CN112516298B

CN112516298B - Application of combination of targeted EpCAM-CART cells and hsBCL9CT-24 in tumor resistance

Info

Publication number: CN112516298B
Application number: CN202011371360.4A
Authority: CN
Inventors: 朱棣
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2020-11-30
Filing date: 2020-11-30
Publication date: 2022-11-01
Anticipated expiration: 2040-11-30
Also published as: CN112516298A

Abstract

The invention discloses an anti-cancer composition, which comprises,comprising as active ingredients the following (a) and (b) components, which can be used simultaneously or separately: (a) CAR-T cells targeting the EpCAM antigen, (b) an inhibitor targeting the BCL9/β -catenin complex, which inhibitor is the dodecapeptide inhibitor hsBCL9_CT-24 or a pharmaceutically acceptable salt thereof. EpCAM-CAR-T and hsBCL9 of the invention_CT-24 synergistic effects enabling hsBCL9_CT-24 increases the penetration of T cells in the tumour tissue without dying the T cells, thus enabling the cold body mass of the patient to be reversed into the hot body mass.

Description

Application of combination of targeted EpCAM-CART cells and hsBCL9CT-24 in tumor resistance

Technical Field

The invention relates to the field of tumor medicine, in particular to novel EpCAM-CART targeted cell therapy and application of combination of EpCAM-CART targeted cell therapy and hsBCL9CT-24 in antitumor preparation.

Background

Colorectal cancer (CRC) is one of common malignant tumors, an effective curing method is lacked at present, and the life quality of a patient is difficult to improve due to strong toxic and side effects of chemical drugs. In recent years, with the development of biotechnology, cancer immunotherapy is gradually used from laboratories to clinics, and has gained wide acceptance.

Adoptive Cell Therapy (ACT) is a highly effective approach to personalized cancer Therapy that utilizes T cells targeting tumor antigens to exert natural effector functions to kill tumor cells. The therapy is to carry out in-vitro amplification, modification and activation on autoimmune cells, and then to re-transfuse the autoimmune cells into a patient body, and the strengthened immune cells can enhance the killing and inhibiting effect on tumor cells. The CAR-T cell therapy mentioned above is among the therapies, and adoptive cell therapy is less toxic and more resistant to patients and does not develop resistance compared to checkpoint blockade immunotherapy. The CAR-T cell therapy in the therapy is selected for research, and the killing effect and influence factors of the CAR-T cells on colon cancer tumor cells are discussed by constructing appropriate CAR-T cells.

Chimeric Antigen Receptors (CARs) are engineered receptors that can transplant any specific antibody onto immune effector cells (T cells) with the aim of redirecting the T cells of a patient or donor to specifically target and kill tumor cells. As shown in figure 1, the four generation CAR-T cell antibody types, collectively, the CAR comprises three parts: an extracellular antigen recognition domain from a single chain fragment variant (scFv) of an antibody, a transmembrane domain, and an intracellular T cell activation domain. The first generation CARs were single chain antibodies (CD 3 ζ or fcsri γ) linked to an Immunoreceptor Tyrosine Activation Motif (ITAM). In second generation CARs, a co-stimulatory molecule (CM 1), such as CD28, is added to the signaling region. The third generation additionally incorporates a co-stimulatory molecule (CM 2) such as CD134 or CD137. The fourth generation is added with key cytokines such as IL-12 and the like on the basis of the second generation.

CAR-T cell therapy has now rapidly progressed from preclinical studies to approved therapeutic approaches for the treatment of leukemia and lymphoma. But at present, CAR-T cell therapy still has room for great progress in its safety and efficacy. For example, various steps in the clinical preparation of CAR-T cells have a potential impact on their efficacy and safety. At present, most of CAR-T clinical tests and treatment thereof adopt autologous T cells, so that the CAR-T clinical tests and treatment thereof are possibly influenced by the quality and quantity of the T cells, and the use of different human T cells also brings the risk of cross contamination, so that the CAR-T is subjected to gene editing by using a multiple CRISPR technology to manufacture allogeneic universal T cells, so that the method for treating tumors is more and more emphasized by people while the allogeneic reaction is reduced.

EpCAM is a tumor-associated antigen, which is first found in colon cancer tissues and is one of the major surface antigens of human colon cancer, and is essentially a membrane protein with adhesion function, and studies show that EpCAM is rarely expressed in normal cells and highly expressed in tumor tissues. However, it has been shown that the extracellular domain of EpCAM promotes colon cancer cell migration, proliferation and tumor growth by activating EGFR and downstream ERK1/2 signaling.

The heterogeneous and complex immune microenvironment of tumors is critical to limit CAR-T efficacy of cancer therapy. Through the combination of the specific CAR-T cell therapy and the immune regulator, the opportunity is provided for solving the problem of poor treatment effect of the solid tumor. In melanoma, the Wnt signaling pathway is activated, preventing T cells from infiltrating tumor cells, thereby reducing the effectiveness of immunotherapy. By inhibiting the Wnt signal channel of the tumor, T cell infiltration can be promoted, immune microenvironment can be improved, and immune response can be promoted. In the work of M Feng et al, they discovered a compound, hsBCL9_CT-24, which inhibits the interaction of β -catenin and BCL9 and exhibits potent antitumor effects, including promotion of cytotoxic T cells in tumorsWhile decreasing regulatory T cells (T regs) and increasing dendritic cells.

Thus, those skilled in the art are working on EpCAM-CAR-T with hsBCL9_CT-24 is used synergistically to enhance antitumor activity.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the technical problem underlying the present invention is how to combine hsBCL9 with EpCAM-CAR-T based therapy for colorectal cancer_CT-24 to achieve better anticancer effect.

In order to achieve the purpose, the invention provides application of CAR-T cells targeting EpCAM antigens and inhibitors targeting BCL 9/beta-catenin complexes in preparation of antitumor drugs.

Further, the inhibitor is a dodecapeptide inhibitor hsBCL9_CT-24 or a pharmaceutically acceptable salt thereof.

Further, the tumor is colorectal cancer.

Further, CAR-T cells targeting the EpCAM antigen are configured to be prepared by viral transfection or electrotransfection.

The present invention also provides an anticancer composition characterized by comprising the following (a) and (b) ingredients as active ingredients, said (a) and (b) ingredients being used simultaneously or separately:

(a) A CAR-T cell that targets the EpCAM antigen,

(b) Inhibitors targeting the BCL 9/beta-catenin complex,

the inhibitor targeting BCL 9/beta-catenin complex is a dodecapeptide inhibitor hsBCL9_CT-24 or a pharmaceutically acceptable salt thereof.

The invention also provides application of the CAR-T cell which is prepared by electrotransfection and targets the EpCAM antigen in preparation of anti-colorectal cancer drugs.

Technical effects

EpCAM-CAR-T and hsBCL9_CT-24 synergistic use can promote hsBCL9_CT-24 increases the penetration of T cells in tumor tissue without T cell death, which can reverse the cold body mass to the hot body mass of the patient.

The electrotransfection of the present invention has higher transfection efficiency than viral transfection and is theoretically safer than viral transfection.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 shows the high titer (2X 10) of example 1 of the present invention⁸TU/mL) lentivirus transfection method to construct EpCAM1-CAR-T flow-type grouping schematic diagram;

FIG. 2 shows the low titer of example 1 of the present invention<1×10⁷TU/mL) lentivirus transfection method to construct EpCAM1-CAR-T flow-type grouping schematic diagram;

FIG. 3 is a schematic diagram of the electrotransfection method for constructing EpCAM1-CAR-T flow-based clustering according to example 1 of the present invention;

FIG. 4 shows green fluorescence of cells after introduction of GFP gene into T cells by the electrotransfection method of example 1 of the present invention;

FIG. 5 is a bar graph of the results of measuring 4 hours after adding CCK8 after the HCT116 and T cell of example 2 of the present invention were co-cultured for 24 hours;

FIG. 6 shows the results of the real-time unlabeled cell Analyzer (RTCA) experiment of viral transfection of example 2 of the present invention (comparison of different numbers of normal T cells);

FIG. 7 is the results of the real-time unlabeled cell analyzer (RTCA) experiment of electrotransfection according to example 2 of the present invention (comparison of different numbers of normal T cells);

FIG. 8 is hsBCL9 of example 3 of the invention_CT-24 results from the synergistic activity of EpCAM-CAR-T on cancer cells in vitro;

FIG. 9 is hsBCL9 of embodiment 4 of the invention_CT-24 results from the synergistic activity of EpCAM-CAR-T on cancer cells in vivo;

FIG. 10 is EpCAM-CAR-T cells and hsBCL9 of example 5 of the present invention on mice_CT-24 performing a security assessment.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

A "CAR-T cell targeting an EpCAM antigen" is denoted herein as "EpCAM-CAR-T".

Example 1 construction of EpCAM1-CAR-T cells

1.1 construction of EpCAM1-CAR-T cells by Lentiviral transfection

Lentivirus transfection of the EpCAM1 gene: transfection with hantah synbiotic packaging virus: virus titer: 2.5X 10⁸TU/ml, PBMC (CD 3+ T cells) were taken and transfected in 24-well plates as shown in Table 1:

TABLE 1

And adding a reagent according to the system, centrifuging for 1h at the normal temperature by 200g, putting the system into an incubator for continuous culture, supplementing the liquid to 500ul after 4h, centrifuging and replacing the liquid the next day, and taking the cell for detecting the EpCAM1-CAR expression condition by flow cytometry.

As shown in fig. 1, the left part is a flow-through clustering map of T cells without EpCAM1-CAR virus transfection, and the right part is a flow-through clustering map of T cells with EpCAM1-CAR virus transfection, it can be clearly observed that the right map shows obvious clustering, compared with the left map, the cell signal is about 40% shifted, which indicates that after EpCAM1-CAR virus transfection, partial cells successfully express chimeric antibody receptor, and the transfection efficiency is about 40%, even if the EpCAM1-CAR-T cells are successfully constructed by virus transfection.

As shown in fig. 2, the left part is a flow-clustering diagram of T cells without EpCAM1-CAR virus transfection, and the right part is a flow-clustering diagram of T cells with EpCAM1-CAR virus transfection, and even if lentivirus is used for infection, the clustering of the lentivirus is not significantly different from that of the uninfected T cells, which indicates that T cells cannot be transfected effectively when the lentivirus titer is small.

1.2 construction of EpCAM1-CAR-T cells by electrotransfection

Electrotransfection of the EpCAM1 gene: according to

Human T Cell

Kit providing step, respectively, to electrotransfect GFP (as control group) and EpCAM1 (experimental group) genes into T cells, observing GFP gene expression in fluorescence microscope the next day, and detecting EpCAM1-CAR expression by flow cytometry. As shown in fig. 3, the left part is a flow-clustering map of the transfected GFP gene into T cells (the green fluorescence produced by the cells can be observed in fig. 4), the right part is a flow-clustering map of the transfected EpCAM1-CAR gene, and it can be clearly observed that the right map has obvious signal migration, compared with the left map, the transfection efficiency of the electrotransfection is about 44%, i.e. it is shown that EpCAM1-CAR-T cells can be successfully constructed by using the electrotransfection method.

Example 2 EpCAM1-CAR-T cytotoxicity assay

CCK8 assay cell killing experiment: 100 μ L of the mixture was mixed at a density of 5X 10⁴Spreading cells/mL HCT116 cell suspension into a 96-well plate, culturing in an incubator, removing supernatant after the cells are completely adhered, adding successfully transfected EpCAM1-CAR-T cells into each well according to different proportions, adding 10 mu L CCK8 solution, mixing the cells uniformly, incubating for a period of time, and measuring the absorbance value at 450nm by using an enzyme-labeling instrument.

RTCA (real time label free cell analyzer) assay cell killing experiment: 100 μ L of the mixture was mixed at a density of 5X 10⁴cells/mL HCT116 cell suspension is paved into a special pore plate with an electronic chip, the cells are cultured in an incubator, the proliferation and adherence conditions of the cells are measured by an RTCA instrument, after the cells are completely adhered to the wall, epCAM1-CAR-T cells which are successfully transfected are added into each pore according to different proportions, and the proliferation condition of the HCT116 cells is continuously measured.

As shown in tables 2-3 and FIG. 5, in the CCK8 experiment, the CAR-T cytotoxicity experiment after lentivirus transfection was detected, and the CAR-T cells were not significantly different from untreated normal T cells by the numerical analysis of variance and histogram, which may be caused by the fact that CAR-T itself also secretes substances binding to CCK8 and the number of CAR-T cells is different, resulting in different background, so CCK8 was judged to be unsuitable for this experiment.

TABLE 2 measurement of HCT116 after 24h coculture with HCT116 and 4h addition of CCK (1, 2,3,4 stands for multiple wells)

TABLE 3 analysis of variance of measurement results after 24h coculture of HCT116 and addition of CCK8 h

ANOVA
							Source of difference	SS	df	MS	F	P-value	F crit
Line of	0.003	3	0.001	0.117	0.947	4.757
							Column(s) of	0.003	2	0.001	0.14	0.872	5.143
Error of the measurement	0.054	6	0.009
							Total of	0.059	11

As shown in fig. 6, which is the result of the CAR-T cell cytotoxicity test by Real-time cell analysis (Real-time cell analysis) technology for lentivirus measurement, it can be clearly observed that, when the ratio of CAR-T to colon cancer cell line HCT116 is large (4, 1, 2. When the proportion of CAR-T and HCT116 is small, CAR-T cells only have inhibitory effect on the proliferation of HCT116 and do not stop the proliferation, and ordinary T cells have lower effect but also have a certain inhibitory effect under the same proportion.

As shown in fig. 7, which is the result of the EpCAM1-CAR-T cytotoxicity experiment constructed by electrotransfection, the results show that the killing effect of the electrotransfection EpCAM1-CAR-T on HCT116 is not obvious, the inhibition on the proliferation of HCT116 is caused only under the condition of a high proportion (10.

Example 3 hsBCL9_CT-24 synergistic activity with EpCAM-CAR-T on cancer cells in vitro.

First, we evaluated hsBCL9_CT-24 effect on colorectal cancer cells. hsBCL9 based on different concentrations of 1.25uM, 2.5uM, 5uM, 10uM_CTCI curves for HCT116 or SW480 cell lines treated with-24 showed the strongest decrease in gradient of 10uM, indicating hsBCL9_CT-24 can inhibit HCT116 or SW480 cells from proliferating in a dose-dependent manner, as shown in figure 8A. Then, we further examined hsBCL9_CT-24 in combination with EpCAM-CAR-T on HCT116, SW480 cells. The results show that the medicine is used for treating EpCAM-CAR-T aloneSignificantly lower CI in target cells from combination therapy compared to therapy, indicating that hsBCL9 is combined_CT-24 treatment can improve the cytotoxicity of EpCAM-CAR-T in vitro, see figures 8B and 8C.

In the cytokine release assay, the cells were tested for INF- γ release by ELISA, following the same co-culture. The results showed that EpCAM-CAR-T cells and hsBCL9_CTThe cytokine INF-gamma levels released by the-24 combination were significantly higher than that of EpCAM-CAR-T cells treated alone, see FIG. 8D, also indicating that hsBCL9_CT-24 can enhance the anti-tumor activity of EpCAM CAR-T cells.

In addition, cell migration assays were performed to investigate hsBCL9_CTWhether-24 affects the migration of EpCAM-CAR-T cells. The lower cavity is respectively provided with a cell-free culture body, a target cell and hsBCL9_CT-24. The results show that hsBCL9 is indicated_CT-24 can increase the in vitro migration of EpCAM-CAR-T cells, see figure 8E.

The above data indicate that hsBCL9_CT-24 can promote EpCAM-CAR-T cell capacity and INF- γ production, as well as migration to antigen.

Example 4 hsBCL9_CT-24 synergistic antitumor activity with EpCAM-CAR-T in vivo.

We continued to evaluate the therapeutic activity of mGFP CAR-T cells/control T cells, epCAM-CAR-T cells and EpCAM-CAR-T cells plus HSBCL9_CT-24 subcutaneous xenograft tumor model established in NSG mice with HCT116, as shown in figure 9A. Consistent with previous results of detecting EpCAM-CAR-T cells against specific tumors in vivo, the volume of EpCAM-CAR-T group tumors was significantly slower than the mGFP CAR-T group. This is because the EpCAM-CAR-T cell group plus hsBCL9_CT-24 significantly inhibited the growth rate of tumors. These results demonstrate EpCAM-CAR-T cells and hsBCL9_CT-24 combined antitumor effect, and hsBCL9_CT-24 can increase the ability of EpCAM CAR-T in the treatment of cancer. Blood was collected from mouse tail vivo vein on day 15 and analyzed by flow cytometry to detect phenotypic changes in T cells in vivo. The results show that, as shown in FIGS. 9B and 9C, the combination treatment resulted in a higher proportion of CD8+ T cells compared to CAR-T cells administered alone, which is consistent with the above resultsIt is consistent to show that CD8+ T cells play a crucial role as effector cells.

Example 5 EpCAM-CAR-T cells and hsBCL9 on mice_CT-24 performing a security assessment

In vivo experiments on mouse hSBCL9_CTThe potential toxic effects induced by-24 and EpCAM-CAR-T cells were evaluated. None of the mice developed graft-versus-host reactions, such as diarrhea or skin rash, during the treatment. Treating H of main internal organs such as heart, liver, kidney, lung and spleen of mice&E staining without overt toxic pathology, see figure 10. These data support the use of EpCAM-CAR-T cells and hsBCL9 in mice_CT-24 security.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims

1. Application of a composition consisting of CAR-T cells targeting EpCAM antigen and a dodecapeptide inhibitor hsBCL9CT-24 in preparation of anti-colorectal cancer drugs.

2. The use of claim 1, wherein the CAR-T cells targeting the EpCAM antigen are configured to be prepared by viral transfection or electrotransfection.