CN116836299B

CN116836299B - Chimeric antigen receptor and uses thereof

Info

Publication number: CN116836299B
Application number: CN202310687031.8A
Authority: CN
Inventors: 胡渊; 张彩; 陈敏华; 王烃; 谢思奇
Original assignee: Shanghai Enkai Cell Technology Co ltd
Current assignee: Shanghai Enkai Cell Technology Co ltd
Priority date: 2023-06-09
Filing date: 2023-06-09
Publication date: 2024-07-09
Anticipated expiration: 2043-06-09
Also published as: CN116836299A

Abstract

The invention provides a chimeric antigen receptor and uses thereof. The chimeric antigen receptor comprises: a first fragment, said first fragment being a NKG2D full-length sequence; a second fragment, wherein the first fragment is a full-length sequence of DAP10, and the first fragment is connected with the second fragment; an intracellular signal transduction domain, the N-terminus of the intracellular signal transduction domain being linked to the C-terminus of the first fragment or the second fragment. The chimeric antigen receptor disclosed by the invention can recognize multiple NKG2D ligands, immune cells expressing the chimeric antigen receptor disclosed by the invention can recognize and kill multiple tumors expressing the NKG2D ligands in a targeted manner, can be used for developing universal immune cell products, and realizes treatment of multiple tumor indications by a CAR-T or CAR-NK product, and has a good clinical application prospect.

Description

Chimeric antigen receptor and uses thereof

Technical Field

The invention relates to the field of biopharmaceuticals, in particular to a chimeric antigen receptor and application thereof, and in particular relates to a chimeric antigen receptor capable of recognizing NKG2D multiple ligands, and corresponding nucleic acid molecules, expression vectors, lentiviral vectors, transgenic immune cells, pharmaceutical compositions and application thereof.

Background

In recent years, chimeric antigen receptor T (CHIMERIC ANTIGEN receptor T, CAR-T) cells have achieved tremendous success in hematological malignancy therapy. CAR-T cells still present a number of challenges in clinical applications, for example, exhibiting low efficacy in the treatment of solid tumors; tumors have off-target effects and drug resistance through immune escape mechanisms that lose antigen. CAR-T cells require autologous adoptive cell transplantation, otherwise allogeneic T cells may lead to Graft Versus Host Disease (GVHD); is easy to produce adverse reactions such as cytokine storm, neurotoxicity and the like, and causes harm to the life of patients. Recent studies indicate that CAR-NK cells may overcome the above-mentioned drawbacks of CAR-T cells and show a significant anti-tumor effect.

In vivo application of the CAR-NK cells generally does not cause cytokine storm, the NK cells do not need strict HLA matching, and the potential of GVHD is not caused; NK cells do not need antigen presentation and are not limited by MHC, so that the NK cells can play a role in directly killing tumor cells; the CAR-NK cells can accurately identify tumors through the installed 'CAR', can also identify corresponding ligand activation signals of the tumor cells through the activation receptor expressed by the CAR-NK cells, kills the tumors in a CAR independent mode, has a broad anti-tumor spectrum, and can prevent the tumors from losing through antigen or MHC molecules to generate off-target effect and immune escape. The CAR-NK cells may also be used in combination with antibodies, whereby the fcγrii (CD 16) on their surface binds to the Fc-segment of the antibody, thereby amplifying the anti-tumor efficacy of the CAR-NK cells by means of antibody-dependent cellular cytotoxicity (ADCC). Therefore, the CAR-NK cells have wide application prospect in anti-tumor treatment and become hot spots in the field of development of cellular immunotherapy.

Traditional CAR-T or CAR-NK technologies mostly target recognition of tumors by expressing single chain antibodies (ScFv) that specifically recognize specific tumor antigens, initiate activation signals of intracellular segments, mediating killing of tumor cells. However, traditional ScFv recognizes a tumor single antigen, off-target effects and therapeutic drug resistance or relapse may occur when the tumor antigen is lost, limiting further applications of CAR-T or CAR-NK technology.

The complex immunosuppressive microenvironment within a solid tumor is also one of the limiting factors that severely affect the anti-tumor effect of CAR-T or CAR-NK cells. Myeloid-derived suppressor cells (Myeloid-derived suppressor cells, MDSCs) are a population of myeloid-derived suppressor cells that suppress the anti-tumor effects of immune cells in the body. Studies have shown that MDSCs down-regulate the expression of NKG2D by membrane binding TGF-beta, thereby inhibiting the function of NK cells. MDSCs can also induce Treg cell expansion, promoting its negative effects on immunity. In addition, MDSCs suppress T cell immune responses and proliferation by, for example, generating reactive oxygen species. Therefore, how to overcome the immunosuppression of MDSCs on immune cells, particularly CAR-T or CAR-NK cells for feedback therapy, within the tumor microenvironment has become a research and development hotspot that breaks through the bottleneck of solid tumor immunotherapy.

Thus, there remains a need to develop new immune cell modification techniques to further enhance the therapeutic effect of CAR-T or CAR-NK.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. Therefore, the chimeric antigen receptor provided by the invention can recognize multiple NKG2D ligands, and the immune cells modified by the chimeric antigen receptor not only can target and recognize and kill multiple tumors expressing the NKG2D ligands, but also can obviously improve the killing efficiency of MDSCs, further eliminate the negative regulation effect of the MDSCs on the immune cells, reverse the tumor immunosuppression microenvironment and ensure that the immune cells can play an anti-tumor role more effectively in organisms. The chimeric antigen receptor can be used for developing a universal immune cell product, realizes the treatment of various tumor indications by a CAR-T or CAR-NK product, and has good clinical application prospect.

The present invention has been completed based on the following work of the inventors:

NK cell receptors have the characteristic of broad specific recognition, and the ligands recognized by their activating receptors are expressed in most blood system tumors and solid tumors, but are hardly expressed in normal cells. Therefore, NK cell receptor is selected as antigen binding domain of chimeric antigen receptor, and immune cells are further modified by the chimeric antigen receptor gene, so that the obtained immune cells may have wider tumor recognition spectrum.

In order to obtain the CAR-immune cells with improved performance, the inventor creatively selects the full-length sequence of NKG2D and the full-length sequence of DAP10 to construct a chimeric antigen receptor structure capable of widely combining with various ligands of NKG 2D; meanwhile, the intracellular segment of the DAP10 molecule is ingeniously used as a chimeric antigen receptor intracellular region activating sequence to exert the effect of a co-stimulatory domain. Based on this, the inventors have obtained a chimeric antigen receptor that can widely recognize NKG2D ligands.

Further test results show that: immune cells (such as NK cells) modified by the chimeric antigen receptor gene can recognize various ligands of NKG2D, target and recognize and kill various hematological tumors and solid tumor cells expressing the NKG2D ligands, and have the characteristics of a general CAR-immune cell product.

MDSCs interact with immune cells (e.g., T cells, NK cells, dendritic cells, macrophages, etc.) and suppress the immune response of the body. In liver cancer mice, MDSCs cells are able to inhibit the expression of NK cell surface receptor NKG2D and IFN- γ production, inducing NK cell disability. The inventors have unexpectedly found that immune cells (e.g., NK cells) modified with the chimeric antigen receptor genes of the invention can not only target killing NKG2D ligand positive tumor cells, but also effectively kill MDSCs. In a mouse model of tumor-bearing human liver cancer H7402 cells containing MDSCs, tail vein reinfusion immune cells or treatment by chimeric antigen receptor immune cells of the invention, the results show that: the chimeric antigen receptor immune cells can effectively eliminate the negative regulation MDSCs cells which play a role in inhibiting immune effector cells in tumor microenvironment, so that the immune cells in the tumor microenvironment are saved from being inhibited by the MDSCs, the immune cells (including the immune cells and the immune cells which are not subjected to genetic modification) can play an anti-tumor role more effectively, and the clinical curative effect of immune cell products is further improved.

Thus, in a first aspect of the invention, the invention provides a chimeric antigen receptor. The chimeric antigen receptor comprises: a first fragment, said first fragment being a NKG2D full-length sequence; a second fragment, wherein the first fragment is a full-length sequence of DAP10, and the first fragment is connected with the second fragment; an intracellular signal transduction domain, the N-terminus of the intracellular signal transduction domain being linked to the C-terminus of the first fragment or the second fragment. The chimeric antigen receptor of the invention has stronger and wider NKG2D ligand binding activity, and immune cells expressing the chimeric antigen receptor of the invention can effectively target and identify and kill various blood tumor and solid tumor cells expressing the NKG2D ligand, and has the characteristics of universal CAR-immune cell products.

In a second aspect of the invention, the invention provides a nucleic acid molecule. The nucleic acid molecule comprises a first nucleic acid molecule encoding the chimeric antigen receptor of the first aspect of the invention. The immune cells carrying the nucleic acid molecules express the chimeric antigen receptor of the first aspect of the invention, and the chimeric antigen receptor can further recognize and bind with various ligands of NKG2D, thereby realizing the targeted recognition of various blood tumor and solid tumor cells expressing the NKG2D ligands by the immune cells and further exerting the killing activity thereof.

In a third aspect of the invention, the invention provides an expression vector. The expression vector carries the nucleic acid molecule of the second aspect of the invention. Thus, the chimeric antigen receptor of the first aspect of the present invention is expressed in cells using the constructed expression vector, and is efficiently expressed and obtained.

In a fourth aspect of the invention, the invention provides a transgenic immune cell. The transgenic immune cell expresses the chimeric antigen receptor of the first aspect of the invention, or carries the nucleic acid molecule of the second aspect of the invention or the expression vector of the third aspect of the invention. The transgenic immune cell can be widely used for targeted recognition and killing of various solid tumors or hematological tumors expressing NKG2D ligands, can be used for effectively killing immunosuppressive cells MDSCs in tumor microenvironment, is a universal immune cell product, has good clinical curative effect, can be used for treating various tumor diseases, and has good clinical application prospect.

In a fifth aspect of the invention, the invention provides a pharmaceutical composition. The pharmaceutical composition comprises the chimeric antigen receptor of the first aspect of the invention, the nucleic acid molecule of the second aspect of the invention, the expression vector of the third aspect of the invention or the transgenic immune cell of the fourth aspect of the invention. The obtained pharmaceutical composition can be further used for preventing or treating various tumor diseases.

In a sixth aspect of the invention, the invention provides a use for preparing a medicament. Use of a chimeric antigen receptor of the first aspect of the invention, a nucleic acid molecule of the second aspect of the invention, an expression vector of the third aspect of the invention, a transgenic immune cell of the fourth aspect of the invention or a pharmaceutical composition of the fifth aspect of the invention in the manufacture of a medicament for the prevention or treatment of a tumor. The chimeric antigen receptor and the corresponding nucleic acid molecules, expression vectors, transgenic immune cells or pharmaceutical compositions can be further prepared into medicines which can be clinically used for preventing or treating diseases.

Those skilled in the art will appreciate that the features and advantages described above for chimeric antigen receptors, nucleic acid molecules, expression vectors, lentiviral vectors, transgenic immune cells and pharmaceutical compositions are equally applicable for this use and will not be described in detail herein.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 is a schematic diagram showing the structure of the genetic elements of the chimeric antigen receptor of example 1 of the present invention;

FIG. 2 is a graph showing the measurement of the expression of MICA/B of the NKG 2D-ligand of the human hepatoma cell line, the breast cancer cell line and the non-small cell lung cancer cell line according to example 2 of the present invention, wherein H7402, huh7 and SMMC-7721 are human hepatoma cell lines, MCF-7 is human breast cancer cell line and A594 is human non-small cell lung cancer cell line;

FIG. 3 is a graph showing the results of examining the in vitro killing activity of the CAR-NK92 cells of the example 2 of the present invention;

FIG. 4 is a graph showing the results of examining the expression levels of CD107a, performin and Granzyme B of the CAR-NK92 cells of example 2 of the present invention;

FIG. 5 is a graph showing the results of examining IFN-. Gamma.secretion levels from CAR-NK92 cells of example 2 of the present invention;

FIG. 6 is a graph showing the results of examining the survival rate of CAR-NK92 cells in example 2 of the present invention;

FIG. 7 is a graph of proliferation of CAR-NK92 cells of example 2 of the present invention, the abscissa indicates the time of cell culture in days;

FIG. 8 is a graph showing the detection of the expression of the NKG 2D-ligands MICA/B and ULBP2/5/6 of the human ovarian cancer cell lines SKOV3 and A1847 of example 3 of the present invention;

FIG. 9 is a graph showing the tumor inhibition effect of the CAR-NK92 cells of the embodiment 3 on a human ovarian cancer SKOV3 cell tumor-bearing mouse model;

FIG. 10 is a graph showing the tumor-inhibiting effect of the CAR-NK92 cells of example 3 of the present invention on a mouse model with tumor cells of human ovarian cancer A1847;

FIG. 11 is a graph showing the tumor-inhibiting effect of peripheral blood derived CAR-NK cells of example 3 of the present invention on a mouse model with tumor cells of human pancreatic cancer HPAF-II, wherein PBS represents PBS group, NK represents a control group of NK cells without genetic modification, and CAR-NK-4E6, CAR-NK-8E6, and CAR-NK-16E6 represent a treatment group of CAR-NK cells with injection doses of 4X 10 ⁶、8×10⁶ and 1.6X10 ⁷ CAR-NK cells/mouse, respectively;

FIG. 12 is a graph of the results of examining the killing activity of MDSCs by CAR-NK92 cells of example 4 of the present invention;

FIG. 13 is a graph showing the tumor-inhibiting effect of the CAR-NK92 cells of example 4 of the present invention on tumor-bearing mice model of human liver cancer H7402 cells containing MDSCs.

Detailed Description

Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.

It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

Terms and definitions

In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.

In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.

As used herein, the term "LTR", which is equivalent to "long TERMINAL REPEATED", and equivalent to "long terminal repeat", refers to a retrovirus having one long terminal repeat (5 '-LTR and 3' -LTR) at each end of its genome. It does not code protein, but contains regulatory elements of promoter and enhancer, etc., LTR in virus genome can be transferred to adjacent place of cell protooncogene, so that these protooncogenes can be activated under the action of LTR strong promoter and enhancer, and can convert normal cell into cancer cell.

As used herein, "super IL-15" is a cytokine that promotes T cell and NK cell survival, proliferation, and is a fusion protein, including IL-15Rα and IL-15.IL-15 and IL-2 share IL-2/15Rβγc receptor, IL-15 and IL-15Rα form dimer and then combine with IL-15Rβγc to activate downstream JAK1/JAK3 and STAT3/STAT5 signal channels, thereby promoting proliferation and activation of NK cells. At present, cytokines such as IL-2, IL-15 and the like are often used clinically to prolong the survival time of NK cells in vivo, but the cytokine support treatment can also bring corresponding side effects. Systemic IL-2 therapy has been shown to induce activation of Treg cells and to bring about severe vascular leak syndrome and neurotoxicity. Whereas systemic administration of IL-15 treatment affects mainly NK cells, γδ T cells and CD8 memory T cells, IL-15 causes symptoms such as hypotension and thrombocytopenia in a dose dependent manner and leads to neutropenia. Thus, IL-15 is a safer option than IL-2, but the systemic toxicity from IL-15 is still not negligible.

Herein, the term "single chain antibody", equivalent to "SINGLE CHAIN FV", equivalent to "scFv", is a small molecule antibody formed by joining an immunoglobulin heavy chain variable region (V _H) and a light chain variable region (V _L) via a linker peptide. The linker peptides used to make scFv must be flexible enough to ensure that V _H and V _L are free to fold, giving the antibody binding region the correct configuration.

In this context, the term "(G ₄S)_n" is identical to "(Gly ₄Ser)_n" means that 4 glycine and 1 serine are repeated N times, and is a class of connecting peptides which are widely used at present, and can be located between the C-terminus of V _H and the N-terminus of V _L, and also between the C-terminus of V _L and the N-terminus of V _H) (G ₄S)₃ is currently commonly used, wherein glycine is the amino acid with the smallest molecular mass and the shortest side chain, which can increase the flexibility of the side chain, serine is the amino acid with the strongest hydrophilicity, which can increase the hydrophilicity of the connecting peptide, (G ₄S)₃ has better stability and vitality).

As used herein, the term "linker" refers to a gap junction basic structural unit, typically consisting of six identical or similar transmembrane proteins, and a gap junction basic structural unit consisting of six identical or similar transmembrane proteins. In some specific cases, this includes but is not limited to "2A peptide". The 2A peptide is a short peptide (about 18-25 amino acids) of viral origin, commonly referred to as a "self-cleaving" peptide, which allows the production of multiple proteins from a single transcript. The 2A peptide does not completely "self-cleave" but rather acts by allowing the ribosome to skip synthesis of glycine and proline peptide bonds at the C-terminus of the 2A element, ultimately resulting in separation of the 2A sequence end and downstream products. Wherein, the C-terminal of the upstream protein will add some additional 2A residues, while the N-terminal of the downstream protein will have additional proline. There are four commonly used 2A peptides, P2A, T2A, E2A and F2A, respectively, derived from four different viruses.

In this context, the term "vector" or "expression vector" generally refers to a nucleic acid molecule capable of insertion into a suitable host for self-replication, which transfers the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having a plurality of functions as described above. The vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell comprising the vector.

The term "pharmaceutical composition" as used herein generally refers to unit dosage forms and may be prepared by any of the methods well known in the pharmaceutical arts. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers, solid carriers, or both.

As used herein, the term "pharmaceutically acceptable excipients" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the particular dosage form of interest. In addition to the extent to which any conventional adjuvant is incompatible with the chimeric antigen receptor, nucleic acid molecule, expression vector or transgenic immune cell of the invention, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, their use is also contemplated by the present invention.

As used herein, the term "administering" refers to introducing a predetermined amount of a substance into a patient by some suitable means. The chimeric antigen receptor, nucleic acid molecule, expression vector or transgenic immune cell or pharmaceutical composition of the invention may be administered by any common route, as long as it reaches the intended tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the invention is not limited to these illustrated modes of administration. Preferably, the compositions of the present invention are administered intravenously.

In this context, the term "treatment" refers to the use to obtain a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease. As used herein, "treating" encompasses diseases in mammals, particularly humans, including: (a) Preventing the occurrence of a disease or disorder in an individual susceptible to the disease but not yet diagnosed with the disease; (b) inhibiting disease, e.g., arresting disease progression; or (c) alleviating a disease, e.g., alleviating symptoms associated with a disease. As used herein, "treating" encompasses any administration of a drug or transgenic immune cell to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a drug comprising a chimeric antigen receptor-containing cell described herein to an individual in need thereof.

Herein, "carbon end" and "C end" are synonymous; "Nitrogen end" and "N end" are synonymous.

The invention provides a chimeric antigen receptor capable of recognizing NKG2D multiple ligands, and corresponding nucleic acid molecules, expression vectors, transgenic immune cells, pharmaceutical compositions and uses thereof, which are respectively described in detail below.

Chimeric antigen receptor

The present invention provides a chimeric antigen receptor. The chimeric antigen receptor comprises: a first fragment, said first fragment being a NKG2D full-length sequence; a second fragment, wherein the first fragment is a full-length sequence of DAP10, and the first fragment is connected with the second fragment; an intracellular signal transduction domain, the N-terminus of the intracellular signal transduction domain being linked to the C-terminus of the first fragment or the second fragment.

According to the embodiments of the present invention, the inventors creatively linked the full-length sequence of NKG2D and the full-length sequence of DAP10 to obtain chimeric antigen receptor with stronger and wider NKG2D ligand binding activity. Therefore, the immune cells expressing the chimeric antigen receptor disclosed by the invention can effectively target and identify and kill various hematological tumors and solid tumor cells expressing NKG2D ligands, have the characteristics of a general CAR-immune cell (such as a CAR-NK product) product, and can effectively kill immunosuppressive cells MDSCs in a tumor microenvironment and effectively reverse the tumor immunosuppression microenvironment.

According to an embodiment of the present invention, the chimeric antigen receptor may further include at least one of the following technical features:

According to an embodiment of the invention, the NKG2D full-length sequence has the sequence set forth in SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1.

According to an embodiment of the invention, the full length DAP10 sequence has the sequence set forth in SEQ ID NO:2, and a polypeptide having the amino acid sequence shown in 2.

According to an embodiment of the invention, the N-terminus of the intracellular signaling domain is linked to the C-terminus of the first fragment.

According to an embodiment of the invention, the intracellular signaling domain is an intracellular segment of a cd3ζ molecule.

According to an embodiment of the invention, the intracellular segment of the CD3 zeta molecule has the sequence as set forth in SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3.

Nucleic acid molecules

The present invention provides a nucleic acid molecule. The nucleic acid molecules include a first nucleic acid molecule encoding the chimeric antigen receptor described above. The immune cells carrying the first nucleic acid molecules can express the chimeric antigen receptor, and after the chimeric conversion receptor recognizes various NKG2D ligands, the immune cells are activated, so that the immune cells can realize the targeted recognition of tumors expressing the NKG2D ligands and can perform killing effect.

According to an embodiment of the invention, the first nucleic acid molecule comprises nucleic acid molecule 1 and nucleic acid molecule 2; said nucleic acid molecule 1 for encoding said first fragment and said intracellular signaling domain, and said nucleic acid molecule 2 for encoding said second fragment; or said nucleic acid molecule 1 is used to encode said first fragment and said nucleic acid molecule 2 is used to encode said second fragment and said intracellular signaling domain.

According to an embodiment of the invention, the nucleic acid molecule 1 has the sequence as set forth in SEQ ID NO:4, said nucleic acid molecule 2 having a nucleotide sequence as set forth in SEQ ID NO:5, a nucleotide sequence shown in seq id no; or the nucleic acid molecule 1 has the sequence as set forth in SEQ ID NO:6, said nucleic acid molecule 2 having a nucleotide sequence as set forth in SEQ ID NO: 7.

According to an embodiment of the invention, the first nucleic acid molecule further comprises a nucleic acid molecule 3, the nucleic acid molecule 3 encoding a first linker, the nucleic acid molecule 1 and the nucleic acid molecule 2 being linked by the nucleic acid molecule 3.

According to an embodiment of the invention, the first linker is selected from at least one of P2A, T2A, E a and F2A.

Thus, immune cells carrying the nucleic acid molecules described above can express the chimeric antigen receptor described above. When the nucleic acid molecule comprises a linker of at least one of P2A, T2A, E a and F2A, the linker and the NKG2D and DAP10 fragments linked by the linker are expressed in immune cells. Further, NKG2D and DAP10 polypeptide fragments are cleaved by the linker polypeptide, thereby assembling a complex with NKG2D ligand binding activity.

According to an embodiment of the present invention, the first linker is P2A.

According to an embodiment of the invention, the first linker has the sequence as set forth in SEQ ID NO:8, and a polypeptide having the amino acid sequence shown in FIG. 8.

In some specific embodiments, the nucleic acid molecule 3 has a sequence as set forth in SEQ ID NO: 9.

In some specific embodiments, the first nucleic acid molecule has a sequence as set forth in SEQ ID NO:10, and a nucleotide sequence shown in seq id no.

According to an embodiment of the invention, the above-mentioned nucleic acid molecule further comprises a second nucleic acid molecule encoding a fusion protein comprising IL-15Rα and IL-15.

According to an embodiment of the invention, the N-terminus of IL-15Rα is linked to the C-terminus of IL-15, or the N-terminus of IL-15 is linked to the C-terminus of IL-15Rα.

According to an embodiment of the invention, the IL-15Rα has the amino acid sequence as set forth in SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no.

According to an embodiment of the invention, the IL-15 has the sequence as set forth in SEQ ID NO:12, and a polypeptide having the amino acid sequence shown in FIG. 12.

According to embodiments of the invention, the IL-15Rα of the invention is an IL-15 receptor α chain comprising a transmembrane region; the IL-15 of the present invention is an IL-15 mature peptide, thereby achieving membrane expression of IL-15Rα and IL-15, and further forming an IL-15Rα and IL-15 complex, abbreviated herein as "super IL-15". Compared with the secretory IL-15 or the secretory super IL-15, the expression quantity in cells is controllable, the expression area is controllable, the range of acting cells is controllable, and side effects caused by systemic administration of recombinant IL-15 or super IL-15 or repeated and repeated administration can be avoided. Whereby, co-expression of said super IL-15 with the chimeric antigen receptor of the invention onto the surface of an immune cell, on the one hand, increases the persistence of said immune cell in vivo and the viability in vivo; on the other hand, unexpectedly, the tumor killing activity of the immune cells can be significantly improved. Makes it possible to develop universal, safe and effective immune cell product.

According to an embodiment of the invention, the fusion protein further comprises a connecting peptide.

According to an embodiment of the invention, the C-terminus of the IL-15Rα is linked to the N-terminus of the linker peptide, which is linked to the N-terminus of the IL-15; or the C-terminal of the IL-15 is connected with the N-terminal of the connecting peptide, and the C-terminal of the connecting peptide is connected with the N-terminal of the IL-15 Ralpha.

According to an embodiment of the invention, the linking peptide is selected from at least one of (G₄S)n、ESGRSGGGGSGGGGS、EGKSSG SGSESKST、EGKSSGSGSESKSTQ、GSTSGSGKSSEGKG、KESGSVSSEQLAQFRSLD、ESGSVSSEELAFRSLD、SGGGSGGGGSGGGGSGGGGSGGGSLQ, n is an integer other than zero.

According to an embodiment of the invention, the linking peptide is selected from (G ₄S)_n, n is any integer between 2 and 6).

According to an embodiment of the invention, the connecting peptide is (G ₄S)₃.

According to an embodiment of the invention, the connecting peptide is SGGGSGGGGSGGGGSGGGGSGGGSLQ.

According to an embodiment of the invention, the fusion protein has the sequence as set forth in SEQ ID NO:13, and a nucleotide sequence shown in seq id no.

In some specific embodiments, the second nucleic acid molecule has a sequence as set forth in SEQ ID NO:14, and a nucleotide sequence shown in seq id no.

Therefore, the expression quantity, the expression region and the action cell range of the super IL-15 are further optimally controlled, and the safety of the obtained genetically modified immune cells is obviously improved.

According to an embodiment of the invention, the nucleic acid molecule further comprises a third nucleic acid molecule encoding a second linker, the first nucleic acid molecule and the second nucleic acid molecule being linked by the third nucleic acid molecule.

According to an embodiment of the invention, the second linker is selected from at least one of P2A, T2A, E a and F2A.

According to an embodiment of the present invention, the second linker is P2A.

According to an embodiment of the invention, the second linker has the sequence as set forth in SEQ ID NO:8, and a polypeptide having the amino acid sequence shown in FIG. 8.

According to an embodiment of the invention, the third nucleic acid molecule has the sequence as set forth in SEQ ID NO: 9.

Therefore, the super IL-15 nucleic acid molecule and the nucleic acid encoding the chimeric antigen receptor of the invention are simultaneously expressed in cells according to fixed proportion, so that the relative expression quantity of the super IL-15 and the chimeric antigen receptor in a single cell is controlled, and the safety of genetically modified immune cells is further improved.

In some specific embodiments, the nucleic acid molecule has a sequence as set forth in SEQ ID NO:15, and a nucleotide sequence shown in seq id no.

It is noted that, for the nucleic acid molecules mentioned herein, one skilled in the art will understand that either one or both of the complementary double strands are actually included. For convenience, although only one strand is shown in most cases herein, the other strand complementary thereto is actually disclosed. In addition, the molecular sequence in the present invention includes a DNA form or an RNA form, and disclosure of one of them means that the other is also disclosed.

Expression vector

The invention provides an expression vector. The expression vector carries the nucleic acid molecule described above. Thus, the expression vector constructed can express the chimeric antigen receptor of the present invention in a receptor cell.

In the case of ligating the above-mentioned nucleic acid molecule to an expression vector, the nucleic acid molecule may be directly or indirectly linked to control elements on the expression vector, as long as these control elements are capable of controlling translation, expression, etc. of the nucleic acid molecule. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e. not from the carrier itself. Of course, the nucleic acid molecule may be operably linked to a control element.

According to an embodiment of the invention, the expression vector is a non-pathogenic viral vector.

According to an embodiment of the invention, the non-pathogenic viral vector is optionally one of a retroviral vector, a chronic viral vector and an adeno-associated viral vector.

According to an embodiment of the invention, the non-pathogenic viral vector is a lentiviral vector.

According to embodiments of the invention, expression of the chimeric antigen receptor of the invention in immune cells may be achieved after introduction of the lentiviral vector into the recipient cell in some embodiments.

As used herein, the term "operably linked" refers to the linkage of a foreign gene to a vector such that control elements within the vector, such as transcription and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene. The usual vectors may be, for example, viral vectors, plasmids, phages and the like. After the expression vector according to some embodiments of the present invention is introduced into a suitable recipient cell, the expression of the nucleic acid molecule described above can be effectively achieved under the mediation of a regulatory system, thereby achieving in vitro mass-production of the protein encoded by the nucleic acid molecule.

Cells

The present invention provides a transgenic immune cell. The transgenic immune cells express the chimeric antigen receptor; or carrying the nucleic acid molecules described above, the expression vectors described above. Thus, the obtained transgenic immune cells have obviously improved tumor killing activity and better safety. Furthermore, the development of a universal, safe and effective immune cell product is possible, and the treatment of various tumor indications by a CAR-T or CAR-NK product is further realized, so that the transgenic immune cell has good clinical application prospect.

According to an embodiment of the invention, the transgenic immune cells express both the chimeric antigen receptor and a fusion protein identical to the fusion protein expressed in the nucleic acid molecule described previously. Therefore, compared with immune cells modified by chimeric antigen receptor genes, the transgenic immune cells express the membrane-binding super IL-15 on the surface of cell membranes, and the obtained transgenic immune cells have the advantages of improved in-vivo and in-vitro proliferation capacity, remarkably enhanced tumor cell killing activity and synergistic effect.

Furthermore, the inventors have further found that at the cellular level, the transgenic immune cells of the invention exhibit significantly increased degranulation levels and killing functions, as well as significantly increased IFN-gamma and TNF-alpha secretion capacity. At animal level, the transgenic immune cells of the invention also show obviously enhanced in vivo tumor inhibiting effect, and can kill the immune suppression cells MDSCs in tumor microenvironment, thereby effectively reversing tumor immune suppression microenvironment. Thus, the expression of the full-length NKG2D sequence and the full-length DAP10 sequence of the invention enables NK cells to recognize tumors expressing the NKG2D ligand more accurately and effectively, while the gene modification strategy of the membrane-bound super IL-15 of the invention can improve the persistence and the viability of the transgenic immune cells of the invention in vivo, and the two gene modification strategies also observe synergistic beneficial effects unexpectedly in the invention. Namely, the gene modification strategy of the invention can obviously improve the tumor killing activity of immune cells, so that the development of a universal, safe and effective immune cell product is possible, and the treatment of various tumor indications by one CAR-T or CAR-NK product is further realized.

According to an embodiment of the invention, the transgenic immune cell is obtained by introducing the expression vector into an immune cell.

According to an embodiment of the present invention, the immune cells are selected from at least one of any one of T cells, NK cells, NKT cells, γδ T cells, macrophages, peripheral blood NK cells, umbilical cord blood NK cells, and iPSC-derived immune cells. The chimeric antigen receptor of the present invention can be expressed on the surface of immune cells such as T, NK, NKT, γδt, macrophage, iPSC, etc., by transduction of these immune cells with an expression vector (lentiviral vector).

In some specific embodiments, the immune cells are selected from at least one of NK cells, peripheral blood NK cells, umbilical cord blood and iPSC-derived NK cells, NK92 cells.

In some more specific embodiments, the immune cells are optionally derived from any one of the NK cell lines.

According to embodiments of the present invention, the immune cells of the present invention have better clinical efficacy and safety and broader clinical applicability than immune cells that sequentially or simultaneously administer single chain antibodies to recognize specific tumor antigens at comparable doses and in the same manner of administration.

Pharmaceutical composition

The invention provides a pharmaceutical composition. The pharmaceutical composition comprises the chimeric antigen receptor, the nucleic acid molecule, the expression vector or the transgenic immune cell. The pharmaceutical composition thus obtained is further used for the treatment of tumors.

According to an embodiment of the present invention, the pharmaceutical composition further comprises: pharmaceutically acceptable auxiliary materials.

Those skilled in the art will appreciate that the features and advantages described above for chimeric antigen receptors, nucleic acid molecules, expression vectors, transgenic immune cells are equally applicable to the pharmaceutical compositions and will not be described in detail herein.

Use of the same

The invention provides the use of the chimeric antigen receptor, the nucleic acid molecule, the expression vector, the transgenic immune cell or the pharmaceutical composition in the preparation of medicaments for preventing or treating tumors.

According to an embodiment of the invention, the tumor is a solid tumor or a hematological tumor.

According to an embodiment of the invention, the solid tumor is a tangible tumor occurring in an organ. The tangible tumors occurring in the viscera include, but are not limited to, at least one of pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, cholangiocarcinoma, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, renal cancer, breast cancer, prostate cancer and melanoma.

According to an embodiment of the invention, the hematological neoplasm comprises at least one selected from the group consisting of acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, and multiple myeloma.

According to some alternative embodiments of the invention, the hematological neoplasm comprises at least one selected from acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, and multiple myeloma within the blood cell and hematopoietic system.

Method for preventing or treating tumor

The present invention provides a method of treating and/or preventing an immune system disorder. According to an embodiment of the invention, the method comprises: administering to a subject a pharmaceutically acceptable amount of the transgenic immune cell described above or the pharmaceutical composition described above.

The effective amount of the transgenic immune cells and pharmaceutical compositions of the present invention may vary depending on the mode of administration, the severity of the disease to be treated, and the like. Preferably, the selection of an effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.

The details of the sequences involved in the present invention are shown in Table 1.

Table 1: nucleotide/amino acid sequence specification table

The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

In the following examples, the chimeric antigen receptor sequences comprise: NKG2D full-length sequence (nucleotide sequence shown as SEQ ID NO: 7), linker protein DAP10 full-length sequence (nucleotide sequence shown as SEQ ID NO: 5) and intracellular signal transduction molecule CD3 zeta (nucleotide sequence shown as SEQ ID NO: 16) and IL-15-linker-IL-15 Ralpha (nucleotide sequence shown as SEQ ID NO: 14) gene fragment connected by P2A (nucleotide sequence shown as SEQ ID NO: 9). The structural schematic diagram of the genetic element is shown in figure 1.

In the following examples, reference STING agonist cGAMP enhances anti-tumor activity of CAR-NK cells against pancreatic cancer.Oncoimmunology.2022Mar 21;11(1):2054105.doi:10.1080/2162402X.2022.2054105.PMID:35371622;PMCID:PMC8967397., detects the killing effect of effector cells on target cells by LDH release.

The "plasmid" and "vector" described in the following examples have the same meaning and are used interchangeably.

EXAMPLE 1 preparation of CAR-NK cells

In this example, the CAR-NK cells of the present invention were prepared as follows.

1.1 Construction of CAR expression plasmids

(1) The nucleotide sequence shown in SEQ ID NO.15 is synthesized through total gene synthesis, cloned to a lentiviral vector pCDG-EF1-MCS-TA 2-copGGFP through enzyme cutting sites XbaI and BamHI, and the pCDH-EF1a-CAR expression plasmid vector is obtained after sequencing verification is correct.

The structural schematic diagram of the genetic element of the chimeric antigen receptor of this example is shown in FIG. 1, and specific sequence information is shown in reference Table 1.

1.2 Packaging of lentiviruses and concentration of viral fluids

293T cells in the logarithmic growth phase were inoculated into 10cm cell culture dishes at 5X 10 ⁶, 10mLDMEM medium was added and incubated overnight in a 5% CO ₂ incubator at 37 ℃. When the cell density in the cell culture dish reached 80%, 10mL of fresh DMEM medium was replaced for virus packaging, and the cell culture dish was kept in an incubator for further use. Preparing a slow virus packaging system, adding psPAX 26 mug of slow virus packaging auxiliary plasmid and 6 mug of target gene carrier plasmid into 250 mug serum-free DMEM culture medium to prepare plasmid mixed solution, and uniformly mixing. Will 15 mu LAdded into 235 mu L of serum-free DMEM medium, and mixed uniformly. Will beThe mixed solution is added into the plasmid mixed solution at one time, mixed evenly and incubated for 15min at room temperature. The mixture was added to 293T cell culture dishes. After 24h, the culture dish was replaced with a 5% CO ₂ incubator at 37℃for 48h, and the cell supernatant was collected, centrifuged at 400 Xg for 5min, the cell debris was removed, and the supernatant was filtered into a 50ml centrifuge tube with a 0.45 μm filter head. Adding 5 XPEG 8000 solution for concentrating virus liquid, reversing the centrifuge tube upside down, mixing, and standing in a refrigerator at 4deg.C overnight. Centrifuging at 4deg.C at 4000 Xg for 20min, discarding supernatant, adding appropriate amount of serum-free DMEM to resuspend virus precipitate, transferring into EP tube, and storing in refrigerator at-80deg.C.

1.3 Lentiviral titer assay

293T cells in the logarithmic growth phase were collected and the concentration was adjusted to 1X 10 ⁵/mL. A24-well plate was used, and 1mL of the cell suspension (1X 10 ⁵/well) was added to each well, and 3 added virus volume gradients were set. The cells were incubated overnight at 37℃in a 5% CO ₂ incubator. The concentrated virus solution is diluted 10 times: a1 mL EP tube was used, 60. Mu.L of the virus concentrate was pipetted into the EP tube, diluted with 540. Mu. LDMEM medium and mixed well. 293T cells were replaced with fresh DMEM medium, 5. Mu.L, 50. Mu.L and 500. Mu.L of diluted virus solution were each aspirated and added to the corresponding wells, labeled, and the plates were returned to 37℃in a 5% CO ₂ incubator. After 24h, the well plates were blotted for virus and 1mL fresh DMEM medium was added. After 72h, cells were harvested by pancreatin digestion, 293T cells were examined for GFP expression using a flow meter, and viral titers were converted according to the formula.

Titer(TU/ml)＝100,000(target cells)×(％of GFP-positive cells/100)×10/volume of supernatant(in mL)。

1.4 Lentivirus infection of human NK92 cells

NK92 cells in the logarithmic phase of growth were aspirated, the cells were harvested by centrifugation at 100 Xg for 5min, and the cells were resuspended in an appropriate amount of alpha-MEM medium to adjust the cell density to 5X 10 ⁵/mL. 5X 10 ⁵ NK92 cells were inoculated into a 24-well plate, and 1mL of the virus concentrate and protamine (final concentration 8. Mu.g/mL) were mixed uniformly. Culturing in a 5% CO ₂ incubator at 37deg.C. After 24h, the cell status was observed, the liquid was changed, the infected cells were transferred into EP tube, centrifuged at 100 Xg for 5min, the cells were resuspended in a small amount of fresh alpha-MEM medium, transferred into cell culture flasks, and cultured for a further 48h with 10mL of fresh alpha-MEM medium and IL-2 (final concentration of 200 IU/mL). Cells were transferred into a inflow tube, 3mL of 1 XPBS solution was added, 100 XPS was centrifuged for 5min, the supernatant was discarded, the cell pellet was sprung, and washed once again with 1 XPBS solution. The expression rate of GFP was measured using a flow meter. And continuing to expand the culture, and adjusting the state of NK92 cells after infection to expand. The infected NK92 cells were sorted by flow meter for GFP-positive CAR-NK92 cells for later experiments.

1.5 Infection of human peripheral blood primary-expanded NK cells with lentiviruses

Isolated Peripheral Blood Mononuclear Cells (PBMC) were inoculated into pre-coated flasks for culture, and induced culture using anti-CD 3 monoclonal antibodies and cytokines such as IL-2, see CN 202310035787.4. Lentiviral infection was performed as described above on day 7 of culture. Culture was continued after the change of fluid on day 9, and cells were harvested for in vivo experimental treatment by day 19.

Example 2 in vitro killing, proliferation and in vitro viability assays of CAR-NK cells

In this example, the inventors examined the killing activity of the CAR-NK cells of the invention obtained in example 1 against NKG2D ligand-positive tumors at the cellular level, proliferation and in vitro survival of the CAR-NK cells of the invention.

2.1CAR genetic modification enhances in vitro killing of NKG2D ligand positive tumors by NK92 cells

First, the expression of several tumor cell lines (hepatoma cell, breast cancer, non-small cell lung cancer) NKG2D ligand MICA/B was examined by flow cytometry, and the results are shown in FIG. 2. The liver cancer cell line H7402 cells, huh7 cells, breast cancer cell line MCF-7 cells and non-small cell lung cancer cell line A549 cells all express high level MICA/B, while the liver cancer cell line SMMC-7721 cells express lower and are basically negative. The inventor selects a MICA/B high-expression H7402 cell line and a SMMC-7721 cell which is basically expressed negatively as target cells to detect the influence of the CAR gene modification on the NK92 cell killing activity.

The specific method comprises the following steps: incubating NK92 and CAR-NK92 cells with liver cancer cell line H7402 or SMMC-7721 cells for 4H respectively, and detecting killing efficiency by an LDH release method; and simultaneously adding NKG2D antibody (Ab blockade) into a killing system for blocking, and respectively setting controls of NK92 and CAR-NK92 cell groups. The ratio of effector cells (NK cells) to target cells (tumor cells) was examined to be 10:1, 5:1 and 2.5:1.

The test results are shown in fig. 3: referring to inventive example 1, the killing efficiency of genetically modified inventive CAR-NK92 cells against MICA/B positive H7402 cells was significantly higher than NK92 cells at an effective target ratio of 10:1 and 5:1. The killing efficiency of the CAR-NK92 and NK92 cells to SMMC-7721 cells and H7402 cells after NKG2D antibody is added into a killing system is obviously reduced.

The test results show that the NKG2D chimeric antigen receptor of the invention has reasonable design and can effectively recognize the NKG2D ligand MICA/B; furthermore, NK cells modified by the NKG2D chimeric antigen receptor and the cell membrane expression IL-15 gene have obviously enhanced killing activity and show synergistic effect.

Further, CAR-NK92 and NK92 cells of the present invention were co-incubated with H7402 cells at an effective target ratio of 5:1, respectively, to examine the expression of NK cell killing effector molecules CD107a, granzyme B and Perforin (Perforin). The IgG group was a negative control group to which no flow antibody staining was added.

The specific method comprises the following steps: the CD107a group was added with 5. Mu.L PE-anti-human CD107a antibody (clone H4A3, biolegend), the positive control group with PMA (Sigma, final concentration 30 ng/mL) and ionomycin (ionomycin, sigma, final concentration 1. Mu.g/mL), the incubation was continued for 1H, and the blockers BFA and monensin (Biolegend, ratio 1:1000) were added and the incubation was continued for 3H. Cells were collected in a centrifuge tube, washed by centrifugation with 1 XPBS solution, resuspended in an appropriate amount of 1 XPBS solution, and examined for CD107a expression by flow cytometry. Granzyme B and Perforin groups, reinforcing fixed solution 100. Mu.L/tube, incubating at room temperature for 15min, adding 1 XPBS solution, centrifuging, washing, adding membrane penetrating solution 100. Mu.L/tube, and adding corresponding antibody (APC-anti-human Perforin, clone No. dG9; alexa)647-Anti-human Granzyme B, clone No. GB11, biolegend) incubated at room temperature for 30min, after centrifugation washing with 1 XPBS solution, appropriate amount of 1 XPBS solution was added to resuspend cells, and flow cytometry was used to detect the expression levels of Granzyme B and Perforin.

The test results are shown in FIG. 4: after co-incubation with MICA/B positive H7402 cells, the expression levels (mean fluorescence intensity MFI) of CAR-NK92 cells CD107a, performin and Granzyme B of the invention were all significantly higher than for NK92 group.

The above test results further verify that: the CAR gene modification strategy can obviously improve the degranulation level and the killing function of NK cells on NKG2D ligand positive tumor cells.

2.2CAR Gene modification enhances cytokine secretion levels in NK92 cells

The CAR-NK92 cells of the invention were further examined for changes in their IFN- γ and TNF- α secretion capacity by flow cytometry. The specific method comprises the following steps: incubating NK92 or CAR-NK92 cells and H7402 cells of a liver cancer cell line positive for NKG2D ligand for 4 hours, collecting NK92 or CAR-NK92 cells in a flow tube, and detecting the expression level of IFN-gamma and TNF-alpha in the cells of the NK92 or the CAR-NK92 cells by flow cytometry after fixation rupture treatment. The IgG group was a negative control group to which no flow antibody staining was added.

The test results are shown in fig. 5: the level of IFN-gamma and TNF-alpha expressed by the CAR-NK92 cells of the invention is obviously higher than that of the NK92 group.

The level of IFN-. Gamma.in the culture supernatant of NK92 cells incubated with H7402 cells was further examined by ELISA. The experimental results also show that the level of IFN-gamma secretion by the CAR-NK92 cells is significantly higher than that of the NK92 group (FIG. 5).

The above test results show that the CAR structure of the invention can obviously improve the secretion capacity of IFN-gamma and TNF-alpha when NK cells are contacted with NKG2D ligand positive tumor cells.

2.3CAR genetic modification promotes survival and proliferation of NK92 cells

The inventors further confirmed the promotion of proliferation and survival of NK92 cells by membrane-expressed IL-15. The specific method comprises the following steps: NK92 and CAR-NK92 cell plates of the same cell number were plated in 96-well plates, respectively, and different IL-2 concentrations (200 IU/ml, 20IU/ml and 0 IU/ml) were set, and flow cytometry was performed every 24 hours to detect apoptosis rate or trypan blue stained cell count every 48 hours. Flow cytometry detection of apoptosis rate was performed according to the procedure of the kit instructions (biankia, cat No. AP 101), briefly: cells were collected in EP tubes, washed once by centrifugation with 1xPBS solution, and resuspended. mu.L of Annexin V-FITC and 10. Mu.L of PI were added to each tube. After gentle vortexing, incubation for 5min at room temperature in the dark, and flow detection of resuspended cells. Cell viability was the proportion of Annexin V-FITC and PI staining double negative.

The results of NK cell viability investigation for 96h of culture are shown in fig. 6: NK92 group in the absence of IL-2 (IL-20 IU/ml), cell viability was significantly reduced from 24 hours to 72 hours when most cells had been apoptotic; while the CAR-NK92 group cells can still keep high cell viability even under the condition of completely removing IL-2, and few cells undergo apoptosis. It is suggested that IL-15 expressed in the membrane in CAR-NK92 plays an important role in promoting the survival of NK cells.

Further observations and cultures were made to day 8, and cell proliferation curves were plotted, with the results shown in fig. 7: the proliferation capacity of the CAR-NK92 cells is obviously higher than that of NK92 cells with the same IL-2 concentration under the condition of different IL-2 concentrations.

The test results above demonstrate that: unexpectedly, the inventors expect that the effect of the membrane-expressed IL-15 element of the invention on promoting NK cell proliferation is significantly enhanced when NK92 cells are cultured in vitro, compared with the effect of adding free IL-2 into a cell culture solution, thereby being more beneficial to NK92 proliferation culture in vitro and the industrialization of immune cell products of the invention.

Example 3CAR-NK cells have stronger anti-tumor ability in vivo

In this example, the inventors examined the killing activity of the CAR-NK cells of the invention obtained in example 1 against NKG2D ligand-positive tumors at animal level.

3.1 In vivo tumor-inhibiting effect of CAR-NK cells on ovarian cancer

The inventors first examined the expression of NKG2D ligands MICA/B and ULBP of human ovarian cancer cell lines SKOV3 and a1847, and the results are shown in fig. 8: SKOV3 expressed predominantly ULBP2/5/6, whereas A1847 cells expressed both MICA/B and ULBP2/5/6 higher. Thus, a human ovarian cancer mouse xenograft model was established with SKOV3 and a1847 cell lines, respectively, to observe the therapeutic effect of CAR-NK92 cells designed and prepared according to the present invention on ovarian cancer.

The specific method comprises the following steps: NCG mice at 6 weeks of age were selected for underarm subcutaneous tumor-bearing at a tumor-bearing dose of 2.5 x 10 ⁶ cells/mouse. NK cell therapy was started on day 7 of tumor bearing. Tumor volume size was measured prior to treatment and randomly divided into PBS group, NK92 cell treatment group and CAR-NK92 cell treatment group according to tumor volume size. Mice in the treated group were given 1X 10 ⁷ effector cells/mouse tail vein, untreated groups were given equal volumes of 1 XPBS, once every other week for 3 total treatments, and tail vein for 3 days for IL-2 (5X 10 ⁴ IU/mouse). Tumor volume was measured every 3 or 4 days and tumor growth curves were drawn.

The test results are shown in fig. 9 and 10: (1) The therapeutic effect on SKOV3 ovarian cancer model showed that CAR-NK92 cells had a stronger tumor growth inhibiting effect than NK92, with a statistical tumor inhibition rate of 64.2% for the CAR-NK92 treated group at day 18 post-treatment, and 32.3% for the NK92 treated group (fig. 9). (2) The therapeutic effect on the a1847 ovarian cancer model showed that CAR-NK92 cells had a stronger tumor growth inhibiting effect than NK92, with a statistical tumor inhibition rate of 55.8% for the CAR-NK92 treated group at day 19 after treatment, and 42.9% for the NK92 treated group (fig. 10).

The above results illustrate: the NKG2D and membrane-bound IL-15-expressing CAR-NK92 cells have obviously enhanced in-vivo tumor inhibiting effect on NKG2D ligand positive tumors. The expression of the NKG2D-CAR enables NK cells to more accurately and effectively recognize tumors expressing the NKG2D ligand, and the membrane expression IL-15 gene modification can improve the persistence of the CAR-NK92 cells in the body and improve the viability of the CAR-NK92 cells in the body; the two are synergistic, and the anti-tumor effect of the CAR-NK92 cells is further enhanced.

3.2 In vivo tumor-inhibiting effect of primary CAR-NK cells on pancreatic cancer

The inventor establishes a human pancreatic cancer mouse xenograft tumor model by using a human pancreatic cancer cell line HPAF-II, prepares peripheral blood-derived CAR-NK cells by infecting human peripheral blood primary NK cells with the CAR lentivirus of the embodiment 1 of the invention, and observes the treatment effect of the CAR-NK cells on the pancreatic cancer model.

The specific method comprises the following steps: 6-week-old NCG mice were selected for underarm subcutaneous tumor loading and NK cell therapy was initiated when tumor volume reached 50mm ³. Tumor volume size was measured before treatment and randomly divided into PBS group, NK cell control group without genetic modification, and three CAR-NK cell treatment groups according to tumor volume size. Mice in the NK cell control group which are not subjected to gene modification are treated 1 time every 2 days for 3 times, the reinfusion dose of each tail vein is 4×10 ⁶CD56⁺ NK cells, and the survival of the NK cells is maintained by intraperitoneal injection of IL-2 every 2 days; the CAR-NK cells are injected into the tail of mice in the CAR-NK cell treatment group intravenously, and the single treatment is carried out for 1 time, the doses are respectively 4 multiplied by 10 ⁶、8×10⁶ and 1.6multiplied by 10 ⁷ CAR-NK cells/mouse, and the CAR-NK cells are modified by IL-15 genes, so that IL-2 injection is not carried out; untreated groups were injected with an equal volume of 1 XPBS. Tumor volume was measured twice weekly, tumor growth curves were drawn, and tumor inhibition was counted.

The test results are shown in FIG. 11: the treatment effect on an HPAF-II pancreatic cancer model shows that the peripheral blood-derived CAR-NK cells prepared based on the invention have a remarkably enhanced effect of inhibiting tumor growth compared with an NK cell control group which is not infected with the CAR lentivirus of the embodiment 1 of the invention; the tumor inhibition rate of the 4 multiplied by 10 ⁶CAR^- NK cell dosage group is 56.78 percent, and the tumor inhibition rate of the 1.6multiplied by 10 ⁷CAR^- NK cell dosage group is 80.97 percent.

Example 4CAR-NK cells were able to effectively kill immunosuppressive cells MSDCs within tumor microenvironment

In this example, the inventors examined the in vitro killing activity of the CAR-NK92 cells of the invention obtained in example 1 against MDSCs and the antitumor activity in tumor-bearing mouse models. In vivo, MDSCs were mixed with tumor cells and mice were treated for tumor-bearing, NK92 or CAR-NK92 cell feedback, and tumor burden was observed by in vivo imaging.

4.1 Induction of MDSCs by human peripheral blood PBMC

The inventors induced MDSCs with PBMCs from human peripheral blood.

The specific method comprises the following steps: human peripheral blood PBMC induced MDSC cells: after isolation of human peripheral blood PBMC, plates were plated and incubated with 100ng/mL IL-4 and 100ng/mL GM-CSF for 24h, followed by additional 25 μg/mL Poly (I: C) stimulation for 24h. The supernatant was discarded and adherent cells were collected by pancreatin digestion, i.e., induced MDSCs.

4.2 In vitro killing effect of CAR-NK92 cells on MDSCs

The inventors detected the killing efficiency of CAR-NK92 cells against MDSCs by LDH method.

The specific method comprises the following steps: incubating NK92 and CAR-NK92 cells with MDSCs for 4 hours respectively, and detecting killing efficiency by an LDH release method; and simultaneously adding NKG2D antibody (Ab blockade) into a killing system for blocking, and respectively setting controls of NK92 and CAR-NK92 cell groups. The ratio of effector cells to target cells was 10:1, 5:1 and 2.5:1.

The test results are shown in FIG. 12: the killing efficiency of the genetically modified CAR-NK92 cells of the invention on MICA/B positive MDSCs is obviously higher than that of NK92 cells when the effective target ratio is 10:1, 5:1 and 2.5:1. After NKG2D antibody is added into the killing system to block, the killing efficiency of NK92 and CAR-NK92 cells is obviously reduced.

4.3 Tumor inhibiting effect of CAR-NK92 cells on tumor-bearing mouse model of human liver cancer H7402 cells containing MDSCs

The specific method comprises the following steps:

The method for establishing the tumor-bearing mouse model of the human liver cancer H7402 cells containing MDSCs comprises the following steps: on day 0, 6-7 week old mice were selected, each mixed with 1x10 ⁶ luciferase-labeled H7402 cells and 6x10 ⁵ MDSCs for underarm subcutaneous tumor bearing. On day 3 after tumor bearing, tail vein reinfusion of NK92 or CAR-NK92 cells is carried out at a dose of 5x10 ⁶ cells/dose, the reinfusion treatment is carried out once every 3 days for 3 times, and meanwhile, the IL-2 solution is injected intraperitoneally every 3 days at a dose of 5x10 ⁴ IU/dose. On day 10, in vivo imaging observations were made.

The test results are shown in fig. 13: as can be seen from the results of live imaging, there was no significant difference in tumor burden in the NK92 cell group compared to the untreated group. The tumor burden of the CAR-NK92 cell group was significantly less than that of the untreated group and the NK92 cell group. This shows that the CAR-NK92 cells can effectively kill MDSCs in the tumor microenvironment, eliminate the inhibition of the MDSCs in the tumor on immune effector cells, and have stronger tumor inhibition effect than NK92 cells.

The test results show that the CAR-NK cells have strong killing effect on the MDSCs in tumors, can resist the immune suppression of the MDSCs on the CAR-NK or the CAR-T cells, exert stronger anti-tumor effect, and break through the bottleneck that immune cell therapy is subject to the immune suppression of the microenvironment of the solid tumor.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A chimeric antigen receptor comprising:

A first fragment, said first fragment being a NKG2D full-length sequence;

a second fragment, wherein the first fragment is a full-length sequence of DAP10, and the first fragment is connected with the second fragment;

An intracellular signaling domain, the N-terminus of the intracellular signaling domain being linked to the C-terminus of the first fragment, the intracellular signaling domain being an intracellular segment of a cd3ζ molecule; the amino acid sequence of the intracellular segment of the CD3 zeta molecule is shown as SEQ ID NO:3 is shown in the figure;

the amino acid sequence of the NKG2D full-length sequence is shown in SEQ ID NO:1 is shown in the specification;

The amino acid sequence of the full-length sequence of the DAP10 is shown as SEQ ID NO:2 is shown in the figure;

Further comprises: a first linker, the first fragment and the second fragment being linked by the first linker; the amino acid sequence of the first connector is shown as SEQ ID NO: shown as 8;

Further comprises: a second linker and a fusion protein, the N-terminus of the second fragment and the C-terminus of the fusion protein being linked by the second linker; the amino acid sequence of the second linker is shown in SEQ ID NO: shown as 8; the amino acid sequence of the fusion protein is shown as SEQ ID NO: shown at 13.

2. A nucleic acid molecule encoding the chimeric antigen receptor of claim 1.

3. The nucleic acid molecule of claim 2, comprising a first nucleic acid molecule, a second nucleic acid molecule, and a third nucleic acid molecule; the first nucleic acid molecule and the second nucleic acid molecule are linked by the third nucleic acid molecule;

The nucleotide sequence of the first nucleic acid molecule is shown as SEQ ID NO:10 is shown in the figure;

the nucleotide sequence of the second nucleic acid molecule is shown as SEQ ID NO: 14;

The nucleotide sequence of the third nucleic acid molecule is shown as SEQ ID NO: shown at 9.

4. The nucleic acid molecule of claim 2, wherein the nucleotide sequence of the nucleic acid molecule is set forth in SEQ ID NO: 15.

5. An expression vector carrying the nucleic acid molecule of any one of claims 2 to 4.

6. The expression vector of claim 5, wherein the expression vector is a non-pathogenic viral vector.

7. The expression vector of claim 6, wherein the non-pathogenic viral vector is optionally one of a retrovirus vector, a chronic virus vector and an adenovirus-associated virus vector.

8. The expression vector of claim 6, wherein the non-pathogenic viral vector is a lentiviral vector.

9. A transgenic immune cell comprising:

Expressing the chimeric antigen receptor of claim 1; or alternatively

Carrying the nucleic acid molecule of any one of claims 2 to 4 or the expression vector of any one of claims 5 to 8.

10. The transgenic immune cell of claim 9, wherein the transgenic immune cell is obtained by introducing the expression vector into an immune cell.

11. The transgenic immune cell of claim 10, wherein the immune cell is selected from at least one of T cells, NK cells, macrophages, and ipscs.

12. The transgenic immune cell of claim 11, wherein the T cell is selected from at least one of NKT cells, γδ T cells, and the NK cell is selected from at least one of peripheral blood NK cells, umbilical cord blood NK cells, iPSC-derived NK cells, NK-92 cells.

13. The transgenic immune cell of claim 10, wherein the immune cell is selected from the group consisting of NK cells.

14. A pharmaceutical composition comprising:

The chimeric antigen receptor of claim 1, the nucleic acid molecule of any one of claims 2 to 4, the expression vector of any one of claims 5 to 8, or the transgenic immune cell of any one of claims 9 to 13.

15. The pharmaceutical composition of claim 14, further comprising: pharmaceutically acceptable auxiliary materials.

16. Use of the chimeric antigen receptor of claim 1, the nucleic acid molecule of any one of claims 2 to 4, the expression vector of any one of claims 5 to 8, the transgenic immune cell of any one of claims 9 to 13, or the pharmaceutical composition of claim 14 or 15 in the preparation of a medicament for preventing or treating a tumor, the tumor being at least one of liver cancer, ovarian cancer, and pancreatic cancer.