WO2024250865A1 - Chimeric antigen receptor and use thereof - Google Patents

Abstract

Provided are a chimeric antigen receptor and the use thereof. The chimeric antigen receptor comprises: a first fragment, the first fragment being a NKG2D full-length sequence; a second fragment, the second fragment being a DAP10 full-length sequence, and the first fragment being linked to the second fragment; and an intracellular signal transduction structural domain, the N terminal of the intracellular signal transduction structural domain being linked to the C terminal of the first fragment or of the second fragment. The chimeric antigen receptor can recognize a plurality of ligands of NKG2D, so that immune cells expressing the chimeric antigen receptor can targetedly recognize and kill a plurality of tumors expressing the NKG2D ligands. Therefore, the chimeric antigen receptor can be used for developing universal immune cell products, and achieves treatment of a plurality of tumor indications simply using one CAR-T or CAR-NK product, and therefore has a good clinical application prospect.

Description

Chimeric antigen receptor and its application Technical Field

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

Background Art

In recent years, chimeric antigen receptor T (CAR-T) cells have made great achievements in the treatment of hematological malignancies. However, CAR-T cells still face a series of challenges in clinical applications. For example, they show very low efficacy in the treatment of solid tumors; the immune escape mechanism of tumors through the loss of antigens causes off-target effects and drug resistance in CAR-T cell therapy. CAR-T cells require autologous adoptive cell transplantation, otherwise allogeneic T cells may cause graft-versus-host disease (GVHD); they are prone to adverse reactions such as cytokine storms and neurotoxicity, which endanger the patient's life. The latest studies have shown that CAR-NK cells may overcome the above-mentioned defects of CAR-T cells and show significant anti-tumor effects.

The in vivo application of CAR-NK cells generally does not cause cytokine storms. NK cells do not require strict HLA matching and have no potential to cause GVHD. NK cells do not require antigen presentation and are not restricted by MHC, and can directly kill tumor cells. CAR-NK cells can not only accurately identify tumors through the installed "CAR", but also recognize the corresponding ligands of tumor cells through the activation receptors expressed by themselves to activate activation signals, killing tumors in a CAR-independent manner, with a wide anti-tumor spectrum, and can prevent tumors from losing antigens or MHC molecules and causing off-target effects and immune escape. CAR-NK cells can also be used in combination with antibodies, through the FcγRⅢ (CD16) on their surface binding to the Fc segment of antibodies, and with the help of antibody-dependent cytotoxic activity (ADCC) to amplify the anti-tumor efficacy of CAR-NK cells. Therefore, CAR-NK cells have broad application prospects in anti-tumor treatment and have become a hot spot in the field of cell immunotherapy research and development.

Traditional CAR-T or CAR-NK technologies mostly target tumors by expressing single-chain antibodies (ScFv) that specifically recognize specific tumor antigens, initiate activation signals in the intracellular segment, and mediate the killing of tumor cells. However, traditional ScFv recognizes a single tumor antigen, and may cause off-target effects and therapeutic resistance or recurrence when the tumor antigen is lost, limiting the further application of CAR-T or CAR-NK technologies.

The complex immunosuppressive microenvironment in solid tumors is also one of the limiting factors that seriously affect the anti-tumor effect of CAR-T or CAR-NK cells. Myeloid-derived suppressor cells (MDSCs) are a group of suppressive cells derived from the bone marrow that can inhibit the anti-tumor effect of the body's immune cells. Studies have shown that MDSCs downregulate the expression of NKG2D through membrane-bound TGF-β, thereby inhibiting the function of NK cells. MDSCs can also induce the proliferation of Treg cells and promote their negative regulatory effects on immunity. In addition, MDSCs inhibit T cell immune responses and proliferation by producing reactive oxygen species and other means. Therefore, how to overcome the immunosuppression of MDSCs in the tumor microenvironment on immune cells, especially CAR-T or CAR-NK cells for reinfusion therapy, has become a research and development hotspot to break through the bottleneck of solid tumor immunotherapy.

Therefore, new immune cell modification technologies still need to be developed to further improve the therapeutic effects of CAR-T or CAR-NK.

Summary of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent. To this end, the present invention provides a chimeric antigen receptor, which can recognize multiple NKG2D ligands. The immune cells modified by it can not only target and kill multiple tumors expressing NKG2D ligands, but also have significantly improved killing efficiency for MDSCs, thereby eliminating the negative regulatory effect of MDSCs on immune cells, reversing the tumor immunosuppressive microenvironment, and ensuring that immune cells can more effectively exert anti-tumor effects in vivo. The chimeric antigen receptor of the present invention can be used to develop universal immune cell products, realize the treatment of multiple tumor indications with a CAR-T or CAR-NK product, and has good clinical application prospects.

It should be noted that the present invention is completed based on the following work of the inventors:

NK cell receptors have the characteristics of broad specific recognition. The ligands recognized by their activated receptors are expressed in the vast majority of hematological tumors and solid tumors, but are almost not expressed in normal cells. Therefore, by selecting NK cell receptors as the antigen binding domain of chimeric antigen receptors and further modifying immune cells with the chimeric antigen receptor gene, the resulting immune cells may have a wider tumor recognition spectrum.

In order to obtain CAR-immune cells with improved performance, the inventors creatively selected the full-length sequences of NKG2D and DAP10 to construct a chimeric antigen receptor structure that can widely bind to multiple NKG2D ligands; at the same time, the intracellular segment of the DAP10 molecule was cleverly used as the activation sequence of the intracellular region of the chimeric antigen receptor to exert the efficacy of the co-stimulatory domain. Based on this, the inventors 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 of the present invention can recognize multiple ligands of NKG2D, target and kill a variety of hematological tumors and solid tumor cells expressing NKG2D ligands, and have the characteristics of universal CAR-immune cell products.

MDSCs interact with immune cells (such as T cells, NK cells, dendritic cells, macrophages, etc.) and inhibit the body's immune response. In mice with liver cancer, MDSCs cells can inhibit the expression of NK cell surface receptor NKG2D and the production of IFN-γ, inducing NK cell dysfunction. The inventor unexpectedly discovered that immune cells (such as NK cells) modified by the chimeric antigen receptor gene of the present invention can not only target and kill NKG2D ligand-positive tumor cells, but also effectively kill MDSCs. In a human liver cancer H7402 cell tumor-bearing mouse model containing MDSCs, immune cells were reinfused into the tail vein or treated with the chimeric antigen receptor immune cells of the present invention. The results showed that the chimeric antigen receptor immune cells of the present invention effectively removed the negatively regulated MDSCs cells that inhibited immune effector cells in the tumor microenvironment, thereby saving immune cells in the tumor microenvironment from the inhibition of MDSCs, so that immune cells (including immune cells of the present invention and unmodified immune cells) can more effectively play an anti-tumor role, thereby improving the clinical efficacy of immune cell products.

Therefore, in the first aspect of the present invention, the present invention proposes a chimeric antigen receptor. The chimeric antigen receptor comprises: a first fragment, the first fragment is the full-length sequence of NKG2D; a second fragment, the first fragment is the full-length sequence of DAP10, the first fragment and the second fragment are connected; an intracellular signal transduction domain, the N-terminus of the intracellular signal transduction domain is connected to the C-terminus of the first fragment or the second fragment. The chimeric antigen receptor of the present invention has stronger and broader NKG2D ligand binding activity, and immune cells expressing the chimeric antigen receptor of the present invention can effectively target, recognize and kill a variety of hematological tumors and solid tumor cells expressing NKG2D ligands, and have the characteristics of a universal CAR-immune cell product.

In the second aspect of the present invention, the present invention proposes a nucleic acid molecule. The nucleic acid molecule includes a first nucleic acid molecule, and the first nucleic acid molecule encodes the chimeric antigen receptor of the first aspect of the present invention. The immune cell carrying the above nucleic acid molecule expresses the chimeric antigen receptor of the first aspect of the present invention, and the chimeric antigen receptor can further recognize and bind to multiple ligands of NKG2D, thereby achieving the targeted recognition of the immune cell to multiple blood tumors and solid tumor cells expressing NKG2D ligands, and further exerting its killing activity.

In the third aspect of the present invention, the present invention provides an expression vector. The expression vector carries the nucleic acid molecule of the second aspect of the present invention. Thus, the constructed expression vector is expressed in cells to effectively express and obtain the chimeric antigen receptor of the first aspect of the present invention.

In the fourth aspect of the present invention, the present invention proposes a transgenic immune cell. The transgenic immune cell expresses the chimeric antigen receptor of the first aspect of the present invention, or carries the nucleic acid molecule of the second aspect of the present invention or the expression vector of the third aspect of the present invention. The transgenic immune cell of the present invention is obtained, which can not only widely target and kill a variety of solid tumors or hematological tumors expressing NKG2D ligands, but also can effectively kill the immunosuppressive cells MDSCs in the tumor microenvironment. It is a universal immune cell product with good clinical efficacy, can be used for the treatment of a variety of tumor diseases, and has good clinical application prospects.

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

In the sixth aspect of the present invention, the present invention proposes a use for preparing a medicine. The chimeric antigen receptor of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the expression vector of the third aspect of the present invention, the transgenic immune cell of the fourth aspect of the present invention, or the pharmaceutical composition of the fifth aspect of the present invention is used in preparing a medicine, and the medicine is used to prevent or treat tumors. The chimeric antigen receptor of the present invention and the corresponding nucleic acid molecule, expression vector, transgenic immune cell or pharmaceutical composition can be further prepared into a medicine, which can be used clinically to prevent or treat diseases.

Those skilled in the art will appreciate that the features and advantages described above for the chimeric antigen receptor, nucleic acid molecule, expression vector, lentiviral vector, transgenic immune cell and pharmaceutical composition are also applicable to this use and will not be described in detail here.

Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

FIG1 is a schematic diagram of the gene element structure of the chimeric antigen receptor of Example 1 of the present invention;

FIG2 is a graph showing the expression of NKG2D ligand MICA/B in human liver cancer cell lines, breast cancer cell lines and non-small cell lung cancer cell lines according to Example 2 of the present invention, wherein H7402, Huh7 and SMMC-7721 are human liver cancer cell lines, MCF-7 is a human breast cancer cell line, and A594 is a human non-small cell lung cancer cell line;

FIG3 is a graph showing the results of investigating the in vitro killing activity of CAR-NK92 cells according to Example 2 of the present invention;

FIG4 is a graph showing the results of investigating the expression levels of CD107a, Perforin and Granzyme B in CAR-NK92 cells according to Example 2 of the present invention;

FIG5 is a graph showing the results of investigating the secretion levels of IFN-γ and TNF-α in CAR-NK92 cells according to Example 2 of the present invention;

FIG6 is a graph showing the results of investigating the survival rate of CAR-NK92 cells in Example 2 of the present invention;

FIG7 is a CAR-NK92 cell proliferation curve of Example 2 of the present invention, where the abscissa represents the cell culture time in days;

FIG8 is a graph showing the expression of NKG2D ligands MICA/B and ULBP2/5/6 in human ovarian cancer cell lines SKOV3 and A1847 according to Example 3 of the present invention;

FIG9 is a diagram showing the tumor inhibition effect of CAR-NK92 cells of Example 3 of the present invention on a human ovarian cancer SKOV3 cell-bearing mouse model;

FIG10 is a diagram showing the tumor inhibition effect of CAR-NK92 cells of Example 3 of the present invention on a mouse model bearing human ovarian cancer A1847 cells;

FIG11 is a diagram showing the tumor inhibition effect of peripheral blood-derived CAR-NK cells of Example 3 of the present invention on a mouse model bearing human pancreatic cancer HPAF-II cells, wherein PBS represents the PBS group, NK represents the NK cell control group without genetic modification, and CAR-NK-4E6, CAR-NK-8E6, and CAR-NK-16E6 represent CAR-NK cell treatment groups with injection doses of 4×10 ⁶ , 8×10 ⁶ , and 1.6×10 ⁷ CAR-NK cells/mouse, respectively;

FIG12 is a graph showing the results of investigating the killing activity of CAR-NK92 cells against MDSCs according to Example 4 of the present invention;

FIG. 13 is a diagram showing the tumor inhibition effect of CAR-NK92 cells of Example 4 of the present invention on a mouse model bearing human liver cancer H7402 cells containing MDSCs.

DETAILED DESCRIPTION

The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

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

The endpoints and any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

Terms and Definitions

In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

In this article, the term "LTR" is equivalent to "long terminal repeated", which refers to the long terminal repeat sequence (5'-LTR and 3'-LTR) at each end of the retrovirus genome. It does not encode protein, but contains regulatory elements such as promoters and enhancers. The LTR in the viral genome can be transferred to the vicinity of cellular proto-oncogenes, so that these proto-oncogenes are activated under the action of LTR strong promoters and enhancers, and normal cells are transformed into cancer cells.

In this article, "super IL-15" is a cytokine that can promote the survival and proliferation of T cells and NK cells. It is a fusion protein including IL-15Rα and IL-15. IL-15 and IL-2 share the IL-2/15Rβγc receptor. After IL-15 and IL-15Rα form dimers, they bind to IL-15Rβγc to activate the downstream JAK1/JAK3 and STAT3/STAT5 signaling pathways, thereby promoting the proliferation and activation of NK cells. At present, cytokines such as IL-2 and IL-15 are often used clinically to prolong the survival time of NK cells in vivo, but cytokine support therapy may also bring corresponding side effects. It has been confirmed that systemic IL-2 therapy can induce the activation of Treg cells, as well as side effects such as severe vascular leakage syndrome and neurotoxicity. Systemic IL-15 therapy mainly affects NK cells, γδT cells and CD8 memory T cells. IL-15 can cause symptoms such as hypotension and thrombocytopenia in a dose-dependent manner, and can lead to a decrease in neutrophils. Therefore, compared with IL-2, IL-15 is a safer choice, but the systemic toxicity caused by IL15 cannot be ignored.

In this article, the term "single-chain antibody" is equivalent to "single chain Fv" and "scFv", which is a small molecule antibody formed by connecting the variable region of the immunoglobulin heavy chain ( _VH ) and the variable region of the light chain ( _VL ) through a connecting peptide. The connecting peptide used to prepare scFv must be flexible enough to ensure that _VH and _VL can fold freely and make the antibody binding region have the correct configuration.

In this article, the term "(G ₄ S) _n " is equivalent to "(Gly ₄ Ser) _n ", which means that 4 glycines and 1 serine are repeated n times. It is a type of widely used connecting peptide, which can be located between the C-terminus of V _H and the N-terminus of V _L , or between the C-terminus of V _L and the N-terminus of V _H. Currently, (G ₄ S) ₃ is commonly used, in which glycine is the amino acid with the smallest molecular weight and the shortest side chain, which can increase the flexibility of the side chain, and serine is the most hydrophilic amino acid, which can increase the hydrophilicity of the connecting peptide. Therefore, (G ₄ S) ₃ has good stability and activity. Connecting peptides of different lengths and sequences can be designed to construct scFvs with different biological functions.

In this article, the term "linker" refers to the basic structural unit of gap junctions, which is usually composed of six identical or similar transmembrane proteins. In some specific cases, it includes but is not limited to "2A peptides". 2A peptides are short peptides (about 18-25 amino acids) derived from viruses. They are usually called "self-cleaving" peptides, which can produce multiple proteins from one transcription product. 2A peptides are not completely "self-cleaving", but work by causing the ribosome to skip the synthesis of glycine and proline peptide bonds at the C-terminus of the 2A element, ultimately resulting in the separation of the 2A sequence end and the downstream product. Among them, the C-terminus of the upstream protein will add some additional 2A residues, and the N-terminus of the downstream protein will have additional proline. There are currently four commonly used 2A peptides, namely P2A, T2A, E2A and F2A, which come from four different viruses.

In this article, the term "vector" or "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicate itself, which transfers the inserted nucleic acid molecule into the host cell and/or between host cells. The vector may include a vector mainly used to insert DNA or RNA into cells, a vector mainly used to replicate The invention relates to a vector for expressing a DNA or RNA, and a vector for expressing the transcription and/or translation of the DNA or RNA. The vector also includes a vector with multiple functions as described above. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Usually, the vector can produce a desired expression product by cultivating a suitable host cell containing the vector.

As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a solid carrier, or both.

In this article, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients that are incompatible with the chimeric antigen receptor, nucleic acid molecule, expression vector or transgenic immune cell of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present invention.

As used herein, the term "administration" refers to the introduction of 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 present invention can be administered by any common route as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous injection.

As used herein, the term "treatment" refers to the use of drugs to obtain the desired pharmacological and/or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and/or may be therapeutic in terms of partially or completely curing a disease and/or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or transgenic immune cell to an individual to treat, cure, alleviate, improve, mitigate or inhibit an individual's disease, including but not limited to administering a drug containing cells containing a chimeric antigen receptor as described herein to an individual in need.

In this document, "carbon terminus" and "C-terminus" are synonymous; "nitrogen terminus" and "N-terminus" are synonymous.

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

Chimeric Antigen Receptor

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

According to an embodiment of the present invention, the inventor creatively connects the full-length sequence of NKG2D and the full-length sequence of DAP10 to obtain a chimeric antigen receptor with stronger and wider NKG2D ligand binding activity. Thus, the immune cells expressing the chimeric antigen receptor of the present invention can not only effectively target and kill a variety of hematological tumors and solid tumor cells expressing NKG2D ligands, but also have the characteristics of universal CAR-immune cells (such as CAR-NK products), and can effectively kill the immunosuppressive cells MDSCs in the tumor microenvironment, effectively reversing the tumor immunosuppressive 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 present invention, the full-length sequence of NKG2D has an amino acid sequence as shown in SEQ ID NO: 1.

According to an embodiment of the present invention, the full-length sequence of DAP10 has an amino acid sequence as shown in SEQ ID NO: 2.

According to an embodiment of the present invention, the N-terminus of the intracellular signal transduction domain is connected to the C-terminus of the first fragment.

According to an embodiment of the present invention, the intracellular signal transduction domain is the intracellular segment of the CD3ζ molecule.

According to an embodiment of the present invention, the intracellular segment of the CD3ζ molecule has an amino acid sequence as shown in SEQ ID NO: 3.

Nucleic acid molecules

The present invention provides a nucleic acid molecule. The nucleic acid molecule includes a first nucleic acid molecule, and the first nucleic acid molecule encodes the chimeric antigen receptor. The immune cell carrying the first nucleic acid molecule can express the chimeric antigen receptor, and the chimeric conversion receptor activates the immune cell after recognizing multiple NKG2D ligands, thereby achieving targeted recognition of the tumor expressing the NKG2D ligand by the immune cell and exerting a killing effect.

According to an embodiment of the present invention, the first nucleic acid molecule includes nucleic acid molecule 1 and nucleic acid molecule 2; the nucleic acid molecule 1 is used to encode the first fragment and the intracellular signal transduction domain, and the nucleic acid molecule 2 is used to encode the second fragment; or the nucleic acid molecule 1 is used to encode the first fragment, and the nucleic acid molecule 2 is used to encode the second fragment and the intracellular signal transduction domain.

According to an embodiment of the present invention, the nucleic acid molecule 1 has a nucleotide sequence as shown in SEQ ID NO: 4, and the nucleic acid molecule 2 has a nucleotide sequence as shown in SEQ ID NO: 5; or the nucleic acid molecule 1 has a nucleotide sequence as shown in SEQ ID NO: 6, and the nucleic acid molecule 2 has a nucleotide sequence as shown in SEQ ID NO: 7. The nucleotide sequence shown in ID NO:7.

According to an embodiment of the present invention, the first nucleic acid molecule further includes a nucleic acid molecule 3, wherein the nucleic acid molecule 3 encodes a first linker, and the nucleic acid molecule 1 and the nucleic acid molecule 2 are connected via the nucleic acid molecule 3.

According to an embodiment of the present invention, the first linker is selected from at least one of P2A, T2A, E2A and F2A.

Thus, the immune cells carrying the above nucleic acid molecules can express the aforementioned chimeric antigen receptor. When the nucleic acid molecule includes a linker of at least one of P2A, T2A, E2A and F2A, the linker and the NKG2D and DAP10 fragments connected by the linker are expressed in immune cells. Further, the NKG2D and DAP10 polypeptide fragments are cleaved by the linker polypeptide and then assembled into a complex having NKG2D ligand binding activity.

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

According to an embodiment of the present invention, the first linker has an amino acid sequence as shown in SEQ ID NO: 8.

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

In some specific embodiments, the first nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 10.

According to an embodiment of the present invention, the above-mentioned nucleic acid molecule further includes a second nucleic acid molecule, wherein the second nucleic acid molecule encodes a fusion protein, and the fusion protein includes IL-15Rα and IL-15.

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

According to an embodiment of the present invention, the IL-15Rα has an amino acid sequence as shown in SEQ ID NO: 11.

According to an embodiment of the present invention, the IL-15 has an amino acid sequence as shown in SEQ ID NO: 12.

According to an embodiment of the present invention, the IL-15Rα described in the present invention is an IL-15 receptor α chain, including a transmembrane region; the IL-15 described in the present invention is an IL-15 mature peptide, thereby achieving membrane expression of IL-15Rα and IL-15, and further forming a complex of IL-15Rα and IL-15, referred to herein as "super IL-15". Compared with secretory IL-15 or secretory super IL-15, the expression amount in cells is controllable, the expression area is controllable, and the range of cells of action is controllable, which can avoid the side effects caused by systemic administration of recombinant IL-15 or super IL-15 or repeated administration. Therefore, the super IL-15 is co-expressed with the chimeric antigen receptor of the present invention on the surface of immune cells. On the one hand, the persistence and viability of the immune cells in the body can be improved; on the other hand, unexpectedly, the tumor killing activity of the immune cells can be significantly improved. It makes it possible to develop universal, safe and effective immune cell products.

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

According to an embodiment of the present invention, the C-terminus of the IL-15Rα is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the IL-15; or the C-terminus of the IL-15 is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the IL-15Rα.

According to an embodiment of the present invention, the connecting peptide is selected from at least one of (G ₄ S) n, ESGRSGGGGSGGGGS, EGKSSGSGSESKST, EGKSSGSGSESKSTQ, GSTSGSGKSSEGKG, KESGSVSSEQLAQFRSLD, ESGSVSSEELAFRSLD, SGGGSGGGGSGGGGSGGGGSGGGSLQ, and n is a non-zero integer.

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

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

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

According to an embodiment of the present invention, the fusion protein has an amino acid sequence as shown in SEQ ID NO: 13.

In some specific embodiments, the second nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 14.

In this way, further optimization and control of the expression level, expression area, and range of active cells of the above-mentioned super IL-15 are achieved, and the safety of the resulting genetically modified immune cells is significantly improved.

According to an embodiment of the present invention, the nucleic acid molecule further includes a third nucleic acid molecule, the third nucleic acid molecule encodes a second linker, and the first nucleic acid molecule and the second nucleic acid molecule are connected via the third nucleic acid molecule.

According to an embodiment of the present invention, the second linker is selected from at least one of P2A, T2A, E2A and F2A.

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

According to an embodiment of the present invention, the second linker has an amino acid sequence as shown in SEQ ID NO: 8.

According to an embodiment of the present invention, the third nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 9.

Thus, the super IL-15 nucleic acid molecule and the nucleic acid encoding the chimeric antigen receptor of the present invention are simultaneously expressed in a fixed ratio in the cell, thereby controlling the relative expression levels of the super IL-15 and the chimeric antigen receptor in a single cell, further improving the safety of genetically modified immune cells.

In some specific embodiments, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 15.

It should be noted that, for the nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or two of the complementary double strands. For convenience, in this article, although only one strand is provided in most cases, 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 disclosing one of them means that the other is also disclosed.

Expression vector

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

When the nucleic acid molecule is connected to the expression vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, the nucleic acid molecule and the control element can be operably connected.

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

According to an embodiment of the present invention, the non-pathogenic viral vector is selected from one of a retroviral vector, a chronic toxic vector and an adenovirus-associated viral vector.

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

According to an embodiment of the present invention, in some specific modes, after the lentiviral vector is introduced into the recipient cell, the chimeric antigen receptor of the present invention can be expressed in the immune cell.

In this article, the term "operably linked" refers to connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, viral vectors, plasmids, bacteriophages, etc. After the expression vectors according to some specific embodiments of the present invention are introduced into suitable recipient cells, the expression of the nucleic acid molecules described above can be effectively achieved under the mediation of the regulatory system, thereby achieving the in vitro acquisition of a large amount of protein encoded by the nucleic acid molecules.

cell

The present invention provides a transgenic immune cell. The transgenic immune cell expresses the chimeric antigen receptor mentioned above; or carries the nucleic acid molecule mentioned above, the expression vector mentioned above. Thus, the transgenic immune cell obtained has significantly improved tumor killing activity and better safety. Furthermore, it makes it possible to develop a universal, safe and effective immune cell product, thereby realizing a CAR-T or CAR-NK product for the treatment of multiple tumor indications. Therefore, the transgenic immune cell of the present invention has good clinical application prospects.

According to an embodiment of the present invention, the transgenic immune cells simultaneously express the chimeric antigen receptor and the fusion protein, and the fusion protein is the same as the fusion protein expressed in the aforementioned nucleic acid molecule. Thus, relative to immune cells modified only with the chimeric antigen receptor gene, the transgenic immune cells of the present invention simultaneously express membrane-bound super IL-15 on the cell membrane surface, and the transgenic immune cells obtained thereby not only have improved in vitro and in vivo proliferation capacity, but also have significantly enhanced tumor cell killing activity, showing a synergistic effect.

Moreover, the inventors further found that at the cellular level, the transgenic immune cells of the present invention showed significantly improved degranulation levels and killing functions, as well as significantly improved IFN-γ and TNF-α secretion capabilities. At the animal level, the transgenic immune cells of the present invention also showed significantly enhanced tumor suppression effects in vivo, and the transgenic immune cells of the present invention can also kill the immunosuppressive cells MDSCs in the tumor microenvironment, effectively reversing the tumor immunosuppressive microenvironment. Thus, the expression of the full-length sequence of NKG2D and the full-length sequence of DAP10 of the present invention enables NK cells to more accurately and effectively identify tumors expressing NKG2D ligands, and the gene modification strategy of the membrane-bound super IL-15 of the present invention, in addition to improving the persistence of the transgenic immune cells of the present invention in vivo and the viability of the transgenic immune cells in vivo, these two gene modification strategies unexpectedly observed synergistic beneficial effects in the present invention. That is, the gene modification strategy of the present invention can significantly improve the tumor killing activity of immune cells, making it possible to develop universal, safe and effective immune cell products, thereby realizing a CAR-T or CAR-NK product for the treatment of multiple tumor indications.

According to an embodiment of the present invention, the transgenic immune cells are obtained by introducing the expression vector into immune cells.

According to an embodiment of the present invention, the immune cells are selected from at least one of T cells, NK cells, NKT cells, γδT cells, macrophages, peripheral blood NK cells, umbilical cord blood NK cells, NK92 cells and any one of iPSC-derived immune cells. The chimeric antigen receptor of the present invention can be transduced to immune cells such as T, NK, NKT, γδT, macrophages, etc. through an expression vector (such as a lentiviral vector or a retrovirus), thereby being expressed on the surface of these immune cells.

According to an embodiment of the present invention, the immune cells are selected from at least one of T cells, NK cells, NKT cells, γδT cells, macrophages, peripheral blood NK cells, umbilical cord blood NK cells, NK92 cells, CD34 ⁺ hematopoietic stem cells or their derived immune cells, iPSCs or their derived immune cells.

In some specific embodiments, the derived immune cells are obtained by differentiation of CD34 ⁺ hematopoietic stem cells or iPSC cells.

In some specific embodiments, the derived immune cells are at least one of T cells, NK cells, NKT cells, γδT cells, and macrophages.

In some specific embodiments, the immune cells are selected from at least one of NK cells, peripheral blood NK cells, umbilical cord blood NK cells, CD34 ⁺ hematopoietic stem cells or iPSC-derived NK cells, and NK92 cells.

In some more specific embodiments, the immune cell is selected from any one NK cell lineage.

According to the embodiments of the present invention, the immune cells of the present invention have better clinical efficacy and safety, as well as wider clinical applicability, compared with immune cells that sequentially or simultaneously administer single-chain antibodies to recognize specific tumor antigens at equivalent doses and the same administration method.

Pharmaceutical composition

The present 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. Thus, the obtained pharmaceutical composition is further used for the treatment of tumors.

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

Those skilled in the art will appreciate that the features and advantages described above for the chimeric antigen receptor, nucleic acid molecule, expression vector, and transgenic immune cell are also applicable to the pharmaceutical composition and will not be described in detail here.

use

The present invention provides a use of the chimeric antigen receptor, the nucleic acid molecule, the expression vector, the transgenic immune cell or the pharmaceutical composition in preparing a drug, wherein the drug is used for preventing or treating tumors.

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

According to an embodiment of the present invention, the solid tumor is a tangible tumor occurring in an organ.

According to an embodiment of the present invention, the solid tumor is at least one selected from pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, kidney cancer, breast cancer, prostate cancer and melanoma.

According to an embodiment of the present invention, the solid tumor is at least one selected from pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, kidney cancer, breast cancer, prostate cancer, thyroid cancer, nasopharyngeal carcinoma, sarcoma, melanoma and skin squamous cell carcinoma.

According to an embodiment of the present invention, the blood tumor includes at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma and multiple myeloma.

According to an embodiment of the present invention, the blood tumor includes at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome and myeloproliferative tumors.

Methods for preventing or treating tumors

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

The effective amount of the transgenic immune cells and pharmaceutical compositions of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. Preferably, the selection of the effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The 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, the patient's weight, the patient's immune status, the route of administration, etc. For example, several divided doses may be administered daily, or the dose may be reduced proportionally, depending on the urgency of the treatment situation.

The sequence descriptions involved in the present invention are shown in Table 1.

Table 1: Nucleotide/amino acid sequence description

The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be purchased commercially.

In the following embodiments, the chimeric antigen receptor sequence comprises: a full-length sequence of NKG2D (nucleotide sequence as shown in SEQ ID NO: 7), a full-length sequence of the adapter protein DAP10 (nucleotide sequence as shown in SEQ ID NO: 5), an intracellular signal transduction molecule CD3ζ (nucleotide sequence as shown in SEQ ID NO: 16), and an IL-15-linker-IL-15Rα (nucleotide sequence as shown in SEQ ID NO: 14) gene fragment connected by P2A (nucleotide sequence as shown in SEQ ID NO: 9). The schematic diagram of the gene element structure is shown in Figure 1.

In the following embodiments, 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., the killing effect of effector cells on target cells was detected by LDH release method.

It should be noted that the "plasmid" and "vector" described in the following embodiments have the same meaning and can be used interchangeably.

Example 1 Preparation of CAR-NK cells

In this example, the CAR-NK cells of the present invention were prepared according to the following method.

1.1 Construction of CAR expression plasmid

(1) The nucleotide sequence shown in SEQ ID NO:15 was synthesized by whole gene synthesis and cloned into the lentiviral vector pCDG-EF1-MCS-TA2-copGFP through the restriction sites XbaI and BamHI. After sequencing verification, the pCDH-EF1a-CAR expression plasmid vector was obtained.

The schematic diagram of the gene element structure of the chimeric antigen receptor of this example is shown in Figure 1, and the specific sequence information is shown in Reference Table 1.

1.2 Lentivirus packaging and virus liquid concentration

Take 5×10 ⁶ 293T cells in the logarithmic growth phase and inoculate them in a 10 cm cell culture dish, add 10 mL of DMEM medium, and culture overnight in a 37°C, 5% CO ₂ incubator. When the cell density in the cell culture dish reaches 80%, replace it with 10 mL of fresh DMEM medium for virus packaging, and continue to place the cell culture dish in the incubator for standby use. Prepare the lentiviral packaging system, add 6 μg of lentiviral packaging auxiliary plasmid psPAX2 and 3 μg of pMD2.G, and 6 μg of the target gene vector plasmid to 250 μL of serum-free DMEM medium to prepare a plasmid mixture, and mix well. Add to 235 μL serum-free DMEM medium and mix well. Add the mixture to the above plasmid mixture at one time, mix well, and incubate at room temperature for 15 minutes. Add the mixture to the 293T cell culture dish. After 24 hours, change the liquid, put the culture dish back into the 37°C, 5% _CO2 incubator, collect the cell supernatant after 48 hours, centrifuge at 400×g for 5 minutes, remove the cell debris, and filter the supernatant with a 0.45μm filter into a 50mL centrifuge tube. Add 5×PEG8000 solution to concentrate the virus solution, mix evenly by inverting the centrifuge tube upside down, and place in a 4°C refrigerator overnight. Centrifuge at 4000×g for 20 minutes at 4°C, discard the supernatant, add an appropriate amount of serum-free DMEM to resuspend the virus precipitate, transfer it to an EP tube, and store it in a -80°C refrigerator.

1.3 Lentiviral titer detection

Take 293T cells in the logarithmic growth phase and adjust the concentration to 1×10 ⁵ /mL. Take a 24-well plate, add 1mL of cell suspension (1×10 ⁵ /well) to each well, and set up 3 gradients of virus volume. Place in a 37°C, 5% CO ₂ incubator for overnight culture. First dilute the concentrated virus solution 10 times: take a 1mL EP tube, draw 60μL of virus concentrate into the EP tube, dilute with 540μL DMEM culture medium, and mix evenly. Replace the 293T cells with fresh DMEM culture medium, draw 5μL, 50μL and 500μL of diluted virus solution into the corresponding wells, mark them, and then put the culture plate back into the 37°C, 5% CO ₂ incubator. After 24h, discard the virus solution in the well plate, and add 1mL of fresh DMEM culture medium. After 72h, harvest the cells with trypsin digestion, use a flow cytometer to detect the GFP expression rate of 293T cells, and convert the virus titer 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 growth phase were harvested by centrifugation at 100×g for 5min, and the cells were resuspended in an appropriate amount of α-MEM medium to adjust the cell density to 5×10 ⁵ /mL. 5×10 ⁵ NK92 cells were inoculated into 24-well plates, 1mL of virus concentrate and protamine (final concentration 8μg/mL) were mixed evenly. Cultured in a 37°C, 5% CO ₂ incubator. After 24h, the cell state was observed, the medium was changed, the infected cells were transferred to an EP tube, centrifuged at 100×g for 5min, a small amount of fresh _α -MEM medium was added to resuspend the cells, the cells were transferred to a cell culture flask, 10mL of fresh _α -MEM medium and IL-2 (final concentration of 200IU/mL) were added, and the culture was continued for 48h. The cells were transferred to a flow tube, 3mL of 1×PBS solution was added, centrifuged at 100×g for 5min, the supernatant was discarded, the cell pellet was flicked, and the 1×PBS solution was used to wash again. The expression rate of GFP was detected using a flow cytometer. Continue to expand the culture, adjust the state of the infected NK92 cells for amplification, and select the GFP-positive CAR-NK92 cells from the infected NK92 cells by flow cytometry for later experiments.

1.5 Lentiviral infection of primary expanded human peripheral blood NK cells

Peripheral blood mononuclear cells (PBMCs) were isolated and inoculated in pre-coated culture flasks for culture. Referring to Chinese patent CN 202310035787.4, anti-CD3 monoclonal antibodies and cytokines such as IL-2 were used for induced culture. Lentivirus infection was performed according to the above method on the 7th day of culture. After changing the medium on the 9th day, the culture was continued, and the cells were harvested on the 19th day for in vivo experimental treatment.

Example 2 Determination of in vitro killing, proliferation and survival ability of CAR-NK cells

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

2.1 CAR gene modification enhances the killing of NK92 cells against NKG2D ligand-positive tumors in vitro

First, flow cytometry was used to detect the expression of NKG2D ligand MICA/B in several tumor cell lines (liver cancer cells, breast cancer cells, and non-small cell lung cancer), and the results are shown in Figure 2. 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 expressed high levels of MICA/B, while liver cancer cell line SMMC-7721 cells expressed low levels and were basically negative. The inventors selected the H7402 cell line with high expression of MICA/B and the SMMC-7721 cells with basically negative expression as target cells to detect the effect of CAR gene modification on the killing activity of NK92 cells.

The specific method is as follows: NK92 and CAR-NK92 cells were co-incubated with liver cancer cell lines H7402 or SMMC-7721 cells for 4 hours, and the killing efficiency was detected by LDH release method; at the same time, NKG2D antibody (Ab blockade) was added to the killing system to block, and controls for NK92 and CAR-NK92 cell groups were set up respectively. The ratios of effector cells (NK cells) to target cells (tumor cells) were investigated to be 10:1, 5:1 and 2.5:1.

The test results are shown in Figure 3: Referring to Example 1 of the present invention, the gene-modified CAR-NK92 cells of the present invention have a significantly higher killing efficiency against MICA/B-positive H7402 cells than NK92 cells when the effector-target ratio is 10:1 and 5:1. The killing efficiency of the CAR-NK92 and NK92 cells of the present invention against SMMC-7721 cells and H7402 cells after adding NKG2D antibody blocking in the killing system is significantly reduced.

The above test results show that the NKG2D chimeric antigen receptor of the present invention is reasonably designed and can effectively recognize the NKG2D ligand MICA/B; further, NK cells modified with the NKG2D chimeric antigen receptor of the present invention and the IL-15 gene expressed on the cell membrane have significantly enhanced killing activity, showing a synergistic effect.

Furthermore, the CAR-NK92 of the present invention and NK92 cells were co-incubated with H7402 cells at an effector-target ratio of 5:1 to detect the expression of NK cell killing effector molecules CD107a, Granzyme B and perforin. The IgG group was a negative control group without flow cytometry antibody staining.

The specific method is as follows: 5 μL PE-anti-human CD107a antibody (clone number H4A3, Biolegend) was added to the CD107a group, and PMA (Sigma, final concentration of 30 ng/mL) and ionomycin (Sigma, final concentration of 1 μg/mL) were added to the positive control group, and the culture was continued for 1 hour. The blocking agent BFA and monensin (Biolegend, ratio 1:1000) were added and the culture was continued for 3 hours. The cells were collected in a centrifuge tube, centrifuged and washed with 1×PBS solution, and then the cells were resuspended in an appropriate amount of 1×PBS solution. The expression of CD107a was detected by flow cytometry. Granzyme B and Perforin groups, 100 μL/tube of fixative solution was added, incubated at room temperature for 15 minutes, and 1×PBS solution was added for centrifugation and washing. Then, 100 μL/tube of transmembrane solution was added and the corresponding antibodies (APC-anti-human Perforin, clone number dG9; Alexa Fluor 647-anti-human Granzyme B, clone number GB11, Biolegend), incubated at room temperature for 30 min, added 1× PBS solution for centrifugation and washing, added appropriate amount of 1× PBS solution to resuspend the cells, and the expression levels of Granzyme B and Perforin were detected by flow cytometry.

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

The above experimental results further verified that the CAR gene modification strategy of the present invention can significantly improve the degranulation level and killing function of NK cells against NKG2D ligand-positive tumor cells.

2.2 CAR gene modification enhances cytokine secretion levels of NK92 cells

The secretion capacity changes of IFN-γ and TNF-α of the CAR-NK92 cells of the present invention were further detected by flow cytometry. The specific method is as follows: NK92 or CAR-NK92 cells were co-incubated with NKG2D ligand-positive liver cancer cell line H7402 cells for 4 hours, NK92 or CAR-NK92 cells were collected in a flow tube, and after fixed membrane treatment, flow cytometry was used to detect the expression levels of intracellular IFN-γ and TNF-α in NK92 or CAR-NK92 cells. The IgG group is a negative control group without the addition of flow antibody staining.

The test results are shown in FIG5 : the levels of IFN-γ and TNF-α expressed by the CAR-NK92 cells of the present invention were significantly higher than those of the NK92 group.

The ELISA method was further used to detect the level of IFN-γ in the culture supernatant of NK92 cells and H7402 cells. The experimental results also showed that the level of IFN-γ secreted by CAR-NK92 cells was significantly higher than that of the NK92 group (Figure 5).

The above test results indicate that the CAR structure of the present invention can significantly improve the secretion capacity of IFN-γ and TNF-α when NK cells come into contact with NKG2D ligand-positive tumor cells.

2.3 CAR gene modification promotes the survival and proliferation of NK92 cells

The inventors further verified the promoting effect of membrane-expressed IL-15 on the in vitro proliferation and survival of NK92 cells. The specific method is as follows: NK92 and CAR-NK92 cells with the same number of cells were plated in 96-well plates, and different IL-2 concentrations (200IU/ml, 20IU/ml and 0IU/ml) were set. The apoptosis rate was detected by flow cytometry every 24 hours or the trypan blue staining cell count was performed every 48 hours. The apoptosis rate was detected by flow cytometry according to the steps in the kit instructions (Lianke Bio, catalog number AP101), which is briefly as follows: collect cells in EP tubes, add 1xPBS solution, centrifuge and wash once, and resuspend the cells. Add 5μL Annexin V-FITC and 10μL PI to each tube. After gentle vortex mixing, incubate at room temperature in the dark for 5 minutes, and resuspend the cells for flow detection. The cell viability is the ratio of double negatives of Annexin V-FITC and PI staining.

The results of the NK cell survival rate test after 96 hours of culture are shown in Figure 6: In the absence of IL-2 (IL-2 0IU/ml), the cell survival rate of the NK92 group decreased significantly from 24 hours, and most cells had undergone apoptosis at 72 hours; while the cells in the CAR-NK92 group could still maintain a high cell survival rate even under the condition of complete withdrawal of IL-2, and few cells underwent apoptosis. This suggests that IL-15 expressed on the membrane of CAR-NK92 plays an important role in promoting the survival of NK cells.

Further observation and culture were performed until the 8th day, and the cell proliferation curve was drawn. The results are shown in Figure 7: The proliferation ability of the CAR-NK92 cells of the present invention under different IL-2 concentrations was significantly stronger than that of the NK92 cells under the same IL-2 concentration.

The above test results show that, contrary to the inventors' expectations, when NK92 cells were cultured in vitro, the effect of the membrane-expressed IL-15 element of the present invention on promoting NK cell proliferation was significantly enhanced compared with adding free IL-2 to the cell culture medium, which was more conducive to the in vitro proliferation culture of NK92 and the industrialization of the immune cell product of the present invention.

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

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

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

The inventors first examined the expression of NKG2D ligands MICA/B and ULBP in human ovarian cancer cell lines SKOV3 and A1847, and the results are shown in Figure 8: SKOV3 mainly expresses ULBP2/5/6, while A1847 cells have high expression of MICA/B and ULBP2/5/6. Therefore, human ovarian cancer mouse xenograft models were established with SKOV3 and A1847 cell lines, respectively, to observe the therapeutic effect of the CAR-NK92 cells designed and prepared by the present invention on ovarian cancer.

The specific method is as follows: 6-week-old NCG mice were selected for subcutaneous tumor bearing in the armpits, and the tumor bearing dose was 2.5×10 ⁶ cells/mouse. NK cell therapy was started on the 7th day of tumor bearing. The tumor volume was measured before treatment, and the mice were randomly divided into PBS group, NK92 cell treatment group and CAR-NK92 cell treatment group according to the tumor volume. The mice in the treatment group were injected with 1×10 ⁷ effector cells/mouse through the tail vein, and the untreated group was injected with an equal volume of 1×PBS, once every week, for a total of 3 treatments, and IL-2 (5×10 ⁴ IU/mouse) was injected through the tail vein every 3 days. The tumor volume was measured every 3 or 4 days, and the tumor growth curve was drawn.

The test results are shown in Figures 9 and 10: (1) The therapeutic effect on the SKOV3 ovarian cancer model showed that CAR-NK92 cells had a stronger effect of inhibiting tumor growth than NK92. On the 18th day after treatment, the tumor inhibition rate of the CAR-NK92 treatment group was 64.2%, while that of the NK92 treatment group was 32.3% (Figure 9). (2) The therapeutic effect on the A1847 ovarian cancer model showed that CAR-NK92 cells had a stronger effect of inhibiting tumor growth than NK92. On the 19th day after treatment, the tumor inhibition rate of the CAR-NK92 treatment group was 55.8%, while that of the NK92 treatment group was 42.9% (Figure 10).

The above results show that the CAR-NK92 cells expressing NKG2D and membrane-bound IL-15 of the present invention have significantly enhanced in vivo tumor inhibition effects on NKG2D ligand-positive tumors. The expression of NKG2D-CAR enables NK cells to more accurately and effectively identify tumors expressing NKG2D ligands, while the membrane-expressed IL-15 gene modification can improve the persistence of CAR-NK92 cells in vivo and improve the viability of CAR-NK92 cells in vivo; the two synergistically enhance the anti-tumor effect of CAR-NK92 cells.

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

The inventors established a human pancreatic cancer mouse xenograft tumor model using the human pancreatic cancer cell line HPAF-II, infected human peripheral blood primary NK cells with the CAR lentivirus of Example 1 of the present invention to prepare peripheral blood-derived CAR-NK cells, and observed the therapeutic effect of CAR-NK cells on the pancreatic cancer model.

The specific method is as follows: 6-week-old NCG mice were selected for subcutaneous tumor bearing in the armpit, and NK cell treatment was started when the tumor volume reached 50 mm ^3. The tumor volume was measured before treatment, and the mice were randomly divided into PBS group, non-genetically modified NK cell control group and three CAR-NK cell treatment groups according to the tumor volume. The mice in the non-genetically modified NK cell control group were treated once every 2 days for a total of 3 times, and the dose of each tail vein reinfusion was 4×10 ⁶ CD56 ⁺ NK cells, and IL-2 was injected intraperitoneally every 2 days to maintain NK cell survival; the mice in the CAR-NK cell treatment group were injected with CAR-NK cells through the tail vein, and the single treatment was 1 time, with a dose of 4×10 ⁶ , 8×10 ⁶ and 1.6×10 ⁷ CAR-NK cells/mouse, respectively. Because CAR-NK cells contain IL-15 gene modification, IL-2 injection was not performed; the untreated group was injected with an equal volume of 1×PBS. The tumor volume was measured twice a week, the tumor growth curve was drawn, and the tumor inhibition rate was statistically calculated.

The test results are shown in Figure 11: The therapeutic effect on the HPAF-II pancreatic cancer model shows that the peripheral blood-derived CAR-NK cells prepared based on the present invention have a significantly enhanced effect of inhibiting tumor growth compared with the NK cell control group that is not infected with the CAR lentivirus of Example 1 of the present invention; and it is dose-dependent, with the tumor inhibition rate of the 4×10 ⁶ CAR ^- NK cell dose group being 56.78%, and the tumor inhibition rate of the 1.6×10 ⁷ CAR ^- NK cell dose group being 80.97%.

Example 4 CAR-NK cells can effectively kill immunosuppressive cells MSDCs in the tumor microenvironment

In this example, the inventors investigated the in vitro killing activity of the CAR-NK92 cells of the present invention against MDSCs and the anti-tumor activity in a tumor-bearing mouse model obtained in Example 1. In vivo, MDSCs were mixed with tumor cells and then mice were subjected to tumor-bearing treatment, and NK92 or CAR-NK92 cells were reinfused for treatment, and tumor burden was observed by in vivo imaging.

4.1 Induction of MDSCs from human peripheral blood PBMCs

The inventors induced MDSCs from human peripheral blood PBMCs.

The specific method is as follows: Human peripheral blood PBMC induces MDSC cells: After isolating human peripheral blood PBMC, plate and add 100ng/mL IL-4 and 100ng/mL GM-CSF for 24h, and then add 25μg/mL Poly (I:C) for 24h. Discard the supernatant and collect the attached cells with trypsin digestion, which are the induced MDSCs.

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

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

The specific method is as follows: NK92 and CAR-NK92 cells were co-incubated with MDSCs for 4 hours, and the killing efficiency was detected by LDH release method; at the same time, NKG2D antibody (Ab blockade) was added to the killing system to block, and the control of NK92 and CAR-NK92 cell groups was set up respectively. The ratio of effector cells to target cells was 10:1, 5:1 and 2.5:1.

The test results are shown in Figure 12: The gene-modified CAR-NK92 cells of the present invention have a significantly higher killing efficiency against MICA/B-positive MDSCs than NK92 cells when the effector-target ratio is 10:1, 5:1 and 2.5:1. After adding NKG2D antibody blocking to the killing system, the killing efficiency of NK92 and CAR-NK92 cells was significantly reduced.

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

The specific method is as follows:

Method for establishing a mouse model bearing human liver cancer H7402 cells containing MDSCs: On day 0, 6-7 week-old mice were selected, and each mouse was subcutaneously implanted with axilla tumors by mixing 1x10 ⁶ luciferase-labeled H7402 cells with 6x10 ⁵ MDSCs. On day 3 after tumor implantation, NK92 or CAR-NK92 cells were reinfused into the tail vein for treatment at a dose of 5x10 ⁶ cells/mouse, and reinfused once every 3 days for a total of 3 times. At the same time, IL-2 solution was intraperitoneally injected every 3 days at a dose of 5x10 ⁴ IU/mouse. On day 10, in vivo imaging observations were performed.

The test results are shown in Figure 13: Through the results of in vivo imaging, it can be observed that there is no significant difference in tumor load between the NK92 cell group and the untreated group. The tumor load of the CAR-NK92 cell group is significantly lower than that of the untreated group and the NK92 cell group. This shows that CAR-NK92 cells can effectively kill MDSCs in the tumor microenvironment and eliminate the inhibitory effect of MDSCs in the tumor on immune effector cells, thus having a stronger tumor inhibition effect than NK92 cells.

The above test results show that the CAR-NK cells of the present invention have a strong killing effect on MDSCs in the tumor, can resist the immunosuppression of MDSCs on CAR-NK or CAR-T cells, exert a more powerful anti-tumor effect, and break through the bottleneck of immune cell therapy being subject to immunosuppression by the solid tumor microenvironment.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (47)

A chimeric antigen receptor, comprising: A first fragment, wherein the first fragment is a full-length sequence of NKG2D; a second fragment, wherein the first fragment is a full-length sequence of DAP10, and the first fragment and the second fragment are connected; An intracellular signal transduction domain, wherein the N-terminus of the intracellular signal transduction domain is connected to the C-terminus of the first fragment or the second fragment. The chimeric antigen receptor according to claim 1 is characterized in that the full-length sequence of NKG2D has an amino acid sequence as shown in SEQ ID NO: 1. The chimeric antigen receptor according to claim 1 is characterized in that the full-length sequence of DAP10 has an amino acid sequence as shown in SEQ ID NO: 2. The chimeric antigen receptor according to any one of claims 1 to 3, characterized in that the N-terminus of the intracellular signal transduction domain is connected to the C-terminus of the first fragment. The chimeric antigen receptor according to claim 4, characterized in that the intracellular signal transduction domain is the intracellular segment of the CD3ζ molecule. The chimeric antigen receptor according to claim 5 is characterized in that the intracellular segment of the CD3ζ molecule has an amino acid sequence as shown in SEQ ID NO: 3. A nucleic acid molecule, characterized in that it comprises a first nucleic acid molecule, wherein the first nucleic acid molecule encodes the chimeric antigen receptor according to any one of claims 1 to 6. The nucleic acid molecule according to claim 7, characterized in that the first nucleic acid molecule comprises nucleic acid molecule 1 and nucleic acid molecule 2; The nucleic acid molecule 1 is used to encode the first fragment and the intracellular signal transduction domain, and the nucleic acid molecule 2 is used to encode the second fragment; or The nucleic acid molecule 1 is used to encode the first fragment, and the nucleic acid molecule 2 is used to encode the second fragment and the intracellular signal transduction domain. The nucleic acid molecule according to claim 8, characterized in that the nucleic acid molecule 1 has a nucleotide sequence as shown in SEQ ID NO: 4, and the nucleic acid molecule 2 has a nucleotide sequence as shown in SEQ ID NO: 5; or The nucleic acid molecule 1 has a nucleotide sequence as shown in SEQ ID NO: 6, and the nucleic acid molecule 2 has a nucleotide sequence as shown in SEQ ID NO: 7. The nucleic acid molecule according to claim 8 is characterized in that the first nucleic acid molecule further comprises a nucleic acid molecule 3, wherein the nucleic acid molecule 3 encodes a first linker, and the nucleic acid molecule 1 and the nucleic acid molecule 2 are connected via the nucleic acid molecule 3. The nucleic acid molecule according to claim 10, characterized in that the first linker is selected from at least one of P2A, T2A, E2A and F2A. The nucleic acid molecule according to claim 11, characterized in that the first linker is P2A. The nucleic acid molecule according to claim 10 is characterized in that the first linker has an amino acid sequence as shown in SEQ ID NO: 8. The nucleic acid molecule according to claim 10 is characterized in that the nucleic acid molecule 3 has a nucleotide sequence as shown in SEQ ID NO: 9. The nucleic acid molecule according to claim 7 is characterized in that the first nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 10. The nucleic acid molecule according to any one of claims 7 to 15, further comprising a second nucleic acid molecule, wherein the second nucleic acid molecule encodes a fusion protein comprising IL-15Rα and IL-15. The nucleic acid molecule according to claim 16, characterized in that the N-terminus of the IL-15Rα is connected to the C-terminus of the IL-15, or the N-terminus of the IL-15 is connected to the C-terminus of the IL-15Rα. The nucleic acid molecule according to claim 16 is characterized in that the IL-15Rα has an amino acid sequence as shown in SEQ ID NO: 11. The nucleic acid molecule according to claim 16 is characterized in that the IL-15 has an amino acid sequence as shown in SEQ ID NO: 12. The nucleic acid molecule according to claim 16, characterized in that the fusion protein further comprises a connecting peptide; The C-terminus of the IL-15Rα is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the IL-15; or The C-terminus of the IL-15 is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the IL-15Rα. The nucleic acid molecule according to claim 20, characterized in that the connecting peptide is selected from at least one of ( _G4S )n, ESGRSGGGGSGGGGS, EGKSSGSGSESKST, EGKSSGSGSESKSTQ, GSTSGSGKSSEGKG, KESGSVSSEQLAQFRSLD, ESGSVSSEELAFRSLD, SGGGSGGGGSGGGGSGGGGSGGGSLQ, and n is a non-zero integer. The nucleic acid molecule according to claim 21, characterized in that the connecting peptide is selected from (G ₄ S) _n , where n is any integer between 2 and 6. The nucleic acid molecule according to claim 22, characterized in that the connecting peptide is (G ₄ S) ₃ . The nucleic acid molecule according to claim 21 is characterized in that the connecting peptide is SGGGSGGGGSGGGGSGGGGSGGGSLQ. The nucleic acid molecule according to claim 16, characterized in that the fusion protein has the amino acid sequence shown in SEQ ID NO: 13. The nucleic acid molecule according to claim 16 is characterized in that the second nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 14. The nucleic acid molecule according to claim 16 is characterized in that the nucleic acid molecule further comprises a third nucleic acid molecule, the third nucleic acid molecule encodes a second linker, and the first nucleic acid molecule and the second nucleic acid molecule are connected through the third nucleic acid molecule. The nucleic acid molecule according to claim 27, characterized in that the second linker is selected from at least one of P2A, T2A, E2A and F2A. The nucleic acid molecule according to claim 28 is characterized in that the second linker is P2A. The nucleic acid molecule according to claim 27 is characterized in that the second linker has an amino acid sequence as shown in SEQ ID NO: 8. The nucleic acid molecule according to claim 27 is characterized in that the third nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 9. The nucleic acid molecule according to claim 7 is characterized in that the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 15. An expression vector, characterized in that it carries the nucleic acid molecule according to any one of claims 7 to 32. The expression vector according to claim 33 is characterized in that the expression vector is a non-pathogenic viral vector. The expression vector according to claim 34 is characterized in that the non-pathogenic viral vector is selected from one of a retroviral vector, a chronic toxic vector and an adenovirus-associated viral vector. A genetically modified immune cell, comprising: Expressing the chimeric antigen receptor according to any one of claims 1 to 6; or Carrying the nucleic acid molecule according to any one of claims 7 to 32 or the expression vector according to any one of claims 33 to 35. The transgenic immune cell according to claim 36 is characterized in that the transgenic immune cell simultaneously expresses the chimeric antigen receptor and the fusion protein, and the fusion protein is the same as the fusion protein encoded by the nucleic acid molecule according to claim 16 or 27. The transgenic immune cell according to claim 36 is characterized in that the transgenic immune cell is obtained by introducing the expression vector into an immune cell. The transgenic immune cell according to claim 36 is characterized in that the immune cell is selected from at least one of T cells, NK cells, NKT cells, γδT cells, macrophages, peripheral blood NK cells, umbilical cord blood NK cells, NK92 cells, CD34 ⁺ hematopoietic stem cells or immune cells derived therefrom, iPSCs or immune cells derived therefrom. A pharmaceutical composition, characterized in that it comprises: The chimeric antigen receptor according to any one of claims 1 to 6, the nucleic acid molecule according to any one of claims 7 to 32, the expression vector according to any one of claims 33 to 35, or the transgenic immune cell according to any one of claims 36 to 39. The pharmaceutical composition according to claim 40 is characterized in that it further comprises: a pharmaceutically acceptable excipient. Use of the chimeric antigen receptor according to any one of claims 1 to 6, the nucleic acid molecule according to any one of claims 7 to 32, the expression vector according to any one of claims 33 to 35, the transgenic immune cell according to any one of claims 36 to 39, or the pharmaceutical composition according to any one of claims 40 to 41 in the preparation of a drug for preventing or treating a tumor. The use according to claim 42, characterized in that the tumor is a solid tumor or a hematological tumor. The use according to claim 43 is characterized in that the solid tumor is a tangible tumor occurring in an organ. The use according to claim 44 is characterized in that the solid tumor includes at least one selected from pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, kidney cancer, breast cancer, prostate cancer, thyroid cancer, nasopharyngeal carcinoma, sarcoma, melanoma and skin squamous cell carcinoma. The use according to claim 43 is characterized in that the blood tumor includes at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome and myeloproliferative tumors. A method for preventing and/or treating tumors, comprising: administering to a subject a pharmaceutically acceptable amount of the transgenic immune cell according to any one of claims 36 to 39 or the pharmaceutical composition according to any one of claims 40 to 41.