Chimeric receptor and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a chimeric receptor containing NKp46 and application of the chimeric receptor. When the selected chimeric receptor is constructed into the immune cell modified by the chimeric receptor, an excellent technical effect can be achieved. The effect capability of the constructed chimeric receptor is obviously superior to that of a traditional chimeric antigen receptor structure. The method has a wide application prospect in adoptive cell therapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a novel chimeric receptor comprising a NKp46 transmembrane region and applications thereof. Background Art

[0002] In recent years, the development of immunotherapy has brought profound changes to the field of tumor treatment, especially immune checkpoint therapy represented by CTLA-4, PD1 / PDL1 pathway inhibitors and adoptive cell therapy represented by chimeric antigen receptor (CAR)-T. Adoptive cell therapy includes TIL, NK, TCR-T, CAR-T / NK / NKT / TIL / Among them, CAR-T therapy targeting CD19 has achieved excellent clinical efficacy in B-cell tumors, and in 2017, two CAR-T products were approved by the FDA for the treatment of B-cell leukemia or lymphoma.

[0003] As an emerging immunotherapy, NK cell therapy has broad application prospects. In cancer treatment, NK cells can directly identify and kill tumor cells, and have potential therapeutic effects on a variety of cancers, including breast cancer, lung cancer, gastric cancer, colorectal cancer and other cancer types. In addition, NK cell therapy can also be used in the treatment of autoimmune diseases. By enhancing the function of NK cells, the balance of the immune system can be effectively regulated, thereby achieving the purpose of treating autoimmune diseases. In addition, NK cell therapy can also be used in the treatment of infectious diseases. NK cells can kill a variety of pathogens, such as bacteria and viruses, so they can be used to treat certain diseases, such as AIDS and hepatitis. Finally, NK cell therapy can also be used for treatment after stem cell transplantation. By improving the body's immune function, diseases such as aplastic anemia that occurs after stem cell transplantation can be prevented and treated. In short, with the in-depth study of NK cell biology and the continuous optimization of CAR structure design, NK cell therapy is expected to become one of the important options for future tumor treatment and expand its application in the fields of autoimmunity, infectious diseases and stem cell transplantation.

[0004] NK cell therapy has certain advantages over traditional T cell therapy. First, NK cells have a broader spectrum of anti-tumor effects. They can quickly identify and attack tumor cells without antigen presentation. Secondly, NK cell therapy is safer and does not cause serious side effects such as cytokine release syndrome or graft-versus-host disease, so it is safer and more reliable. Finally, since NK cells can be used immediately and do not need to be customized for each patient, treatment time is saved and it is more suitable for the application of spot-based therapies. Although NK cell therapy is relatively new, its unique advantages have made it a focus of attention in tumor treatment. With the in-depth study of NK cell biology and the continuous optimization of CAR structural design, NK cell therapy is expected to become one of the important options for future tumor treatment.

[0005] Traditional chimeric antigen receptor (CAR) is a synthetic transmembrane receptor that enables immune cells to specifically recognize and attack target cells by fusing the antigen recognition domain and the signal transduction domain. At present, the traditional CAR structure is mainly designed for T cells. Because T cells have strong cytotoxicity and the ability to secrete cytokines, they are widely used in immunotherapy such as tumor treatment. The application effect of existing CAR structures in NK cells is not ideal. At present, there is an urgent need to design a CAR structure suitable for NK cells to improve the killing activity of NK cells and their specific recognition ability of tumor cells. Summary of the invention

[0006] Since NK cells play an important role in the immune system, but the chimeric antigen receptor CAR structure currently used is mainly designed for T cells, it is necessary to redesign a chimeric receptor structure suitable for NK cells to better utilize the killing activity and specific recognition ability of NK cells. The new chimeric receptor structure can optimize the surface molecular distribution and signal transduction pathways of NK cells to improve their ability to recognize and kill tumor cells, and can better regulate the effector function of NK cells to enhance their killing effect on target cells. Therefore, the development of a new chimeric receptor structure suitable for NK cells is of great significance for its application in tumors, infections, autoimmune diseases, and aging-related diseases.

[0007] The purpose of the present invention is to provide a chimeric receptor structure based on NKp46 suitable for NK cells to improve the killing activity of NK cells and their specific recognition ability for tumor cells, and to solve some problems existing in existing NK cell therapy and improve the effect and safety of tumor immunotherapy.

[0008] In one aspect, the present invention provides a chimeric antigen receptor, which comprises an extracellular recognition region, a hinge region and a transmembrane region, wherein the transmembrane region comprises a NKp46 transmembrane region.

[0009] The present invention also provides nucleic acid molecules encoding the chimeric receptor, vectors comprising the nucleic acid molecules, cells comprising the chimeric receptor, nucleic acid molecules, vectors, compositions comprising the chimeric receptor, nucleic acid molecules, vectors, or cells, and uses of the chimeric receptor, nucleic acid molecules, vectors, or cells in the preparation of drugs for preventing or treating tumors, viral infections, aging or autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0011] Figure 1 Schematic diagram of the structure of CD70 chimeric receptor based on NKp44 or NKp46;

[0012] Figure 2 The results are flow cytometric detection results of the expression level of NKp44 or NKp46 chimeric receptors on the HEK293 cell membrane;

[0013] Figure 3 is the level of Raji-Luc killing of target cells by each CD70 CAR-NK cell;

[0014] Figure 4 It is the detection of IFN-γ secretion when each CD70 CAR-NK cell is co-incubated with the target cell Raji-Luc;

[0015] Figure 5 Schematic diagram of the CLDN18.2 chimeric receptor structure based on NKp46, the level of CLDN18.2 CAR-NK cell killing of corresponding target cells, and the detection of IFN-γ secretion when co-incubated with corresponding target cells;

[0016] Figure 6 Schematic diagram of the structure of CD19 chimeric receptor based on NKp46, the level of CD19 CAR-NK cells killing corresponding target cells, and the detection of IFN-γ secretion when co-incubated with corresponding target cells;

[0017] Figure 7 Schematic diagram of the CD20 chimeric receptor structure based on NKp46, the level of CD20CAR-NK cell killing of corresponding target cells, and the detection of IFN-γ secretion when co-incubated with corresponding target cells. DETAILED DESCRIPTION

[0018] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0019] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0020] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0021] As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0022] Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0023] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0024] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0025] As used herein, the "region" or "domain" contained in the chimeric antigen receptor refers to a region in a polypeptide that can fold into a specific structure independently of other regions. These "regions" or "domains" can be sequences of mouse or other animal origin, preferably human sequences. In addition, when "regions" or "domains" are not specifically distinguished or emphasized, they should be understood as known sequences, which can be full-length or partially active segments.

[0026] The application effect of existing chimeric antigen receptor CAR structures in NK cells is not ideal, mainly because the surface molecular distribution of NK cells is quite different from that of T cells, so the chimeric receptor structure needs to be redesigned to better utilize the killing activity of NK cells. In addition, the signal transduction pathway of NK cells is also different from that of T cells, so a more effective chimeric receptor structure needs to be designed based on the characteristics of NK cells.

[0027] It is possible to explore the synergistic effect achieved by activating multiple signal transduction pathways of NK cells at the same time, thereby improving the killing activity and persistence of NK cells. The design of chimeric receptor structures is not only to improve the recognition and killing ability of NK cells, but also to achieve better tumor treatment effects by regulating the effector functions of NK cells. For example, it is possible to explore the use of chimeric receptor structures to induce effector functions such as cytokine secretion and granzyme release of NK cells to enhance the killing effect on tumor cells. Safety issues are very important when designing and applying chimeric receptor structures of NK cells. It is necessary to pay attention to whether the chimeric receptor structure will cause self-activation or over-activation of NK cells, and whether it will lead to the production of adverse toxic side effects.

[0028] On the one hand, the present invention constructs a chimeric receptor, which comprises an extracellular recognition region, a hinge region, and a transmembrane region, wherein the transmembrane region comprises a NKp46 transmembrane region. The chimeric receptor of the present application can couple the activation signaling pathway of NK cells through the NKp46 transmembrane region, and endow the immune cells transducing the chimeric receptor with a stronger function of killing target cells.

[0029] The NKp46 transmembrane region described herein may be a natural NKp46 transmembrane region, or a variant or fragment that retains the signal transduction function of the transmembrane region; preferably, the NKp46 transmembrane region is a human NKp46 transmembrane region; more preferably, the amino acid sequence of the NKp46 transmembrane region is: MGLAFLVLVALVWFLVEDWLS (SEQ ID NO: 13).

[0030] The chimeric receptors in this article have a wider range than traditional chimeric antigen receptors. The chimeric receptors in this article can specifically recognize antigens, receptors or ligands, that is, the extracellular recognition region of the chimeric receptors in this article is selected from antibodies or antibody fragments, ligands or ligand fragments, receptors or receptor fragments, or fusion proteins formed by two or three of antibodies, ligands, and receptors.

[0031] In some embodiments, the extracellular recognition region is an antibody or an antibody fragment; in some embodiments, the antibody or antibody fragment specifically binds to a tumor antigen.

[0032] Tumor antigens include both tumor-specific antigens and tumor-associated antigens. In some embodiments, the tumor antigen is selected from: α-fetoprotein, BMCA, carbonic anhydrase IX, CD4, CD5, CD19, CD20, CD30, CD44, CD46, CD70, CD79a, CD79b, CD133, CD138, CLDN family proteins, colon-specific antigen p, CEA, CEACAM-6, c-Met, DAM, EGFR, EGFRvIII, Ep-CAM, fibroblast activation protein α, fibroblast growth factor, Flt-1, Flt-3, folate receptor, GD2, gp100, HLA-DR, HER2 / neu, IL-13 At least one of R, Le-Y, LDR / FUT, MAGE, MART1, MART-2, NY-ESO-1, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, mesothelin, pancreatic cancer mucin, PD1 receptor, prostate stem cell antigen, placental growth factor, p53, PLAGL2, PRAME, PSMA, SAGE, S100, survivin, survivin-2B, TAC, TAG-72, tenascin, Tn antigen, Thomson-Fredenreich antigen, tumor necrosis antigen, VEGFR, WT-1, 17-1A antigen and Kras.

[0033] In some embodiments, the extracellular recognition region of the chimeric receptor is CD70 scFv, CLDN18.2 VHH, CD19 scFv (FMC63) or CD20 scFv. In some specific embodiments, the extracellular recognition region comprises an anti-CD70 scFv with an amino acid sequence as shown in SEQ ID NO: 2, an anti-CLDN18.2 VHH with an amino acid sequence as shown in SEQ ID NO: 8, an anti-CD19 scfv with an amino acid sequence as shown in SEQ ID NO: 11 (FMC63), and an anti-CD20 scfv with an amino acid sequence as shown in SEQ ID NO: 12.

[0034] The extracellular recognition region of the chimeric receptor herein is connected by a hinge region and a transmembrane region, and the hinge region enables the extracellular recognition region to obtain space to display its own structure. In some embodiments, the hinge region is selected from the hinge region of CD8, CD28, IgG1, IgG4, 4-1BB, ICOS, OX40, CD40, CD80, CD7, NKp46, or CH3, CH2-CH3 constant region; preferably, it is the NKp46 hinge region. The hinge region can be a hinge region, a variant or a fragment thereof of a natural protein or polypeptide; preferably, it is a hinge region of a human protein or polypeptide. In a specific embodiment, the hinge region is a NKp46 hinge region, and the amino acid sequence is: GDIENTSLAPEDPTFPADTWGTYLLTTETGLQKDHALWDHTAQNL LR (SEQ ID NO: 14).

[0035] In some embodiments, the chimeric receptor further comprises an intracellular region; preferably, the intracellular region is the NKp46 intracellular region; preferably, the intracellular region sequence is RKRTRERASRASTWEGRRRLNTQTL (SEQ ID NO: 15).

[0036] Some conventional chimeric antigen receptor structures include a CD8 hinge region and a CD8 transmembrane region (SEQ ID NO: 3):

[0037] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

[0038] Intracellular regions of some traditional chimeric antigen receptor structures: 4-1BB ICD+CD3ζ (SEQ ID NO: 4):

[0039] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0040] Some conventional chimeric antigen receptor structures include a CD28 hinge region and a CD28 transmembrane region (TM) (SEQ ID NO: 9)

[0041] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSL LVTVAFIIFWV

[0042] Some conventional chimeric antigen receptor structures include the intracellular domain: CD28 ICD + CD3ζ (SEQ ID NO: 10)

[0043] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0044] In some embodiments of the present application, the hinge region, transmembrane region and intracellular region amino acid sequences of the chimeric receptor respectively comprise the amino acid sequences of positions 212-258, 259-279 and 280-304 of NKp46, and the specific amino acid sequences are as follows:

[0045] GDIENTSLAPEDPTFPADTWGTYLLTTETGLQKDHALWDHTAQNLLRMGLAFLV LVALVWFLVEDWLSRKRTRERASRASTWEGRRRLNTQTL(SEQ ID NO: 5)

[0046] Variants of SEQ ID NO: 5 are also within the scope of the present invention, and the variants may have, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO: 5. The variants may also have conservative substitutions as described above.

[0047] In some embodiments, the intracellular region further comprises a co-stimulatory signal region; in some embodiments, the co-stimulatory signal region is selected from 2B4, DNAM1, CD28, 4-1BB, OX40, ICOS, CD27, MYD88, KIR2DS2, DAP10, DAP12, CD3ζ, TLRs, CD2, LFA-1, CD8α, CD40, CD80, CD3ε or a combination thereof or a mutant thereof.

[0048] In a specific embodiment, the hinge region (Hinge) + transmembrane region (TM) + intracellular region is NKp46 hinge + TM + cytoplasmic, and further comprises a 4-1BB costimulatory structure, and the specific amino acid sequence is as follows:

[0049] GDIENTSLAPEDPTFPADTWGTYLLTTETGLQKDHALWDHTAQNLLRMGLAFLV LVALVWFLVEDWLSRKRTRERASRASTWEGRRRLNTQTLKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCEL(SEQ IDNO: 6)

[0050] Exemplary costimulatory signal regions that can be used in the present invention and corresponding exemplary sequences are shown in the following table:

[0051]

[0052]

[0053]

[0054] In some embodiments, the chimeric receptor or control chimeric antigen receptor further comprises the following signal peptide sequence:

[0055] Signal peptide: CD8a (SEQ ID NO: 1): MALPVTALLLPLALLLHAARP.

[0056] Signal peptide: GM-CSF (SEQ ID NO: 7): MLLLVTSLLLCELPHPAFLLIP.

[0057] According to yet another aspect of the present invention, it also relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the chimeric receptor as described above.

[0058] In this article, nucleic acid includes variants of its conservative substitution (e.g., substitution of degenerate codons) and complementary sequences, and also includes variants optimized by codons to be more efficiently expressed in desired host cells. Nucleic acid is typically RNA or DNA, including genes, cDNA molecules, mRNA molecules, and fragments thereof such as oligonucleotides. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively connected". For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is effectively connected to the coding sequence. It is preferred to use DNA nucleic acids when it is linked to a vector.

[0059] The present invention also relates to a vector comprising the nucleic acid molecule as described above.

[0060] The term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; CRISPR / CAS plasmids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0061] The present invention also relates to cells expressing the chimeric antigen receptor as described above, or cells comprising the chimeric receptor, nucleic acid molecule, vector as described above. In some embodiments, the cells are immune cells, such as one or more of T cells, B cells, NK cells, macrophages, dendritic cells, etc.

[0062] In some embodiments, the immune cells are T cells.

[0063] The T cells may be subtypes well known in the art, such as one or more of helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, MAIT cells, NKT cells, and γδ T cells.

[0064] In some embodiments, the immune cells are NK cells.

[0065] In some embodiments, the immune cells are further modified to contain or express a T cell receptor (TCR) and / or a chimeric antigen receptor (CAR).

[0066] According to yet another aspect of the present invention, it also relates to a pharmaceutical composition comprising the chimeric receptor, nucleic acid molecule, vector or cell as described above.

[0067] The pharmaceutical composition may also include a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any material that allows the component to maintain biological activity when combined with the active ingredient and does not react with the subject's immune system. Examples include, but are not limited to, any of standard pharmaceutical carriers (such as phosphate buffered saline solutions, water, emulsions (such as oil / water emulsions)) and various types of wetting agents. Exemplary diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are prepared by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 21st edition, Mack Publishing, 2005).

[0068] According to another aspect of the present invention, it also relates to the use of the chimeric receptor, nucleic acid molecule, vector, cell or composition as described above in the preparation of a drug for preventing or treating tumors, viral infections, aging or autoimmune diseases.

[0069] In some embodiments, the medicament is used for preventing or treating tumors.

[0070] According to yet another aspect of the present invention, it also relates to a method for treating a tumor in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the chimeric receptor, nucleic acid molecule, vector, cell or composition as described above.

[0071] Tumors can be solid tumors or hematological tumors. In the present invention, "solid tumors" include: tumors generated by lesions in any of the following: bones, bone connections, muscles, lungs, trachea, heart, spleen, arteries, veins, capillaries, lymph nodes, lymphatic vessels, lymph fluid, oral cavity, pharynx, esophagus, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum, testicles, vas deferens, penis, eyes, ears, nose, tongue, skin, brain, brainstem, medulla oblongata, spinal cord, cerebrospinal fluid, nerves, thyroid gland, parathyroid gland, adrenal gland, pituitary gland, pineal gland, pancreatic islets, thymus gland, gonads, sublingual gland and parotid gland. 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, multiple myeloma, etc. in blood cells and the hematopoietic system.

[0072] A "patient" is a mammal, including but not limited to humans, monkeys, pigs and other farm animals, sports animals, pets, primates, horses, dogs, cats, giant pandas, rodents (including mice, rats, guinea pigs), etc.

[0073] Embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0074] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0075] Example

[0076] 1. Construction and screening of novel chimeric receptor structures

[0077] In order to construct a chimeric receptor structure suitable for NK cells, different chimeric receptor molecular structures were constructed based on NKp44 and NKp46 NK cell activating receptors. In the NKp44 chimeric receptor structure, the extracellular Ig-like domain of NKp44 was replaced with a scFv sequence that binds to a specific antigen; in the NKp46 chimeric receptor structure, the extracellular Ig-like domain 1 or Ig-like domains 1 and 2 of NKp46 were removed and replaced with a scFv sequence that binds to a specific antigen; optionally, the intracellular signaling regions CD28, CD28-CD3ζ, 4-1BB, and 4-1BB-CD3ζ were added to the above structures. The specific structures are as follows: Figure 1 23222 is a traditional chimeric antigen receptor structure. The specific amino acid sequences of each structure are as follows:

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] The above structure was constructed into a eukaryotic expression vector: first, the above gene fragment was obtained by gene synthesis, and then the above gene fragment was constructed into a eukaryotic expression vector pCMV by a classical gene cloning method. Gene sequencing verification showed that each plasmid sequence was consistent with the target sequence.

[0088] Since synthetic artificial proteins may have problems such as misfolding and aggregation during gene expression, resulting in protein instability or poor expressibility, we first detected the expression of the above-mentioned chimeric receptor structures on the cell membrane in HEK293 cells. The above-mentioned plasmids were transfected into HEK293 cells by chemical transfection, and the expression of each chimeric receptor structure on the cell membrane was detected by flow cytometry 24 to 48 hours after transfection. The research results are as follows Figure 2 A shows that, except for NKp44(121-276), all chimeric receptor structures based on NKp44 cannot be normally displayed on the cell membrane. However, the chimeric receptor based on NKp46 can detect the expression of chimeric receptor structures, and the expression level of chimeric receptor structures based on NKp46(212~304) is higher than that of chimeric receptor structures based on NKp46(119~304), such as Figure 2 B. Further analysis found that the addition of CD28 intracellular region would result in the inability to display the chimeric receptor structure, while 4-1BB had little effect on the expression of the chimeric receptor structure. Further addition of CD3ζ would further affect the display of the chimeric receptor structure. The results are shown in Figure 2 A and 2B. The above results indicate that the chimeric receptor structure constructed based on the NK natural activating receptor NKp46 (212-304) can be normally expressed and displayed on the cell membrane surface.

[0089] 2. In vitro effector function study of NKp46-based chimeric receptor structure targeting CD70

[0090] In order to study the functions of the above-mentioned chimeric receptor structures, we constructed the above-mentioned chimeric receptor structures and traditional CAR structures into NK92 cells by electroporation and studied their ability to kill target cells in vitro and secrete cytokines.

[0091] Preparation of CAR-NK cells: First, NK92 cells were revived and cultured. After the cell state was restored, each group of cells was electroporated separately, and the blank control was not subjected to the transfer of chimeric receptor structure. During the electroporation process, 3ug plasmid / 2E6 was used, and the plasmid concentration should be greater than 500ng / ul. The electroporated cells were transferred into a complete medium without antibiotics for continued culture, and a new complete medium was replaced in about 24 hours. After about 48 hours, the electroporation efficiency can be detected, and CAR-NK is successfully prepared and can be used for subsequent in vitro experiments. Continue to expand the culture to about day 8, and test the electroporation stability every 3 days. When the expression of CAR-NK is stable, it can be used for subsequent functional experiments and the cells can be frozen for standby use.

[0092] Detection of CAR-NK killing target cells and IFN-γ secretion ability: First, collect target Raji cells, wash and resuspend them in culture medium, add them to the round bottom plate at 5E4 / 100ul / well, two replicates per group, and place 2 more wells of control target cell self-release group without effector cells. Next, we collect effector cells, stain them with CFSE, wash them twice with culture medium and resuspend them, and add them to the corresponding well plates at E:T=0.1:1 and 0.3:1. We mix the cells and centrifuge them, centrifuge them at 200g for 2 minutes, culture them in a 37℃ incubator, and incubate them for about 4 hours. After incubation, aspirate 140ul supernatant and use ELISA method for IFN-γ detection. Then, centrifuge to remove the supernatant, wash once with PBS, centrifuge at 400g for 5 minutes, remove the supernatant, add 2ul Annexin V+100ul binding buffer premixed to each well to resuspend the cells. Finally, we incubate them at room temperature for 15 minutes, add 100ul / well binding buffer, and detect them on the machine. The killing efficiency of CAR-T cells was evaluated by calculating the ratio of the live cell ratio of the experimental group to the target cell self-release group. Using the live cell ratio of the target cell self-release group as the denominator, killing efficiency = 1-experimental group ÷ target cell self-release. In this way, we can effectively detect the killing efficiency of CAR-NK cells.

[0093] The research results are as follows Figure 3 and Figure 4 The results showed that the chimeric receptor structure based on NKp46 (23223) had a higher target cell apoptosis ratio and IFN-γ release level compared with the CAR-NK constructed with the traditional CAR structure (23222). The above results indicate that the chimeric receptor structure based on NKp46 can better activate the effector function of NK cells.

[0094] 3. Construction and screening of new chimeric receptor structures targeting CLDN18.2 and study of in vitro effector functions.

[0095] Based on the expression of the NKp46-based CD70-targeting CAR structure and the in vitro function of CD70 CAR-NK in Example 1-2, in order to further verify the scalability of the CAR structure on other targets, we constructed a new chimeric receptor targeting CLDN18.2 based on NKp46. The specific structure is as follows Figure 5 A. The specific amino acid sequences of the traditional CAR and the constructed new chimeric receptor are as follows:

[0096] 24182 Sequence:

[0097] Signal peptide: GM-CSF (SEQ ID NO: 7)

[0098] VHH: CLDN18.2 VHH (SEQ ID NO: 8)

[0099] Hinge region + transmembrane region (TM): CD28 hinge + CD28 TM (SEQ ID NO: 9)

[0100] Intracellular region: CD28 ICD + CD3ζ (SEQ ID NO: 10)

[0101] 24186 Sequence:

[0102] Signal peptide: GM-CSF (SEQ ID NO: 7)

[0103] VHH: CLDN18.2 VHH (SEQ ID NO: 8)

[0104] Hinge region (Hinge) + transmembrane region (TM) + intracellular region: NKp46 hinge + TM + cytoplasmic (SEQ ID NO: 5)

[0105] 24190 Sequence:

[0106] Signal peptide: GM-CSF (SEQ ID NO: 7)

[0107] VHH: CLDN18.2 VHH (SEQ ID NO: 8)

[0108] Hinge region (Hinge) + transmembrane region (TM) + intracellular region: NKp46 hinge + TM + cytoplasmic + 4-1BB ICD (SEQ ID NO: 6)

[0109] The above structure was constructed into a eukaryotic expression vector: first, the above gene fragment was obtained by gene synthesis, and then the above gene fragment was constructed into a eukaryotic expression vector pCMV by a classical gene cloning method. Gene sequencing verification showed that each plasmid sequence was consistent with the target sequence.

[0110] In order to study the function of the above-mentioned chimeric receptor structure on the CLDN18.2 target, we constructed the above-mentioned chimeric receptor structures and traditional CAR structures into NK92 cells by electroporation, and studied their ability to kill target cells in vitro and secrete cytokines. The method and process refer to Example 2.

[0111] The research results are as follows Figure 5 As shown, CAR-NK cells targeting CLDN18.2 constructed based on NKp46 (24186) and NKp46+4-1BB (24190) can detect the normal expression of CLDN18.2 CAR structure on the cell surface by flow cytometry ( Figure 5 B), when killing target cells NUGC4, compared with the CAR-NK constructed with the traditional chimeric receptor structure (24182), it has a higher target cell apoptosis ratio ( Figure 5 C) and IFN-γ release levels ( Figure 5 D).

[0112] 4. Construction and screening of novel chimeric receptor structures targeting CD19 and study of in vitro effector functions

[0113] In order to further verify the scalability of the CAR structure on other targets, we constructed a new chimeric receptor targeting CD19 based on NKp46. Figure 6 A. The specific amino acid sequences of the traditional CAR and the constructed new chimeric receptor are as follows:

[0114] 24183 Sequence:

[0115] Signal peptide: GM-CSF (SEQ ID NO: 7)

[0116] scFv: FMC63 (SEQ ID NO: 11)

[0117] Hinge region + transmembrane region (TM): CD28 hinge + CD28 TM (SEQ ID NO: 9)

[0118] Intracellular region: CD28 ICD + CD3ζ (SEQ ID NO: 10)

[0119] 24187 Sequence:

[0120] Signal peptide: GM-CSF (SEQ ID NO: 7)

[0121] scFv: FMC63 (SEQ ID NO: 11)

[0122] Hinge region (Hinge) + transmembrane region (TM) + intracellular region: NKp46 hinge + TM + cytoplasmic (SEQ ID NO: 5)

[0123] 24191 Sequence:

[0124] Signal peptide: GM-CSF (SEQ ID NO: 7)

[0125] scFv: FMC63 (SEQ ID NO: 11)

[0126] Hinge region (Hinge) + transmembrane region (TM) + intracellular region: NKp46 hinge + TM + cytoplasmic + 4-1BB ICD (SEQ ID NO: 6)

[0127] The above structure was constructed into a eukaryotic expression vector: first, the above gene fragment was obtained by gene synthesis, and then the above gene fragment was constructed into a eukaryotic expression vector pCMV by a classical gene cloning method. Gene sequencing verification showed that each plasmid sequence was consistent with the target sequence.

[0128] In order to study the function of the above-mentioned chimeric receptor structure on the CD19 target, we constructed the above-mentioned chimeric receptor structures and traditional CAR structures into NK92 cells by electroporation, and studied their ability to kill target cells and secrete cytokines in vitro. The method and process refer to Example 2.

[0129] The research results are as follows Figure 6 As shown, the CAR-NK cells constructed based on the novel chimeric receptor structures 24187 and 24191 targeting CD19 and the traditional chimeric receptor structure (24183) can be detected by flow cytometry to show normal expression of CD19 CAR structure on the cell surface ( Figure 6 B), when killing target cells Raji, the CAR-NK constructed with the traditional chimeric receptor structure (24182) has a higher target cell apoptosis ratio ( Figure 6 C) and IFN-γ release levels ( Figure 6 D).

[0130] 5. Construction and screening of novel chimeric receptor structures targeting CD20 and in vitro effector function studies

[0131] In order to further verify the scalability of the CAR structure on other targets, we constructed a new chimeric receptor targeting CD20 based on NKp46. Figure 7 A. The specific amino acid sequences of the traditional CAR and the constructed new chimeric receptor are as follows:

[0132] Construction and screening of novel chimeric receptor structures of CD19 and CD20 The above-mentioned embodiments only express several embodiments of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims, and the description and drawings can be used to interpret the content of the claims.

[0133] Based on the expression of the NKp46-based CD70-targeting CAR structure and the in vitro function of CD70 CAR-NK in Example 1-2, in order to further verify the scalability of the CAR structure on other targets, we constructed a new chimeric receptor targeting CLDN18.2, CD19 and CD20 based on NKp46. The specific structure is as follows Figure 5 The specific amino acid sequences of the traditional CAR and the constructed new chimeric receptor are as follows:

[0134] 24182 Sequence:

[0135] Signal peptide: GM-CSF (SEQ ID NO: 7)

[0136] VHH: CLDN18.2 VHH (SEQ ID NO: 8)

[0137] Hinge region + transmembrane region (TM): CD28 hinge + CD28 TM (SEQ ID NO: 9)

[0138] Intracellular region: CD28 ICD + CD3ζ (SEQ ID NO: 10)

[0139] 24186 Sequence:

[0140] Signal peptide: GM-CSF (SEQ ID NO: 7)

[0141] VHH: CLDN18.2 VHH (SEQ ID NO: 8)

[0142] Hinge region (Hinge) + transmembrane region (TM) + intracellular region: NKp46 hinge + TM + cytoplasmic (SEQ ID NO: 5)

[0143] 24190 Sequence:

[0144] Signal peptide: GM-CSF (SEQ ID NO: 7)

[0145] VHH: CLDN18.2 VHH (SEQ ID NO: 8)

[0146] Hinge region (Hinge) + transmembrane region (TM) + intracellular region: NKp46 hinge + TM + cytoplasmic + 4-1BB ICD (SEQ ID NO: 6)

[0147] 24183 Sequence:

[0148] Signal peptide: GM-CSF (SEQ ID NO: 7)

[0149] scFv: FMC63 (SEQ ID NO: 11)

[0150] Hinge region + transmembrane region (TM): CD28 hinge + CD28 TM (SEQ ID NO: 9)

[0151] Intracellular region: CD28 ICD + CD3ζ (SEQ ID NO: 10)

[0152] 24187 Sequence:

[0153] Signal peptide: GM-CSF (SEQ ID NO: 7)

[0154] scFv: FMC63 (SEQ ID NO: 11)

[0155] Hinge region (Hinge) + transmembrane region (TM) + intracellular region: NKp46 hinge + TM + cytoplasmic (SEQ ID NO: 5)

[0156] 24191 Sequence:

[0157] Signal peptide: GM-CSF (SEQ ID NO: 7)

[0158] scFv: FMC63 (SEQ ID NO: 11)

[0159] Hinge region (Hinge) + transmembrane region (TM) + intracellular region: NKp46 hinge + TM + cytoplasmic + 4-1BB ICD (SEQ ID NO: 6)

[0160] 24184 Sequence:

[0161] Signal peptide: GM-CSF (SEQ ID NO: 7)

[0162] scFv:CD20 SCFV (SEQ ID NO: 12)

[0163] Hinge region + transmembrane region (TM): CD8 hinge + CD8 TM (SEQ ID NO: 3)

[0164] Intracellular region: 4-1BB ICD+CD3ζ (SEQ ID NO: 4)

[0165] 24192 Sequence:

[0166] Signal peptide: GM-CSF (SEQ ID NO: 7)

[0167] scFv:CD20 SCFV (SEQ ID NO: 12)

[0168] Hinge region (Hinge) + transmembrane region (TM) + intracellular region: NKp46 hinge + TM + cytoplasmic + 4-1BB ICD (SEQ ID NO: 6)

[0169] The above structure was constructed into a eukaryotic expression vector: first, the above gene fragment was obtained by gene synthesis, and then the above gene fragment was constructed into a eukaryotic expression vector pCMV by a classical gene cloning method. Gene sequencing verification showed that each plasmid sequence was consistent with the target sequence.

[0170] In order to study the function of the above-mentioned chimeric receptor structure on the CD20 target, we constructed the above-mentioned chimeric receptor structures and traditional CAR structures into NK92 cells by electroporation, and studied their ability to kill target cells in vitro and secrete cytokines. The method and process refer to Example 2.

[0171] The research results are as follows Figure 7 We further verified that the CAR-NK cells constructed by the novel chimeric receptor structure 24192 and the traditional chimeric receptor structure (24184) on the CD20 target can detect the normal expression of the CD20 CAR structure on the cell surface by flow cytometry ( Figure 7 B), and also had a higher target cell apoptosis ratio when killing target cells Raji ( Figure 7 C) and IFN-γ release levels ( Figure 7 D).

[0172] The above results indicate that the novel chimeric receptor structures based on NKp46 and NKp46+4-1BB can better activate the effector function of NK cells, and have similar functional characteristics at different targets, proving that the novel chimeric receptor structures have high application potential at different targets.

[0173] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A chimeric receptor comprising an extracellular recognition region, a hinge region and a transmembrane region, wherein the transmembrane region comprises a NKp46 transmembrane region.

2. The chimeric receptor according to claim 1, wherein the extracellular recognition region is selected from an antibody or an antibody fragment, a ligand or a ligand fragment, a receptor or a receptor fragment, or a fusion protein formed between two or three of an antibody, a ligand, and a receptor.

3. The chimeric receptor according to claim 2, wherein the extracellular recognition region is an antibody or an antibody fragment; preferably, the antibody or antibody fragment specifically binds to a tumor antigen.

4. The chimeric receptor of claim 3, wherein the tumor antigen is selected from the group consisting of: alpha-fetoprotein, BMCA, carbonic anhydrase IX, CD4, CD5, CD19, CD20, CD30, CD44, CD46, CD70, CD79a, CD79b, CD133, CD138, CLDN family proteins, colon-specific antigen p, CEA, CEACAM-6, c-Met, DAM, EGFR, EGFRvIII, Ep-CAM, fibroblast activation protein alpha, fibroblast growth factor, Flt-1, Flt-3, folate receptor, GD2, gp100, HLA-DR, HER2 / ne u, IL-13R, Le-Y, LDR / FUT, MAGE, MART1, MART-2, NY-ESO-1, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, mesothelin, pancreatic cancer mucin, PD1 receptor, prostate stem cell antigen, placental growth factor, p53, PLAGL2, PRAME, PSMA, SAGE, S100, survivin, survivin-2B, TAC, TAG-72, tenascin, Tn antigen, Thomson-Fredenreich antigen, tumor necrosis antigen, VEGFR, WT-1, 17-1A antigen and Kras.

5. The chimeric receptor according to any one of claims 1 to 4, characterized in that The hinge region is selected from the hinge region of CD8, CD28, IgG1, IgG4, 4-1BB, ICOS, OX40, CD40, CD80, CD7, NKp46, or CH3, CH2-CH3 constant region; preferably, it is the NKp46 hinge region.

6. The chimeric receptor according to any one of claims 1 to 5, further comprising an intracellular region; preferably, the intracellular region is the NKp46 intracellular region.

7. The chimeric receptor according to any one of claims 1 to 6, wherein the NKp46 transmembrane region comprises the amino acid sequence of positions 259 to 279 of NKp46, as shown in SEQ ID NO: 13; and / or The hinge region comprises the amino acid sequence of positions 212-258 of NKp46, as shown in SEQ ID NO: 14; and / or The intracellular region comprises the amino acid sequence of positions 280-304 of NKp46, as shown in SEQ ID NO:

15.

8. The chimeric receptor according to any one of claims 1-7, wherein the intracellular region further comprises a co-stimulatory signaling region.

9. The chimeric receptor of claim 8, wherein the co-stimulatory signal region is selected from 2B4, DNAM1, CD28, 4-1BB, OX40, ICOS, CD27, MYD88, KIR2DS2, DAP10, DAP12, CD3ζ, TLRs, CD2, LFA-1, CD8α, CD40, CD80, CD3ε or a combination thereof; preferably, the co-stimulatory signal region is 4-1BB.

10. A nucleic acid molecule comprising a nucleic acid sequence encoding the chimeric receptor according to any one of claims 1 to 9. A vector comprising the nucleic acid molecule according to claim 10 .

12. A cell comprising the chimeric receptor of any one of claims 1 to 9, the nucleic acid molecule of claim 11, or the vector of claim 11.

13. The cell according to claim 12, which is an immune cell, preferably, the immune cell is selected from T, NK, DC, monocyte, macrophage or B cell, more preferably, the immune cell is a NK cell.

14. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the chimeric receptor according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, the vector according to claim 11, or the cell according to claim 12 or 13.

15. Use of the chimeric receptor according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, the vector according to claim 11, or the cell according to claim 12 or 13 in the preparation of a drug for preventing or treating tumors, viral infections, aging or autoimmune diseases.

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