Long-acting meso-b7h3 dual-target chimeric antigen receptor and uses thereof

Abstract

The application discloses a long-acting Meso-B7H3 double-target chimeric antigen receptor and application thereof, wherein the Meso-B7H3 double-target chimeric antigen receptor comprises a CAR chain composed of two independent transmembrane proteins, a first CAR chain targets scFv of a first target point, and an intracellular signal comprises a second signal and an intracellular transduction signal or only the intracellular transduction signal; a second CAR chain targets scFv of a second target point, and an intracellular signal comprises a costimulatory signal and a JAK enzyme activation transduction domain. The CAR-T cell prepared by the application has a strong and persistent killing effect on tumor cells simultaneously expressing the first target point and the second target point, and can be used for anti-tumor treatment of solid tumors.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and immunotherapy technology, specifically relating to long-acting dual-target chimeric antigen receptors, nucleic acid molecules, recombinant vectors, cells, and their applications. This invention is a divisional application with application number 2022111797820, entitled "Long-acting Dual-target Chimeric Antigen Receptors, Nucleic Acid Molecules, Recombinant Vectors, Cells, and Their Applications." Background Technology

[0002] CAR-T cell technology is based on the theory of immune system recognition and activation. Through genetic engineering, elements that specifically recognize tumor antigens (single-chain antibodies scFv) and initiate immune activity are integrated into a single gene. This gene is then introduced into the patient's own T lymphocytes in vitro using methods such as viruses, amplified, and then infused back into the patient. This allows the patient to regain the ability to specifically recognize tumor cells, activate their own T cells, and specifically attack and kill the recognized tumor cells. CAR-T cell technology has advantages such as not being limited by the major histocompatibility complex in tumor killing, and has achieved encouraging results in patients with refractory or relapsed B-cell leukemia and lymphoma. In recent years, research on CAR-T cell therapy in solid tumors such as glioma, prostate cancer, and lung cancer has also made significant progress, and it is considered one of the most promising cancer treatments. To date, more than 300 CAR-T cell therapies have entered clinical trials worldwide. The core of CAR-T cell technology is the use of genetic engineering to modify T cells, using CAR molecules to recognize tumor cells and simultaneously activate T cells, thereby exerting a powerful tumor-killing effect. However, existing CAR-T cell therapy for tumors has inherent limitations. This is because it is still difficult to find tumor-specific targets at this stage. The main approach is to recognize tumor cells through tumor-associated antigens, which inevitably leads to off-target toxicity and even serious death.

[0003] Many types of tumor cells exhibit two target proteins simultaneously on their surface. For example, some ovarian cancer tissues highly express both mesothelin and B7H3, while the probability of normal tissues simultaneously expressing both targets is very low. If these two target proteins are designed as dual-target CARs, CAR-T cells will be fully activated and exert their killing function only when they simultaneously target the two targets highly expressed by tumor cells. When normal cells express only the target as the first activation signal or only the target as the second activation signal, CAR-T cells will have no killing effect or only a weak killing effect. In this way, we can greatly reduce off-target toxicity in vivo through appropriate dual-target CAR design.

[0004] Furthermore, existing CAR-T cell therapy for tumors generally suffers from drawbacks such as insufficient in vivo expansion capacity and limited lifespan, leading to limited clinical efficacy and a high relapse rate. To address this issue, various cytokines are typically used to achieve sustained CAR-T cell expansion, such as IL-2, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, and GM-CSF-R. These cytokines work by binding to cytokine receptors on CAR-T cells, activating downstream intracellular signal transduction. For example, IL-15 first binds to the α subunit of the IL-15 membrane receptor, then forms an immune synapse with IL2R-γc / IL-15Rβ on effector CAR-T cells, activating intracellular JAK1 / JAK3 and STAT3 / STAT5 pathways, thereby promoting CAR-T cell differentiation and proliferation. Currently, there are two ways to achieve this goal. The first is to directly inject these cytokines into the body simultaneously with CAR-T cell therapy. However, to achieve the desired CAR-T cell expansion, this method requires high doses of cytokines, which inevitably triggers reactions from other immune cells, leading to unpredictable side effects and even death. The second method involves designing CARs to secrete cytokines while activating and expanding CAR-T cells. This can effectively prevent the depletion of CAR-T cells in the body. However, these secreted cytokines do not only act on the CAR cells themselves but also enter the body fluids, causing severe reactions from other immune cells and leading to unpredictable side effects. Summary of the Invention

[0005] Purpose of the invention: In view of 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. The dual-target chimeric antigen receptor can reduce off-target toxic side effects in vivo and enhance the amplification capacity and persistence of CAR-T in vivo by introducing a third signal.

[0006] Another technical problem to be solved by the present invention is to provide a nucleic acid molecule that encodes the aforementioned long-acting dual-target chimeric antigen receptor.

[0007] Another technical problem that this invention aims to solve is to provide a vector or a recombinant virus containing it.

[0008] Another technical problem to be solved by the present invention is to provide the application 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 a drug for treating solid tumors with positive expression of anti-target proteins.

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

[0010] Objective of the Invention: To address the aforementioned technical problems, this invention provides a long-acting dual-target chimeric antigen receptor. This long-acting dual-target chimeric antigen receptor comprises two independent transmembrane proteins. 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 for 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. The JAK enzyme activation signal transduction domain can capture and activate JAK enzymes, including JAK1, JAK2, or JAK3. The Gene ID of JAK1 is 3716, the Gene ID of JAK2 is 3717, the Gene ID of JAK3 is 3718, and the Gene ID of Tyk2 is... The ID is 7297, and the first chimeric antigen receptor and the second chimeric antigen receptor are connected by a linker.

[0011] The JAK enzyme activation signaling domain recruits JAK kinase and phosphorylates tyrosine residues under its catalysis, thereby activating STAT signals and triggering intracellular gene transcription and expression. Therefore, it plays a role similar to that of cytokines. The sequence of any enzyme that can be captured, such as JAK1, JAK2, JAK3 or Tyk2, is within the protection scope of this invention.

[0012] Preferably, the JAK enzyme is a JAK1 enzyme, and the activation signal transduction domain of the JAK1 enzyme has an amino acid sequence with 90-99% identity selected from one or more of the following sequences: 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), SEQ ID No. 9 (JAKAcS9), and SEQ ID No. 10 (JAKAcS10). All of these sequences include the binding site and activation signal of JAK1 kinase. These sequences can capture JAK1 kinase and catalyze the phosphorylation of its amino acid residues to activate the STAT signal, thereby initiating intracellular gene transcription and expression.

[0013] Preferably, 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 a JAK3 enzyme, and the domains that can also activate JAK3 enzymes are also within the scope of protection of this invention.

[0015] Preferably, the intracellular signal transduction domain of the first target CAR includes intracellular signal transduction domains 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 with it, 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. The first and second targets include 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 and combined according to the expression patterns on the surface of different tumor cells. In one embodiment of the present invention, the first target is Claudin18.2, whose nucleotide sequence is shown in SEQ ID No. 22, and the second target is B7H3, whose nucleotide sequence is shown in SEQ ID No. 23. In another embodiment of the present invention, the first chimeric antigen receptor is a first-generation, second-generation, or third-generation CAR-chain designed with the first target Mesothelin, and the nucleotide sequence of Mesothelin is shown in SEQ ID No. 24; the second chimeric antigen receptor has an extracellular region consisting of a scFv targeting B7H3 and an intracellular region consisting of a second signal and a JAK enzyme activation signal transduction domain, wherein the extracellular region of the CAR-chain is composed of a scFv targeting mesothelin, and the intracellular region is composed of a CD3ζ intracellular region.

[0017] The signal peptide can guide the translocation of the antigen recognition region and hinge region to the extracellular space. Any suitable signal peptide or combination of signal peptides can achieve the purpose of this invention. The first and second signal peptides include signal peptides of the α and β chains of T cell receptors, CD3, CD4, CD5, CD8, CD28, CD33, CD45, CD80, CD86, CD134, CD137, ICOS, GM-CSF, immunoglobulin heavy chain, or immunoglobulin light chain. Preferably, the signal peptide is selected from CD8α, such as the signal peptide shown in SEQ ID No. 25.

[0018] In this invention, the target-binding domain is connected to its encoded transmembrane region via a hinge region, and any suitable hinge region sequence can achieve the purpose of this invention. The first and second hinge domains include the hinge regions of the following molecules: IgG, CD8α, CD28, and IL-2 receptors. 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 following: the α, β, or ζ chain 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 transmembrane regions of CD8, with nucleotide sequences as shown in SEQ ID No. 27.

[0020] Wherein, the first intracellular costimulatory domain and the second intracellular costimulatory domain are functional signaling domains obtained by means of one or more of the following proteins or amino acid sequences having 90-99% or the same as 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. The invention can achieve its objective even without a co-stimulatory domain at the first target site, and is within the scope of protection of the invention. In one preferred embodiment, the intracellular co-stimulatory domains of both the first and second targets are CD137, and its nucleotide sequence is shown in SEQ ID No. 28. In another preferred embodiment, the intracellular co-stimulatory domain of the first target is CD28, and its nucleotide sequence is shown in SEQ ID No. 29. The intracellular co-stimulatory 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 appropriate positions between the antigen recognition region, hinge region, transmembrane region and intracellular signaling region, and the peptide chain can be an oligopeptide or a polypeptide.

[0023] In one embodiment of the present invention, the inventors obtained the binding domain of the target using a chemical synthesis method.

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

[0025] The preparation methods for the aforementioned nucleic acid molecules can be based on the base sequences of the target recognition region, hinge region, transmembrane region, and intracellular signaling region, as well as other structural domains, and can be achieved through known techniques such as chemical synthesis or PCR amplification. Typically, the codons encoding the amino acids of these structural domains can be optimized to improve their expression in host cells.

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

[0027] In this invention, the 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 transposons. The vector may contain regulatory sequences such as promoters and terminators, as well as marker sequences such as drug resistance genes and reporter genes. The viral vector can be a retroviral vector, a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, etc. In one embodiment of this invention, a lentiviral expression vector is used.

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

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

[0030] In one embodiment of the present invention, the cells described above are human T cells. The T cells may be derived from peripheral blood, bone marrow, or tissues such as the spleen, thymus, and lymph nodes, and are obtained after separation and purification. Furthermore, the T cells may be CD4+ T cells, CD8+ T cells, or γδ T cells. The T cells may be appropriately replaced with NK cells, NKT cells, helper T cells, or macrophages, which are also considered to be included 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 application of the dual-target chimeric antigen receptor, the nucleic acid molecule, the recombinant vector or recombinant virus, and the recombinant cell in the preparation of a drug for treating solid tumors.

[0032] 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, neurobladder cancer, or prostate cancer.

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

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

[0035] Similarly, the pharmaceutical compositions of the present invention can also be used in combination with other suitable anticancer agents. For example, vincristine, daunorubicin, aspartate aminotransferase, cyclophosphamide, prednisone, etc.

[0036] Another aspect of the present invention is to provide the use of the above-described pharmaceutical composition in the preparation of a drug for solid tumors that positively express anti-target proteins.

[0037] In summary, the dual-target chimeric antigen receptor of this invention simultaneously expresses two transmembrane protein chains, one of which is a first-generation, second-generation, or third-generation CAR design (CAR-chain); the second chain consists of an extracellular domain targeting tumor antigens and an intracellular domain consisting of a second signal and a 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 in this invention is that the extracellular binding region of the CAR-chain consists of scFv targeting the first target, while the intracellular region consists of the intracellular region of the co-stimulatory molecule and the intracellular region of the first signal transduction domain. The extracellular binding region of the Cos-chain consists of scFv targeting the second target, while the intracellular region consists of the intracellular region of the co-stimulatory molecule and the JAK enzyme activation signal transduction domain. This design allows positive tumor cells on the surface of tumor cells that highly express the first target to bind to the first target scFv and provide T cells with the first and second signals, initiating T cell killing of tumor cells. Positive tumor cells on the surface of tumor cells that highly express the second target to bind to the second target scFv and provide T cells with the second and third signals, enhancing the function and persistence of T cells in killing tumor cells. When normal cells do not express both target proteins or only express the second target, T cells do not have killing function, reducing the off-target toxicity of CAR-T cells. Another advantage of the CAR design in this invention is that the intracellular region of the CAR is enriched with JAK enzyme activation signal transduction elements in CAR-T cells through the structural design of the second target CAR. When the extracellular region scFv of the second CAR binds to the antigen on the tumor cell, it activates the JAK / STAT signal transduction pathway in CAR-T cells, etc., promoting the differentiation and proliferation of CAR-T cells, thereby preventing the depletion 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 this CAR design is that positive tumor cells that highly express the first target Mesothelin on their surface bind to the first target scFv and provide T cells with first and second signals, initiating T cells to kill tumor cells. Positive tumor cells that highly express the second target B7H3 on their surface bind to the second target scFv and provide T cells with second and third signals, enhancing the function and persistence of T cells in killing 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 killing function, greatly reducing the off-target toxicity of CAR-T cells. Another advantage is that the intracellular region of the CAR is enriched with JAK enzyme activation signal transduction binding elements in CAR-T cells through the structural design of the second target CAR. When the extracellular scFv of the second CAR binds to the antigen on the target cell, it activates the JAK / STAT signal transduction pathway in CAR-T cells, promoting the differentiation and proliferation of CAR-T cells, thereby preventing the depletion of CAR-T cells.

[0040] Beneficial Effects: The CAR of this invention has the following advantages over existing similar products: First, the CAR does not secrete cytokines while exerting cytokine effects, thus avoiding excessive activation of other immune cells and side effects. Second, only the binding of the scFv of the second target CAR to the antigen of tumor cells can stimulate the expansion of CAR-T cells, ensuring that CAR-T cell expansion is concentrated near tumor cells. This not only enhances the tumor-killing effect but also stops CAR-T cell expansion once the tumor cells are eliminated, further ensuring in vivo safety. Examples of this invention show that CAR-T cells prepared from the CAR designed in this invention have a significantly greater ability to repeatedly kill tumor cells than third-generation CAR-T cells. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the structure of a long-acting dual-target CAR.

[0042] Figure 2 The image shows the GFP expression fluorescence of CLDN18.2 / B7H3 CART cells in Example 4. GFP is the fluorescent labeling 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 The GFP dynamic fluorescence statistical value of tumor cells after co-culturing CLDN18.2 / B7H3 CART cells and target cells (effect-to-target ratio of 1:1) for 40 hours in Example 5 is shown.

[0044] Figure 4 The results are obtained by ELISA detection of IL2 and IFN-γ release after CLDN18.2 / B7H3 CART cells and target cells were co-cultured for 24 h in Example 5.

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

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

[0047] Figure 7 The growth and expansion curves of Meso-B7H3 CAR-T cells and the changes in T cell size are shown.

[0048] Figure 8 The GFP dynamic fluorescence statistical value of tumor cells after co-culturing Meso-B7H3 CAR-T cells and target cells (effect-to-target ratio of 1:1) for 40 hours in Example 10.

[0049] Figure 9 The results are obtained by ELISA detection of IL2 and IFN-γ release after Meso-B7H3 CAR-T cells and target cells were co-cultured for 24 h in Example 10.

[0050] Figure 10 Example 10 of this invention uses dual-target Meso-B7H3 CAR-T cells, third-generation CAR-T cells (targeting Meso scFv-CD28-CD137-CD3ζ), and unmodified T cells at the same dose (10 5 Results of repeated killing experiments on target cells (number of cells). Detailed Implementation

[0051] This invention discloses the construction and application of long-acting dual-target CARs. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. Furthermore, those skilled in the art can clearly modify or appropriately alter and combine the content described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

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

[0053] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be 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 CLDN18.2scFv-CD8αhinge-CD8TM-CD3ζ-T2A-signal peptide-second target B7H3scFv-CD8α hinge-CD8TM-CD137-JAKAcS1) expression vector

[0055] The first target was selected as Claudin18.2 and the second target was selected as B7H3 to construct a dual-target CAR: signal peptide - first target CLDN18.2scFv-CD8α hinge-CD8TM-CD3ζ-T2A - signal peptide - second target B7H3scFv-CD8ahinge-CD8TM-CD137-JAKAcS1.

[0056] The nucleic acid sequence encoding the above-mentioned CLDN18.2 / B7H3 CAR was synthesized by GenScript, and the sequence is SEQ ID No. 32.

[0057] Linearization of the vector and fragment was achieved by double digestion of Plvx-EF1a-IRES-ZsGreen1, CLDN18.2 / B7H3 CAR with BamHI-MluI. The digestion system is as follows:

[0058]

[0059] The chimeric antigen receptor coding fragment with sticky ends and the linearized Plvx-EF1a-IRES-ZsGreen1 vector were ligated and transformed using T4 DNase.

[0060] The Plvx-EF1α-IRES-ZsGreen1, CLDN18.2 / B7H3 CAR system is as follows:

[0061]

[0062] The conversion steps for linking the recombinant products are as follows:

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

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

[0065] 3) Perform an accurate heat stress at 42℃ for 45 seconds, then immediately place the device on ice for 2 minutes.

[0066] 4) Add 500 μL of antibiotic-free LB medium and incubate at room temperature for 1 hour (270 rpm).

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

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

[0069] 7) Incubate upside down in a 37℃ incubator for 12-16 hours.

[0070] Example 2 Lentiviral Packaging of Dual-Target CLDN18.2 / B7H3 CAR

[0071] The CLDN-B7H3 CAR expression plasmid was extracted from the bacterial culture obtained by picking single colonies of the recombinant bacteria in Example 1 using the EndoFree Plasmid Mid plasmid extraction kit (Omega). The CLDN18.2 / B7H3 CAR expression plasmid and the packaging plasmids pSPAX and pMD2.G were transfected into HEK293T cells at a ratio of 4:3:1 using the calcium phosphate method. Fresh culture medium was added 12 hours after transfection. Viral supernatant was collected at 24 and 48 hours, centrifuged at 3,000 rpm for 15 minutes at 4°C, filtered through a 0.45 μm filter, and then 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 peripheral blood was collected from a healthy donor in the Department of Hematology, Zhongda Hospital, Southeast University. PBMCs were isolated using Ficoll-Paque PLUS (GE HealthCare) (specific steps followed the instructions). Anti-CD3 / CD28 magnetic beads (GibCo) were added at a 1:1 ratio of cells to magnetic beads, and the cells were cultured for 24 hours to obtain pre-transfection T cells.

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

[0075] The viral supernatant prepared in Example 2 was removed from -80°C and thawed at room temperature, yielding a viral titer of 5.6 x 10⁻⁶. 7 Based on a multiplicity of infection (MOI) of 5, 68 μL of virus concentrate was added, meaning the required volume of virus concentrate was 1 * 10⁻⁶. 6 (Number of cells to be infected) * 5 / 5.6 * 10 7 Add 10 mg / ml of polybrene to a final concentration of 10 pg / ml. Centrifuge at 2,000 rpm for 2 hours at 30°C, then transfer to a 5% CO2 incubator at 37°C to culture and 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. Results are as follows: Figure 2 As shown, by Figure 2 As can be seen, the positivity rate of CLDN18.2 / B7H3 CART cells was 27.7%.

[0077] Example 5: Killing effect of dual-target CLDN18.2 / B7H3 CART cells on tumor cells with dual-target positive expression.

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

[0079] After 24 hours, the culture supernatant was collected and analyzed (the specific operating procedures were performed according to the ELISA kit instructions). The results are as follows: Figure 4 As shown in the results, the levels of IL-2 and IFN-γ cytokines in the supernatant of SGC-7901 cells expressing Claudin18.2 and B7H3 co-cultured with dual-target CLDN18.2 / B7H3 CART cells were significantly higher than those in the normal T co-culture group (P < 0.001), indicating that dual-target CLDN18.2 / B7H3 CART cells have a significant killing effect on target cells expressing both targets. However, the killing effect on SGC-7901 target cells with knockdown of Claudin18.2 is comparable to that of T cells, while the killing activity on SGC-7901 target cells with knockdown of B7H3 is lower. These results indicate that the specificity and killing function of dual-target CLDN18.2 / B7H3 CART have achieved the expected goals.

[0080] The dual-target CLDN18.2 / B7H3 CAR-T cells designed in this invention were compared with third-generation CAR-T cells (i.e., target CLDN18.2scFv-CD28-CD137-CD3ζ, sequence as shown in SEQ ID No. 33) at the same dose (105 Multiple repeated killing experiments were conducted on target cells SGC-7901 (number of cells), and the results were as follows: Figure 5 As shown in the figure. The results indicate that the dual-target CLDN18.2 / B7H3 CART designed in this invention has a more sustained killing ability than third-generation CAR-T.

[0081] Example 6

[0082] The nucleotide sequences JAKAcS1 in SEQ ID No. 32 of Example 1 were replaced with JAKAcS2, JAKAcS3, JAKAcS4, JAKAcS5, JAKAcS6, JAKAcS7, JAKAcS8, and JAKAcS9, respectively, to obtain the corresponding nucleotide sequences. These sequences were then synthesized and the corresponding recombinant strains were obtained following the specific steps described above. The experimental procedures described in Examples 2-5 were repeated to obtain the corresponding dual-target CAR-T cells. The positive rate of the dual-target CAR-T cells was tested, and the positive rate reached 20-40%, exhibiting a sustained ability to kill target cells.

[0083] Example 7: Construction of a dual-target Meso-B7H3 CAR (signal peptide-first target Meso scFv-CD8α hinge-CD8TM-CD3ζ-P2A-signal peptide-second target B7H3 scFv-CD8α hinge-CD8TM-CD137-JAKAcS1) expression vector

[0084] The nucleic acid sequence encoding the above-mentioned dual-target Meso-B7H3 CAR was synthesized by GenScript, and the sequence is SEQ ID No. 34.

[0085] Linearization of the vector and fragment was achieved by double digestion of Plvx-EF1α-IRES-ZsGreen1 and Meso-B7H3 CAR with BamHI-MluI. The digestion system is as follows:

[0086]

[0087] The chimeric antigen receptor coding fragment with sticky ends and the linearized Plvx-EF1α-IRES-ZsGreen1 vector were ligated and transformed using T4 DNase.

[0088] The Plvx-EF1α-IRES-ZsGreen1, Meso-B7H3 CAR system is connected as follows:

[0089]

[0090] The conversion steps for linking the recombinant products are as follows:

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

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

[0093] 3) Perform an accurate heat stress at 42℃ for 45 seconds, then immediately place the device on ice for 2 minutes.

[0094] 4) Add 500 μL of antibiotic-free LB medium and incubate at room temperature for 1 hour (270 rpm).

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

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

[0097] 7) Incubate upside down in a 37℃ incubator for 12-16 hours.

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

[0099] The recombinant bacteria obtained in Example 6 were cultured using the EndoFree Plasmid Mid plasmid extraction kit (Omega). Single colonies were then extracted from the culture to obtain the dual-target Meso-B7H3 CAR expression plasmid. The dual-target Meso-B7H3 CAR expression plasmid and packaging plasmids pSPAX and pMD2.G were transfected into HEK293T cells at a ratio of 4:3:1 using the calcium phosphate method. Fresh culture medium was added 12 hours after transfection. Viral supernatant was collected at 24 and 48 hours, centrifuged at 3,000 rpm for 15 minutes at 4°C, filtered through a 0.45 μm filter, and then 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 peripheral blood was collected from a healthy donor in the Department of Hematology, Zhongda Hospital, Southeast University. PBMCs were isolated using Ficoll-Paque PLUS (GE HealthCare) (specific steps followed the instructions). Anti-CD3 / CD28 magnetic beads (GibCo) were added at a 1:1 ratio of cells to magnetic beads, and the cells were cultured for 24 hours to obtain pre-transfection T cells.

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

[0103] The viral supernatant was removed from -80℃ and thawed at room temperature; the viral titer was 6.3*10⁻⁶. 7 Based on a multiplicity of infection (MOI) of 5, 79 μL of virus concentrate was added, meaning the required volume of virus concentrate is 1 * 10⁻⁶. 6 (Number of cells to be infected) * 5 / 6.3 * 10 7 Add 10 mg / ml of polybrene to a final concentration of 10 pg / ml. Centrifuge at 2,000 rpm for 2 hours at 30°C, then transfer to a 5% CO2 incubator at 37°C for incubation.

[0104] Flow cytometry was used to detect the positivity rate of Meso-B7H3 CAR-T cells: Cells were collected, labeled with rabbit anti-mouse IgG-F(ab')2 antibody, and the expression of F(ab')2 and GFP in T cells was analyzed by flow cytometry. Results are as follows: Figure 6 As shown in the figure, the positive rate of Meso-B7H3 CAR-T cells was 40.4%.

[0105] Example 11: Killing effect of dual-target Meso-B7H3 CAR-T cells on tumor cells expressing dual targets.

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

[0107] After 24 hours, collect the culture supernatant and perform detection on the co-culture supernatant (follow the ELISA kit instructions for specific procedures). The results are as follows: Figure 9As shown in the results, the levels of IL-2 and IFN-γ cytokines in the supernatant of SKOV3 cells expressing Mesothelin and B7H3 co-cultured with dual-target Meso-B7H3 CAR-T cells were significantly higher than those in the normal T co-culture group (P < 0.001), indicating that dual-target Meso-B7H3 CAR-T cells have a significant killing effect on target cells expressing both targets. However, the killing effect on SKOV3 target cells with knockdown of Mesothelin is comparable to that of T cells, while the killing activity on SKOV3 target cells with knockdown of B7H3 is lower. These results indicate that the specificity and killing function of dual-target Meso-B7H3 CAR-T cells have achieved the expected goals.

[0108] The dual-target Meso-B7H3 CAR-T cells of this invention and third-generation CAR-T cells (target MesoscFv-CD28-CD137-CD3ζ, sequence as shown in SEQ ID No. 35) were used in repeated killing experiments against the same dose (10 / ^5) of target cells SKOV3. The results are as follows: Figure 10 As shown in the figure. The results indicate that the dual-target Meso-B7H3CAR-T designed in this invention has a more sustained killing ability than third-generation CAR-T.

[0109] The nucleotide sequences JAKAcS1 in SEQ ID No. 33 of Example 7 were replaced with JAKAcS2, JAKAcS3, JAKAcS4, JAKAcS5, JAKAcS6, JAKAcS7, JAKAcS8, JAKAcS9, and JAKAcS10 respectively to obtain the corresponding nucleotide sequences. These sequences were then synthesized and the corresponding recombinant strains were obtained following the specific steps described above. The experimental procedures described in Examples 8-11 were repeated to obtain the corresponding dual-target CAR-T cells. The positive rate of the dual-target CAR-T cells was detected, showing a positive rate of 20-40%, and they exhibited persistent target cell killing ability.

Claims

1. A recombinant vector, characterized in that, The construction of the recombinant vector includes: linearizing the vector and fragment by double digestion of Plvx-EF1α-IRES-ZsGreen1 and CLDN18.2 / B7H3 CAR with BamHI and MluI, and then ligating and transforming the chimeric antigen receptor coding fragment containing sticky ends and the linearized Plvx-EF1α-IRES-ZsGreen1 vector with T4 DNase. The nucleotide sequence of the CLDN18.2 / B7H3 CAR is shown in SEQ ID No.

32.

2. A recombinant vector, characterized in that, The construction of the recombinant vector includes: linearizing the vector and fragment by double digestion of Plvx-EF1α-IRES-ZsGreen1 and Meso-B7H3 CAR with BamHI and MluI, and then ligating and transforming the chimeric antigen receptor coding fragment containing sticky ends and the linearized Plvx-EF1α-IRES-ZsGreen1 vector with T4 DNase. The nucleotide sequence of the Meso-B7H3 CAR is shown in SEQ ID No.

34.

3. A recombinant virus, characterized in that, The recombinant virus comprises the recombinant vector as described in claim 1 or 2.

4. A recombinant cell, characterized in that, The recombinant cells comprise the recombinant vector of claim 1 or 2 or the recombinant virus of claim 3.

5. The use of the recombinant vector of claim 1 or 2, the recombinant virus of claim 3, and the recombinant cell of claim 4 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, kidney cancer, glioma, bone cancer, and pancreatic cancer.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the recombinant vector of claim 1 or 2 or the recombinant virus of claim 3, and a pharmaceutically acceptable vector.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition further includes one or more of the following: physiological saline, cell culture medium, glucose, water for injection, glycerol, ethanol, stabilizer, surfactant, preservative, and isotonic agent.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also includes other anticancer agents.

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