CN109971714B

CN109971714B - Chimeric antigen receptor modified T cells from PD-1 antibody expression and targeting mesothelin and uses thereof

Info

Publication number: CN109971714B
Application number: CN201711458257.1A
Authority: CN
Inventors: 钱其军; 金华君; 江芏青; 刘祥箴; 何周; 王超; 崔连振; 李林芳
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: CN109971714A; WO2019128998A1

Abstract

The present invention provides chimeric antigen receptor-modified T cells that self-express PD-1 antibodies and target mesothelin and uses thereof. T cells of the invention self-express PD1 antibodies and target mesothelin, preferably (1) contain coding sequences that express chimeric antigen receptors that recognize mesothelin and coding sequences for PD-1 antibodies; and/or (2) express chimeric antigen receptor recognizing mesothelin and PD-1 antibody. The T cells inhibit the PD-1/PD-L1 pathway of immune check points while adoptive immune cell treatment, can activate the functions of residual tumor specific T cells in situ, increase the anti-tumor killing effect of endogenous cytotoxic T cells, promote the proliferation of CAR-T cells in vivo, and further improve the curative effect of specifically killing tumors.

Description

Chimeric antigen receptor modified T cells from PD-1 antibody expression and targeting mesothelin and uses thereof

Technical Field

The invention belongs to genetic engineering and immunology, and relates to a chimeric antigen receptor modified T cell targeting mesothelin and application thereof.

Background

Cancer is now the first killer of human health, and rapid life rhythm, huge working pressure, unhealthy eating habits and poor environment are all the help of cancer occurrence, so that the high incidence and the younger trend of cancer are more and more obvious. The current commonly used treatment methods have very limited effects, and a more effective treatment method is still needed to be explored to improve the survival rate and the survival quality of cancer patients.

Immunotherapy against malignant tumors has been rapidly developed in recent years, and has achieved remarkable clinical effects. Since 2011, nature and journal of clinical tumor top-grade JCO respectively published a review article (Nature.2011; 480 (7378): 480;J Clin Oncol.2011;29 (36): 4828) of the same title, "tumor immunotherapy" and a new round of research on tumor immune cell therapy have come.

Chimeric antigen receptor T cell therapy is taken as one of important branches of tumor immunotherapy, has achieved very good curative effects in malignant blood tumors, and has a complete remission rate of over 90% for relapsed refractory B cell leukemia. In month 2017, the U.S. FDA approved Tisamgenlecieucel chimeric antigen receptor T cell (CAR-T cell) therapy for the treatment of Acute Lymphoblastic Leukemia (ALL) in pediatric and young adult patients, the first approved marketed CAR-T drug. Immediately following 10 months of the year, the U.S. FDA announced approval of Kite Pharma's CAR-T therapy Yescanta on the market for treatment of adult patients suffering from a particular type of large B cell lymphoma. The sequential acquisition of CAR-T drugs has led to a new step in CAR-T therapy.

A chimeric antigen receptor is an artificial synthetic receptor that generally comprises an extracellular antigen binding domain, a transmembrane hinge region, and an intracellular signaling region. Gene recombination is performed in vitro by combining a single-chain variable region (scFv) of an antibody recognizing a tumor-associated antigen (tumor associated antigen, TAA) with an intracellular signaling domain "immunoreceptor tyrosine activation motif (immunoreceptor tyrosine-based activation motifs, ITAM)". The resulting genetically engineered T cells are then introduced into T cells by a viral or other vector system, and are referred to as 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.

However, the efficacy of CAR-T cells in treating solid tumors is currently inadequate. The main reasons include: 1. solid tumors are high in heterogeneity 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).

PD1 (Programmed Death 1, reprogrammed cell Death receptor 1) is a member of the CD28 family of regulatory T cells belonging to the immunoglobulin superfamily of receptors. PD-1 and its ligand PD-L1/PD-L2 play an important role in co-suppression and failure of T cells, and their interaction inhibits proliferation of T cells and cytokine secretion regulated by co-stimulatory molecules, down regulates expression of anti-apoptotic molecule BCL-xl, weakens the function of tumor-specific T cells, and results in some tumor patients unable to completely eliminate tumors. Thus, inhibition of the immune checkpoint PD-1/PD-L1 pathway is a new direction and target for current treatment of lymphomas. The PD-1 antibody blocking treatment has a certain treatment effect on advanced or refractory melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, colorectal cancer, hodgkin lymphoma, ovarian cancer and the like, but the higher production cost and response rate of the antibody blocking treatment do not limit the clinical application of the antibody blocking treatment.

Disclosure of Invention

Provided herein is a T cell that self-expresses a PD1 antibody and targets mesothelin.

In one or more embodiments, the T cell genome incorporates an expression cassette for a PD1 antibody and an expression cassette for a chimeric antigen receptor that recognizes mesothelin.

In one or more embodiments, the PD1 antibody has an amino acid sequence as set forth in amino acid residues 21-495 of SEQ ID NO. 2, or as set forth in SEQ ID NO. 2.

In one or more embodiments, the PD1 antibody has a coding sequence as set forth in SEQ ID NO. 4 at positions 61-1488 or as set forth 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 a CD8 hinge region or an IgG4CH2CH3 hinge region; preferably, the amino acid sequence of the hinge region of the IgG4CH2CH3 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 the coding sequence of the PD1 antibody or its complement 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 the coding sequence of a PD1 antibody or its complement for integration of the expression cassette into the genome of a host cell.

The invention also provides a pharmaceutical composition, which contains the T cells or the T cells and the PD1 antibodies expressed by the T cells.

The invention also provides the use of the T cells described herein or the T cells and their expressed PD1 antibodies in the manufacture of a medicament for the treatment or prevention of malignancy. 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: PD-1 antibody and mesothelin chimeric antigen receptor gene structural pattern.

Fig. 2A: three CAR-T cell positive rates of meso3 CAR-2A-anti-iPD 1, anti-iPD 1-IRES-meso3CAR, meso3 CAR-anti-iPD 1.

Fig. 2B: three CAR-T cell positive rate antibody secretion amounts of meso3CAR-2A-anti pid 1, anti pid 1-IRES-meso3CAR, meso3CAR-anti pid 1.

Fig. 3A: comparison of positive rates of meso3CAR-antiPD1T cells constructed under conditions of different ratios of CAR to PD1 antibody plasmid.

Fig. 3B: comparison of the amount of antibody secretion of meso3CAR-anti pid 1T cells constructed under different ratios of CAR to PD1 antibody plasmid.

Fig. 4: flow detection of meso3CAR T and meso3CAR-anti PD1T cell surface PD1 expression.

Fig. 5: killing cervical cancer cells Hela, ovarian cancer cells SK-OV-3 and gastric cancer cells HGC-27 by the meso3CAR-antiPD1T cells.

Fig. 6: alterations in secretion of IL-2, IL-4, IL-6, IL-10, TNF- α and IFN- γ cytokines by meso3 CAR-anti-iPD 1 under stimulation of the mesothelin antigen.

Fig. 7: therapeutic effects of meso3CAR T cells and meso3CAR-anti pd1T 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, gene coding sequences, and PolyA tailing signal 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 to (or has specificity for) an antigen means that the antibody binds to or has specificity for an antigen in an amount of less than about 10 ^-5 M, e.g. less than about 10 ^-6 M、10 ^-7 M、10 ^-8 M、10 ^-9 M or 10 ^-10 M or less affinity (KD) binds the antigen. "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. The antibody may be secreted or membrane anchored.

In order to improve the curative effect of the meso3CAR-T cells, the meso3CAR T cells which express the PD-1 antibody are constructed, and the immune check point PD-1/PD-L1 passage is inhibited while adoptive immune cells are treated, so that the functions of the residual tumor specific T cells can be activated in situ, the anti-tumor killing effect of endogenous cytotoxic T cells is improved, and the proliferation of the CAR-T cells in vivo is promoted, thereby improving the curative effect of specifically killing tumors. In addition, the Fc fragment of the PD-1 antibody designed by the invention is mutant IgG4Fc, so that the gamma-2 receptor combined with the surface of the dendritic cell is prevented from being recognized and phagocytized by macrophages, and the CAR-T cell of the PD-1 antibody can function and does not cause AICD reaction.

Accordingly, the present invention provides a PD-1 antibody comprising an anti-PD-1 single chain antibody and an IgG4Fc. In certain embodiments, the amino acid sequence of the IgG4Fc is shown as amino acid residues 267-495 of SEQ ID NO. 2; preferably, the coding sequence is shown as the base sequence of 799-1485 of SEQ ID NO. 4.

In certain embodiments, the anti-PD-1 single chain antibody (scFv) has an antibody light chain variable region (VL region) amino acid sequence as set forth in amino acid residues 21-131 of SEQ ID NO. 2; preferably, the coding sequence is shown as 61-393 base sequence of SEQ ID NO. 4. In certain embodiments, the heavy chain variable region (VH region) amino acid sequence of the anti-PD-1 single-chain antibody is shown as 147-266 amino acid sequences of SEQ ID NO. 2; preferably, the coding sequence is shown in the base sequence of 439-798 of SEQ ID NO. 4. In certain embodiments, the anti-PD-1 single-chain antibody has an amino acid sequence as set forth in amino acid residues 21-266 of SEQ ID NO. 2; preferably, the coding sequence is shown as 61-798 base sequences of SEQ ID NO. 4.

In certain embodiments, the PD-1 antibody further comprises a light chain signal peptide. In certain embodiments, the PD-1 antibody comprises, from N-terminus to C-terminus, a light chain signal peptide, an anti-PD-1 single chain antibody, and an 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 PD-1 antibody has an amino acid sequence as set forth in SEQ ID NO. 2 at amino acid positions 21-495 or as set forth in SEQ ID NO. 4.

The invention also includes the coding sequence of the PD-1 antibody or its complement, which comprises at least the coding sequence of IgG4Fc described herein or its complement. In certain embodiments, the coding sequence of the PD-1 antibody comprises the sequence set forth in base sequence positions 61-1495 of SEQ ID NO. 4, preferably the sequence set forth in SEQ ID NO. 4.

The invention also includes a nucleic acid construct comprising the coding sequence of the PD-1 antibodies 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 PD-1 antibodies, their coding sequences or complementary sequences, nucleic acid constructs, and host cells in the preparation of a method for treating or preventing a malignancy, particularly a PD-1-associated neoplasm, including, but not limited to, the various malignancies described herein.

The invention also provides a T cell modified by the meso3CAR gene and capable of expressing the PD-1 antibody, the T cell can stably express the meso3CAR gene and the PD-1 antibody at a high level, the exogenously expressed meso3CAR gene can accurately target mesothelin, the proliferation capacity and the secretion of cytokines of the T cell are enhanced, the killing of the CAR-T cell on tumor cells is enhanced, and the anti-tumor effect is exerted by enhancing the immune response. Meanwhile, the exogenously expressed PD-1 antibody can eliminate the immune escape of tumor cells, restore the phagocytosis of macrophages to the tumor cells, promote the apoptosis of the tumor cells and play an anti-tumor immune response. In addition, the exogenous meso3CAR gene and the PD-1 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 PD-1 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 IgG1Fc CH2CH3 hinge region, the IgD hinge region, the extracellular hinge region of CD28, the IgG4Fc 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 IgG4Fc CH2CH3 hinge region is used herein. The amino acid residues 273-500 of the amino acid sequence SEQ ID NO. 1 of an exemplary IgG4FcCH2CH3 hinge region, and the coding sequence of an exemplary IgG4FcCH2CH3 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 IgG4Fc 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 PD-1 antibody described herein, and one or more regulatory sequences operably linked to these sequences. In certain embodiments, the nucleic acid construct is an expression cassette.

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 PD-1 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 PD-1 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 PD-1 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, comprising a corresponding promoter sequence, a coding sequence for the CAR or antibody, and 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 expression cassette of 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 of the PD-1 antibody and the coding sequence of a signal peptide (e.g., the coding sequence of 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 PD-1 antibody can be transformed into: a vector comprising a transposase coding sequence for integration into the expression cassette of a chimeric antigen receptor in the T cell genome, and a vector comprising no transposase coding sequence for integration into the expression cassette of a PD-1 antibody described herein in the T cell genome.

Preferably, the T cells are transformed with a vector comprising a chimeric antigen receptor coding sequence constructed with the pNB328 vector as a scaffold vector and a vector comprising a PD-1 antibody coding sequence constructed with the pS328 vector (without the 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 PD-1 antibody is shown as the 61 st to 1488 th base sequence of SEQ ID NO. 4. In certain embodiments, the signal peptide of the PD-1 antibody is a light chain signal peptide in the vector comprising the coding sequence of the PD-1 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 coding sequence-containing vector having a chimeric antigen receptor coding sequence integrated into the T cell genome comprises, in order, a 5'ltr, a promoter, a light chain peptide coding sequence, a coding sequence that recognizes an scFv for a mesothelin antigen (preferably a coding sequence that recognizes an scFv for mesothelin Region III), a coding sequence for an IgG4Fc CH2CH3 hinge Region, a coding sequence for a CD8 transmembrane Region, a coding sequence for a CD28 intracellular domain, a coding sequence for a CD3 zeta intracellular signal domain, a polyA tailing signal sequence, a coding sequence for a 3' ltr and a transposase, and promoters thereof; the vector without transposase coding sequence, which incorporates the coding sequence of the PD-1 antibodies described herein in the T cell genome, contains a promoter, a coding sequence for a light chain signal peptide, a coding sequence for the PD-1 antibody, and a polyA 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 PD-1 antibody coding sequence is 1-7 during transfection: 1 to 3, 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 the chimeric antigen receptor expression cassette described herein and a vector comprising the expression cassette of the PD-1 antibody 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 expression cassette described herein and a vector comprising the expression cassette of the PD-1 antibody described herein, or a composition described herein. The kit may also be provided with reagents or instruments for transferring the vector into cells.

It is to be understood that the expression cassettes described herein contain at least a suitable promoter and polyA tailing signal sequence in addition to the coding sequences for the CARs or antibodies described herein.

The invention also provides a pharmaceutical composition comprising a T cell as described herein or a PD-1 antibody expressed by the T cell. 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 the T cells described herein or the T cells and their expressed PD-1 antibodies or pharmaceutical compositions thereof in the manufacture of a medicament for the treatment or prevention of a malignancy. 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. "Meso3CAR" and "Meso3CAR" are used throughout and in the drawings to mean the same.

Example 1: construction of recombinant plasmids pNB328-meso3CAR, pS 328-anti-iPD 1, pNB328-meso3 CAR-2A-anti-iPD 1 and pNS 328-anti-iPD 1-IRES-meso3CAR

The coding sequence of the meso3CAR (SEQ ID NO: 3), the coding sequence of the anti-iPD 1 (SEQ ID NO: 4), the coding sequence of the meso3 CAR-2A-anti-iPD 1 (SEQ ID NO:5, amino acid sequence of the 2A) and the coding sequence of the anti-iPD 1-IRES-meso3CAR (IRES, SEQ ID NO: 7) were artificially synthesized by commercial companies, respectively, and the structural modes thereof are shown in FIG. 1. Each sequence was placed between the EcoRI and SalI cleavage sites of the pNB328, pS328 vectors (the structure and sequence of pNB328 is referred to as CN 201510638974.7, the entire contents of which are incorporated herein by reference; pS328 lacks PB transposons in comparison to pNB328, and other elements are identical to pNB 328), and the constructed recombinant plasmids were named pNB328-meso3CAR, pS328-antiPD1, pNB328-meso3CAR-2A-antiPD1, pNB328-antiPD1-IRES-meso3CAR, respectively. 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 mesothelin-targeted CAR T cells from PD1 antibody-expressing CAR T cells

Peripheral Blood Mononuclear Cells (PBMCs) are isolated by Ficoll isolation. 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, numbered a, b, c,1200rmp were centrifuged for 3min, the supernatant was discarded, the electrotransfer kit (from Lonza corporation) was taken, 100ul total of electrotransfer reagents was added to the a, b, c tubes, 4ug each of constructed recombinant plasmids pNB328-meso3CAR and pS328-antiPD1 was added to the a tube, 6ug of pNB328-meso3CAR-2A-antiPD1 plasmid was added to the b tube, and 6ug of pNB328-antiPD1-IRES-meso3CAR plasmid was added to the c tube; transferring the mixed solution to an electric rotating cup, putting the electric rotating cup into an electric rotating instrument, selecting a required program, and performing electric shock; transferring the electrotransformed cell suspension to a six-well plate (AIM-V culture solution containing 2% FBS) added with a culture solution by using a micropipette in a kit, uniformly mixing, placing the mixture in a 37 ℃ and 5% CO2 incubator for culture, adding the stimulating factors IL-2 and meso/anti-CD28 after six hours, culturing the mixture at 37 ℃ and 5% CO2 for 3-4 days, observing the growth condition of T cells, and obtaining meso3 CAR-anti-iPD 1T cells, meso3 CAR-2A-anti-iPD 1T cells and anti-IRES-meso 3CAR T cells which express PD1 antibodies and target mesothelin.

Furthermore, 5X 10 is added to another centrifuge tube ⁶ The individual cells, numbered d,1200rmp were centrifuged for 3min, the supernatant was discarded, the electrotransfer kit (from Lonza corporation) was taken, 100ul total of electrotransfer reagent was added in proportion, and 6ug control plasmid (pNB 328-meso3 CAR) was added, and the cells were constructed as described aboveControl T cells, i.e., meso3CAR T cells.

Example 3: comparison of the positive rates of three mesothelin-targeted CAR T cells from PD 1-expressing antibodies and antibody secretion

1. Flow detection of CAR T cell positive rate

Collecting the meso3 CAR-anti-iPD 1T cells, the meso3 CAR-2A-anti-iPD 1T cells and the anti-iPD 1-IRES-meso3CAR T cells obtained in the examples, dividing into two parts each of 1X 10 ⁶ Cells were washed twice with saline, resuspended in 100ul saline, one portion added with 1ug of mesothelin-biotin and the other portion without, and incubated at 4℃for 30 min. 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 checking on the machine, wherein only secondary antibody is added as a control, and the result is shown in figure 2A.

2. ELISA (enzyme-Linked immuno sorbent assay) for detecting expression quantity of anti-iPD 1 antibodies of three T cells

(1) PD1 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 is measured at 450nm on an enzyme labeling instrument, a standard curve is drawn, and PD-1 antibody concentration is calculated.

The results are shown in FIG. 2B.

Example 4: proportioning of pNBS328-meso3CAR and pS328-antiPD1 plasmids

The amounts of pNBS328-meso3CAR and pS328-antiPD1 plasmids 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 antibody secretion amount of CAR T cells constructed under 7 ratios were measured by the method described in example 3, and the results are shown in FIGS. 3A and 3B, respectively.

Example 5: blocking of autologous PD1 by meso3CAR-antiPD1T cells

The meso3CAR T cells and meso3CAR-anti pid 1T cells constructed in example 2 were collected, and expression of PD1 was detected using the streaming antibody PE-murine anti human CD279 of BD, streaming method example 3.

The results are shown in fig. 4, in which meso3CAR-anti pid 1T from the PD1 antibody can block its own PD1 expression.

Example 6: comparison of killing function of meso3CAR T and meso3CAR-anti pid 1T cells

The killing effect of the meso3CAR T and the meso3CAR-antiPD1T cells constructed in example 2 on tumor cells in vitro was detected by using a real-time label-free cell function analyzer.

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 (all 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 were cultured for 24 hours, step 2 was suspended, effector cells were added, 50. Mu.l per well, the effective target ratio was set to 4:1, and the co-culture was continued for 24 hours after starting step 3 with Mock T cells without plasmids as a control, and cell proliferation curves were observed.

As shown in fig. 5, the meso3CAR-anti pid 1T cells from which PD1 antibodies were expressed were substantially identical to the meso3CAR T cell killing function alone, and antibody expression did not affect CAR-T function.

Example 7: comparison of cytokine release by meso3CAR with meso3CAR-anti pid 1T 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, and adding 1×10 respectively ⁵ The meso3CAR, meso3CAR-antiPD1T cells and control Mock T cells constructed in example 2 (transferred into, cultured for 24h and then the cell supernatant was collected. BD was used) ^TM The CBA Human Th1/Th2Cytokine Kit II detects the secretion of cytokines after the three T cells are stimulated by the mesothelin antigen, and the specific steps are as follows:

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

(2) 50ul of human Th1/Th2 cytokine standard (dilution of power ratio 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) Cells were resuspended by adding 300ul of wash buffer per tube and transferred to flow tubes and fluorescence values were detected by flow cytometry.

The results are shown in fig. 6, with no significant difference in cytokine secretion from the meso3CAR-anti PD1T cells expressing the PD1 antibody versus the meso3CAR T cells alone.

Example 8: therapeutic effects of meso3CAR and meso3 CAR-anti-ipd 1T cells on ovarian cancer mouse engraftment tumor model

1. 25 NSG complete immunodeficiency mice of 4-6 weeks old, average weight 22-27 g, were raised by SPF grade animal laboratory supplied by Bai Osai Biolabs.

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 SK-OV-3-luc cells, 0.1 ml/mouse dorsum of the right rib. After 7 days of inoculation, fluorescence intensity was observed by a biopsy imager and NSG immunodeficient mice were randomly divided into 5 groups. Each group was injected with 1×10 corresponding T cells ⁷ 100ul of PBS was administered to the PBS group; 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 figure 7, in SK-OV-3 ovarian cancer mouse engraftment tumor model, the Meso3 CAR-anti-ipd 1T cells had significantly better therapeutic effect than the Meso3CAR T cells, while the Meso3 CAR-wt-anti-ipd 1T cells were substantially ineffective.

Although specific embodiments of the invention have been described in detail. Those skilled in the art will understand. Numerous modifications and substitutions of details are possible in light of all the teachings disclosed, and such modifications are contemplated as falling within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Sequence listing

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

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<211> 2046

<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

cgctga 2046

<210> 4

<211> 1488

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60

gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 120

ctctcctgca gggccagcaa aggtgtcagt acatctggct atagttattt gcactggtat 180

caacagaaac ctggccaggc tcccaggctc ctcatctatc ttgcatccta cctagaatct 240

ggcgtcccag ccaggttcag tggtagtggg tctgggacag acttcactct caccatcagc 300

agcctagagc ctgaagattt tgcagtttat tactgtcagc acagcaggga ccttccgctc 360

acgttcggcg gagggaccaa agtggagatc aaaggtggag gcggttcagg cggaggtggc 420

agcggcggtg gcgggtcgca ggtgcagctg gtgcagtccg gcgtggaggt gaagaagcct 480

ggcgcctccg tcaaggtgtc ctgtaaggcc tccggctaca ccttcaccaa ctactacatg 540

tactgggtgc ggcaggcccc aggccaggga ctggagtgga tgggcggcat caacccttcc 600

aacggcggca ccaacttcaa cgagaagttc aagaaccggg tgaccctgac caccgactcc 660

tccaccacaa ccgcctacat ggaactgaag tccctgcagt tcgacgacac cgccgtgtac 720

tactgcgcca ggcgggacta ccggttcgac atgggcttcg actactgggg ccagggcacc 780

accgtgaccg tgtcctccga gtccaaatat ggtcccccat gcccaccatg cccagcacct 840

gagttcgagg ggggaccatc agtcttcctg ttccccccaa aacccaagga cactctcatg 900

atctcccgga cccctgaggt cacgtgcgtg gtggtggacg tgagccagga agaccccgag 960

gtccagttca actggtacgt ggatggcgtg gaggtgcata atgccaagac aaagccgcgg 1020

gaggagcagt tccagagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1080

tggctgaacg gcaaggagta caagtgcaag gtctccaaca aaggcctccc gtcctccatc 1140

gagaaaacca tctccaaagc caaagggcag ccccgagagc cacaggtgta caccctgccc 1200

ccatcccagg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1260

taccccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1320

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcag gctaaccgtg 1380

gacaagagca ggtggcagga ggggaatgtc ttctcatgct ccgtgatgca tgaggctctg 1440

cacaaccact acacacagaa gagcctctcc ctgtctctgg gtaaatga 1488

<210> 6

<211> 78

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

cgtaggaaac gaggcagcgg cgccacaaac ttctctctgc taaagcaagc aggtgatgtt 60

gaagaaaacc ccgggcct 78

<210> 7

<211> 26

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

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> 8

<211> 197

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

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> 9

<211> 94

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

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> 10

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

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> 11

<211> 240

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

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

Claims

1. A T cell that self-expresses a PD-1 antibody and targets mesothelin, wherein the T cell:

(1) Comprising coding sequences for a chimeric antigen receptor that recognizes mesothelin and coding sequences for a PD-1 antibody; and/or

(2) Expressing chimeric antigen receptor recognizing mesothelin and PD-1 antibody;

wherein, the chimeric antigen receptor for recognizing mesothelin contains a CD8 signal peptide, an anti-mesothelin III region single-chain antibody with an amino acid sequence shown as amino acid residues 23-272 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;

the PD-1 antibody comprises an anti-PD-1 single-chain antibody and an IgG4Fc, wherein the amino acid sequence of the IgG4Fc is shown as 267 th to 495 th amino acid residues of SEQ ID NO. 2, the amino acid sequence of a light chain variable region of the anti-PD-1 single-chain antibody is shown as 21 st to 131 th amino acid residues of SEQ ID NO. 2, and the amino acid sequence of a heavy chain variable region is shown as 147 th to 266 th amino acid sequences 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 PD-1 antibody and an expression cassette for a 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 intracellular domain of CD28 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;

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 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.

10. 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.

11. The T cell of claim 1, wherein the PD-1 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 anti-PD-1 single chain antibody has an amino acid sequence as set forth in amino acid residues 21-266 of SEQ ID No. 2.

14. The T cell of claim 1, wherein the anti-PD-1 single chain antibody has a coding sequence as set forth in base sequence nos. 61-798 of SEQ ID No. 4.

15. The T cell of claim 1, wherein the PD-1 antibody has an amino acid sequence as set forth in SEQ ID No. 2 at amino acid positions 21-495 or as set forth in SEQ ID No. 2.

16. The T cell of claim 1, wherein the PD-1 antibody has a coding sequence as set forth in amino acid residues 61-1485 of SEQ ID No. 4 or as set forth in SEQ ID No. 4.

17. 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 the coding sequence of a PD-1 antibody as defined in any one of claims 1 and 11-16, or the complement thereof, for integration of the expression cassette into the genome of a host cell.

18. A pharmaceutical composition comprising the T cell of any one of claims 1-16 or comprising the T cell and its expressed PD-1 antibody.

19. Use of the T cell of any one of claims 1-16 or the T cell and its expressed PD-1 antibody 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.

20. The use of claim 19, 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.

21. The use of claim 19, wherein the cancer is a cancer in which mesothelin and CA125/MUC16 are simultaneously highly expressed.

22. The use of claim 20, wherein the lung cancer is non-small cell lung cancer.

23. The use of claim 19, wherein the cancer is an adenocarcinoma.