Cell membrane GRP94-targeting chimeric antigen receptor, engineered cell and use

Abstract

A cancer neoantigen-expressing cell membrane GRP94-targeting chimeric antigen receptor, comprising a signal peptide region, a cell membrane GRP94-targeting antigen binding domain, a hinge region, a transmembrane domain, and a signal transduction activation domain which are connected in sequence from the amino terminus to the carboxyl terminus, wherein the nucleotide sequence and amino acid sequence of the signal peptide region are as shown in SEQ ID No: 1 and SEQ ID No: 2 respectively; and the antigen binding domain is a portion of an antibody itself that targets cancer neoantigen-expressing cell membrane GRP94, or formed between portions of the antibody itself or between a portion thereof and an antibody region for another target. A novel chimeric antigen receptor (CAR)-engineered NK immune cell and a cancer drug can rapidly induce apoptosis in novel cancer cells having potentially cancer antigen-expressing cell membrane GRP94 protein, generate no toxic and side effects such as cytokine storms, and have off-the-shelf universal applicability, thereby providing a new choice for cell immunotherapy for cancers and also laying a foundation for accurate individualized treatment for cancer treatment.

Description

A chimeric antigen receptor targeting cell membrane GRP94, engineered cells and applications Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a novel type of chimeric antigen receptor (CAR) engineered NK immune cells based on cancer neoantigen cell membrane GRP94 and their use in the preparation / treatment of cancer drugs. Background Technology

[0002] Chimeric antigen receptor T (CAR-T) cells are a type of cell immunotherapy that has emerged in recent years. CAR-T cells are prepared by transfecting the patient's own T lymphocytes with a virus in vitro, integrating CAR molecules targeting tumors into the T cells. These T cells then express chimeric antibodies that recognize target cells, and through their specific recognition of antigens on the target cell surface, they specifically kill those cells. This approach is considered one of the most promising therapies for conquering cancer.

[0003] Currently, CD19, as a specific antigen for cancers such as acute lymphoblastic leukemia and lymphoma, has attracted much attention. CAR-T cells targeting CD19 offer new hope for cancer treatment. Regarding specific antigens, some cancer-related antigens have been identified, such as CD33 and CD123 associated with acute myeloid leukemia. However, because CD33 and CD123 are also expressed in normal myeloid precursor cells, CAR cell therapy targeting these antigens can severely inhibit the survival of normal myeloid cells, leading to off-target effects or "targeted non-cancer" toxic side effects. Therefore, it is urgent to find suitable and specific new targets to improve the specificity and safety of CAR technology applications.

[0004] Studies have shown that GRP94, which is continuously expressed on the surface of various cancer cells such as breast cancer and acute myeloid leukemia, can serve as an effective target for cancer treatment. Therefore, constructing CAR-NK cells targeting GRP94 will be an important breakthrough in the treatment of autoimmune diseases and / or malignant tumors. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies and provide a class of novel chimeric antigen receptor (CAR) engineered NK immune cells based on the novel cancer neoantigen cell membrane GRP94 that can induce rapid apoptosis in cancer cells expressing the novel cancer antigen GRP94 protein without producing toxic side effects such as cytokine storms, has high safety, and is readily available and universally applicable, as well as its use in cancer drugs.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A chimeric antigen receptor for GRP94 on cell membranes targeting neocancer antigen expression includes a signal peptide region, an antigen-binding domain targeting GRP94 on the cell membrane, a hinge region, a transmembrane domain, and a signal transduction activation domain, sequentially linked from the amino terminus to the carboxyl terminus. The nucleotide and amino acid sequences of the signal peptide region are shown in SEQ ID No: 1 and SEQ ID No: 2, respectively. The nucleotide sequence of SEQ ID No: 1 is as follows:

[0008] The amino acid sequence of SEQ ID No:2 is ATMALPTVALLLPLALLLHAARP; the antigen-binding domain is a portion of the antibody itself targeting the cell membrane GRP94 of the cancer neoantigen expression site, or a portion of the antibody itself or with antibody regions targeting other different targets, which can be tandemly linked by a linker sequence. The nucleotide sequence of the antibody is shown in SEQ ID No:3 (CAGGTGCAGCTGGTGC).

[0009] AGTCCGGCGCCGAGGTGAAGAAGCCCGGCGCTTCCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACATTCACAAGCTACGCCATGCACTGGGTGAGACAGGCCCTGGCCAGAGGCTGGAGTGGATGGGCTGGATCAACGCCGGCAACGGCAACCAAGTACTCC CAGAAGTTCCAGGGCAGAGTGACCATCACCAGAGATACCTCCGCCTCCAGCCTACATGGAGCTGTCCAGCCTGAGGAGCGAGGATACCGCCGTGTACTACTGCGCCAGGGCCCACTTCGACTACTGGGGCCAGGGCACACTGGTGACAGTGAGCGCC), or as shown in SEQ ID No: 4(GAGATCGAGCTGACACAGTCCCCTAGCTCCCTGTCCGCCTCCGTGG

[0010] as shown in SEQ ID No: 4 (GCGACAGGGTGACCATCACCTGCAGAGCCTCCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGTCCGGCGTGCCTAGCAGATTTTCCGGCTCCGGCAGCGGCACAGACTTTACACTGACCATCTCCAGCCTGCAGCCCGAGGATTTCGCCACCTACTACTGTCAGCAGAGCTACAGCACACCTCCTACCTTTGGCCAGGGCACAAAGGTGGAGATCAAG), or as SEQ ID No: 5 (GACGTGCAGCTGCAGGAGAG

[0011] CGGACCCGACCTGGTGAAACCCAGCCAGAGCCTGTCCCTGACATGCACAGTGACCGGCCACAGCATTACCAGCGACTATACCTGGCACTGGATTAGACAGTTTCCCGGCAACAAGCTGGAGTGGATGGGCTATATCCACTACAGCGGCAACACCTACTACAACCCCAGCCTGAAGAGCAGAATCTCCATCACCCGGGACACCAGCAAGAACCAGTTCTTCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCACCTACTACTGCGCCAGAGTGGGCTACTGGGGCCAGGGAACCCTGGTGACCGTGAGCGC

[0012] C), or as SEQ ID No: 6 (GACATTGTGATGACCCAGAGCCCCAGCAGCCTGGCTCTGAGTGTGGGGCAGAAAGTGACC

[0013] As shown in SEQ ID No: 7 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARAHFDYWGQGTLVTVSA), or as shown in SEQ ID No: 8 (EIELTQSPSSLSASVG

[0014] DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK), or as shown in SEQ ID No: 9 (DVQLQESGPDLVKPSQSLSLTCTVTGHSITSDYTWHWIRQFPGN

[0015] KLEWMGYIHYSGNTYYNPSLKSRISITRDTSKNQFFLQLNSVTPEDTATYYCARVGYWGQGTLVTVSA) or as shown in SEQ ID No: 10 (DIVMTQSPSSLALSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGS

[0016] GTDFTLTISSVQAEDLADYFCQQHYMPPLTFGAGTKLELK).

[0017] Furthermore, the nucleotide and amino acid sequences of the tandem regions of the connecting sequences are shown in SEQ ID No: 11 (GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT) and SEQ ID No: 12 (GGGGSGGGGSGGGGS).

[0018] Furthermore, the hinge region is a nucleotide sequence such as SEQ ID No: 13 (ACCACGACGCCAGCGCCGCGACCACCAACACCGG

[0019] CGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC), and the amino acid sequence is as shown in SEQ ID No: 14 (TTTPAPRPPTPAPTIASQPLSLRPEACRP

[0020] As shown in AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC), or the nucleotide sequence as shown in SEQ ID No:15 (ACC

[0021] The amino acid sequence is shown in ACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT), and the amino acid sequence is shown in SEQ ID No:16 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD).

[0022] And / or, the transmembrane domain is a nucleotide sequence or amino acid sequence such as SEQ ID No: 17 (AGGAGTAAGAGGAGCAGG

[0023] CTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCCACCACGCGACTTCGCAGCCTATCGCTCC); SEQ ID No: 18 (RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR

[0024] DFAAYRS),SEQ ID No:19(TTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGAC

[0025] AGTGGCCTTCATCATCTTTTTGGGTGAGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCTACCGGAGC),SEQ ID The CD28 transmembrane and 4-1BB costimulatory domain shown in No: 20 (FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS);

[0026] And / or, the nucleotide or amino acid sequence of the 4-1BB co-stimulatory domain is as follows: SEQ ID No: 21 (AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG), SEQ ID No: 22 (KRGRKKLLYIF

[0027] As shown in KQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL);

[0028] And / or, the nucleotide or amino acid sequence of CD3ζ ITAMS1 as shown in SEQ ID No: 23(AGAGTGAAGTTCAGCAGGA)

[0029] GCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAAT GAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC), SEQ ID No: 24(RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY

[0030] As shown in NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR);

[0031] And / or, the nucleotide or amino acid sequence of the desired T2A for co-expression of other protein molecules, such as SEQ ID No: 25 (GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT), SEQ ID No: 26 (EGR

[0032] As shown in GSLLTCGDVEENPGP).

[0033] The present invention also provides an isolated nucleic acid comprising a nucleotide sequence and an amino acid sequence fragment for expressing the GRP94 chimeric antigen receptor on the cell membrane targeting the neoantigen of cancer.

[0034] The present invention also provides a recombinant vector comprising the isolated nucleic acid as described in claim 4.

[0035] The present invention provides a CAR-NK cell containing the isolated nucleic acid as described in claim 4, or a cell transformed by the recombinant vector as described in claim 5.

[0036] The present invention relates to the application of a chimeric antigen receptor for GRP94 on a cell membrane targeting cancer neoantigen expression in a drug, wherein the chimeric antigen receptor for GRP94 on a cell membrane targeting cancer neoantigen expression, the isolated nucleic acid, the recombinant vector, or the CAR-NK cells are used in the preparation of a drug for treating cancer-related diseases.

[0037] A pharmaceutical composition of the present invention comprises an expression vector for expressing the GRP94 chimeric antigen receptor of a cell membrane targeting a neoantigen as described in any one of claims 1 to 3, or CAR-NK cells as described in claim 6.

[0038] The cancer-related diseases described in this invention include acute myeloid leukemia with FLT3-ITD+ mutation.

[0039] The CAR-NK cells described in this invention can be used in combination with PD-L1 / PD-1 immune checkpoint inhibitors.

[0040] The sources of NK immune cells described in this invention include, but are not limited to, cell lines NK-92, NK-92MI, HANK-1, KHYG-1, NK-YS, NKG, NK101, NK3.3, YTS, NKL, autologous or allogeneic peripheral blood, umbilical cord blood, induced pluripotent stem cells (iPSCs), and hematopoietic stem cells (HSCs).

[0041] This invention can be extended to different immune cell types besides NK cells, including T cells, macrophages, etc.

[0042] The organic components of the chimeric antigen receptor of this invention particularly include all antibody sequences (including scFv), nanobodies, polypeptide sequences, small chemical molecules and their possible derivatives (amino acid sequences, nucleotide sequences, chemical molecules, etc.) capable of recognizing cell membrane GRP94, a potential cancer-associated antigen, and related sequences that activate immune cell activity. These are necessary for constructing engineered NK immune cells (including but not limited to NK cells, as well as other types of cells with immune activity), containing various expression plasmids (including various promoters, enhancers, guide peptides, etc., for expressing cell membrane GRP94 chimeric antigen receptors targeting cancer neoantigens), and transiently expressed mRNA sequences. The invention includes the nucleotide or amino acid sequences mentioned herein, and any continuous sequence with more than 60% repetition at any position is within the scope of protection of this invention. This invention can also be further extended to other cell drugs, antibody drugs, tumor vaccines, etc., besides NK cell drugs targeting cell membrane GRP94.

[0043] The present invention has the following beneficial effects:

[0044] 1. GRP94, as a molecular chaperone, is mainly distributed in the endoplasmic reticulum of normal cells. This invention uses GRP94 expressed on the cell membrane of cancer cells such as FLT3-ITD+ acute myeloid leukemia as an effective target to construct CAR-NK cells and anticancer drugs targeting cell membrane GRP94. The construction of CAR-NK cells targeting cell membrane GRP94 provides a new option for cancer cell immunotherapy and lays the foundation for precise individualized treatment of cancer.

[0045] 2. The CAR-NK cells constructed using the NK92 cell line in this invention have high safety. Multiple phase I / II clinical trials have shown that, unlike CAR-T cells, CAR-NK-92 cells that have been pre-treated with radiation do not have obvious clinical toxic side effects such as cytokine storms and neurotoxicity, and do not require "custom-made" products. This makes it possible for universal "commercial off-the-shelf" cell therapy products.

[0046] 3. By employing viral and non-viral transgenic methods, this invention enables CAR to be easily expressed in cell lines, peripheral blood, human embryonic stem cells, and / or iPSC-derived NK cells. It can also provide a reference for the CAR cell engineering modification of other types of immune cells, such as macrophages and T cells.

[0047] 4. The CAR-NK cells constructed in this invention have an organic component of the chimeric antigen receptor CAR, namely the antibody recognition sequence, which is the scFv of a conventional monoclonal antibody. This lays the foundation for the next step of replacing and modifying dual-target antibodies, nanobodies, antigen recognition peptide sequences, etc., as well as the logic gate circuit control of CAR and cell state, thereby creating a new generation of CAR cells.

[0048] 5. The CAR-NK cells constructed in this invention, when used in combination with other drugs such as atezolizumab and PD-L1 / PD-1 immune checkpoint inhibitors, can significantly enhance the killing effect on cancer cells. Attached Figure Description

[0049] Figure 1 shows the screening results of acute lymphoblastic leukemia cancer cells and acute myeloid leukemia cancer cells using cell membrane mass spectrometry in Example 1 of the present invention.

[0050] Figure 2 shows the flow cytometry (left) and immunofluorescence imaging (right) verification results of membrane CD19 in Example 1 of the present invention;

[0051] Figure 3 shows the verification results of membrane GRP94 flow cytometry (left) and immunofluorescence imaging (right) in Example 1 of the present invention;

[0052] Figure 4 shows the expression of membrane GRP94 in healthy peripheral blood cells (left) and T cells (right) in Example 1 of the present invention.

[0053] Figure 5 shows the verification of the relationship between membrane GRP94 and oncogene activity in Example 1 of the present invention;

[0054] Figure 6 shows the verification of GRP94 expression in the cell membrane of other AML cell lines in Example 1 of the present invention;

[0055] Figure 7 shows the expression of GRP94 in the blood cell membrane of patients with FLT3-ITD+AML leukemia in Example 1 of the present invention;

[0056] Figure 8 shows the construction and verification of GRP94 CAR NK-92 cells targeting the cell membrane in Example 2 of the present invention;

[0057] Figure 9 shows the lysis of target cells by GRP94 CAR NK-92 cells targeting the cell membrane in Example 3 of the present invention (6 hours);

[0058] Figure 10 shows the lysis of target cells by GRP94 CAR NK-92 cells targeting the cell membrane in Example 3 of the present invention (24 hours);

[0059] Figure 11 is a schematic diagram of the induction of apoptosis in target cells by GRP94 CAR NK-92 cells targeting the cell membrane in Example 3 of the present invention and the apoptosis of target cells MV4-11.

[0060] Figure 12 shows the real-time apoptosis-inducing effect of GRP94 CAR NK-92 cells targeting cell membranes on target cells MV4-11 in Example 3 of the present invention;

[0061] Figure 13 shows the transcriptome changes during the killing of target cells MV4-11 by GRP94 CAR NK-92 cells targeting the cell membrane in Example 3 of the present invention (left image shows target cells, right image shows effector cells);

[0062] Figure 14 shows the apoptosis induced by GRP94 CAR NK-92 cells targeting the cell membrane in Molm13 cells in Example 3 of the present invention.

[0063] Figure 15 shows the process of GRP94 CAR NK-92 cells killing target cells in Example 3 of the present invention, and the expression of CD107a on the surface of NK92 cells (left figure shows the percentage of positive cells, right figure shows the quantitative analysis of expression level).

[0064] Figure 16 shows the secretion of IFN-γ (left) and granzyme B (GZMB, right) during the killing of target cells by GRP94 CAR NK-92 cells targeting the cell membrane in Example 3 of the present invention.

[0065] Figure 17 shows the apoptosis-inducing effect of GRP94 CAR NK-92 cells targeting the cell membrane on primary CLL-1+ AML cancer cells in Example 3 of the present invention;

[0066] Figure 18 shows the combination of CAR-NK92 cells targeting cell membrane GRP94 with other drugs such as atezolizumab and PD-L1 / PD-1 immune checkpoint inhibitors in Example 4 of the present invention (the left figure is a representative flow cytometry plot, and the right figure is a quantitative statistical plot).

[0067] Figure 19 shows the killing effect of GRP94 CAR NK-92 cells targeting cell membranes on target cells MV4-11 in zebrafish in Example 5 of the present invention.

[0068] Figure 20 shows the safety of GRP94 CAR NK-92 cells targeting the cell membrane in mice in Example 6 of the present invention (A. Mouse body weight dynamics, B. Mouse survival rate, C. Mouse body weight 1 week after CAR NK-92 cell injection);

[0069] Figure 21 is a schematic diagram of the CAR structure and anti-cancer principle of the present invention. Detailed Implementation

[0070] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0071] Example 1: Cell membrane GRP94 is a potential specific antigen for cancer cells such as FLT3-ITD+AML.

[0072] To identify more cancer-specific antigens, we used MV4-11, an acute myeloid leukemia (AML) FLT3-ITD+ AML cancer cell line, as an example. By comparing it with RS4-11, an acute lymphoblastic leukemia (AML) cancer cell line, we extracted their respective membrane protein components (Minute™ Plasma Membrane Protein Isolation and Cell Fractionation Kit, Invent, SM-005) and analyzed them using protein mass spectrometry. We found that CD19 and other proteins were expressed on the RS4-11 cell membrane (Figure 1), and GRP94 was found on the MV4-11 cell membrane. Further confirmation using immunofluorescence laser confocal imaging and immunofluorescence flow cytometry indicated that CD19 and other proteins were expressed on the RS4-11 cell membrane, and GRP94 was found on the MV4-11 cell membrane (18229, Abcam; MABT196, Millpore) (Figures 2 and 3). As shown in Figure 4, the results showed that GRP94 was not found in peripheral blood mononuclear cells (PBMCs) and CD3+ T cells from healthy controls. Flow cytometry analysis also showed no expression of GRP94 on the surface of healthy PBMCs (including CD3+ T cells such as CD3 mAb, 16669, and Abcam). As shown in Figure 5, the level of GRP94 expression in MV4-11 cells decreased with decreasing FLT3 kinase activity (24 hours of kinase inhibitor treatment), indicating its association with oncogene activity. As shown in Figure 6, the results showed that other AML cell lines, such as Molm13, also express GRP94. As shown in Figure 7, GRP94 expression was detected on the surface of other cancer cells, such as some primary AML cancer cells. Therefore, GRP94 is a potential cancer cell-associated antigen for certain AML subtypes and could serve as a potential target for cancer therapy.

[0073] The specific experimental method is as follows:

[0074] 1) Membrane protein extraction and protein proteometry detection

[0075] RS4;11 cells and MV4-11 cells were cultured and expanded in RPMI 1640 medium (Giboco; with additional 10% FBS, 100 U / ml penicillin, and 0.1 mg / ml streptomycin).

[0076] Cell membrane proteins were extracted and separated using the Minute™ Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent, SM-005) following the instructions.

[0077] Take 300 μg of protein sample, dilute it to 100 μl with 50 mM NH4HCO3, add 4 times the volume of pre-cooled acetone, and let it precipitate overnight;

[0078] Centrifuge at 15000g for 30 minutes at 4℃, then discard the supernatant;

[0079] Wash once with 500 μl of cold acetone, once with 500 μl of 70% cold ethanol, and once with 500 μl of cold acetone. Centrifugation was performed at 4°C.

[0080] Freeze-dry the precipitate for about 3 minutes, then resuspend it in 50 μl of UA buffer (it can usually be dissolved by shaking at room temperature on a ThermoMixer for about two hours, during which time it can be blown away with a pipette).

[0081] DTT: Add 2 μl of 50 mM DTT (final DTT concentration 2 mM) and incubate at 30°C for 1.5 hours (1 M DTT = 0.154 g / ml, dilute 20 times before use);

[0082] IAA: Add 13 μl of 50 mM IAA (final IAA concentration 10 mM) and place in the dark for 40 min (500 mM IAA = 0.0925 g / mL, diluted 10 times before use);

[0083] Dilute with 50 mM NH4HCO3 to 600 μl (final urea concentration approximately 0.7 M), add Trypsin (approximately 1:80, w / w), and incubate at 37°C for 4 hours. Note: Resuspend Trypsin in 10 mM acetic acid; recommended concentration is 0.25 μg / μl.

[0084] Add Trypsin (approximately 1:80, w / w) and incubate overnight at 37°C. Note: The optimal pH for Trypsin hydrolysis is 7-8.5. It is recommended to measure the pH of 1 μl of protein solution after adding trypsin.

[0085] The reaction was terminated by acidification with 10% TFA to a final concentration of 0.4%.

[0086] Wash the C18 desalting column with 20 μl of methanol, repeat once;

[0087] The C18 desalting column was activated twice with buffer B (80% ACN + 0.1% FA), with 20 μl added each time;

[0088] The C18 desalting column was equilibrated twice with buffer A (H2O + 0.1% FA), with 20 μl added each time;

[0089] Dissolve the sample in 20 μl buffer A, gently tap to completely dissolve the peptides, transfer to the column, gently tap the column, let it stand for a while, and then use a syringe to push the liquid into the original sample tube.

[0090] After centrifuging the sample tube and loading it onto the column once more, the solution is pushed out into the sample tube. At this point, the peptide has bound to the column.

[0091] Wash the column once with buffer A, push the solution into a new sample tube, and wash the column tip with water.

[0092] Elute with 20 μl of 50% ACN + 0.5% FA, then transfer to a new PCR tube. Elute with 20 μl of lbuffer B, then transfer to the PCR tube mentioned above.

[0093] The PCR tubes were dried, and the samples were dissolved in a peptide dissolving solution containing H2O and 1% FA (formic acid) for mass spectrometry detection.

[0094] 2) Cell membrane target protein immunofluorescence confocal microscopy imaging experiment

[0095] Cells pre-placed on poly-L-lysine-pretreated slides (cell membranes can be pre-labeled with WGA, etc.);

[0096] Fix with 4% paraformaldehyde for 10 minutes;

[0097] After rinsing three times with PBS;

[0098] 0.5% Triton X-100 perforation treatment for 15 minutes;

[0099] Rinse three times with PBS;

[0100] Block with 1% BSA for 30 minutes;

[0101] Add the corresponding primary antibody and hybridize at 37°C for 2 hours, then wash three times with PBS.

[0102] Add the corresponding secondary antibody and hybridize at 37°C for 1 hour, then wash three times with PBS (if the primary antibody is coupled with fluorescence, omit this step);

[0103] Add 5 μg / ml DAPI and stain for 2 minutes;

[0104] After mounting with anti-quenching mounting medium, the slides were observed under an immunofluorescence microscope.

[0105] The above steps can also be combined with fluorescent probes for organelles such as cell membranes for the localization and observation of target proteins;

[0106] If only the protein expression at the cell membrane is observed, the above fixation, perforation and other steps can be omitted.

[0107] 3) Flow cytometry immunofluorescence labeling experiment

[0108] After rinsing with cold PBS, adjust the cell concentration to 1-5 × 10⁶ cells / mL;

[0109] Then, block with 1% BSA for 30 minutes;

[0110] Add an appropriate concentration of monoclonal antibody against the target protein and incubate in the dark for 30 minutes.

[0111] Rinse three times with PBS;

[0112] Add the appropriate fluorescent secondary antibody and incubate at 37°C for 1 hour;

[0113] Rinse three times with PBS;

[0114] Perform flow cytometry experiments and analyze the results;

[0115] Each cell type has a control, namely an isotype control antibody.

[0116] Example 2: Preparation and validation of CAR-NK92 cells targeting cell membrane GRP94

[0117] 1) Construct a CAR expression plasmid using the scFv sequence of a monoclonal antibody that recognizes cell membrane GRP94 or other sequences that can recognize cell membrane GRP94 using conventional cloning methods (using co-expression of GFP protein as an expression signal). Then, use conventional lentiviral or retroviral methods (① Culture HEK293T cells in a culture dish. Transfect the cells 12-24 hours after seeding (when the cells reach 70-80% confluency): Add 200 μl (500 μl) of Opti-MEM to each of two 1.5 ml sterile EP tubes. Add 2 μg (4 μg) of the target expression plasmid, 2 μg of the packaging vector plasmid, and 2 μg of the envelope plasmid to one tube, and vortex to mix; add Lipo... ① Add 12 μl (16 μl) of the Plus transfection reagent and vortex to mix. Let stand at room temperature for 5 min. ② Mix the two solutions, vortex to mix, and let stand at room temperature for 20 min. ③ Add the above solution to a culture dish, gently shake to mix, and incubate in an incubator. ④ Replace with fresh culture medium after 6-12 h and continue culturing. ④ After culturing for 24 h, observe the 293T cells. If the cell condition is poor, with about 20-30% of the cells dying, it indicates that lentivirus has been produced. Otherwise, continue to wait until 48 h. ⑤ Use an ultrafiltration membrane or centrifuge at 1250 rpm × 5 min to obtain the virus suspension (can be used directly or stored at 4℃). ⑥ Infect target cells: Seed cells in 60 mm culture dishes in advance. When the cell density is 70-80%, replace with fresh culture medium and add the virus suspension. Infect for 24-48 h, passage once, and culture in fresh culture medium for 24 h. ⑦ Infect NK92 cells. NK92 cells were cultured in a specific NK92 medium (Procell, CM-0530) supplemented with recombinant human interleukin-2 (IL-2) before and after transfection. The medium consisted of MEMα (PM150422) + 0.2 mM Inositol + 0.1 mM β-mercaptoethanol (PB180633) + 0.02 mM Folic Acid + 100-200 U / mL recombinant IL-2 + 12.5% ​​HS (164215) + 12.5%. FBS (164210-50) + 1% P / S (PB180120) was used for culture and expansion. (① NK-92 cells grow in suspension, with most cells clustered together and a few scattered cells, and many dead cells and cell debris in the intercellular spaces; ② NK-92 cells are sensitive to IL-2. IL-2 degradation in the culture medium will lead to a deterioration in cell condition. Freshly prepared culture medium should be used as soon as possible. IL-2 is kept on hand in the laboratory. If poor cell condition, increased scattered cells, or no cell growth are observed, IL-2 is added at a concentration of 200 U / ml. The cells will recover after 2-3 days of culture; ③ NK-92 cells are sensitive to centrifugation.)During normal culture, the medium change cycle is 2-3 days. It is recommended to alternate between half-volume medium changes and centrifugal medium changes, that is, 2-3 half-volume medium changes followed by a full-volume centrifugal medium change to minimize the number of centrifugations. ④ Medium change methods: a. Replenishment method: Add an appropriate amount (1-2 ml) of fresh culture medium every 2-3 days, and simultaneously add 200 U / ml IL-2. After 2-3 replenishments, centrifuge to change the entire medium. b. Half-replenishment method: Taking a T25 bottle as an example, the bottle contains 5 ml of culture medium. Stand the bottle upright and let it stand for a period of time (tapping the bottle before standing it upright to make the cell clusters that are lightly attached to the bottom float up). After the cells settle to the bottom (observe the cell settling with the naked eye), carefully aspirate 2.5 ml of culture medium, transfer it to a centrifuge tube, centrifuge at 1000 rpm for 3-5 minutes, and check for any precipitation to avoid cell loss. Add 2.5 ml of fresh culture medium to the original bottle. If there are cells in the centrifuge tube, resuspend them with fresh culture medium and put them back into the original bottle, avoiding vigorous pipetting. ⑤ During cell growth, the cell clusters gradually increase in size. Normal cell clusters appear white and transparent under a microscope. If too many cells are clustered together, the center of the cell cluster may appear dark, indicating that the cell cluster may need to be passaged. After viral transfection, GFP-positive cells are detected using flow cytometry. The GFP-positive cell population is then sorted and collected for amplification.

[0118] 2) After multiple rounds of cell culture and sorting of GFP-positive cell populations, CAR-NK92 cells were tested and identified: (a) Compared with ordinary NK92, the GFP positivity rate of CAR-NK92 cells was over 90%; (b) Compared with ordinary NK92, protein L (RPL-PP2H2, ACRO) can specifically recognize scFv sequences; ① After washing with cold PBS, the cell concentration was adjusted to 1-5×106 cells / mL; ② An appropriate concentration of fluorescently conjugated protein L was added, and the cells were incubated in the dark for 30 min; ③ The cells were washed three times with PBS; ④ Flow cytometry showed that the positive cells were also over 90%, and the cells also expressed GFP fluorescent protein, as shown in Figure 8. The preparation of NK92 cells targeting cell membrane GRP94 was completed and their expression of CAR was verified.

[0119] Example 3: Selective Targeted Killing of FLT3-ITD+AML by CAR-NK92 Cells Targeting Cell Membrane GRP94

[0120] 1) Luciferase assay: FLT3-ITD+ AML cancer cells MV4-11 with stable luciferase expression were co-cultured with CAR-NK92 cells targeting GRP94 in different ratios (1:2; 1:5; 1:10; 1:20) for 6 hours, followed by luciferase assay (Bright-Glo luciferase assay system, E2620, Promega). The results, obtained using a SpectraMax iD5 microplate reader from Molecular Devices, showed that most cells were killed and lysed, as shown in Figure 9. Furthermore, after 24 hours of co-culture, almost all cancer cells were killed and lysed, as shown in Figure 10.

[0121] 2) Flow cytometry experiment on apoptosis: FLT3-ITD+AML cancer cells MV4-11 were stained with the non-toxic red fluorescent dye CM-DiI, and then co-cultured with ordinary NK92 cells or CAR-NK92 cells targeting cell membrane GRP94 at a ratio of 1:2 for 6 hours. Apoptotic cells were then detected using an apoptosis kit (CellEvent Caspase-3 / 7 green ready probes reagent R37111, ThermoFisher). The results were analyzed by flow cytometry. The results showed that CAR-NK92 cells targeting cell membrane GRP94 led to more cancer cell apoptosis, as shown in Figure 11.

[0122] 3) Real-time cancer cell killing experiment: We stained FLT3-ITD+AML cancer cells MV4-11 with the non-toxic red fluorescent dye CM-DiI (this dye can penetrate the plasma membrane and enter the cell without cytotoxicity, and does not affect cell viability and proliferation). Then, we co-cultured them with CAR-NK92 cells targeting cell membrane GRP94 at a ratio of 1:5. Apoptosis was detected using an apoptosis kit (CellEvent Caspase-3 / 7 green ready probes reagent R37111, ThermoFisher), and real-time imaging was performed, as shown in Figure 12. The results showed that CAR-NK92 cells targeting cell membrane GRP94 were activated upon contact with target cells and killed cancer cells. The cancer cells exhibited apoptosis within a few hours (the red fluorescently labeled target cells turned green intracellularly).

[0123] 4) Molecular level experiment of cancer cell killing: CM-DiL red fluorescently labeled MV4-11 cells were co-incubated with csGRP94-CAR NK92 cells for 6 h (E:T = 2:1). Then, effector cells and target cells were sorted by flow cytometry and compared with cells that were not co-incubated. RNS-Seq sequencing analysis showed that genes such as GZMB in csGRP94-CAR NK92 cells were significantly upregulated, while oncogenes such as flt3, myc, and bcl2 in cancer cells were significantly downregulated, indicating that NK cells were activated and cancer cells were killed and entered apoptosis, as shown in Figure 13.

[0124] 5) Flow cytometry experiments on apoptosis in other cancer cell lines: We stained FLT3-ITD+AML cancer cells Molm13 with the non-toxic red fluorescent dye CM-DiI, and then co-cultured them with ordinary NK92 cells or CAR-NK92 cells targeting cell membrane GRP94 at a ratio of 1:2 for 6 hours. Apoptotic cells were then detected using a kit (CellEvent Caspase-3 / 7 green ready probes reagent R37111, ThermoFisher) and analyzed by flow cytometry. The results showed that CAR-NK92 cells targeting cell membrane GRP94 led to more cancer cell apoptosis, as shown in Figure 14.

[0125] 6) Detection of CD107a expression level on the surface of immune cells: We stained FLT3-ITD+AML cancer cells MV4-11 or Molm13 with the non-toxic red fluorescent dye CM-DiI, and then co-cultured them with CAR-NK92 cells targeting cell membrane GRP94 at a ratio of 1:0.5 for 24 hours. Then, we used CD107a antibody (65051, Proteintech) to detect the level of CD107a expression on the surface of NK cells. The results showed that the expression level of CD107a membrane was significantly increased, indicating that co-incubation with cancer cells significantly activated the killing activity of CAR NK92, as shown in Figure 15.

[0126] 7) ELISA assay for cytokine levels in cancer cell killing experiments: We co-cultured GRP94-positive cancer cells MV4-11 or Molm13, and GRP94-negative RS4;11 cells with GRP94-targeting CAR-NK92 cells at a ratio of 1:0.5 for 24 hours. Then, we used ELISA kits (E-EL-H1617c, E-EL-H0108c, Elabscience) to detect changes in IFN-γ and granzyme B (GZMB) levels in the cell culture medium. We found that the fold increase in MV4-11 or Molm13 co-incubation system was greater than that in RS4;11, indicating that co-incubation with cancer cells significantly activated the killing activity of CAR NK92, as shown in Figure 16.

[0127] 8) Primary cancer cell killing experiment: We sorted primary AML cancer cells using CLL-1 antibody (57950, CST), and then co-cultured them with ordinary NK92 cells or CAR-NK92 cells targeting cell membrane GRP94 at a ratio of 1:2 for 24 hours. Apoptotic cells were then detected using an apoptosis kit (CellEvent Caspase-3 / 7 green ready probes reagent R37111, ThermoFisher), and the results were analyzed by flow cytometry. The results showed that CAR-NK92 cells targeting cell membrane GRP94 induced more apoptosis in primary cancer cells compared to ordinary NK92 cells, as shown in Figure 17.

[0128] Example 4: CAR-NK92 cells targeting cell membrane GRP94, combined with other drugs such as atezolizumab and PD-L1 / PD-1 immune checkpoint inhibitors, can significantly enhance the killing effect on cancer cells.

[0129] Atezolizumab (25 μg / ml) was used in a co-culture system. FLT3-ITD+ AML cancer cells MV4-11 were stained with the non-toxic red fluorescent dye CM-DiI, and then co-cultured with CAR-NK92 cells targeting GRP94 at a 2:1 ratio for 24 hours (with or without atezolizumab). Apoptosis was detected using an apoptosis kit (CellEvent Caspase-3 / 7 green ready probes reagent R37111, ThermoFisher), and analyzed by flow cytometry. The results showed that atezolizumab, in combination with CAR-NK92 cells targeting GRP94, led to more cancer cell apoptosis, as shown in Figure 18.

[0130] Example 5: In vivo anticancer effect of CAR-NK92 cells targeting cell membrane GRP94

[0131] MV4-11-mCherry cells, stably expressing the red fluorescent protein mCherry, were co-injected into the yolk sacs of zebrafish that had developed to 2 days. After 24 hours, almost no cancer cell signals were detected in the zebrafish co-injected with CAR-NK92 cells containing cell membrane GRP94, as shown in Figure 19. These results indicate that this type of CAR-NK92 cell targeting cell membrane GRP94 also possesses a certain anti-cancer effect in animals.

[0132] Example 6: Relative safety of CAR-NK92 cells targeting cell membrane GRP94

[0133] Five × 10⁶ cells (dissolved in physiological saline) were injected intravenously into 6-8 week old NSG mice twice a week for a total of four injections. Results showed no significant change in mouse body weight during the administration period (Figure 20A); one week after the last administration, the survival rate remained 100% (Figure 20B); and there was still no significant change in mouse body weight one week after the last administration (Figure 20C). These results indicate that this type of CAR-NK92 cell targeting cell membrane GRP94 is relatively safe.

Claims

1. A chimeric antigen receptor for GRP94 on the cell membrane targeting cancer neoantigen expression, characterized in that, The antibody comprises a signal peptide region, an antigen-binding domain targeting cell membrane GRP94, a hinge region, a transmembrane domain, and a signal transduction activation domain, sequentially linked from the amino terminus to the carboxyl terminus. The nucleotide and amino acid sequences of the signal peptide region are shown in SEQ ID No: 1 and SEQ ID No: 2, respectively. The antigen-binding domain is a portion of the antibody itself targeting cell membrane GRP94 expressing a cancer neoantigen, or a portion of the antibody itself or an antibody region targeting other different targets, which can be tandemly linked by a linker sequence. The nucleotide sequence of the antibody is shown in SEQ ID No: 3, or SEQ ID No: 4, or SEQ ID No: 5, or SEQ ID No: 6, and the corresponding amino acid sequence is shown in SEQ ID No: 7, or SEQ ID No: 8, or SEQ ID No: 9, or SEQ ID No:

10.

2. The chimeric antigen receptor for GRP94 on the cell membrane targeting cancer neoantigen expression cells according to claim 1, characterized in that, The nucleotide and amino acid sequences of the tandem regions of the linking sequence are shown in SEQ ID No: 11 and SEQ ID No: 12, respectively.

3. The chimeric antigen receptor for GRP94 on the cell membrane targeting cancer neoantigen expression cells according to claim 1, characterized in that, The hinge region is a CD8 hinge domain with a nucleotide sequence as shown in SEQ ID No: 13 and an amino acid sequence as shown in SEQ ID No: 14, or a nucleotide sequence as shown in SEQ ID No: 15 and an amino acid sequence as shown in SEQ ID No:

16. And / or, the transmembrane domain is a nucleotide or amino acid sequence such as the CD28 transmembrane and 4-1BB co-stimulatory domain shown in SEQ ID No: 17; SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20; And / or, the nucleotide or amino acid sequence of the 4-1BB co-stimulatory domain is as shown in SEQ ID No: 21, SEQ ID No: 22; And / or, the nucleotide or amino acid sequence of CD3ζ ITAMS1 is as shown in SEQ ID No: 23, SEQ ID No: 24; And / or, the required nucleotide or amino acid sequence of T2A for co-expression of other protein molecules, as shown in SEQ ID No: 25, SEQ ID No:

26.

4. An isolated nucleic acid, characterized in that, The isolated nucleic acid includes a nucleotide sequence and an amino acid sequence fragment for expressing the cell membrane GRP94 chimeric antigen receptor for targeting cancer neoantigens as described in any one of claims 1 to 3.

5. A recombinant vector, characterized in that, The recombinant vector comprises the isolated nucleic acid as described in claim 4.

6. A CAR-NK cell, characterized in that, The CAR-NK cells contain the isolated nucleic acid as described in claim 4, or are cells transformed by the recombinant vector as described in claim 5.

7. The application of a chimeric antigen receptor targeting cancer neoantigen expression cell membrane GRP94 in a drug, characterized in that, The use of GRP94 chimeric antigen receptor on cell membranes expressing cancer neoantigens as described in any one of claims 1 to 3, the isolated nucleic acid as described in claim 4, the recombinant vector as described in claim 5, or the CAR-NK cells as described in claim 6 in the preparation of drugs for treating cancer-related diseases.

8. A pharmaceutical composition, characterized in that, This includes expression vectors for expressing the GRP94 chimeric antigen receptor on the cell membrane of a target cancer neoantigen as described in any one of claims 1 to 3, or CAR-NK cells as described in claim 6.

9. The application according to claim 7, characterized in that, The cancer-related diseases include acute myeloid leukemia with FLT3-ITD+ mutation.

10. The application according to claim 9, characterized in that, The CAR-NK cells can be used in combination with PD-L1 / PD-1 immune checkpoint inhibitors.

11. The application according to claim 9, characterized in that, The sources of the NK immune cells include, but are not limited to, cell lines NK-92, NK-92MI, HANK-1, KHYG-1, NK-YS, NKG, NK101, NK3.3, YTS, NKL, autologous or allogeneic peripheral blood, umbilical cord blood, induced pluripotent stem cells (iPSCs), and hematopoietic stem cells (HSCs).

Images