Immune cell for regulating and controlling CD226 and TIGIT receptors as well as preparation method and application of immune cell

Abstract

The invention belongs to the technical field of cell biology, and particularly relates to an immune cell for regulating CD226 and TIGIT receptors as well as a preparation method and application of the immune cell. The preparation method comprises the following steps: preparing a nucleotide sequence of a target gene; wherein the target gene comprises CD226OE, antiTIGIT or a CD226OE-antiTIGIT fusion gene, the nucleotide sequence of the CD226OE is as shown in SEQ ID NO: 7, the nucleotide sequence of the antiTIGIT is as shown in SEQ ID NO: 8, and the nucleotide sequence of the CD226OE-antiTIGIT fusion gene is as shown in SEQ ID NO: 9; constructing a recombinant plasmid containing a target gene; performing in-vitro amplification on the primary immune cells; and delivering the recombinant plasmid carrying the target gene to the primary immune cell to obtain the engineered immune cell. A delivery technology system of various gene vectors is developed, expression schemes of different gene structures in the NK cells are provided, and the developed gene modified NK cells show remarkably enhanced anti-tumor effects in in-vitro function experiments.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell biology, and specifically relates to an immune cell that regulates CD226 and TIGIT receptors, and a preparation method and application thereof. Background Art

[0002] With the successful application of chimeric antigen receptor T cell therapy (CAR-T) in B-cell malignancies, natural killer (NK) cells have gradually entered the clinical research echelon due to their ability to nonspecifically recognize and kill tumor cells, and their therapeutic potential in hematological malignancies and solid tumors has gradually attracted attention. However, the dynamic regulation of NK cell functional receptors has a significant impact on their anti-tumor efficacy, especially the regulation of the CD226 / TIGIT-CD155 signaling axis, which plays a key role in NK cell functional exhaustion and tumor immune escape. CD226 and TIGIT are both members of the immunoglobulin superfamily. Among them, CD226, also known as DNAX accessory molecule-1 (DNAM-1), is an adhesion molecule and co-stimulatory activating receptor that promotes NK cell activation by binding to CD155. T cell immunoreceptor with Ig and ITIMdomains (TIGIT) is an inhibitory receptor for NK cells and T cells. It binds to CD155 with high affinity, blocks the activation signal of CD226, and transmits the inhibitory function of NK cells.

[0003] Despite the inherent immune advantages of NK cells, their function is dynamically regulated by the CD226 / TIGIT-CD155 axis within the TME. Recent studies have demonstrated that high CD155 expression and overactivation of TIGIT signaling lead to NK cell exhaustion, while impaired CD226 binding further impairs anti-tumor responses. Intervention strategies targeting this axis require further exploration. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides an immune cell that regulates CD226 and TIGIT receptors, and a preparation method and application thereof.

[0005] The first object of the present invention is to provide a method for preparing immune cells that regulate CD226 and TIGIT receptors, comprising: preparing a nucleotide sequence of a target gene; wherein the target gene includes CD226 OE , antiTIGIT, or CD226 OE -antiTIGIT fusion gene, CD226OE The nucleotide sequence of antiTIGIT is shown in SEQ ID NO: 7, the nucleotide sequence of CD226 is shown in SEQ ID NO: 8, OE -The nucleotide sequence of the antiTIGIT fusion gene is shown in SEQ ID NO: 9; constructing a recombinant plasmid containing the target gene; amplifying primary immune cells in vitro; delivering the recombinant plasmid carrying the target gene to the primary immune cells to construct / obtain engineered immune cells.

[0006] The second object of the present invention is to provide an immune cell prepared by the above method.

[0007] The third object of the present invention is to provide a use of immune cells for killing at least one of the following tumor cells: blood tumor cells expressing the ligand CD155, solid tumor cell lines, and primary tumor cells.

[0008] Compared with the prior art, the present invention has the following beneficial effects:

[0009] 1. The present invention regulates the expression of CD226 and TIGIT by gene modification, and constructs a CD226 overexpression (CD226 OE ) and / or block the nucleotide sequence of TIGIT (antiTIGIT), and by intervening in this axis, restore the activation signal of NK cells or block the inhibitory signal, successfully construct engineered NK cells, achieve genetic modification of primary NK cells, enhance the anti-tumor effect of NK cells, overcome their functional exhaustion, and improve the immunotherapy potential of NK cells.

[0010] 2. The present invention aims to solve the problem that the existing technology uses exogenous CD226 agonistic antibodies or TIGIT inhibitory antibodies to unidirectionally regulate the expression of CD226 / TIGIT, which results in imbalance of immune signals and insufficient anti-tumor function. The present invention proposes a bidirectional synergistic regulation strategy. The bidirectional synergistic regulation strategy of the present invention is based on the function of NK cell receptors, with the activating receptor CD226 and the inhibitory receptor TIGIT as the core, and designs CD226 OE The -antiTIGIT fusion gene achieves endogenous overexpression of CD226 in NK cells to enhance activation signals and secreted anti-TIGIT scFv to continuously block inhibitory signals, thereby simultaneously optimizing the activation / inhibitory signal status of NK cells.

[0011] 3. The present invention has developed a delivery technology system for multiple gene vectors and provides expression schemes for different gene structures in NK cells. On the one hand, molecular cloning technology is used to construct in vitro transcription recombinant plasmids, and mRNA is introduced into NK cells by electrofection technology to obtain NK cells that transiently express the target gene. On the other hand, a reverse transcription recombinant plasmid is constructed, and a highly efficient and concentrated retrovirus is successfully packaged. NK cells that stably express the target gene are obtained by viral transduction. Compared with existing NK cell gene modification methods, the electrofection and retroviral transduction schemes used in the present invention improve the efficiency of gene modification and overcome technical bottlenecks such as the difficulty in transducing NK cells and the low efficiency of exogenous gene introduction.

[0012] 4. The gene-modified NK cells developed by the present invention showed significantly enhanced anti-tumor effects in in vitro functional experiments. Luciferase killing experiments confirmed that CD226 OE Compared to unmodified NK cells, NK cells modified with the anti-TIGIT gene have significantly increased their tumor cell-killing efficiency. Furthermore, mechanistic studies have shown that this effect is primarily achieved by enhancing the binding of NK cell surface CD226 to the tumor cell ligand CD155 and blocking the binding of NK cell surface TIGIT to the tumor cell ligand CD155. The cytotoxicity is particularly pronounced against tumor cell lines with high CD155 expression. This strategy is expected to be expanded to personalized immune cell therapy for patients with tumors that overexpress CD155, and has significant clinical translational value. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0014] Figure 1 Two in vitro transcription plasmid maps. A is pT7-CD226 OE A is the in vitro transcription plasmid map and B is the in vitro transcription plasmid map of pT7-antiTIGIT.

[0015] Figure 2 A is a map of an in vitro transcription plasmid and a map of a reverse transcription plasmid. A is pT7-CD226 OE -AntiTIGIT in vitro transcription plasmid map, B is pMSCV-CD226 OE -Reverse transcription plasmid map of antiTIGIT.

[0016] Figure 3 pMSCV-CD226 OE -Reverse transcription plasmid map of antiTIGIT-mbIL-15.

[0017] Figure 4Agarose gel electrophoresis diagram of in vitro transcription recombinant plasmid construction. A, pT7-EGFP vector double restriction enzyme digestion (MluI and XbaI), Vector represents the linearized vector fragment (about 5000bp), EGFP fragment is about 700bp; B, PCR amplification of the target gene fragment CD226 gene PCR amplification product; C, antiTIGIT gene PCR amplification product; D, CD226 OE -antiTIGIT fusion gene PCR amplification product; E, pT7-CD226 OE Plasmid clone identification, 1 to 5 represent CD226 fragments amplified from different monoclonal strains; F, pT7-antiTIGIT plasmid clone identification, 1 to 5 represent antiTIGIT fragments amplified from different monoclonal strains; G, pT7-CD226 OE -anti-TIGIT plasmid clone identification, 1 to 5 represent CD226-anti-TIGIT fusion fragments amplified from different monoclonal strains.

[0018] Figure 5 In vitro synthesis of CD226 OE mRNA, antiTIGIT mRNA, and CD226 OE -Agarose gel electrophoresis of antiTIGIT mRNA. A, pT7-CD226 OE Plasmid linearized fragment; B, pT7-antiTIGIT plasmid linearized fragment; C, pT7-CD226 OE -antiTIGIT plasmid linearized fragment; D, CD226 OE Electrophoresis results of mRNA samples; E, electrophoresis results of antiTIGIT mRNA samples; F, CD226 OE -antiTIGIT-mRNA sample electrophoresis results.

[0019] Figure 6 Electroporation of CD226 into NK cells OE mRNA, antiTIGIT mRNA, and CD226 OE -Flow cytometry histogram of antiTIGIT mRNA. A, TIGIT expression blocking rate of NK cells after electroporation of antiTIGIT mRNA; B, CD226 OE CD226 positive expression rate of NK cells after mRNA; C, CD226 electroporated OE -NK cell TIGIT expression blocking rate after anti-TIGIT mRNA; D, electroporated CD226 OECD226 positive expression rate of NK cells after anti-TIGIT mRNA expression. Control represents the electroporated NK cell control group.

[0020] Figure 7 For electroporation of CD226 OE mRNA, antiTIGIT mRNA, and CD226 OE -antiTIGIT mRNA cytotoxicity against target cells. A, Cytotoxicity against K562 cells (E:T = 2:1 and 1:1); B, Cytotoxicity against U937 cells (E:T = 2:1 and 1:1); C, Cytotoxicity against MOLM-13 cells (E:T = 2:1, 1:1, 0.5:1, and 0.25:1). Control represents the electroporated NK cell control group. *P < 0.05, **P < 0.01, ***P < 0.001. ns indicates P > 0.05.

[0021] Figure 8 CD226 OE Flow cytometry analysis of gene expression in NK cells transduced with anti-TIGIT retrovirus. A, CD226 positivity; B, TIGIT blockade rate. Control represents untransduced NK cells.

[0022] Figure 9 CD226 OE Flow cytometry analysis of gene expression in NK cells transduced with the anti-TIGIT-mbIL-15 retrovirus. A, CD226 positivity; B, TIGIT blockade rate; C, IL-15 positivity. Control represents untransduced NK cells.

[0023] Figure 10 For transduction of CD226 OE -antiTIGIT and CD226 OE Cytotoxicity of NK cells against AML cells after the anti-TIGIT-mbIL-15 retrovirus was expressed (E:T = 1:1). A, K562 cell line; B, U937 cell line; C, MOLM-13 cell line. *P < 0.05, **P < 0.01.

[0024] Figure 11Agarose gel electrophoresis diagrams showing the construction of CD155 expression plasmids. A, pLVX-EF1α-IRES-Neo vector double-digested with EcoRI and BamHI; the single band (Vector) represents the linearized vector (approximately 9000 bp); B, PCR amplification product of the CD155 gene; C, pLVX-EF1α-CD155-IRES-Neo plasmid clone identification; 1-8 represent CD155 fragments amplified from different monoclonal strains.

[0025] Figure 12 Analysis of CD155 expression levels in AML cell lines. A, K562; B, U937; C, MOLM-13 cell lines; fluorescence intensity distribution in isotype control group (Isotype), cell background control group (Parental), and CD155 overexpression experimental group (CD155-OE).

[0026] Figure 13 For transduction of CD226 OE -antiTIGIT and CD226 OE Cytotoxicity of NK cells transfected with anti-TIGIT-mbIL-15 retrovirus against AML cells expressing high levels of CD155 (E:T = 1:1). A, K562 cell line; B, U937 cell line; C, MOLM-13 cell line. *P < 0.05, ***P < 0.001. DETAILED DESCRIPTION

[0027] In order to better illustrate the purpose, technical process and benefits of the present invention, so that those skilled in the art can better understand the technical solution of the present invention and implement it, the present invention will be further described below with reference to specific examples and drawings. In the description of the present invention, unless otherwise specified, the reagents used are commercially available and the methods used are conventional techniques in the art. The present invention is further described in detail by the following exemplary embodiments, in which immune cells are NK cells as an example and tumor cells are AML cell lines as an example.

[0028] Example 1: Preparation of immune cells that simultaneously regulate CD226 and TIGIT receptors

[0029] 1. Obtain CD226 OE -antiTIGIT fusion gene coding region sequence

[0030] (1) Search the human CD226 amino acid sequence on the UniProt website: The CD226 structure is mainly composed of an extracellular domain (ECD) and an intracellular domain (ICD), which are connected by a transmembrane domain (TM). The signal peptide (SP) of the CD8A molecule is used to promote its expression, thereby realizing the target recognition and signal transduction functions of CD226. The human CD226 amino acid sequence is shown in SEQ ID NO: 1.

[0031] (2) Screening of human anti-TIGIT amino acid sequences: Two anti-TIGIT monoclonal antibody drugs were selected by reviewing the literature. After screening, the drug with the best blocking effect was determined. Based on the patent, the single-chain variable fragment (scFv) structure of the antibody that binds to the TIGIT receptor was obtained. This antigen-binding fragment is composed of the heavy chain variable region (VH) and light chain variable region (VL) of the antibody connected by a short peptide linker. The SP of the CD8A molecule was used to promote its expression. The human anti-TIGIT amino acid sequence is shown in SEQ ID NO: 2.

[0032] (3)CD226 OE -Design of antiTIGIT fusion gene construct: CD226 OE The amino acid sequence of the antiTIGIT fusion gene is formed by concatenating the human CD226 amino acid sequence and the antiTIGIT amino acid sequence through T2A (Thosea asignavirus 2A peptide) in the 2A peptide sequence family. The specific amino acid sequence is shown in SEQ ID NO: 3. Compared with the amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 2, SEQ ID NO: 3 achieves co-expression of CD226 and antiTIGIT structures through fusion design. Among them, the NCBI accession number of the human CD226 gene sequence is NM_006566.4. The nucleotide sequence found on NCBI was codon optimized on the website http: / / sg.idtdna.com / CodonOpt to ensure that it is more suitable for gene expression while the encoded amino acid sequence remains unchanged. The optimized human CD226 nucleotide sequence is shown in SEQ ID NO: 7, which is the full gene coding region sequence of CD226. The optimized human antiTIGIT nucleotide sequence is shown in SEQ ID NO: 8, which is the full gene coding region sequence of antiTIGIT. The optimized human CD226 OE-antiTIGIT nucleotide sequence is shown in SEQ ID NO: 9 (corresponding amino acid sequence is SEQ ID NO: 3), which is CD226 OE -The complete gene coding region sequence of antiTIGIT.

[0033] 2.pT7-CD226 OE Construction of antiTIGIT recombinant plasmid

[0034] (1) The nucleotide sequence SEQ ID NO: 9 was synthesized by a gene company and constructed into a cloning vector pUC57. (2) The pT7-EGFP vector was linearized by double digestion with MluI and XbaI. After double digestion, the vector produced a long sequence fragment and a short sequence fragment. The long sequence fragment after double digestion was recovered by gel running to obtain the double linearized plasmid after enzyme digestion. Figure 4 A. (3) Human CD226 synthesized by Gene Company OE -antiTIGIT nucleotide sequence was used as a template and homologous recombination was used to design pT7-CD226 OE -antiTIGIT recombinant plasmid primers, perform PCR amplification, run gel to recover PCR amplification products, and purify them. Figure 4 (4) Use seamless cloning reagent to connect the linearized pT7-EGFP plasmid and CD226 OE -PCR amplification product of antiTIGIT nucleotide sequence. (5) The above ligated products were transformed into E. coli and single clones were identified, and sent for first generation sequencing to obtain the correct plasmid vector. Figure 4 G. pT7-CD226 OE -antiTIGIT recombinant plasmid (in vitro transcription vector) map see Figure 2 A.

[0035] human CD226 OE -The primers for the antiTIGIT nucleotide sequence (SEQ ID NO: 9) are as follows:

[0036] F1:5'-AGACCACAACGGTTTTCCCACGCGTGCACCATGGCACTG-3', SEQ ID NO:13;

[0037] R1: 5'-GTAATCCAGAGGTTGATTTCTAGACTACTTGATCTCCACCTTAGTTC-3', SEQ ID NO: 14.

[0038] 3.pMSCV-CD226 OEConstruction of antiTIGIT recombinant plasmid

[0039] (1) Using pMSCV-puro as the vector backbone, the pMSCV-puro plasmid was linearized by double enzyme digestion with BglII and ClaI. After double enzyme digestion, the plasmid produced a long sequence fragment and a short sequence fragment. The long sequence fragment after double enzyme digestion was recovered by gel running to obtain the linearized plasmid after double enzyme digestion. (2) The above human CD226 OE -antiTIGIT nucleotide sequence was used as a template and homologous recombination was used to design pMSCV-CD226 OE -antiTIGIT recombinant plasmid primers, perform PCR amplification, run gel recovery and purification of PCR amplification products. (3) Use seamless cloning reagent to connect the above linearized pMSCV-puro plasmid with CD226 OE -antiTIGIT nucleotide sequence PCR amplification product. The ligated product was transformed into E. coli and the monoclonal bacterial solution was identified and sent for first generation sequencing to obtain the correct plasmid vector, which can be obtained as pMSCV-CD226. OE -antiTIGIT recombinant plasmid, which is a reverse transcription recombinant plasmid. For the map, see Figure 2 B.

[0040] CD226 OE -The primers for the antiTIGIT nucleotide sequence (SEQ ID NO: 9) are as follows:

[0041] F2:5'-CGCCGGAATTAGATTCTATGGCACTGCCAGTGACTG-3', SEQ ID NO:15;

[0042] R2: 5'-TCTTTTATTTTATCGATCTACTTGATCTCCACCTTAGTTCCACC-3', SEQ ID NO: 16.

[0043] 4.Contains CD226 OE -Construction of recombinant plasmid containing antiTIGIT-mbIL-15 fusion gene

[0044] Obtaining CD226 OE -antiTIGIT-mbIL-15 fusion gene coding region sequence

[0045] (1) mbIL-15 anchors IL-15 to the membrane surface of effector cells through the CD8A transmembrane region or the native IL-15Rα transmembrane region, providing a sustained secretion signal and enhancing cell proliferation and survival. The human mbIL-15 amino acid sequence is found on the UniProt website; the human mbIL-15 amino acid sequence is shown in SEQ ID NO:4. The human mbIL-15 gene sequence is a combination of NCBI accession numbers NM_000585.5 and NM_001256765.1; the human mbIL-15 nucleotide sequence is shown in SEQ ID NO:10.

[0046] (2)CD226 OE -Design of antiTIGIT-mbIL-15 fusion gene construct: Based on the CD226 OE Based on the anti-TIGIT fusion gene structure, the mbIL-15 structure was connected through T2A. The amino acid sequence after connection is shown in SEQ ID NO: 5. OE -antiTIGIT-mbIL-15 nucleotide sequence is shown in SEQ ID NO: 11, which is CD226 OE -The complete gene coding region sequence of antiTIGIT-mbIL-15.

[0047] 4.2.pMSCV-CD226 OE Construction of antiTIGIT-mbIL-15 recombinant plasmid

[0048] The pMSCV-CD226 constructed in Example 1 was OE -antiTIGIT recombinant plasmid and mbIL-15 nucleotide sequence SEQ ID NO: 10 were synthesized by the company and cloned into pMSCV-CD226 OE -antiTIGIT recombinant plasmid and add mbIL-15 gene. OE -antiTIGIT-mbIL-15 recombinant plasmid CD226 OE -The sequencing results of the antiTIGIT-mbIL-15 fusion gene were compared with the amino acid sequence SEQ ID NO: 5 and the nucleotide sequence SEQ ID NO: 11 to ensure the correctness of the plasmid. Figure 3 .

[0049] 5. Expression and Functional Verification of mRNA Electroporated NK Cells

[0050] Compared to T cells, many vector systems have lower gene uptake efficiency and transgene expression levels in NK cells, and may be associated with reduced NK cell activity and increased risk of apoptosis. To verify the effects of different constructs in this invention on NK cell function, we used mRNA electroporation technology to genetically modify NK cells and validate its feasibility under specific conditions.

[0051] 5.1.CD226 OE Preparation of anti-TIGIT mRNA

[0052] (1) Linearized plasmid: pT7-CD226 was digested with HindⅢ enzyme. OE -antiTIGIT recombinant plasmid, the enzyme digestion product showed a single band by agarose gel electrophoresis, the size of which was consistent with the theoretical length of the plasmid. The enzyme digestion product was recovered to obtain the linearized plasmid template. Figure 5 C.

[0053] (2) In vitro transcription and tailing: using mMESSAGE Kit and Poly(A) Tailing Kit for linearized pT7-CD226 OE -antiTIGIT plasmid was used for in vitro transcription and tailing. TM CD226 was purified by transcription purification kit OE -antiTIGIT mRNA.

[0054] (3) Verify the purity and quality of mRNA: Confirm that the mRNA product band is single and clear (no degradation) by 1g / 100mL denaturing agarose gel electrophoresis and imaging, see Figure 5 The concentration and purity of F. mRNA were detected using an ultra-micro-volume spectrophotometer.

[0055] 5.2.CD226 OE -antiTIGIT mRNA electroporated into NK cells

[0056] (1) Culture of primary NK cells: Prepare peripheral blood mononuclear cells (PBMC) or purified NK cells from healthy donors, and use feeder cells (K562-4-1BBL-mbIL-21 / -15) for activation and expansion. During the culture process, use human PE-CD45 antibody, FITC-CD3 antibody, APC-CD56 antibody and PE-Cy7-CD19 antibody to measure the proportion of lymphocyte subsets and observe the effect of NK cell expansion. (2) Electroporation of NK cells: Collect NK cells expanded in vitro to day 9, and prepare 1×10 6NK cells were washed once with serum-free and antibiotic-free NK cell culture medium at a concentration of 100 μL / mL. After centrifugation, the supernatant was discarded as much as possible. 10 μL of each electroporation solution A and solution B (Celetrix, USA) were taken to prepare a 20 μL / tube system, for a total of 2 tubes. OE -antiTIGIT mRNA was added to 20 μL of electroporation solution system, and the remaining 1 tube was an electroporation system without mRNA as the control group. Use the above electroporation system to resuspend NK cells, add them to the electrode tube, and place them in the electroporator (Celetrix, USA). Electrotransfection was performed under 520V conditions and transferred to a 24-well plate for culture. (3) Detection of gene expression: After 24 hours, the electroporated cells were collected, washed twice with MACS buffer, and centrifuged for 5 minutes at a speed of 350g / min. After resuspending the cells with MACS buffer, the antibody PI-PerCP was added to remove dead cells, the antibodies CD3-FITC and CD56-APC were used to circle the NK cells, and the antibodies CD226-PE-Cy7 and antiTIGIT-APC-Cy7 were used to detect the positive rate of CD226 and the blocking rate of TIGIT, respectively, see Figure 6 C and D.

[0057] 5.3.CD226 OE -Direct killing effect of antiTIGIT mRNA on tumor cells after electroporation of NK cells

[0058] (1) Cell preparation: Luciferase release assay was used to detect the killing efficiency of NK cells. The experiment included at least 3 independent repeated experiments of 3 donors. The effector cells were electroporated CD226 OE -NK cells after anti-TIGIT mRNA injection. Target cells are hematologic malignancy cell lines K562-luc, U937-luc, and MOLM-13-luc.

[0059] (2) Co-culture of NK cells and tumor cells: The effector-target ratio (E:T) was set to 2:1, 1:1, 0.5:1, and 0.25:1. The target cell density was adjusted to 2×10 3 15 μL / well was inoculated into a 384-well culture plate with a white bottom and a transparent cover. The density and number of effector cells were adjusted according to the number of target cells, and 15 μL / well was added to the corresponding target cells according to the set effector-target ratio, with a total volume of 30 μL / well. A negative control group (only culture medium was added) and a positive control group (only target cells were added) were set up. Three replicates were set up for the negative control, positive control, and each experimental group. The culture plate was placed in a 37°C, 5% CO2 incubator for 15 hours.

[0060] (3) Detection results: Add 30 μL of ONE-Glo™ luciferase detection reagent to each well, incubate at room temperature in the dark for 3 minutes, and use a microplate reader to detect the luminescence reading. Calculation formula: NK cell killing efficiency (%) = [(positive control reading - negative control reading) - (experimental group reading - negative control reading)] / (positive control reading - negative control reading) × 100%. For results, see Figure 7 , electroporated CD226 OE -antiTIGITmRNA can enhance the killing activity of NK cells against tumor cell lines.

[0061] 6. Expression and Functional Verification of Retroviral-transduced NK Cells

[0062] Primary NK cells were genetically modified using electrofection to successfully express the target gene structure. However, due to the limited duration of gene expression under electrofection, it is difficult to meet the requirements of efficient NK cell engineering. In contrast, the retroviral system has higher stability and transduction efficiency. Therefore, we constructed pMSCV-CD226 OE -antiTIGIT and pMSCV-CD226 OE -antiTIGIT-mbIL-15 reverse transcription plasmid, followed by retroviral packaging and transduction, for stable gene modification of NK cells.

[0063] 6.1.pMSCV-CD226 OE -antiTIGIT and pMSCV-CD226 OE -antiTIGIT-mbIL-15 retroviral packaging

[0064] (1) Preparation of packaging cells: 293T Clone 2 packaging cells were cultured in a 10 cm dish using 10% complete FBS-DMEM medium until they were in the logarithmic growth phase and had a cell density of 90%. The cells were replaced with 5 mL of DMEM medium containing 2% FBS and no antibiotics. (2) Configuration system: A single plasmid system (pMSCV-CD226 OE -antiTIGIT or pMSCV-CD226 OE-antiTIGIT-mbIL-15) Prepare plasmid dilutions, add each plasmid dilution to the corresponding PEI solution to make the final concentration of the plasmid 1μg / μL, mix well, let stand at room temperature for 15 minutes, obtain a mixed solution, and add it dropwise to 293T Clone2 cells. (3) Cultivate cells: 6 hours after transfection, discard the culture medium, replace with 10mL of fresh DMEM complete medium, and continue to culture at 32℃. (4) Harvest and concentrate virus: 72 hours after transfection, collect the supernatant of 293T Clone 2 cell culture medium, centrifuge at 1500g for 10 minutes at 4℃, filter the supernatant with a 0.22μm filter, and add the filtrate to an ultracentrifuge tube. Centrifuge at 100,000g for 2 hours at 4℃. After centrifugation, discard the supernatant, add 100μL DPBS solution to each tube, dissolve the lentiviral precipitate at 4℃ overnight, and aliquot the virus suspension into virus tubes the next day and freeze at -80℃.

[0065] 6.2.pMSCV-CD226 OE -antiTIGIT and pMSCV-CD226 OE -antiTIGIT-mbIL-15 retroviral transduction of NK cells

[0066] (1) Culture primary NK cells as in “5.2(1)”. Prepare NK cells for expansion on day 5 and adjust the cell density to 1×10 6 / mL. Non-Treated 24-well plates were coated with 5μg / mL RetroNectin working solution (500μL / well) and incubated at room temperature for 2h. The plates were left standing at room temperature for 30min with 2% BSA blocking solution (500μL / well), and washed once with DPBS. (2) Viral transduction: Determine the multiplicity of infection (MOI) of NK cells: MOI is the ratio of the number of virus particles to the number of target cells. Calculate the amount of virus according to the virus titer: Virus amount = number of cells × MOI / virus titer. After adding the virus and mixing, centrifuge at 32℃ and 900g for 60min to improve the viral transduction efficiency of NK cells, and continue to culture in an incubator. (3) Culture and detection: After the virus is transduced, continue to culture for 72h, and detect the positive rate of CD226, the blocking rate of TIGIT, and the positive rate of IL-15 respectively as described in "5.2(3)". For specific transduction efficiency, see Figure 8 and Figure 9 .

[0067] 6.3.pMSCV-CD226 OE -antiTIGIT and pMSCV-CD226 OE -Direct killing effect of antiTIGIT-mbIL-15 retrovirally transduced NK cells on tumor cells

[0068] Prepare effector cells and target cells according to the method in "5.3". After co-culture, use a microplate reader to detect the results and calculate the killing efficiency of each group of effector cells. The results are shown in Figure 10 .

[0069] 7. CD226 / TIGIT bidirectionally regulates the killing effect of NK cells on CD155-overexpressing tumor cells

[0070] The gene regulation strategy of the present invention is based on the molecular mechanism that the activating receptor CD226 and the inhibitory receptor TIGIT transmit opposite signals by competitively binding to the same ligand (especially CD155). To verify the functional characteristics of immune cells that regulate CD226 and TIGIT receptors in an environment with high CD155 expression, we constructed an AML cell line that overexpresses CD155 based on an AML cell line that is positive for CD155 expression. Luciferase killing experiments confirmed that the NK cells constructed by the present invention mainly exert anti-tumor effects through the CD226 / TIGIT-CD155 axis.

[0071] 7.1. Construction and Validation of AML Cell Lines Overexpressing CD155

[0072] (1) Construction and synthesis of CD155 lentiviral expression plasmid: CD155 lentiviral plasmid (Miaoling platform: P53061) was purchased and the CD155 sequence was aligned using SnapGene software to ensure correctness. The amino acid sequence of CD155 is shown in SEQ ID NO: 6, and the nucleotide sequence is shown in SEQ ID NO: 12. The plasmid powder was dissolved in 20 μL of sterile water for transformation, plasmid amplification and extraction. The lentiviral plasmid pLVX-EF1α-IRES-Neo was selected as the vector of CD155 expression plasmid and linearized with EcoRⅠ restriction enzyme. The primers of pLVX-EF1α-CD155-IRES-Neo recombinant plasmid were designed using homologous recombination method, PCR amplification was performed, and the PCR amplification product was recovered and purified by gel running.

[0073] The primers for the CD155 nucleotide sequence (SEQ ID NO: 12) are as follows:

[0074] F3:5'-ATTAGCTTGGTACGAATTCGCCACCATGGCCCGAGCC-3', SEQ ID NO:17;

[0075] R3: 5'-AGGGAGAGGGGCGGGATCCTCACCTTGTGCCCTCTGTCTG-3', SEQ ID NO: 18.

[0076] Use seamless cloning reagent to connect the linearized pLVX-EF1α-IRES-Neo plasmid and the PCR amplification product of CD155 nucleotide sequence, transform E. coli and identify the single clone, send it for first-generation sequencing, and obtain the correct plasmid vector. Figure 11 , the pLVX-EF1α-CD155-IRES-Neo recombinant plasmid can be obtained.

[0077] (2) pLVX-EF1α-CD155-IRES-Neo lentiviral packaging: 293T cells were selected after about 20 generations of recovery. According to the lentiviral three-plasmid packaging system, the expression plasmid pLVX-EF1α-CD155-IRES-Neo, the packaging plasmid psPAX2, and the envelope plasmid pMD2.G were prepared. They were added to 1 mL of serum-free and antibiotic-free DMEM in a mass ratio of 4:3:2. After gentle mixing, the cells were allowed to stand at room temperature for 5 minutes. The transfection reagent Lipofectamine 2000 (1 μg / μL) was dissolved in 1 mL of serum-free and antibiotic-free DMEM, mixed, and allowed to stand at room temperature for 5 minutes. The plasmid solution after standing was slowly added to the transfection reagent solution and allowed to stand at room temperature for 20 minutes. The solution was evenly added to the culture dish and placed in a 37°C, 5% CO2 incubator for continued cultivation. After 6 hours of transfection, the culture medium was discarded and replaced with 10 mL of fresh DMEM complete culture medium and the culture was continued. The viral supernatant was collected and concentrated according to 6.1 of the present invention.

[0078] (3) Infection of AML cell lines: K562, U937, and MOLM-13 cells were adjusted to 2×10 5 12-well plates were inoculated with pLVX-EF1α-CD155-IRES-Neo virus (MOI = 30). Protamine sulfate (40 μg / mL) was added to promote infection and the cells were cultured in an incubator. Fresh medium was replaced after 24 hours, and 500 μg / mL G418 was added for selection after 48 hours. Uninfected cells died after 7 days, and successfully infected stably transfected cell lines were cultured again, with the selection concentration maintained at 500 μg / mL.

[0079] (4) Detection of CD155 expression: K562, U937, and MOLM-13 cells that were not transduced with the gene, as well as K562, U937, and MOLM-13 cells that were transduced with CD155 and selected with antibiotics were collected. The cells were resuspended in MACS buffer and washed twice. Human CD155-PE antibody and isotype control were used for labeling and detection on the microscope. Figure 12 .

[0080] (5) Prepare effector cells and target cells according to the method in “5.3”. After co-culture, use a microplate reader to detect the results and calculate the killing efficiency of each group of effector cells. Figure 13 .

[0081] Example 2 Preparation Method of Immune Cells Regulating CD226 Receptors

[0082] The operation is basically the same as that of Example 1, except that:

[0083] 1. Obtaining the gene coding region sequence: The human CD226 nucleotide sequence is shown in SEQ ID NO: 7, which is used to construct pT7-CD226 OE Recombinant plasmid.

[0084] 2.pT7-CD226 OE Recombinant plasmid: The gene coding sequence of human CD226 was ligated into the MluI and XbaI sites of the pT7-EGFP vector to form pT7-CD226 OE Recombinant plasmids, see Figure 1 A.

[0085] The PCR amplification sequence templates and primers used correspond to the following:

[0086] The primers for the human CD226 nucleotide sequence (SEQ ID NO: 7) are as follows:

[0087] F4:5'-ACAACGGTTTTCCCACGCGTTGCCACCATGGCACTG-3', SEQ ID NO:19;

[0088] R4: 5'-CCAGAGGTTGATTTCTAGACTAGACTCTGGTCTTTGGTCTGC-3', SEQ ID NO: 20.

[0089] 3.pMSCV-CD226 OE Construction of recombinant plasmid: Using pMSCV-puro as the vector backbone, the gene coding sequence of human CD226 was connected between BglII and ClaI of pMSCV-puro to obtain pMSCV-CD226 OE Recombinant plasmid;

[0090] 4. mRNA expression and functional verification of NK cells electroporated

[0091] 4.1.CD226 OE Preparation of mRNA: pT7-CD226 in Example 2 was digested with HindIII. OE Through in vitro transcription, tailing and purification experiments, CD226 was obtained. OE mRNA.

[0092] 4.2.CD226OE mRNA electroporation into NK cells: CD226 OE NK cells were electroporated with mRNA using an electroporator. 24 hours later, the electroporated cells were harvested and the CD226 positivity rate was determined using flow cytometry.

[0093] 4.3.CD226 OE Direct killing effect of mRNA on tumor cells after electroporation of NK cells: Prepare cells and co-culture as in Example 1, use a microplate reader to detect experimental values, and calculate the NK cell killing efficiency.

[0094] The accompanying drawings related to this embodiment are shown in FIG. Figure 1 A, Figure 4 B, E, Figure 5 A, D, Figure 6 B, Figure 7 .

[0095] Example 3 Preparation of immune cells that regulate TIGIT receptors

[0096] The operation is basically the same as that of Example 1, except that:

[0097] 1. Obtaining the gene coding region sequence: The human antiTIGIT nucleotide sequence is shown in SEQ ID NO: 8, which is used to construct the pT7-antiTIGIT recombinant plasmid.

[0098] 2. pT7-antiTIGIT recombinant plasmid: Connect the gene coding sequence of human antiTIGIT into the MluI and XbaI sites of the pT7-EGFP vector to obtain the pT7-antiTIGIT recombinant plasmid.

[0099] The PCR amplification sequence templates and primers used correspond to the following:

[0100] The primers for the human antiTIGIT nucleotide sequence (SEQ ID NO: 8) are as follows:

[0101] F5:5'-ACAACGGTTTTCCCACGCGTGCCCACCATGGCTTTGCCT-3', SEQ ID NO:21;

[0102] R5: 5'-CCAGAGGTTGATTTCTAGACTACTTGATCTCCACCTTAGTTCCAC-3', SEQ ID NO: 22.

[0103] 3. Construction of pMSCV-antiTIGIT recombinant plasmid: Using pMSCV-puro as the vector backbone, the gene coding sequence of human antiTIGIT was connected between BglII and ClaI of pMSCV-puro to obtain the pMSCV-antiTIGIT recombinant plasmid.

[0104] 4. mRNA expression and functional verification of NK cells electroporated

[0105] 4.1. Preparation of antiTIGIT mRNA: The pT7-antiTIGIT in Example 2 was digested with HindIII and antiTIGIT mRNA was obtained through in vitro transcription, tailing, and purification.

[0106] 4.2. Electrotransfection of NK cells with antiTIGIT mRNA: Electrotransfect NK cells with antiTIGIT mRNA using an electroporator. Harvest the electrotransfected cells 24 hours later, and measure the TIGIT blocking rate using flow cytometry.

[0107] 4.3. Direct killing effect of antiTIGIT mRNA on tumor cells after electroporation of NK cells: Cells were prepared and co-cultured as in Example 1, and the experimental values ​​were detected using a microplate reader to calculate the NK cell killing efficiency.

[0108] The accompanying drawings related to this embodiment are shown in FIG. Figure 1 B, Figure 4 C, F, Figure 5 B, E, Figure 6 A, Figure 7 .

[0109] Some sequences of the present invention are specifically shown below:

[0110] SEQ ID NO:1(CD226 OE Amino acid sequence):

[0111] MALPVTALLLPLALLLHAARPEEVLWHTSVPFAENMSLECVYPSMGILTQVEWFKIGTQQDSIAIFSPTHGMVIRKPYAERVYFLNSTMASNNMTLFFRNASEDDVGYYSCSLYTYPQGTWQKVIQVVQSDSFEAAVPSNSHIVSEPGKNVTLTCQPQMTWPVQAVRWEKIQPRQIDL LTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQYTLFVAGGTVLLLLFVISITTIIVIFLNRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKTRV*, where * represents the stop codon TAA.

[0112] SEQ ID NO: 2 (antiTIGIT amino acid sequence):

[0113] MALPPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVAYITKGGGSTYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARQTNYDFTMDYWGQGTLVTVSS GGGSGGGSGGGSGGGSEIVMTQSPATLSVSPGERATLSCKASQDVGTSVAWYQQKPGQAPRLLIYWASARHTGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYSSYPLTFGGGTKVEIK*, where * represents the stop codon TAA.

[0114] SEQ ID NO:3(CD226 OE -amino acid sequence corresponding to the antiTIGIT fusion gene):

[0115] MALPVTALLLPLALLLHAARPEEVLWHTSVPFAENMSLECVYPSMGILTQVEWFKIGTQQDSIAIFSPTHGMVIRKPYAERVYFLNSTMASNNMTLFFRNASEDDVGYYSCSLYTYPQGTWQKVIQVVQSDSFEAAVPSNSHIVSEPGKNVTLTCQPQMTWPVQAVRWEKIQPRQIDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQYTLFVAGGTVLLLLFVISITTIIVIFLNRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKTRVGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVAYITKGGGSTYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARQTNYDFTMDYWGQGTLVTVSSGGGSGGGSGGGSGGGSEIVMTQSPATLSVSPGERATLSCKASQDVGTSVAWYQQKPGQAPRLLIYWASARHTGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYSSYPLTFGGGTKVEIK*, where * represents the stop codon TAA.

[0116] SEQ ID NO:4 (Amino acid sequence of mbIL-15):

[0117] MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGSGGGSGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL*, where * represents the stop codon TAA.

[0118] SEQ ID NO:5(CD226 OE

[0119] SEQ ID NO: 6 (CD155 amino acid sequence):

[0120] MARAMAAAWPLLLVALLVLSWPPPGTGDVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWHSDLGGMPNTSQVPGFLSGTVTVTSLWILVPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNVTNALGARQAELTVQVKEGPPSEHSGMSRNAIIFLVLGILVFLILLGIGIYFYWSKCSREVLWHCHLCPSSTEHASASANGHVSYSAVSRENSSSQDPQTEGTR*, where * represents the stop codon TGA.

[0121] SEQ ID NO: 7 (CD226 OE nucleotide sequence):

[0122]

[0123] SEQ ID NO:8 (antiTIGIT nucleotide sequence):

[0124] ATGGCTTTGCCTGTTACAGCTCTGCTCTTGCCACTGGCTCTGCTGTTGCACGCAGCAAGACCAGAGGTGCAGCTCGTGGAGTCTGGAGGCGGCTTGGTCCAGCCTGGAGGTTCACTGAGACTGAGCTGTGCCGCTTCCGGATTCACCTTCAGCGACTATTACATGTACTGGGTTCGCCAGGCTCCAGGCAAAGGCCTGGAGTGGGTGGCCTACATCACTAAAGGCGGAGGCTCCACCTACTATCCAGACTCCGTGAAGGGCCGGTTCACCATCAGCAGGGATAACGCCAAGAACACACTGTATCTCCAGATGAACAGCCTCAGGGCTGAGGATACCGCCGTTTATTACTGCGCCAGACAGACCAACTACGATTTCACTATGGATTACTGGGGTCAGGGTACACTGGTGACAGTCAGCTCTGGAGGTGGTTCTGGTGGCGGCAGCGGTGGTGGCAGTGGCGGCGGCTCCGAGATTGTGATGACCCAGTCCCCTGCCACTCTGAGCGTCTCACCTGGTGAACGCGCCACACTGTCATGCAAGGCAAGCCAAGATGTCGGCACATCTGTGGCTTGGTATCAGCAGAAGCCTGGTCAGGCCCCTAGACTGCTGATTTATTGGGCCAGTGCAAGACACACCGGCATACCTGCTAGATTCAGCGGTAGTGGCTCCGGCACAGAGTTTACTCTCACAATCAGCTCACTTCAGTCCGAAGACTTCGCCGTGTATTATTGTCAGCAGTACAGCAGCTACCCTCTGACCTTCGGCGGTGGAACTAAGGTGGAGATCAAG*, where * represents the stop codon TAA.

[0125] SEQ ID NO:9 (CD226 OE

[0126] SEQ ID NO: 10 (mbIL-15 nucleotide sequence):

[0127]

[0128] SEQ ID NO:11(CD226 OE -nucleotide sequence of antiTIGIT-mbIL-15 fusion gene):

[0129]

[0130] SEQ ID NO: 12 (nucleotide sequence of CD155):

[0131]

[0132] It should be noted that the present invention should by no means be interpreted as being limited to the above embodiments, but rather should be interpreted as a method that those skilled in the art can refer to the contents of this document and appropriately improve the process parameters for implementation, but all similar replacements and modifications to implement and apply the technology of the present invention will be deemed to be included in the present invention.

Claims

1. A method for preparing immune cells that regulate CD226 and TIGIT receptors, characterized in that: include: Prepare the nucleotide sequence of the target gene, the target gene includes CD226 OE , antiTIGIT, or CD226 OE -antiTIGIT fusion gene, CD226 OE The nucleotide sequence of antiTIGIT is shown in SEQ ID NO: 7, the nucleotide sequence of CD226 is shown in SEQ ID NO: 8, OE -The nucleotide sequence of the antiTIGIT fusion gene is shown in SEQ ID NO: 9; Construct a recombinant plasmid containing the target gene; In vitro expansion of primary immune cells; The recombinant plasmid carrying the target gene is delivered to primary immune cells to obtain engineered immune cells.

2. The method for preparing immune cells that regulate CD226 and TIGIT receptors according to claim 1, characterized in that: The recombinant plasmid containing the target gene is an in vitro transcription vector, which is constructed by cloning the target gene between the MluI and XbaI restriction sites of the pT7-EGFP vector.

3. The method for preparing immune cells that regulate CD226 and TIGIT receptors according to claim 2, characterized in that: Delivering the recombinant plasmid carrying the target gene to primary immune cells means that the mRNA obtained after in vitro transcription and polyA tailing of the in vitro transcription vector is introduced into the primary immune cells through electro-transfection.

4. The method for preparing immune cells that regulate CD226 and TIGIT receptors according to claim 3, characterized in that: The voltage parameters for electrofection are 500V to 540V, and the culture time is 24h to 48h.

5. The method for preparing immune cells that regulate CD226 and TIGIT receptors according to claim 1, characterized in that: The recombinant plasmid containing the target gene is a reverse transcription recombinant plasmid, the vector skeleton of the reverse transcription recombinant plasmid is pMSCV-puro, and the target gene is connected between the BglII and ClaI sites of pMSCV-puro.

6. The method for preparing immune cells that regulate CD226 and TIGIT receptors according to claim 5, characterized in that: The target gene is also connected with the nucleotide sequence of mbIL-15.

7. The method for preparing immune cells that regulate CD226 and TIGIT receptors according to claim 5 or 6, characterized in that: Delivering the recombinant plasmid carrying the target gene to primary immune cells means: using liposome transfection to transfect the reverse transcription recombinant plasmid into the packaging cell line, collecting the supernatant containing viral particles after 48h to 72h of culture, concentrating it by ultracentrifugation to obtain the retrovirus, and performing viral transduction during the logarithmic growth phase of the immune cells.

8. Immune cells prepared by the preparation method according to any one of claims 1 to 7.

9. The use of immune cells according to claim 8, characterized in that: The immune cells are used to kill at least one of the following tumor cells: a blood tumor cell line expressing the ligand CD155, a solid tumor cell line, and a primary tumor cell.

10. The use of immune cells according to claim 9, characterized in that: The blood tumor cells include at least one of K562 cells, U937 cells, MOLM-13 cells and HEL cells; The solid tumor cell line includes at least one of HCC 1954 cells and MDA-MB-231 cells; The primary tumor cells include at least one of the original cells of the bone marrow of patients with acute myeloid leukemia.

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