Novel chimeric receptor compositions, recombinant vectors, cells, and their applications

Abstract

A novel chimeric receptor composition, recombinant vector, cell and their application. The novel chimeric receptor composition comprises a conventional chimeric antigen receptor and an NKG2D chimeric receptor comprising the full-length sequence or truncated fragment of NKG2D, DAP10 and / or DAP12, and is abbreviated as CAR with SNR. The chimeric receptor composition provided by the present invention allows conventional CAR-T cells to express the NKG2D extracellular domain and intracellular signal domain, broadening the CAR-T antigen recognition spectrum, resolving tumor heterogeneity, enhancing the killing ability of CAR-T against tumor cells expressing the target antigen, while achieving a relatively low level of factor release, reducing the possibility of the occurrence of cytokine storm, and improving the safety of CAR-T.

Description

Technical Field

[0001] [Cross - reference to Related Applications] This application claims the priority of a Chinese patent with application number 202110793058.6, titled "Novel Chimeric Receptor Composition, Recombinant Vector, Cell and Their Applications", filed with the China National Intellectual Property Administration on July 14, 2021, and all of its contents are incorporated herein by reference.

[0002] The present invention relates to the technical field of biopharmaceuticals, and specifically, to novel chimeric receptor compositions, recombinant vectors, cells and their applications.

Background Art

[0003] Conventional tumor treatment methods include surgery, radiotherapy, chemotherapy, targeted therapy, etc. However, the development of immunotherapy in recent years has brought profound changes to the field of tumor treatment, especially immunotherapy represented by immune checkpoint therapy typified by CTLA - 4, PD - 1 / PD - L1 pathway inhibitors and adoptive cell therapy typified by CAR - T. Adoptive cell therapy includes TIL, NK, TCR - T, CAR - T / NK / NKT / TIL / Mφ, etc. Among them, the CAR - T therapy targeting CDi9 has achieved excellent clinical effects in B - cell tumors, and in 2017, two CAR - T products were approved by the FDA for use in the treatment of B - cell leukemia or lymphoma.

[0004] While CAR-T therapy has made significant progress in hematological malignancies, the incidence of side effects such as cytokine storms is relatively high in the treatment of hematological malignancies, and most patients require the use of adjuncts to mitigate the side effects caused by cytokine storms during CAR-T therapy. CAR-T therapy is less effective in the treatment of solid tumors, partly due to tumor heterogeneity, the relatively weak ability of CAR-T cells to kill solid tumors, and the inability to completely eliminate tumor cells. Furthermore, CAR-T therapy in solid tumors has a higher incidence and level of cytokine storms than in hematological malignancies, the side effects are more severe, and the ratio of benefits to clinical risks is lower, all of which affect the clinical acceptance of CAR-T therapy by patients with solid tumors.

[0005] Current solutions typically involve using dual targets to address tumor heterogeneity, intervening during or before treatment, symptomatically treating cytokine storms with antibodies, or designing safety switches on CAR-T cells to mitigate CAR-T toxic side effects by neutralizing cytokines or removing CAR-T cells with antibodies if cytokine storms or CAR-T hyperproliferation occur.

[0006] NK cells express NKG2D on their surface and, upon recognizing its ligands, such as MICA and MICB, activate downstream signaling molecules such as DAP10 and DAP12 to kill target cells. T cells, on the other hand, express NKG2D on their surface and DAP10 intracellularly, but not DAP12. Under normal circumstances, when surface NKG2D on T cells recognizes its ligand, they are unable to effectively activate intracellular signaling molecules and, consequently, cannot kill target cells. This invention aims to design conventional CAR-T cells to express both the extracellular and intracellular signaling regions of NKG2D, thereby expanding the CAR-T antigen recognition spectrum while simultaneously resolving tumor heterogeneity. [Overview of the project]

[0007] To overcome the shortcomings of the prior art, the present invention provides novel chimeric receptor compositions or fusion proteins, recombinant vectors, cells, and applications thereof that expand the CAR-T antigen recognition spectrum, resolve tumor heterogeneity, reduce the likelihood of cytokine storms, and enhance the safety of CAR-T.

[0008] To achieve the above objectives, the present invention employs the following technical approach: A first aspect of the present invention provides a novel chimeric receptor composition or fusion protein comprising a chimeric antigen receptor and an NKG2D chimeric receptor comprising the full-length sequence or cleaved fragments of NKG2D, DAP10, and / or DAP12.

[0009] Furthermore, the chimeric receptor composition or fusion protein comprises a chimeric antigen receptor and an NKG2D chimeric receptor, wherein the NKG2D chimeric receptor includes an NKG2D extracellular domain and a DAP12 intracellular domain sequence.

[0010] Furthermore, the NKG2D chimeric receptor further contains a costimulatory molecule, which is selected from CD28, 4-1BB, DAP10, ICOS, OX40, and CD40.

[0011] Furthermore, the above-mentioned chimeric antigen receptor includes an extracellular recognition region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region.

[0012] Furthermore, the extracellular recognition region described above includes an antibody or antibody fragment that recognizes a tumor-associated antigen or a tumor-specific antigen.

[0013] Furthermore, the above tumor-associated antigens or tumor-specific antigens include CD19, BCMA, CD22, CD20, CD123, CD30, CD38, CD138, CD56, CD7, CLL-1, CD10, CD34, CS1, CD16, CD4, CD5, IL-1-RAP, ITGB7, k-IgG, TAC1, TRBC1, MUC1, NKG2D, PD-L1, CD133, CD177, LeY, CD70, ROR1, AFP, AXL, and C. Selected from D80, CD86, DLL3, DR5, FAP, LMP1, MAGE-A1, MAGE-A4, MG7, MUC16, PMEL, ROR2, VEGFR2, CD171, Claudin18.2, Claudin6, EphA2, ErbB, Fra, PSCA, cMet, IL13Ra2, EPCAM, EGFR, PSMA, EGFRvIII, GPC3, CEA, HER2, GD2, and Mesothelin.

[0014] Furthermore, the sequence of the hinge region is derived from at least one of CD8α, CD28, 4-1BB, ICOS, OX40, CD40, CD80, and IgG; the sequence of the transmembrane region is derived from at least one of CD2, CD27, LFA-1 (CD11a / CD18), CD8α, CD28, 4-1BB, ICOS, OX40, CD40, CD80, CD3ζ, and CD3ε; and the sequence of the intracellular signaling region is derived from at least one of Toll-like receptors, CD2, CD27, LFA-1 (CD11a / CD18), CD8α, CD28, 4-1BB, ICOS, OX40, CD40, CD80, DAP10, DAP12, CD3ζ, and CD3ε.

[0015] Furthermore, the above chimeric receptor composition further comprises a linking peptide that connects the chimeric antigen receptor and the chimeric receptor, wherein the linking peptide is a self-cleaving polypeptide 2A peptide, and the 2A peptide comprises F2A, P2A, T2A and E2A, preferably P2A, and its amino acid sequence is Sequence ID No. 9.

[0016] Furthermore, the amino acid sequence of the above-mentioned chimeric receptor is selected from one of the sequences of SEQ ID NOs: 1 to 8, preferably SEQ ID NOs: 3 or 6.

[0017] A second aspect of the present invention provides nucleic acids encoding the above-mentioned chimeric receptor composition or fusion protein.

[0018] A third aspect of the present invention provides a vector containing the above-mentioned nucleic acid.

[0019] A fourth aspect of the present invention provides cells that express the above-mentioned chimeric receptor composition or fusion protein and contain the above-mentioned nucleic acid or vector.

[0020] Furthermore, the above cells are selected from among T cells, NK cells, DC cells, and macrophages.

[0021] Furthermore, the T cells mentioned above are selected from αβT cells, γδT cells, or NKT cells.

[0022] A fifth aspect of the present invention provides a biologic for tumor treatment, comprising the above-mentioned cells as the main active ingredient.

[0023] A sixth aspect of the present invention provides a method for producing the cells, comprising the step of transfecting the cells with the vector.

[0024] A seventh aspect of the present invention provides applications of the above-mentioned chimeric receptor composition, nucleic acid, vector, or cell in the production of an antitumor drug.

[0025] This invention adopts the above-mentioned technical proposal and has the following technical advantages compared to the prior art: The chimeric receptor composition or fusion protein provided by the present invention enables conventional CAR-T cells to express the NKG2D extracellular region and intracellular signal region, expands the CAR-T antigen recognition spectrum, resolves tumor heterogeneity, enhances the killing ability of CAR-T against tumor cells expressing the target antigen, while achieving relatively low levels of factor release, reducing the possibility of cytokine storm generation, and improving the safety of CAR-T.

Brief Description of the Drawings

[0026] [Figure 1] The expression status of CLDN18.2 and NKG2D Ligands mRNA in PDX models derived from gastric cancer (A), pancreatic cancer (B), and esophageal cancer (C) in one embodiment of the present invention. [Figure 2] The expression status of CLDN18.2 and NKG2D ligands at the protein level in a gastric cancer tissue chip in one embodiment of the present invention. [Figure 3] The schematic diagram of the structure of the chimeric receptor composition with SEQ ID Nos. 21007, 21067 - 21074 in one embodiment of the present invention. [Figure 4] The expression results of CAR-T cells anti-CLDN18.2-CAR and NKG2D constructed in one embodiment of the present invention are shown. [Figure 5] The high expression of CLDN18.2 by the cell line 293-CLDN18.2 constructed in one embodiment of the present invention is shown. [Figure 6] The relatively high level of expression of the NKG2D ligand MICA / B by 293T in one embodiment of the present invention is shown. [Figure 7] The killing effect of CAR-T cells with SNR on the cell line 293-CLDN18.2 in one embodiment of the present invention is shown. [Figure 8] The growth multiple of CAR-T by co-incubation with the cell line 293-CLDN18.2 in one embodiment of the present invention is shown. [Figure 9]This demonstrates the killing effect of CAR-T cells equipped with SNR in one embodiment of the present invention on 293T cells. [Figure 10] This embodiment of the present invention demonstrates that CAR-T cells equipped with SNR have lower factor secretion levels when killing target cells 293-CLDN18.2, of which Figures A to D show the secretion levels of IL-2, IFN-γ, IL-6, and TNF-α, respectively. [Figure 11] This example demonstrates that CAR-T cells equipped with SNR in one embodiment of the present invention release moderate levels of cytokines when killing target cells expressing MICA / B, with Figures A-D showing the secretion levels of IFN-γ, TNF-α, IL-6, and IL-2, respectively. [Figure 12] This is a schematic diagram of a CLDN18.2 CAR equipped with an SNR according to one embodiment of the present invention. [Figure 13] This is a diagram showing the expression results of ULBP2 / 5 / 6 in A431 in one embodiment of the present invention. [Figure 14] This figure shows the results of an in vitro euthanasia and cytokine secretion experiment in one embodiment of the present invention. In Figures C and D, the columns are unT, 21047, 21326, 21327, 21328, and 21329, in order from left to right. [Figure 15] This describes the effect of SNR on CAR-T cell proliferation, memory phenotype, and depletion in one embodiment of the present invention. [Figure 16] This is the result of an in vivo efficacy study experiment of CLDN18.2 uniform expression in one embodiment of the present invention. [Figure 17] This is an A431 rechallenge experiment in one embodiment of the present invention. [Figure 18] This is a drug efficacy experiment of the A431 tumor model in one embodiment of the present invention. In Figure B, the columns from left to right are PBS, UNT, 21047, 21327, 21328, and 21329, with the horizontal axis coinciding with the PBS group. [Figure 19]This is the result of a drug efficacy experiment on a CLDN18.2 heterogeneous tumor model in one embodiment of the present invention. [Figure 20] This is a schematic diagram of an EPCAM CAR and a CD19 CAR equipped with an SNR in one embodiment of the present invention. [Figure 21] The expression results of CAR-T cells anti-EPCAM-CAR, anti-CD19-CAR, and NKG2D constructed in one embodiment of the present invention are shown. [Figure 22] This figure shows the experimental results of in vitro euthanasia and IFN-γ secretion of EPCAM CAR-T in one embodiment of the present invention. [Figure 23] This figure shows the experimental results of in vitro euthanasia of CD19 CAR-T and IFN-γ secretion in one embodiment of the present invention. [Modes for carrying out the invention]

[0027] The present invention involves constructing a chimeric receptor composition or fusion protein, increasing the NKG2D chimeric receptor, which contains full-length sequences or cleaved fragments of NKG2D, DAP10, and / or DAP12, based on a conventional chimeric antigen receptor expression vector, packaging the vector as a lentivirus, and transducing the lentivirus into cells to produce immune cells that target CLDN18.2 while simultaneously expressing NKG2D, thereby improving the safety of immunotherapy.

[0028] "Full-length sequences of NKG2D, DAP10, and / or DAP12" means that the full-length sequence of NKG2D must be included, and that the full-length sequence of either DAP10 or DAP12, or both, must be included.

[0029] In some embodiments, this comprises a cleavage fragment of the NKG2D chimeric receptor and includes either or both of the full-length sequence of DAP10 or the full-length sequence of DAP12. The cleavage fragment of the NKG2D chimeric receptor may be an intracellular cleavage fragment (ICD) or an extracellular cleavage fragment (ECD) of NKG2D.

[0030] One embodiment of the present invention provides a novel chimeric receptor composition or fusion protein comprising a chimeric antigen receptor and an NKG2D chimeric receptor comprising the full-length sequence or cleaved fragments of NKG2D, DAP10, and / or DAP12.

[0031] In one preferred embodiment of the present invention, the chimeric receptor composition or fusion protein comprises a chimeric antigen receptor and an NKG2D chimeric receptor, wherein the NKG2D chimeric receptor comprises an NKG2D extracellular domain and a DAP12 intracellular domain sequence.

[0032] In one preferred embodiment of the present invention, the NKG2D chimeric receptor further comprises a costimulatory molecule selected from CD28, 4-1BB, DAP10, ICOS, OX40, and CD40.

[0033] In one preferred embodiment of the present invention, the chimeric antigen receptor comprises an extracellular recognition region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region.

[0034] In one preferred embodiment of the present invention, the extracellular recognition region comprises an antibody or antibody fragment that recognizes a tumor-associated antigen or a tumor-specific antigen.

[0035] In one preferred embodiment of the present invention, the tumor-associated antigen or tumor-specific antigen is CD19, BCMA, CD22, CD20, CD123, CD30, CD38, CD138, CD56, CD7, CLL-1, CD10, CD34, CS1, CD16, CD4, CD5, IL-1-RAP, ITGB7, k-IgG, TAC1, TRBC1, MUC1, NKG2D, PD-L1, CD133, CD177, LeY, CD70, ROR1, AFP, AXL, Selected from, but not limited to, CD80, CD86, DLL3, DR5, FAP, LMP1, MAGE-A1, MAGE-A4, MG7, MUC16, PMEL, ROR2, VEGFR2, CD171, Claudin18.2, Claudin6, EphA2, ErbB, Fra, PSCA, cMet, IL13Ra2, EPCAM, EGFR, PSMA, EGFRvIII, GPC3, CEA, HER2, GD2, and Mesothelin.

[0036] In one preferred embodiment of the present invention, the sequence of the hinge region is derived from at least one of CD8α, CD28, 4-1BB, ICOS, OX40, CD40, CD80, and IgG; the sequence of the transmembrane region is derived from at least one of CD2, CD27, LFA-1 (CD11a / CD18), CD8α, CD28, 4-1BB, ICOS, OX40, CD40, CD80, CD3ζ, and CD3ε; and the sequence of the intracellular signaling region is derived from at least one of Toll-like receptors, CD2, CD27, LFA-1 (CD11a / CD18), CD8α, CD28, 4-1BB, ICOS, OX40, CD40, CD80, DAP10, DAP12, CD3ζ, and CD3ε.

[0037] In one preferred embodiment of the present invention, the amino acid sequence of the chimeric antigen receptor is SEQ ID NO: 19.

[0038] In one preferred embodiment of the present invention, the chimeric receptor composition or fusion protein further comprises a linking peptide that links the chimeric antigen receptor and the chimeric receptor, wherein the linking peptide is a self-cleaving polypeptide 2A peptide, and the 2A peptide comprises F2A, P2A, T2A and E2A, preferably P2A, and its amino acid sequence is Sequence ID No. 9.

[0039] In one preferred embodiment of the present invention, the amino acid sequence of the chimeric receptor is selected from one of the sequences of SEQ ID NOs: 1 to 8, preferably SEQ ID NO: 3 or SEQ ID NO: 6.

[0040] One embodiment of the present invention provides a nucleic acid encoding the above-mentioned chimeric receptor composition or fusion protein.

[0041] In this specification, nucleic acids include variants of their conserved substitutions (e.g., degenerate codon substitutions) and complementary sequences, as well as variants that are more efficiently expressed in desired host cells through codon optimization. Nucleic acids are typically RNA or DNA, including genes, cDNA molecules, mRNA molecules, and fragments thereof such as oligonucleotides. Nucleic acid molecules may be single-stranded or double-stranded, but are preferably double-stranded DNA. When nucleic acids are functionally related with another nucleic acid, the nucleic acids are "effectively ligated". For example, when a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is effectively ligated to the coding sequence. When ligated into a vector, it is preferable to use DNA nucleic acids.

[0042] One embodiment of the present invention provides a vector containing the above-mentioned nucleic acid.

[0043] The term "vector" refers to a nucleic acid vehicle into which polynucleotides can be inserted. A vector is called an expression vector if it enables the expression of a protein encoded by the inserted polynucleotide. A vector can be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements it contains can be 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 (YACs), bacterial artificial chromosomes (BACs), or artificial chromosomes derived from P1 (PACs), phages such as lambda phages or M13 phages, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomabaculoviruses (e.g., SV40). In some embodiments, the vectors described in the present invention include regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRESs), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences).

[0044] One embodiment of the present invention provides cells that express the above-mentioned chimeric receptor composition or fusion protein and contain the above-mentioned nucleic acid or vector.

[0045] The cells should preferably be immune killer cells.

[0046] In one preferred embodiment of the present invention, the cells are selected from one of T cells, NK cells, DC cells, and macrophages.

[0047] In the present invention, T cells may include one or more of the well-known subclasses in the art, such as helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, MAIT cells, NKT cells, and γδT cells. In one preferred embodiment of the present invention, the T cells are selected from αβT cells, γδT cells, or NKT cells.

[0048] One embodiment of the present invention provides a biologic for tumor treatment that comprises the above-mentioned cells as the main active ingredient, and preferably further comprises a pharmaceutically acceptable vector, diluent, or excipient.

[0049] As used herein, “pharmaceutically acceptable vector, diluent or excipient” includes any material that, when combined with an active ingredient, enables the ingredient to maintain its biological activity and not react with the target immune system. Examples include, but are not limited to, any one of standard drug carriers (e.g., phosphate-buffered saline, water, emulsions (e.g., oil / water emulsions)) and various types of wetting agents. Exemplary diluents used 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 (e.g., Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990, and Remington, The Science and Practice of Pharmacy, 21st edition, Mack Publishing, 2005).

[0050] One embodiment of the present invention provides a method for producing the above-mentioned cells, comprising the step of transfecting the cells with the above-mentioned vector.

[0051] One embodiment of the present invention provides an application of the above-mentioned chimeric receptor composition, nucleic acid, vector, or cell in the production of an antitumor drug.

[0052] In a preferred embodiment, the antitumor agent is used to treat heterogeneous tumors.

[0053] In one preferred embodiment of the present invention, the heterogeneous tumor comprises at least NKG2DL-positive tumor cells and tumor cells targeted by the chimeric antigen receptor.

[0054] In order to better understand the present invention, the present invention will be described in detail and specifically with reference to concrete examples and drawings below, but the following examples do not limit the scope of the present invention.

[0055] Unless otherwise specified, the methods described in the examples are carried out by conventional methods, and unless otherwise specified, the reagents used are commercially available reagents that are commonly used or reagents prepared according to conventional methods. [Examples]

[0056] Detection of CLDN18.2 and NKG2D ligand expression in gastric cancer tissue The inventors first analyzed the expression status of CLDN18.2 and NKG2DL mRNA in PDX tissues derived from gastric, pancreatic, and esophageal cancers using a PDX database (CrownBio). As shown in Figure 1, the positive rates for CLDN18.2 in PDX tissues from gastric, pancreatic, and esophageal cancers were 63%, 82%, and 26%, respectively, and most tissues expressed at least one NKG2D ligand or CLDN18.2. Similarly, the inventors detected the expression of CLDN18.2 and NKG2D ligands (MICA, MICB, ULBP1-6) in gastric cancer tissue chips (purchased from Zhongke Guanghua). The results were consistent with the expression status in the PDX database, with most tissues expressing at least one NKG2D ligand or CLDN18.2 (Figure 2). The above results indicate that the use of targeted NKG2DL may be able to solve the difficult problem of tumor heterogeneity faced by single-target products such as CLDN18.2. [Examples]

[0057] Design of Synthetic Natural Killer Receptors (SNRs) This embodiment provides a novel chimeric receptor composition, which is hereinafter referred to as Dual CAR, also referred to as SNR or CAR molecule with SNR, and its corresponding CAR-T cell is referred to as Dual CAR-T or CAR-T with SNR). This composition includes the amino acid sequence of anti-CLDN18.2-CAR (conventional second-generation Claudin18.2 CAR molecule, hereinafter referred to as 21007) of Sequence ID No. 1 of the Chinese patent application published under publication number CN113621073A on November 9, 2021, and the chimeric receptor sequence shown in Sequence IDs 1 to 8 (the structural sequence is shown in Figure 3). The specific amino acid sequence corresponds to sequence names 21067 to 21074 in Table 1 below.

[0058] [Table 1] TIFF0007894434000002.tif250166TIFF0007894434000003.tif38165

[0059] As shown in Figure 3, the above chimeric receptor composition further includes the following sequence information in addition to the 21007 chimeric antigen receptor: (1) P2A, its amino acid sequence is as follows: GSGATNFSLLKQAGDVEENPGP (Sequence ID 9), (2) DAP12ICD (intracellular region), its amino acid sequence is as follows: YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK(Sequence ID 10), (3) DAP10ICD, its amino acid sequence is as follows: LCARPRRSPAQEDGKVYINMPGRG (Sequence ID 11), (4) NKG2DFL (full sequence), its amino acid sequence is as follows: MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVTIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV (Sequence ID 12), (5) The CD8 hinge region has the following amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (Sequence ID 13), (6) CD8™ (transmembrane region), its amino acid sequence is as follows: IYIWAPLAGTCGVLLLSLVITLYC (Sequence ID 14), (7) DAP10FL, its amino acid sequence is as follows: MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRG (Sequence ID 15), (8) DAP12FL, its amino acid sequence is as follows: MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK(Sequence ID 16), (9) DAP12ECD+TM (extracellular and transmembrane regions), its amino acid sequence is as follows: LRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAV (Sequence ID 17), (10) T2A, its amino acid sequence is as follows: GSGEGRGSLLTCGDVEENPGP (Sequence ID 18). [Examples]

[0060] Plasmid construction and viral packaging of CAR molecules equipped with synthetic natural killer receptors (SNRs) This embodiment provides CAR-T cells equipped with SNR expressing the above-mentioned chimeric receptor, and the method for constructing them includes the following steps: 1. Construction of Expression Vectors: The CAR molecules with SNRs and the backbone portions of the lentiviral vectors were constructed by CRO using whole-gene synthesis technology. The CAR fragments with SNRs were cloned into lentiviral vectors using conventional molecular cloning methods, and the names of the constructed plasmids are shown in Table 1 above. The success of each plasmid construction was confirmed by verifying the complete sequencing of the inserted fragments.

[0061] 2. Lentivirus packaging and titer measurement Day 1: 1) Inoculate 293T cells into a T75 culture flask, and the quantity is 5 × 10 6 The culture volume is 20 mL. Day 2: 2) Before encapsulating the virus, confirm that the confluence of 293T cells is approximately 70% to 80%, and perform an equivolute fluid exchange. 3) Preparation and transfection of the transfection complex, Tube A and Tube B were prepared separately, and the system was as shown below. They were then mixed uniformly by inversion or low-speed shaking. Tube A: Opti-MEM (2 mL) + Lipo3000 (55 μL), Tube B: Opti-MEM (2 mL) + P3000 (46 μL) + Helper plasmid granules 18 μg + CAR master plasmid with SNR 6 μg Add Tube A to Tube B, shake to mix uniformly, and incubate at room temperature for 15 minutes. In Step 3, rotate the culture flask after the liquid exchange so that the culture medium is on the opposite side of the flask, add the Tube A+B mixture, shake gently to mix evenly, then slowly return the culture flask to the front, and continue culturing for 48 hours. Day 4: 4) After transfection is complete and the cells are incubated for 2 days, the supernatant is collected, centrifuged at 500 g for 10 min, filtered through a 0.45 μm filter and transferred to a 50 mL centrifuge tube, sealed with a sealing film, and centrifuged overnight at 10000 g and 4°C until a white virus precipitate is observed. After discarding the supernatant, the centrifuge tube is inverted until no more supernatant remains, the precipitate is dissolved in 200 μL of AIM-V medium, 2 μL is taken and the titer is measured according to the subsequent steps, and the remainder is stored at -80°C.

[0062] 5) Dilute the virus by adding 2 μL of resuspended viral supernatant to 198 μL of 1640 medium, and then place 2 μL, 10 μL, and 50 μL of the diluted virus into a 24-well plate, 2 × 10⁶ wells per well. 5 Three wells were added with a total of [number] Jurkat T cells, and Polybrene helper virus infection at a final concentration of 5 μg / mL was added.

[0063] Day 6: 6) The viral titer was detected using the Anti-VHH-FITC antibody, and the titer was 2.5 × 10⁻⁶. 7 ~1.2 × 10 8 It was within the range of conventional in vitro CAR-T construction experiments and was able to meet the requirements.

[0064] 3. Construction of CAR-T cells equipped with synthetic natural killer receptors (SNRs) Figure 4 shows the results of infecting T cells with the above-described packaged lentivirus and measuring the expression of anti-CLDN18.2-CAR and NKG2D on the T cells.

[0065] Figure 4 shows that anti-CLDN18.2-CAR-T cells have relatively low levels of NKG2D expression, while CAR-T cells with SNR have relatively high levels of NKG2D expression compared to anti-CLDN18.2-CAR-T cells, and simultaneously express anti-CLDN18.2-CAR normally. This indicates that the NKG2D chimeric receptor is successfully expressed on the surface of CAR-T cells with SNR. [Examples]

[0066] This example verifies the killing effect of CAR-T cells equipped with SNR on target cells, and the specific steps and results are as follows: (1) A cell line 293-CLDN18.2 that highly expresses the CLDN18.2 antigen was constructed based on 293 cells, and this cell line highly expressed CLDN18.2 (shown in Figure 5).

[0067] (2) Detection of NKG2D ligand MICA / B expression in cell lines: MICA / B expression levels were identified in the 239T cell line, and the 293T cell line expressed relatively high levels of MICA / B (shown in Figure 6).

[0068] (3) CAR-T cells and 293-CLDN18.2 were mixed at effect-to-target ratios of 2:1 and 0.5:1, incubated for 5 hours, and the killing effect of CAR-T cells on target cells was detected by Annexin V flow cytometry. Anti-CLDN18.2-CAR-T cells and CAR-T cells with SNR exhibited a stronger killing effect against 293-CLDN18.2 compared to unT (untransduced T) cells. Of these, cells 21069 and 21072 exhibited a stronger killing effect compared to conventional CAR-T cells (21007) at an effect-to-target ratio of 2:1 (shown in Figure 7). Furthermore, when 293-CLDN18.2 was co-incubated with CAR-T cells, the proliferation factor of CAR-T cells with SNRs 21069 and 21072 was significantly higher than that of conventional CAR-T cells (21007) (shown in Figure 8).

[0069] CAR-T cells and 293T cells were mixed at effect-to-target ratios of 2:1 and 0.5:1, incubated for 5 hours, and the killing effect of CAR-T cells on target cells was detected by Annexin V flow cytometry. Anti-CLDN18.2-CAR-T and CAR-T cells with SNR exhibited a stronger killing effect against MICA / B-expressing target cells compared to unT (untransduced T) cells. Of these, cells 21069 and 21072 exhibited a stronger killing effect compared to conventional CAR-T cells at an effect-to-target ratio of 2:1 (shown in Figure 9). [Examples]

[0070] This example verifies that CAR-T cells with SNR have lower factor secretion levels when killing target cells 293-CLDN18.2, and the specific steps and results are as follows: Using the cell line 293-CLDN18.2 constructed in Example 4, CAR-T cells and 293-CLDN18.2 were mixed in an effect-to-target ratio of 2:1, incubated for 24 hours, and CAR-T cytokine release was detected by flow cytometry. Using Biolegend's multifactor detection kit, 15 μL / sample of the supernatant from the incubation of effector cells with target cells was transferred to a new V-type 96-well plate, 15 μL / sample of mixed and diluted magnetic beads and 15 μL / sample of assay buffer were added, the plate was sealed with seal film, shaken at 500 rpm at room temperature for 2 hours, centrifuged at 250 g for 5 minutes, the supernatant was removed, 200 μL / sample of wash buffer was added, centrifuged at 250 g for 5 minutes, the supernatant was removed, 15 μL / sample of detection antibody was added, shaken at 500 rpm at room temperature for 1 hour, 15 μL / sample of SA-PE was added, shaken at 500 rpm at room temperature for 30 minutes, and 250 The cells were centrifuged at 250 g for 5 minutes, the supernatant was removed, 200 μL / sample of wash buffer was added, the cells were centrifuged again at 250 g for 5 minutes, the supernatant was removed, 150 μL / sample of Wash Buffer was added, and the cells were placed in the instrument for detection. The results showed that when anti-CLDN18.2-CAR-T and CAR-T cells with SNR killed 293-CLDN18.2 target cells, CAR-T cells with SNRs such as 21067, 21068, 21069, 21070, and 21072 had significantly lower levels of IFN-γ, IL2, IL6, and TNF-α secretion (shown in Figure 10). [Examples]

[0071] This example verifies that CAR-T cells with SNR release moderate levels of cytokines when killing target cells expressing MICA / B, and the specific steps and results are as follows: CAR-T cells and 293T cells were mixed in a 2:1 effect-to-target ratio and incubated for 24 hours. Flow cytometry was used to detect CAR-T cytokine release. When anti-CLDN18.2-CAR-T cells and CAR-T cells with SNRs killed 293T target cells, CAR-T cells with SNRs such as 21068, 21069, 21070, and 21072 exhibited moderate levels of IFN-γ secretion and relatively low levels of TNF-α, IL2, and IL6 secretion (shown in Figure 11). [Examples]

[0072] Construction of CLDN18.2 CAR-T cells with SNR Based on the above results, the inventors selected intramolecular SNR structures such as molecules 21067, 21070, 21071, and 21072 for further verification. The inventors constructed several of the above SNRs into the second-generation CAR molecule 21047 to form new CAR molecules 21326, 21327, 21328, and 21329. Their structures and sequences are shown in Figure 12 and the table below. Referring to the method in Example 2, the inventors constructed each of the above molecules as plasmids, lentiviruses, and CAR-T cells.

[0073] [Table 2] TIFF0007894434000005.tif62166 [Examples]

[0074] Detection of the ability of CLDN18.2 CAR-T cells to kill target cells with SNR To verify whether SNR successfully kills NKG2DLs-positive cells, the inventors used ULBPs-positive tumor cells A431 to detect the killing capacity provided by SNR. The specific steps and results are as follows: (1) Detection of NKG2D ligand ULBP2 / 5 / 6 expression in cell lines: The expression levels of NKG2D ligands in the A431 cell line were identified, and the A431 cell line expressed relatively high levels of ULBP2 / 5 / 6 (shown in Figure 13).

[0075] (2) CAR-T cells were mixed with NUGC4 and A431 at effect-to-target ratios of 3:1, 1:1, and 0.3:1, incubated for 5 hours, and the killing effect of CAR-T cells on target cells was detected by Annexin V flow cytometry. As shown in the following figure, cells 21327 and 21329 were more efficient at killing NUGC4 than 21047 at high effect-to-target ratios, and 21047 was unable to kill A431, while cells 21326-21329 were all able to effectively kill A431. Multifactor detection showed that when killing NUGC4, the IFN-γ secreted by 21047 was higher than that secreted by 21326-21329, while when killing A431, the IFN-γ levels secreted by 21327-21329 were generally lower than those secreted when killing NUGC4 (shown in Figure 14). These results showed that all four SNR designs could target and kill CLAUDIN18.2 and NKG2DLs-positive cells, and that the levels of cytokine secretion were relatively low. [Examples]

[0076] SNR improved the proliferative capacity and memory level of CAR-T cells while simultaneously reducing their depletion level. The inventors further analyzed the in vitro effects of SNR on CAR-T cells. As shown in Figure 15, the proliferation rates of each molecule were clearly increased compared to 21047, while the expression of each depletion marker (PD1, LAG3, TIM3) was clearly decreased. Further analysis of the proportion of memory center memory cells showed that the proportion of Tscm cells for the 21327 molecule was clearly increased compared to the levels of the other molecules. The above advantages may be related to the function of the signaling regions used in SNR. [Examples]

[0077] Efficacy study of an in vivo model of homogeneous expression of CLDN18.2 To study the in vivo efficacy of each of the above molecules, the inventors constructed a subcutaneous tumor model of NUGC4 (CLDN18.2+, NKG2DLs-) with tumors measuring 100 mm 3 Each CAR-T was administered when the target was reached. The results of the study showed that 21327 had better efficacy compared to 21047, as it was able to remove tumor tissue approximately 10 days earlier. In contrast, the efficacy of 21326 and 21329 was not as good as that of 21047, and the efficacy of 21328 could not be concluded because the mice died prematurely (Figure 16A). The above results indicate that the 21327 molecule has a stronger ability to remove tumors with homogeneous CLDN18.2 expression compared to the conventional second-generation CAR-T 21047.

[0078] Further analysis of mouse body weight revealed that molecule 21328 caused significant weight loss and mouse death during the study period, demonstrating clear in vivo toxicity. While molecule 21047 caused weight loss in one mouse during the study period, no mouse deaths occurred. Conversely, molecules 21326, 21327, and 21329 did not cause any apparent toxicity throughout the study period (Figure 16B). Consistent with this, further analysis of cytokine expression levels in mouse blood showed that molecules 21326, 21327, and 21329 secreted lower levels of IFN-gamma (Figure 16C) and TNF-alpha (Figure 16D) in vivo, a result consistent with the safety findings.

[0079] In summary, the 21327 molecule demonstrated superior efficacy and lower toxicity compared to conventional second-generation CAR-T receptors. The SNR3 design effectively improved the efficacy and safety of CAR-T receptors.

[0080] To further investigate the ability of CAR-T cells to combat CLDN18.2-negative recurrence after removal of CLDN18.2-positive tumors, the inventors conducted rechallenge experiments using A431 cells (CLDN18.2-, NKG2DLs+) in tumor-removed mice in groups 21047 and 21327. The results showed that the 21327 molecule significantly inhibited the growth of A431 tumors compared to 21047 (Figure 17). These results suggest that the addition of SNR may suppress recurrence in CLDN18.2-negative tumors. [Examples]

[0081] Drug efficacy experiments on the NKG2DL+ tumor model of SNR To further investigate the tumor-inhibiting ability of each group's SNR, the inventors constructed a mouse subcutaneous A431 tumor transplantation model (CLDN18.2-, NKG2DLs+) with tumors measuring 60 mm 3 Each CAR-T receptor was administered when the cells had grown to a certain stage, and the results are shown in Figure 18. This data showed that 21327 and 21329 could inhibit the proliferation of A431, and that 21329 had a better tumor inhibitory effect than 21327. 21047 had little tumor inhibitory effect, and at the same time, the SNR structures of 21327 and 21329 were shown to specifically recognize NKG2DL and initiate a killing effect. [Examples]

[0082] Drug efficacy experiments on the CLDN18.2 heterogeneous tumor model To further investigate whether SNR can enhance the ability of CAR-T cells to combat heterogeneous tumors, the inventors constructed the A431-18.2 cell line and mixed it with A431 to form an A431 / A431-18.2 tumor model. CAR-T administration was performed approximately 7 days after tumor inoculation. The results showed that, in the heterogeneous tumor model, conventional second-generation CAR-T molecules, although capable of proliferating and secreting IFN-gamma in vivo, were unable to clearly inhibit tumor growth, suggesting that CLDN18.2-positive tumor cells were killed while the tumor in the CLDN18.2-negative region could still proliferate normally. In contrast, the 21327 molecule with an SNR structure effectively inhibited the proliferation of mixed tumor cells. Analysis of IFN-γ secretion levels showed no significant difference between 21327 and 21047 in vivo (Figure 19), and these results suggest that the addition of SNR effectively helps CAR-T cells combat heterogeneous tumor tissue. [Examples]

[0083] Construction of EPCAM CAR-T and CD19 CAR-T cells with SNR Based on the above results, the inventors constructed a new CAR molecule 22162 by constructing SNR 21327 into a second-generation EPCAM CAR molecule (21002), and simultaneously constructed 22163 into a second-generation CD19 CAR molecule (21144) by constructing SNR 21329 (Figure 20). The sequences are shown in the table below. Referring to the method in Example 2, the inventors constructed each of the above molecules into plasmids, lentiviruses, and CAR-T cells. The packaged lentiviruses were used to infect T cells, and the expression of anti-EPCAM-CAR, anti-CD19 CAR, and NKG2D on the T cells was measured. The results are shown in Figure 21.

[0084] Figure 21 shows that anti-EPCAM-CAR-T cells have relatively high levels of NKG2D expression, and anti-CD19-CAR-T cells with SNR also have relatively high levels of NKG2D expression, while simultaneously expressing EPCAM CAR and CD19 CAR normally. This indicates that the NKG2D chimeric receptor is successfully expressed on the surface of CAR-T cells with SNR.

[0085] [Table 3] TIFF0007894434000007.tif244165TIFF0007894434000008.tif81165 [Examples]

[0086] Detection of cytokine secretion levels in EpCAM CAR-T cells with SNR To verify the ability of EpCAM CAR-T cells with SNR to kill heterogeneous tumors, we studied cytokine release levels during the process of killing EpCAM target-positive (HCT116) or target-negative (MIA PaCa2) cells.

[0087] CAR-T cells were mixed with HCT116 or MIA PaCa2 in a 1:1 effect-to-target ratio, incubated for 24 hours, and the supernatant was removed. Cytokine secretion levels were measured using ELISA. The results of IFN-γ detection are shown in Figure 22. When HCT116 was killed, the IFN-γ secreted by 22162 was 50,000-60,000 pg / mL, which is equivalent to the secretion level of conventional second-generation CAR-T molecules. In contrast, during the process of killing EpCAM target-negative cells MIA PaCa2, SNR increased the secretion level of IFN-γ by CAR-T cells. Thus, SNR enhanced the ability of EpCAM CAR-T cells to kill EpCAM-negative cells. [Examples]

[0088] Detection of the ability of CD19 CAR-T cells with SNR to kill target cells. To verify the ability of CD19 CAR-T cells with SNR to kill heterogeneous tumors, we studied cytokine release levels during the process of killing CD19 target-positive (Raji) or target-negative (MIA PaCa2) cells.

[0089] CAR-T cells were mixed with Raji or MIA PaCa2 in a 1:1 effect-to-target ratio, incubated for 24 hours, and the supernatant was removed. Cytokine secretion levels were measured using ELISA. IFN-γ detection results showed that when Raji was killed, IFN-γ secreted by 22163 was 30,000–40,000 pg / mL, significantly higher than the secretion levels of conventional second-generation CAR-T molecules. During the process of killing CD19-target-negative cells (MIA PaCa2), SNR increased the secretion level of IFN-γ by CAR-T cells. Therefore, SNR enhanced the ability of CD19 CAR-T cells to kill CD19-negative cells.

[0090] Although specific embodiments of the present invention have been described in detail above, these are merely illustrative, and the present invention is not limited to the specific embodiments described above. To those skilled in the art, any equivalent modification or substitution made to the present invention falls within the scope of the present invention. Accordingly, any equivalent transformation or modification made without departing from the spirit or scope of the present invention should fall within the scope of the present invention.

Claims

1. A novel chimeric receptor composition or fusion protein comprising a chimeric antigen receptor and an NKG2D chimeric receptor containing an NKG2D extracellular domain and a DAP12 intracellular domain sequence, wherein the extracellular recognition region of the chimeric antigen receptor is selected from an antibody that recognizes CLDN18.2, an antibody that recognizes EpCAM, and an antibody that recognizes CD19, and the amino acid sequence of the NKG2D chimeric receptor is selected from the sequences of SEQ ID NOs: 1 to 6.

2. The chimeric antigen receptor composition or fusion protein according to claim 1, characterized in that the chimeric antigen receptor comprises an extracellular recognition region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region.

3. The chimeric receptor composition or fusion protein according to claim 2, characterized in that the sequence of the hinge region is derived from at least one of CD8α, CD28, 4-1BB, ICOS, OX40, CD40, CD80, and IgG; the sequence of the transmembrane region is derived from at least one of CD2, CD27, LFA-1, CD8α, CD28, 4-1BB, ICOS, OX40, CD40, CD80, CD3ζ, and CD3ε; and the sequence of the intracellular signaling region is derived from at least one of Toll-like receptor, CD2, CD27, LFA-1, CD8α, CD28, 4-1BB, ICOS, OX40, CD40, CD80, DAP10, DAP12, CD3ζ, and CD3ε.

4. The chimeric receptor composition or fusion protein according to claim 1, further comprising a linking peptide that links the chimeric antigen receptor and the chimeric receptor, wherein the linking peptide is a self-cleaving polypeptide 2A peptide, and the 2A peptide comprises F2A, P2A, T2A and E2A.

5. The chimeric receptor composition or fusion protein according to claim 4, characterized in that the 2A peptide is P2A and its amino acid sequence is SEQ ID NO:

9.

6. A nucleic acid encoding a chimeric receptor composition or fusion protein according to any one of claims 1 to 5.

7. A vector comprising the nucleic acid described in claim 6.

8. A cell expressing the chimeric receptor composition or fusion protein described in claim 1.

9. The cell according to claim 8, characterized in that the cell is a T cell.

10. The cell according to claim 9, characterized in that the T cell is an αβT cell, a γδT cell, or an NKT cell.

11. A biological agent for tumor treatment, characterized by containing the cells described in any one of claims 8 to 10 as the main active ingredient.

12. A method for producing cells according to any one of claims 8 to 10, comprising the step of transfecting the cells with the vector described in claim 7.

13. An antitumor agent comprising a chimeric receptor composition or fusion protein according to any one of claims 1 to 5, a nucleic acid according to claim 6, a vector according to claim 7, or a cell according to any one of claims 8 to 10.

14. An antitumor agent according to claim 13, used for treating heterogeneous tumors.

15. The antitumor agent according to claim 14, wherein the heterogeneous tumor comprises at least NKG2DL-positive tumor cells and tumor cells targeted by the chimeric antigen receptor.

Images