Biological preparation containing lymphocytes without adhesion g protein-coupled receptor f5 with Anti-tumor activity

Abstract

The object of the invention is a biological preparation containing 0.5 to 1 ×106 CD4+ subpopulation T cells lacking ADGRF5 for use as a medication, and its use in neoplastic disorders of the gastrointestinal tract, specifically solid tumors.

Description

Biological preparation containing lymphocytes without adhesion G Protein-coupled receptor F5 with anti-tumor activity

[0001] The subject of the invention is a biological preparation containing lymphocytes lacking an adhesion G protein-coupled receptor F5 with anti-tumor activity.

[0002] Immunotherapy includes numerous treatment options, depending on the therapeutic strategy adopted. One of the most promising therapeutic strategies is adoptive immunotherapy, which is based on selected activated and / or modified immune cells that are manipulated in vitro, and then administered to patients.

[0003] Despite abundant evidence on the crucial importance of immune cells and the immune response modulated through them, immunotherapy targeting patients with neoplastic disorders has limited efficacy.

[0004] Observational and experimental studies suggest that ADGRF5 appears to be involved in the pathogenesis of numerous solid tumors.

[0005] Tang, X. et al. "GPR116, an adhesion G-protein-coupled receptor, promotes breast cancer metastasis via the Gαq-p63RhoGEF-Rho GTPase pathway", Cancer Res, 2013. 73(20): p. 6206-18 demonstrated that downregulation of ADGRF5 expression in breast cancer cells reduces the migratory potential of tumor cells in both in vitro and in vivo models.

[0006] In another publication by Yang, L., et al, titled: ”High expression of GPR116 indicates poor survival outcome and promotes tumor progression in colorectal carcinoma”. Oncotarget, 2017. 8(29): p. 47943-47956, the authors showed that ADGRF5 is involved in colorectal cancer development involving the AKT / ERK signaling pathway.

[0007] However, the available literature lacks evidence of specific ADGRF5 ligands, particularly ADGRF5 inhibitors capable of blocking its activity and inhibiting the development of solid tumors. This prompted the inventors to seek effective anti-tumor therapies using immune cells lacking ADGRF5.

[0008] Over the course of their research, the inventors discovered that CD4+ T cells lacking ADGRF5 exhibit significantly stronger anti-tumor activity, compared to unmodified CD4+ T cells.Technical Problem

[0009] The technical problem to be addressed by the present invention is to provide effective cancer immunotherapy, especially against solid tumors.Solution to Problem

[0010] The object of the invention is a biological preparation containing 0.5 to 1 × 106CD4+ subpopulation T cells lacking ADGRF5 for use as a medication.

[0011] The biological preparation for use according to the invention is intended for treatment of neoplastic disorders of the gastrointestinal tract, specifically solid tumors.

[0012] The biological preparation for use according to the invention is in the form of a suspension of 0.5 to 1 × 106CD4+ subpopulation T cells devoid of ADGRF5, in a pharmaceutically acceptable buffer or in a 0.9% sodium chloride solution.

[0013] Preferably, the biological preparation for use according to the invention is administered subcutaneously into the tumor area.

[0014] Ongoing research work has found that CD4+ T cells lacking ADGRF5 have significantly stronger anti-tumor effects compared to unmodified CD4+T cells.

[0015] It should be noted that the anti-tumor effect of CD4+ T cells lacking ADGRF5 is possible not only in colorectal gastrointestinal cancers but can be demonstrated when applied to solid tumors. Moreover, it is highly likely that the modification to eliminate ADGRF5 from lymphocytes could lead to their enhanced anti-tumor effects on other lymphocyte subpopulations.

[0016] The object of the invention is illustrated in the Figures of the drawing, wherein:

[0017] shows the relative expression of IFN-γ (A) at the mRNA level in activated unmodified CD4+ T cells and CD4+ T cells lacking ADGRF5;

[0018] shows the relative expression of GZMB (B) at the mRNA level in activated unmodified CD4+ T cells and CD4+ T cells lacking ADGRF5;

[0019] shows the relative expression of PRF1 (C) at the mRNA level in activated unmodified CD4+ T cells and CD4+ T cells lacking ADGRF5.

[0020] The values in Figures 1A, 1B, 1C are presented as mean ± SEM, while statistical significance was determined in relation to activated unmodified CD4+ T cells and denoted by *: * for p<0.05 and *** for p<0.001, respectively.

[0021] shows the concentration of IL-10 (A) secreted from activated unmodified CD4+ T cells and CD4+ T cells lacking ADGRF5;

[0022] shows the concentration of IL-4 (B) secreted from activated unmodified CD4+ T cells and CD4+ T cells lacking ADGRF5;

[0023] shows the concentration of IL-5 (C) secreted from activated unmodified CD4+ T cells and CD4+ T cells lacking ADGRF5.

[0024] The values in Figures 2A, 2B, 2C are presented as mean ± SEM, while statistical significance was determined in relation to activated unmodified CD4+ T cells and denoted by *: ** for p<0.01 and *** for p<0.001, respectively.

[0025] shows the % of apoptotic cells in intestinal cancer cell cultures and inactivated or activated unmodified CD4+ T cells and CD4+ T cells lacking ADGRF5.

[0026] Values inare presented as mean ± SEM, while statistical significance was determined in relation to inactivated unmodified CD4+ T cells or inactivated CD4+ T cells lacking ADGRF5 and marked with * or #: ** for p<0.01 and **** for p<0.0001 or #### for p<0.0001, respectively.

[0027] shows the concentration of IFN-γ in cultures of intestinal cancer cells and activated unmodified CD4+ T cells or activated CD4+ T cells lacking ADGRF5.

[0028] The values inare presented as mean ± SEM, while statistical significance was determined in relation to intestinal cancer cells and denoted by *: ** for p<0,01, respectively.

[0029] shows time-dependent tumor size in untreated mice (control), in mice treated with 0.5 × 106unmodified CD4+ T cells, in mice treated with 0.5 × 106CD4+ T cells lacking ADGRF5;

[0030] shows tumor size at day 21 in untreated mice (control), mice treated with 0.5 × 106unmodified CD4+ T cells, in mice or treated with 0.5 × 106CD4+ T cells lacking ADGRF5.

[0031] Values in Figures 5A, 5B are presented as mean ± SEM, while statistical significance was determined relative to controls or mice treated with unmodified CD4+ T cells and denoted by * or #: ** for p<0.01 and **** for p<0.0001; or # for p<0.05; ## for p<0.01 and #### for p<0.001, respectively.

[0032] shows time-dependent tumor size in untreated mice (control), in mice treated with 1 ×106unmodified CD4+ T cells, in mice treated with 1 ×106CD4 +T cells lacking ADGRF5;

[0033] shows tumor size at day 21 in untreated mice (control), in mice treated with 1 ×106unmodified CD4+ T cells, in mice treated with 1 ×106CD4 +T cells lacking ADGRF5.

[0034] Values in Figures 6A, 6B are presented as mean ± SEM, while statistical significance was determined relative to controls or mice treated with unmodified CD4+ T cells and denoted by * or #: * for p<0.05 and **** for p<0.0001; or # for p<0.05; ## for p<0.01 and #### for p<0.001, respectively.

[0035] In the detailed embodiment of the invention presented below, a description of the experimental work carried out by the inventors is presented, and the results presented do not limit the scope of the invention.Example 1

[0036] CD4+ subpopulation T cells lacking ADGRF5 were obtained from spleen cells of ADGRF5 knockout mice using CD4+ subpopulation isolation kit from STEMCELL Technologies. Cells obtained in this way are characterized by CD4 marker expression, specific for CD4+ subpopulation T cells, and the absence of ADGRF5 expression.

[0037] The phenotype of wild-type and ADGRF5 knockout mice was analyzed by polymerase chain reaction (PCR). A fragment of the mouse tail constituted the test material, which was placed in a test tube and buffer containing 25 mM NaOH and 0.2 mM EDTA, pH 12, was added and incubated at 95°C for 30 minutes. After this time, the samples were mixed in a vortex mixer and incubated at 95°C for 30 minutes. 40 mM Tris / HCl, pH 5, was added to the samples and mixed using a vortex mixer. Subsequently, DNA was taken from the mixture (100 ng / µL) and the PCR reaction was carried out using the OneTaq® Quick-Load® 2X Master Mix with Standard Buffer kit from New England Biolabs, and a set of primers synthesized by Merck, including a forward primer with a sequence of 5’-GGAGGCTCTGTGCGTTTC-3’, reverse primer 1 with a sequence of 5’-CTGTGGACATGATGAAGGGTG-3’, and reverse primer 2 with a sequence of 5’-CTCCCTGAATCATAGTCTAGTCTCC-3’. The reaction was carried out at 94°C for 30 seconds, followed by 30 reaction cycles, including incubation at 94°C for 15 seconds, incubation at 55°C for 30 seconds, and incubation at 68°C for 1 minute. The final process step involved incubation at 68°C for 5 minutes. The reaction products were subjected to electrophoretic separation in a 3% agarose gel with ethidium bromide in the presence of a reference DNA marker within the range of 50 – 3000 base pairs. The separation was carried out in a buffer containing 40 mM Tris, pH 8.0, 20 mM acetic acid and 1 mM EDTA at 100 V for 60 minutes at room temperature. The resulting electrophoretic separation images were obtained using an inGenius camera and GeneSnap software.

[0038] To verify the phenotype of the obtained cell lymphocytes, specific anti-CD4 antibodies complexed with PE (Thermo Fisher Scientific) were used by flow cytometry. The acquired lymphocytes were resuspended in phosphate-buffered saline at pH 7.4, containing 10% bovine serum albumin, and non-specific antibody binding sites were blocked by incubating the cells in 2% normal rat serum for 15 minutes. The CD4-PE antibody was then added to the cells and incubated at 4°C for 1 hour. After this time, phosphate-buffered saline at pH 7.4 containing 10% bovine serum albumin was added to the cells and centrifuged at 300 × g for 5 minutes. This stage was repeated twice. Samples prepared in this way were analyzed by flow cytometry using a FACSymphony™ A1 analytical cytometer (BD Bioscience).

[0039] The anti-tumor effect of a biological preparation containing ADGRF5-deficient CD4+ T cells was determined by in vivo tests conducted on a mouse model of gastrointestinal cancers. For this purpose, mice of the C57BL / 6 strain were transplanted with allogeneic colon cancer cells – MC38 – using methods known in the state of the art.

[0040] The animals were divided into three cohorts comprising five mice each, treated with the biological preparation according to the invention, that is, ADGRF5-deficient CD4+ T cells as the test group, and control mice treated with unmodified CD4+ T cells, or 0.9% sodium chloride solution as the control group.

[0041] The cohorts were given ADGRF5-deficient CD4+ T cells or unmodified CD4+ T cells or 0.9% sodium chloride solution subcutaneously near the tumor on days 1, 8 and 15 of the experiment. Then, on day 21, mice from the three groups were euthanized, and the dissected tumors were further analyzed. The size of the tumors was determined using the equation: tumor size = (tumor width × tumor width × tumor length) / 2.

[0042] To confirm the anti-tumor activity of the ADGRF5-deficient CD4+ T-cell preparation, an in vitro assay was conducted, wherein MC38 tumor cells were cultured in the presence of unmodified CD4+ T-cells (control) or ADGRF5-deficient CD4+ T-cells (test group), followed by quantitative analysis of apoptotic cells. In a 96-well plate, 5 × 104MC38 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, and a mixture of penicillin and streptomycin. After 1 hour of incubation, 1 × 105wild-type CD4+ T-cells or 1 × 105 CD4+ T-cells with ADGRF5 absent were added to MC38 cells. In addition, selected MC38 cells co-cultured with CD4+ cells were treated with lymphocyte activators (Dynabeads™ Mouse T-Activator CD3 / CD28 for T-Cell Expansion and Activation from Gibco). In vitro cultures were conducted for 18 hours in a humid atmosphere at 37°C and in the presence of 5% CO2. After that time, the culture medium was removed, and the cells were washed with phosphate-buffered saline at pH 7.4, and subjected to a trypsinization process that allowed the cells to detach from the medium. Then, cells were transferred to tubes for staining and further analysis using flow cytometry. Acquired cells were resuspended in phosphate-buffered saline at pH 7.4 containing 10% bovine serum albumin, and non-specific antibody binding sites were blocked by incubating the cells in 2% normal rat serum for 15 minutes. The CD4-PE antibody was then added to the cells and incubated at 4°C for 1 hour. After this time, phosphate-buffered saline at pH 7.4 containing 10% bovine serum albumin was added to the cells and centrifuged at 300 × g for 5 minutes. This stage was repeated twice. Staining buffer and Hoechst 33342 stain were added to the cells and incubated at 4°C for 30 minutes, as recommended by the manufacturer of the apoptosis level analysis kit (Apoptosis and Necrosis Assay Kit from MedChemExpress). Samples prepared in this way were analyzed by flow cytometry using a FACSymphony™ A1 analytical cytometer (BD Bioscience).

[0043] Proteomic tests were also carried out to determine the levels of selected cytokines in media obtained from CD4+ T-cell cultures and co-cultures of MC38 CD4+T-cells. These tests were conducted using the Mouse ProcartaPlex™ Simplex Kit from Thermo Fisher Scientific and the MAGPIX System device (Luminex) according to the manufacturer's recommendations.Results

[0044] In vitro characterization of activated CD4+ T cells lacking ADGRF5 showed that these cells overexpress anti-tumor factors such as interferon-γ (IFN-γ) (), granzyme B (GZMB) () and perforin-1 (PRF1) (), compared to activated unmodified CD4+ T cells. Further analysis of both cell populations showed that CD4+ T cells lacking ADGRF5 were capable of enhanced secretion of the anti-tumor cytokine IFN-γ. Importantly, this population of CD4+ T cells is also characterized by a significant decrease in the production of immunosuppressive cytokines, such as interleukin-10 (IL-10), (), interleukin-4 (IL-4), () and interleukin-5 (IL-5), ().

[0045] The results show that a biologic preparation containing CD4+ T cells lacking ADGRF5 exhibits much stronger anti-tumor activity than unmodified CD4+T cells. According to the data shown in, activated CD4+ T cells lacking ADGRF5 are capable of much stronger apoptosis induction than activated unmodified CD4+ T cells. Importantly, proteomic tests have shown that it is the activated CD4+ T cells lacking ADGRF5, rather than the unmodified lymphocytes of this population, that are responsible for the enhanced IFN-γ secretion ().

[0046] As depicted in,and,, animals treated with a biological preparation containing 0.5 ×106and 1 ×106CD4+ T cells lacking ADGRF5 have a statistically significant decrease in tumor size.

Claims

Biological preparation containing 0.5 to 1 ×106CD4+ subpopulation T cells lacking ADGRF5 for use as a medicament.Biological preparation for use according to claim 1 in neoplastic disorders of the gastrointestinal tract, specifically solid tumors.Biological preparation for use according to claims 1 or 2, characterized in that it is in the form of a suspension of 0.5 to 1 × 106CD4+ subpopulation T cells lacking ADGRF5 in a pharmaceutically acceptable buffer or 0.9% sodium chloride solution.Biological preparation for use according to claim 3, characterized in that it is administered subcutaneously into the tumor area.

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