CN113087805A

CN113087805A - Preparation and application of chimeric antigen receptor T cell of co-expression immune regulatory molecule

Info

Publication number: CN113087805A
Application number: CN201911422121.4A
Authority: CN
Inventors: 谭炳合; 史秀娟; 杜冰; 刘明耀; 张亮; 席在喜
Original assignee: Shanghai Bangyao Biological Technology Co ltd; East China Normal University
Current assignee: Shanghai Bangyao Biological Technology Co ltd; East China Normal University
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2021-07-09
Also published as: WO2021136040A1

Abstract

The invention provides a preparation method and application of a chimeric antigen receptor T cell co-expressing an immune regulatory molecule, and particularly provides a CAR specifically targeting a tumor cell surface antigen and a CAR-T cell thereof, wherein the chimeric antigen receptor CAR comprises an antigen binding domain and the immune regulatory molecule. The CAR-T cell has good tumor killing effect.

Description

Preparation and application of chimeric antigen receptor T cell of co-expression immune regulatory molecule

Technical Field

The invention relates to the field of biomedicine, in particular to preparation and application of a chimeric antigen receptor T cell co-expressing an immune regulatory molecule.

Background

Chimeric Antigen Receptor (CAR) T cell therapy is a new cellular immunotherapy technology that has been developed very rapidly in recent years, and is a technology that combines high affinity of antigen antibody with killing effect of T lymphocytes to construct a specific Chimeric antigen receptor, inserts a gene encoding the Chimeric antigen receptor into T lymphocytes through a certain route, allows the T lymphocytes to express the Chimeric antigen receptor, then purifies the genetically modified T cells by in vitro amplification, and transfects the T cells into a body, and CAR-T cells specifically recognize a target antigen in the body, and performs a series of immune reactions, T cell activation and amplification, and cytokine secretion, and specificity of the target cells in a non-MHC (Major histocompatibility complex) limited manner. For tumors, a tumor-associated antigen chimeric antigen receptor is constructed, and the chimeric antigen receptor is expressed after being transferred into T cells, so that the antigen on the surface of the tumor cells can be specifically identified, the T cells are activated to play a role in cellular immunity, the tumor cells are eliminated, and the purpose of resisting tumors is achieved.

To date, the CAR-T technology has enjoyed a promising breakthrough in the treatment of hematological tumors, but the efficacy of CAR-T technology in the treatment of solid tumors, which dominate the majority of malignancies, is not satisfactory. CAR-T therapy works poorly in the treatment of solid tumors, being intimately associated with the solid tumor tissue barrier, high heterogeneity of tumor cells, lack of good tumor-specific or tumor-associated antigens, highly suppressed tumor immune microenvironment, and activation signals of CAR-T cells themselves. In the process of treating solid tumors, after the CAR-T cells break through physical barriers and recognize tumor cells, the CAR-T cells play a role in efficient tumor cell killing and rely on overcoming the highly-inhibited tumor microenvironment and maintaining stable activation state and functional persistence. However, although the current CAR structural design can enable CAR-T cells to better recognize related antigens and to be rapidly activated, under the action of strong inhibitory signals of a tumor microenvironment, CAR-T cells often have the conditions of insufficient activation strength, poor amplification capacity, poor persistence, rapid progression to failure, apoptosis and the like, and the treatment effect of solid tumors is severely restricted.

Moreover, in the prior art, cytokines or chemokines are integrated and expressed in the CAR molecule, and although the expression of the cytokines with anti-tumor effect can be improved while the CAR is expressed, the expression mode is systemic, and one of the important side effects of the CAR-T cells in the treatment process is cytokine storm, and the risk of the cytokine storm related to CAR-T treatment is undoubtedly improved by the systemic improvement of the expression of the cytokines.

The integration of different co-stimulatory molecules expressed in the intracellular activation domain of the CAR molecule, while able to provide the co-stimulatory signals required for CAR-T cell activation, relies on the expression of tumor-associated or tumor-specific antigens and is unable to remodel, improve the tumor microenvironment through interaction with other immune cells in the tumor microenvironment, thus having very limited therapeutic effect on solid tumors.

Therefore, there is an urgent need in the art to develop a novel chimeric antigen receptor molecule, which is capable of effectively resisting the inhibitory action of tumor immune microenvironment after being expressed in T cells, maintaining or improving the effector function of CAR-T cells and the amplification capacity and persistence capacity thereof, and can show a therapeutic effect obviously superior to that of the existing CAR structure in the treatment of solid tumors.

Disclosure of Invention

The invention aims to provide a novel chimeric antigen receptor molecule, which can effectively resist the inhibitory action of a tumor immune microenvironment after being expressed in a T cell, maintain or improve the effector function, the amplification capacity and the persistence capacity of a CAR-T cell, and can show a treatment effect obviously superior to that of the existing CAR structure in the treatment of solid tumors.

The present invention provides in a first aspect a chimeric antigen receptor CAR comprising: an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain specifically binds to a tumor cell surface antigen;

and said chimeric antigen receptor CAR further comprises: an immunomodulatory molecule linked to and co-expressible with the intracellular domain.

In another preferred embodiment, the tumor cell antigens include cell surface antigens of various solid and non-solid tumors.

In another preferred embodiment, the tumor cell surface antigen is selected from the group consisting of: CD19, BCMA, CD38, PSMA, HER2, GPC3, Mesothelin, claudin18.2, EGFR, EGFRVIII, CEA, GD2, IL13R, FAP, CD171, or a combination thereof.

In another preferred embodiment, the tumor cell surface antigen comprises CD19, and/or PSMA.

In another preferred embodiment, the immune modulatory molecule is selected from the group consisting of: GITRL, 4-1BBL, CD40, LIGHT, B7.1, B7.2, OX40L, CD70, or a combination thereof.

In another preferred embodiment, the immunomodulatory molecule comprises GITRL.

In another preferred embodiment, the antigen binding domain is an antibody or antigen binding fragment.

In another preferred embodiment, the antigen binding fragment is a Fab or scFv or a single domain antibody sdFv.

In another preferred embodiment, the CAR has the structure shown in formula I below:

Z1-T-H-TM-C-Z2-(Z3-P)m(I)

in the formula (I), the compound is shown in the specification,

each "-" is independently a linker peptide or a peptide bond;

z1 is a null or signal peptide sequence;

t is an antibody single-chain variable region sequence of a targeted tumor cell surface antigen;

h is a null or hinge region;

TM is a transmembrane domain;

c is a costimulatory signal molecule;

z2 is a cytoplasmic signaling sequence derived from CD3 ζ;

z3 is a self-cleaving protein;

p is an immunomodulatory molecule;

m is 1, 2, 3, or 4.

In another preferred embodiment, the structure of the antibody single chain variable region sequence targeting tumor cell surface antigen is represented by formula a1 or a 2:

V_L1-V_H1(A1) (ii) a Or

V_L2-V_H2(A2)；

Wherein, V_L1、V_L2A light chain variable region of an antibody against a tumor cell surface antigen; v_H1、V_H2A heavy chain variable region of an antibody against a tumor cell surface antigen; "-" is a linker peptide (or flexible linker) or peptide bond.

In another preferred embodiment, V is_L1And V_H1Connected by a flexible joint.

In another preferred embodiment, the flexible linker is 1-5 (preferably 2-4) consecutive sequences of SEQ ID NO. 4 (GGGGS).

In another preferred embodiment, V_L1Has the amino acid sequence shown in the 22 nd-128 th position of SEQ ID No. 2 (CAR of PSMA), and V_H1The amino acid sequence of (1) is shown in the position 144-258 of SEQ ID NO. 2.

In another preferred embodiment, V_L2Has the amino acid sequence shown in the 22 nd-128 th position of SEQ ID NO. 3 (CAR of CD 19), and V_H2The amino acid sequence of (1) is shown in the position 144-263 of SEQ ID NO. 3.

In another preferred embodiment, the structure of the CAR is represented by formula II:

Z1-V_L1-V_H1-H-TM-C-Z2-(Z3-P)m(II)

wherein each element is as described above.

In another preferred embodiment, the CAR has the structure shown in formula III:

Z1-V_L2-V_H2-H-TM-C-Z2-(Z3-P)m(III)

wherein each element is as described above.

In another preferred embodiment, Z1 is a signal peptide of a protein selected from the group consisting of: CD8a, CSF1R, or a combination thereof.

In another preferred embodiment, said H is a hinge region of a protein selected from the group consisting of: CD8a, IgG, CD28, or a combination thereof.

In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD8a, CD4, CD28, or a combination thereof.

In another preferred embodiment, C is a costimulatory signal molecule for a protein selected from the group consisting of: 4-1BB (CD137), OX40, CD28, CD30, CD40, CD70, CD134, PD1, Dap10, CDS, ICAM-1, HVEM, GITR, or a combination thereof.

In another preferred embodiment, the C comprises a co-stimulatory signaling molecule from 4-1 BB.

In another preferred embodiment, said self-cleaving protein of Z3 is selected from the group consisting of: T2A, P2A, E2A, F2A, or a combination thereof.

In another preferred embodiment, the self-cleaving protein of Z3 comprises T2A.

In another preferred embodiment, the amino acid sequence of the self-cleavage protein of Z3 is shown in SEQ ID No. 7.

In another preferred embodiment, the immunomodulatory molecule comprises a wild-type immunomodulatory molecule and a mutant immunomodulatory molecule, or an active fragment thereof.

In another preferred embodiment, the GITRL has the amino acid sequence shown in SEQ ID No. 1.

In another preferred embodiment, the amino acid sequence of GITRL is as shown in SEQ ID NO. 1.

In another preferred embodiment, the amino acid sequence of the CAR is as shown in SEQ ID No. 2 or 3.

In a second aspect, the invention provides a nucleic acid molecule encoding a Chimeric Antigen Receptor (CAR) according to the first aspect of the invention.

In another preferred embodiment, the nucleic acid molecule encoding the Chimeric Antigen Receptor (CAR) of claim 1 is as set forth in SEQ ID No.:5 or 6.

In a third aspect, the invention provides a vector comprising a nucleic acid molecule according to the second aspect of the invention.

In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, or combinations thereof.

In another preferred embodiment, the vector is a lentiviral vector.

In a fourth aspect, the invention provides a host cell comprising a vector or chromosome of the third aspect of the invention into which has been integrated an exogenous nucleic acid molecule of the second aspect of the invention.

In another preferred embodiment, the cell is an isolated cell, and/or the cell is a genetically engineered cell.

In another preferred embodiment, the cell is a mammalian cell, preferably a human cell.

In another preferred embodiment, the host cell comprises an engineered immune cell.

In another preferred embodiment, the immune cell further expresses a foreign immunomodulatory molecular protein.

In another preferred embodiment, the exogenous immunomodulatory molecule protein is independently expressed and/or co-expressed with a CAR that targets a tumor cell surface antigen.

In another preferred embodiment, said co-expression with a CAR targeting a tumor cell surface antigen comprises tandem expression of an immunomodulatory molecule protein and a CAR targeting a tumor cell surface antigen.

In another preferred embodiment, the engineered immune cells include T cells, NK cells, or macrophages.

In another preferred embodiment, the cell is a T cell.

In another preferred embodiment, the engineered immune cell is selected from the group consisting of:

(i) a chimeric antigen receptor T cell (CAR-T cell);

(ii) chimeric antigen receptor NK cells (CAR-NK cells); or

(iii) Exogenous T Cell Receptor (TCR) T cells (TCR-T cells).

In another preferred embodiment, the immune cells are autologous.

In another preferred embodiment, the immune cells are allogeneic.

In another preferred embodiment, the cell is a CAR-T cell that expresses the chimeric antigen receptor CAR of claim 1.

In a fifth aspect, the invention provides a method of making an engineered immune cell expressing a CAR according to the first aspect of the invention, wherein the method comprises the steps of: transducing the nucleic acid molecule of the second aspect of the invention or the vector of the third aspect of the invention into an immune cell, thereby obtaining the engineered immune cell.

In another preferred embodiment, the introducing includes introducing simultaneously, sequentially or sequentially.

In another preferred embodiment, the immune cell is a T cell or NK cell.

In another preferred embodiment, the method further comprises the step of performing functional and effective detection on the obtained engineered immune cells.

In a sixth aspect, the invention provides a pharmaceutical composition comprising a CAR according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, or a host cell according to the fourth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

In another preferred embodiment, the engineered immune cell is (i) a chimeric antigen receptor T cell (CAR-T cell); or (ii) a chimeric antigen receptor NK cell (CAR-NK cell).

In another preferred embodiment, the concentration of the cells in the pharmaceutical composition is 1 × 10³-1×10⁸Individual cell/mL, preferably 1X 10⁶-1×10⁷Individual cells/mL.

In another preferred embodiment, the pharmaceutical composition further comprises an immunomodulatory molecular agonist.

In another preferred embodiment, the immunomodulatory molecular agonist is selected from the group consisting of: antibodies, small molecule compounds, synthetic or recombinant polypeptides, or combinations thereof.

In another preferred embodiment, the pharmaceutical composition further comprises other drugs (such as antibody drugs, chemotherapeutic drugs or other CAR-T drugs) for killing tumor cells.

In a seventh aspect, the invention provides a use of a CAR according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, or a pharmaceutical composition according to the sixth aspect of the invention, for the manufacture of a medicament or formulation for killing tumor cells.

In another preferred embodiment, the tumor cells comprise CD19 positive tumor cells.

In another preferred embodiment, the tumor cells comprise PSMA-positive tumor cells.

In another preferred embodiment, the tumor cell is derived from a solid tumor.

In another preferred embodiment, the solid tumor is selected from the group consisting of: breast cancer, pancreatic cancer, colon cancer, gastric cancer, lung cancer, renal cell carcinoma, liver cancer, ovarian cancer, esophageal adenocarcinoma, prostate cancer, cervical cancer, multiple sarcoma, melanoma, nasopharyngeal cancer, or a combination thereof.

In an eighth aspect, the invention provides a kit for killing a tumor cell, the kit comprising a container, and in the container, a CAR of the first aspect of the invention, a nucleic acid molecule of the second aspect of the invention, a vector of the third aspect of the invention, or a host cell of the fourth aspect of the invention.

In another preferred embodiment, the kit further comprises a label or instructions for use.

The ninth aspect of the present invention provides a method for killing tumor cells, comprising:

administering to a subject in need thereof a safe and effective amount of a CAR of the first aspect of the invention, a nucleic acid molecule of the second aspect of the invention, a vector of the third aspect of the invention, a host cell of the fourth aspect of the invention, or a pharmaceutical composition of the sixth aspect of the invention.

In another preferred embodiment, the subject comprises a human or non-human mammal.

In another preferred embodiment, the non-human mammal includes a rodent (e.g., mouse, rat, rabbit), primate (e.g., monkey).

In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

In a tenth aspect, the present invention provides a method of treating cancer or a tumor, comprising:

In another preferred embodiment, the tumor comprises a solid tumor.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. For reasons of space, they will not be described in detail.

Drawings

Figure 1 shows the GITRL-CAR expression framework.

FIG. 2 shows that GITRL-CAR is highly expressed in human primary T cells.

FIG. 3 shows that the antitumor effect and proliferative capacity of GITRL-CART are significantly superior to those of the existing second-generation CART.

Figure 4 shows that GITRL promotes differentiation of Th9 subpopulations in CAR-T cells.

FIG. 5 shows that GITRL-CART depletion was reduced with a significant increase in persistence.

FIG. 6 shows that GITRL-CART has a stronger antitumor effect in vivo.

Detailed Description

The present inventors have unexpectedly found, through extensive and intensive studies and extensive screening, that co-expression of a CAR and an immunomodulatory molecule (such as GITRL) in T cells or NK cells can significantly improve antitumor (especially solid tumor) activity. The Chimeric Antigen Receptors (CARs) of the invention are useful in the treatment of PSMA or CD19 positive tumor patients. On this basis, the present inventors have completed the present invention.

The present invention is representatively illustrated in detail for the engineered immune cells of the present invention, taking CAR-T cells as an example. The engineered immune cells of the invention are not limited to the CAR-T cells described above and below, and the engineered immune cells of the invention have the same or similar technical features and benefits as the CAR-T cells described above and below. Specifically, when the immune cell expresses the chimeric antigen receptor CAR, the NK cell is identical to a T cell (or a T cell can replace an NK cell); when the immune cell is a T cell, the TCR is identical to the CAR (or the CAR can be replaced with a TCR).

Term(s) for

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

As used herein, a "Chimeric Antigen Receptor (CAR)" is a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain derived from a different polypeptide than the extracellular domain, and at least one intracellular domain. "Chimeric Antigen Receptors (CARs)" are also referred to as "chimeric receptors", "T-bodies" or "Chimeric Immunoreceptors (CIRs)". The term "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide capable of binding an antigen. "intracellular domain" refers to any oligopeptide or polypeptide known to be a domain that transmits signals to activate or inhibit biological processes in a cell.

As used herein, "domain" refers to a region of a polypeptide that is independent of other regions and folds into a specific structure.

As used herein, "tumor antigen" refers to an antigenic biomolecule, the expression of which results in cancer.

As used herein, the terms "administration" and "treatment" refer to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells, and contacting the reagent with a fluid, and contacting the fluid with the cells. "administering" and "treating" also mean treating in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, animal or study subject refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnosis; including contact of an anti-human LAG-3 antibody with a human or animal, subject, cell, tissue, physiological compartment, or physiological fluid.

As used herein, the term "treatment" refers to the administration of a therapeutic agent, either internally or externally, comprising any of the CARs of the invention and compositions thereof, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (therapeutically effective amount).

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may, but need not, be 1, 2 or 3.

"sequence identity" as referred to herein means the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions. The sequence identity between a sequence described in the present invention and a sequence with which it is identical may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

Immunomodulatory molecules

Immunoregulatory molecules are substances produced by immune cells or other cells that exert a modulating effect on the immune response, including antibodies, lymphokines, polysaccharides, polypeptides, lysozymes, and even certain small molecule compounds.

In a preferred embodiment, the immunomodulatory molecules of the invention include, but are not limited to: GITRL, 4-1BBL, CD40, LIGHT, B7.1, B7.2, OX40L, CD 70.

In a preferred embodiment, the immunomodulatory molecule of the invention comprises GITRL.

In a preferred embodiment, the amino acid sequence of GITRL of the present invention is as shown in SEQ ID No. 1.

Tumor cell surface antigens

Tumor cell surface antigens of the invention include, but are not limited to, CD19, BCMA, CD38, PSMA, HER2, GPC3, Mesothel in, claudin18.2, EGFR, EGFRVIII, CEA, GD2, IL13R, FAP, CD 171.

Take CD19 and PSMA as examples.

CD19 is one of the important membrane antigens involved in B cell activation and proliferation, is a surface marker common to all B cells, does not disappear after B cell activation, is the most important B cell marker factor, and CD19 is also a component of the signaling complex on the B cell surface. CD 19-targeted CAR-T is mainly applied to the field of B cell malignancy treatment. CD19 can be widely expressed on the surface of various B cell malignant tumor cells, but is not expressed in other tissues and blood cells, and the existence of CD19 soluble protein in blood is not detected. It is therefore considered to be an ideal target for CAR-T treatment of B cell tumors. The clinical test result shows that the cure rate of CD19 CAR-T on acute B-lymphocyte leukemia (B-ALL) reaches 90%.

PSMA is a prostate specific membrane surface antigen, is highly expressed in most prostate cancer tissues, and is a novel tumor-associated antigen of prostate cancer and a diagnostic marker.

Antigen binding domains

In the present invention, the antigen binding domain of the chimeric antigen receptor CAR specifically binds to a tumor cell surface antigen.

In a preferred embodiment, the antigen binding domain of the chimeric antigen receptor CAR of the invention targets CD19 and/or PSMA.

Hinge region and transmembrane region

For the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domains may be selected, or modified by amino acid substitutions, to avoid binding such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex.

The transmembrane domain may be derived from natural sources or synthetic sources. In natural sources, the domain may be derived from any membrane bound or transmembrane protein. Preferably, the hinge region in the CAR of the invention is that of CD8a and the transmembrane region of the invention is that of CD8 a.

Intracellular domains

The intracellular domain or additional intracellular signaling domain of the CAR of the invention is responsible for the activation of at least one normal effector function of the immune cell in which the CAR has been placed. The term "effector function" refers to a cell's exclusive function. For example, the effector function of a T cell may be cytolytic activity or helper activity involving secretion of cytokines. The term "intracellular signaling domain" thus refers to a portion of a protein that transduces effector function signals and directs a cell to perform a proprietary function. Although the entire intracellular signaling domain may generally be used, in many instances, the entire strand need not be used. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used in place of the entire chain, so long as it transduces effector function signals. The term intracellular signaling domain thus refers to any truncated portion of an intracellular signaling domain that includes sufficient signal transduction of effector function.

Preferred examples of intracellular signaling domains for the CARs of the invention include cytoplasmic sequences of the T Cell Receptor (TCR) and co-receptors that act synergistically to initiate signal transduction upon antigen receptor binding, as well as any derivative or variant of these sequences and any synthetic sequence with the same functional capacity.

In preferred embodiments, the cytoplasmic domain of the CAR can be designed to itself include the CD 3-zeta signaling domain, or can be associated with any other desired cytoplasmic domain(s) useful in the context of the CARs of the invention. For example, the cytoplasmic domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. A costimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of the costimulatory molecule. Costimulatory molecules are cell surface molecules required for effective response of lymphocytes to antigens, rather than antigen receptors or their ligands. Preferably, 4-1BB (CD137) and the like are included.

The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention can be linked to each other randomly or in a defined order. Optionally, short oligopeptide or polypeptide linkers, preferably between 2 and 10 amino acids in length, can form the linkage. Glycine-serine doublets provide particularly suitable linkers.

In one embodiment, the cytoplasmic domain in the CAR of the invention is designed to include the signaling domain of 4-1BB (co-stimulatory molecule) and the signaling domain of CD3 ζ.

Chimeric Antigen Receptor (CAR)

Chimeric immune antigen receptors (CARs) consist of an extracellular antigen recognition region, usually a scFv (single-chain variable fragment), a transmembrane region, and an intracellular costimulatory signal region. The design of CARs goes through the following process: the first generation CARs had only one intracellular signaling component, CD3 ζ or Fc γ RI molecule, and, because of the single activation domain in the cell, it caused only transient T cell proliferation and less cytokine secretion, and did not provide long-term T cell proliferation signaling and sustained in vivo anti-tumor effects, and therefore did not achieve good clinical efficacy. The second generation CARs introduce a costimulatory molecule such as CD28, 4-1BB, OX40 and ICOS on the basis of the original structure, and compared with the first generation CARs, the function of the second generation CARs is greatly improved, and the persistence of CAR-T cells and the killing capability of the CAR-T cells on tumor cells are further enhanced. On the basis of the second generation CARs, a plurality of novel immune co-stimulatory molecules such as CD27 and CD134 are connected in series, and the development is three-generation and four-generation CARs.

The extracellular domain of CARs recognizes a specific antigen and subsequently transduces this signal through the intracellular domain, causing activated proliferation, cytolytic toxicity and cytokine secretion of the cell, thereby clearing the target cell. Autologous cells from the patient (or a heterologous donor) are first isolated, activated and genetically engineered to produce immune cells for CAR production, and then injected into the same patient. In this way, the probability of graft versus host disease is very low and antigens are recognized by immune cells in a non-MHC restricted manner.

CAR-immune cell therapy has achieved very high clinical response rates in the treatment of hematological malignancies, which rates were previously unattainable by any therapeutic approach, and have triggered a hot surge of clinical research in the world.

Specifically, the Chimeric Antigen Receptors (CARs) of the invention include an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding member (also referred to as an antigen-binding domain). The intracellular domain includes a costimulatory signaling region and/or a zeta chain moiety. The costimulatory signaling region refers to a portion of the intracellular domain that includes the costimulatory molecule. Costimulatory molecules are cell surface molecules required for efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.

A linker may be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular domain or a cytoplasmic domain of a polypeptide chain. The linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

The CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds its associated antigen, it affects the tumor cells, causing the tumor cells to not grow, to be driven to death, or to otherwise be affected, and causing the patient's tumor burden to shrink or be eliminated. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecules and/or the zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the 4-1BB signaling domain and/or the CD3 zeta signaling domain combination.

As used herein, "antigen binding domain" and "single chain antibody fragment" each refers to an Fab fragment, an Fab "fragment, an F (ab") 2 fragment, or a single Fv fragment having antigen binding activity. Fv antibodies contain the variable regions of the antibody heavy chain, the variable regions of the light chain, but no constant regions, and have the smallest antibody fragment of the entire antigen binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding. The antigen binding domain is typically a scFv (single-chain variable fragment). The size of the scFv is typically 1/6 for a whole antibody. Single chain antibodies are preferably a sequence of amino acids encoded by a single nucleotide chain. In a preferred embodiment of the invention, the scFv comprises an antibody, preferably a single chain antibody, that specifically recognizes the tumor highly expressed antigens CD19 and PSMA.

In the present invention, the scFv of the present invention also includes conservative variants thereof, which means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced with amino acids having similar or similar properties as compared with the amino acid sequence of the scFv of the present invention to form a polypeptide.

In the present invention, the number of amino acids to be added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total number of amino acids in the original amino acid sequence.

In the present invention, the number of the amino acids to be added, deleted, modified and/or substituted is usually 1, 2, 3, 4 or 5, preferably 1 to 3, more preferably 1 to 2, and most preferably 1.

The extracellular domain of the CAR of the invention includes an antibody single chain variable region sequence that targets a tumor cell surface antigen, preferably an antibody single chain variable region sequence having a specific sequence that targets a tumor cell surface antigen.

In the present invention, the intracellular domains in the CAR of the invention include the transmembrane region of CD8a, the costimulatory factor of 4-1BB, and the signaling domain of CD3 zeta.

In a preferred embodiment of the invention, the amino acid sequence of the CAR (the amino acid sequence of the CAR comprising an immunomodulatory molecule (such as GITRL)) is as set forth in SEQ ID No.:2 or 3:

PSMA CAR amino acid sequence containing an immunomodulatory molecule (such as GITRL):

MALPVTALLLPLALLLHAARPDIVMTQSPSSLSASVGDRVTITCKASQDCGTAVDWYQQKPGKAPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISSLQPEDFADYFCQQYNSYPLTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKISCKTSGYTFTEYTIHWVKQASGKGLEWIGNINPNNGGTTYNQKFEDRATLTVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPTLHPSPITCEFLFSTALISPKMCLSHLENMPLSHSRTQGAQRSSWKLWLFCSIVMLLFLCSFSWLIFIFLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFIS(SEQ ID NO.:2)

CD19 CAR amino acid sequence containing an immune modulatory molecule (such as GITRL):

MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPTLHPSPITCEFLFSTALISPKMCLSHLENMPLSHSRTQGAQRSSWKLWLFCSIVMLLFLCSFSWLIFIFLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFIS(SEQ ID NO.:3)

in a preferred embodiment of the invention, the nucleotide sequence of the CAR (the nucleotide sequence of the CAR comprising an immune modulatory molecule such as GITRL) is as set forth in SEQ ID No.:5 or 6:

nucleotide sequence of PSMA CAR containing an immunomodulatory molecule (such as GITRL):

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgGACATCGTGATGACCCAGTCCCCCTCCTCCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCACATGCAAGGCCTCCCAGGATTGTGGCACCGCCGTGGACTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTGGGCCTCCACCAGACACACCGGCGTGCCTGACAGATTCACCGGCTCCGGCTCTGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGGACTTCGCCGACTACTTCTGCCAGCAGTACAACTCCTACCCTCTGACCTTCGGCGGAGGCACCAAGCTGGAAATCAAAGGCGGAGGCGGATCAGGTGGTGGCGGATCTGGAGGTGGCGGAAGCGAAGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCCTGCAAGACCTCCGGCTACACCTTCACCGAGTACACCATCCACTGGGTGAAACAGGCCTCCGGCAAGGGCCTGGAATGGATCGGCAACATCAACCCTAACAACGGCGGCACCACCTACAACCAGAAGTTCGAGGACCGGGCCACCCTGACCGTGGACAAGTCCACCTCCACCGCCTACATGGAACTGTCCTCCCTGCGGTCTGAGGACACCGCCGTGTACTACTGCGCCGCTGGCTGGAACTTCGACTACTGGGGCCAGGGCACCACAGTGACAGTCTCGAGCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCACAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCgagggcagaggcagtctgctgacatgcggtgacgtggaagagaatcccggccctACATTGCATCCTTCACCCATCACTTGTGAATTTTTGTTTTCCACAGCTCTCATTTCTCCAAAAATGTGTTTGAGCCACTTGGAAAATATGCCTTTAAGCCATTCAAGAACTCAAGGAGCTCAGAGATCATCCTGGAAGCTGTGGCTCTTTTGCTCAATAGTTATGTTGCTATTTCTTTGCTCCTTCAGTTGGCTAATCTTTATTTTTCTCCAATTAGAGACTGCTAAGGAGCCCTGTATGGCTAAGTTTGGACCATTACCCTCAAAATGGCAAATGGCATCTTCTGAACCTCCTTGCGTGAATAAGGTGTCTGACTGGAAGCTGGAGATACTTCAGAATGGCTTATATTTAATTTATGGCCAAGTGGCTCCCAATGCAAACTACAATGATGTAGCTCCTTTTGAGGTGCGGCTGTATAAAAACAAAGACATGATACAAACTCTAACAAACAAATCTAAAATCCAAAATGTAGGAGGGACTTATGAATTGCATGTTGGGGACACCATAGACTTGATATTCAACTCTGAGCATCAGGTTCTAAAAAATAATACATACTGGGGTATCATTTTACTAGCAAATCCCCAATTCATCTCCTAA(SEQ ID NO.：5)

nucleotide sequence of CD19 CAR comprising an immunomodulatory molecule (such as GITRL):

ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGCTCCACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCGGCCGCAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCACAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCgagggcagaggcagtctgctgacatgcggtgacgtggaagagaatcccggccctACATTGCATCCTTCACCCATCACTTGTGAATTTTTGTTTTCCACAGCTCTCATTTCTCCAAAAATGTGTTTGAGCCACTTGGAAAATATGCCTTTAAGCCATTCAAGAACTCAAGGAGCTCAGAGATCATCCTGGAAGCTGTGGCTCTTTTGCTCAATAGTTATGTTGCTATTTCTTTGCTCCTTCAGTTGGCTAATCTTTATTTTTCTCCAATTAGAGACTGCTAAGGAGCCCTGTATGGCTAAGTTTGGACCATTACCCTCAAAATGGCAAATGGCATCTTCTGAACCTCCTTGCGTGAATAAGGTGTCTGACTGGAAGCTGGAGATACTTCAGAATGGCTTATATTTAATTTATGGCCAAGTGGCTCCCAATGCAAACTACAATGATGTAGCTCCTTTTGAGGTGCGGCTGTATAAAAACAAAGACATGATACAAACTCTAACAAACAAATCTAAAATCCAAAATGTAGGAGGGACTTATGAATTGCATGTTGGGGACACCATAGACTTGATATTCAACTCTGAGCATCAGGTTCTAAAAAATAATACATACTGGGGTATCATTTTACTAGCAAATCCCCAATTCATCTCCTAA(SEQ ID NO.:6)。

chimeric antigen receptor T cells (CAR-T cells)

As used herein, the terms "CAR-T cell", "CAR-T cell of the invention" all refer to a CAR-T cell of the invention, which can target a tumor surface antigen (e.g., CD19, PSMA) for the treatment of tumors, especially solid tumors, that are highly expressed or positive for a tumor cell surface antigen (e.g., CD19, PSMA).

CAR-T cells have the following advantages over other T cell-based therapies: (1) the action process of the CAR-T cell is not limited by MHC; (2) given that many tumor cells express the same tumor antigen, CAR gene construction for a certain tumor antigen can be widely utilized once it is completed; (3) the CAR can utilize tumor protein antigens and glycolipid non-protein antigens, so that the target range of the tumor antigens is expanded; (4) the use of patient autologous cells reduces the risk of rejection; (5) the CAR-T cell has an immunological memory function and can survive in vivo for a long time.

In the present invention, the CAR of the invention comprises (i) an extracellular domain comprising an antibody single chain variable region sequence that targets a tumor cell surface antigen; (ii) a transmembrane domain; (iii) a co-stimulatory factor; and (iv) the signaling domain of CD3 ζ; and; (v) a self-cleaving protein; (vi) immunomodulatory molecules (such as GITRL, 4-1BBL, CD40, LIGHT, B7.1, B7.2, OX40L, CD 70).

Chimeric antigen receptor NK cells (CAR-NK cells)

As used herein, the terms "CAR-NK cell", "CAR-NK cell of the invention" all refer to a CAR-NK cell of the invention. The CAR-NK cells of the invention can target tumor surface antigens (such as CD19 and PSMA) and are used for treating tumors with high expression or positive tumor cell surface antigens (such as CD19 and PSMA), especially solid tumors.

Natural Killer (NK) cells are a major class of immune effector cells that protect the body from viral infection and tumor cell invasion through non-antigen specific pathways. By engineering (genetically modifying) NK cells it is possible to obtain new functions, including the ability to specifically recognize tumor antigens and having an enhanced anti-tumor cytotoxic effect.

CAR-NK cells also have the following advantages compared to autologous CAR-T cells, for example: (1) directly kills tumor cells by releasing perforin and granzyme, but has no killing effect on normal cells of an organism; (2) they release very small amounts of cytokines and thus reduce the risk of cytokine storm; (3) is easy to be amplified in vitro and can be developed into ready-made products. Otherwise, similar to CAR-T cell therapy.

Exogenous T cell antigen receptor

As used herein, a foreign T cell antigen receptor (TCR) is a TCR that is exogenously transferred into a T cell by means of genetic engineering, using lentivirus or retrovirus as a vector, by cloning the α chain and β chain of the TCR from a tumor-reactive T cell by gene transfer technique.

The exogenous TCR modified T cell can specifically recognize and kill tumor cells, and affinity of the T cell and tumor can be improved and anti-tumor effect can be improved by optimizing affinity of TCR and tumor specific antigen.

Carrier

Nucleic acid sequences encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.

The present invention also provides a vector into which the expression cassette of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, since they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.

In brief summary, an expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence.

The expression constructs of the invention may also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the invention provides a gene therapy vector.

The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and Molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors comprise an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

Additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50bp apart, and activity begins to decline. Depending on the promoter, it appears that the individual elements may function cooperatively or independently to initiate transcription.

An example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1 α (EF-1 α). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40(SV40) early promoter, the mouse mammary cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr (Epstein-Barr) virus immediate early promoter, the rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

To assess the expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cells can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in a host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, the reporter gene is the following: which is not present in or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al, 2000FEBS Letters479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, the construct with the minimum of 5 flanking regions that showed the highest level of reporter gene expression was identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate the ability of an agent to modulate promoter-driven transcription.

Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vector may be readily introduced into a host cell by any method known in the art, e.g., mammalian, bacterial, yeast or insect cells. For example, the expression vector may be transferred into a host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing the polynucleotide into a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.

Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).

In the case of non-viral delivery systems, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for use to introduce nucleic acids into host cells (ex vivo or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution comprising the lipid, mixed with the lipid, associated with the lipid, contained as a suspension in the lipid, contained in or complexed with a micelle, or otherwise associated with the lipid. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. For example, they may be present in bilayer structures, either as micelles or with a "collapsed" structure. They may also simply be dispersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include fatty droplets that occur naturally in the cytoplasm as well as such compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

In a preferred embodiment of the invention, the vector is a lentiviral vector.

Preparation

The invention provides a CAR according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention or a host cell according to the fourth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the CAR-T cells are present in the formulation at a concentration of 1X 10³-1×10⁸One cell/Kg body weight, more preferably 1X 10⁶-1×10⁷One cell/Kg body weight.

In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The formulations of the present invention are preferably formulated for intravenous administration.

Therapeutic applications

The invention includes therapeutic applications of cells (e.g., T cells) transduced with Lentiviral Vectors (LV) encoding expression cassettes of the invention. The transduced T cells can target tumor cell markers (such as CD19, and/or PSMA) proteins, synergistically activate T cells, elicit a cellular immune response, and thereby significantly increase their killing efficiency against tumor cells from malignant tumors.

Accordingly, the present invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal comprising the steps of: administering to the mammal the CAR-T cells of the invention.

In one embodiment, the invention includes a class of cell therapy in which autologous T cells (or allogeneic donors) from a patient are isolated, activated, genetically engineered to produce CAR-T cells, and subsequently injected into the same patient. In this way, the probability of graft versus host disease is very low and antigens are recognized by T cells in an MHC-unrestricted manner. Furthermore, one CAR-T can treat all cancers expressing this antigen. Unlike antibody therapy, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

In one embodiment, the CAR-T cells of the invention can undergo robust in vivo T cell expansion and can last for an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-modified T cell induces an immune response specific to the antigen binding domain in the CAR. For example, CAR-T cells that are markers of tumor cells (such as CD19, and/or PSMA) elicit a specific immune response against cells that express markers of tumor cells (such as CD19, and/or PSMA).

Although the data disclosed herein specifically disclose lentiviral vectors comprising an antibody single chain variable region sequence targeting a tumor cell surface antigen, a hinge and transmembrane region, and 4-1BB and CD3 zeta signaling domains, T2A, immunomodulatory molecules (such as GITRL, 4-1BBL, CD40, LIGHT, B7.1, B7.2, OX40L, CD70), the invention should be construed to include any number of variations on each of the construct components.

Treatable cancers include tumors that are not vascularized or have not substantially vascularized, as well as vascularized tumors. The cancer may comprise a non-solid tumor (such as a hematological tumor, e.g., leukemia and lymphoma) or may comprise a solid tumor. The types of cancer treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, such as sarcomas, carcinomas and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematological) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, granulo-monocytic, monocytic and erythrocytic leukemias), chronic leukemias (such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, hodgkin's disease, non-hodgkin's lymphoma (indolent and higher forms), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

A solid tumor is an abnormal mass of tissue that generally does not contain cysts or fluid regions. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.

The CAR-modified T cells of the invention may also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals. Preferably, the mammal is a human.

For ex vivo immunization, at least one of the following occurs in vitro prior to administration of the cells into a mammal: i) expanding the cell, ii) introducing a nucleic acid encoding the CAR into the cell, and/or iii) cryopreserving the cell.

Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cells can be autologous with respect to the recipient. Alternatively, the cells may be allogeneic, syngeneic (syngeneic), or xenogeneic with respect to the recipient.

In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

The invention provides a method of treating a tumor comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the invention.

The CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components or other cytokines or cell populations. Briefly, a pharmaceutical composition of the invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.

The pharmaceutical compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease-although the appropriate dosage may be determined by clinical trials.

When referring to an "immunologically effective amount", "an anti-tumor effective amount", "a tumor-inhibiting effective amount", or a "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, extent of infection or metastasis, and individual differences in the condition of the patient (subject). It can be generally pointed out that: pharmaceutical compositions comprising T cells described herein mayTo be at 10⁴To 10⁹Dosage of individual cells/kg body weight, preferably 10⁵To 10⁶Doses of individual cells per kg body weight (including all integer values within those ranges) are administered. The T cell composition may also be administered multiple times at these doses. Cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al, New Eng.J.of Med.319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject composition may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by i.v. injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.

In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) any number of relevant treatment modalities, including but not limited to treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or efavirenz therapy for psoriasis patients or other therapy for PML patients. In further embodiments, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In a further embodiment, the cell composition of the invention is administered to the patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) bone marrow transplantation with a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an injection of the expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered pre-or post-surgery.

The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The proportion of doses administered to a human can be effected in accordance with accepted practice in the art. Typically, 1X 10 may be administered per treatment or per course of treatment ⁶1 to 10¹⁰A subject modified T cell (e.g., a CAR-T cell of the subject invention) is administered to a patient, for example, by intravenous infusion.

The main advantages of the invention include:

(1) the engineered immune cells of the invention can specifically target the antibody single-chain variable region sequence of tumor cell surface antigens (such as CD19 and PSMA), thereby efficiently killing tumors (especially solid tumors).

(2) The present invention is the first discovery that CARs expressing immunomodulatory molecules (such as GITRL) can more specifically kill tumor cells, particularly tumor cells that are highly expressed or positive for CD19, and/or PSMA.

(3) The invention discovers for the first time that expression of an exogenous immune modulatory molecule (such as GITRL) with a CAR in CAR-modified T cells or NK cells can significantly increase tumor suppression activity with synergistic effects.

(4) The invention develops a novel chimeric antigen receptor molecule for the first time, the CAR molecule can effectively resist the inhibition of tumor immune microenvironment after being expressed in T cells, maintains or improves the effector function, the amplification capacity and the persistence capacity of the CAR-T cells, and the CAR molecule has a treatment effect which is obviously superior to that of the existing CAR structure in the treatment of solid tumors represented by the prostate cancer.

(5) The invention discovers for the first time that fusion expression is carried out in a 2A-linked manner by expressing an expression sequence of a glucocorticoid-induced tumor necrosis factor receptor (GITR) ligand GITRL in a CAR molecule. Since activated T cells highly express GITR, the co-expressed GITRL enhances the respective effector functions through the cooperation between CAR-T cells.

(6) The invention discovers for the first time that expression of GITRL can remarkably promote differentiation of Th9 subgroup in CAR-T cells, inhibit formation of regulatory T cell Treg, remarkably improve subgroup composition of CAR-T cells, and embody stronger anti-tumor effect function.

(7) According to the invention, an important immunoregulation molecule GITRL is introduced into a CAR molecule for the first time, and is co-expressed with CAR in a 2A connection mode, the molecule promotes CAR-T cell activation, enhances differentiation of CD4+ T cells to Th9, inhibits differentiation and formation of Treg T cells, plays an important role in overcoming solid tumor immunosuppressive microenvironment, shows an effect which is obviously superior to the existing CAR-T technology in prostate cancer CAR-T treatment targeting PSMA, and has huge application potential in immunotherapy of various solid tumors.

(8) The CAR-T cell co-expressing GITRL provided by the invention has the function of regulating the functions of other immune cells expressing the receptor GITR, wherein the immune cells include but are not limited to B cells, macrophages, natural killer cells, granulocytes and mast cells, and the effect of enhancing the anti-tumor immune response of a patient is achieved by comprehensively and diversely regulating the tumor immune microenvironment.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Unless otherwise specified, materials and reagents used in examples of the present invention are commercially available products.

Example 1: GITRL-CART cell preparation and CAR expression identification

GITRL-CAR was constructed in the following order (fig. 1): antigen recognition region → junction/transmembrane region → 4-1BB costimulation → CD3 ζ → P2A → GITRL coding sequence, and then integrated between the 5 and 3 terminal LTR sequences of pELPS lentiviral vector, a CAR expression master plasmid was constructed, followed by lentiviral packaging.

1. Lentiviral packaging

Cell culture before transfection

One day before transfection, 293T cells in good growth status were digested, centrifuged at 1200rpm for 3min and the supernatant discarded. The cells were resuspended by adding the medium, the cell suspension was inoculated uniformly into a 10cm cell culture dish containing 9ml of DMEM complete medium, and the dish was gently shaken to distribute the cells uniformly therein. Culturing at 37 deg.C in 5% CO2 incubator. The growth density and the state of the cells are regularly observed, and the cell fusion rate reaches about 70 to 80 percent, so that the cells can be used for transfection. The 293T cells used for transfection should be subcultured for no more than 24 hours.

Transfection

1) The three plasmids (psPAX2, pMD2.G and the plasmid of interest with the CAR-GITRL expression cassette integrated) were diluted, and psPAX 25. mu.g, pMD2. G3. mu.g and the plasmid of interest 5. mu.g were added to 500. mu.L of serum-free medium DMEM and gently mixed. Incubating at room temperature for 5 min;

2) the transfection reagent PEI was diluted and 50. mu.L of PEI was added to 450. mu.L of DMED medium without serum and mixed gently. Incubating at room temperature for 5 min;

3) and dropwise adding the incubated PEI diluent into the diluted three plasmids, and lightly blowing, beating and uniformly mixing. Standing and incubating for 20min at room temperature, preparing a three-plasmid-PEI mixture, and marking according to different added plasmids;

4) the plasmid mixture was added dropwise to 293T cells for transfection, and cultured in a 5% CO2 incubator at 37 ℃. After culturing for 6-8 h, the cells are replaced by fresh complete medium DMEM (marked as transfection for 0 h). The culture was continued at 37 ℃ with 5% CO 2.

Harvesting lentiviruses

After 48h of transfection, cell supernatants were collected separately in clean 50mL centrifuge tubes, labeled, and the dishes were supplemented with 10mL of complete Medium DMEM and cultured at 37 deg.C with 5% CO 2. After 72h of transfection, the cell culture supernatant was again collected and the collected virus stock was placed in a 250ml centrifuge tube. In the process of continuous culture, the growth state of the cells is regularly observed, and the cell toxicity production efficiency is ensured.

Concentration and purification of lentivirus

1) And (3) ultrafiltration: the collected virus stock was added to a 100kD ultrafiltration tube, trimmed and centrifuged at 4000rpm for 30 minutes.

2) Super separation: transferring the virus concentrated solution after ultrafiltration into a sterilized quick-sealing ultracentrifuge tube, strictly balancing, and centrifuging at 40000rpm at 4 ℃ for 3 hours.

3) And (3) purification: after the virus liquid is centrifuged, the waste liquid is discarded, 1-3ml of blank X-vivo culture medium is added into an ultracentrifuge tube, the virus is concentrated by about 100 times and 300 times, and the virus is blown and beaten until the virus precipitate is completely dissolved. 250ul of each tube was dispensed into sterile EP tubes and stored in an ultra low temperature freezer at-80 ℃.

Lentiviral titer determination

Taking HEK-293T cells with good growth state, counting after digestion, uniformly spreading the HEK-293T cells into a 24-pore plate according to 2 x 10^5 pores, culturing for about 8 hours until the cells adhere to the wall, adding virus concentrated solutions with different volumes into the cell culture supernatant of the 24-pore plate according to a dilution gradient of 3 times, setting 8 gradients in total, infecting the cells for 48 hours in an incubator, and digesting and collecting the cells after infecting the cells for 48 hours.

Flow-through detection of percentage of positive cells

1) Collection 1 x 10⁵The 293T cells after about infection are placed in a clean 1.5ml EP tube, rinsed twice with PBS at 1200rpm and centrifuged for 3 min;

2) discarding the supernatant, adding 100 μ L of flow-type sample buffer, adding 0.6 μ L of PSMA Protein-Human, Recombinant (His tag) with Protein concentration of 0.15mg/mL, pipetting, mixing, incubating at 4 deg.C in dark for 45min

3) Rinsing twice with PBS, 1200rpm, centrifuging for 3 min;

4) the supernatant was discarded, 100. mu.L of flow loading buffer was added to resuspend the cell pellet, and 0.6ul of APC-Anti-Streptavidin flow antibody was added. Incubating at 4 deg.C in dark for 30 min;

5) rinsing twice with PBS, 1200rpm, centrifuging for 3 min;

6) discard the supernatant, add 300. mu.L of flow loading buffer to resuspend the cell pellet

7) And (5) detecting on a flow type computer.

And calculating the virus titer according to the following formula:

titer ═ percentage of cells positive at plating × 1000/volume of virus solution added (μ L) TU/mL.

2. GITRL-CART cell preparation and flow identification

Sorting and activating T cells

X-VIVO complete medium: X-VIVO basal medium + 10% FBS + 1% P/S +10 mug/mLIL-2

1) Strictly sterilizing a blood collection bag, a joint and the like by using 75% alcohol, transferring the blood collection bag into a biological safety cabinet, cutting the blood collection bag by using sterile scissors, transferring a blood sample into a clean 50mL centrifuge tube, and marking;

2) taking a clean 50mL centrifuge tube, adding 15mL of lymphocyte separation liquid, and marking;

3) slowly adding 30mL of blood sample into the centrifuge tube, wherein the action is slow as much as possible when the blood sample is added to keep the interface clear, centrifuging for 25min at 800g, and slowly ascending and descending during centrifugation, wherein the ascending speed is 1 and the descending speed is 0;

4) after the centrifugation is finished, carefully absorbing the middle white membrane layer into a new 50mL centrifuge tube by using a pipette gun, marking, taking care to avoid absorbing impurities as far as possible, adding PBS to 50mL, lightly blowing and uniformly beating, and centrifuging for 10min at 500 g;

5) after centrifugation, the supernatant was discarded, the cell pellet was resuspended in 50ml PBS, blown down evenly, and the WBC number was read by the hemocyte analyzer;

6) centrifuging the cells at 300g for 10 min;

7) after centrifugation, the supernatant was discarded. According to the end result of the counter, every 10⁷Adding 70 μ l buffer solution (x-vivo basal medium + 10% FBS) into each cell, resuspending the cell precipitate, gently pumping, mixing, transferring into a clean 15mL centrifugal tube, and marking;

8) according to the number of cells, every 10⁷20. mu.l of each of C was added to the cellsD4+ and CD8+ magnetic beads, and incubating for 15min at 4 ℃;

9) after the incubation is finished, adding a buffer solution into the cell suspension to 15mL, marking, gently blowing and uniformly mixing by using a pipettor, washing the cells, and centrifuging for 5min at 1200 g;

10) centrifuging, removing supernatant, and adding 500. mu.L buffer solution to resuspend cells;

11) putting the separation column into a magnetic field, connecting the lower end of the separation column with a 15mL core tube, rinsing the separation column with 3mL of buffer solution, slowly adding the cell suspension into the separation column in batches, slowly dripping the liquid, and adding 3mL of buffer solution to rinse for 2 times when the liquid level of the cell suspension is close to the upper end of the magnetic column;

12) after washing, removing the column from the magnetic field, adding 5mL of buffer solution, quickly pushing the cells into a new 15mL centrifuge tube by using a piston, marking, and centrifuging for 10min at 300 g;

13) after the centrifugation is finished, removing the supernatant, adding 5mL of X-vivo complete culture medium, and counting;

14) based on the counting results of the cell counter, the cells were suspended in X-vivo complete medium containing 0.1% IL-2 and 1% of the complex of CD3 and CD28, and the cell density was adjusted to 1X 10⁶Inoculating the cells/mL into a T75 culture bottle, gently blowing and uniformly mixing, and putting into an incubator for culture;

15) after 48h incubation, cells were pipetted into a clean 50mL centrifuge tube, 1200rpm, and centrifuged for 3 min.

Infection of T cells

Infecting and separating the obtained GITRL virus concentrated solution in the experiment according to MOI (molar equivalent of identity) 50 to obtain primary T cells, centrifuging and changing the solution 12 hours after infection, and continuously culturing for 48 hours, wherein the positively infected T cells are the target CAR-T cells.

Cells were harvested and the corresponding percentage of CAR-T cells was flow-tested

1) Collection 1 x 10⁵Placing the infected T cells in a clean 1.5ml EP tube, rinsing twice with PBS at 1200rpm, and centrifuging for 3 min;

2) discarding the supernatant, adding 100 μ L of flow-type sample buffer, adding 0.6 μ L of PSMA Protein, Human, recombinant (His tag) and Protein concentration of 0.15mg/mL, simultaneously adding 1ul of PE-anti-Human GITRL antibody, pipetting and mixing, and incubating at 4 deg.C in dark for 45 min;

3) rinsing twice with PBS, 1200rpm, centrifuging for 3 min;

4) the supernatant was discarded, 100. mu.L of flow loading buffer was added to resuspend the cell pellet, and 0.6. mu.l of APC-Anti-Streptavidin flow antibody was added. Incubating at 4 deg.C in dark for 30 min;

5) rinsing twice with PBS, 1200rpm, centrifuging for 3 min;

7) Expression of PSMA-CAR and GITRL was detected on a flow-through machine (FIG. 2).

Example 2: GITRL-CART cell luciferase killing detection and amplification detection

Co-cultivation

1. And taking each group of GITRL-CAR-T with good growth state, the control CART cells and the target cells PC-3, counting after centrifuging, and adding T cells into CAR-T according to the positive rate difference of the CAR-T cells of each group to adjust the positive rate of the CAR-T cells of each group to be consistent.

2. Adjusting the target cell density to 2 x 10^ 5/mL, designing different effective target ratios according to the experimental purpose, adjusting the densities of T cells and each group of CAR-T cells, and carrying out the co-culture experiment by referring to the following table.

3. According to the effector cell: target cell ratio (effective target ratio), calculating the number of effector cells required by each hole, adding into a low adsorption 96-well plate, and making three multiple holes for each effective target ratio to make the system be 150 microliter system. Co-culture experiment effective target ratio 2: 1 was performed as in table 1.

TABLE 1

4. After the desired number of cells was added to each group, the cells were placed in an incubator and cultured for a corresponding period of time (12 hours, 16 hours, 20 hours, 24 hours, etc.).

5. And (3) blowing and uniformly mixing cells corresponding to each hole in the 96-hole plate, sucking 100 mu L of cell suspension, and transferring the cell suspension to a new 96-hole fluorescent ELISA plate.

6. Luciferase substrate was added at 10. mu.L per well.

7. And (4) measuring fluorescence reading values.

8. The kill rate calculation formula (target cell fluorescence value-effector cell fluorescence value-target cell and effector cell co-incubation fluorescence value)/(target cell fluorescence value-effector cell fluorescence value) × 100%.

Multi-round killing detection

1) The cell co-culture plating mode was performed with reference to the above method for detecting one round of killing, with a potency to target ratio of 0.5: 1, laying three 96-well plates (one is an ultra-low adsorption 96-well plate, the other two are normal wall-adhering 96-well plates and are respectively numbered as plate one, plate two and plate three)

2) After 24 hours of co-incubation, taking out the plate I, and respectively calculating the killing rate of the control CAR-T cells and the GITRL-CART cells to the target cells according to a cell luciferase killing determination method, namely the round of killing rate

3) The suspended cells in plate two were transferred to another new ultra low adsorption 96 well plate (numbered plate four) and then plated according to 0.5: 1 ratio of effective targets in the corresponding wells, PC-3 cells were added, the suspension cells in plate three were transferred in the same manner to a normally adherent 96-well plate (numbered plate five), and the corresponding target cells were added

4) After 24 hours of co-culture, the cells in the fourth plate are subjected to killing measurement according to the cell luciferase to calculate the killing rate of the target cells, namely the second round of killing rate

5) The suspended cells in the corresponding wells of plate five were all transferred to another new ultra-low adsorption 96-well plate (numbered plate 6) and PC-3 cells were added

6) After 24 hours of co-culture, the cells in the plate 6 were subjected to killing assay by luciferase to calculate the killing rate of the target cells, i.e., the three rounds of killing rate.

GITRL-CART cell background amplification

Control CART and GITRL-CART cells obtained by culturing two days after infection with the corresponding viruses and T cells obtained by sorting the same batch were cultured strictly in the same culture conditions (x-vivo medium + 10% FBS + 1% P/S + 10. mu.g/mL IL-2).

1) Adjusting the initial cell density to 1 x 10^6/mL, making three multiple wells, adjusting the initial cell number to 2 x 10^6

2) Supplementing or replacing liquid to cells in time according to the growth state of the cells to ensure that the cells grow in a good nutritional environment

3) Every two days, cells were centrifuged and then counted in a resuspension mix (cytometric instrument), recorded, plotted and a cell growth curve was plotted.

Amplification of GITRL-CART cells after co-incubation with target cells

1) According to the effective target ratio of 2: 1, regulating the number of target cells PC-3 and control T cells, control CART and GITRL-CART cells, respectively to 1X 10^6 and 2X 10^6, adding X-VIVO complete culture medium, placing into six-well plate, culturing

2) After 24 hours of co-incubation, the cells suspended in the wells were counted after centrifugation and transferred to a new plate for culture

3) Supplementing or replacing liquid to cells in time according to the growth state of the cells to ensure that the cells grow in a good nutritional environment

Every two days, cells were counted after centrifugation in a resuspension mix (cytometric instrument), recorded, plotted, and cell growth curves plotted (fig. 3).

Example 3: detection of IL-9 cytokine expression after Co-culture of GITRL-CART cells and target cells

Enzyme-linked immunosorbent assay (ELISA)

Transferring cell supernatants of the control T, control CART and GITRL-CART cells after 24 hours of co-culture with the PC-3 target cells into a new centrifuge tube, centrifuging at 4000rpm for 10 minutes, removing cell debris, transferring into a new centrifuge tube, subpackaging, and storing at-80 ℃.

1) Before use, all reagents are fully mixed to avoid generating foams.

2) The number of slats required was determined based on the number of experimental wells (blank and standard). Samples (with standards) and blanks should be done in triplicate.

3) Coating: mu.L/well diluted coating antibody (overnight at 4 ℃) was added

4) Washing the plate: deducting liquid in the hole, and adding 1 × Washing buffer working solution into 300 μ L/well; after 1 minute of residence, the liquid in the wells was discarded. Repeat 3 times, each time buckling dry on filter paper.

5) Sample adding: add diluted IL-9 standard to the standard wells at 100. mu.L/well, sample to the sample wells at 100. mu.L/well, Dilution buffer (1X) to the blank wells at 100. mu.L/well. The plate-sealing membrane was covered and incubated at room temperature (18-25 ℃) for 2 hours.

6) Washing the plate: and (4) repeating the step.

7) Adding a detection antibody: add 100. mu.L/well of detection antibody working solution. The plate-sealing membrane was covered and incubated at room temperature (18-25 ℃) for 1 hour.

8) Washing the plate: and (4) repeating the step.

9) Adding an enzyme: 100 μ L/well were added to Streptavidin-HRP working solution. The plate-sealing membrane was covered and incubated at room temperature (18-25 ℃) for 30 minutes.

10) Washing the plate: and (4) repeating the step.

11) Color development: TMB was added to 100. mu.L/well, incubated at room temperature (18-25 ℃) for 5-30 minutes in the absence of light, and the termination was judged by the shade of color in the wells (dark blue). Good results can be achieved by developing for 10-20 minutes.

12) And (3) terminating the reaction: the reaction was stopped by adding 100. mu.L/well quickly to Stop solution.

13) Reading a plate: within 10 minutes after termination, the reading is carried out at a detection wavelength of 450 nm.

Flow cytometry

1) 1 x 10 per tube collection⁵The co-incubated cells were placed in a clean 1.5ml EP tube, rinsed twice with PBS, 1200rpm, and centrifuged for 3 min;

2) discarding the supernatant, adding 100 μ L of membrane rupture fixing solution, gently blowing uniformly, and incubating at 4 deg.C in dark for 2 h;

3) rinsing twice with PBS, and centrifuging at 1200rpm for 3 min;

4) sequentially adding 0.6ul of PE-anti-human IL-9 and 1ul of APC-anti-human CD4 antibody, blowing, mixing, and incubating at 4 deg.C in dark for 45 min;

5) discard the supernatant, add 100. mu.L of flow loading buffer (PBS containing 2% fetal bovine serum) to resuspend the cell pellet;

6) rinsing twice with PBS, 1200rpm, centrifuging for 3 min;

7) discarding the supernatant, and adding 300. mu.L of flow-type loading buffer solution to resuspend the cell pellet;

8) co-expression of IL-9 and CD4 was detected by flow-on-machine (FIG. 4).

Example 4: GITRL-CART cell exhaustion, index detection

Co-cultivation

1) And taking control T cells, control CART, GITRL-CART cells and target cells PC-3, counting after centrifuging, and adding T cells into CAR-T according to the difference of the positive rates of various CAR-T cells until the positive rates of various CAR-T cells are consistent.

2) According to the effective target ratio of 1: 1 ratio, 1 x 10^6 effector and target cells were added to each well of a six-well plate, three replicates of each CAR-T were made, and the initial cell density was adjusted to 1 x 10^ 6/mL.

3) After 24 hours of co-incubation, various CAR-T cells were transferred to new culture plates for culture.

4) After 7 days of normal culture, flow staining was performed with PD-1, TIM 3.

Marker for flow detection of GITRL-CART cell exhaustion

1) Collecting about 3 x 10^5 cells, placing the cells in a clean EP tube with 1.5ml, rinsing twice with PBS (phosphate buffer solution), 1200rpm, and centrifuging for 3 min;

2) discarding the supernatant, adding 100 μ L of loading buffer solution, sequentially adding 0.6 μ L of APC-anti-human PD-1 and PE-anti-human TIM3 flow antibodies, blowing, mixing, and incubating at 4 deg.C in dark for 45 min;

3) rinsing twice with PBS, 1200rpm, centrifuging for 3 min;

4) discard the supernatant, add 300. mu.L of flow loading buffer to resuspend the cell pellet

5) And (5) detecting on a flow type computer.

6) Flowjo flow analysis software analyzed the expression of PD-1 and TIM3 after co-culture of various GITRL-CART cells with PC-3 (FIG. 5).

Example 5: GITRL-CART animal in vivo function evaluation

The evaluation tool used in this example was a 6-8 week old NSG mouse, which was bred in an SPF-grade laminar flow chamber, and all items related to mice, such as standard pellet breeding and padding, were sterilized. The evaluation of the in vivo function of GITRL-CART was carried out as follows.

1) Prepare 1640 complete medium, digest and count the target cells of cultured PC-3-luciferase, adjust the cell concentration to 2 x 10^7/mL (cells were resuspended in sterile PBS + matrigel ═ 3: 1).

2) NSG mice were selected for 6-8 weeks, and each mouse was anesthetized with 400. mu.L of sodium pentobarbital and the back hair was removed with a razor.

3) Mice were swab sterilized on the right dorsal side using an alcohol cotton swab, and then 100 μ L (2X 10^6 cells) of PC-3-luciferase cell suspension was injected subcutaneously into the right dorsal side using a 1mL syringe slowly, and the needle was withdrawn after several seconds of withdrawal. When the mice were soon to wake up, the mice were returned to their cages.

4) After the injection is finished, the medical cotton is used for stopping bleeding.

5) Cutting toes of the mice, numbering in sequence, and continuing feeding.

6) Fifteen days after the target cells are injected, a living body imaging instrument is used for carrying out living body imaging on the mice, the growth condition of the tumor is observed, the mice which are not successfully constructed are removed according to the imaging result, and the rest mice are randomly grouped according to the experimental arrangement and marked.

Mouse living body imaging IVIS (fig. 6)

Weighing a substrate D-Luciferin potassium salt, dissolving in PBS, storing in dark place, and entering an SPF (specific pathogen free) system through a transfer window. Intraperitoneal injection of substrate was performed according to the weight of 3 m/mouse, and 3min later, isoflurane anesthesia was performed on the mouse. After the mouse is anesthetized, the mouse is placed into an imager to be imaged and photographed, the pictures are counted and processed, and the read fluorescence value is recorded.

Mouse tail intravenous injection of CAR-T effector cells

1) According to the groups, mice were again imaged in vivo and body weights were taken into account before injecting effector CAR-T cells.

2) Preparing cell suspensions of Mock T, control CART and GITRL-CART, adjusting the cell density to 5 x 10^7/mL by using normal saline, subpackaging the cell suspensions into EP tubes, and filling the EP tubes into ice boxes for later use.

3) Tail vein injections of the corresponding CAR-T effector cells, 100 μ L each, were marked.

4) And performing living body imaging detection every 3-4 days, and recording and counting data.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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Shanghai Bangyao Biological Technology Co.,Ltd.

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245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

305 310 315 320

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

325 330 335

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

340 345 350

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Arg Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn

485 490 495

Pro Gly Pro Thr Leu His Pro Ser Pro Ile Thr Cys Glu Phe Leu Phe

500 505 510

Ser Thr Ala Leu Ile Ser Pro Lys Met Cys Leu Ser His Leu Glu Asn

515 520 525

Met Pro Leu Ser His Ser Arg Thr Gln Gly Ala Gln Arg Ser Ser Trp

530 535 540

Lys Leu Trp Leu Phe Cys Ser Ile Val Met Leu Leu Phe Leu Cys Ser

545 550 555 560

Phe Ser Trp Leu Ile Phe Ile Phe Leu Gln Leu Glu Thr Ala Lys Glu

565 570 575

Pro Cys Met Ala Lys Phe Gly Pro Leu Pro Ser Lys Trp Gln Met Ala

580 585 590

Ser Ser Glu Pro Pro Cys Val Asn Lys Val Ser Asp Trp Lys Leu Glu

595 600 605

Ile Leu Gln Asn Gly Leu Tyr Leu Ile Tyr Gly Gln Val Ala Pro Asn

610 615 620

Ala Asn Tyr Asn Asp Val Ala Pro Phe Glu Val Arg Leu Tyr Lys Asn

625 630 635 640

Lys Asp Met Ile Gln Thr Leu Thr Asn Lys Ser Lys Ile Gln Asn Val

645 650 655

Gly Gly Thr Tyr Glu Leu His Val Gly Asp Thr Ile Asp Leu Ile Phe

660 665 670

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

675 680 685

Leu Leu Ala Asn Pro Gln Phe Ile Ser

690 695

<210> 3

<211> 709

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 3

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser

20 25 30

Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser

35 40 45

Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly

50 55 60

Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr

85 90 95

Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu

165 170 175

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu

180 185 190

Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser

195 200 205

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

210 215 220

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

225 230 235 240

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ala Ala Thr Thr

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

325 330 335

Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Glu Gly Arg

485 490 495

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

500 505 510

Leu His Pro Ser Pro Ile Thr Cys Glu Phe Leu Phe Ser Thr Ala Leu

515 520 525

Ile Ser Pro Lys Met Cys Leu Ser His Leu Glu Asn Met Pro Leu Ser

530 535 540

His Ser Arg Thr Gln Gly Ala Gln Arg Ser Ser Trp Lys Leu Trp Leu

545 550 555 560

Phe Cys Ser Ile Val Met Leu Leu Phe Leu Cys Ser Phe Ser Trp Leu

565 570 575

Ile Phe Ile Phe Leu Gln Leu Glu Thr Ala Lys Glu Pro Cys Met Ala

580 585 590

Lys Phe Gly Pro Leu Pro Ser Lys Trp Gln Met Ala Ser Ser Glu Pro

595 600 605

Pro Cys Val Asn Lys Val Ser Asp Trp Lys Leu Glu Ile Leu Gln Asn

610 615 620

Gly Leu Tyr Leu Ile Tyr Gly Gln Val Ala Pro Asn Ala Asn Tyr Asn

625 630 635 640

Asp Val Ala Pro Phe Glu Val Arg Leu Tyr Lys Asn Lys Asp Met Ile

645 650 655

Gln Thr Leu Thr Asn Lys Ser Lys Ile Gln Asn Val Gly Gly Thr Tyr

660 665 670

Glu Leu His Val Gly Asp Thr Ile Asp Leu Ile Phe Asn Ser Glu His

675 680 685

Gln Val Leu Lys Asn Asn Thr Tyr Trp Gly Ile Ile Leu Leu Ala Asn

690 695 700

Pro Gln Phe Ile Ser

705

<210> 4

<211> 5

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 4

Gly Gly Gly Gly Ser

1 5

<210> 5

<211> 2094

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 5

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacatcg tgatgaccca gtccccctcc tccctgtctg cctccgtggg cgacagagtg 120

accatcacat gcaaggcctc ccaggattgt ggcaccgccg tggactggta tcagcagaag 180

cctggcaagg cccctaagct gctgatctac tgggcctcca ccagacacac cggcgtgcct 240

gacagattca ccggctccgg ctctggcacc gacttcaccc tgaccatctc cagcctgcag 300

cctgaggact tcgccgacta cttctgccag cagtacaact cctaccctct gaccttcggc 360

ggaggcacca agctggaaat caaaggcgga ggcggatcag gtggtggcgg atctggaggt 420

ggcggaagcg aagtgcagct ggtgcagtct ggcgccgaag tgaagaaacc tggcgcctcc 480

gtgaagatct cctgcaagac ctccggctac accttcaccg agtacaccat ccactgggtg 540

aaacaggcct ccggcaaggg cctggaatgg atcggcaaca tcaaccctaa caacggcggc 600

accacctaca accagaagtt cgaggaccgg gccaccctga ccgtggacaa gtccacctcc 660

accgcctaca tggaactgtc ctccctgcgg tctgaggaca ccgccgtgta ctactgcgcc 720

gctggctgga acttcgacta ctggggccag ggcaccacag tgacagtctc gagcaccacg 780

acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcacagcc cctgtccctg 840

cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 900

gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca 960

ctggttatca ccctttactg caaacggggc agaaagaaac tcctgtatat attcaaacaa 1020

ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca 1080

gaagaagaag aaggaggatg tgaactgaga gtgaagttca gcaggagcgc agacgccccc 1140

gcgtacaagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1200

tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 1260

aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1320

agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1380

ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 1440

cgcgagggca gaggcagtct gctgacatgc ggtgacgtgg aagagaatcc cggccctaca 1500

ttgcatcctt cacccatcac ttgtgaattt ttgttttcca cagctctcat ttctccaaaa 1560

atgtgtttga gccacttgga aaatatgcct ttaagccatt caagaactca aggagctcag 1620

agatcatcct ggaagctgtg gctcttttgc tcaatagtta tgttgctatt tctttgctcc 1680

ttcagttggc taatctttat ttttctccaa ttagagactg ctaaggagcc ctgtatggct 1740

aagtttggac cattaccctc aaaatggcaa atggcatctt ctgaacctcc ttgcgtgaat 1800

aaggtgtctg actggaagct ggagatactt cagaatggct tatatttaat ttatggccaa 1860

gtggctccca atgcaaacta caatgatgta gctccttttg aggtgcggct gtataaaaac 1920

aaagacatga tacaaactct aacaaacaaa tctaaaatcc aaaatgtagg agggacttat 1980

gaattgcatg ttggggacac catagacttg atattcaact ctgagcatca ggttctaaaa 2040

aataatacat actggggtat cattttacta gcaaatcccc aattcatctc ctaa 2094

<210> 6

<211> 2130

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 6

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccagaca tccagatgac acagactaca tcctccctgt ctgcctctct gggagacaga 120

gtcaccatca gttgcagggc aagtcaggac attagtaaat atttaaattg gtatcagcag 180

aaaccagatg gaactgttaa actcctgatc taccatacat caagattaca ctcaggagtc 240

ccatcaaggt tcagtggcag tgggtctgga acagattatt ctctcaccat tagcaacctg 300

gagcaagaag atattgccac ttacttttgc caacagggta atacgcttcc gtacacgttc 360

ggagggggga ctaagttgga aataacaggc tccacctctg gatccggcaa gcccggatct 420

ggcgagggat ccaccaaggg cgaggtgaaa ctgcaggagt caggacctgg cctggtggcg 480

ccctcacaga gcctgtccgt cacatgcact gtctcagggg tctcattacc cgactatggt 540

gtaagctgga ttcgccagcc tccacgaaag ggtctggagt ggctgggagt aatatggggt 600

agtgaaacca catactataa ttcagctctc aaatccagac tgaccatcat caaggacaac 660

tccaagagcc aagttttctt aaaaatgaac agtctgcaaa ctgatgacac agccatttac 720

tactgtgcca aacattatta ctacggtggt agctatgcta tggactactg gggtcaagga 780

acctcagtca ccgtctcctc agcggccgca accacgacgc cagcgccgcg accaccaaca 840

ccggcgccca ccatcgcgtc acagcccctg tccctgcgcc cagaggcgtg ccggccagcg 900

gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatatcta catctgggcg 960

cccttggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccct ttactgcaaa 1020

cggggcagaa agaaactcct gtatatattc aaacaaccat ttatgagacc agtacaaact 1080

actcaagagg aagatggctg tagctgccga tttccagaag aagaagaagg aggatgtgaa 1140

ctgagagtga agttcagcag gagcgcagac gcccccgcgt acaagcaggg ccagaaccag 1200

ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 1260

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1320

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1380

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1440

acctacgacg cccttcacat gcaggccctg ccccctcgcg agggcagagg cagtctgctg 1500

acatgcggtg acgtggaaga gaatcccggc cctacattgc atccttcacc catcacttgt 1560

gaatttttgt tttccacagc tctcatttct ccaaaaatgt gtttgagcca cttggaaaat 1620

atgcctttaa gccattcaag aactcaagga gctcagagat catcctggaa gctgtggctc 1680

ttttgctcaa tagttatgtt gctatttctt tgctccttca gttggctaat ctttattttt 1740

ctccaattag agactgctaa ggagccctgt atggctaagt ttggaccatt accctcaaaa 1800

tggcaaatgg catcttctga acctccttgc gtgaataagg tgtctgactg gaagctggag 1860

atacttcaga atggcttata tttaatttat ggccaagtgg ctcccaatgc aaactacaat 1920

gatgtagctc cttttgaggt gcggctgtat aaaaacaaag acatgataca aactctaaca 1980

aacaaatcta aaatccaaaa tgtaggaggg acttatgaat tgcatgttgg ggacaccata 2040

gacttgatat tcaactctga gcatcaggtt ctaaaaaata atacatactg gggtatcatt 2100

ttactagcaa atccccaatt catctcctaa 2130

<210> 7

<211> 18

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 7

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

1 5 10 15

Gly Pro

Claims

1. A chimeric antigen receptor CAR, comprising: an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain specifically binds to a tumor cell surface antigen;

2. The chimeric antigen receptor CAR of claim 1, wherein said immunomodulatory molecule is selected from the group consisting of: GITRL, 4-1BBL, CD40, LIGHT, B7.1, B7.2, OX40L, CD70, or a combination thereof.

3. The chimeric antigen receptor CAR of claim 1, wherein said CAR has the structure of formula I:

Z1-T-H-TM-C-Z2-(Z3-P)m (I)

in the formula (I), the compound is shown in the specification,

each "-" is independently a linker peptide or a peptide bond;

z1 is a null or signal peptide sequence;

h is a null or hinge region;

TM is a transmembrane domain;

c is a costimulatory signal molecule;

z2 is a cytoplasmic signaling sequence derived from CD3 ζ;

z3 is a self-cleaving protein;

p is an immunomodulatory molecule;

m is 1, 2, 3, or 4.

4. A nucleic acid molecule encoding the Chimeric Antigen Receptor (CAR) of claim 1.

5. A vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the vector or chromosome of claim 5 into which has been integrated an exogenous nucleic acid molecule of claim 4.

7. A method of making an engineered immune cell expressing the CAR of claim 1, wherein said method comprises the steps of: transferring the nucleic acid molecule of claim 4 or the vector of claim 5 into an immune cell, thereby obtaining the engineered immune cell.

8. A pharmaceutical composition comprising the CAR of claim 1, the nucleic acid molecule of claim 4, the vector of claim 5, or the host cell of claim 6, and a pharmaceutically acceptable carrier, diluent, or excipient.

9. Use of the CAR of claim 1, the nucleic acid molecule of claim 4, the vector of claim 5, the host cell of claim 6, or the pharmaceutical composition of claim 8, for the preparation of a medicament or formulation for killing tumor cells.

10. A kit for killing a tumor cell, comprising a container, and the CAR of claim 1, the nucleic acid molecule of claim 4, the vector of claim 5, or the host cell of claim 6 disposed within the container.