WO2021136040A1

WO2021136040A1 - Preparation and applications of chimeric antigen receptor t-cell co-expressing immunomodulatory molecule

Info

Publication number: WO2021136040A1
Application number: PCT/CN2020/138691
Authority: WO
Inventors: 谭炳合; 史秀娟; 杜冰; 刘明耀; 席在喜
Original assignee: 华东师范大学; 上海邦耀生物科技有限公司
Priority date: 2019-12-31
Filing date: 2020-12-23
Publication date: 2021-07-08
Also published as: CN113087805A

Abstract

Provided in the present invention are a preparation and applications of a chimeric antigen receptor T-cell co-expressing an immunomodulatory molecule. Specifically, provided in the present invention are a CAR and the CAR-T-cell thereof specifically targeting a tumor cell surface antigen, the chimeric antigen receptor (CAR) comprises an antigen-binding structural domain and an immunomodulatory molecule. The CAR-T-cell of the present invention provides excellent tumor-killing effects.

Description

Preparation and application of a chimeric antigen receptor T cell co-expressing immunoregulatory molecules

Technical field

The present invention relates to the field of biomedicine, in particular to the preparation and application of a chimeric antigen receptor T cell co-expressing immunoregulatory molecules.

Background technique

Chimeric antigen receptor (CAR) T cell therapy is a new cellular immunotherapy technology that has developed very rapidly in recent years. This technology combines the high affinity of antigen and antibody with the killing effect of T lymphocytes. Combine to construct a specific chimeric antigen receptor, insert the gene encoding the chimeric antigen receptor into T lymphocytes through a certain way, make the T lymphocytes express this chimeric antigen receptor, and then expand and purify the chimeric antigen receptor in vitro The genetically modified T cells are imported into the body. CAR-T cells specifically recognize the target antigen in the body and undergo a series of immune responses. The T cells activate and expand and secrete cytokines. The capacitive complex specifically kills target cells in a restrictive manner. For tumors, construct a tumor-associated antigen chimeric antigen receptor, which can be transferred to T cells to express the chimeric antigen receptor, which can specifically recognize the antigen on the surface of tumor cells, thereby activating T cells to exert cellular immunity and eliminate tumors. Cells, to achieve the purpose of anti-tumor.

Up to now, although CAR-T technology has achieved gratifying breakthroughs in the treatment of hematomas, its efficacy in the treatment of solid tumors, which account for the vast majority of malignant tumors, has not been satisfactory. CAR-T therapy is not effective in the treatment of solid tumors. It is related to solid tumor tissue barriers, tumor cells are highly heterogeneous, lacks good tumor-specific antigens or tumor-related antigens, highly suppressed tumor immune microenvironment, and CAR-T The cell's own activation signal is closely related. In the process of treating solid tumors, after CAR-T cells break through the physical barrier to recognize tumor cells, they must play an effective tumor cell killing effect. It depends on overcoming the highly inhibited tumor microenvironment and maintaining a stable activation state and functional continuity. . Although the current CAR structure design allows CAR-T cells to better recognize related antigens and activate quickly, under the strong inhibitory signal of the tumor microenvironment, CAR-T cells often have insufficient activation strength, poor expansion ability, and continuous activation. Conditions such as weak sex, rapid exhaustion and apoptosis have severely restricted the therapeutic effect of solid tumors.

In addition, the prior art integrates and expresses cytokines or chemokines in CAR molecules, although it can increase the expression of cytokines with anti-tumor effects while expressing CAR, but this expression mode is systemic, and CAR-T One of the important side effects of cells in the treatment process is cytokine storm. Systemically increasing cytokine expression will undoubtedly increase the risk of cytokine storm related to CAR-T treatment.

Integrating and expressing different costimulatory molecules in the intracellular activation domain of CAR molecules can provide costimulatory signals required for CAR-T cell activation, but the activation process depends on the expression of tumor-related or tumor-specific antigens and cannot By interacting with other immune cells in the tumor microenvironment to reshape and improve the tumor microenvironment, the therapeutic effect on solid tumors is very limited.

Therefore, there is an urgent need in the art to develop a new type of chimeric antigen receptor molecule, which can effectively resist the inhibitory effect of the tumor immune microenvironment after being expressed on T cells, and maintain or improve the effector function and expansion of CAR-T cells. Increased capacity and sustained capacity can show a significantly better therapeutic effect than the existing CAR structure in the treatment of solid tumors.

Summary of the invention

The purpose of the present invention is to provide a new type of chimeric antigen receptor molecule, which can effectively resist the inhibition of tumor immune microenvironment after being expressed on T cells, and maintain or improve the effector function and expansion of CAR-T cells Ability and sustainability, in the treatment of solid tumors, it can show a therapeutic effect that is significantly better than the existing CAR structure.

The first aspect of the present invention provides a chimeric antigen receptor CAR, the chimeric antigen receptor CAR comprising: an antigen binding domain, a transmembrane domain and an intracellular domain, wherein the antigen binding domain is specific Binding to tumor cell surface antigen;

And the chimeric antigen receptor CAR also includes: an immunomodulatory molecule that is connected to the intracellular domain and can be co-expressed.

In another preferred embodiment, the tumor cell antigens include cell surface antigens of various solid tumors 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, Claudin 18.2, EGFR, EGFRVIII, CEA, GD2, IL13R, FAP, CD171, Or a combination.

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

In another preferred embodiment, the immunomodulatory 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 includes GITRL.

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

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

In another preferred embodiment, the structure of the CAR is shown in the following formula I:

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

Where

Each "-" is independently a connecting peptide or a peptide bond;

Z1 is no or signal peptide sequence;

T is an antibody single-chain variable region sequence targeting tumor cell surface antigen;

H is no or hinge area;

TM is the transmembrane domain;

C is a costimulatory signal molecule;

Z2 is the cytoplasmic signal transduction sequence derived from CD3ζ;

Z3 is a self-cleaving protein;

P is an immunomodulatory molecule;

m is 1, 2, 3, or 4.

In another preferred example, the structure of the single-chain variable region sequence of the antibody targeting the tumor cell surface antigen is as shown in formula A1 or A2:

V _L1 -V _H1 (A1); or

V _L2 -V _H2 (A2);

Wherein, V _L1 and V _L2 are the light chain variable regions of antibodies against tumor cell surface antigens; V _H1 and V _H2 are the heavy chain variable regions of antibodies against tumor cell surface antigens; "-" is the connecting peptide (or Flexible linker) or peptide bond.

In another preferred embodiment, the V _L1 and V _H1 are connected by a flexible joint.

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

In another preferred example, _{the amino acid sequence of V L1} is as shown in SEQ ID NO.: 2 (CAR of PSMA) at positions 22-128, and _{the amino acid sequence of V H1} is as shown in SEQ ID NO.: 2 at positions 144-128. 258 shows.

In another preferred example, _{the amino acid sequence of V L2} is as shown in SEQ ID NO.: 3 (CAR of CD19) at positions 22-128, and _{the amino acid sequence of V H2} is as shown in SEQ ID NO.: 3 at positions 144-128. 263 shows.

In another preferred example, the structure of the CAR is shown in formula II:

Z1-V _L1 -V _H1 -H-TM-C-Z2-(Z3-P)m (II)

In the formula, each element is as described above.

In another preferred example, the structure of the CAR is shown in formula III:

Z1-V _L2 -V _H2 -H-TM-C-Z2-(Z3-P)m (III)

In the formula, each element is as described above.

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

In another preferred embodiment, the H is the 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 example, the C is a costimulatory signal molecule of 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 includes a costimulatory signal molecule derived from 4-1BB.

In another preferred embodiment, the 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 includes T2A.

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

In another preferred embodiment, the immunomodulatory molecules include wild-type immunomodulatory molecules and mutant immunomodulatory molecules, or active fragments thereof.

In another preferred embodiment, the GITRL has an amino acid sequence as shown in SEQ ID NO.:1.

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

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

The second aspect of the present invention provides a nucleic acid molecule that encodes the chimeric antigen receptor (CAR) of the first aspect of the present invention.

In another preferred embodiment, the nucleic acid molecule encoding the chimeric antigen receptor (CAR) of claim 1 is shown in SEQ ID NO.: 5 or 6.

The third aspect of the present invention provides a vector containing the nucleic acid molecule according to the second aspect of the present invention.

In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.

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

The fourth aspect of the present invention provides a host cell that contains the vector of the third aspect of the present invention or the nucleic acid molecule of the second aspect of the present invention integrated into the chromosome.

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

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

In another preferred embodiment, the host cells include engineered immune cells.

In another preferred embodiment, the immune cells also express exogenous immune regulatory molecular proteins.

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

In another preferred embodiment, the co-expression with the CAR targeting the tumor cell surface antigen includes the tandem expression of an immunomodulatory molecule protein and the CAR targeting the 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 cells are selected from the following group:

(i) Chimeric antigen receptor T cells (CAR-T cells);

(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, and the CAR-T cell expresses the chimeric antigen receptor CAR of claim 1.

The fifth aspect of the present invention provides a method for preparing engineered immune cells that express the CAR according to the first aspect of the present invention, wherein the method includes the steps of: The nucleic acid molecule or the vector described in the third aspect of the present invention is transferred into immune cells to obtain the engineered immune cells.

In another preferred embodiment, the introduction includes simultaneous, sequential, or sequential introduction.

In another preferred embodiment, the immune cells are T cells or NK cells.

In another preferred embodiment, the method further includes the step of performing function and effectiveness testing on the obtained engineered immune cells.

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

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

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

In another preferred embodiment, the host cell includes engineered immune cells.

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

In another preferred embodiment, in the pharmaceutical composition, the concentration of the cells is 1×10 ³ -1×10 ⁸ cells/mL, preferably 1×10 ⁶ -1×10 ⁷ cells/mL .

In another preferred embodiment, the pharmaceutical composition further includes an immunomodulatory molecule agonist.

In another preferred embodiment, the immunomodulatory molecule 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 also contains other drugs that kill tumor cells (such as antibody drugs, chemotherapeutic drugs or other CAR-T drugs).

The seventh aspect of the present invention provides a CAR according to the first aspect of the present invention, a nucleic acid molecule according to the second aspect of the present invention, a vector according to the third aspect of the present invention, and a host cell according to the fourth aspect of the present invention Or the use of the pharmaceutical composition of the sixth aspect of the present invention to prepare drugs or preparations for killing tumor cells.

In another preferred embodiment, the tumor cells include CD19-positive tumor cells.

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

In another preferred embodiment, the tumor cells are derived from solid tumors.

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 Osteosarcoma, melanoma, nasopharyngeal carcinoma, or a combination thereof.

The eighth aspect of the present invention provides a kit for killing tumor cells, the kit containing a container, and the CAR according to the first aspect of the present invention and the nucleic acid molecule according to the second aspect of the present invention in the container , The vector according to the third aspect of the present invention, or the host cell according to the fourth aspect of the present invention.

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

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

To a subject in need of treatment is administered a safe and effective amount of the CAR according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, the vector according to the third aspect of the present invention, and the host according to the fourth aspect of the present invention Cell, or the pharmaceutical composition according to the sixth aspect of the present invention.

In another preferred embodiment, the subject includes humans or non-human mammals.

In another preferred embodiment, the non-human mammals include rodents (such as mice, rats, rabbits) and primates (such as monkeys).

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

The tenth aspect of the present invention provides a method for treating cancer or tumor, including:

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

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them here.

Description of the drawings

Figure 1 shows the GITRL-CAR expression framework.

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

Figure 3 shows that the anti-tumor effect and proliferation ability of GITRL-CART is significantly better than the existing second-generation CART.

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

Figure 5 shows that the depletion capacity of GITRL-CART is reduced and the continuity is significantly enhanced.

Figure 6 shows that GITRL-CART has a stronger anti-tumor effect in vivo.

Detailed ways

Through extensive and in-depth research and extensive screening, the inventors unexpectedly discovered that the co-expression of CAR and immunomodulatory molecules (such as GITRL) in T cells or NK cells can significantly improve anti-tumor (especially solid tumors) activity. The chimeric antigen receptor (CAR) of the present invention can be used for the treatment of PSMA or CD19-positive tumor patients. On this basis, the inventor completed the present invention.

The present invention takes CAR-T cells as an example, and representatively describes the engineered immune cells of the present invention in detail. The engineered immune cells of the present invention are not limited to the CAR-T cells described in the context, and the engineered immune cells of the present invention have the same or similar technical features and beneficial effects as the CAR-T cells described in the context. Specifically, when immune cells express chimeric antigen receptor CAR, NK cells are equivalent to T cells (or T cells can replace NK cells); when immune cells are T cells, TCR is equivalent to CAR (or CAR can be replaced by TCR ).

the term

In order to make the present disclosure easier to understand, first define certain terms. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meaning given below. Other definitions are stated throughout the application.

The term "about" may refer to a value or composition within an acceptable error range of a specific value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

As used herein, "chimeric antigen receptor (CAR)" is a fusion protein comprising an extracellular domain capable of binding antigen, a transmembrane domain derived from a different polypeptide from the extracellular domain, and at least one cell Inner domain. "Chimeric antigen receptor (CAR)" is also called "chimeric receptor", "T-body" or "chimeric immune receptor (CIR)". The "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to a certain antigen. "Intracellular domain" refers to any oligopeptide or polypeptide known as a domain that transmits signals to activate or inhibit biological processes in a cell.

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

As used herein, "tumor antigen" refers to a biological molecule with antigenicity, the expression of which leads to cancer.

As used herein, the terms "administration" and "treatment" refer to the application of exogenous drugs, therapeutic agents, diagnostic agents or compositions to animals, humans, subjects, cells, tissues, organs, or biological fluids. "Administration" and "treatment" can refer to treatment, pharmacokinetics, diagnosis, research, and experimental methods. The treatment of cells includes contact between reagents and cells, contact between reagents and fluids, and contact between fluids and cells. "Administration" and "treatment" also mean treatment by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo. "Treatment" when applied to humans, animals or research subjects, refers to treatment, preventive or preventive measures, research and diagnosis; including anti-human LAG-3 antibodies and humans or animals, subjects, cells, tissues , Physiological compartment or physiological fluid contact.

As used herein, the term "treatment" refers to the administration of an internal or external therapeutic agent, including any one CAR of the present invention and a composition thereof, to a patient who has one or more disease symptoms, and the therapeutic agent is known to These symptoms have a therapeutic effect. Generally, the patient is administered in an amount (therapeutically effective amount) of a therapeutic agent effective to alleviate one or more disease symptoms.

As used herein, the term "optional" or "optionally" means that the event or situation described later can occur but does not have to occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence can have but not necessarily have, and can be 1, 2, or 3.

The "sequence identity" in the present invention refers to 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 the sequence described in the present invention and its identical sequence 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 molecule

Immunomodulatory molecules are substances produced by immune cells or other cells that regulate the immune response, including antibodies, lymphokines, polysaccharides, polypeptides, lysozyme and even some small molecule compounds.

In a preferred embodiment, the immunomodulatory molecules of the present invention include but are not limited to: GITRL, 4-1BBL, CD40, LIGHT, B7.1, B7.2, OX40L, CD70.

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

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

Tumor cell surface antigen

The tumor cell surface antigens of the present invention include but are not limited to CD19, BCMA, CD38, PSMA, HER2, GPC3, Mesothelin, Claudin 18.2, EGFR, EGFRVIII, CEA, GD2, IL13R, FAP, CD171.

Take CD19 and PSMA as examples.

CD19 is one of the important membrane antigens involved in the activation and proliferation of B cells. It is a surface marker shared by all B cells. B cells do not disappear after activation. It is the most important B cell marker factor. At the same time, CD19 is also a signal complex on the surface of B cells. The constituent parts of the body. CAR-T targeting CD19 is mainly used in the treatment of B cell malignant tumors. CD19 can be widely expressed on the surface of a variety of B-cell malignant tumor cells, but it is not expressed in other tissues and blood cells, and the presence of CD19 soluble protein has not been detected in the blood. Therefore, it is considered as an ideal target for CAR-T treatment of B-cell tumors. Clinical trial results show that the cure rate of CD19 CAR-T for acute B-lymphocytic leukemia (B-ALL) has reached 90%.

PSMA is a prostate-specific membrane surface antigen, which is highly expressed in most prostate cancer tissues. It is a tumor-associated antigen and a new diagnostic marker for prostate cancer.

Antigen binding domain

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

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

Hinge region and transmembrane region

For the hinge region and the 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 cases, transmembrane domains can 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 the interaction with the receptor complex. Interaction of other members.

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

Intracellular domain

The intracellular domain or another intracellular signaling domain of the CAR of the present 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 the exclusive function of the cell. For example, the effector function of T cells may be cytolytic activity or auxiliary activity including cytokine secretion. Therefore, the term "intracellular signal transduction domain" refers to the part of the protein that transduces effector function signals and directs the cell to perform specific functions. Although the entire intracellular signaling domain can generally be used, in many cases, the entire chain need not be used. In terms of using truncated portions of intracellular signaling domains, such truncated portions can be used to replace the complete chain as long as it transduces effector function signals. The term intracellular signaling domain therefore refers to any truncated portion of the intracellular signaling domain that is sufficient to transduce effector function signals.

Preferable examples of the intracellular signal transduction domain used in the CAR of the present invention include the cytoplasmic sequence of T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction after antigen receptor binding, and these sequences Any derivative or variant of and any synthetic sequence with the same functional capabilities.

In a preferred embodiment, the cytoplasmic domain of the CAR can be designed to include the CD3-ζ signaling domain itself, or can be combined with any other desired cytoplasmic domain (a Or multiple) joint. For example, the cytoplasmic domain of CAR may include a CD3ζ chain portion and a costimulatory signal transduction region. The costimulatory signal transduction region refers to a part of CAR that includes the intracellular domain of costimulatory molecules. Co-stimulatory molecules are cell surface molecules required for effective response of lymphocytes to antigens, not antigen receptors or their ligands. Preferably, it includes 4-1BB (CD137) and the like.

The cytoplasmic signal transduction sequences in the cytoplasmic signal transduction portion of the CAR of the present invention can be connected to each other randomly or in a prescribed order. Optionally, short oligopeptide or polypeptide linkers, preferably between 2 and 10 amino acids in length, can form the link. The glycine-serine doublet provides a particularly suitable linker.

In one embodiment, the cytoplasmic domain in the CAR of the present invention is designed to include the signaling domain of 4-1BB (costimulatory molecule) and the signaling domain of CD3ζ.

Chimeric Antigen Receptor (CAR)

Chimeric antigen receptors (CARs) are composed of extracellular antigen recognition regions, usually scFv (single-chain variable fragment), transmembrane regions and intracellular co-stimulatory signal regions. The design of CARs has gone through the following process: The first-generation CAR has only one intracellular signal component CD3ζ or FcγRI molecule. Since there is only one activation domain in the cell, it can only cause transient T cell proliferation and less cytokine secretion. , And cannot provide long-term T cell proliferation signals and sustained anti-tumor effects in vivo, so it has not achieved good clinical effects. The second-generation CARs introduce a costimulatory molecule based on the original structure, such as CD28, 4-1BB, OX40, and ICOS. Compared with the first-generation CARs, the function has been greatly improved, which further strengthens the persistence of CAR-T cells and the effect on tumor cells. The lethality. On the basis of the second-generation CARs, some new immunostimulatory molecules such as CD27 and CD134 are connected in series to develop into the third-generation and fourth-generation CARs.

The extracellular segment of CARs can recognize a specific antigen, and then transduce the signal through the intracellular domain to cause cell activation, proliferation, cytolytic toxicity, and cytokine secretion, thereby eliminating target cells. First, the patient’s autologous cells (or heterologous donors) are isolated, activated and genetically modified to produce CAR immune cells, and then injected into the same patient. In this way, the probability of graft-versus-host disease is extremely low, and the antigen is recognized by immune cells in a non-MHC-restricted manner.

CAR-immune cell therapy has achieved a very high clinical response rate in the treatment of hematological malignancies. Such a high response rate could not be achieved by any previous treatment method. It has triggered an upsurge of clinical research in the world.

Specifically, the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes target-specific binding elements (also called antigen binding domains). The intracellular domain includes a costimulatory signal transduction region and/or a zeta chain portion. The costimulatory signal transduction region refers to a part of the intracellular domain including costimulatory molecules. Co-stimulatory molecules are cell surface molecules required for effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

A linker can 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 connect the transmembrane domain to the extracellular or 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.

When the CAR of the present invention is expressed in T cells, it can perform antigen recognition based on the antigen binding specificity. When it binds to its associated antigen, it affects tumor cells, resulting in tumor cells not growing, being promoted to die or being affected in other ways, and causing the patient's tumor burden to shrink or eliminate. The antigen binding domain is preferably fused with an intracellular domain from one or more of the costimulatory molecule and/or zeta chain. Preferably, the antigen binding domain is fused with the intracellular domain combined with the 4-1BB signaling domain and/or the CD3ζ signaling domain.

As used herein, "antigen-binding domain" and "single-chain antibody fragment" all refer to Fab fragments, Fab" fragments, F(ab")2 fragments, or single Fv fragments that have antigen-binding activity. The Fv antibody contains the variable region of the heavy chain and the variable region of the light chain, but does not have the constant region, and has the smallest antibody fragment with all the antigen binding sites. Generally, an Fv antibody also contains a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding. The antigen binding domain is usually scFv (single-chain variable fragment). The size of scFv is generally 1/6 that of a complete antibody. The single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain. As a preferred mode of the present invention, the scFv includes an antibody that specifically recognizes the tumor highly expressed antigens CD19 and PSMA, preferably a single-chain antibody.

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

In the present invention, the number of added, deleted, modified and/or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, It is more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

In the present invention, the number of added, deleted, modified and/or substituted amino acids is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2, The best is one.

The extracellular domain of the CAR of the present invention includes an antibody single-chain variable region sequence targeting a tumor cell surface antigen, preferably an antibody single-chain variable region sequence targeting a tumor cell surface antigen with a specific sequence.

In the present invention, the intracellular domain in the CAR of the present invention includes the transmembrane region of CD8a, the costimulatory factor of 4-1BB, and the signal transduction domain of CD3ζ.

In a preferred embodiment of the present invention, the amino acid sequence of the CAR (the amino acid sequence of the CAR containing an immunomodulatory molecule (such as GITRL)) is shown in SEQ ID NO.: 2 or 3:

The amino acid sequence of PSMA CAR containing immunomodulatory molecules (such as GITRL):

The amino acid sequence of CD19 CAR containing immunomodulatory molecules (such as GITRL):

In a preferred embodiment of the present invention, the nucleotide sequence of the CAR (the nucleotide sequence of the CAR containing an immunomodulatory molecule (such as GITRL)) is shown in SEQ ID NO.: 5 or 6:

The nucleotide sequence of PSMA CAR containing immunomodulatory molecules (such as GITRL):

The nucleotide sequence of CD19 CAR containing immunomodulatory molecules (such as GITRL):

Chimeric antigen receptor T cells (CAR-T cells)

As used herein, the terms "CAR-T cell", "CAR-T" and "CAR-T cell of the present invention" all refer to the CAR-T cell of the present invention. The CAR-T cell of the present invention can target tumor surface antigens. (Such as CD19, PSMA), used to treat tumors with high expression or positive tumor cell surface antigens (such as CD19, PSMA), especially solid tumors.

CAR-T cells have the following advantages over other T cell-based therapies: (1) The action process of CAR-T cells is not restricted by MHC; (2) In view of the fact that many tumor cells express the same tumor antigen, they are targeted at a certain type of tumor. Once the CAR gene construction of the antigen is completed, it can be widely used; (3) CAR can use both tumor protein antigens and glycolipid non-protein antigens, expanding the target range of tumor antigens; (4) using the patient's own body Cells reduce the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.

In the present invention, the CAR of the present invention comprises (i) an extracellular domain, which comprises an antibody single-chain variable region sequence targeting a tumor cell surface antigen; (ii) a transmembrane domain; (iii) a costimulatory factor; and (iv) the signal transduction domain of CD3ζ; and; (v) self-cleaving protein; (vi) immunomodulatory molecules (such as GITRL, 4-1BBL, CD40, LIGHT, B7.1, B7.2, OX40L, CD70) .

Chimeric antigen receptor NK cells (CAR-NK cells)

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

Natural killer (NK) cells are a major type of immune effector cells that protect the body from virus infection and tumor cell invasion through non-antigen-specific ways. The engineered (gene modified) NK cells may acquire new functions, including the ability to specifically recognize tumor antigens and enhanced anti-tumor cytotoxicity.

Compared with autologous CAR-T cells, CAR-NK cells also have the following advantages, for example: (1) They directly kill tumor cells by releasing perforin and granzyme, but have no killing effect on normal cells in the body; (2) They release A small amount of cytokines reduces the risk of cytokine storm; (3) It is easy to expand and develop into "off-the-shelf" products in vitro. Otherwise, it is similar to CAR-T cell therapy.

Foreign T cell antigen receptor

As used herein, the foreign T cell antigen receptor (T cell receptor, TCR) is the cloning of the α chain and β chain of TCR from tumor-reactive T cells through gene transfer technology. Retroviruses are vectors that are transferred exogenously into TCR in T cells.

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

Carrier

The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by using standard Technology to separate directly from the cells and tissues that contain the gene. Alternatively, the gene of interest can 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 to achieve long-term gene transfer because 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 because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

In a brief summary, the expression cassette or nucleic acid sequence of the present invention is usually operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration of eukaryotic cells. A typical cloning vector contains transcription and translation terminators, initial sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

The expression construct of the present invention can also use standard gene delivery protocols for nucleic acid immunization and gene therapy. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety. In another embodiment, the present 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 such vectors, which include, but are 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 can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, 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, retrovirus, adenovirus, adeno-associated virus, herpes virus, and lentivirus. Generally, a suitable vector contains an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers (e.g., WO01/96584; WO01/29058; and U.S. Patent 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 retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. Many adenovirus vectors are known in the art. In one embodiment, a lentiviral vector is used.

Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Generally, these are located in the 30-110 bp region upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible in order to maintain promoter function 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 50 bp before the activity begins to decrease. Depending on the promoter, it appears that individual elements can act 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 to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Ruth sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter , Myosin promoter, heme promoter and creatine kinase promoter. Further, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered part of the invention. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter and tetracycline promoter.

In order to evaluate the expression of the CAR polypeptide or part thereof, the expression vector introduced into the cell may also contain either or both of the selectable marker gene or the reporter gene, so as to facilitate the search for the cell population to be transfected or infected by the viral vector. To identify and select expressing cells. In other aspects, the selectable marker can be carried on a single piece of DNA and used in the co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences so that they can be expressed in the 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. Generally, a reporter gene is a gene that does not exist in or is expressed by a recipient organism or tissue, and it encodes a polypeptide whose expression is clearly indicated by some easily detectable properties such as enzyme activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is measured at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79 -82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, a construct with a minimum of 5 flanking regions that shows the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be linked to a reporter gene and used to evaluate the ability of the reagent to regulate the promoter-driven transcription.

Methods of introducing genes into cells and expressing genes into cells are known in the art. In the content of the expression vector, the vector can be easily introduced into a host cell by any method in the art, for example, a mammalian, bacterial, yeast, or insect cell. For example, the expression vector can be transferred into the 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 so on. Methods of producing cells including vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). The preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used method of inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, adeno-associated virus, and so on. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

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

Where a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. Consider using lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with lipids. Lipid-associated nucleic acids can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, and attached via linking molecules associated with both liposomes and oligonucleotides To liposomes, trapped in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in micelles or Complexed with micelles, or otherwise associated with lipids. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any specific structure in the solution. For example, they may exist in a bilayer structure, as micelles or have a "collapsed" structure. They can also simply be dispersed in the solution, possibly forming aggregates of uneven size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which occur naturally in the cytoplasm and in such compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

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

preparation

The present invention provides a CAR according to the first aspect of the present invention, a nucleic acid molecule according to the second aspect of the present invention, a vector according to the third aspect of the present invention, or a host cell according to the fourth aspect of the present invention, and A pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the preparation is an injection. Preferably, the concentration of the CAR-T cells in the preparation is 1×10 ³ -1×10 ⁸ cells/Kg body weight, more preferably 1×10 ⁶ -1×10 ⁷ cells/Kg body weight.

In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids such as glycine ; Antioxidant; Chelating agent such as EDTA or glutathione; Adjuvant (for example, aluminum hydroxide); and Preservative. The formulations of the invention are preferably formulated for intravenous administration.

Therapeutic application

The present invention includes therapeutic applications with cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of the present invention. The transduced T cells can target tumor cell markers (such as CD19, and/or PSMA) proteins to synergistically activate T cells and cause cellular immune responses, thereby significantly improving their killing efficiency on tumor cells from malignant tumors.

Therefore, the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal, which comprises the following steps: administering the CAR-T cell of the present invention to the mammal.

In one embodiment, the present invention includes a type of cell therapy in which the patient's autologous T cells (or heterologous donors) are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the same patient. In this way, the probability of suffering from graft-versus-host disease is extremely low, and the antigen is recognized by T cells in a non-MHC-restricted manner. In addition, one CAR-T can treat all cancers that express the antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.

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

Although the data disclosed herein specifically discloses antibody single-chain variable region sequences, hinges and transmembrane regions, and 4-1BB and CD3ζ signaling domains, T2A, immunomodulatory molecules (such as GITRL, 4-1BBL, CD40, LIGHT, B7.1, B7.2, OX40L, CD70), but the present invention should be interpreted as including any number of changes to each of the construct components.

Cancers that can be treated include tumors that have not been vascularized or have not been substantially vascularized, as well as vascularized tumors. The cancer may include non-solid tumors (such as hematological tumors such as leukemia and lymphoma) or may include solid tumors. The types of cancer treated with the CAR of the present invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcoma, carcinoma, and melanoma. It also includes adult tumors/cancers and childhood tumors/cancers.

Hematological cancer is cancer of the blood or bone marrow. Examples of hematological (or hematogenic) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, myelomonocytic type , Monocytic and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocyte) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin’s disease, non- Hodgkin's lymphoma (painless and high-grade form), 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 does not usually contain a cyst or fluid area. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell type that formed them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcoma and cancer include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, and ovarian cancer.

The CAR-modified T cells of the present invention can also be used as a type of vaccine for ex vivo immunity 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 before administering the cells into the mammal: i) expanding the cells, ii) introducing the CAR-encoding nucleic acid into the cells, and/or iii) cryopreserving the cells.

In vitro procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from mammals (preferably humans) and genetically modified (ie, transduced or transfected in vitro) with a vector expressing the CAR disclosed herein. CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefits. The mammalian recipient can be a human, and the CAR-modified cell can be autologous relative to the recipient. Alternatively, the cell may be allogeneic, syngeneic, or xenogeneic relative to the recipient.

In addition to the use of 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 present invention provides a method for treating tumors, which comprises administering to a subject in need thereof a therapeutically effective amount of the CAR-modified T cells of the present invention.

The CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and/or with other components or other cytokines or cell populations. Briefly, the pharmaceutical composition of the present invention may include the 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, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelate Mixtures such as EDTA or glutathione; adjuvants (for example, aluminum hydroxide); and preservatives. The composition of the invention is preferably formulated for intravenous administration.

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

When referring to "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by the physician, who considers the patient (subject ) Individual differences in age, weight, tumor size, degree of infection or metastasis, and disease. May generally indicated: including those described herein, the pharmaceutical compositions of T cells may be ¹⁰⁴ to ¹⁰⁹ doses cells / kg body weight, preferably ¹⁰⁵ to ¹⁰⁶ cells / kg body weight doses (including all integers within that range Value) application. The T cell composition can also be administered multiple times at these doses. The cells can be administered by using injection techniques well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a specific patient can be easily determined by those skilled in the medical field by monitoring the patient's signs of disease and adjusting the treatment accordingly.

The administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intracutaneously, intratumorally, intranodal, intraspinal, intramuscular, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by i.v. injection. The composition of T cells can be injected directly into tumors, lymph nodes or sites of infection.

In certain embodiments of the present invention, cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are combined with any number of relevant treatment modalities (e.g., previous , At the same time or after) administration to the patient, the treatment modality includes, but is not limited to, treatment with the following agents: the agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known It is ARA-C) or natalizumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for PML patients. In a further embodiment, the T cells of the present invention can be used in combination with chemotherapy, radiation, immunosuppressants, such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies Or other immunotherapeutics. In a further embodiment, the cell composition of the present invention is administered to bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide (for example, before, simultaneously, or after). patient. For example, in one embodiment, the subject may undergo the standard treatment of high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered before or after surgery.

The dosage of the above treatment administered to the patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio for human administration can be implemented according to the practice accepted in the art. ^{Generally, 1×10 6} to 1×10 ¹⁰ modified T cells of the present invention (for example, CAR-T cells of the present invention) can be injected into each treatment or course of treatment by, for example, intravenous infusion, Apply to the patient.

The main advantages of the present invention include:

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

(2) The present invention found for the first time that CARs expressing immunomodulatory molecules (such as GITRL) can more specifically kill tumor cells, especially tumor cells with high expression or positive CD19 and/or PSMA.

(3) The present invention found for the first time that in CAR-modified T cells or NK cells, expression of exogenous immunomodulatory molecules (such as GITRL) together with CAR can significantly improve tumor suppressor activity and has a synergistic effect.

(4) The present invention developed a new chimeric antigen receptor molecule for the first time. After the CAR molecule is expressed on T cells, it can effectively resist the inhibitory effect of the tumor immune microenvironment, and maintain or improve the effector function of CAR-T cells. Expansion capacity and continuity capacity, in the treatment of solid tumors represented by prostate cancer, show a significantly better therapeutic effect than the existing CAR structure.

(5) The present invention finds for the first time that the expression sequence of tumor necrosis factor receptor (GITR) ligand GITRL induced by glucocorticoids is expressed in CAR molecules and expressed in a fusion manner in a 2A connection. Since activated T cells highly express GITR, the co-expressed GITRL enhances their respective effector functions through the mutual cooperation between CAR-T cells.

(6) The present invention found for the first time that the expression of GITRL can significantly promote the differentiation of Th9 subgroups in CAR-T cells, while inhibiting the formation of regulatory T cells Treg, significantly improve the subgroup composition of CAR-T cells, and show stronger Anti-tumor effect function.

(7) The present invention introduces the important immunomodulatory molecule GITRL into the CAR molecule for the first time, and it is co-expressed with CAR through 2A connection. This molecule promotes the activation of CAR-T cells and enhances the differentiation of CD4+ T cells into Th9, and at the same time inhibits The differentiation and formation of Treg T cells play an important role in overcoming the immunosuppressive microenvironment of solid tumors. It is significantly better than the existing CAR-T technology in the treatment of prostate cancer CAR-T targeting PSMA. The immunotherapy of solid tumors has huge application potential.

(8) The CAR-T cells co-expressing GITRL provided by the present invention have the function of regulating the function of other immune cells expressing its receptor GITR. The immune cells include but are not limited to B cells, macrophages, and natural immune cells. Killer cells, granulocytes, and mast cells can enhance the anti-tumor immune response of patients by comprehensively and multi-directionally regulating the tumor immune microenvironment.

The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing The conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and parts by weight.

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

Example 1: Preparation of GITRL-CART cells and identification of CAR expression

The GITRL-CAR was constructed in the following order (Figure 1): antigen recognition area → junction/transmembrane area → 4-1BB costimulation → CD3ζ → P2A → GITRL coding sequence, and then integrated into the 5 and 3 end LTR of the pELPS lentiviral vector Between the sequences, the CAR expression master plasmid is constructed, followed by lentivirus packaging.

1. Lentivirus packaging

Cell culture before transfection

The day before transfection, digest 293T cells in good growth condition, centrifuge at 1200 rpm for 3 min, and discard the supernatant. Add culture medium to resuspend the cells, so that the cell suspension is evenly inoculated into a 10 cm cell culture dish containing 9ml of DMEM complete medium, and the culture dish is gently shaken to make the cells evenly distributed in the culture dish. Cultivate in a 37°C, 5% CO2 incubator. Regularly observe the growth density and state of the cells, and the cell fusion rate can reach about 70% to 80% before being used for transfection. The subculture of 293T cells used for transfection should not exceed 24 hours.

Transfection

1) Dilute the three plasmids (psPAX2, pMD2.G and the target plasmid integrated with CAR-GITRL expression cassette), add psPAX25 μg, pMD2.G3 μg and the target plasmid 5 μg to 500 μL serum-free medium DMEM, and mix gently. Incubate at room temperature for 5 minutes;

2) Dilute the transfection reagent PEI, add 50 μL of PEI to 450 μL of serum-free DMED medium, and mix gently. Incubate at room temperature for 5 minutes;

3) Add the incubated PEI diluent dropwise to the diluted three plasmids, and gently pipette to mix. Incubate at room temperature for 20 minutes, prepare a three-plasmid-PEI mixture, and label according to the different plasmids added;

4) The plasmid mixture is evenly added dropwise to the 293T cells used for transfection, and cultured in an incubator at 37°C and 5% CO2. After culturing for 6-8 hours, the cells were replaced with fresh complete medium DMEM (denoted as transfection 0h). Continue to incubate at 37°C and 5% CO2.

Harvest lentivirus

After 48 hours of transfection, the cell supernatants were collected into clean 50ml centrifuge tubes, labeled, and 10mL complete medium DMEM was added to the petri dish, and the culture was continued at 37°C and 5% CO2. After 72 hours of transfection, the cell culture supernatant was collected again, and the collected virus stock solution was placed in a 250 ml centrifuge tube. In the process of continuing the culture, regularly observe the cell growth state to ensure the cell toxin production efficiency.

Concentrated and purified lentivirus

1) Ultrafiltration: Add the collected virus stock to a 100KD ultrafiltration tube, balance it and centrifuge at 4000rpm for 30 minutes.

2) Ultra-isolation: The virus concentrate after ultrafiltration is transferred to a sterilized quick-sealing ultracentrifuge tube, and after strict trimming, it is centrifuged at 40000 rpm and 4 degrees Celsius for 3 hours.

3) Purification: After the virus liquid is centrifuged, discard the waste liquid, add 1-3 ml of blank X-vivo medium to the ultracentrifuge tube, concentrate the virus about 100-300 times, and pipette until the virus precipitate is completely dissolved. Dispense 250ul each tube into a sterile EP tube and store in an ultra-low temperature refrigerator at -80 degrees Celsius.

Lentivirus titer determination

Take HEK-293T cells in good growth condition, count them after digestion, spread them evenly into a 24-well plate according to 2*10^5 wells, culture for about 8 hours until the cells adhere to the wall, press 3 times into the cell culture supernatant of the 24-well plate The dilution gradient was added with different volumes of virus concentrates, a total of 8 gradients were set, and the infection was carried out in the incubator for 48 hours. After 48 hours of infection, the cells were digested and collected.

Percentage of positive cells in flow cytometry

1) Collect about 1*10 ⁵ infected 293T cells and place them in a clean 1.5ml EP tube, rinse twice with PBS, 1200rpm, and centrifuge for 3min;

2) Discard the supernatant, add 100μL of flow loading buffer, add 0.6μl of PSMA Protein-Human, Recombinant (His Tag), the protein concentration is (0.15mg/mL), mix by pipetting, and incubate at 4℃ for 45min in the dark

3) Rinse twice with PBS, 1200rpm, centrifuge for 3min;

4) Discard the supernatant, add 100 μL of flow cytometry loading buffer to resuspend the cell pellet, and add 0.6 ul of APC-Anti-Streptavidin flow cytometry antibody. Incubate in the dark at 4°C for 30min;

5) Rinse twice with PBS, 1200rpm, centrifuge for 3min;

6) Discard the supernatant and add 300μL of flow loading buffer to resuspend the cell pellet

7) Streaming test on the machine.

And calculate the virus titer according to the following formula:

Titer=number of cells at the time of plating*percentage of positive cells*1000/volume of virus solution added (μL)TU/mL.

2. GITRL-CART cell preparation and flow cytometric identification

Sorting and activation of T cells

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

1) Strictly disinfect the blood collection bag and interface with 75% alcohol and transfer it to the biological safety cabinet. Use sterile scissors to cut the blood collection bag, transfer the blood sample to a clean 50mL centrifuge tube, and mark it;

2) Take a clean 50mL centrifuge tube, add 15mL lymphocyte separation solution, and make a mark;

3) Slowly add 30mL blood sample to the above centrifuge tube. Note that the action should be as slow as possible when adding to keep the interface clear. Centrifuge at 800g for 25min, and slowly rise and fall during centrifugation, with the speed rising to 1 and the speed falling to 0;

4) After centrifugation, use a pipette to carefully draw the middle buffy coat layer into a new 50mL centrifuge tube, mark it, and be careful not to draw impurities as much as possible, add PBS to 50mL, gently pipette evenly, and centrifuge at 500g for 10 minutes;

5) After centrifugation, discard the supernatant, resuspend the cell pellet with 50 mL PBS, blow well, and read the number of WBCs with a blood cell analyzer;

6) Cell centrifugation, 300g centrifugation for 10 minutes;

7) After centrifugation, discard the supernatant. According to the end result of the counter ^{, add 70μl buffer (x-vivo basal medium + 10% FBS) to every 10 7} cells to resuspend the cell pellet, gently pipette to mix, and transfer to a clean 15mL centrifuge tube. mark;

8) The number of cells, ¹⁰⁷ cells per 20μl added to each of the CD4 + and CD8 + magnetic beads, incubated for 15min 4 ℃;

9) After the incubation is over, add buffer to the cell suspension to 15mL, make a mark, gently pipette to mix, wash the cells, and centrifuge at 1200g for 5min;

10) Discard the supernatant after centrifugation and add 500μL of buffer to resuspend the cells;

11) Put the separation column into the magnetic field, connect a clean 15mL heart tube to the lower end, rinse the separation column with 3mL buffer solution, slowly add the cell suspension to the separation column in batches, let the liquid drop slowly, and wait until the cell suspension level approaches Add 3mL buffer solution to the upper end of the magnetic column and rinse twice;

12) After washing, remove the column from the magnetic field, add 5 mL of buffer solution, quickly push the cells into a new 15 mL centrifuge tube with a piston, mark them, and centrifuge at 300 g for 10 minutes;

13) After centrifugation, discard the supernatant, add 5 mL of X-vivo complete medium, and count;

14) According to the counting results of the cell counter, resuspend the cells in X-vivo complete medium containing 0.1% IL-2 and 1% CD3 and CD28 complexes, adjust the cell density to 1×10 ⁶ cells/mL, and inoculate them on T75 In the culture flask, gently pipette to mix, and put it in the incubator for culture;

15) After culturing for 48 hours, suck the cells into a clean 50 mL centrifuge tube, centrifuge at 1200 rpm for 3 min.

T cell infection

The GITRL virus concentrate obtained in the above experiment was used to infect the isolated primary T cells at MOI=50, centrifuged for 12 hours after infection, and continued to culture for 48 hours. The positively infected T cells were the target CAR-T cells.

Collect the cells and detect the percentage of CAR-T cells by flow cytometry

1) Collect 1*10 ⁵ infected T cells and place them in a clean 1.5ml EP tube, rinse twice with PBS, 1200rpm, and centrifuge for 3min;

2) Discard the supernatant, add 100μL flow loading buffer, add 0.6μl PSMA Protein, Human, Recombinant (His Tag), the protein concentration is (0.15mg/mL), and add 1ul PE-anti-human GITRL antibody at the same time , Mix by pipetting, and incubate at 4°C for 45min in the dark;

3) Rinse twice with PBS, 1200rpm, centrifuge for 3min;

4) Discard the supernatant, add 100 μL of flow loading buffer to resuspend the cell pellet, and add 0.6 μl of APC-Anti-Streptavidin flow cytometry antibody. Incubate in the dark at 4°C for 30min;

5) Rinse twice with PBS, 1200rpm, centrifuge for 3min;

7) Flow cytometry to detect the expression of PSMA-CAR and GITRL (Figure 2).

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

Co-culture

1. Take each group of GITRL-CAR-T, control CART cells and target cell PC-3 that are in good growth condition, and count them after centrifugation. According to the difference in the positive rate of CAR-T cells in each group, add T cells to CAR-T to adjust to The positive rate of CAR-T cells in each group remained consistent.

2. Adjust the target cell density to 2*10^5 cells/mL, according to the purpose of the experiment, design different effective target ratios, adjust the density of T cells and CAR-T cells in each group, the co-culture experiment can refer to the following table.

3. According to the ratio of effector cell: target cell (efficiency target ratio), calculate the number of effector cells required per well, add it to a low-adsorption 96-well plate, and make three replicate wells for each effect target ratio to make the system 150 microliters. system. The co-cultivation experiment has a target ratio of 2:1 as shown in Table 1.

Table 1

4. After each group has added the required number of cells, put it in the incubator for the corresponding time (12 hours, 16 hours, 20 hours, 24 hours, etc.).

5. After pipetting and mixing the cells corresponding to each well in each well of the 96-well plate, suck 100μL of cell suspension and transfer it to a new 96-well fluorescence microtiter plate.

6. Add 10 μL of luciferase substrate to each well.

7. Determination of fluorescence reading.

8. Killing 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%.

Multiple rounds of kill detection

1) The cell co-cultivation method refers to the method of detecting a round of killing, and the effective target ratio = 0.5:1. At the same time, three identical 96-well plates (one is an ultra-low adsorption 96-well plate, and the other two are normal plates). The wall 96-well plates are numbered as plate one, plate two, plate three)

2) After 24 hours of incubation, take the plate out and calculate the killing rate of control CAR-T cells and GITRL-CART cells to target cells according to the cell luciferase killing assay method, that is, one round of killing rate

3) Transfer the suspended cells in plate two to another new ultra-low adsorption 96-well plate (numbered plate four), and then add PC-3 cells to the corresponding wells according to the 0.5:1 efficiency-to-target ratio. Transfer the suspended cells in the same way to a normally adherent 96-well plate (numbered plate five), and add the corresponding target cells

4) After 24 hours of co-cultivation, calculate the killing rate of the target cells in accordance with the cell luciferase killing assay method for the cells in plate 4, which is the second round of killing rate

5) Transfer all the suspended cells in the corresponding wells of plate five to another new ultra-low adsorption 96-well plate (numbered plate 6), and add PC-3 cells

6) After 24 hours of co-cultivation, the cells in the plate 6 were killed according to the cell luciferase killing assay method to calculate the killing rate to the target cells, which is the three-round killing rate.

GITRL-CART cell background expansion

The control CART and GITRL-CART cells obtained by culturing for two days after infection with the corresponding virus and the T cells obtained from the same batch were cultured strictly in accordance with the same culture conditions (x-vivo medium + 10% FBS + 1% P /S+10μg/mL IL-2).

1) Adjust the initial cell density to 1*10^6 cells/mL, make three replicate holes, and adjust the initial number of cells to 2*10^6 cells

2) According to the cell growth status, timely replenish or change the cell fluid to ensure that the cells grow in a good nutrient environment

3) Every two days, the cells are centrifuged and then resuspended and mixed to count (cell counter), record, graph, and trace the cell growth curve.

GITRL-CART cells are incubated with target cells and then expanded

1) Adjust target cells PC-3 cells and control T cells, control CART and GITRL-CART cells according to the condition of effective target ratio=2:1. The numbers of target cells and effector cells are 1*10^6 and 2*10^, respectively. 6 pieces, add X-VIVO complete medium and put them in a six-well plate for culture

2) After 24 hours of co-incubation, centrifuge the suspended cells in the wells and count them, and then transfer them to a new culture plate for culture

3) According to the cell growth status, timely replenish or change the cell fluid to ensure that the cells grow in a good nutrient environment

Every two days, the cells were centrifuged and resuspended and mixed and counted (cytometer), recorded, graphed, and depicted the cell growth curve (Figure 3).

Example 3: Detection of IL-9 cytokine expression after GITRL-CART cells and target cells are co-cultured

Enzyme-linked immunosorbent assay (ELISA)

Transfer the cell supernatant from control T, control CART and GITRL-CART cells to PC-3 target cells after 24 hours of co-cultivation with PC-3 target cells, transfer to a new centrifuge tube, centrifuge at 4000 rpm for 10 minutes, remove cell debris, and transfer to a new centrifuge tube Medium, aliquot and store at -80°C.

1) Before use, mix all reagents thoroughly to avoid foaming.

2) Determine the number of slats required according to the number of experimental holes (blank and standard). Samples (including standards) and blanks should be made three replicates.

3) Coating: 100μL/well add diluted coating antibody (4℃ overnight)

4) Wash the plate: deduct the liquid in the well, add 300μL/well to 1×Washing buffer working solution; leave it for 1 minute and discard the liquid in the well. Repeat 3 times, buckle dry on the filter paper each time.

5) Add sample: add 100μL/well of diluted IL-9 standard to the standard well, 100/well to add sample to the sample well, and 100μL/well to add Dilutation buffer (1×) to the blank control well. Cover with sealing film and incubate at room temperature (18-25°C) for 2 hours.

6) Wash plate: Repeat step 4.

7) Add detection antibody: 100μL/well is added to the detection antibody working solution. Cover with sealing film and incubate at room temperature (18-25°C) for 1 hour.

8) Wash plate: Repeat step 4.

9) Add enzyme: add 100μL/well to Streptavidin-HRP working solution. Cover with sealing film and incubate at room temperature (18-25°C) for 30 minutes.

10) Wash the plate: Repeat step 4.

11) Color development: 100μL/well was added to TMB, incubated at room temperature (18-25°C) in the dark for 5-30 minutes, and the reaction was terminated according to the color depth (dark blue) in the well. Usually 10-20 minutes of color development can achieve good results.

12) Stop the reaction: 100μL/well is quickly added to Stop solution to stop the reaction.

13) Reading the plate: within 10 minutes after termination, use the detection wavelength of 450nm to read the value.

Flow cytometry

1) Collect 1*10 ⁵ co-incubated cells in each tube and place them in a clean 1.5ml EP tube, rinse twice with PBS, 1200rpm, and centrifuge for 3min;

2) Discard the supernatant, add 100 μL of rupture membrane fixative, blow gently, and incubate for 2 hours at 4°C in the dark;

3) Rinse twice with PBS, centrifuge at 1200rpm for 3min;

4) Add 0.6ul of PE-anti-human IL-9 and 1ul of APC-anti-human CD4 antibody in sequence, mix by pipetting, and incubate for 45 minutes at 4°C in the dark;

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

6) Rinse twice with PBS, 1200rpm, centrifuge for 3min;

7) Discard the supernatant and add 300 μL of flow loading buffer to resuspend the cell pellet;

8) Flow cytometry to detect the co-expression of IL-9 and CD4 (Figure 4).

Example 4: GITRL-CART cell exhaustion, index detection

Co-culture

1) Take control T cells, control CART, GITRL-CART cells and target cell PC-3, count them after centrifugation. According to the difference in the positive rate of various CAR-T cells, add T cells to CAR-T to adjust to various CAR-T cells. The positive rate of T cells remains the same.

2) According to the ratio of efficiency to target = 1:1, add 1*10^6 effector cells and target cells to the wells of the six-well plate. Make three replicate wells for each CAR-T, and adjust the initial cell density. It is 1*10^6/mL.

3) After 24 hours of co-incubation, transfer various CAR-T cells to a new culture plate for culture.

4) After 7 days of normal culture, perform PD-1 and TIM3 flow cytometry staining.

Flow cytometry marker for GITRL-CART cell depletion

1) Collect about 3*10^5 cells in a clean 1.5ml EP tube, rinse twice with PBS, 1200rpm, and centrifuge for 3min;

2) Discard the supernatant, add 100μL of loading buffer, add 0.6μl of APC-anti-human PD-1, PE-anti-human TIM3 flow cytometry antibody in turn, pipette to mix, and incubate at 4°C for 45min in the dark;

3) Rinse twice with PBS, 1200rpm, centrifuge for 3min;

4) Discard the supernatant and add 300μL of flow loading buffer to resuspend the cell pellet

5) Streaming on-board testing.

6) Flowjo flow analysis software analyzes the expression of PD-1 and TIM3 after co-cultivation of various GITRL-CART cells and PC-3 (Figure 5).

Example 5: GITRL-CART animal function evaluation in vivo

The evaluation tool mice used in this example are 6-8 week old NSG mice, raised in an SPF-class laminar flow room, standard pellets, litter and other items related to mice are sterilized. Carry out the in vivo function evaluation of GITRL-CART according to the following steps.

1) Prepare 1640 complete medium, digest and count the PC-3-luciferase target cells in the culture, and adjust the cell concentration to 2*10^7/mL (cells are resuspended in sterile PBS+ Matrigel=3:1).

2) Select 6-8 weeks old NSG mice, each mouse is intraperitoneally injected with 400 μL of sodium pentobarbital to anesthetize the mice, and remove the hair on the back of the mice with a razor.

3) Wipe and disinfect the right back of the mouse with an alcohol cotton ball, then use a 1mL syringe to slowly inject 100μL (2*10^6 cells) of PC-3-luciferase cell suspension into the right back subcutaneously, stop the needle for a few seconds Withdraw the needle. When the mouse is about to wake up, return the mouse to the cage.

4) After the injection, use medical cotton to stop the bleeding.

5) Cut the toes of the mice, number them sequentially, and continue to raise them.

6) Fifteen days after the target cell injection, use the in vivo imager to perform in vivo imaging of the mice to observe the tumor growth, and eliminate the unsuccessful mouse models based on the imaging results. The remaining mice are randomly grouped according to the experimental arrangement and marked .

Mouse in vivo imaging IVIS (Figure 6)

Weigh the substrate D-Luciferin potassium salt, dissolve it in PBS, store it in the dark, and enter the SPF-level system through the delivery window. The substrate was injected intraperitoneally according to the mouse body weight of 3m/each, and the mice were subjected to isoflurane anesthesia after 3 minutes. After the mice are anesthetized, they are put into the imager for imaging and taking pictures, the pictures are counted and processed, and the fluorescence values read are recorded at the same time.

Mouse tail vein injection of CAR-T effector cells

1) According to the grouping, before the effector CAR-T cells are injected, the mice will be imaged in vivo again, and the mouse body weight will be included.

2) Prepare Mock T, control CART and GITRL-CART cell suspensions, adjust the cell density to 5*10^7 cells/mL with physiological saline, distribute them into EP tubes, put them in an ice box, and set aside.

3) Inject the corresponding CAR-T effector cells into the tail vein, each injection of 100μL, and make a label.

4) In-vivo imaging detection, recording and statistical data are carried out every 3-4 days.

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

A chimeric antigen receptor CAR, characterized in that the chimeric antigen receptor CAR comprises: 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 the chimeric antigen receptor CAR also includes: an immunomodulatory molecule that is connected to the intracellular domain and can be co-expressed.
The chimeric antigen receptor CAR of claim 1, wherein the immunomodulatory molecule is selected from the group consisting of GITRL, 4-1BBL, CD40, LIGHT, B7.1, B7.2, OX40L, CD70, Or a combination.
The chimeric antigen receptor CAR of claim 1, wherein the structure of the CAR is shown in the following formula I:

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

Where

Each "-" is independently a connecting peptide or a peptide bond;

Z1 is no or signal peptide sequence;

T is an antibody single-chain variable region sequence targeting tumor cell surface antigen;

H is no or hinge area;

TM is the transmembrane domain;

C is a costimulatory signal molecule;

Z2 is the cytoplasmic signal transduction sequence derived from CD3ζ;

Z3 is a self-cleaving protein;

P is an immunomodulatory molecule;

m is 1, 2, 3, or 4.
A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the chimeric antigen receptor (CAR) according to claim 1.
A vector, characterized in that the vector contains the nucleic acid molecule of claim 4.
A host cell, characterized in that the host cell contains the vector of claim 5 or the nucleic acid molecule of claim 4 integrated into the chromosome.
A method for preparing engineered immune cells, characterized in that the engineered immune cells express the CAR according to claim 1, wherein the method comprises the step of: converting the nucleic acid molecule according to claim 4 or claim 5 The vector is transferred into immune cells to obtain the engineered immune cells.
A pharmaceutical composition, characterized in that the pharmaceutical composition contains 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.
A 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, which It is characterized in that it is used to prepare drugs or preparations for killing tumor cells.
A kit for killing tumor cells, characterized in that the kit contains a container, and the CAR according to claim 1, the nucleic acid molecule according to claim 4, and the CAR according to claim 5 in the container. A vector, or the host cell of claim 6.