CN113663076A

CN113663076A - Cancer combination treatment composition

Info

Publication number: CN113663076A
Application number: CN202010416770.XA
Authority: CN
Inventors: 刘�文; 夏琳; 郑早早; 刘珺懿; 陈宇洁
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2020-05-15
Filing date: 2020-05-15
Publication date: 2021-11-19
Anticipated expiration: 2040-05-15
Also published as: CN113663076B

Abstract

The invention discloses a cancer combination therapy composition, the effective components comprise CAR-T cells and epigenetic regulators, wherein the CAR-T cells can recognize at least one cancer specific antigen or cancer associated antigen; an epigenetic modulator comprising at least one of a CDK7 inhibitor and a BRD4 inhibitor. The treatment modalities of CAR-T in combination with epigenetic modulators of the invention have broader suppression of the tumor microenvironment than CAR-T in combination with inhibitors or antibodies that block a single immune escape pathway, while also being easier to manipulate and implement than other approaches that block multiple immune escape pathways in CAR-T therapy.

Description

Cancer combination treatment composition

Technical Field

The invention belongs to the technical field of cancer treatment, and particularly relates to a cancer combination treatment composition.

Background

Over the past few decades, immunotherapy has proven to be an effective method of cancer treatment and has become an important component of many treatment regimens, providing new hopes for better treatment. Immunotherapy is generally divided into two categories, the "active" immunotherapy and the "passive" immunotherapy. Among them, the "active" immunotherapy is the enhancement of the activation of the immune system by modulating endogenous immune mechanisms (regulation and/or activation mechanisms), including cancer vaccines and the like. "passive" immunotherapy is the direct attack of cancer cells by effector cells/molecules of the immune system, and includes antibody-targeted therapies and their derivatives (e.g., antibody-drug conjugates), cytokine therapies, and adoptive immune cell therapies (e.g., genetically engineered CAR-T or TCR-T). Several immunotherapies have been FDA approved for some cancer treatments in recent years, the most representative examples being the DC therapy-based product Provenge (sipuleucel-T) approved in 2010, the CTLA-4 antibody yervoy (ipilimumab) approved in 2011, the PD-1 antibody keytruda (pembrolizumab) approved in 2014, and the opdivo (nivolumab), the PD-L1 antibody tecentiq (atezolizumab) approved in 2016, and the targeted CD19 CAR-T products kymeriah and ycarta approved in 2017. CAR-T, a chimeric antigen receptor T cell (CAR-T), is a cell with anti-cancer activity formed by expressing a CAR structure capable of specifically recognizing a cancer surface antigen on the surface of a T cell using genetic engineering techniques. After the CAR-T is infused into a patient, the CAR-T can directly recognize and bind to a cancer antigen without MHC restriction, and then specifically activate T cells to exert an anti-cancer effect. In addition to the results obtained in hematological tumors targeting CAR-ts such as CD19(NCT02348216), BCMA (NCT02658929), some CAR-ts have also been shown to be somewhat responsive to cancer, such as HER2 targeted CAR-T for HER2 positive sarcoma (NCT00902044), CEA targeted CAR-T for metastatic colorectal cancer (NCT02349724), IL12R α 2 targeted CAR-T for neuroblastoma (NCT01975701), c-MET targeted CAR-T for metastatic breast cancer (NCT01837602), and others. The above suggests that immunotherapy will have an increasingly important role in the treatment of cancer.

However, as immunotherapy progresses, scientists have found that immune activation does not always lead to regression of cancer (particularly cancer), and studies have shown that the presence of fully activated cancer-specific T cells in peripheral blood is not correlated with regression of cancer or a better prognosis for cancer patients. These phenomena are explained with the finding of an inhibitory characterization of the cancer locally: there are a number of mechanisms in cancer and its microenvironment that delay, alter or even suppress the immunity against cancer, i.e., immune evasion mechanisms. Immune evasion mechanisms include, but are not limited to: (1) insufficient infiltration of immune cells; (2) aggregation of regulatory T cells (tregs); (3) the presence of cancer-associated macrophages (TAMs) and Myeloid Derived Suppressor Cells (MDSCs); (4) up-regulation of immune checkpoints, immunosuppressive molecules, cytokines, metabolites, and down-regulation of immunostimulatory molecules. In the presence of these mechanisms, immunotherapy is often limited in action and even suppressed, resulting in treatment unresponsiveness or treatment tolerance, and thus strategies that simply enhance the immune response do not achieve therapeutic goals and need to be improved by blocking immune evasion mechanisms. The best evidence for this is the effective use of immune checkpoint PD-1/PD-L1 blocking therapy in cancer therapy, and anti-PD-1/PD-L1 therapy is currently approved by the FDA for the treatment of metastatic melanoma, lung cancer, head and neck cancer, renal cell carcinoma, transitional cell carcinoma, liver cancer, gastric cancer, hodgkin's lymphoma, mercker's cell carcinoma, large B-cell lymphoma, cervical cancer, and MSI-positive cancers.

The above cancer immune escape mechanisms also exist for the treatment of CAR-T therapy. In a clinical study in 2017, investigators treated 10 patients with glioma with EGFRvIII-targeted CAR-T, and only 1 patient reached stable disease (> 18 months) while CAR-T cell infiltration at the cancer site was detected, and at the same time EGFRvIII was detected within the cancerDown-regulated expression, notably up-regulation of the immunosuppressive molecule IDO1, the immune checkpoint PDL1, the inflammatory factor IL10, and an increase in the number of tregs were detected in the cancer microenvironment. In addition, in a 2010 study, ERBB2 over-expressed cancer patients were found to detect a large increase in cytokines such as IFN gamma, GM-CSF, TNF alpha, IL6 and IL10 in serum after intravenous injection of CAR-T targeting ERBB2^[6]. The PD-1/PD-L1 pathway is known to be the earliest discovery and is one of characteristic immune escape mechanisms, and can mediate T cell apoptosis, exhaustion, induction of suppressive cytokines, Tregs and the like. While the immunosuppressive molecule IDO1 also plays an important role in the tolerance of cancer immunity in the cancer microenvironment. In addition, inflammatory factors such as IL6, IL8, and IL10 also have the effects of suppressing immune response and promoting cancer development in cancer microenvironment. IL6 has been shown to promote the growth of cancers such as multiple myeloma and colon cancer, and its target gene is also involved in the development of cell cycle and the inhibition of apoptosis, and has important cancer-promoting effect. The research shows that the principle that IL8 can promote migration and stem cell performance of cancer by inducing epithelial-to-mesenchymal transition (EMT) of cancer cells and can promote a series of immune escape mechanisms to generate tolerance after cancer treatment tolerance CAR-T treatment by enhancing immune suppression microenvironment is not clearly reported, but a plurality of evidences show that the IL8 may exist. CAR-T can directly recognize and activate cancer cells and then secrete large amounts of cytokines such as IFN γ, TNF α, etc. to kill cancer cells, however these cytokines are actually a double-edged sword. Studies have shown that when CD8+ T cells infiltrate cancer tissues, prolonged IFN γ signaling can induce up-regulation of PD-L1, IDO1 expression, driving immunosuppressive responses; on the other hand, research also shows that IFN gamma and TNF alpha can also play a role in a cancer chronic inflammation environment as proinflammatory molecules, and can inhibit T cell reaction and cytotoxicity of activated macrophages to a certain extent, and TNF alpha generated in a cancer microenvironment can promote the survival of cancer cells through anti-apoptosis molecules at the downstream of an NF kappa B channel. It can therefore be speculated that CAR-T, while killing cancer, may also induce immune escape in the cancer microenvironmentThe escape mechanism, in turn, promotes the survival and proliferation of incompletely understood cancer cells, as well as inhibits the persistence of the immune response, ultimately resulting in acquired tolerance. This may be a major factor in the poor efficacy of CAR-T in cancer, and there is a need for a new therapeutic regimen to normalize the immune response in CAR-T therapy by inhibiting the immune escape mechanism while maintaining the immune response, thereby improving the therapeutic effect on cancer.

To address this dilemma, in recent years in a number of preclinical studies, researchers have attempted to treat cancer by combining CAR-T with antibodies, inhibitors, or other genetic engineering approaches. The combination regimen used in these studies was primarily directed to immune checkpoints, in particular PD-1/PD-L1. One result showed that CAR-T targeting GD2 had significantly reduced cytokine production after long-term co-culture with melanoma cells, which PD-1 antibodies could reverse; likewise, one result shows that PD-1 antibodies delay CAR-T depletion and retain their effector function; in addition, several results demonstrate that the combination of these two treatment modalities produces a highly synergistic effect that effectively extends survival in vivo experiments. However, the clinical results still do not perform well, do not yield significant advantages in combination with PD-1 antibody blocking therapy in a small phase I clinical trial against cancer, and also do not address the issue of inflammatory factor release. Indeed, with the discovery of more and more molecules involved in immune escape mechanisms, meaning that the PD-1/PD-L1 mechanism is only a small part of the cancer microenvironment, simple PD-1/PD-L1 blockade is difficult to be a complete or universal immune escape limiting means. For other immune escape pathways, there are some studies using antibodies to the inflammatory factor pathway to ameliorate the cytokine release syndrome of CAR-T. There have also been studies to use IDO1 inhibition to boost CAR-T's anti-cancer immunity. However, these approaches usually block only one immune escape pathway, and the immune microenvironment is often a multi-pathway interaction, so the current combination strategies are likely to have limited effects.

Although CAR-T therapy has now proven to have therapeutic efficacy and therapeutic potential for the treatment of cancer, particularly for hematological neoplasms. There are still many difficulties in the treatment of cancer, of which it is important to be limited by the presence of an immunosuppressive microenvironment. The current mainstream method of combining CAR-T therapy with PD-1/PD-L1 blockade did not significantly improve efficacy. CN105153315A discloses an immunosuppressive receptor combined with a cancer antigen chimeric receptor and application thereof, cancer is recognized through the cancer antigen chimeric receptor, and a recombinant immunosuppressive receptor is combined with an immunosuppressive factor, however, the in vitro killing is not obviously improved; CN107073138A discloses compositions and methods for reducing immune tolerance associated with CAR-T cell therapy, expressing both CAR and a modified PD-1, while effectively reducing the rate of inhibition of CAR-T by PD-L1, still do not exhibit good therapeutic effects; CN107325185A discloses a PSCA and PDL1 double-targeting chimeric antigen receptor based on OCST-CAR, which solves the defects of low efficiency and long period of simultaneous expression of two groups of receptors, but the in vitro killing rate of cancer cells is not more than 60%. On one hand, the treatment effect of the above patents is still limited, on the other hand, the above patents are generally only operated aiming at the PD-1/PD-L1 pathway, and if other immune escape mechanisms are further inhibited, the problems of difficult construction, complex design and high cost are faced, so that a better combination treatment scheme is urgently needed to be provided, the immune inhibition in CAR-T treatment is solved, and the anti-cancer effect of CAR-T is improved.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a cancer combination treatment composition.

The technical scheme of the invention is as follows:

a cancer combination therapy composition comprising CAR-T cells and epigenetic modulators as an active ingredient, wherein

CAR-T cells that can recognize at least one cancer specific antigen or cancer associated antigen;

an epigenetic modulator comprising at least one of a CDK7 inhibitor and a BRD4 inhibitor.

The other technical scheme of the invention is as follows:

use of a CAR-T cell together with an epigenetic modulator in the preparation of a composition for the treatment of cancer,

wherein

In a preferred embodiment of the invention, the administration of the epigenetic modulator is performed after the administration of the CAR-T cells.

Further preferably, the CAR-T cells are administered once daily for two consecutive days and once on the third day with the epigenetic modulator.

The other technical scheme of the invention is as follows:

wherein

The invention has the beneficial effects that:

1. after the CAR-T is combined with a specific epigenetic regulator, the invention can down-regulate CAR-T induced inhibitory immune checkpoints (PD-L1, PD-L2 and the like), inhibitory inflammatory factors (IL6, IL8, IL10 and the like), immunosuppressive molecules (IDO1, VEGF and the like), chemokines (CXCL2, CCRL2 and the like), thereby relieving the tumor microenvironment inhibition caused by the simple use of the CAR-T, improving the anti-tumor immune response of the CAR-T and effectively solving the immune tolerance problem in CAR-T treatment.

2. The combined treatment of CAR-T and a specific epigenetic regulator in the invention can effectively play a role in vivo synergistic anti-tumor, and provides a more novel and effective choice for tumor treatment.

3. The cancer treatment compositions of the invention in which CAR-T is combined with a specific epigenetic modulator have a broader inhibitory effect on the tumor microenvironment than CAR-T in combination with an inhibitor or antibody that blocks a single immune escape pathway while also being easier to manipulate and implement than other approaches to blocking multiple immune escape pathways in CAR-T treatment compositions.

Drawings

FIG. 1 is a map of an EGFR-CAR recombinant transfer vector based on a pCDH vector in example 1 of the present invention; including IL 2R-derived Signal Peptide (SP), EGFR-targeting scFv (EGFR-scFv), IgG1 Fc hinge region (IgG1 Fc), CD 28-derived transmembrane region (CD28TM), CD 28-derived intracellular signaling domain (CD28), 4-1 BB-derived intracellular signaling domain (4-1BB), and CD3 ζ intracellular signaling domain (CD3 z).

FIG. 2 shows the inhibition of different epigenetic regulators of the immune suppressor molecules (PD-L1 and IDO1) on MDA-MB-231, which is induced by CAR-T and is up-regulated in triple-negative breast cancer cells, as measured by Westernblot in example 2 of the present invention.

FIG. 3 shows the inhibition of epigenetic regulators at the transcriptional level at different targets of the escape-related genes (PD-L1, PD-L2, IDO1, IL6, etc.) induced by CAR-T in triple-negative breast cancer cells MDA-MB-231(A) and glioma cells U87(B) by qPCR in example 2 of the present invention.

FIG. 4 shows the transcriptional level inhibition of the CAR-T-induced upregulated immune escape-related genes (PD-L1, PD-L2, IDO1, IL6, etc.) in triple-negative breast cancer cells MDA-MB-231(A) and glioma cells U87(B) using conventional epigenetic regulators targeting CDK7 and BRD4 as measured by qPCR in example 3 of the present invention.

FIG. 5 is a heatmap of CAR-T induced upregulation of immune escape-related genes (PD-L1, PD-L2, IDO1, IL6, etc.) in triple negative breast cancer cells MDA-MB-231(A) and MDA-MB-468(B) as measured by qPCR in example 3 of the present invention, expressed downregulated at the transcriptional level following combination with the epigenetic regulator THZ 1.

FIG. 6 is a heatmap of CAR-T induced upregulation of immune escape-related genes (PD-L1, PD-L2, IDO1, IL6, etc.) at the transcriptional level in association with the epigenetic regulator JQ1, as measured by qPCR in example 3 of the invention, for brain glioma cells U87(A) and primary GBM cells (B) from patients.

FIG. 7 is an immunoblot graph of normalization of protein expression levels after combination with the epigenetic regulator THZ1 for the CAR-T induced up-regulated immunosuppressive molecule (IDO1), the inhibitory immune checkpoint (PD-L1) in MDA-MB-231(A) and MDA-MB-468(B) triple negative breast cancer cells tested by Westernblot in example 4 of the present invention.

FIG. 8 is a Western blot analysis of normalization of protein expression levels after association with the epigenetic regulator JQ1 of the immunosuppressive molecule induced by CAR-T and up-regulated protein expression levels (IDO1) and the inhibitory immune checkpoint (PD-L1) in glioma cells U87 in example 4 of the present invention.

FIG. 9 is a bar graph of increased secretion of the inflammatory factors IL6, IL8 and IDO1 induced by CAR-T at different effect-to-target ratios in triple negative breast cancer cells MDA-MB-231, as measured by ELISA in example 5 of the invention, after combination with the epigenetic regulator THZ 1.

FIG. 10 is a bar graph of increased secretion of the inflammatory factors IL6, IL8 and IDO1 induced by CAR-T in brain glioma cells U87, after combination with the epigenetic regulator JQ1, as measured by ELISA in example 5 of the present invention.

FIG. 11 is a photograph showing the live images of subcutaneous transplants of triple negative breast cancer MDA-MB-231 from the beginning of treatment (D1) to the end of observation (D130) in each group of mice in example 6 of the present invention.

FIG. 12 is a graph showing the trend of the fluorescence of the subcutaneous transplanted tumor of triple negative breast cancer MDA-MB-231 during the period from the start of treatment (D1) to the end of observation (D130) in each group of mice in example 6 of the present invention.

FIG. 13 is a graph showing the volume growth trend of subcutaneous transplantable triple negative breast cancer MDA-MB-231 tumors from the beginning of treatment (D1) to the end of observation (D130) in each group of mice in example 6 of the present invention.

FIG. 14 is a graph showing the survival of groups of mice loaded with triple negative breast cancer MDA-MB-231 subcutaneous transplantable tumor in example 6 of the present invention.

FIG. 15 is a graph showing the immunohistochemical staining results of tumor, lung and liver proliferation marker Ki67(A), tumor associated antigen EGFR (B) and T cell marker CD8(C) in each group of mice dissected after the treatment of triple negative breast cancer MDA-MB-231 in example 6 of the present invention.

FIG. 16 is a graph showing immunohistochemical staining results of tumor, lung, and suprahepatic suppressive immune checkpoints PD-L1(A) and PD-L2(B), inflammatory factors IL6(C) and IL8(D) in each group of mice dissected after treatment of triple negative breast cancer MDA-MB-231 in example 6 of the present invention.

FIG. 17 is a photograph showing in vivo imaging of brain glioma U87 transplants in situ from the beginning of treatment (D1) to the end of observation (D40) in each group of mice in example 7 of the present invention.

FIG. 18 is a fluorescence trend graph (A) of brain glioma U87 orthotopic transplantations tumors and a fluorescence histogram (B) of the endpoints of D40 during the period from the beginning of treatment (D1) to the end of observation (D40) for each group of mice in example 7 of the present invention.

Fig. 19 is a graph showing the immunohistochemical staining results of the proliferation marker Ki67(a), the tumor-associated antigen egfr (b), and the T cell marker CD8(C) on the tissues of the brain, lung, and liver of each group of mice dissected after the treatment of brain glioma U87 in example 7 of the present invention.

FIG. 20 is a graph showing the immunohistochemical staining results of the inhibitory immune checkpoints PD-L1(A) and PD-L2(B), the inflammatory factors IL6(C) and IL8(D), and the immunosuppressive molecule IDO1(E) on the brain, lung and liver after each group of mice dissected after the treatment of brain glioma U87 in example 7 of the present invention.

FIG. 21 is a photograph showing in vivo imaging of brain glioma GBM orthotopic transplantations tumors from the beginning (D1) to the end of observation (D35) of each group of mice in example 7 of the present invention.

FIG. 22 is a fluorescence trend graph (A) of brain glioma GBM orthotopic transplantation tumors and a fluorescence histogram (B) of D40 during the period from the start of treatment (D1) to the end of observation (D40) in each group of mice in example 7 of the present invention.

FIG. 23 shows the survival of mice in each group loaded with brain glioma GBM orthotopic transplantation tumor in example 7 of the present invention.

Detailed Description

The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.

The tumor specific antigen or tumor associated antigen includes CD19, CD20, CEA, GD2 (also known as B4GALNT1 beta-1, 4-N-acetyl-galactosamine transferase1), FR (Flavingreductase), PSMA (pro-specific membrane antigen), gp100(PMELpremelanosome protein), CA9(carbonic anhydrase IX), CD171/L1-CAM, IL-13R alpha 2, MART-1 (also known as melan-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cane/testins antigen 1B), MAGE (melanocated antigen E1) family protein, BAGE B family antigen, GAMMA family antigen, VEGFR2, VEGFR 8, CD22, VEGFR2, VEGFR 6385, VE, FBP, GD3 (also known as ST8SIA1, ST8alpha-N-acetyl-neuraminide alpha-2, 8-sialyltransferase 1), PSCA (pro state stem cell antigen), FSA (also known as KIAA1109), PSA (also known as KLK3, lyophilized peptide 3), HMGA2, tertiary acetyl chloride receptor (tertiary-AChR), LeY (also known as FUT3), CAM, MSLN (mesothelin), IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125 (also known as MUC16, mucidin 16, cellular afocal), CA 36-3, CA19-9, CA72-4, CA242, CA50, CYC 8538, CYC 16, nuclear fusion protein, SCC-GCA 2, GCA-1, GCA-5, GCA-5-3, GCA-5-9, CPG-2-GCA-5, CPG-C-III, CPG-2, CPG-C-III, CPG-G-2, CPG-C, CPG-III, CPG-G-2, CPG, at least one of glycolipids F77, GD-2, NY-ESO-1 TCR.

The epigenetic modulators include targeted inhibitors of at least one of DNMT, DNMT3, TET, SNF (also known as SMARCB, INI or BAF), ARID1, ARID, PBRM (also known as BAF180 or PB), BRGG (also known as SMARCA), BRM (also known as SMARCA), SMARCD (BAF 60), SMARCE (also known as BAF), CHD, ATRX (also known as RAD), XX, MLL (also known as KMT 2), MLL, EZH, SUZ, EED, SETD (also known as HYPB), NSD, KDM1 (also known as LSD), KDM2, KDM4, KDM5, HDAC 5, KDM6 (HDAC), HDAC, PHFH,

HDAC

3, 300, 3, 300, 3, three; preferably comprises at least one of a CDK7 inhibitor, CDK9 inhibitor, BRD4 inhibitor, p300 inhibitor, TIP60 inhibitor, MOF inhibitor and KDM5 inhibitor; more preferably at least one of a CDK7 inhibitor and a BRD4 inhibitor.

The cancer comprises lung cancer, hepatocellular carcinoma, lymphoma, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, renal cancer, brain glioma, melanoma, pancreatic cancer and prostate cancer, and is preferably triple negative breast cancer and brain glioma

In a preferred embodiment of the invention, the at least one cancer specific antigen or cancer associated antigen comprises EGFR.

Further preferably, the CAR in the CAR-T cell is: IL2R signal peptide-Anti-EGFR scFv-IgG1 Fc hinge region-CD 28 transmembrane region-CD 28 intracellular signal domain-4-1 BB intracellular signal domain-CD 3 ζ intracellular signal domain (see CN110845623A for details).

Still further preferably, the therapeutically effective amount of the CAR-T cells is 1.25X 10⁸-5×10⁸Individual cell/kg, more preferably 2.5X 10⁸Individual cells/kg.

In a preferred embodiment of the invention, the inhibitor of CDK7 comprises THZ 1.

Further preferably, the therapeutically effective amount of THZ-1 is 8-15mg/kg, even more preferably 10 mg/kg.

In a preferred embodiment of the invention, the BRD4 inhibitor comprises JQ 1.

Further preferably, the therapeutically effective amount of JQ1 is 20-30mg/kg, more preferably 25 mg/kg.

Example 1

This example is a method of making the CAR-T cells of the invention.

(1) Packaging and preparation of lentiviruses: two days prior to transfection, 293T was applied at 2.5X 10⁶Spread in 10cm plates using 8mL complete medium DMEM + 10%FBS is cultured. The 293T medium was changed to 7mL complete medium RMPI 1640+ 10% FBS 2-4h prior to transfection. Dissolving 84 mu L of PEI into 600 mu L of basal medium RMPI 1640 during transfection, and standing for 2 min; next, 42. mu.g of the plasmid was dissolved in 600. mu.L of basal medium RPMI 1640 with a 4: 3: 1 ratio of EGFR-CAR recombinant transfer vector based on pCDH vector (see CN110845623A, as shown in FIG. 1), psPAX and pMD2G, followed by adding PEI solution to the plasmid solution, shaking immediately for 8s, and after standing for 8min, adding to 293T cells. 12h after transfection, 293T medium was changed to 12mL complete medium DMEM + 10% FBS. Cell supernatants were harvested 60h after transfection, centrifuged at 3500rpm for 5min, and the supernatants were filtered through 0.22 μm filters and subsequently concentrated 20-40 fold in Millipore Ultrafiltration tubes to obtain lentiviruses. The Lentivirus Titer determined by qPCR Lentivirus Titration (Titer) Kit from abm was up to 1X 10⁹IU/mL-1×10¹⁰IU/mL. Then subpackaging and storing at-80 ℃.

(2) Isolation and culture of human primary T lymphocytes: lymphocytes were obtained by separating human peripheral blood lymphocyte separation medium Ficoll (manufactured by Tianjin Yangyi Co., Ltd.), and adjusting the cell density to 1X 10 using X-VIVO (manufactured by LONZA Co., Ltd.) medium containing 10% FBS, CD3, CD28 antibody and cytokine as T cell medium⁶The cells/mL are cultured, and the subsequent experiment is carried out after stimulating the culture for 72 h.

(3) Lentivirus infection of human primary T cells: cultured human primary T lymphocytes were plated at 2X 10 per well⁵Inoculating each cell into a 24-well plate, adding the lentivirus prepared in the step (1) into the 24-well plate with the MOI of 100-400, uniformly mixing, centrifuging at 800 Xg and 32 ℃ for 50-60min in a plate centrifuge for infection, then culturing in a 37 ℃ and 5% CO2 incubator for 12h, taking out the cells, centrifuging at 1000rpm, discarding supernatant, suspending each well with 500 mu L of fresh T cell culture medium, adding the suspended cells into the 24-well plate or re-infecting the cells (adding the lentivirus prepared in the step (1), uniformly mixing, centrifuging at 800 Xg and 32 ℃ for 50-60min in the plate centrifuge for infection, and then infecting the cells at 37 ℃ and 5% CO for 50-60min₂Culturing for 12h in incubator, taking out cells, centrifuging at 1000rpm, discarding supernatant, suspending each well with 500 μ L fresh T cell culture medium, adding into 24-well plate), and changing the culture solution every other dayThe culture method (2) is carried out for 4-7 days.

Example 2

This example is the screening of epigenetic modulators in combination therapy for different tumors.

Tumor cells cultured in vitro (triple negative breast cancer cells MDA-MB-231 or glioma cells U87) were digested and plated in 6-well plates overnight, and the following day when the cell confluence reached 80%, the media was changed and CAR-T cells prepared in example 1 (effective target ratio E: T1: 2, suspended using X-VIVO medium) were added (effective target ratio E: T ═ 1: 2), and/or different epigenetic regulators (250nM THZ1, 100nM JQ1, 20 μ M C646, 200 μ M MG149, 5 μ M KDM5-C70, 5 μ M ICDK9, dissolved using DMSO) were added. After the cells were cultured for 48 hours, suspended CAR-T cells were removed, and Total RNA of tumor cells was extracted with Eastep Super Total RNA Extraction Kit (manufactured by Promega corporation) and subjected to RT-qPCR; or after co-culturing for 72h, removing suspended CAR-T cells, lysing tumor cells by RIPA lysate, performing ultrasonic treatment, centrifuging to obtain supernatant, measuring concentration, quantifying, boiling, and performing Western blot detection.

Results as shown in fig. 2 and 3, CAR-T alone induced up-regulation of expression of multiple immune escape-related molecules at the transcriptional and protein levels, while the addition of epigenetic regulators may regulate these molecules to some extent. The Western blot results in FIG. 2 show that the protein expression levels of PD-L1 and IDO1 in MDA-MB-231 are significantly increased after EGFR-targeting CAR-T is added to MDA-MB-231 cells, while the simultaneous addition of certain epigenetic regulators improves this phenomenon, in particular the protein expression levels of PD-L1 and IDO1 in MDA-MB-231 are greatly reduced when a combination of THZ1(CDK7 inhibitor) and CAR-T is added; the RT-qPCR results in FIG. 3 also show that the combined use of epigenetic regulators and CAR-T in MDA-MB-231 and U87 cells reduces the expression of multiple CAR-T induced upregulated immune escape related molecules at the transcriptional level, with a stronger effect of the combination of THZ1 and JQ 1. The results show that the CDK7 inhibitor THZ1 and BRD4 inhibitor JQ1 have good inhibitory action on immune escape related molecules induced by CAR-T targeting EGFR, and the effects are better than those of C646(p300 inhibitor), MG149(TIP60 and MOF inhibitor), KDM5-C70(KDM5 inhibitor) and ICDK9(CDK9 inhibitor).

This example illustrates the beneficial effects of epigenetic modulators on CAR-T therapy, with epigenetic modulators targeting CDK7 or BRD4 being preferred.

Example 3

This example examined the down-regulation of immune escape-related molecules at the transcriptional level by a combination of CAR-T and epigenetic modulators targeting CDK7 or BRD4 against a variety of inhibitory immune checkpoints, inflammatory factors, immunosuppressive molecules and chemokines. Triple negative breast cancer cells (MDA-MB-231 or MDA-MB468) and glioma cells (U87 or GBM-PDX) cultured in vitro were digested and plated in 6-well plates for overnight culture, and the following day when the cell confluence reached 80%, the media was changed and CAR-T cells prepared in example 1 were added (effective to target ratio E: T ═ 1: 2, suspended using X-VIVO medium), and/or different epigenetic regulators were added: common epigenetic regulators of CDK7 (250nM THZ1, 350nM THZ2, 1. mu.M BS-181, 1. mu.M CT7001) and BRD4 (100nM JQ1, 1. mu.M IBET-151, 500nM Molibrescib, 500nM Mivebresib, 500nM INCB057643, 75nM Birabrescib, 1. mu.M MS 417). After the incubation for 48 hours, the suspended CAR-T cells were removed, Total RNA of tumor cells was extracted with Eastep Super Total RNA Extraction Kit (manufactured by Promega), and RT-qPCR detection was performed using the corresponding primers and GoScript Reverse Transcription System (manufactured by Promega). The results are shown in FIGS. 4-6. FIG. 4 shows that the expression of CAR-T-induced immune escape related molecules (e.g., PD-L1, PD-L2, IL6, IDO1, etc.) can be significantly down-regulated at the transcriptional level when a commonly used epigenetic regulator targeting CDK7 or BRD4 is used together with CAR-T in triple negative breast cancer cells MDA-MB-231 and glioma cells U87. Figures 5 and 6 illustrate the transcriptional regulation of CAR-T-induced immune escape-related molecules by epigenetic regulators targeting CDK7 or BRD4 in different tumors, as exemplified by the combination of CAR-T with THZ1 or JQ1, respectively.

This example further demonstrates that the combination of an epigenetic regulator targeting CDK7 or BRD4 and CAR-T provides good control of the expression of multiple immune escape-related molecules simultaneously at the transcriptional level.

Example 4

This example examined the down-regulation of protein levels of various inhibitory immune checkpoints, inflammatory factors, immunosuppressive molecules, chemokines, etc., by a combination of CAR-T and epigenetic modulators targeting CDK7 or BRD 4.

The procedure was carried out according to the co-culture method described in example 3, and after 72h of co-culture, suspended CAR-T cells were removed, tumor cells were lysed with RIPA lysate, and the supernatant was ultrasonically treated, centrifuged, quantified by assay, and boiled for Western blot assay.

Results as shown in figures 7 and 8, the combination strategy of THZ1+ CAR-T in triple negative breast cancer cells (MDA-MB-231 and MDA-MB-468), or JQ1+ CAR-T in glioma cells (U87), down-regulated the expression of CAR-T induced immune escape related molecules (e.g. PD-L1, IDO1) at the protein level. This example also demonstrates at the protein level the role of compositions of epigenetic modulators targeting CDK7 or BRD4 and CAR-T in normalizing the expression of immune escape-related molecules.

Example 5

This example examined the reduction in secretion of various inflammatory factors following use of CAR-T in combination with epigenetic modulators targeting CDK7 or BRD 4.

The operation was carried out according to the co-cultivation method shown in example 3, using the epigenetic regulator THZ1 or JQ 1. After the co-culture for 72 hours, cell culture supernatants were collected, centrifuged at 3000rpm for 30min, and ELISA was performed using a Human IL6 ELISA Kit (produced by Proteitech corporation), a Human IL8 ELISA Kit (produced by ExCell Bio Inc.) and a Human Indexamine 2, 3-dioxygenase/IDO ELISA Kit (produced by Novus Biological Inc.).

As shown in FIGS. 9 and 10, ELISA results showed that the secretion of IL6, IL8, IDO1 could be effectively reduced by using the combination of THZ1 and CAR-T in MDA-MB-231 or JQ1 and CAR-T in U87.

Example 6

This example demonstrates the in vivo anti-tumor activity of CAR-T in combination with an epigenetic modulator targeting CDK 7.

4-8 week-old SCID immunodeficient mice were injected subcutaneously with 5X 10⁵The MDA-MB-231 cells were transfected and screened for over-expression of the luciferase gene. After the tumor grows for 3-4 days, the fluorescence intensity generated by the tumor cells is detected by living body imaging after injecting a fluorescein substrate into the abdominal cavity. When the fluorescence intensity reaches 1X 10⁷-5×10⁷p/sec/cm²At/sr, mice were divided into four groups of three mice per group: control group, THZ1 treatment alone group, CAR-T treatment alone group, THZ1+ CAR-T combination treatment group. Wherein the control mice were not treated at all; THZ1 group each mouse was intraperitoneally injected with THZ1 at a therapeutic dose of 10 mg/kg; CAR-T group at 2.5X 10 per mouse⁸Tail vein injection of CAR-T at therapeutic dose per cell/kg; THZ1+ CAR-T combination treatment group 2.5X 10/mouse⁸A therapeutic dose of individual cells/kg was tail vein injected with CAR-T, followed by intraperitoneal injection of THZ1 at a therapeutic dose of 10 mg/kg. The treatment was performed at intervals according to the above treatment strategy, and the fluorescence intensity of the tumors of each group of mice was continuously measured by in vivo imaging, and the volume of subcutaneous tumors (volume: length × width) of each group of mice was measured²2) and observing the survival of the mice. When the mice reached the end of the experiment (subcutaneous tumor volume over 1500 mm)³) The mice are sacrificed by cervical dislocation, and liver, lung, spleen and tumor tissues are dissected out and are fixed by formaldehyde, dehydrated, embedded by paraffin and sliced to obtain tissue sections. When all of the THZ1 groups died, the surviving mice were sacrificed by cervical dislocation and tissue sections were obtained in the same manner. Finally, the expression of proliferation marker Ki67, tumor-associated antigen EGFR, effector T cell marker CD8, inhibitory immune checkpoints PD-L1 and PD-L2, and inflammatory factors IL6 and IL8 are detected by immunohistochemical staining.

The results are shown in FIGS. 11-16. The mouse in vivo images and tumor growth trend plots of FIGS. 11-13 show that treatment with THZ1 alone can only inhibit the growth of triple negative breast cancer MDA-MB-231 in the early phase, with eventual tumor progression and mouse death; while CAR-T alone only one mouse had regressed but relapsed after 70 days, while one mouse was tolerated by treatment and the other mouse eventually developed tumor progression; tumor regression was achieved in all mice after THZ1+ CAR-T combination treatment with no recurrence for 130 days. The survival curves in figure 14 also reflect that the survival rate of mice under combination treatment can reach 100%, and death occurs in all other groups of mice. The immunohistochemistry graphs of fig. 15 and 16 show that the control, THZ1 and CAR-T group mice all detected tumor proliferation and metastasis, while the CAR-T group detected high expression of immune escape related molecules (PD-L1, PD-L2, IL6, IL8) despite the detection of infiltration of CD8 positive T cells; however, no metastasis of tumor and no immunosuppressive molecules were detected in the organs of mice after the combination treatment.

The embodiment shows that the combination of THZ1+ CAR-T can produce good treatment effect when treating triple negative breast cancer MDA-MB-231, the curative effect is obviously superior to that of single treatment of THZ1 or CAR-T, and simultaneously, the combination can effectively normalize the immunosuppressive environment induced by CAR-T treatment, and the composition of the epigenetic regulator targeting CDK7 and CAR-T is proved to have good in vivo anti-tumor potential.

Example 7

This example demonstrates the in vivo anti-tumor activity of CAR-T in combination with an epigenetic modulator targeting BRD 4.

Using 6-8 week old SCID immunodeficient mice, intracranial injection of 3X 10⁵U87 cells transfected and selected to overexpress the luciferase Gene or 5X 10⁵The transfected and selected GBM-PDX cells overexpressing the luciferase gene. After the tumor grows for 3-4 days, the fluorescence intensity generated by the tumor cells is detected by living body imaging after injecting a fluorescein substrate into the abdominal cavity. When the fluorescence intensity reaches 1X 10⁷-5×10⁷p/sec/cm²At/sr, mice were divided into four groups of three mice per group: control, JQ1 treatment alone, CAR-T treatment alone, JQ1+ CAR-T combination treatment. Wherein the control mice were not treated at all; JQ1 treatment group alone each mouse was given an intraperitoneal injection of JQ1 at a therapeutic dose of 25 mg/kg; CAR-T monotherapy group at 2.5X 10 per mouse⁸Tail vein injection of CAR-T at therapeutic dose per cell/kg; JQ1+ CAR-T combination treatment group 2.5X 10 mice per mouse⁸A therapeutic dose of individual cells/kg was tail vein injected with CAR-T, followed by intraperitoneal injection of JQ1 at a therapeutic dose of 25 mg/kg. The above-mentioned treatmentTreatment strategies were spaced apart and the fluorescence intensity of the tumors of each group of mice was continuously measured by in vivo imaging. For the experiment carried out in the brain glioma U87 orthotopic transplantation tumor model, after the mice are continuously observed for 40 days, the mice are killed by cervical dislocation, liver, lung, spleen and brain tissues are dissected out to be fixed by formaldehyde, dehydrated, embedded by paraffin and sectioned to obtain tissue sections, and then the expression conditions of a proliferation marker Ki67, a tumor-associated antigen EGFR, an effector T cell marker CD8, inhibitory immune check points PD-L1 and PD-L2, inflammatory factors IL6 and IL8 and an immune inhibitory molecule IDO1 are detected by immunohistochemical staining. For the experiment carried out in the brain glioma GBM orthotopic transplantation tumor model, when the mouse reaches the end point of the experiment (the mouse has neurological symptoms such as limb weakness, paralysis, imbalance and the like), the mouse is killed by a cervical dislocation method, and the survival condition of the mouse is recorded.

The results are shown in FIGS. 17-23. The mouse in vivo imaging and tumor growth trend plots in figures 17, 18 and 21, 22 show that neither JQ1 treatment alone nor CAR-T treatment alone had a poor therapeutic effect on brain gliomas (U87 or GBM-PDX), but that the therapeutic effect was greatly enhanced with the combination of JQ1+ CAR-T. The mouse survival curves of figure 23 show that using the combination of JQ1+ CAR-T increased the overall survival of mice from 50 days in the control group, 52 days in the JQ1 group, and 56 days in the CAR-T group to 75 days in the GBM-PDX model. In addition, the immunohistochemistry results of figures 19, 20 show that the combination of JQ1+ CAR-T also reduced tumor metastasis while normalizing the expression of CAR-T induced immune escape related molecules (PD-L1, PD-L2, IL6, IL8, IDO 1).

This example also demonstrates that compositions of an epigenetic modulator targeting BRD4 and CAR-T can improve the in vivo anti-tumor activity of CAR-T.

The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.

Claims

1. A cancer combination therapy composition characterized by: the effective components at least comprise CAR-T cells and epigenetic regulators, wherein

2. The cancer combination therapy composition of claim 1, wherein: the at least one cancer specific antigen or cancer associated antigen comprises EGFR.

3. The cancer combination therapy composition of claim 1, wherein: the CDK7 inhibitor comprises THZ 1.

4. The cancer combination therapy composition of claim 1, wherein: the BRD4 inhibitor includes JQ 1.

5. The cancer combination therapy composition of claim 1, wherein: the administration of the epigenetic modulator is performed after the administration of the CAR-T cells.

6. The cancer combination therapy composition of claim 5, wherein: the CAR-T cells are administered once daily for two consecutive days and once on the third day with the epigenetic modulator.

Use of CAR-T cells and an epigenetic modulator together in the preparation of a composition for the treatment of cancer,

wherein

8. The use of claim 7, wherein: the at least one cancer specific antigen or cancer associated antigen comprises EGFR.

9. The use of claim 7, wherein: the CDK7 inhibitor comprises THZ 1.

10. The use of claim 7, wherein: the BRD4 inhibitor includes JQ 1.