Pharmaceutical composition for improving treatment effect of CAR-T (chimeric antigen receptor T) cells

Abstract

The invention relates to the field of biological pharmacy, in particular to a medicine composition for improving the treatment effect of CAR-T cells. The invention provides a composition and application of the composition in preparation of medicines for treating cancers. The composition comprises the chemotherapeutic drug and the CAR-T cells, the chemotherapeutic drug and the CAR-T cells are administered according to a specific sequence, tumor can be effectively resisted, and the treatment effect of the CAR-T cells is improved.

Description

Technical Field

[0001] This application relates to the field of biopharmaceuticals, and in particular to drug combinations that enhance the efficacy of CAR-T cell therapy. Background Technology

[0002] Chimeric antigen receptor T (CAR-T) cell therapy involves genetically modifying patient or donor T lymphocytes in vitro to express tumor-specific antigen receptors. These receptors bind to specific antigens on the surface of tumor cells, exerting potent anti-tumor activity through the secretion of cytotoxic cytokines and the induction of immune responses. While CAR-T cell therapy has achieved excellent efficacy in hematologic therapy, breakthroughs are still needed in the treatment of solid tumors.

[0003] Solid tumors, such as lung cancer, breast cancer, and liver cancer, differ significantly from hematologic malignancies in their biological characteristics and treatment response. The microenvironment of solid tumors is complex, containing numerous immunosuppressive cells, stromal cells, and a dense vascular network. These factors collectively constitute a natural barrier for CAR-T cell infiltration and function. Furthermore, solid tumor cells exhibit high antigenic heterogeneity; even within the same type of tumor, tumor cells from different patients or different sites within the same patient may express different antigens, making it difficult for single-target CAR-T cells to comprehensively cover all tumor cells. Moreover, solid tumor cells often develop various "immune escape" mechanisms, such as reducing antigen expression and secreting immunosuppressive factors, to evade the immune system's attack. Therefore, new cell therapy products suitable for solid tumors are needed.

[0004] Currently, researchers are actively exploring various innovative strategies to develop CAR-T cell therapy products suitable for solid tumors. For example, combining immune checkpoint inhibitors, cytokines, oncolytic viruses, targeted drugs, or radiotherapy can effectively improve the tumor microenvironment, promote CAR-T cell infiltration and proliferation, and enhance therapeutic efficacy. Furthermore, local administration methods, such as direct injection of CAR-T cells into the tumor site, can reduce CAR-T cell loss in circulation and increase their concentration and activity in tumor tissue. There are also reports of using DNA demethylating agents to reprogram CAR T cells, significantly increasing the CD4 / CD8 ratio in CAR T cells without affecting the expression of the corresponding CAR, thereby promoting the tumor-killing ability of CAR-T cells; however, this report was still validated using hematologic malignancies.

[0005] Therefore, there is still an urgent need to develop cell therapy products suitable for solid tumors. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a drug combination that improves the efficacy of CAR-T cell therapy.

[0007] The drug combination for improving the effectiveness of CAR-T cells provided by the present invention includes at least one of decitabine, azacitidine, or chidamide.

[0008] In some embodiments, the drugs that enhance CAR-T efficacy are decitabine, azacitidine, or chidamide, or the drug combination consists of azacitidine and chidamide, or decitabine and chidamide.

[0009] The present invention also provides the use of the drug combination as described above in the preparation of formulations that enhance the efficacy of CAR-T.

[0010] In this invention, improving CAR-T efficacy includes infusing CAR-T cells after administering any one of the drugs in the drug combination.

[0011] In this invention, after the CAR-T cells are reinfused, the procedure further includes administering any one of the drugs from the drug combination again.

[0012] In some embodiments, the method includes sequentially administering decitabine and CAR-T cells;

[0013] In other embodiments, the method includes sequentially administering decitabine, CAR-T cells, and chidamide;

[0014] In other embodiments, the method includes sequentially administering azacitidine, CAR-T cells, and chidamide.

[0015] The composition provided by this invention includes chemotherapy drugs and CAR-T cells;

[0016] The chemotherapy drug is selected from at least one of decitabine, azacitidine, or chidamide.

[0017] For example, the composition includes decitabine and CAR-T cells;

[0018] Alternatively, the composition may include azacitidine and CAR-T cells;

[0019] Alternatively, the composition may include chidamide and CAR-T cells;

[0020] Alternatively, the composition may include decitabine, CAR-T cells, and azacitidine;

[0021] Alternatively, the composition may include decitabine, CAR-T cells, and chidamide;

[0022] Alternatively, the composition may include azacitidine, CAR-T cells, and decitabine;

[0023] Alternatively, the composition may include azacitidine, CAR-T cells, and chidamide;

[0024] Alternatively, the composition may include chidamide, CAR-T cells, and decitabine;

[0025] Alternatively, the composition may include chidamide, CAR-T cells, and chidamide.

[0026] This invention combines chemotherapy drugs and CAR-T cells for the treatment of tumors, and has shown good therapeutic effects, especially on solid tumors.

[0027] Based on this, the present invention provides the use of the composition as described above in the preparation of a medicament for treating cancer.

[0028] The experiments of this invention have found that the order of administration of decitabine (DAC) or HDAC inhibitors to CAR-T has an impact on the final anti-tumor efficacy. Compared with other regimens, decitabine (DAC) administered before CAR-T infusion can better increase the efficacy of CAR-T. On this basis, administering HDAC inhibitors after CAR-T infusion can have a better effect.

[0029] In some embodiments, the composition is decitabine and CAR-T cells; the treatment includes administering decitabine followed by infusion of CAR-T cells.

[0030] In some specific embodiments, the treatment includes the following steps:

[0031] Decitabine should be used continuously for 3-6 days.

[0032] Discontinue administration for 1-3 days;

[0033] CAR-T cells were reinfused.

[0034] In this embodiment, decitabine is administered before CAR-T infusion. Compared to regimens that administer chidamide or azacitidine before CAR-T infusion, the decitabine infusion regimen is more effective in inhibiting tumor proliferation. Preferably, decitabine is administered for 5 to 6 days, for example, for 5 or 6 days. Preferably, CAR-T cells are infused 1, 2, or 3 days after decitabine discontinuation, for example, CAR-T cells are infused 3 days after decitabine discontinuation.

[0035] In this invention, administering an HDAC inhibitor after CAR-T cell infusion can achieve better results. Preferably, the HDAC inhibitor is chidamide.

[0036] In some embodiments, the composition is decitabine, CAR-T cells, and chidamide; the treatment includes administering decitabine first, then reinfusing CAR-T cells, and finally administering chidamide.

[0037] In some specific embodiments, the treatment includes the following steps:

[0038] Decitabine should be used continuously for 3-6 days.

[0039] CAR-T cells were reinfused.

[0040] Administer Chidamide 1-3 days later.

[0041] More specifically, decitabine is administered for 5 to 6 consecutive days, for example, decitabine is administered for 5 or 6 days. Preferably, CAR-T cells are infused 1, 2, or 3 days after decitabine discontinuation, for example, CAR-T cells are infused 3 days after decitabine discontinuation. Preferably, chidamide is administered twice a week, or for five consecutive days.

[0042] In some embodiments, the composition is azacitidine, CAR-T cells, and chidamide; the treatment includes administering azacitidine first, then reinfusing CAR-T cells, and finally administering chidamide.

[0043] In some specific embodiments, the treatment includes the following steps:

[0044] Azacitidine was administered continuously for 5-6 days.

[0045] CAR-T cells were reinfused.

[0046] Administer Chidamide 1-3 days later.

[0047] More specifically, azacitidine is administered for 5 to 6 consecutive days, for example, for 5 or 6 days. Preferably, CAR-T cells are infused 1, 2, or 3 days after azacitidine discontinuation, for example, CAR-T cells are infused 3 days after azacitidine discontinuation. Preferably, chidamide is administered twice a week with a 2-day interval for three consecutive weeks, or for five consecutive days.

[0048] In an embodiment of the present invention, the dose of decitabine (DAC) administered to mice is equivalent to 1 mg / kg of chidamide, which translates to a dose of 20 mg / m² for humans. 2 -40mg / m 2 As a preferred method, the dosage of decitabine is 40 mg / m². 2 .

[0049] In this embodiment of the invention, the dosage of azacitidine is 1 μM to 5 μM; preferably, for example, the dosage of azacitidine is 1 μM, 2.5 μM, or 5 μM. In this embodiment of the invention, the dosage of azacitidine (AZA) given to mice is 2-2.5 mg / kg, preferably 2.5 mg / kg, which is equivalent to a human dosage of 75 mg / kg. 2 -100mg / m 2 Preferably, the dosage of azacitidine is 100 mg / m². 2 As a preferred option, the dosage of azacitidine is 93.75 mg / m². 2 .

[0050] In this embodiment of the invention, the dosage of chidamide is 10 mg / kg or 40 mg / m². 2 .

[0051] In this embodiment of the invention, the cancer is a solid tumor. The solid tumors in this invention include: colorectal cancer, pancreatic head cancer, cervical cancer, ovarian cancer, endometrial cancer, vulvar cancer, prostate cancer, parotid gland cancer, thyroid cancer, salivary gland cancer, lung cancer, fibrosarcoma, skin cancer, nasopharyngeal carcinoma, tongue cancer, oral cavity cancer, laryngeal cancer, brain tumor, gallbladder cancer, kidney cancer, gingival cancer, testicular cancer, penile cancer, multiple myeloma, and / or malignant melanoma.

[0052] In this embodiment of the invention, the cancer treatment includes inhibiting tumor cell proliferation, inhibiting tumor cell volume, improving patient survival rate, and / or prolonging patient survival time.

[0053] The present invention also provides a method for treating cancer, comprising administering drugs and CAR-T cells according to the protocol described above.

[0054] This invention provides a composition and its use in the preparation of a medicament for treating cancer. The composition comprises a chemotherapeutic agent and CAR-T cells, wherein the chemotherapeutic agent and CAR-T cells are administered in a specific sequence to more effectively fight tumors and improve the therapeutic effect of CAR-T cells. Attached Figure Description

[0055] Figure 1 : Expression of CEA in colorectal cancer cell lines after treatment with different concentrations of DAC;

[0056] Figure 2 The killing effect of the combination of DAC and CAR-T on tumors in vivo;

[0057] Figure 3 The effect of DAC on CEA protein expression in tumor cells with decreased CEA expression after CAR-T therapy;

[0058] Figure 4The effect of DAC on the intensity of CEA protein in tumor cells with decreased CEA expression after CAR-T therapy;

[0059] Figure 5 Effect of DAC on CEA gene expression in tumor cells. The horizontal axis represents time, specifically day 1, day 6, day 10, and day 15, with day 6 being the peak.

[0060] Figure 6A : Expression rate of CEA after treatment with different concentrations of CHI in colorectal cancer cell lines;

[0061] Figure 6B : Expression rate of CEA after treatment with different concentrations of AZA in colorectal cancer cell lines;

[0062] Figure 6C The expression intensity of CEA in colorectal cancer cell lines after treatment with different concentrations of AZA or CHI;

[0063] Figure 7A Tumor fluorescence images after treatment with different drug combinations;

[0064] Figure 7B Tumor proliferation curves after treatment with different drug combinations;

[0065] Figure 8A Tumor fluorescence images after treatment with different drug combinations;

[0066] Figure 8B Tumor proliferation curves after treatment with different drug combinations. Detailed Implementation

[0067] This invention provides a drug combination to improve the efficacy of CAR-T cell therapy. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired effect. 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. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0068] the term

[0069] Adoptive cell therapy (ACT) is a treatment method that uses cells from the body's own immune system, which are cultured and modified externally before being reinfused into the body to eliminate disease.

[0070] Chimeric antigen receptors (CARs): A CAR is a group of engineered peptides or proteins that, when present in immune effector cells, bind to specific antigens on target cells and generate intracellular signals upon recognition of the antigen, activating downstream pathways in the cell containing the receptor to initiate the killing action of the immune effector cells on the target cells. CARs typically include at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain. The extracellular antigen-binding domain specifically recognizes antigens. Non-limiting examples include single-chain variable fragments (scFv) derived from antibodies, fragment antigen-binding regions (Fab) selected from libraries, single-domain fragments or natural ligands that bind to their homologous receptors, artificially designed target-specific recognition domains such as fibronectin type III (FN3) domain combinations, and designed ankyrin repeat proteins (DARPins) that recognize specific targets.

[0071] The "signal transduction domain" typically contains an immune-receptor tyrosine-based activation motif (ITAM), whose basic composition is YXXL / V. Here, Y represents tyrosine, L / V refers to leucine or valine, and X can be any amino acid. When the receptor binds to its corresponding ligand, the tyrosine residue in the ITMA is phosphorylated by a protein tyrosine kinase (PTK) linked to the cell membrane. This phosphorylation recruits other free intracellular protein kinases or adaptor proteins to transduce activation signals into the cell.

[0072] In some implementations, the "signal transduction domain" is selected as an intracellular signal transduction domain of TCRζ (CD3ζ) or FcεRIγ.

[0073] As used herein, the “co-stimulatory domain” is also referred to as the “co-stimulatory signaling domain”, primarily used to provide co-stimulatory signals to enhance the capabilities of immune cells, including, for example, enhancing the proliferation, survival, and / or development of memory cells. In some embodiments, the “co-stimulatory domain” is selected from intracellular domains of CD28, 4-1BB (CD137), OX40 (CD134), etc.

[0074] As used herein, a "transmembrane domain," also known as a "transmembrane region," refers to a thermodynamically stable protein structural region anchored within the cell membrane. Transmembrane domains can be obtained from natural proteins, such as transmembrane domains derived from the T-cell receptor (TCR). In some embodiments, the transmembrane domain is selected from the transmembrane domains of CD4, CD8α, CD28, and CD3ζ.

[0075] In some embodiments, the extracellular antigen-binding region of the "chimeric antigen receptor" or "CAR" structure can recognize target molecules expressed on the surface of solid tumor or hematologic malignancy cells / tissues, said target molecules including, but not limited to: CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, T rop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), or TAG-72, etc.

[0076] "Chimeric antigen receptors" or "CARs" can have various structures, such as those containing secretible or membrane-expressed cytokines or antibody gene sequences; and those containing structures that can be regulated for activation or inactivation, including: suicide switches such as inducible caspase-9 (iCasp9), thymidine kinase (HSV-TK) and suicide epitopes in herpes simplex virus, truncated EGFR (EGFRt), and Fas-FasL apoptosis structures; and inducible CAR structures such as: peptide neo-epitope (PNE), fluorescein (FITC), 10 amino acids (5B9 tag), FITC-HM-3 bifunctional molecule (FHBM) and scFv, leucine ZipFv linked to antibody, streptavidin 2 (mSA2) biotin-binding domain, VIPER CAR inducible structures, and biotin-biding immune receptors. The receptor (BBIR) system; the "logic gate" regulatory system that binds to the SynNotch receptor, etc.

[0077] Decitabine (DAC): also known as 5-aza-2'-deoxycytidine, is an adenosine analog of natural 2'-deoxycytidine. It inhibits the proliferation of tumor cells and prevents the development of drug resistance by inhibiting DNA methyltransferases and reducing DNA methylation.

[0078] Azacitidine (AZA): Also known as 5-azacytidine nucleoside or 5-azacytidine, it is an organic compound with the chemical formula C8H. 12 N4O5 is primarily used as an antitumor drug. It can directly integrate into DNA, inhibiting DNA and RNA synthesis and killing cells in the S phase. Its antitumor effect is achieved by inducing DNA demethylation and through direct cytotoxic effects on abnormal hematopoietic cells in the bone marrow.

[0079] HDAC inhibitors (HDACi, histone deacetylase inhibitors) are compounds that have the potential to inhibit cell proliferation and induce apoptosis. They are currently used clinically to treat various tumors. HDAC inhibitors are related to Zn... 2+ The chemical structures of the bound functional groups can be classified into four main categories: hydroxamic acids, cyclic tetrapeptides, short-chain fatty acids, and benzamides. Chidamide (CHI) described in this application is an HDAC inhibitor.

[0080] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0081] There are various CAR-T preparation methods. CAR-T cell preparation involves T cell activation, gene modification, and T cell expansion. In some embodiments, activation and gene modification can be performed separately; in others, they can be performed simultaneously. Those skilled in the art know that the steps of T cell activation and gene modification do not affect the technical problem solved or the technical effect obtained by the present invention. In the following embodiment, taking the CEA target as an example, the CAR-T preparation method is as follows: T cell activation is performed within 12-24 hours, and the activated T cells are transduced using a lentiviral vector to target the CAR gene. This example illustrates the effect of this embodiment. The CEA CAR structure targeting the CEA target involves the following amino acid sequence: The CEA CAR sequence is as follows:

[0082] DIQMTQSPSSSLSASVGDRVTITCSTSSSSVSYMHWYQQKPGKAPRLLIYSTSNLASGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQWSSYPTFGQGTKVEIKGSTSGSGKPGSG EGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFTISSGYSWHWVRQPPGRGLEWIGYIQYSGITNYNPSLKSRVTMLVDTSKNQFSLRLSSVTAADTAVYYCAREDYDYHWYFDVWGQ GSTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO.1).

[0083] The present invention will be further illustrated below with reference to the embodiments:

[0084] Example 1: CAR-T preparation

[0085] Mononuclear cells were isolated or T cells were generated from mononuclear cells through sorting (or cells that were thawed after cryopreservation). The T cells were activated using CD3 / CD28 antibody-conjugated magnetic beads (Dynabeads™ CD3 / CD28, 40203D). After activation, CEA CAR-T cells were prepared by transducing a lentiviral vector targeting CEA. The cells were then cultured in a combination of 5 ng / ml IL-7 and 25 ng / ml IL-21 cytokines for 7–21 days before harvesting.

[0086] The specific product codes for the above factor combinations are: IL-2 (human interleukin-2 for injection, Shandong Gangquan Pharmaceutical), IL-7 (Recombinant Human IL-7, AF-200-07), and IL-21 (Recombinant Human IL-21, AF-200-21). The basal culture medium is SCGM medium from CellGenix, Germany, 20802-0500.

[0087] According to the above preparation method, CAR-T cells cultured for 11 days were collected, the cells were resuspended in cell cryopreservation solution, and the resuspended cells were cryopreserved at ultra-low temperature.

[0088] Example 2: Decitabine can increase the expression of CEA molecules in colorectal cancer cells.

[0089] LOVO colorectal cancer cell line was cultured in 1640 medium supplemented with 10% fetal bovine serum (FBS). After 3 days of culture, LOVO cells were collected, centrifuged to remove the supernatant, and 1E6 cells were cultured in 1640 medium supplemented with 10% FBS containing 10 nM, 100 nM, 500 nM, and 1000 nM DAC, respectively. After 3 days of culture, cells were collected, centrifuged to remove the supernatant, resuspended in PBS, and labeled with Invitrogen anti-CEA antibody 2604441. After incubation at 4°C for 30 minutes, 1 ml of PBS was added, centrifuged, and the supernatant was discarded for two rounds of washing. After resuspending in 100 μL of PBS, Invitrogen IgG secondary antibody labeled with 647 fluorescently was incubated at 4°C for 30 minutes, 1 ml of PBS was added, centrifuged, and the supernatant was discarded for two rounds of washing. Flow cytometry was then performed.

[0090] The results are as follows Figure 1 As shown, the horizontal axis represents the concentration of DAC added, where vehicle refers to LOVO cells cultured in 10% FBS with 100 nM glycerol (DMSO, the medium for dissolving DAC, serving as a negative control), and the vertical axis represents the intensity of CEA expression, denoted by MFI.

[0091] The results showed that DAC (decitabine) of 10 nM-1000 nM could promote the expression intensity of CEA in LOVO colorectal cancer cell line, and the effect of 100 nM-500 nM on the enhancement of CEA expression intensity was more significant.

[0092] Example 3: Decitabine used before CAR-T infusion can enhance the efficacy of CAR-T therapy.

[0093] Six- to eight-week-old female NCG mice were subcutaneously (sc) inoculated with LOVO-Luc-GFP (human colorectal cancer cells) tumor cells expressing CEA at a dose of 1.0E+06 per mouse to establish a subcutaneous solid tumor model. Mice were divided into four groups: CT group (infused with T lymphocytes without CAR transduction structure), CAR-T group (infused with CEA CAR-T cells only), DAC+CAR-T group (two weeks after tumor bearing, mice were given 1 mg / kg DAC for 5 consecutive days, followed by CAR-T infusion 3 days after drug withdrawal), and CAR-T+DAC group (two weeks after tumor bearing, mice were infused with CAR-T cells, followed by 1 mg / kg DAC for 5 consecutive days 3 days after CAR-T cell infusion). CAR-T cell resuscitation in Example 1 was performed, with a re-infusion dose of 2.0E+06 CAR+ per mouse via subcutaneous infusion, following the above protocol. In vivo imaging was conducted weekly to compare the in vivo anti-tumor effect of CAR-T cells. Blood samples were collected from mice, and RT-PCR was used to detect the survival of CAR-T cells in the blood.

[0094] The results are as follows Figure 2 As shown, the horizontal axis represents the number of days since CAR-T infusion, and the vertical axis represents the tumor volume (mm). 3 The results showed that DAC can enhance the efficacy of CAR-T therapy, and that DAC administered before CAR-T infusion resulted in more significant efficacy.

[0095] Example 4: Decitabine can promote the expression of CEA in tumor cells that escape after CAR-T therapy.

[0096] In Example 3, the tumors of mice 2104 that had not been completely eliminated were isolated and tested using the method in Example 2. It was found that CEA expression was significantly lower than that of LOVO cells bearing tumors.

[0097] The isolated tumor cells were cultured in 1640 medium containing 10% FBS with added amounts of 10 nM, 50 nM, 100 nM, and 500 nM decitabine, respectively. After 6 days of culture, CEA expression was detected using the protocol described in Example 2. The results are as follows... Figure 3 and Figure 4 As shown, Figure 3 The x-axis represents different DAC addition groups, and the y-axis represents the expression rate of CEA. Figure 4 The horizontal axis represents different DAC addition groups, and the vertical axis represents the expression abundance of CEA.

[0098] The results showed that CEA expression in tumor cells was significantly reduced after CAR-T therapy. However, this reduction was a tumor cell "escape" mechanism, where tumor cells regulated and reduced CEA expression to avoid CAR-T killing. After treatment with DAC (50 nM-500 nM), the regulation of CEA expression in tumor cells could be restored, and the expression intensity and abundance of CEA could be increased.

[0099] The day DAC ended was designated D0. Tumor samples were collected on D1, D6, D10, and D15. The samples were homogenized using TRIzol reagent lysis buffer to separate RNA from proteins, releasing the RNA into solution. RNA was further extracted using chloroform or 1-bromo-3-chloropropane. The extracted RNA was reverse transcribed using reverse transcriptase to obtain cDNA for qPCR. 1 μg of RNA was added to HiScript® III All-in-one RT SuperMix to prepare the qPCR system. The cDNA was diluted to 200 μl, mixed, and 4.5 μl was added to a 96-well PCR plate containing the qPCR system.

[0100] Primer sequence: CEA-174-F: CTTATCGCCAAAATCACGC,

[0101] CEA-174-R:CCAACCAGCACTCCAATCAT,

[0102] The internal reference gene was GAPDH. After centrifugation for 20 seconds in a microcentrifuge, the sample was placed in a real-time PCR instrument and the relative expression fold was obtained based on the Ct value and the relative quantification formula 2^-△△Ct.

[0103] The results are as follows Figure 5 As shown, the horizontal axis represents the number of days of DAC stimulation, and the vertical axis represents gene expression. The results show that after treatment with DAC: 500 nM, the regulation of CEA expression in tumor cells can be restored, and the expression of the CEA gene can be increased.

[0104] Example 5: Chidamide (CHI) and azacitidine (AZA) can promote the expression of CEA in colorectal cancer cells.

[0105] Using the culture and detection protocol of Example 2, cells were cultured for 3 days (72 hours) with medium supplemented with 0.125 μM, 0.25 μM, and 0.5 μM chidamide (CHI) or 1 μM, 2.5 μM, and 5 μM AZA, respectively. Cells were then collected for CEA expression detection. The results are as follows: Figures 6A-6C As shown, Figure 6A To investigate the effect of different CHI concentrations on CEA expression rate in colorectal cancer cells. Figure 6BTo investigate the effect of different AZA concentrations on CEA expression rate in colorectal cancer cells. Figure 6C The effect of different concentrations of CHI or AZA on the expression abundance (MFI) of CEA in colorectal cancer cells is shown. The horizontal axis represents different concentrations of CHI or AZA, and the vertical axis represents the expression rate or expression abundance.

[0106] Example 6: The combination of Chidamide (CHI) and DAC can enhance the efficacy of CAR-T therapy.

[0107] The in vivo experimental protocol of Example 3 was used, with the following groups: CT group (infusion of T lymphocytes without CAR transduction structure), CAR-T group (infusion of CEA CAR-T cells only), DAC+CAR-T group (two weeks after tumor bearing, mice were given 1 mg / kg DAC for 5 consecutive days, followed by CAR-T infusion 3 days after stopping administration), CAR-T+CHI group (two weeks after tumor bearing, mice were given 10 mg / kg CHI twice a week with a 2-day interval, for three consecutive weeks), CHI+CAR-T group (two weeks after tumor bearing, mice were given 10 mg / kg CHI twice a week with a 2-day interval, followed by CAR-T infusion 3 days after stopping administration), CHI+CAR-T+CHI group (two weeks after tumor bearing, mice were given 10 mg / kg CHI twice a week with a 2-day interval, followed by CAR-T infusion 3 days after stopping administration, followed by 10 mg / kg CHI infusion 3 days after CAR-T infusion). CHI (twice a week with a two-day interval, for three consecutive weeks) and DAC+CAR-T+CHI group (two weeks after tumor bearing, mice were given 1 mg / kg DAC for 5 consecutive days, CAR-T was reinfused 3 days after stopping the administration, and CAR-T was reinfused 3 days after CAR-T was reinfused, mice were given 10 mg / kg CHI twice a week with a two-day interval, for three consecutive weeks).

[0108] The results are as follows Figure 7A and 7B As shown, Figure 7A Fluorescence imaging images of mouse tumors under different treatment regimens. Figure 7B This is the tumor proliferation curve.

[0109] The results showed that the DAC+CAR-T group significantly improved the in vivo efficacy of CAR-T, and the DAC+CAR-T+CHI group exhibited even better in vivo efficacy. Furthermore, CHI should be administered after CAR-T; administering it before CAR-T may actually negatively impact the efficacy of CAR-T.

[0110] Example 7: The combination of chidamide (CHI) and DAC can enhance the therapeutic effect of pancreatic head cancer.

[0111] Besides colorectal cancer, pancreatic head cancer also expresses CEA. We constructed a PDX mouse model using pancreatic head cancer patient samples: human pancreatic head cancer tissue was transplanted into tumors via PDX, and the tumor volume was increased to 100 mm. 3 When the time is right, administer the medication (DAC / AZA is administered intraperitoneally before CAR-T infusion, with DAC at 1 mg / kg and AZA at 2.5 kg / mg for 5 consecutive days. After CAR-T infusion, administer 10 mg / kg Chi twice a week with a 2-day interval, for 3 consecutive weeks).

[0112] The results are as follows Figures 8A-8B As shown in Figure 8, the tumor volume growth curve is... Figure 8B The study focused on CAR-T copy number detection. Results showed that the combination of DAC / AZA and Chi with CAR-T enhanced the in vivo anti-tumor effect of CAR-T. The combination of DAC and Chi significantly stimulated CAR-T proliferation in vivo and exhibited better tumor growth inhibition.

[0113] According to the formula provided on page 71 of the Pharmacological Experimental Methodology textbook, the dosage of a drug for human use can be converted from the dosage in mice as described above. The conversion formula is: Human dosage (mg / m²). 2 )*8% / 3mg / kg.

[0114] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A pharmaceutical combination for improving the effectiveness of CAR-T cells, comprising at least one of decitabine, azacitidine or chidamide.

2. The pharmaceutical combination according to claim 1, characterized in that, consisting of azacitidine and chidamide, or consisting of decitabine and chidamide.

3. Use of the pharmaceutical combination of claim 1 or 2 in the preparation of a preparation for improving the effectiveness of CAR-T cells.

4. Use according to claim 3, characterized in that, The improvement of the effectiveness of CAR-T cells comprises reinfusing CAR-T cells after administering any of the pharmaceutical combinations of claim 1 or 2.

5. Use according to claim 4, characterized in that, The reinfusion of CAR-T cells further comprises the step of administering any of the pharmaceutical combinations of claim 1 or 2 again.

6. A composition comprising the pharmaceutical combination of claim 1 or 2 and CAR-T cells.

7. Use of the composition of claim 6 in the preparation of a medicament for treating cancer.

8. The use of claim 7, wherein, the composition is decitabine and CAR-T cells; and the treatment comprises administering decitabine first and then reinfusing CAR-T cells.

9. Use according to claim 8, characterized in that, the treatment comprises the following steps: administering decitabine continuously for 3-6 days, stopping administration for 1-3 days; and reinfusing CAR-T cells.

10. The use of claim 7, wherein, the composition is decitabine, CAR-T cells and chidamide; and the treatment comprises administering decitabine first and then reinfusing CAR-T cells and finally administering chidamide.

11. Use according to claim 10, characterized in that, the treatment comprises the following steps: administering decitabine continuously for 3-6 days, reinfusing CAR-T cells, administering chidamide 1-3 days later.

12. The use of claim 7, wherein, the composition is azacitidine, CAR-T cells and chidamide; and the treatment comprises administering decitabine first and then reinfusing CAR-T cells and finally administering chidamide.

13. Use according to claim 12, characterized in that, the treatment comprises the following steps: administering azacitidine continuously for 5-6 days, reinfusing CAR-T cells, administering chidamide 1-3 days later.

14. The use according to any one of claims 7 to 13, characterized in that, the cancer is a solid tumor.

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