Long-acting dual-target chimeric antigen receptor, nucleic acid molecule, recombinant vector, cell and its application

Abstract

The present invention provides a long-acting dual-target chimeric antigen receptor, nucleic acid molecule, recombinant vector, cell, and application thereof. The dual-target chimeric antigen receptor comprises two independent transmembrane protein chains, wherein the first CAR chain targets a scFv of the first target, and the intracellular signal comprises a second signal and an intracellular transduction signal or only an intracellular transduction signal; the second CAR chain targets a scFv of the second target, and the intracellular signal comprises a costimulatory signal and a JAK enzyme activation transduction domain. The CAR-T cells prepared by the present invention have a strong and persistent killing effect on tumor cells that simultaneously express the first and second targets, and can be used for the anti-tumor treatment of solid tumors.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and immunotherapy technology, and specifically relates to a long-acting dual-target chimeric antigen receptor, a nucleic acid molecule, a recombinant vector, a cell and applications thereof. Background Art

[0002] CAR-T cell technology is based on the theory of immune system recognition and activation. Through genetic engineering, components that specifically recognize tumor antigens (single-chain antibody scFv) and initiate immune activity are integrated into a single gene. This gene is then transduced into the patient's own T lymphocytes in vitro through viral methods and amplified. After infusion back into the patient, the cells regain the ability to specifically recognize tumor cells, activate their own T cells, and attack and kill them in a targeted manner. CAR-T cell technology offers advantages such as being unrestricted by major histocompatibility complex (MHC) levels. It has demonstrated promising results in patients with refractory and relapsed B-cell leukemia and lymphoma. In recent years, CAR-T cell therapy has also made significant progress in solid tumors such as glioma, prostate cancer, and lung cancer, and is considered one of the most promising cancer treatments. To date, over 300 CAR-T cell therapies have entered clinical trials worldwide. The core of CAR-T cell technology is the genetic engineering of T cells, enabling them to recognize and activate tumor cells through the CAR molecule, thereby exerting a powerful anti-tumor effect. However, the existing CAR-T cell technology for treating tumors has inherent defects. This is because it is still difficult to find tumor-specific targets at this stage. Tumor cells are mainly identified through tumor-associated antigens, which inevitably leads to off-target toxicity and even serious lethality.

[0003] Many types of tumor cells have two target proteins on their surfaces at the same time. For example, some ovarian cancer tissues highly express both mesothelin and B7H3. However, the probability of normal tissues highly expressing both targets at the same time is very low. If these two target proteins are designed into a dual-target CAR, so that CAR-T cells can simultaneously target both targets highly expressed by tumor cells, the CAR-T cells will be fully activated and exert their killing function. When normal cells only express the target as the first activation signal or only express the target as the second activation signal, the CAR-T cells will have no killing effect or only a weak killing effect. In this way, we can greatly reduce off-target toxic side effects in the body through appropriate dual-target CAR design.

[0004] Furthermore, existing CAR-T cell technologies for treating tumors generally suffer from insufficient in vivo expansion and survival, leading to limited clinical efficacy and a high risk of relapse after treatment. To address this issue, various cytokines are typically used to achieve sustained expansion of CAR-T cells, such as IL-2, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, and GM-CSF-R. These cytokines bind to cytokine receptors on CAR-T cells and activate downstream intracellular signaling pathways. For example, IL-15 first binds to the α subunit of the IL-15 membrane receptor, then forms an immune synapse with the IL2R-γc / IL-15Rβ on effector CAR-T cells, activating the JAK1 / JAK3 and STAT3 / STAT5 pathways within the cell, thereby promoting the differentiation and proliferation of CAR-T cells. There are currently two ways to achieve this goal. The first is to directly inject these cytokines into the body at the same time as CAR-T cell therapy. However, if this method is to achieve the effect of expanding CAR-T cells, the cytokines must be in large doses, which will inevitably cause reactions from other immune cells, resulting in unpredictable side effects and even death. Secondly, some researchers have designed CARs to secrete cytokines while activating and expanding CAR-T cells, which can effectively prevent CAR-T exhaustion in the body. However, these secreted cytokines will not only act on the body's own CAR cells, but will also be released into the body fluids, causing a violent reaction from other immune cells in the body, resulting in unpredictable side effects. Summary of the Invention

[0005] Purpose of the invention: In response to the problems existing in CAR-T cell technology, the technical problem to be solved by the present invention is to provide a long-acting dual-target chimeric antigen receptor, which can reduce off-target toxic side effects in the body and enhance the expansion ability and persistence of CAR-T in the body by introducing a third signal.

[0006] The technical problem that the present invention also aims to solve is to provide a nucleic acid molecule that encodes the long-acting dual-target chimeric antigen receptor.

[0007] Another technical problem to be solved by the present invention is to provide a vector or a recombinant virus containing the same.

[0008] The technical problem that the present invention also aims to solve is to provide the use of the long-acting dual-target chimeric antigen receptor, the nucleic acid molecule, the vector or its recombinant virus, or its cells in the preparation of drugs for treating solid tumors that positively express anti-target proteins.

[0009] The final technical problem to be solved by the present invention is to provide a pharmaceutical composition, which comprises the above-mentioned nucleic acid molecule, the above-mentioned chimeric antigen receptor, the above-mentioned vector or the above-mentioned cell, and a pharmaceutically acceptable carrier.

[0010] Purpose of the invention: In order to solve the above technical problems, the present invention provides a long-acting dual-target chimeric antigen receptor, which includes a chimeric antigen receptor composed of two independent transmembrane proteins, wherein the first chimeric antigen receptor includes a first signal peptide, a binding domain for a first target tumor antigen, a first hinge domain, a first transmembrane domain, a first intracellular co-stimulatory domain and an intracellular signal transduction domain of a first target CAR; the second chimeric antigen receptor includes a second signal peptide, a binding domain for a second target tumor antigen, a second hinge domain, a second transmembrane domain, a second intracellular co-stimulatory domain and a JAK enzyme activation signal transduction domain, wherein the JAK enzyme activation signal transduction domain can capture and activate the JAK enzyme, wherein the JAK enzyme includes JAK1, JAK2 or JAK3, wherein the Gene ID of the JAK1 is 3716, the Gene ID of the JAK2 is 3717, the Gene ID of the JAK3 is 3718, and the Gene ID of the Tyk2 is 3719. The ID is 7297, and the first chimeric antigen receptor and the second chimeric antigen receptor are connected by a linker.

[0011] Among them, the JAK enzyme activation signal domain recruits JAK kinase and phosphorylates tyrosine residues under its catalysis, subsequently activating STAT signal, and then triggering intracellular gene transcription and expression. Therefore, it plays a role similar to that of cytokines. Once the sequence of any enzyme in JAK1, JAK2, JAK3 or Tyk2 can be captured, it falls within the scope of protection of the present invention.

[0012] Preferably, the JAK enzyme is a JAK1 enzyme, and the JAK1 enzyme activates a signal transduction domain, and its amino acid sequence with 90%-99% identity is selected from one of the following or a superposition of two or more thereof: SEQ ID No.1 (JAKAcS1), SEQ ID No.2 (JAKAcS2), SEQ ID No.3 (JAKAcS3), SEQ ID No.4 (JAKAcS4), SEQ ID No.5 (JAKAcS5), SEQ ID No.6 (JAKAcS6), SEQ ID No.7 (JAKAcS7), SEQ ID No.8 (JAKAcS8), SEQID No.9 (JAKAcS9), and SEQ ID No.10 (JAKAcS10), all of which include the binding site and activation signal of JAK1 kinase; the above sequences can capture JAK1 kinase and catalyze the phosphorylation of amino acid residues to activate STAT signals, thereby triggering intracellular gene transcription and expression.

[0013] Among them, as a preference, the nucleotide sequence of the JAK1 enzyme activation signal transduction domain is as follows: SEQ ID No.11 (JAKAcS1), SEQ ID No.12 (JAKAcS2), SEQ ID No.13 (JAKAcS3), SEQ ID No.14 (JAKAcS4), SEQ ID No.15 (JAKAcS5), SEQ ID No.16 (JAKAcS6), SEQ ID No.17 (JAKAcS7), SEQ ID No.18 (JAKAcS8), SEQ ID No.19 (JAKAcS9), SEQ ID No.20 (JAKAcS10).

[0014] Preferably, the JAK enzyme is JAK3 enzyme, and the domain that can activate JAK3 enzyme is also within the protection scope of the present invention.

[0015] Preferably, the intracellular signal transduction domain of the first target CAR includes the intracellular signaling domain of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD40L, CD45, CD66d, CD79, CD80, CD86, CD278, DAP10, DAP12, FcγR or Zap70, such as the CD3ζ signal transduction domain, or an amino acid sequence signal transduction domain having 90%-99% identity thereto, such as the nucleotide sequence contained in the CD3ζ signal transduction domain as shown in SEQ ID No.21.

[0016] Preferably, the binding domain of the first target tumor antigen comprises a single-chain antibody scFv that highly expresses the first target on the surface of tumor cells, and the binding domain of the second target tumor antigen comprises a single-chain antibody scFv that highly expresses the second target on the surface of tumor cells, and the first target and the second target comprise any one of Claudin18.2, GPC3, B7H3, PD-L1, MUC1, Mesothelin, Her2, EGFR, PSMA, CEA, GD2, EpCAM, EGFRvIII, CD70, CD20, CD133, CD177, AFP, AXL, CD171, CD117, C-MET, FAP, MUC16, NKG2D, NY-ESO-1, PSCA, VEGFR-2, Lewis-Y, Gp100, FAP, or EPHA2. The above targets are selected for combination according to their expression on the surface of different tumor cells. In one embodiment of the present invention, the first target is Claudin18.2, the nucleotide sequence of which is shown in SEQ ID No. 22, and the second target is B7H3, the nucleotide sequence of which is shown in SEQ ID No. 23. In another embodiment of the present invention, the first chimeric antigen receptor is a first-generation or second-generation CAR or a third-generation CAR design (CAR-chain) whose first target is Mesothelin, and the nucleotide sequence of Mesothelin is shown in SEQ ID No. 24; the second chimeric antigen receptor is composed of an extracellular scFv targeting B7H3 and an intracellular second signal and JAK enzyme activation signal transduction domain, wherein the extracellular region of the CAR-chain is composed of an scFv targeting mesothelin and the intracellular region is composed of the CD3ζ intracellular region.

[0017] Among them, the signal peptide can guide the antigen recognition region and hinge region to be transferred to the extracellular space, and any suitable signal peptide or combination of signal peptides can achieve the purpose of the present invention. The first signal peptide and the second signal peptide include signal peptides of the α chain and β chain of the T cell receptor, CD3, CD4, CD5, CD8, CD28, CD33, CD45, CD80, CD86, CD134, CD137, ICOS, GM-CSF, immunoglobulin heavy chain or immunoglobulin light chain. As a preferred signal peptide, the signal peptide of CD8α is selected as shown in SEQ ID No. 25.

[0018] The target binding domain of the present invention is connected to the transmembrane region encoded by it via a hinge region, and any suitable hinge region sequence can achieve the purpose of the present invention. The first hinge domain and the second hinge domain include the hinge regions of the following molecules: IgG, CD8α, CD28, and IL-2 receptor. For example, the nucleotide sequence of the hinge region of CD8α is shown in SEQ ID No. 26.

[0019] The first and second transmembrane domains comprise one or more of the α, β, or ζ chains of the T cell receptor, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In a preferred embodiment, the first and second transmembrane domains are the transmembrane regions of CD8, the nucleotide sequence of which is shown in SEQ ID No. 27.

[0020] Wherein, the first intracellular co-stimulatory domain and the second intracellular co-stimulatory domain are selected from one or more of the following proteins or functional signaling domains obtained by amino acid sequences having 90%-99% or identical identity with the proteins: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules, activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD137, CDS, ICAM-1, LFA-1, CLAUDIN, CD278 or GITR. Among them, the first target does not contain a costimulatory domain and can also achieve the purpose of the present invention, which belongs to the protection scope of the present invention; in a preferred embodiment, the intracellular costimulatory domains of the first target and the second target are both CD137, whose nucleotide sequence is shown in SEQ ID No. 28; in another preferred embodiment, the intracellular costimulatory domain of the first target is CD28, whose nucleotide sequence is shown in SEQ ID No. 29; the intracellular costimulatory domain of the second target is CD137.

[0021] The linker is selected from any one of P2A, T2A, E2A, F2A, and IRES. A preferred embodiment is P2A, whose nucleotide sequence is shown in SEQ ID No. 30; another preferred embodiment is T2A, whose nucleotide sequence is shown in SEQ ID No. 31.

[0022] In addition, any peptide chain can be inserted as a spacer at a suitable position between the above-mentioned antigen recognition region, hinge region, transmembrane region and intracellular signaling region. The peptide chain can be an oligopeptide or a polypeptide.

[0023] In one embodiment of the present invention, the inventors used chemical synthesis methods to obtain the target binding domain.

[0024] The present invention also includes a nucleic acid molecule encoding the dual-target chimeric antigen receptor.

[0025] The above-mentioned nucleic acid molecules can be prepared by known techniques such as chemical synthesis or PCR amplification based on the base sequences of the two target recognition regions, hinge region, transmembrane region, and intracellular signaling region. Typically, the codons encoding the amino acids in the above-mentioned domains can be optimized to optimize their expression in host cells.

[0026] The present invention also includes a vector or a recombinant virus containing the vector, wherein the vector contains the nucleic acid molecule.

[0027] In the present invention, the above-mentioned vector can be a linear vector or a circular vector. It can be a non-viral vector such as a plasmid, a viral vector, or a vector utilizing a transposon. The vector can contain regulatory sequences such as promoters and terminators, as well as marker sequences such as drug-resistance genes and reporter genes. The above-mentioned viral vector can be a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or the like. In one embodiment of the present invention, a lentiviral expression vector is used.

[0028] The present invention also includes a method for constructing a vector, which comprises synthesizing the entire gene sequence of a dual-target chimeric antigen receptor using conventional biosynthesis methods, ligating the synthesized chimeric antigen receptor to a plasmid vector, amplifying the chimeric antigen receptor coding sequence using PCR primers containing homology arms, and inserting the chimeric antigen receptor coding sequence into a viral vector by homologous recombination. The viral vector is selected from one or more combinations of DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, and other gene transfer systems, wherein a preferred lentiviral vector is Plvx-EF1α-MCS-(PGK-puro).

[0029] The present invention also includes a recombinant cell, which expresses the nucleic acid molecule, the dual-target chimeric antigen receptor, or the vector, and the recombinant cell includes a modified T cell.

[0030] In one embodiment of the present invention, the cells are human T cells. The T cells can be isolated and purified from peripheral blood, bone marrow, spleen, thymus, lymphoid tissue, or other tissues. Furthermore, the T cells can be CD4+ T cells, CD8+ T cells, or γδ T cells. The T cells can be replaced with NK cells, NKT cells, helper T cells, or macrophages in a suitable manner, which are also considered to be within the scope of protection of the present invention.

[0031] The present invention also includes an application, which includes any one of the following: the use of the dual-target chimeric antigen receptor, the nucleic acid molecule, the recombinant vector or recombinant virus, or the recombinant cell in the preparation of a drug for treating solid tumors.

[0032] Among them, the solid tumors include gastric cancer, lung cancer, liver cancer, esophageal cancer, colorectal cancer, melanoma, intrahepatic bile duct cancer, ovarian cancer, kidney cancer, glioma, head and neck cell carcinoma, bone cancer, brain cancer, pancreatic cancer, breast cancer, malignant mesothelioma, thyroid cancer, cervical cancer, neuroblastoma or prostate cancer.

[0033] The present invention also provides a pharmaceutical composition comprising the above nucleic acid molecule, the above chimeric antigen receptor, the above vector or the above cell, and a pharmaceutically acceptable carrier.

[0034] In addition to the above ingredients, the pharmaceutical composition of the present invention may also contain any pharmaceutically acceptable additives, such as physiological saline, cell culture medium, glucose, water for injection, glycerol, ethanol and combinations thereof, stabilizers, surfactants, preservatives, isotonic agents, etc.

[0035] Likewise, the pharmaceutical composition of the present invention can also be used in combination with other suitable anticancer agents, such as vincristine, daunorubicin, asparaginase, cyclophosphamide, prednisone, and the like.

[0036] Another aspect of the present invention provides a use of the above-mentioned pharmaceutical composition in the preparation of a drug for solid tumors with positive expression of target proteins.

[0037] In summary, the dual-target chimeric antigen receptor of the present invention simultaneously expresses two transmembrane protein chains, one of which is a first-generation or second-generation CAR or third-generation CAR design (CAR-chain); the extracellular portion of the second chain is composed of a tumor antigen-binding domain, and the intracellular portion is composed of a second signal and JAK enzyme activation signal transduction domain (costimulatory chain, Cos-chain); the extracellular binding domains of the two chains can be domains targeting the same antigen epitope, or binding domains targeting different epitopes of the same antigen, or binding domains targeting different antigens.

[0038] One advantage of the CAR design of the present invention is that the extracellular binding region of the CAR-chain is composed of an scFv targeting the first target, and the intracellular region is composed of the intracellular region of the costimulatory molecule and the intracellular region of the first signal for intracellular signal transduction. The extracellular binding region of the Cos-chain is composed of an scFv targeting the second target, and the intracellular region is composed of the intracellular region of the costimulatory molecule and the JAK enzyme activation signal transduction domain. This design allows positive tumor cells that highly express the first target on the surface of tumor cells to bind to the first target scFv and provide the first signal and the second signal to T cells, initiating T cells to kill tumor cells. Positive tumor cells that highly express the second target on the surface of tumor cells to bind to the second target scFv and provide the second and third signals to T cells, enhancing the function and persistence of T cells in killing tumor cells. When normal cells do not express the two target proteins on their surface or only express the second target, T cells do not have the killing function, reducing the off-target toxic side effects of CAR-T cells. Another advantage of the CAR design of the present invention is that the intracellular region of the CAR is directly enriched with JAK enzyme activation signal transduction elements in CAR-T cells through the structural design of the second target CAR. When the second CAR extracellular region scFv binds to the antigen on the tumor cell, it activates the JAK / STAT signal transduction pathway in the CAR-T cell, promotes the differentiation and proliferation of CAR-T cells, and thus prevents the exhaustion of CAR-T cells.

[0039] For example, in one embodiment of the present invention, the first target is Mesothelin and the second target is B7H3. One advantage of its CAR design is that positive tumor cells that highly express the first target Mesothelin on the surface of tumor cells provide the first signal and the second signal to T cells after binding to the first target scFv, thereby initiating T cells to kill tumor cells. Positive tumor cells that highly express the second target B7H3 on the surface of tumor cells provide the second and third signals to T cells after binding to the second target scFv, thereby enhancing the function and persistence of T cells to kill tumor cells. When normal cells do not express both target proteins or only express the second target B7H3 on their surface, T cells do not have the killing function, thereby greatly reducing the off-target toxic side effects of CAR-T cells. Another advantage is that the structural design of the second target CAR directly enriches the intracellular region of the CAR with JAK enzyme activation signal transduction binding elements in CAR-T cells. When the second CAR extracellular region scFv binds to the antigen on the target cell, it activates the JAK / STAT signal transduction pathway in the CAR-T cell, promotes the differentiation and proliferation of CAR-T cells, and thus prevents the exhaustion of CAR-T cells.

[0040] Beneficial effects: The advantages of the CAR of the present invention over existing similar products are: First, CAR does not secrete cytokines while exerting its cytokine effects, and will not cause excessive activation of other immune cells, thus avoiding side effects. Secondly, only when the scFv of the second target CAR binds to the antigen of the tumor cell can the expansion of the CAR-T cells be stimulated, so that the expansion of the CAR-T cells is concentrated only near the tumor cells, which not only enhances the effect of killing tumor cells, but also stops the expansion of the CAR-T cells once the tumor cells are cleared, further ensuring in vivo safety. The examples of the present invention show that the CAR-T cells prepared by the CAR designed by the present invention have a significantly higher ability to repeatedly kill tumor cells than the third-generation CAR-T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of the structure of long-acting dual-target CAR.

[0042] Figure 2 This is a fluorescence image of GFP expression in the CLDN18.2 / B7H3 CART cells of Example 4, where GFP is a fluorescent marker protein of CLDN18.2 / B7H3 CART, and the positive expression level of CLDN18.2 / B7H3 CART cells was detected by flow cytometry.

[0043] Figure 3 GFP dynamic fluorescence statistics of tumor cells after co-culture of the CLDN18.2 / B7H3 CART cells and target cells (effector-target ratio of 1:1) for 40 hours in Example 5.

[0044] Figure 4 These are the ELISA test results of IL2 and IFN-γ release after the CLDN18.2 / B7H3 CART cells of Example 5 were co-cultured with target cells for 24 hours.

[0045] Figure 5 The dual-target CLDN18.2 / B7H3 CART designed in Example 5, the third-generation CAR-T cells (i.e., the first target scFv-CD28-CD137-CD3ζ), and the unmodified T cells were tested for the same dose (10 5 The killing results of target cells were compared repeatedly.

[0046] Figure 6 For Example 9, the positive expression level of CAR on the seventh day of growth of Meso-B7H3 CAR-T cells was detected by flow cytometry.

[0047] Figure 7 Figure 3 shows the growth expansion curve of Meso-B7H3 CAR-T cells and the changes in T cell size.

[0048] Figure 8 Statistical values ​​of GFP dynamic fluorescence of tumor cells after co-culture of Meso-B7H3 CAR-T cells and target cells (effector-target ratio of 1:1) for 40 hours in Example 10.

[0049] Figure 9 These are the ELISA test results for IL2 and IFN-γ release after the Meso-B7H3 CAR-T cells of Example 10 were co-cultured with target cells for 24 hours.

[0050] Figure 10 The dual-target Meso-B7H3 CAR-T cells designed in Example 10 of the present invention, the third-generation CAR-T cells (i.e., the target Meso scFv-CD28-CD137-CD3ζ), and the unmodified T cells were tested for the same dose (10 5 The results of repeated killing experiments were performed on target cells. DETAILED DESCRIPTION

[0051] The present invention discloses the construction and application of long-acting dual-target CAR. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention, and relevant personnel can obviously modify or appropriately change and combine the contents described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0052] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.

[0053] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is further described in detail below with reference to specific embodiments.

[0054] Example 1 Construction of a dual-target CLDN18.2 / B7H3 CAR (signal peptide-first target CLDN18.2 scFv-CD8αhinge-CD8TM-CD3ζ-T2A-signal peptide-second target B7H3 scFv-CD8αhinge-CD8 TM-CD137-JAKAcS1) expression vector

[0055] Claudin18.2 was selected as the first target and B7H3 was selected as the second target to construct a dual-target CAR: signal peptide-first target CLDN18.2 scFv-CD8αhinge-CD8TM-CD3ζ-T2A-signal peptide-second target B7H3 scFv-CD8αhinge-CD8TM-CD137-JAKAcS1.

[0056] GenScript was commissioned to synthesize the nucleic acid sequence encoding the above-mentioned CLDN18.2 / B7H3 CAR, the sequence of which is SEQ ID No. 32.

[0057] Plvx-EF1α-IRES-ZsGreen1 and CLDN18.2 / B7H3 CAR were linearized by double digestion with BamHI-MluI. The enzyme digestion system is as follows:

[0058]

[0059] The chimeric antigen receptor encoding fragment containing sticky ends and the linearized Plvx-EF1α-IRES-ZsGreen1 vector were ligated and transformed using T4 DNA enzyme.

[0060] Connect Plvx-EF1α-IRES-ZsGreen1 and the CLDN18.2 / B7H3 CAR system as follows:

[0061]

[0062] The transformation steps for ligating the recombinant product are as follows:

[0063] 1) Thaw 50 μL of competent cells XL1-Blue on ice for 5-10 minutes.

[0064] 2) Add 2 μL of the recombinant product to 50 μL of competent cells, gently tap the tube to mix, and let it stand on ice for 30 minutes.

[0065] 3) Heat stress at 42°C for 45 seconds, then immediately place on ice for 2 minutes.

[0066] 4) Add 500 μL of LB medium without antibiotics and shake at room temperature for 1 h (270 rpm).

[0067] 5) Preheat the ampicillin-resistant LB solid culture medium in a 37°C incubator for 30 minutes.

[0068] 6) Centrifuge at 5000 rpm for 3 min, discard the supernatant, resuspend the cells in the remaining 30 μL of LB medium, and spread evenly on a plate containing kanamycin using a sterile spreader.

[0069] 7) Incubate the cells upside down at 37°C for 12-16 hours.

[0070] Example 2 Lentiviral packaging of dual-target CLDN18.2 / B7H3 CAR

[0071] The dual-target CLDN-B7H3 CAR expression plasmid was extracted from a single colony of the recombinant bacteria obtained in Example 1 using the EndoFree Plasmid Mid Plasmid Extraction Kit (Omega). HEK293T cells were transfected with the dual-target CLDN18.2 / B7H3 CAR expression plasmid and the packaging plasmids pSPAX and pMD2.G at a ratio of 4:3:1 using the calcium phosphate method. Fresh culture medium was replaced 12 hours after transfection. Viral supernatants were harvested 24 and 48 hours after transfection and centrifuged at 3,000 rpm for 15 minutes at 4°C. The supernatants were filtered through a 0.45 μm filter and concentrated by ultracentrifugation at 100,000 g at 4°C for 1.5 hours.

[0072] Example 3 Preparation of T cells

[0073] 20 ml of fresh human peripheral blood was obtained from a healthy donor in the Department of Hematology at Zhongda Hospital, Southeast University. PBMCs were isolated using Ficoll-Paque PLUS (GE Healthcare) (specific steps were as per the manufacturer's instructions). Anti-CD3 / CD28 magnetic beads (GibCo) were added at a 1:1 cell:bead ratio and cultured for 24 hours to obtain T cells prior to transfection.

[0074] Example 4 Lentivirus infection of T cells and culture of infected T cells

[0075] The supernatant of the virus solution prepared in Example 2 was taken out from -80°C and thawed at room temperature. The virus titer was 5.6*10 7 , according to the virus infection multiplicity MOI = 5, add 68 μL of virus concentrate, that is, the required virus concentrate volume = 1*10 6 (Number of cells to be infected)*5 / 5.6*10 7 10 mg / ml polybrene was added to a final concentration of 10 pg / ml. The cells were centrifuged at 2,000 rpm at 30°C for 2 hours and then transferred to a 5% CO2-37°C incubator for culture to obtain dual-target CLDN18.2 / B7H3 CART cells.

[0076] Flow cytometry was used to detect the positive rate of CLDN18.2 / B7H3 CART cells: cells were collected, labeled with rabbit anti-mouse IgG-F(ab')2 antibody, and the expression of F(ab')2 and GFP in CLDN18.2 / B7H3 CART cells was analyzed by flow cytometry. Figure 2 As shown by Figure 2 As can be seen from the figure, the positive rate of CLDN18.2 / B7H3 CART cells was 27.7%.

[0077] Example 5: Cytotoxicity of Dual-Target CLDN18.2 / B7H3 CART Cells against Dual-Target Positively Expressed Tumor Cells

[0078] SGC-7901 cell line was purchased from ATCC, USA. It highly expresses Claudin18.2 and B7H3 on its surface. SGC-7901 cells were plated at 10 5 The cells were seeded in 48-well plates and co-cultured with CLDN18.2 / B7H3 CART cells or T cells at an effector-target ratio of 1:1. The dynamic changes in GFP fluorescence values ​​of SGC-7901 cells during co-culture were recorded 40 hours after co-culture. Figure 3 As shown, the results showed that after 40 hours of co-culture of dual-target CLDN18.2 / B7H3 CART cells with SGC-7901 cells overexpressing Claudin18.2 and B7H3, the fluorescence value of the target cells gradually decreased over time, indicating that the number of target cells was decreasing and the dual-target CLDN18.2 / B7H3 CART could specifically kill the target cells. In the ordinary T cell group, the fluorescence value of SGC-7901 cells increased over time because ordinary T cells did not kill the target cells. SGC-7901 cells grew and expanded normally. Compared with ordinary T cells, dual-target CLDN18.2 / B7H3 CART cells had a significant killing effect on SGC-7901 cells positive for Claudin18.2 and B7H3.

[0079] After 24 hours, the culture supernatant was collected and tested (the specific operation steps were carried out according to the instructions of the ELISA test kit). Figure 4 The results showed that the levels of IL-2 and IFN-γ cytokines in the supernatant of co-culture of SGC-7901 cells expressing Claudin18.2 and B7H3 with dual-target CLDN18.2 / B7H3 CART cells were significantly higher than those in the co-culture group with ordinary T cells (P<0.001), indicating that dual-target CLDN18.2 / B7H3 CART cells had a significant killing effect on target cells expressing dual targets, while the killing effect on SGC-7901 target cells with knockdown of Claudin18.2 was comparable to that on T cells, and the killing activity on SGC-7901 target cells with knockdown of B7H3 was lower. These results indicate that the specificity and killing function of dual-target CLDN18.2 / B7H3 CART achieved the expected purpose.

[0080] The dual-target CLDN18.2 / B7H3 CART cells designed by the present invention were compared with the third-generation CAR-T cells (i.e., target CLDN18.2 scFv-CD28-CD137-CD3ζ, sequence as shown in SEQ ID No. 33) at the same dose (105 The target cell SGC-7901 was subjected to repeated killing experiments. The results are as follows Figure 5 The results showed that the dual-target CLDN18.2 / B7H3 CART designed by the present invention has a more sustained killing ability than the third-generation CAR-T.

[0081] Example 6

[0082] The JAKAcS1 in the sequence SEQ ID No. 32 in Example 1 was replaced with JAKAcS2, JAKAcS3, JAKAcS4, JAKAcS5, JAKAcS6, JAKAcS7, JAKAcS8, and JAKAcS9 to obtain the corresponding nucleotide sequences, which were then synthesized and operated according to the above specific steps to obtain the corresponding recombinant strains. According to the specific experimental methods of Examples 2-5, the above experimental process was repeated to obtain the corresponding dual-target CART cells. The positive rate of the above dual-target CART cells was tested, and the positive rate could reach 20%-40%, and they had a long-lasting ability to kill target cells.

[0083] Example 7 Construction of Dual-Target Meso-B7H3 CAR (Signal Peptide-First Target Meso scFv-CD8αhinge-CD8TM-CD3ζ-P2A-Signal Peptide-Second Target B7H3 scFv-CD8αhinge-CD8TM-CD137-JAKAcS1) Expression Vector

[0084] GenScript was commissioned to synthesize the nucleic acid sequence encoding the dual-target Meso-B7H3 CAR, the sequence of which is SEQ ID No. 34.

[0085] Plvx-EF1α-IRES-ZsGreen1 and Meso-B7H3 CAR were linearized by double digestion with BamHI-MluI. The enzyme digestion system is as follows:

[0086]

[0087] The chimeric antigen receptor encoding fragment containing sticky ends and the linearized Plvx-EF1α-IRES-ZsGreen1 vector were ligated and transformed using T4 DNA enzyme.

[0088] Connect Plvx-EF1α-IRES-ZsGreen1 and Meso-B7H3 CAR system as follows:

[0089]

[0090] The transformation steps for ligating the recombinant product are as follows:

[0091] 1) Thaw 50 μL of competent cells XL1-Blue on ice for 5-10 minutes.

[0092] 2) Add 2 μL of the recombinant product to 50 μL of competent cells, gently tap the tube to mix, and let it stand on ice for 30 minutes.

[0093] 3) Heat stress at 42°C for 45 seconds, then immediately place on ice for 2 minutes.

[0094] 4) Add 500 μL of LB medium without antibiotics and shake at room temperature for 1 h (270 rpm).

[0095] 5) Preheat the ampicillin-resistant LB solid culture medium in a 37°C incubator for 30 minutes.

[0096] 6) Centrifuge at 5000 rpm for 3 min, discard the supernatant, resuspend the cells in the remaining 30 μL of LB medium, and spread evenly on a plate containing kanamycin using a sterile spreader.

[0097] 7) Incubate the cells upside down at 37°C for 12-16 hours.

[0098] Example 8 Dual-target Meso-B7H3 CAR Lentiviral Packaging

[0099] The EndoFree Plasmid Mid plasmid extraction kit (Omega) was used to extract the bacterial solution from a single colony of the recombinant bacteria obtained in Example 6. The dual-target Meso-B7H3 CAR expression plasmid was then transfected into HEK293T cells using the calcium phosphate method with the dual-target Meso-B7H3 CAR expression plasmid and packaging plasmids pSPAX and pMD2.G at a ratio of 4:3:1. Fresh culture medium was replaced 12 hours after transfection, and viral supernatants were collected 24 and 48 hours later. The supernatants were centrifuged at 3,000 rpm for 15 minutes at 4°C, filtered through a 0.45 μm filter, and concentrated by ultracentrifugation at 100,000 g at 4°C for 1.5 hours.

[0100] Example 9 Preparation of T cells

[0101] 20 ml of fresh human peripheral blood was obtained from a healthy donor in the Department of Hematology at Zhongda Hospital, Southeast University. PBMCs were isolated using Ficoll-Paque PLUS (GE Healthcare) (specific steps were as per the manufacturer's instructions). Anti-CD3 / CD28 magnetic beads (GibCo) were added at a 1:1 cell:bead ratio and cultured for 24 hours to obtain T cells prior to transfection.

[0102] Example 10 Lentivirus infection of T cells and culture of infected T cells

[0103] The virus supernatant was taken out from -80℃ and thawed at room temperature. The virus titer was 6.3*10 7 , according to the virus infection multiplicity MOI = 5, add 79 μL of virus concentrate, that is, the required virus concentrate volume = 1*10 6 (Number of cells to be infected)*5 / 6.3*10 7 , and add 10mg / ml polybrene to a final concentration of 10pg / ml. Centrifuge at 2,000rpm at 30℃ for 2 hours and transfer to a 5% CO2-37℃ incubator for culture.

[0104] Flow cytometry was used to detect the positive rate of Meso-B7H3 CAR-T cells: cells were collected, labeled with rabbit anti-mouse IgG-F(ab')2 antibody, and the expression of T cell F(ab')2 and GFP was analyzed by flow cytometry. Figure 6 As shown in the figure, it can be seen that the positive rate of Meso-B7H3 CAR-T cells is 40.4%.

[0105] Example 11: Cytotoxicity of Dual-Target Meso-B7H3 CAR-T Cells against Dual-Target Positive Tumor Cells

[0106] SKOV3 cell line was purchased from ATCC, USA. It has low expression of Mesothelin on its surface and high expression of B7H3. SKOV3 cells were plated at 10 5 The cells were seeded in 48-well plates and co-cultured with Meso-B7H3 CAR-T cells or T cells at an effector-target ratio of 1:1. The dynamic changes in GFP fluorescence values ​​of SKOV3 cells during the co-culture period were recorded 40 hours after co-culture. Figure 8 As shown in the figure, after 40 hours of co-culture of dual-target Meso-B7H3 CAR-T cells with SKOV3 cells overexpressing mesothelin and B7H3, the fluorescence value of the target cells gradually decreased over time, indicating that the number of target cells was decreasing and the dual-target CAR-T cells were able to specifically kill the target cells. In contrast, in the control T cell group, the fluorescence value of the SKOV3 cells increased over time because the control T cells did not kill the target cells, and the SKOV3 cells grew and expanded normally. Compared with control T cells, the dual-target Meso-B7H3 CAR-T cells had a significant killing effect on SKOV3 cells positive for mesothelin and B7H3.

[0107] After 24 hours, the culture supernatant was collected and tested (the specific operation steps were carried out according to the instructions of the ELISA test kit). Figure 9The results showed that the levels of IL-2 and IFN-γ cytokines in the supernatant of co-culture of SKOV3 cells expressing mesothelin and B7H3 with dual-target Meso-B7H3 CAR-T cells were significantly higher than those in the co-culture group with ordinary T cells (P<0.001), indicating that dual-target Meso-B7H3 CAR-T cells had a significant killing effect on target cells expressing dual targets, while the killing effect on SKOV3 target cells with mesothelin knockdown was comparable to that of T cells, and the killing activity on SKOV3 target cells with B7H3 knockdown was lower. These results indicate that the specificity and killing function of dual-target Meso-B7H3 CAR-T have achieved the expected purpose.

[0108] The dual-target Meso-B7H3 CAR-T cells of the present invention and the third-generation CAR-T cells (i.e., target Meso scFv-CD28-CD137-CD3ζ, sequence as shown in SEQ ID No. 35) were subjected to repeated killing tests on the same dose (10^5) of target cells SKOV3. The results are as follows: Figure 10 The results showed that the dual-target Meso-B7H3 CAR-T designed in the present invention has a more sustained killing ability than the third-generation CAR-T.

[0109] The JAKAcS1 in the sequence SEQ ID No. 33 in Example 7 was replaced with JAKAcS2, JAKAcS3, JAKAcS4, JAKAcS5, JAKAcS6, JAKAcS7, JAKAcS8, JAKAcS9, and JAKAcS10 to obtain the corresponding nucleotide sequences, which were then synthesized and operated according to the above specific steps to obtain the corresponding recombinant strains. According to the specific experimental methods of Examples 8-11, the above experimental process was repeated to obtain the corresponding dual-target CART cells. The positive rate of the above dual-target CART cells was tested, and the positive rate was between 20% and 40%, and it had a persistent ability to kill target cells.

Claims

1. A long-acting dual-target chimeric antigen receptor, characterized in that: The long-acting dual-target chimeric antigen receptor includes a chimeric antigen receptor composed of two independent transmembrane proteins, wherein the first chimeric antigen receptor includes, in sequence, a first signal peptide, a binding domain for a first target tumor antigen, a first hinge domain, a first transmembrane domain, a first intracellular co-stimulatory domain, and an intracellular signal transduction domain of a first target CAR; the second chimeric antigen receptor includes, in sequence, a second signal peptide, a binding domain for a second target tumor antigen, a second hinge domain, a second transmembrane domain, a second intracellular co-stimulatory domain, and a JAK enzyme activation signal transduction domain, wherein the JAK enzyme activation signal transduction domain can capture and activate the JAK enzyme, the first chimeric antigen receptor and the second chimeric antigen receptor are connected by a linker, the JAK enzyme is a JAK1 enzyme, the first target is Claudin18.2 or Mesothelin, and the second target is B7H3, and the nucleic acid sequence of the long-acting dual-target chimeric antigen receptor is SEQ ID No. 32 or SEQ ID No.

34.

2. A nucleic acid molecule encoding the dual-target chimeric antigen receptor according to claim 1.

3. A carrier, characterized in that The vector comprises the nucleic acid molecule according to claim 2.

4. A recombinant virus, characterized in that The recombinant virus comprises the vector according to claim 3.

5. The method for constructing the recombinant virus according to claim 4, characterized in that: The method comprises synthesizing the gene of the dual-target chimeric antigen receptor and then introducing it into a plasmid vector, amplifying the chimeric antigen receptor coding sequence using PCR primers containing homology arms, and inserting the chimeric antigen receptor coding sequence into a viral vector by homologous recombination.

6. A recombinant cell, wherein the recombinant cell expresses the dual-target chimeric antigen receptor of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, or the recombinant virus of claim 4, wherein the recombinant cell comprises a modified T cell.

7. Use of the dual-target chimeric antigen receptor according to claim 1, the nucleic acid molecule according to claim 2, the vector according to claim 3 or the recombinant virus according to claim 4, or the recombinant cell according to claim 6 in the preparation of a medicament for treating solid tumors, wherein the solid tumor is selected from gastric cancer, lung cancer, liver cancer, esophageal cancer, colorectal cancer, melanoma, ovarian cancer, renal cancer, glioma, bone cancer, pancreatic cancer, breast cancer, malignant mesothelioma, thyroid cancer, cervical cancer, or prostate cancer.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the dual-target chimeric antigen receptor according to claim 1, the nucleic acid molecule according to claim 2, the vector according to claim 3 or the recombinant virus according to claim 4, and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition further comprises one or more of physiological saline, cell culture medium, glucose, water for injection, glycerol, ethanol, stabilizer, surfactant, preservative, and isotonic agent.

10. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition also includes other anticancer agents.

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