CN109971717B

CN109971717B - T cells co-expressing CD40 antibody and mesothelin specific chimeric antigen receptor and uses thereof

Info

Publication number: CN109971717B
Application number: CN201711459160.2A
Authority: CN
Inventors: 钱其军; 金华君; 唐熙; 江芏青; 何周; 张志伟; 李林芳; 王超
Original assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Current assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Priority date: 2017-12-28
Filing date: 2017-12-28
Publication date: 2023-06-20
Anticipated expiration: 2037-12-28
Also published as: WO2019129173A1; CN109971717A

Abstract

The present invention relates to T cells co-expressing a CD40 antibody and a mesothelin-specific chimeric antigen receptor and uses thereof. The T cells of the invention contain coding sequences for chimeric antigen receptors that recognize mesothelin and coding sequences for CD40 activating antibodies; and/or express chimeric antigen receptor and CD40 activating antibody that recognize mesothelin. The externally expressed CAR gene in the T cells can accurately target mesothelin antigen, enhance the proliferation capacity of the T cells and secretion of cytokines, and the expressed CD40 antibody can help the CAR-T cells to break through inhibition of tumor microenvironment, so that the survival rate and efficacy of the CAR-T cells are improved, the killing of the CAR-T cells to the tumor cells is enhanced, and the anti-tumor effect is exerted by enhancing immune response.

Description

T cells co-expressing CD40 antibody and mesothelin specific chimeric antigen receptor and uses thereof

Technical Field

The invention belongs to genetic engineering and immunology, and relates to T cells co-expressing a CD40 antibody and a mesothelin specific chimeric antigen receptor and application thereof.

Background

Tumor immunotherapy is one of the most promising research directions in the current tumor treatment field, and journal of science evaluates tumor immunotherapy as the first breakthrough of ten science in 2013. Tumor immunotherapy is a wide variety of, and to date, the hottest, most effective, immunotherapy that has the greatest potential to combat tumor disease is chimeric antigen receptor T cells (CAR-T). Both the Kymriah and Kite Yescanta products of North have been marketed in batches.

A Chimeric Antigen Receptor (CAR) is an artificial receptor that generally comprises an extracellular antigen binding domain, a transmembrane hinge region, and an intracellular signaling region. Recombinant plasmids were generated by gene recombination in vitro of the single chain variable region (scFv) of an antibody recognizing a tumor associated antigen (tumor associated antigen, TAA) and the intracellular signaling domain "immunoreceptor tyrosine activation motif (immunoreceptor tyrosine-based activation motifs, ITAM)". The plasmid is transferred into T cells by a gene transduction method, and the T cells subjected to gene modification are called CAR-T cells. After large-scale expansion in vitro, CAR-T cells are infused back into patients and can exhibit potent anticancer effects in a non-MHC restricted mode.

220 CAR-T cell assays have been recorded at the end of 2016, with 133 for hematological malignancies and 78 for solid tumors in the current assay. CAR-T cells have met with great success in the treatment of hematological cancers, for example, with the most commonly used antigen MESO, with an average objective response rate of over 60%. However, hematological tumors are only a small fraction of many cancers, and solid tumors account for 90% of the tumors, whereas CAR-T has far different therapeutic effects in hematological tumors than solid tumors. The main reasons include: 1. solid tumors are highly heterogeneous and lack cell surface targets suitable for CAR-T treatment. 2. Solid tumors have a microenvironment that strongly inhibits immunity.

Mesothelin is a glycoprotein anchored to the cytoplasmic membrane by phosphatidylinositol (GPI), is highly expressed in a variety of tumor tissues, and is expressed in small amounts in mesothelial cells of normal pleura, pericardium and peritoneum. The mesothelin gene encodes a 69kDa precursor protein which is processed to form a 40kDa membrane-bound protein and a 31kDa shedding fragment called megakaryocyte stimulating factor (MPF) which is released extracellularly, and we refer to the fragment anchored to the membrane and can be divided into three regions, region I, region II and Region III, depending on the protein structure. On one hand, the GPI structural domain can activate the signal channels in NF kappa B, MAPK and PI3K cells, promote cell proliferation and resist apoptosis; on the other hand, the interaction with the receptor CA125/MUC16 leads to abnormal cell adhesion and promotes cancer cell metastasis. Mesothelin is a potential tumor-specific therapeutic target because of its limited distribution in normal tissues and its overexpression in various malignant tumors (mesothelioma, ovarian cancer, pancreatic cancer, gastric cancer, cholangiocarcinoma, etc.).

At present, small molecule drugs and antitoxins with mesothelin as targets have obtained good effects, and CAR-T cell researches with mesothelin as targets are well developed mainly aiming at pancreatic cancer (NCT 01897415, NCT 02465983), mesothelioma (NCT 01355965, NCT 02414269), lung cancer and breast cancer (NCT 02414269).

The CD40 antigen is one kind of cell surface molecule of TNFR superfamily, is one kind of I type transmembrane glycoprotein with molecular weight of 48KD, and is widely expressed in T cell, antigen presenting cell, hemopoietic cell, granulocyte, etc. CD40L is a type II transmembrane glycoprotein belonging to the TNF superfamily and is predominantly expressed in CD4+ helper T cells (Th cells). CD40-CD40L is a co-stimulatory molecule of great importance in the immune response, with a wide range of biological effects. CD40-CD40L interactions transmit signals, which cause the up-regulation of IL-12 levels, activate DC cells, enhance the presentation of APCs to antigens, and at the same time, CD40-CD40L can promote the secretion of a large amount of cytokines such as GM-CSF, IL-4, TNF-a, IFN-gamma by T cells, thereby enhancing CTL effects of CD8+ T cells and enhancing killing effects on tumors. Researchers at the university of hadfield developed a method of targeted therapy with CD40, which they activated with a ligand for CD40, followed by intravenous injection. The method was patented by university of Hardsiffeld (Oncogene, 2017May 4;36 (18): 2515-2528). APX005M (NCT 02482168) developed by Apexigen, a biopharmaceutical development company in the united states has a good inhibitory effect on non-small cell cancer, melanoma, urothelial cancer, high frequency microsatellite instability (MSI-H), head and neck cancer, and the like.

Disclosure of Invention

The present invention provides a T cell that self-expresses a CD40 activating antibody and targets mesothelin.

In one or more embodiments, the T cell genome incorporates an expression cassette for a CD40 activating antibody and an expression cassette for a chimeric antigen receptor that recognizes mesothelin.

In one or more embodiments, the amino acid sequence of the CD40 activating antibody is shown as amino acid residues 21-497 of SEQ ID NO. 2, or as SEQ ID NO. 2.

In one or more embodiments, the CD40 activating antibody has a coding sequence as shown in SEQ ID NO. 4 at positions 61-1491 or as shown in SEQ ID NO. 4.

In one or more embodiments, the chimeric antigen receptor that recognizes mesothelin contains, in order from N-terminus to C-terminus, an optional signal peptide, an anti-mesothelin III single chain antibody, a hinge region, a transmembrane region, an intracellular co-stimulatory signaling domain, and an intracellular signaling domain.

In one or more embodiments, the signal peptide is a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide, or a light chain signal peptide; more preferably a CD8 signal peptide; preferably, the amino acid sequence of the CD8 signal peptide is shown as amino acid residues 1-22 of SEQ ID NO. 1.

In one or more embodiments, the amino acid sequence of the scFv is shown as amino acid residues 23-272 of SEQ ID NO. 1.

In one or more embodiments, the hinge region is selected from the group consisting of an extracellular hinge region of CD8, an IgG1 Fc CH2CH3 hinge region, an IgD hinge region, an extracellular hinge region of CD28, an IgG4 Fc CH2CH3 hinge region, and an extracellular hinge region of CD 4; preferably a CD8 hinge region or an IgG4 CH2CH3 hinge region; preferably, the amino acid sequence of the hinge region of the IgG4 CH2CH3 is shown as amino acid residues 273-500 of SEQ ID NO. 1.

In one or more embodiments, the transmembrane region is one of a CD28 transmembrane region, a CD8 transmembrane region, a cd3ζ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region; preferably a CD8 transmembrane region, preferably having an amino acid sequence as shown in amino acid residues 501-528 of SEQ ID NO. 1.

In one or more embodiments, the intracellular co-stimulatory signaling domain includes an intracellular domain of a co-stimulatory signaling molecule, including an intracellular domain of CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell co-stimulatory factor (ICOS), and DNAX activator protein 10; preferably, the intracellular co-stimulatory signaling domain is the intracellular domain of CD 28; preferably, the amino acid sequence of the CD28 is shown as amino acid residues 529-569 of SEQ ID NO. 1.

In one or more embodiments, the intracellular signaling domain is a cd3ζ intracellular signaling domain or an fcsriy intracellular signaling domain; preferably a CD3 zeta intracellular signal domain, preferably the amino acid sequence of said CD3 zeta intracellular signal domain is shown as amino acid residues 570-681 of SEQ ID NO. 1.

In one or more embodiments, the chimeric antigen receptor has an amino acid sequence as shown in amino acid residues 23-681 of SEQ ID NO. 1, or as shown in SEQ ID NO. 1; preferably, the coding sequence of the chimeric antigen receptor is shown as the 67 th to 2043 rd bases of SEQ ID NO. 3 or as the SEQ ID NO. 3.

The present invention also provides a composition comprising: a vector comprising an expression cassette for a chimeric antigen receptor of the invention for integration of the expression cassette into the genome of a host cell; and a vector comprising an expression cassette for a CD40 activating antibody for integration of the expression cassette into the genome of a host cell.

The invention also provides a kit comprising:

(1) A vector comprising an expression cassette for a chimeric antigen receptor of the invention for integration of the expression cassette into the genome of a host cell; and

(2) A vector comprising an expression cassette for a CD40 activating antibody for integration of said expression cassette into the genome of a host cell.

The invention also provides a pharmaceutical composition containing the T cells.

The invention also provides the use of a T cell as described herein in the manufacture of a medicament for the treatment or prophylaxis of a malignant neoplasm. Preferably, the malignancy is a cancer whose cancer cell surface abnormally expresses mesothelin; preferably, the cancer is selected from: adenocarcinoma, mesothelioma, lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gall bladder cancer, esophageal cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, rectal cancer, hodgkin's lymphoma, pancreatic cancer, or prostate cancer; more preferably, the cancer is a cancer in which both mesothelin and CA125/MUC16 are highly expressed.

Drawings

Fig. 1: gene structure pattern of pNB328-mesoCAR, pS328- αcd40, pNB328-mesoCAR-2A- αcd40, pNB328- αcd 40-IRES-mesoCAR.

Fig. 2A: three CAR-T cell positive rates of mesoCAR-2A- αcd40, αcd40-IRES-mesoCAR, mesoCAR- αcd40 constructed by different methods.

Fig. 2B: three CAR-T cell positive rate antibody secretion amounts of mesoCAR-2A- αcd40, αcd40-IRES-mesoCAR, mesoCAR- αcd40 constructed by different methods.

Fig. 3A-3B: comparison of the positive rate and the secretion amount of the mesoCAR-alpha CD 40T cells constructed under the condition of different ratios of the CAR and the CD40 antibody plasmid.

Fig. 4: killing cervical cancer cells Hela, ovarian cancer cells SK-OV-3 and gastric cancer cells HGC-27 by mesoCAR-alpha CD 40T cells.

Fig. 5: alterations in IL-2, IL-4, IL-6, IL-10, TNF- α and IFN- γ cytokine secretion by mesoCAR- αCD40 under stimulation of mesothelin antigen.

Fig. 6: proliferation assay of mesoCAR T cells with mesoCAR- αcd 40T cells.

Fig. 7: therapeutic effects of mesoCAR T cells and mesoCAR-oc CD 40T cells on SK-OV-3 ovarian cancer mouse engraftment tumor model.

Detailed Description

The following is a description of some of the terms involved in the present invention.

In the present invention, the term "expression cassette" refers to the complete elements required for expression of a gene, including promoters and gene coding sequences.

The term "coding sequence" is defined herein as that portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (e.g., CAR, single chain antibody, hinge region, and transmembrane region). The boundaries of the coding sequence are typically determined by a ribosome binding site (for prokaryotic cells) immediately upstream of the open reading frame at the 5 'end of the mRNA and a transcription termination sequence immediately downstream of the open reading frame at the 3' end of the mRNA. Coding sequences may include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

The term "Fc", i.e., the crystallizable section of an antibody (fragment crystallizable, fc), refers to the peptide section comprising the CH2 and CH3 domains of the heavy chain of an antibody at the end of the stem of the "Y" structure of an antibody molecule, which is the site of interaction of the antibody with an effector molecule or cell.

The term "costimulatory molecule" refers to a molecule that is present on the surface of an antigen presenting cell and that is capable of binding to a costimulatory molecule receptor on a Th cell to produce a costimulatory signal. Proliferation of lymphocytes requires not only antigen binding but also signal of the co-stimulatory molecule. The co-stimulatory signal is transmitted to the T cell primarily through the co-stimulatory molecule CD80, CD86 expressed on the surface of the antigen presenting cell binding to the CD28 molecule on the surface of the T cell. B cells receive costimulatory signals through common pathogen components such as LPS, or through complement components, or through activated antigen-specific CD40L on Th cell surfaces.

The term "linker" or hinge is a polypeptide fragment that connects between different proteins or polypeptides in order to maintain the connected proteins or polypeptides in their respective spatial conformations in order to maintain the function or activity of the protein or polypeptide. Exemplary linkers include linkers comprising G and/or S, and for example Furin 2A peptides.

The term "specific binding" refers to a reaction between an antibody or antigen binding fragment and an antigen against which it is directed. In certain embodiments, an antibody that specifically binds (or has specificity for) an antigen refers to an antibody that binds the antigen with an affinity (KD) of less than about 10-5M, such as less than about 10-6M, 10-7M, 10-8M, 10-9M, or 10-10M or less. "specific recognition" has similar meaning.

The term "pharmaceutically acceptable excipients" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers. For example, pH modifiers include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

The term "effective amount" refers to the amount that achieves treatment, prevention, alleviation and/or relief of a disease or condition of the present invention in a subject.

The term "disease and/or disorder" refers to a physical state of the subject that is associated with the disease and/or disorder of the present invention.

The term "subject" or "patient" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., receiving a pharmaceutical composition of the invention for treating, preventing, alleviating and/or alleviating a disease or condition described herein.

The term "chimeric antigen receptor" (CAR) is an engineered receptor capable of anchoring a specific molecule (e.g., an antibody) that recognizes a tumor cell surface antigen to an immune cell (e.g., a T cell), allowing the immune cell to recognize a tumor antigen or viral antigen and kill a tumor cell or virus-infected cell. The CAR typically comprises, in order, an optional signal peptide, a polypeptide that binds to a tumor cell membrane antigen, such as a single chain antibody, a hinge region, a transmembrane region, and an intracellular signal region. In general, polypeptides that bind tumor cell membrane antigens are capable of binding with moderate affinity to membrane antigens that are widely expressed by tumor cells. The polypeptide that binds to a tumor cell membrane antigen may be a natural polypeptide or an artificial polypeptide; preferably, the synthetic polypeptide is a single chain antibody or Fab fragment.

The term "single chain antibody" (scFv) refers to an antibody fragment having the ability to bind antigen, which is formed by the amino acid sequence of the light chain variable region (VL region) and the amino acid sequence of the heavy chain variable region (VH region) of an antibody, which are joined by a hinge. In certain embodiments, the single chain antibody of interest (scFv) is from an antibody of interest. The antibody of interest may be a human antibody, including a human murine chimeric antibody and a humanized antibody. Antibodies may be secreted or membrane anchored; preferably of the membrane anchor type.

The inventor has unexpectedly found that the proliferation capability and killing capability of the CAR-T are obviously improved by adding the CD40 antibody in the research process of the CAR-T, and in view of the fact that the single anti-production of the CD40 relates to a complex production, preparation and purification process, the cost is high, the treatment cost is high, the inventor expresses the CD40 antibody on the existing mesothelin CAR-T cell, can start a co-stimulation signal after being combined with the CD40 antigen, promotes the activation and proliferation of the CAR-T cell in vivo, and increases the anti-tumor killing effect of the cytotoxic T cell, thereby improving the curative effect of specifically killing tumors.

Studies show that the IgG4 Fc fragment of the CD40 activating antibody is easily recognized by mononuclear/macrophages to be phagocytosed, and the CD40 activating antibody IgG4 Fc fragment is subjected to base mutation modification to meet the requirement of the CD40 activating antibody expressed by T cells, so that the CD40 activating antibody can well function and does not cause ADCC reaction.

Accordingly, the present invention provides a CD40 activating antibody comprising an anti-CD 40 single chain antibody and IgG4Fc. In certain embodiments, the amino acid sequence of the IgG4Fc is shown as amino acid residues 269-497 of SEQ ID NO. 2; preferably, the coding sequence is shown as the base sequence of 805-1491 of SEQ ID NO. 4.

In certain embodiments, the amino acid sequence of the light chain variable region (VL region) of said anti-CD 40 single chain antibody (scFv) is as shown in amino acid residues 21-146 of SEQ ID NO. 2; preferably, the coding sequence is shown as the 64 th to 438 th base sequence of SEQ ID NO. 4. In certain embodiments, the heavy chain variable region (VH region) amino acid sequence of said anti-CD 40 single chain antibody is as shown in amino acid sequences 161-268 of SEQ ID NO. 2; preferably, the coding sequence is shown as the 481-804 base sequence of SEQ ID NO. 4. In certain embodiments, the anti-CD 40 single chain antibody has an amino acid sequence as shown in amino acid residues 21-268 of SEQ ID NO. 2; preferably, the coding sequence is shown as 61-804 base sequences of SEQ ID NO. 4.

In certain embodiments, the CD40 antibody further comprises a light chain signal peptide. In certain embodiments, the CD40 antibody comprises, from N-terminus to C-terminus, a light chain signal peptide, an anti-CD 40 single chain antibody, and IgG4Fc, in that order. In certain embodiments, the amino acid sequence of the light chain signal peptide is as shown in amino acid residues 1-20 of SEQ ID NO. 2; preferably, the coding sequence of the light chain signal peptide is shown as the 1 st to 60 th base sequence of SEQ ID NO. 4.

In certain embodiments, the CD40 activating antibody has an amino acid sequence as set forth in SEQ ID NO. 2 at amino acid positions 21-497, or as set forth in SEQ ID NO. 2.

The invention also includes the coding sequence of the CD40 antibody, or the complement thereof, comprising at least the coding sequence of IgG4Fc, or the complement thereof, as described herein. In certain embodiments, the coding sequence of the CD40 antibody comprises the sequence shown in base sequence No. 61-1491 of SEQ ID NO. 4, preferably the sequence shown in SEQ ID NO. 4.

The invention also includes a nucleic acid construct comprising the coding sequence of the CD40 antibody of the invention or the complement thereof. Preferably, the nucleic acid construct is an expression vector or an integration vector for integrating the coding sequence or the complement thereof into a host cell.

The invention also provides a host cell comprising a nucleic acid construct as described herein.

The invention also provides the use of the CD40 antibodies, their coding sequences or complementary sequences, nucleic acid constructs, and host cells in the preparation of a method for treating or preventing malignant tumors, particularly those associated with CD40, including but not limited to the various malignant tumors described herein.

The invention also provides a T cell modified by the meso3CAR gene and capable of expressing the CD40 activating antibody, the T cell can express the CAR gene and the CD40 antibody stably at a high level, the externally expressed CAR gene can accurately target the mesothelin antigen, the proliferation capacity of the T cell and the secretion of cytokines are enhanced, the expressed CD40 antibody can help the CAR-T cell to break through the inhibition of tumor microenvironment, and the survival rate and the efficacy of the CAR-T cell are improved, so that the CAR-T cell can kill the tumor cell, and the anti-tumor effect can be exerted by enhancing the immune response. In addition, the exogenous meso3CAR gene and CD40 activating antibody gene can be integrated into the genome of T cells via the PB transposase system, thereby stabilizing sustained expression in T cells. The T cells capable of stably expressing the meso3CAR gene and the CD40 activating antibody gene at high level can be used for treating various malignant tumors with high mesothelin expression.

The CARs of the invention generally contain an optional signal peptide sequence, an scFv that recognizes an mesothelin antigen, a hinge region, a transmembrane region, an intracellular co-stimulatory signaling domain, and an intracellular signaling domain.

The signal peptide is a short peptide chain (5-30 amino acids in length) that directs the transfer of a newly synthesized protein to the secretory pathway, often referred to as the N-terminal amino acid sequence (sometimes not necessarily at the N-terminus) of the newly synthesized polypeptide chain that directs the transmembrane transfer (localization) of the protein, which is responsible for directing the protein into subcellular organelles of the cell containing different membrane structures. The signal peptide may be a secretory signal peptide or a membrane-bound signal peptide. In certain embodiments of the invention, the signal peptide is a CD8 signal peptide, a CD28 signal peptide, or a CD4 signal peptide or a light chain signal peptide; more preferably a CD8 signal peptide. The amino acid sequence of the CD8 signal peptide can be shown as the 1 st to 22 nd amino acid residues of SEQ ID NO. 1; in certain embodiments, the coding sequence is shown as bases 1-66 of SEQ ID NO. 3.

The scFv that recognizes the mesothelin antigen described herein can be a single chain antibody directed against the mesothelin antigen as known in the art. Preferably, the light chain variable region amino acid sequence and the heavy chain variable region amino acid sequence of the single chain antibody are derived from an antibody directed against the juxtamembrane terminal amino acid sequence of mesothelin. Preferably, the anti-mesothelin single chain antibodies described herein are single chain antibodies to Region I or III of mesothelin. Preferably, the light chain variable Region amino acid sequence and the heavy chain variable Region amino acid sequence of the single chain antibody are derived from an antibody directed against the amino acid sequence of mesothelin Region I or III. In certain embodiments, the amino acid sequence of mesothelin Region I is as shown in SEQ ID NO. 8; the amino acid sequence of the mesothelin Region III is shown as SEQ ID NO. 9. An exemplary single chain antibody against mesothelin Region I has the amino acid sequence shown in SEQ ID NO. 10. The amino acid sequence of the exemplary anti-mesothelin Region III single-chain antibody is shown as amino acid residues 23-272 of SEQ ID NO. 1, and the exemplary coding sequence is shown as nucleotide sequences 67-816 of SEQ ID NO. 3. Herein, if not specifically stated, mesothelin refers to a mesothelin fragment anchored to a membrane.

The hinge region, as used herein, refers to the region between the functional regions of the heavy chains CH1 and CH2 of an immunoglobulin which is rich in proline, does not form an alpha helix, and is subject to stretching and some degree of warping, which facilitates complementary binding between the antigen binding site of the antibody and the epitope. Hinge regions suitable for use herein may be selected from any one or more of the extracellular hinge region of CD8, the IgG1 Fc CH2CH3 hinge region, the IgD hinge region, the extracellular hinge region of CD28, the IgG4 Fc CH2CH3 hinge region, and the extracellular hinge region of CD 4. The hinge region is preferably a hinge region that is more than 50 amino acid residues in length, more preferably more than 80 amino acids in length. In certain embodiments, a CD8 a hinge region or an IgG4 Fc CH2CH3 hinge region is used herein. The amino acid residues 273-500 of the amino acid sequence SEQ ID NO. 1 of an exemplary IgG4 FcCH2CH3 hinge region, and the coding sequence of an exemplary IgG4 FcCH2CH3 hinge region is shown as SEQ ID NO. 3 817-1500.

The transmembrane region may be one of a CD28 transmembrane region, a CD8 transmembrane region, a cd3ζ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region; preferably CD8, and preferably has an amino acid sequence as shown in SEQ ID NO. 1, 501-528; in certain embodiments, the coding sequence is shown as bases 1501-1584 of SEQ ID NO. 3.

Intracellular costimulatory signaling domains the intracellular domain comprising the costimulatory signaling molecule may be selected from the group consisting of the intracellular domains of CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), inducible T cell costimulatory factor (ICOS) and DNAX activator protein 10 (DAP 10). In certain embodiments, the intracellular domain of the costimulatory signaling molecule is the intracellular domain of CD28, preferably having the amino acid sequence shown as amino acid residues 529-569 of SEQ ID NO. 1, and exemplary coding sequences shown as bases 1585-1707 of SEQ ID NO. 3.

The intracellular signaling domain is preferably an immunoreceptor tyrosine-activating motif, which may be a cd3ζ intracellular signaling domain or an fcsriy intracellular signaling domain; preferably a CD3 zeta intracellular signal domain, preferably the amino acid sequence of said CD3 zeta intracellular signal domain is as described in amino acid residues 570-681 of SEQ ID NO. 1; in certain embodiments, the coding sequence is as set forth in SEQ ID NO. 3, nos. 1708-2043.

In certain embodiments, the chimeric antigen receptor comprises, in order from N-terminus to C-terminus: optionally a CD8 signal peptide, an scFv against mesothelin Region III, an IgG4 Fc CH2CH3 hinge Region, a CD8 transmembrane Region, an intracellular domain of CD28, and a CD3 zeta intracellular signal domain; preferably, the amino acid sequence of the chimeric antigen receptor is shown as amino acid residues 23-681 of SEQ ID NO. 1. In certain embodiments, the chimeric antigen receptor further comprises a CD8 signal peptide, preferably the chimeric antigen receptor has an amino acid sequence as set forth in amino acid residues 1-22 of SEQ ID NO. 1.

It is to be understood that the present invention also includes chimeric antibody receptors described herein and coding sequences thereof.

The above-described portions forming the chimeric antigen receptor herein, such as the signal peptide, the light chain variable region and heavy chain variable region of the anti-mesothelin single-chain antibody, the hinge region, the transmembrane region, the intracellular co-stimulatory signaling domain, the intracellular signaling domain, and the like, may be directly linked to each other or may be linked by a linker sequence. The linker sequences may be linker sequences suitable for antibodies as known in the art, such as G and S containing linker sequences. The length of the linker may be 3 to 25 amino acid residues, for example 3 to 15, 5 to 15, 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a glycine linker sequence. The number of glycine in the linker sequence is not particularly limited, and is usually 2 to 20, for example 2 to 15, 2 to 10, 2 to 8. In addition to glycine and serine, other known amino acid residues may be contained in the linker, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), etc.

It will be appreciated that in gene cloning operations, it is often necessary to design suitable cleavage sites, which tend to introduce one or more unrelated residues at the end of the expressed amino acid sequence, without affecting the activity of the sequence of interest. To construct fusion proteins, facilitate expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside of the host cell, or facilitate purification of recombinant proteins, it is often desirable to add some amino acid to the N-terminus, C-terminus, or other suitable region within the recombinant protein, including, for example, but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like. Thus, the amino-or carboxy-terminus of a CAR herein can also contain one or more polypeptide fragments as protein tags. Any suitable label may be used herein. For example, the tag may be FLAG, HA, HA1, c-Myc, poly-His, poly-Arg, strep-TagII, AU1, EE, T7,4A6, ε, B, gE, and Ty1. These tags can be used to purify proteins.

Also included herein are polynucleotide sequences encoding the chimeric antigen receptors. The polynucleotide sequences herein may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded.

The polynucleotide sequences described herein can generally be obtained using PCR amplification methods. Specifically, primers can be designed based on the nucleotide sequences disclosed herein and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order. For example, in certain embodiments, the polynucleotide sequence encoding the fusion proteins described herein is set forth in SEQ ID NO. 3.

Also included herein are nucleic acid constructs comprising a polynucleotide sequence encoding the chimeric antigen receptor or a polynucleotide sequence encoding the CD40 activating antibody described herein, and one or more regulatory sequences operably linked to these sequences. In certain embodiments, the nucleic acid constructs of the invention are expression cassettes.

The regulatory sequence may be a suitable promoter sequence. The promoter sequence is typically operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

The regulatory sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used herein.

In certain embodiments, the nucleic acid construct is a vector. In particular, the coding sequence of the CAR or the coding sequence of the CD40 activating antibody herein can be cloned into many types of vectors, for example, such types of vectors include, but are not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. The vector may be an expression vector. The expression vector may be provided to the cell as a viral vector. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

In general, suitable vectors comprise an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers. For example, in certain embodiments, the invention uses a retroviral vector comprising a replication initiation site, a 3'LTR, a 5' LTR, the coding sequences for CARs described herein or the coding sequences for CD40 activating antibodies, and optionally a selectable marker.

Suitable promoters include, but are not limited to, the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV 40) early promoter, the mouse mammary carcinoma virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the epstein barr virus immediate early promoter, the ruses sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, the myosin promoter, the heme promoter, and the creatine kinase promoter. Further, the use of inducible promoters is also contemplated. The use of an inducible promoter provides a molecular switch that is capable of switching on expression of a polynucleotide sequence operably linked to the inducible promoter when expressed for a period of time and switching off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

In certain embodiments, various promoter sequences published by CN201510021408.1 can be used, including but not limited to the CCEF promoter comprising the mCMV enhancer, the hCMV enhancer and the EF 1. Alpha. Promoter shown in SEQ ID NO. 5 of this application; the TCEF promoter shown in SEQ ID NO. 7 and containing the CD3e enhancer, the mCMV enhancer, the hCMV enhancer and the EF1 alpha promoter; the CCEFI promoter shown in SEQ ID NO. 8 and containing the mCMV enhancer, the hCMV enhancer and the EF1 alpha promoter containing the intron; the TEFI promoter shown in SEQ ID NO. 3 and containing a CD3e enhancer and an EF1 alpha promoter containing an intron; and the TCEFI promoter shown in SEQ ID NO. 3 and containing the CD3e enhancer, the mCMV enhancer, the hCMV enhancer and the EF1 alpha promoter containing the intron. The entire contents of this application are incorporated herein by reference.

Selectable markers include either or both selectable marker genes or reporter genes to facilitate identification and selection of expressing cells from a population of cells infected with the viral vector. Useful selectable marker genes include, for example, antibiotic resistance genes, such as neo and the like. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein genes.

In certain embodiments, the coding sequences for the chimeric antigen receptor and the coding sequences for the CD40 activating antibody described herein are separately cloned into vectors (also referred to as integration vectors), particularly transposon vectors, for integration of the nucleic acid sequence of interest into the genome of the host cell. In certain embodiments, the transposon vector is a eukaryotic expression vector containing a transposable element selected from piggybac, sleep reliability, frog priority, tn5, or Ty. Such transposon vectors contain the 5 'inverted terminal repeat (5' LTR) of the corresponding transposon and the 3 'inverted terminal repeat (3' LTR) of the corresponding transposon. The transposase may be a transposase from a piggybac, sleep bearing, frog priority, tn5 or Ty transposase system. When transposases from different transposition systems are used, the sequences of the 5'LTR and 3' LTR in the vector are also changed accordingly to sequences that fit the transposition system, as can be readily determined by one skilled in the art. Between the 5'ltr and the 3' ltr is an expression cassette for a CAR or antibody of the invention, including the corresponding promoter sequence, coding sequence for the CAR or antibody, and e.g., a polyA tailing signal sequence.

In certain embodiments, the transposase is a transposase from the piggybac transposable system. Thus, in these embodiments, the transposon 5 'inverted terminal repeat and 3' inverted terminal repeat are the 5 'inverted terminal repeat and 3' inverted terminal repeat, respectively, of the piggybac transposon. In certain embodiments, the transposon 5' inverted terminal repeat is as shown in CN 201510638974.7 (the contents of which are incorporated herein by reference) SEQ ID No. 1. In certain embodiments, the transposon 3' inverted terminal repeat is as shown in CN 201510638974.7SEQ ID NO:4. In certain embodiments, the piggybac transposase is a transposase comprising a c-myc nuclear localization signal coding sequence. In certain embodiments, the coding sequence of the piggybac transposase is as set forth in CN 201510638974.7SEQ ID NO:5.

Promoters of the transposase coding sequence may be any of the promoters known in the art for controlling the expression of the transposase coding sequence. In certain embodiments, the expression of the transposase coding sequence is controlled using a CMV promoter. The sequence of the CMV promoter may be as shown in CN 201510638974.7SEQ ID NO:6.

In certain embodiments, the vector of the invention comprising the coding sequence for the chimeric antigen receptor is the pNB328 vector disclosed in CN 201510638974.7. The coding sequences for the chimeric antigen receptor of the invention can be prepared by methods conventional in the art and cloned into a suitable vector.

In certain embodiments, the vector for integrating the gene of interest into the genome of the host cell does not contain a transposase coding sequence. For example, such vectors may be obtained by removing the transposase coding sequence from the pNB328 vector. Typically, such vectors are used to integrate the coding sequence for a CD40 activating antibody and the coding sequence for a signal peptide (e.g., the coding sequence for a light chain signal peptide) into the genome of a host cell. Exemplary light chain signal peptides have the amino acid sequence shown in SEQ ID NO:2 at amino acid residues 1-20, the coding sequence of an exemplary light chain signal peptide is shown in SEQ ID NO:4 1-60 bases.

In certain embodiments, a T cell modified by a meso3CAR gene described herein and capable of expressing a CD40 activating antibody can be transformed into: a vector comprising a transposase coding sequence for integration into the T cell genome of a chimeric antigen receptor coding sequence, and a vector comprising no transposase coding sequence for integration into the T cell genome of a coding sequence of a CD40 activating antibody as described herein.

Preferably, the T cells are transformed with a vector comprising a chimeric antigen receptor coding sequence constructed using the pNB328 vector as a scaffold vector and a vector comprising a CD40 activating antibody coding sequence constructed using the pS328 vector (which does not comprise a transposase coding sequence as compared to pNB 328) as a scaffold vector. In certain embodiments, the chimeric antigen receptor has a coding sequence as set forth in SEQ ID NO. 3; the coding sequence of the CD40 activating antibody is shown as the 61 st-1491 st base sequence of SEQ ID NO. 4. In certain embodiments, the signal peptide of the CD40 activating antibody is a light chain signal peptide in the vector comprising the coding sequence of the CD40 activating antibody. The amino acid sequence of the exemplary light chain signal peptide can be shown as amino acid residues 1-20 of SEQ ID NO. 1; an exemplary light chain signal peptide has a coding sequence shown in nucleotide sequences 1-60 of SEQ ID NO. 4. More specifically, in certain embodiments, the transposase-containing coding sequence-containing vector that incorporates the chimeric antigen receptor coding sequence in the T cell genome contains a 5'ltr, a promoter, a light chain signal peptide coding sequence, a coding sequence that recognizes the scFv of a mesothelin antigen (preferably a coding sequence that recognizes the scFv of mesothelin Region III), a coding sequence of the IgG4 Fc CH2CH3 hinge Region, a coding sequence of the CD8 transmembrane Region, a coding sequence of the CD28 intracellular domain, a coding sequence of the CD3 zeta intracellular signal domain, a polyA tailing signal sequence, a coding sequence of the 3' ltr and transposase, and promoters thereof; the vector without transposase coding sequence, which incorporates the coding sequence of the CD40 activating antibody described herein in the T cell genome, contains a promoter, a coding sequence of a light chain signal peptide, a coding sequence of a CD40 activating antibody, and a polA tailing signal sequence in that order between the 5'ltr and the 3' ltr.

Preferably, the mass ratio of the vector containing the chimeric antigen receptor coding sequence to the vector containing the CD40 activating antibody coding sequence at the time of transfection is 1 to 7:1 to 7, preferably 1: 3-1: 3, more preferably 1:1 to 3, more preferably 1:1 to 2, more preferably 1:1.

methods of transfection are conventional in the art and include, but are not limited to: viral transduction, microinjection, particle bombardment, gene gun transformation, electrotransformation, and the like. In certain embodiments, electrotransfection is used to transfect the vector into a cell of interest.

The cells of interest may be a variety of T cells well known in the art, including but not limited to T cells of mixed cell populations such as peripheral blood T lymphocytes, cytotoxic killer T Cells (CTLs), helper T cells, suppressor/regulatory T cells, γδ T cells, and cytokine-induced killer Cells (CIKs), tumor Infiltrating Lymphocytes (TILs), and the like. In certain embodiments, the T cells may be derived from PBMCs of B cell malignancy patients. In certain embodiments, the T cell is a primary culture T cell.

The invention also provides a composition comprising a vector comprising a chimeric antigen receptor coding sequence as described herein and a vector comprising a coding sequence for a CD40 activating antibody as described herein. Suitable agents may also be included in the composition, including but not limited to agents for transfection.

The invention also provides a kit comprising a vector comprising the chimeric antigen receptor coding sequence described herein and a vector comprising the coding sequence of a CD40 activating antibody described herein, or a composition described herein. The kit may also be provided with reagents or instruments for transferring the vector into cells.

The expression cassette, as described herein, contains at least a suitable promoter and polyA tailing signal sequence in addition to the coding sequence for the chimeric antigen receptor or CD40 activating antibody.

The invention also provides a pharmaceutical composition comprising the T cells described herein. The pharmaceutical composition may contain suitable pharmaceutically acceptable carriers or excipients. The pharmaceutical composition contains a therapeutically or prophylactically effective amount of T cells. The therapeutically or prophylactically effective amount of T cells can be determined based on factors such as the patient's condition.

The invention also provides the use of a T cell or a pharmaceutical composition thereof as described herein in the manufacture of a medicament for the treatment or prophylaxis of a malignant tumor. The invention also provides a method of treating or preventing a malignancy, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a T cell of the invention. Cancers suitable for treatment or prophylaxis of T cells described herein are preferably cancers in which mesothelin is abnormally expressed on the surface of cancer cells; preferably, the cancer is selected from: adenocarcinoma, mesothelioma, lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gall bladder cancer, esophageal cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, rectal cancer, hodgkin's lymphoma, pancreatic cancer, or prostate cancer; more preferably, the cancer is a cancer in which both mesothelin and CA125/MUC16 are highly expressed.

Embodiments of the present invention will be described in detail below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples, and are carried out according to techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, ind. Molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1: construction of recombinant plasmids pNB328-mesoCAR, pS328- αCD40-wt, PNS328- αCD40-IRES-mesoCAR, pNB328-mesoCAR-2A- αCD40 and acquisition of chimeric antigen receptor-modified T cells

The construction modes of the mesoCAR (the nucleotide sequence is shown as SEQ ID NO:3, the amino acid sequence is shown as SEQ ID NO: 1), the anti-CD40 (the αCD40, the nucleotide sequence is shown as SEQ ID NO:4, the amino acid sequence is shown as SEQ ID NO: 2), the anti-CD40-wt (the αCD40-wt, the nucleotide sequence is shown as SEQ ID NO: 12), the mesoCAR-2A- αCD40 (the nucleotide sequence of 2A is shown as SEQ ID NO:5, the amino acid sequence is shown as SEQ ID NO: 6), the αCD40-IRES-mesoCAR gene (the nucleotide sequence of IRES is shown as SEQ ID NO: 7) are respectively filled between pNB328, pS328 vector EcoRI and SalI cleavage sites, and the constructed recombinant plasmids are named pNB328-mesoCAR, pS328- αCD40-wt, pNB-328- αCD40, p328- αCD40, and pIRES- αCD40.

For structure and sequence, pNB328 is referred to CN 201510638974.7, the entire contents of which are incorporated herein by reference; in contrast to pNB328, pS328 lacks PB transposon sequences, and the other elements are identical to pNB328 vectors. The promoter sequence and polyA tailing signal sequence are not shown in the structural schematic diagrams, and are located between the 5'LTR and the signal peptide sequence and before the 3' LTR, respectively.

Example 2: construction of CAR T cells

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from Shanghai cell therapy production centers. Culturing PBMC for 2-4h in an adherence way, wherein non-adherence suspension cells are initial T cells, collecting the suspension cells into a 15ml centrifuge tube, centrifuging for 3min at 1200rmp, discarding the supernatant, adding physiological saline, centrifuging for 3min at 1200rmp, discarding the physiological saline, and repeating the steps; four 1.5ml centrifuge tubes were taken and 5X 10 added to each tube ⁶ The individual cells, accession number a, b, c, d,1200rmp were centrifuged for 3min, the supernatant was discarded, the electrotransfer kit (from Lonza corporation) was taken, 100ul of electrotransfer reagent was added in proportion to a, b, c, d tubes, 4ug of each of the constructed recombinant plasmids pNB328-mesoCAR and pS328- αcd40 was added to a tube, 6ug of pNB328-mesoCAR-2A- αcd40 plasmid was added to b tube, 6ug of pNB328- αcd40-IRES-mesoCAR plasmid was added to c tube, and 6ug of the control plasmid (pNB 328 blank plasmid) was added to d tube; transferring the mixed solution into an electric rotating cup, placing into an electric rotating instrument, selecting a required program, and performing Electric shock; transferring the electrotransformed cell suspension into six-well plate (AIM-V culture solution containing 2% FBS) with micropipette in kit, mixing, standing at 37deg.C, and 5% CO ₂ Culturing in incubator for six hours, adding stimulating factors IL-2 and meso/anti-CD28, 37 deg.C, 5% CO ₂ Culturing for 3-4 days, observing the growth of T cells, and obtaining mesoCAR-alpha CD 40T cells, mesoCAR-2A-alpha CD 40T cells and alpha CD40-IRES-mesoCAR T cells which express CD40 antibody and target mesothelin.

The same procedure was used to construct mesoCAR T cells using pNB328-mesoCAR (6 ug) constructed in example 1. Mock T cells were constructed using pNB328 blank plasmid (6 ug); mesoCAR- αCD40-wt T cells were constructed using pNB328-mesoCAR (4 ug) and pS328- αCD40-wt (4 ug) constructed in example 1.

Example 3: CAR T cell positive rate and antibody secretion

1. Flow detection of CAR T cell positive rate

The mesoCAR- αCD 40T cells, the mesoCAR-2A- αCD 40T cells and the αCD40-IRES-mesoCAR T cells prepared in example 2 were collected and divided into two parts each of 1X 10 ⁶ Cells were washed twice with saline, resuspended in 100ul saline, one portion added with 1ug of meso-biotin and the other portion without, and incubated at 4℃for 30 minutes. The cells were resuspended in 100ul of saline, and 1ul of streptomycin-PE antibody was added and incubated at 4℃for 30 min. Washing twice with physiological saline, and detecting in the presence of the machine, wherein only the secondary antibody is added as a control, and the result is shown in figure 2A.

ELISA three T cell antibody expression levels obtained in example 2 were measured.

(1) CD40 antigen was diluted to 0.5ug/ml (5 ul+1ml coating) with coating solution, and the enzyme-labeled reaction plate was coated at 100 ul/well overnight at 4 ℃.

(2) The cells were washed 5 times with PBST for 3 minutes each, and then dried with absorbent paper at 200 ul/well.

(3) 100ul of blocking solution was added to each well and incubated at 37℃for 1 hour.

(4) The cells were washed 5 times with PBST for 3 minutes each, and then dried with absorbent paper at 200 ul/well.

(5) Samples and standards were added, 100 ul/well, multiplex wells and control wells were set, and incubated for 1 hour at 37 ℃.

(6) The cells were washed 5 times with PBST for 3 minutes each, and then dried with absorbent paper at 200 ul/well.

(7) Blocking solution IgG F4HRP1: diluted at 30000, 100 ul/well, incubated at 37℃for 45 min.

(8) The cells were washed 5 times with PBST for 3 minutes each, and then dried with absorbent paper at 200 ul/well.

(9) Adding the color development liquid TMB,100 ul/hole, and developing for 10-15min at 37 ℃ in dark.

The reaction was stopped by adding a stop solution, 50 ul/well.

OD value was measured at 450nm on a microplate reader, a standard curve was drawn, and CD40 antibody concentration was calculated.

The results are shown in FIG. 2B.

Example 4: pNB328-mesoCAR and pS 328-alpha CD40 different plasmid ratio test

The amounts of pNB328-mesoCAR and pS 328-. Alpha.CD40 plasmids obtained in example 1 were set to 7 ratios of 1ug+7ug, 2ug+6ug, 3ug+5ug, 4ug+4ug, 5ug+3ug, 6ug+2ug, 7ug+1ug, respectively, and CAR T cell construction was performed in the same manner as in example 2. The positive rate and the antibody secretion amount of the CAR T cells constructed and tested under 7 ratios were detected respectively (the method is the same as in example 3), and the results are shown in FIGS. 3A and 3B.

Example 5: comparison of killing function of mesoCAR T and mesoCAR-alpha CD 40T cells

The real-time label-free cell function analyzer detects the killing effect of the mesoCAR T cells and the mesoCAR- αcd 40T cells constructed in example 2 on tumor cells in vitro.

Specifically, effector cells and target cells matched with MHC class I in a typing way are selected, and the real-time label-free cell function analyzer (RTCA) of the Aisen company is used for detecting the in-vitro killing activity of the two CAR-T cells, and the specific steps are as follows:

(1) Zeroing: adding 50 μl of DMEM or 1640 culture solution into each well, placing into instrument, selecting step 1, and zeroing;

(2) Target cell plating: cervical cancer cell Hela, ovarian cancer cell SK-OV-3, gastric cancer HGC-27 (purchased)Purchased from American type culture Collection ATCC) at 10 per well ⁴ Spreading the cells/50 μl in a plate containing detection electrodes, standing for several minutes, standing for stabilization, placing into instrument, starting step 2, and culturing cells;

(3) Adding effector cells: after the target cells are cultured for 24 hours, step 2 is suspended, effector cells are added, 50 mu l of effector cells are added in each hole, the effective target ratio is set to be 4:1, mock T cells are used as a control, step 3 is started, and after co-culture is continued for 24 hours, a cell proliferation curve is observed;

As shown in fig. 4, the mesoCAR- αcd 40T cells from which the CD40 antibodies were expressed were substantially identical to the mesoCAR T cell killing function alone, and antibody expression did not affect CAR-T function.

Example 6: comparison of cytokine release by mesoCAR with mesoCAR- αcd 40T cells under specific stimulation of mesothelin antigen

Coating 96-well plate with 2ug/ml mesothelin antigen, coating overnight at 4deg.C, washing 3 times with PBS, adding 1×10 ⁵ The resulting meso3CAR T cells and mesoCAR- αcd 40T cells and control MockT cells were constructed, and cell supernatants were collected after 24h of culture. The secretion of cytokines after the stimulation of the three T cells by the mesothelin antigen is detected by using CBA Human Th1/Th2Cytokine Kit II of BD, and the specific steps are as follows:

(1) Mixing human IL-2, IL-4, IL-6, IL-10, TNF and IFN-gamma capturing magnetic beads, vortex oscillating and mixing the capturing magnetic beads, and adding 50ul of the capturing magnetic beads after mixing into each tube;

(2) 50ul of human Th1/Th2cytokine standard (dilution of magnification 5000pg/ml, 2500pg/ml, 1250pg/ml, 625pg/ml, 312.5pg/ml, 156pg/ml, 80pg/ml, 40pg/ml, 20pg/ml, 0 pg/ml) and 50ul of sample to be tested (2-fold dilution with diluent) were added.

(3) 50ul of human Th1/Th2-II-PE detection antibody was added to each tube;

(4) Incubating for 3 hours at room temperature in a dark place;

(5) Adding 1ml of washing buffer solution into each tube, centrifuging for 5min at 200 min, and discarding the supernatant;

(6) Adding 300ul of washing buffer solution into each tube to resuspend the cells, transferring the cells into a flow tube, and detecting fluorescence values by using a flow cytometer;

the results are shown in figure 5, with no significant difference in cytokine secretion from the mesoCAR- αcd 40T cells expressing CD40 antibodies versus the mesoCAR T cells alone.

Example 6: mesoCAR and mesoCAR- αcd 40T cell proliferation assay

Each taking 3×10 ⁵ mesoCAR, mesoCAR-. Alpha.CD40T cells and Mock-T cells on day 8 of the culture of example 2 were placed in 12-well plates for culture in 1ml volumes.

2. A96-well white opaque plate was prepared, and 100. Mu.L of each cell-containing culture solution was added to each well from the three groups of cells, while the cell-free culture solution was used as a blank. 100. Mu. LCellTiter-Glo reagent was added to each well, mixed well on a shaker for 2min, incubated at room temperature for 10min, and the Luc fluorescence values were read by an ELISA reader. The CellTiter-Glo Luminescent Cell Viability Assay kit used was purchased from Promega corporation.

3. Cells cultured in 12-well plates were sampled daily on

days

9, 10, 11, 12, and 13 of culture, and cell proliferation curves were plotted based on fluorescence values as measured by the above procedure.

The results are shown in figure 6, mesoCAR-oc CD 40T cells have better proliferative effects than mesoCAR T cells.

Example 7: therapeutic effects of mesoCAR and mesoCAR-oc CD 40T cells on ovarian cancer mouse engraftment tumor model

1: the NSG complete immunodeficiency mice with the age of 4-6 weeks have the average weight of 22-27 g and are fed by SPF grade animal laboratories provided by biological company of Baioci.

2: culturing human ovarian cancer cells SK-OV-3-luc in vitro, taking adherent growth cells in logarithmic growth phase, digesting with 0.25% pancreatin, centrifuging, collecting cells, re-suspending with PBS, centrifuging at 1000rmp room temperature for 2 min, discarding supernatant, re-suspending with PBS, centrifuging, collecting cells, and adjusting cell suspension concentration to 5×10 ⁷ And each ml.

3: the mouse was inoculated subcutaneously with OVCAR-3-luc cells, 0.1 ml/mouse dorsum of the right rib. After 7 days of inoculation, the fluorescence intensity can be observed by a living body imager, and the tumor size can be measured by a vernier caliperNSG immunodeficient mice were randomly divided into 5 groups, PBS group (100 ul) and Mock T group, mesoCART group, mesoCAR- αCD40-wt T group, mesoCAR- αCD 40T group (1×10) ⁷ Individual cells/cell). The administration route is tail vein injection.

4: mice were observed daily for their state of life and observed every 4 days for tumor changes by a biopsy imager.

The results are shown in FIG. 7.

Sequence listing

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SHANGHAI ENGINEERING RESEARCH CENTER FOR CELL THERAPY GROUP Co.,Ltd.

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Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

465 470 475 480

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

485 490 495

Lys

<210> 3

<211> 2043

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgagcgagg tgcagctggt ggagtccggg ggaggcctgg tccagcctgg gggatccctg 120

agactctcct gcgcagcctc tggattcgac ctcggtttct acttttacgc ctgttgggtc 180

cgccaggctc cagggaaggg cctggagtgg gtctcatgca tttatactgc tggtagtggt 240

agcacgtact acgcgagctg ggcgaaaggc cgattcacca tctccagaga caattcgaag 300

aacacgctgt atctgcaaat gaacagtctg agagccgagg acacggccgt gtattactgt 360

gcgagatcta ctgctaatac tagaagtact tattatctta acttgtgggg ccaaggcacc 420

ctggtcaccg tctcctcagg cggaggcgga tcaggtggtg gcggatctgg aggtggcgga 480

agcgacatcc agatgaccca gtctccatcc tccctgtctg catctgtggg agacagagtc 540

accatcactt gccaggccag tcagaggatt agtagttact tatcctggta tcagcagaaa 600

ccagggaaag ttcccaagct cctgatctat ggtgcatcca ctctggcatc tggggtcccc 660

tcgcggttca gtggcagtgg atctgggaca gatttcactc tcaccatcag cagcctgcag 720

cctgaagatg ttgccactta ctactgtcag agttatgctt attttgatag taataattgg 780

catgctttcg gcggagggac caaggtggag atcaaagagt ccaaatatgg tcccccatgc 840

ccaccatgcc cagcacctcc cgtggccgga ccatcagtct tcctgttccc cccaaaaccc 900

aaggacactc tcatgatctc ccggacccct gaggtcacgt gcgtggtggt ggacgtgagc 960

caggaagacc ccgaggtcca gttcaactgg tacgtggatg gcgtggaggt gcataatgcc 1020

aagacaaagc cgcgggagga gcagttccag agcacgtacc gtgtggtcag cgtcctcacc 1080

gtcctgcacc aggactggct gaacggcaag gagtacaagt gcaaggtctc caacaaaggc 1140

ctcccgtcct ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agagccacag 1200

gtgtacaccc tgcccccatc ccaggaggag atgaccaaga accaggtcag cctgacctgc 1260

ctggtcaaag gcttctaccc cagcgacatc gccgtggagt gggagagcaa tgggcagccg 1320

gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 1380

agcaggctaa ccgtggacaa gagcaggtgg caggagggga atgtcttctc atgctccgtg 1440

atgcatgagg ctctgcacaa ccactacaca cagaagagcc tctccctgtc tctgggtaaa 1500

cccttttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt gctagtaaca 1560

gtggccttta ttattttctg ggtgaggagt aagaggagca ggctcctgca cagtgactac 1620

atgaacatga ctccccgccg ccccgggccc acccgcaagc attaccagcc ctatgcccca 1680

ccacgcgact tcgcagccta tcgctccaga gtgaagttca gcaggagcgc agacgccccc 1740

gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1800

tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 1860

aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1920

agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1980

ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 2040

cgc 2043

<210> 4

<211> 1491

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 120

tcctgcaagg cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc 180

cctggacaag ggcttgagtg gatgggatgg atcaaccctg acagtggtgg cacaaactat 240

gcacagaagt ttcagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 300

atggagctga acaggctgag atctgacgac acggccgtgt attactgtgc gagagatcag 360

cccctaggat attgtactaa tggtgtatgc tcctactttg actactgggg ccagggaacc 420

ctggtcaccg tctcctcagg tggaggcggt tcaggcggag gtggcagcgg cggtggcggg 480

tcggacatcc agatgaccca gtctccatct tccgtgtctg catctgtagg agacagagtc 540

accatcactt gtcgggcgag tcagggtatt tacagctggt tagcctggta tcagcagaaa 600

ccagggaaag cccctaacct cctgatctat actgcatcca ctttacaaag tggggtccca 660

tcaaggttca gcggcagtgg atctgggaca gatttcactc tcaccatcag cagcctgcaa 720

cctgaagatt ttgcaactta ctattgtcaa caggctaaca ttttcccgct cactttcggc 780

ggagggacca aggtggagat caaagagtcc aaatatggtc ccccatgccc accatgccca 840

gcacctgagt tcgagggggg accatcagtc ttcctgttcc ccccaaaacc caaggacact 900

ctcatgatct cccggacccc tgaggtcacg tgcgtggtgg tggacgtgag ccaggaagac 960

cccgaggtcc agttcaactg gtacgtggat ggcgtggagg tgcataatgc caagacaaag 1020

ccgcgggagg agcagttcca gagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 1080

caggactggc tgaacggcaa ggagtacaag tgcaaggtct ccaacaaagg cctcccgtcc 1140

tccatcgaga aaaccatctc caaagccaaa gggcagcccc gagagccaca ggtgtacacc 1200

ctgcccccat cccaggagga gatgaccaag aaccaggtca gcctgacctg cctggtcaaa 1260

ggcttctacc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 1320

tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaggcta 1380

accgtggaca agagcaggtg gcaggagggg aatgtcttct catgctccgt gatgcatgag 1440

gctctgcaca accactacac acagaagagc ctctccctgt ctctgggtaa a 1491

<210> 5

<211> 78

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

cgtaggaaac gaggcagcgg cgccacaaac ttctctctgc taaagcaagc aggtgatgtt 60

gaagaaaacc ccgggcct 78

<210> 6

<211> 26

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

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

1 5 10 15

Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

20 25

<210> 7

<211> 197

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

ccggcgggtt tctgacatcc ggcgggtttc tgacatccgg cgggtttctg acatccggcg 60

ggtttctgac atccggcggg tttctgacat ccggcgggtt tctgacatcc ggcgggtttc 120

tgacatccgg cgggtttctg acatccggcg ggtttctgac atccggcggg tgactcacaa 180

ccccagaaac agacata 197

<210> 8

<211> 94

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

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

1 5 10 15

Asp Glu Ser Leu Ile Phe Tyr Lys Lys Trp Glu Leu Glu Ala Cys Val

20 25 30

Asp Ala Ala Leu Leu Ala Thr Gln Met Asp Arg Val Asn Ala Ile Pro

35 40 45

Phe Thr Tyr Glu Gln Leu Asp Val Leu Lys His Lys Leu Asp Glu Leu

50 55 60

Tyr Pro Gln Gly Tyr Pro Glu Ser Val Ile Gln His Leu Gly Tyr Leu

65 70 75 80

Phe Leu Lys Met Ser Pro Glu Asp Ile Arg Lys Trp Asn Val

85 90

<210> 9

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

Tyr Pro Lys Ala Arg Leu Ala Phe Gln Asn Met Asn Gly Ser Glu Tyr

1 5 10 15

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

20 25 30

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

35 40 45

Lys Leu Arg Thr Asp Ala Val Leu Pro Leu Thr Val Ala Glu Val Gln

50 55 60

Lys Leu Leu Gly Pro His Val Glu Gly Leu Lys Ala Glu Glu Arg His

65 70 75 80

Arg Pro Val Arg Asp Trp Ile Leu Arg Gln Arg Gln Asp Asp Leu Asp

85 90 95

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

100 105 110

<210> 10

<211> 240

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

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

130 135 140

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

145 150 155 160

Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser

165 170 175

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

180 185 190

Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile

195 200 205

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

210 215 220

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

225 230 235 240

<210> 11

<211> 497

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

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

20 25 30

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

35 40 45

Phe Thr Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly

50 55 60

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

65 70 75 80

Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile

85 90 95

Ser Thr Ala Tyr Met Glu Leu Asn Arg Leu Arg Ser Asp Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Asp Gln Pro Leu Gly Tyr Cys Thr Asn Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ile Phe Pro

245 250 255

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Glu Ser Lys Tyr

260 265 270

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

465 470 475 480

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

485 490 495

Lys

<210> 12

<211> 1431

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcaaccctg acagtggtgg cacaaactat 180

gcacagaagt ttcagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240

atggagctga acaggctgag atctgacgac acggccgtgt attactgtgc gagagatcag 300

cccctaggat attgtactaa tggtgtatgc tcctactttg actactgggg ccagggaacc 360

ctggtcaccg tctcctcagg tggaggcggt tcaggcggag gtggcagcgg cggtggcggg 420

tcggacatcc agatgaccca gtctccatct tccgtgtctg catctgtagg agacagagtc 480

accatcactt gtcgggcgag tcagggtatt tacagctggt tagcctggta tcagcagaaa 540

ccagggaaag cccctaacct cctgatctat actgcatcca ctttacaaag tggggtccca 600

tcaaggttca gcggcagtgg atctgggaca gatttcactc tcaccatcag cagcctgcaa 660

cctgaagatt ttgcaactta ctattgtcaa caggctaaca ttttcccgct cactttcggc 720

ggagggacca aggtggagat caaagagtcc aaatatggtc ccccatgccc accatgccca 780

gcacctgagt tcctgggggg accatcagtc ttcctgttcc ccccaaaacc caaggacact 840

ctcatgatct cccggacccc tgaggtcacg tgcgtggtgg tggacgtgag ccaggaagac 900

cccgaggtcc agttcaactg gtacgtggat ggcgtggagg tgcataatgc caagacaaag 960

ccgcgggagg agcagttcaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 1020

caggactggc tgaacggcaa ggagtacaag tgcaaggtct ccaacaaagg cctcccgtcc 1080

tccatcgaga aaaccatctc caaagccaaa gggcagcccc gagagccaca ggtgtacacc 1140

ctgcccccat cccaggagga gatgaccaag aaccaggtca gcctgacctg cctggtcaaa 1200

ggcttctacc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 1260

tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaggcta 1320

accgtggaca agagcaggtg gcaggagggg aatgtcttct catgctccgt gatgcatgag 1380

gctctgcaca accactacac acagaagagc ctctccctgt ctctgggtaa a 1431

Claims

1. A T cell that self-expresses a CD40 activating antibody and targets mesothelin; wherein the T cell:

(1) Comprising a coding sequence for a chimeric antigen receptor that recognizes mesothelin and a coding sequence for a CD40 activating antibody; and/or

(2) Expressing a chimeric antigen receptor that recognizes mesothelin and a CD40 activating antibody;

the chimeric antigen receptor for recognizing mesothelin sequentially comprises a CD8 signal peptide, an anti-mesothelin III region single-chain antibody with an amino acid sequence shown as 23 rd to 272 th amino acid residues of SEQ ID NO. 1, an IgG4 CH2CH3 hinge region, a CD8 transmembrane region, an intracellular domain of CD28 and a CD3 zeta intracellular signal domain from the N end to the C end;

The CD40 activating antibody comprises an anti-CD 40 single-chain antibody and an IgG4Fc, wherein the amino acid sequence of the IgG4Fc is shown as amino acid residues 269-497 of SEQ ID NO. 2, the amino acid sequence of a light chain variable region of the anti-CD 40 single-chain antibody is shown as amino acid residues 21-146 of SEQ ID NO. 2, and the amino acid sequence of a heavy chain variable region is shown as amino acid sequences 161-268 of SEQ ID NO. 2.

2. The T cell of claim 1, wherein the T cell has integrated into its genome an expression cassette for the CD40 activating antibody and an expression cassette for the chimeric antigen receptor that recognizes mesothelin.

3. The T cell of claim 1, wherein the amino acid sequence of the CD8 signal peptide is set forth in amino acid residues 1-22 of SEQ ID No. 1.

4. The T cell of claim 1, wherein the amino acid sequence of the hinge region of IgG4 CH2CH3 is shown as amino acid residues 273 to 500 of SEQ ID No. 1.

5. The T cell of claim 1, wherein the amino acid sequence of the CD8 transmembrane region is shown as amino acid residues 501-528 of SEQ ID No. 1.

6. The T cell of claim 1, wherein the CD28 intracellular domain has an amino acid sequence as set forth in amino acid residues 529 to 569 of SEQ ID No. 1.

7. The T cell of claim 1, wherein the amino acid sequence of the cd3ζ intracellular signaling domain is depicted as amino acid residues 570-681 of SEQ ID No. 1.

8. The T cell of claim 1, wherein the chimeric antigen receptor has one or more of the following characteristics:

the coding sequence of the CD8 signal peptide is shown as 1 st to 66 th base of SEQ ID NO. 3;

the coding sequence of the anti-mesothelin III region single-chain antibody is shown as the 67 th to 816 th nucleotide sequence of SEQ ID NO. 3;

the coding sequence of the IgG4 CH2CH3 hinge region is shown in 817-1500 th position of SEQ ID NO. 3;

the coding sequence of the CD8 transmembrane region is shown as the 1501 th to 1584 th bases of SEQ ID NO. 3;

the coding sequence of the CD28 intracellular domain is shown as 1585 th to 1707 th bases of SEQ ID NO. 3; and

the coding sequence of the CD3 zeta intracellular signal domain is shown as 1708-2043 of SEQ ID NO. 3.

9. The T cell of claim 1, wherein the chimeric antigen receptor has a coding sequence as set forth in SEQ ID No. 3 at bases 67-2043 or as set forth in SEQ ID No. 3.

10. The T cell of claim 1, wherein the chimeric antigen receptor has an amino acid sequence as set forth in amino acid residues 23-681 of SEQ ID No. 1, or as set forth in SEQ ID No. 1.

11. The T cell of claim 1, wherein the CD40 activating antibody further comprises a light chain signal peptide.

12. The T cell of claim 11, wherein the amino acid sequence of the light chain signal peptide is set forth in amino acid residues 1-20 of SEQ ID No. 2.

13. The T cell of claim 1, wherein the amino acid sequence of the CD40 activating antibody is shown as amino acid residues 21-497 of SEQ ID No. 2 or as SEQ ID No. 2.

14. The T cell of claim 1, wherein the coding sequence of said CD40 activating antibody is as set forth in SEQ ID No. 4 at nucleotide sequences 61-1491 or as set forth in SEQ ID No. 4.

15. A composition or kit comprising:

(1) A vector comprising an expression cassette for a chimeric antigen receptor as defined in any one of claims 1 and 3 to 10 for integration of said expression cassette into the genome of a host cell; and

(2) A vector comprising an expression cassette for a CD40 activating antibody as defined in any one of claims 1 and 11-14 for integration of said expression cassette into the genome of a host cell.

16. A pharmaceutical composition comprising the T cell of any one of claims 1-14.

17. Use of the T cell of any one of claims 1-14 in the manufacture of a medicament for the treatment or prevention of a malignancy; wherein the malignant tumor is cancer of which the cancer cell surface abnormally expresses mesothelin.

18. The use of claim 17, wherein the cancer is selected from the group consisting of: mesothelioma, lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct cancer, gall bladder cancer, esophageal cancer, melanoma, renal cell carcinoma, head and neck squamous cell carcinoma, rectal cancer, hodgkin's lymphoma, pancreatic cancer or prostate cancer.

19. The use of claim 18, wherein the lung cancer is non-small cell lung cancer.

20. The use of claim 17, wherein the cancer is an adenocarcinoma.

21. The use of claim 17, wherein the cancer is a cancer in which mesothelin and CA125/MUC16 are simultaneously highly expressed.