP4 inhibitors to enhance the chemotaxis of CAR-T cells towards tumors.

Example 4 Effect of DPP4 inhibitor in combination with CAR-T cells on mouse tumor proliferation

Mouse transplantation tumor models of human CEA positive and human PSCA tumor cell lines are respectively established for verifying the anti-tumor effect of the CAR-T cells and the DPP4 inhibitor sitagliptin in the animal model.

Cg-PrkdcscidiI2rgtm1Sug/Jiccrl, NOG mouse for short, is bred by Mamoru Ito of the Japanese institute of Experimental animals (CIEA), and is the most common strain for CAR-T related in vivo tumor formation experiments internationally. In vivo verification the tumor target cells used were LoVo cells and Hela cells, respectively. Female NOD/SCID mice of 6-8 weeks old were selected, labeled with ear number, and placed on the back of the mice at 1X 10⁶Cell size LoVo or Hela cells were injected subcutaneously. Tumor volumes of mice were measured on day 6 of neoplasia and were randomly divided into three groups (LoVo, 6 mice per group) or a blank group, DPP4i (DPP inhibitor) group, CAR-T group and a combined group (Hela, 5 mice per group) according to tumor volume. The mice feeds of the DPP4i group and the combined group were changed to a medicated feed supplemented with sitagliptin (1.2%), and the blank group and CAR-T group were continued to be given ordinary feeds. Injecting CAR-T cells into tail vein of CAR-T group and combination group mice at 7 days of tumor formation, each cell amount is 1 × 10⁷The blank group and the DPP4i group were given the same volume of physiological saline.

Tumor volumes were measured weekly in each group of mice, and the results are shown in fig. 4 and 6. FIG. 4 shows that the combination therapy effect of CEA-CAR-T and DPPI inhibitor is better when LoVo tumor-bearing mice are tested; FIG. 6 shows that the combined treatment effect of PSCA-CAR-T and DPPI is better when Hela tumor-bearing mice are tested. In conclusion, in vivo animal experiment results show that the CAR-T cells and the DPP4 inhibitor have better curative effect in tumor treatment.

Example 5 detection of the Effect of DPP4 inhibitors on CAR-T cell tumor tissue infiltration

(1) Single cell isolation

Collecting mouse subcutaneous tumor, cutting into pieces with dissecting scissors in culture dish, adding digestive juice at 37 deg.C CO₂The incubator was digested for 30min, and digestion was stopped by adding 10mL of DMEM medium containing 10% FBS. After filtering the cell suspension through a 70-micron filter membrane, the cell suspension is centrifuged at 680g for 15min and washed twice with PBS, and then the new PBS is used for resuspension to obtain single cell suspension.

(2) Flow assay for tumor-infiltrating CAR-T cell content

Blowing and uniformly mixing the single cell suspension separated in the step (1), and taking 1 × 10⁶Adding a blocking antibody for resisting a mouse into living cells, blocking the living cells for 15min in a dark place at room temperature, removing non-specific binding, adding an anti-human CD3-FITC antibody, uniformly mixing, incubating at 4 ℃ for 30min, finally centrifuging, washing for 2 times by using PBS to remove unbound antibody, resuspending by using 200 mu L of PBS, and detecting on a flow cytometer. Results as shown in fig. 5A and fig. 7A, fig. 5A is an experimental result of LoVo tumor-bearing mice, and the CAR-T combined DPP4 inhibitor group (combination group) tumor tissue has a significantly higher proportion of CAR-T cells than the CAR-T group infused back alone; FIG. 7A is an experimental result of Hela tumor-bearing mice, and the CAR-T combined with DPP4 inhibitor group (combination group) has significantly higher CAR-T cell ratio in tumor tissues than the CAR-T group infused back alone. Taken together, CAR-T combined with DPP4 inhibitor group (combination group) the number of CAR-T cells in tumor tissue was significantly higher than that of CAR-T group reinfused alone, DPP4 inhibitor could promote infiltration of CAR-T cells into tumor tissue.

(3) qRT-PCR detection of CAR Gene copy number

Weighing subcutaneous tumor tissue of mouse, placing about 10mg of tissue into Ep tube, adding 500 μ L Extraction Buffer and 10 μ L proteinase K in turn, reversing and mixing, and digesting and cracking the tissue completely. Adding 4 mu L of RNase for digestion at 70 ℃ for 10min, adding the prepared NaCl solution, violently shaking and centrifuging, transferring the supernatant into a new EP tube, adding precooled absolute ethyl alcohol, slightly reversing and uniformly mixing, centrifuging again, discarding the supernatant, adding 1mL of 70% ethanol, reversing and uniformly mixing, centrifuging again and discarding the supernatant, drying the Ep tube, adding 100 mu L of TE buffer solution to resuspend DNA, and obtaining the tumor tissue genome.

The DNA obtained above was used for qRT-PCR:

reaction system:

name of reagent	Volume of
		SYBR premix ExTaq II(2×)	10μL
Upstream primer (10. mu.M)	0.5μL
		Downstream primer (10. mu.M)	0.5μL
DNA sample	1μL
		ddH₂O	8μL
Total	20μL

Reaction conditions are as follows:

experimental results as shown in fig. 5B and 7B, fig. 5B is an experimental result of LoVo tumor-bearing mice, and the CAR-T combined DPP4 inhibitor group (combination group) tumor tissue has significantly higher CAR copy number than the CAR-T group infused back alone; FIG. 7B is an experimental result of Hela tumor-bearing mice, and the CAR-T combined with DPP4 inhibitor group (combination group) has significantly higher CAR copy number in tumor tissues than the CAR-T group infused back alone. Taken together, CAR copy number in CAR-T combined DPP4 inhibitor group (combination group) tumor tissues was significantly higher than that of CAR-T group infused back alone, suggesting that DPP4 inhibitor may promote CAR-T cell infiltration into tumor tissues.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

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

100 105 110

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

115 120 125

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

130 135 140

Cys Thr Val Ser Gly Phe Thr Ile Ser Ser Gly Tyr Ser Trp His Trp

145 150 155 160

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

165 170 175

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

180 185 190

Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser

195 200 205

Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Asp Tyr

210 215 220

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

225 230 235 240

Val Ser Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

<210> 13

<211> 237

<212> PRT

<213> PSCA-ScFv

<400> 13

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Arg Phe Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr Tyr Ile His

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235

<210> 14

<211> 506

<212> PRT

<213> PSCA-CAR

<400> 14

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Arg Phe Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr Tyr Ile His

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505

<210> 15

<211> 18

<212> PRT

<213> Linker

<400> 15

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

<210> 16

<211> 15

<212> PRT

<213> Linker

<400> 16

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

1 5 10 15

<210> 17

<211> 120

<212> PRT

<213> VH

<400> 17

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

1 5 10 15

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

20 25 30

Tyr Ser Trp His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Gln Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

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

85 90 95

Ala Arg Glu Asp Tyr Asp Tyr His Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Ser Thr Val Thr Val Ser Ser

115 120

<210> 18

<211> 121

<212> PRT

<213> VH

<400> 18

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

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 19

<211> 119

<212> PRT

<213> VH

<400> 19

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Pro Val Thr Val Ser Ser

115

<210> 20

<211> 112

<212> PRT

<213> VH

<400> 20

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Phe Val Pro Lys Phe

50 55 60

Gln Gly Arg Ala Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

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

85 90 95

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

100 105 110

<210> 21

<211> 122

<212> PRT

<213> VH

<400> 21

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

1 5 10 15

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

20 25 30

Thr Met Ser Trp Val Arg Arg Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Tyr Ile His Asn Gly Gly Gly His Thr Tyr Tyr Pro Asp Thr Ile

50 55 60

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

65 70 75 80

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

85 90 95

Thr Arg Arg Met Tyr Tyr Gly Asn Ser His Trp Tyr Phe Asp Val Trp

100 105 110

Gly Ala Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 22

<211> 105

<212> PRT

<213> VL

<400> 22

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile Tyr

35 40 45

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

50 55 60

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

65 70 75 80

Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 23

<211> 111

<212> PRT

<213> VL

<400> 23

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

1 5 10 15

Gln Arg Ala Thr Met Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe

20 25 30

Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Val

50 55 60

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

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Thr Asn

85 90 95

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

100 105 110

<210> 24

<211> 106

<212> PRT

<213> VL

<400> 24

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

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

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 25

<211> 106

<212> PRT

<213> VL

<400> 25

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Arg Phe Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 26

<211> 107

<212> PRT

<213> VL

<400> 26

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Asp Ile Ser Asn Tyr

20 25 30

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

35 40 45

Tyr Tyr Thr Leu Lys Leu Asn Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Lys Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

Claims

1. A CAR-T cell expressing a chimeric antigen receptor comprising an extracellular antigen-recognition region and an intracellular activation signal domain, the extracellular antigen-recognition region of the chimeric antigen receptor comprising an ScFv, a hinge structure and a transmembrane structure, the amino acid sequence of the hinge structure being as set forth in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4 or SEQ ID No. 5.

2. The CAR-T cell of claim 1, wherein the amino acid sequence of the transmembrane region of the chimeric antigen receptor is as set forth in SEQ ID No.6 or SEQ ID No. 7.

3. The CAR-T cell of claim 1, wherein the extracellular antigen-recognition region of the chimeric antigen receptor has an amino acid sequence as set forth in SEQ ID No.11 or SEQ ID No. 13.

4. The CAR-T cell of claim 1, wherein the intracellular activation signaling domain of the chimeric antigen receptor comprises CD28 and/or CD137 and CD 3.

5. The CAR-T cell of claim 4, wherein the amino acid sequence of the intracellular activation signal domain of the chimeric antigen receptor is a tandem of SEQ ID No.8, SEQ ID No.9 and SEQ ID No. 10.

6. The CAR-T cell of claim 1, wherein the amino acid sequence of the chimeric antigen receptor is set forth in SEQ ID No.12 or SEQ ID No. 14.

7. The CAR-T cell according to any one of claims 1 to 6, wherein the CAR-T cell is targeted to CEA or PSCA.

Use of a DPP4 inhibitor in combination with a CAR-T cell according to any one of claims 1-7 for the preparation of a medicament for inhibiting tumor proliferation, wherein the DPP4 inhibitor includes but is not limited to sitagliptin, vildagliptin, saxagliptin, alogliptin and linagliptin.

9. The use of claim 8, wherein the tumor is a malignancy, including but not limited to lymphoma, prostate cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, lung cancer, renal cancer, liver cancer, brain cancer, and skin cancer.

10. A pharmaceutical composition comprising a CAR-T cell and the DPP4 inhibitor.

11. The pharmaceutical composition of claim 10, wherein: the pharmaceutical composition is used for inhibiting tumor proliferation.