A CAR that enhances tumor antigen action and its application

Abstract

The present invention discloses a CAR that enhances the action of tumor antigens and its application. The CAR includes a signal peptide, a target scFv, a hinge domain, a transmembrane domain, and an intracellular co-stimulatory domain. The chimeric antigen receptor also includes a JAK enzyme activation signal transduction domain. The chimeric antigen receptor provided by the present invention can enhance the long-term killing ability of tumor antigen-activated killer T cells against tumor cells expressing the target antigen, and has no killing effect on non-tumor cells expressing the target antigen. The present invention can be used for the anti-tumor treatment of solid tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering and immunotherapy, and specifically relates to a CAR that enhances the effect of tumor antigens and its application. Background Art

[0002] With the development of tumor immunology theory and technology, tumor immunotherapy has made rapid progress. Among them, chimeric antigen receptor T (CAR-T) cell therapy has made the most rapid progress, and tumor vaccine technology targeting tumor neoantigens is also gaining momentum, bringing new hope to cancer patients.

[0003] 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 methods such as viruses 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 in tumor killing. 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 basically identified by tumor-associated antigens, which inevitably leads to off-target toxicity and even serious lethality.

[0004] Tumor vaccine technology involves introducing tumor antigens into the patient's body in various forms, such as tumor cells, tumor-associated proteins or peptides, or genes expressing tumor antigens. This approach aims to overcome the immunosuppressive state caused by the tumor, enhance immunogenicity, and activate the patient's own immune system, thereby achieving the goal of controlling or eliminating the tumor. Because tumor vaccines primarily utilize neoantigens to mediate innate immune responses and induce adaptive immune responses, this therapeutic technology offers high specificity, good safety, and no side effects. Therefore, tumor vaccines are often used in combination with radiotherapy, chemotherapy, and surgery in clinical practice. However, tumor vaccines rely on the patient's existing immune cells to produce tumor-killing effects, and the functional activity of immune cells in cancer patients is generally low, thus limiting the therapeutic effectiveness of tumor vaccines.

[0005] How to combine the respective advantages of CAR-T technology and tumor vaccine technology to develop new tumor immunotherapy technologies with better efficacy urgently needs further research by those skilled in the art. Summary of the Invention

[0006] Purpose of the invention: To address the technical problems existing in both CAR-T cell technology and tumor vaccine technology, the present invention proposes a CAR that can enhance the effect of tumor antigens and its application.

[0007] Technical solution: In order to solve the above technical problems, the present invention provides a chimeric antigen receptor, which includes a signal peptide, a target scFv, a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain and a JAK enzyme activation signal transduction domain. The chimeric antigen receptor also includes a JAK enzyme activation signal transduction domain, which can capture and activate the JAK enzyme. The JAK enzyme includes JAK1, JAK2, JAK3 or Tyk2, 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 7297.

[0008] 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. 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.

[0009] 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).

[0010] 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.

[0011] The extracellular region comprises a single-chain antibody structure region (scFv) of a target highly expressed on the surface of tumor cells, wherein the scFv of the target comprises the scFv of a target highly expressed on the surface of tumor cells, and the target comprises any one or more of Claudin18.2, Mesothelin, B7H3, PD-L1, GPC3, MUC1, 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, and EPHA2.

[0012] In one embodiment of the present invention, the nucleotide sequence of the single-chain antibody against the selected target B7H3 is shown in SEQ ID No. 21.

[0013] In the present invention, positive tumor cells that highly express the target protein on their cell surface can co-stimulate T cells after binding to scFv; while negative cells that do not express the target protein on their cell surface cannot co-stimulate T cells.

[0014] In the present invention, target-positive tumor cells can co-stimulate T cells after binding to the anti-target scFv; however, target-negative cells cannot co-stimulate T cells. In the present invention, the amino acid sequence of the anti-target scFv can be subjected to random or engineered point mutations in an appropriate manner, the purpose of which can be, for example, to obtain better affinity and / or dissociation properties, and these mutated amino acid sequences are all included in the scope of protection of the present invention.

[0015] The signal peptide of the present invention can guide the transfer 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 the present invention. Among them, the signal peptide includes the signal peptide of the α chain or 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, or an amino acid sequence having 90-99% identity with the signal peptide.

[0016] More preferably, the signal peptide is the signal peptide in CD8α, and the sequence is the signal peptide shown in SEQ ID No. 22.

[0017] In one embodiment of the present invention, the target binding domain encoded by the nucleic acid molecule of the present invention is connected to the transmembrane region encoded by the nucleic acid molecule of the present invention via a hinge region. Any suitable hinge region sequence can achieve the objectives of the present invention. Preferably, in one embodiment of the present invention, the hinge region is CD8α, the nucleotide sequence of which is shown in SEQ ID No. 23.

[0018] In the present invention, the nucleic acid molecule further encodes a transmembrane domain. Any suitable transmembrane domain can achieve the purpose of the present invention. Preferably, the transmembrane domain is selected from the transmembrane domain of the following proteins or an amino acid sequence having 90-99% identity with the proteins: 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 transmembrane domain is the transmembrane region of CD8, the nucleotide sequence of which is shown in SEQ ID No. 24.

[0019] The intracellular co-stimulatory domain is selected from one or more of the following proteins or functional signaling domains obtained from an amino acid sequence having 90%-99% identity with the protein: MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, lymphocyte activation signaling molecule, activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD137, CDS, ICAM-1, LFA-1, CLAUDIN, CD278 or GITR.

[0020] More preferably, in one embodiment of the present invention, the co-stimulatory factor is CD28 or CD137 or an amino acid sequence having 90%-99% identity thereto. A preferred embodiment is CD137, the nucleotide sequence of which is shown in SEQ ID No. 25.

[0021] 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.

[0022] The present invention also includes a method for constructing the chimeric antigen receptor, comprising the following steps: synthesizing the chimeric antigen receptor according to the gene sequence of the structure of the chimeric antigen receptor using conventional biosynthesis methods, presenting the chimeric antigen receptor on a plasmid vector after synthesis, 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.

[0023] The viral vector is selected from one or more combinations of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, and other gene transfer systems.

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

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

[0026] In one embodiment of the present invention, the inventors used chemical synthesis to obtain the target binding domain. In one embodiment of the present invention, the inventors used chemical synthesis to obtain the B7H3 binding domain.

[0027] The present invention also includes expression cassettes, recombinant expression vectors, recombinant viruses, and recombinant cells, which contain the nucleic acid molecules.

[0028] In the present invention, the vector can be a linear or 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.

[0029] The viral vector may 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. A preferred lentiviral vector is Plvx-EF1α-MCS-(PGK-puro).

[0030] The cells contain the aforementioned nucleic acid molecules, the aforementioned chimeric antigen receptors, or the aforementioned vectors. In one embodiment of the present invention, the cells are human T cells. The T cells may be isolated and purified from peripheral blood, bone marrow, or spleen, thymus, lymphoid tissue, or other tissues. Furthermore, the T cells may be CD4+ T cells, CD8+ T cells, or γδ T cells. The T cells may be replaced with NK cells in an appropriate manner, which is also considered to be within the scope of protection of the present invention.

[0031] The present invention also includes a pharmaceutical composition or cell preparation for target-CAR to enhance the effect of tumor antigens, which contains the chimeric antigen receptor, the nucleic acid molecule, the expression cassette, the recombinant expression vector, the recombinant virus, and the recombinant cell.

[0032] Wherein, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0033] The present invention also includes a method for preparing the target-CAR composition for enhancing tumor antigen effect, comprising the following steps:

[0034] 1) Collect mononuclear cells from a peripheral vein or extract anticoagulated blood to isolate mononuclear cells, and then culture them to obtain antigen-presenting cells and T cells;

[0035] 2) preparing tumor antigens, co-incubating the tumor antigens with antigen-presenting cells, and preparing mature tumor antigen-presenting cells under the action of maturation-promoting factors;

[0036] 3) Sensitizing T cells with mature tumor antigen presenting cells, and then using the nucleic acid molecule or the recombinant vector to introduce the sensitized T cells to obtain the target-CAR composition that enhances the effect of tumor antigens; or first introducing the nucleic acid molecule or the recombinant vector into T cells to obtain target-CAR-modified T cells, and then using mature tumor antigen presenting cells to sensitize CAR-modified T cells to obtain the target-CAR composition that enhances the effect of tumor antigens.

[0037] As a preparation method of the present invention, the preparation method of the composition for enhancing the effect of tumor antigen immunotherapy by B7H3-CAR includes:

[0038] 1) Collect mononuclear cells from a peripheral vein or extract anticoagulated blood to isolate mononuclear cells, and then culture them to obtain antigen-presenting cells and T cells;

[0039] 2) preparing tumor antigens, co-incubating the tumor antigens with antigen-presenting cells, and preparing mature tumor antigen-presenting cells under the action of maturation-promoting factors;

[0040] 3) Sensitizing T cells with mature tumor antigen presenting cells, and then introducing the nucleic acid molecule encoding the CAR or the recombinant vector into the sensitized T cells to obtain the B7H3-CAR composition for enhancing the tumor antigen immunotherapy effect; or first introducing the nucleic acid molecule encoding the CAR or the recombinant vector into T cells to obtain B7H3-CAR-modified T cells, and then sensitizing the CAR-modified T cells with mature tumor antigen presenting cells to obtain the B7H3-CAR composition for enhancing the tumor antigen immunotherapy effect.

[0041] Wherein, the peripheral blood in step 1) is autologous or allogeneic, and the allogeneic meets the requirement of having at least 3 identical HLA matching sites; the antigen-presenting cells include professional antigen-presenting cells and / or non-professional antigen-presenting cells.

[0042] The tumor antigens in step 2) are derived from the patient's own tumor tissue lysate, or tumor cell lysate cultured from tumor tissue, or one or more of personalized tumor neoantigen peptides prepared by autologous gene sequencing and bioinformatics prediction; or tumor cell line lysate, etc., depending on the feasibility of clinical sample acquisition.

[0043] As a further preferred method, the preparation of the tumor antigen in step 2) can be carried out by directly lysing the obtained patient tumor tissue, or digesting the patient tumor tissue and isolating and culturing the obtained tumor cells, or lysing the tumor cell line and then preparing the tumor antigen. The tumor antigen can also be prepared by sequencing the patient tumor tissue using high-throughput sequencing technology to obtain mutation sites, using bioinformatics methods to predict tumor neoantigen polypeptides at the mutation sites, and synthesizing these tumor neoantigen polypeptides as tumor antigens.

[0044] Furthermore, a preferred solution for preparing tumor antigens of the present invention is to oxidize or repeatedly freeze-thaw the lysate of tumor tissue or tumor cells.

[0045] Furthermore, the tumor neoantigen peptides predicted by gene sequencing and bioinformatics generally include a group of 1-50 polypeptide compositions, each of which is an MHC-I restricted polypeptide with a length of 8-13 amino acids.

[0046] Furthermore, each peptide segment in the tumor neoantigen peptide can be prepared by conventional methods well known in the art, including but not limited to chemical synthesis followed by separation of the synthesis product from the by-products via a chromatographic column, and intracellular expression of a nucleic acid encoding a polypeptide comprising an antigenic fragment in the predicted tumor neoantigen polypeptide, or translation of the above encoding nucleic acid by an in vitro cell-free translation system followed by purification to obtain the predicted tumor neoantigen peptide fragments, which are then mixed to prepare tumor antigens.

[0047] Furthermore, the tumor antigens can be used alone or in combination.

[0048] In the present invention, antigen presenting cells (APCs) are used to load tumor antigens, and the antigen presenting cells have an HLA typing that matches the T lymphocytes used, which is a complete match or a partial match. The antigen presenting cells are professional antigen presenting cells and non-professional antigen presenting cells. The professional antigen presenting cells are dendritic cells (DCs), B lymphocytes, macrophages, preferably dendritic cells, macrophages or lymphocytes, more preferably DCs. DCs can be of autologous or allogeneic origin, DCs can also be DC cell lines with similar biological properties to natural DCs, artificially constructed DC cell lines, DC cells differentiated from DC precursor cells or induced pluripotent stem cells or embryonic stem cells, DCs can also be derived from CD34+ hematopoietic precursor cells differentiated from umbilical cord blood, more preferably autologous DC cells. Non-professional antigen presenting cells are antigen presenting cells that express class I HLA.

[0049] In the present invention, the T lymphocytes can be autologous T cells, donor T cells, or genetically modified T cells. The T cells can be isolated and purified from peripheral blood, bone marrow, spleen, thymus, lymphoid tissue, etc.

[0050] In step 2), the tumor antigen is co-incubated with DC cells to sensitize and activate DC cells, and maturation-promoting factors are added to obtain mature tumor antigen-presenting DC cells, and the mature DC cells are used to stimulate and activate T cells or CAR-modified T cells.

[0051] In one embodiment of the present invention, the method for collecting and isolating and culturing DC cells and T cells is as follows:

[0052] 1. Draw 50-100 ml of peripheral anticoagulated blood and obtain mononuclear cells by conventional density gradient centrifugation. 7 / cm 2 The mononuclear cells were inoculated into a culture flask and incubated in a 37°C, 5% carbon dioxide incubator for 2 hours.

[0053] 2. The adherent cells after incubation are used for DC cell culture. The suspended cells are transferred to another culture flask for T cell culture. The adherent cells are used as DC precursor cells.

[0054] 3. Add DC cell-specific culture medium, recombinant human interleukin-4 (IL-4) at a final concentration of 1000 IU, and recombinant human granulocyte colony-stimulating factor (GM-CSF) at a final concentration of 1000 IU to the DC precursor cells. Directedly induce and expand DC precursor cells in a 37°C, 5% CO2 incubator for 3-8 days, and harvest immature DC cells.

[0055] 4. After adding magnetic beads to the suspended cells to sort out CD3+ cells, or directly add CD3 / CD28 immunomagnetic beads and culture medium containing a final concentration of 30 IU / ml IL-2, change half the medium every other day, and harvest T cells.

[0056] In the present invention, the above-mentioned tumor antigens are used to sensitize and activate antigen-presenting cells to obtain mature antigen-presenting cells.

[0057] Furthermore, a preferred embodiment of the present invention is to sensitize and activate DC cells with tumor antigens and activators, add the prepared tumor antigens to the DC cells at a ratio of approximately 1:1, and simultaneously add a certain concentration of maturation-promoting factors, such as poly I-1 at a final concentration of 0.1-1 μg / ml and TNF-α at a final concentration of 0.05-1 μg / ml, and incubate at room temperature to 37°C for 8-24 hours to obtain mature tumor antigen-presenting DC cells. For polypeptide combination antigens, the polypeptide combination is co-incubated with DC cells at a concentration of 10-100 μg / ml, and simultaneously add a certain concentration of maturation-promoting factors, such as poly I-1 at a final concentration of 0.1-1 μg / ml and TNF-α at a final concentration of 0.05-1 μg / ml, and incubate at room temperature to 37°C for 5-72 hours to obtain mature polypeptide combination tumor antigen-presenting DC cells.

[0058] Furthermore, optionally, for tumor neoantigen peptides, nucleic acids encoding polypeptides containing antigenic fragments in predicted tumor neoantigen peptides are directly transfected into APC cells for translation of the neoantigen peptides encoding the nucleic acids, and assisted by the action of activators, these neoantigen peptides are presented on the MHC molecules of APC cells, and mature antigen-presenting cells are directly obtained.

[0059] In the present invention, the composition for enhancing the tumor antigen immunotherapy effect of the target CAR can be obtained by sensitizing T cells through mature antigen-presenting cells, and then using a nucleic acid molecule or recombinant vector encoding CAR to introduce the sensitized T cells.

[0060] In the present invention, the B7H3-CAR composition for enhancing the effect of tumor antigen immunotherapy can be obtained by sensitizing T cells through mature antigen-presenting cells, and then introducing the sensitized T cells using a nucleic acid molecule or recombinant vector encoding CAR.

[0061] Furthermore, in a preferred embodiment of the present invention, mature antigen-presenting DC cells and T cells are mixed at a ratio of 1:3-100, culture medium is added to a culture flask, and the cells are incubated at 37°C, 5% CO2 incubator. Recombinant human IL-2 is added the next day for continued culture, and then, according to the cell growth and expansion conditions, individualized expansion medium containing recombinant human IL-2 is added in a timely and appropriate amount for expansion culture. After culture for 2-7 days, the cell suspension is collected, anti-human CD3 monoclonal antibody, anti-human CD28 monoclonal antibody, and recombinant human IL-2 are added to continue stimulation and culture for 2-7 days, and then a lentiviral vector expressing the above-mentioned CAR of the present invention is added and culture is continued for 7-14 days. The cell suspension is collected and washed twice by centrifugation, and a sample is retained for quality control; the obtained cell suspension is resuspended with 100 ml of physiological saline at a certain concentration, and 0.5-1% human serum albumin is added for intravenous drip; or 30 ml or 2 ml is resuspended according to clinical needs for human thoracic or peritoneal injection, or local injection of part of the tumor.

[0062] In the present invention, the composition for enhancing the tumor antigen immunotherapy effect of the target CAR can also be obtained by first introducing the nucleic acid molecule or recombinant vector encoding CAR into T cells to obtain the target CAR-modified T cells, and then using mature antigen-presenting cells to sensitize the CAR-modified T cells to obtain the composition.

[0063] In the present invention, the B7H3-CAR composition for enhancing the effect of tumor antigen immunotherapy can also be obtained by first introducing a nucleic acid molecule or recombinant vector encoding CAR into T cells to obtain B7H3-CAR-modified T cells, and then using mature antigen-presenting cells to sensitize the CAR-modified T cells to obtain the composition.

[0064] Furthermore, in a preferred embodiment of the present invention, T cells are first cultured for 2-7 days using anti-human CD3 monoclonal antibody, anti-human CD28 monoclonal antibody, and recombinant human IL-2 to stimulate the cells, and then a lentiviral vector expressing the CAR of the present invention is added, and the cells are cultured for 7-14 days. The cell suspension is collected and washed to obtain CAR-modified T cells.

[0065] Furthermore, in a preferred embodiment of the present invention, T cells are first cultured for 2-7 days using anti-human CD3 monoclonal antibody, anti-human CD28 monoclonal antibody, and recombinant human IL-2 to stimulate the cells, and then a lentiviral vector expressing the CAR of the present invention is added, and the cells are cultured for 7-14 days. The cell suspension is collected and washed to obtain B7H3-CAR-modified T cells.

[0066] Furthermore, the mature antigen-presenting cells and the obtained CAR-modified T cells are co-cultured in vitro to obtain the composition.

[0067] Furthermore, the mature antigen-presenting cells and the obtained B7H3-CAR-modified T cells are co-cultured in vitro to obtain the composition.

[0068] Furthermore, mature antigen-presenting DC cells and CAR-modified T cells are mixed at a ratio of 1:3-100, placed in a culture flask, and culture medium is added. The cells are incubated in a 37°C, 5% CO2 incubator. The next day, anti-human CD3 monoclonal antibody, anti-human CD28 monoclonal antibody, and recombinant human IL-2 are added and culture is continued. Then, according to the cell growth and expansion, individualized expansion medium containing recombinant human IL-2 is added in a timely and appropriate amount for expansion culture. After culture for 7-14 days, the cell suspension is collected and centrifuged and washed twice, and a sample is retained for quality control. The obtained cell suspension is resuspended in 100 ml of physiological saline at a certain concentration, and 0.5-1% human serum albumin is added for intravenous drip. Alternatively, 30 ml or 2 ml is resuspended according to clinical needs for injection into the human chest cavity, peritoneal cavity, or local injection into some tumors.

[0069] Furthermore, mature antigen-presenting DC cells and B7H3-CAR-modified T cells are mixed at a ratio of 1:3-100, placed in a culture flask, and culture medium is added. The cells are incubated in a 37°C, 5% CO2 incubator. The next day, anti-human CD3 monoclonal antibody, anti-human CD28 monoclonal antibody, and recombinant human IL-2 are added and culture is continued. Then, according to the cell growth and expansion, individualized expansion medium containing recombinant human IL-2 is added in a timely and appropriate amount for expansion culture. After culture for 7-14 days, the cell suspension is collected and centrifuged and washed twice, and a sample is retained for quality control. The obtained cell suspension is resuspended in 100 ml of physiological saline at a certain concentration, and 0.5-1% human serum albumin is added for intravenous drip. Alternatively, 30 ml or 2 ml is resuspended according to clinical needs for injection into the human chest cavity, peritoneal cavity, or local injection into some tumors.

[0070] The present invention also includes the use of the chimeric antigen receptor, the nucleic acid molecule, the expression cassette, the recombinant expression vector, the recombinant virus, the recombinant cell, the composition or preparation, and the preparation method in preparing drugs or pharmaceutical compositions for treating tumor diseases.

[0071] The present invention also includes the application of the target-CAR enhanced tumor antigen effect composition to the tumor patient by reinfusion or local injection. The present invention can be applied to the anti-tumor treatment of various tumors.

[0072] Wherein, the tumor is a solid tumor, and the solid tumor includes one or more of gastric cancer, liver cancer, lung cancer, melanoma, esophageal cancer, colorectal cancer, 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, neuro-bladder cancer or prostate cancer.

[0073] In summary, the present invention first uses mature antigen-presenting cells to sensitize and activate T cells to obtain T cells that specifically kill tumors. The anti-human target scFv coding sequence is synthesized by chemical synthesis, and the coding gene of the CAR of the present invention is cloned into a lentiviral vector by fusion PCR and homologous recombination. The lentivirus is used to infect tumor-specific killing T cells, causing the cells to express the chimeric antigen receptor. Such T cells can not only target tumor cells that highly express the target protein, but also have the potential to activate intracellular signaling through CAR molecules, that is, they have a lasting killing effect only on tumor cells that express the target protein, reducing the risk of off-target in vivo.

[0074] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0075] 1. One advantage of the present invention is that it organically combines the respective advantages of tumor neoantigen technology and CAR-T technology. On the one hand, it uses the p-MHC presented by tumor neoantigens unique to tumor cells to provide the first activation signal for T cells. On the other hand, it uses the target scFv of CAR to recognize the target highly expressed on tumor cells and provide the second activation signal for T cells at the same time. This not only solves the side effects caused by off-target, but also enhances the effect of the patient's T cells in killing tumor cells.

[0076] 2. Another advantage of the present invention is that the intracellular region of the CAR is enriched with cytokine receptor binding elements in the cell through the structural design of the CAR. When the scFv of the extracellular region of the CAR binds to the antigen on the target cell, it activates the JAK / STAT signal transduction pathway in the T cell, promotes the differentiation and proliferation of cytotoxic T cells, and thus prevents the exhaustion of cytotoxic T cells. The advantages over existing similar CAR designs are: first, CAR does not secrete cytokines under the premise of initiating cytokine action, and will not cause excessive activation of other immune cells, thus avoiding side effects. Secondly, only the binding of the scFv of the cytotoxic T cell to the antigen of the tumor cell can stimulate the expansion of the cytotoxic T cell, so that the expansion of the cytotoxic T cell is concentrated only near the target cell, which not only enhances the effect of killing tumor cells, but also stops the expansion of the cytotoxic T cell once the tumor cells are cleared, ensuring safety in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 Schematic diagram of the CAR structure used to enhance the effect of tumor antigens.

[0078] Figure 2 This is the result of the DC cell isolation and culture characterization in Example 3. The DC cell purity reached over 99%.

[0079] Figure 3 This is the T cell characterization result after co-culture of DC cells loaded with tumor antigens and T cells in Example 7. The CD8+ ratio is over 40%.

[0080] Figure 4 This is the result of flow cytometry detection of the positive rate of CART cells in Example 8.

[0081] Figure 5 These are the ELISA test results of IL2 and IFN-γ release after co-culture of the CART cells sensitized with enhanced tumor antigens in Example 9 with target cells for 24 hours.

[0082] Figure 6 These are the anti-tumor effects of the tumor antigen-enhanced CART (CAR-E-Vac), tumor antigen-sensitized T cells (DC-T), unsensitized CART (CAR) cells, and unmodified T cells in PDX mice of Example 9. DETAILED DESCRIPTION

[0083] The present invention is a method and application of enhancing the effect of tumor antigen immunotherapy by CAR. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be noted that all similar replacements 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 make changes or appropriate changes and combinations to 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. 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. In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below with reference to specific embodiments.

[0084] Example 1 Target selection B7H3, construction of CAR vector (signal peptide-B7H3scFv-CD8αhinge-CD8TM-CD137-JAKAcS1)

[0085] Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize the nucleic acid sequence encoding the above-mentioned CAR, and the sequence is shown in SEQ ID No. 26.

[0086] Plvx-EF1α-IRES-ZsGreen1 and B7H3 CAR were linearized by double digestion with BamHI-MluI. The enzyme digestion system is as follows:

[0087]

[0088] 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.

[0089] Connect Plvx-EF1α-IRES-ZsGreen1 and B7H3 CAR system as follows:

[0090]

[0091] The transformation steps for ligating the recombinant product are as follows:

[0092] 1) Thaw 50 μL of competent cells XL1-Blue on ice for 5-10 minutes.

[0093] 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.

[0094] 3) Heat stress at 42°C for 45 seconds, then immediately place on ice for 2 minutes.

[0095] 4) Add 500 μL of LB medium without antibiotics and shake at room temperature for 1 h (270 rpm).

[0096] 5) Preheat the ampicillin-resistant LB solid culture medium in a 37°C incubator for 30 minutes.

[0097] 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.

[0098] 7) Incubate the plate upside down at 37°C for 16 hours.

[0099] Example 2 Lentiviral packaging of B7H3-CAR vector

[0100] The strain of Example 1 was extracted using the EndoFree Plasmid Mid plasmid extraction kit (Omega) to obtain the B7H3-CAR expression plasmid. The B7H3-CAR expression plasmid and packaging plasmids pSPAX and pMD2.G were transfected into HEK293 T cells at a ratio of 4:3:1 using the calcium phosphate method. Fresh culture medium was replaced 12 hours after transfection, and the virus supernatant was collected 24 hours and 48 hours later, centrifuged at 4°C, 3,000 rpm, for 15 minutes, filtered through a 0.45 μm filter, and concentrated by ultracentrifugation at 100,000g, 4°C, for 1.5 hours. The virus solution was frozen at -80°C for later use.

[0101] Example 3 Isolation and culture of DC cells from patient peripheral blood

[0102] 50 ml of fresh peripheral blood donated by ovarian cancer patients from Zhongda Hospital of Southeast University was collected and mononuclear cells were obtained by conventional density gradient centrifugation. 7 / cm 2 The mononuclear cells were inoculated into a culture flask and incubated in a 37°C, 5% carbon dioxide incubator for 2 hours.

[0103] The adherent cells after incubation were used for DC cell culture, the suspended cells were transferred to another culture flask for T cell culture, and the adherent cells were used as DC precursor cells.

[0104] RPMI1640 medium containing 10% FBS, recombinant human interleukin-4 (IL-4) at a final concentration of 1000 IU, and recombinant human granulocyte colony-stimulating factor (GM-CSF) at a final concentration of 1000 IU were added to the DC precursor cells. The DC precursor cells were induced and expanded in a 37°C, 5% CO2 incubator for 6 days. Immature DC cells were harvested and the DC cell purity reached more than 99% ( Figure 2 ).

[0105] Example 4: Isolation and culture of T cells from peripheral blood

[0106] 20 ml of fresh peripheral blood donated by the ovarian cancer patient in Example 3 was collected and PBMCs were isolated using Ficoll-Paque PLUS (GE Healthcare) (specific steps were as per the instructions). Anti-CD3 / CD28 magnetic beads (GibCo) were added at a cell:magnetic bead ratio of 1:1 and cultured for 24 hours to obtain T cells before transfection.

[0107] Example 5: Preparation of tumor cells and tumor antigens

[0108] The ovarian cancer tumor tissue sample was taken from the ovarian cancer patient in Example 3, and the size was about 5 cm. 3After 1 minute, remove fat, connective tissue and necrotic parts, wash three times with Hanks solution in a petri dish, mince the tissue and cut into small pieces of about 10 cubic millimeters. Use a cell disperser to prepare a single-cell suspension of tumor cells, count them, and set aside.

[0109] HClO solution (60 mM) was prepared by diluting NaClO reagent with HBSS and added to tumor cells. The final cell density was 8*10 5 Incubate with gentle shaking every 30 minutes for 1 hour to allow the tumor cells to fully oxidize and die.

[0110] Oxidative dead tumor cells were collected and washed twice with HBSS at a concentration of 1*10 7 Tumor cells / ml were resuspended in RPMI1640 medium containing 10% FBS, placed in dry ice for 20 minutes or in a -80°C refrigerator for 1 hour and then thawed at room temperature. This freeze-thaw cycle was repeated 6 times until the tumor cells were completely fragmented, and then tumor antigens of tumor cell oxidative lysates were prepared for use.

[0111] Example 6 Tumor antigen pulse activation of DC cells

[0112] The tumor cells obtained in Example 5 and the DC cells obtained in Example 3 were added to the DC cells in a ratio of approximately 1:1. At the same time, poly I-cell at a final concentration of 0.5 μg / ml and TNF-α maturation-promoting factors at a final concentration of 0.5 μg / ml were added. The cells were incubated at room temperature to 37°C for 10 hours to obtain mature tumor antigen-presenting DC cells.

[0113] Example 7 Tumor Antigen Sensitization of T Cells

[0114] The mature antigen-presenting DC cells obtained in Example 6 were mixed with the T cells obtained in Example 4 at a ratio of 1:50, and RPMI 1640 medium was added to a culture flask and incubated in a 37°C, 5% CO2 incubator. IL-2 (10 ng / mL) was added the next day and culture was continued. Then, RPMI 1640 basal medium containing 10% fetal bovine serum (FBS) with a final concentration of 10 ng / mL IL-2 was added every 48 hours for expansion culture. After culture for 5 days, the cell suspension was collected, anti-CD3 (5 μg / mL), anti-CD28 (1 μg / mL), and IL-2 (10 ng / mL) were added, and stimulation culture was continued for 3 days. Sensitized T cells were harvested, and the CD8+ cell ratio was 47.3% ( Figure 3 ).

[0115] Example 8 Lentivirus-Infected Sensitized T Cells and Their Culture

[0116] The sensitized T cells obtained in Example 7 were added to anti-CD3 / CD28 magnetic beads (GibCo) at a ratio of 1:1 using magnetic beads and cultured for 24 hours to obtain the T cells before infection.

[0117] The virus supernatant was taken out from -80℃ and thawed at room temperature. The virus titer was 5.6*10 7 , according to the virus infection multiplicity MOI = 5, add 100 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 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 incubator at 37°C for 14 days.

[0118] Flow cytometry was used to detect the positive rate of CART cells: cells were collected, labeled with rabbit anti-mouse IgG-F(ab')2 antibodies, and flow cytometry was used to analyze the expression of T cell F(ab')2 and GFP. The positive rate was 38.7% ( Figure 4 ).

[0119] Example 9: Enhanced Tumor Antigen-Inhibiting Effects of CART Cells

[0120] The tumor cells prepared in Example 5 were placed in each well at 10 5 The cells were inoculated into 48-well plates, and sensitized CART cells, unsensitized CART cells (i.e., T cells obtained by infecting T cells with the virus obtained in Example 2), sensitized T cells, and unmodified T cells were co-cultured at an effector-target ratio of 1:1. After 24 hours, the culture supernatant was taken and the co-culture supernatant was tested (the specific operation steps were carried out according to the instructions of the ELISA test kit). The results are as follows: Figure 5 As shown in the results, the levels of IL-2 and IFN-γ cytokines in the supernatant of co-culture of tumor cells and CART (CAR-E-Vac) were significantly higher than those of tumor antigen-sensitized T cells (DC-T) and unmodified T cells (P<0.001), and the killing effect of unsensitized CART (CAR) cells was equivalent to that of unmodified T cells. These results indicate that the design of CAR to enhance the effect of tumor antigens has achieved the expected purpose.

[0121] Figure 6These are the experimental results of detecting anti-tumor effects using a mouse transplant tumor model. Tumor antigen-enhanced CART (CAR-E-Vac) has a stronger tumor inhibitory effect than tumor antigen-sensitized T cells (DC-T) and unmodified T cells. The tumor inhibitory effect of unsensitized CART (CAR) cells is similar to that of unmodified T cells. The results show that the CAR designed by the present invention can only inhibit tumors when combined with tumor antigens, thereby enhancing in vivo safety.

[0122] Example 10

[0123] The JAKAcS1 in the sequence SEQ ID No. 26 in Example 1 was replaced with JAKAcS2, JAKAcS3, JAKAcS4, JAKAcS5, JAKAcS6, JAKAcS7, JAKAcS8, JAKAcS9, and JAKAcS10, respectively, to obtain the corresponding nucleotide sequences, which were then synthesized and operated according to the above specific steps to obtain the corresponding recombinant strains. The above experimental process was repeated according to the specific experimental methods of Examples 2-9 to obtain the corresponding CART cells. The positive rate of the above CART cells was tested, and the positive rate was between 30% and 50%, which also had the anti-tumor effect of enhancing the tumor vaccine.

Claims

1. A chimeric antigen receptor comprising a signal peptide, a target scFv, a hinge domain, a transmembrane domain, and an intracellular co-stimulatory domain, wherein: The chimeric antigen receptor also includes a JAK enzyme activation signal transduction domain, which can capture and activate the JAK enzyme. The JAK enzyme is JAK1 enzyme, the target is B7H3, the single-chain antibody nucleotide sequence of the target B7H3 is shown in SEQ ID No. 21, and the nucleic acid sequence of the chimeric antigen receptor is shown in SEQ ID No.

26.

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

3. An expression cassette, a recombinant expression vector, a recombinant virus or a recombinant cell comprising the nucleic acid molecule of claim 2.

4. A composition or cell preparation for enhancing the effect of a target-CAR on a tumor antigen, comprising the chimeric antigen receptor of claim 1, the nucleic acid molecule of claim 2, the expression cassette, recombinant expression vector, recombinant virus or recombinant cell of claim 3.

5. The method for preparing the target-CAR composition for enhancing tumor antigen effect according to claim 4, characterized in that: The following steps are involved: 1) Collect mononuclear cells from a peripheral vein or extract anticoagulated blood to isolate mononuclear cells, and then culture them to obtain antigen-presenting cells and T cells; 2) preparing tumor antigens, co-incubating the tumor antigens with antigen-presenting cells, and preparing mature tumor antigen-presenting cells under the action of maturation-promoting factors; 3) Sensitizing T cells with mature tumor antigen-presenting cells, and then introducing the nucleic acid molecule described in claim 2 or the recombinant expression vector described in claim 3 into the sensitized T cells to obtain the target-CAR composition for enhancing the effect of tumor antigens; or first introducing the nucleic acid molecule described in claim 2 or the recombinant expression vector described in claim 3 into T cells to obtain target-CAR-modified T cells, and then sensitizing the CAR-modified T cells with mature tumor antigen-presenting cells to obtain the target-CAR composition for enhancing the effect of tumor antigens.

6. The preparation method according to claim 5, characterized in that The tumor antigen is derived from the patient's own tumor tissue lysate, or tumor cell lysate cultured from tumor tissue, or one or more of personalized tumor neoantigen peptides prepared by autologous gene sequencing and bioinformatics prediction.

7. The method for preparing the composition according to claim 5, wherein The tumor antigens described in step 2) are co-incubated with DC cells to sensitize and activate DC cells, and maturation-promoting factors are added to obtain mature tumor antigen-presenting DC cells, which are then used to stimulate and activate T cells or CAR-modified T cells.

8. Use of the chimeric antigen receptor according to claim 1, the nucleic acid molecule according to claim 2, the expression cassette, recombinant expression vector, recombinant virus or recombinant cell according to claim 3, the composition or preparation according to claim 4, or the preparation method according to any one of claims 5 to 7 in preparing a medicament or pharmaceutical composition for treating a tumor disease, wherein the tumor is a solid tumor, and the solid tumor is one or more of gastric cancer, liver cancer, lung cancer, melanoma, esophageal cancer, colorectal cancer, ovarian cancer, kidney cancer, glioma, bone cancer, pancreatic cancer, breast cancer, malignant mesothelioma, thyroid cancer, cervical cancer or prostate cancer.

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