Efficacy of novel allogeneic cancer cells vaccine to treat colorectal cancer

Abstract

Colorectal cancer (CRC) remains a significant global health burden, emphasizing the need for innovative treatment strategies. 95% of the CRC population are microsatellite stable (MSS), insensitive to classical immunotherapies such as anti-PD-1; on the other hand, responders can become resistant and relapse. Recently, the use of cancer vaccines enhanced the immune response against tumor cells. In this context, we developed a therapeutic vaccine based on Stimulated Tumor Cells (STC) platform technology. This vaccine is composed of selected tumor cell lines stressed and haptenated in vitro to generate a factory of immunogenic cancer-related antigens validated by a proteomic cross analysis with patient's biopsies. This technology allows a multi-specific education of the immune system to target tumor cells harboring resistant clones. Here, we report safety and antitumor efficacy of the murine version of the STC vaccine on CT26 BALB/c CRC syngeneic murine models. We showed that one cell line (1CL)-based STC vaccine suppressed tumor growth and extended survival. In addition, three cell lines (3CL)-based STC vaccine significantly improves these parameters by presenting additional tumor-related antigens inducing a multi-specific anti-tumor immune response. Furthermore, proteomic analyses validated that the 3CL-based STC vaccine represents a wider quality range of tumor-related proteins than the 1CL-based STC vaccine covering key categories of tumor antigens related to tumor plasticity and treatment resistance. We also evaluated the efficacy of STC vaccine in an MC38 anti-PD-1 resistant syngeneic murine model. Vaccination with the 3CL-based STC vaccine significantly improved survival and showed a confirmed complete response with an antitumor activity carried by the increase of CD8+ lymphocyte T cells and M1 macrophage infiltration. These results demonstrate the potential of this technology to produce human vaccines for the treatment of patients with CRC.

Keywords: antigens; cancer vaccine; colorectal cancer; haptenation; immune response; immunotherapy; proteomics; stimulated tumor cells.

Figure 1

Manufacturing flowchart. Representation showing vaccines production steps for study A (one cell line-based vaccine: CT26) and study B & C (three cell lines-based vaccine: CT26, CMT-93 and LTPA). -S, stressed cells; -H, haptenated cells; -SH, stressed and haptenated cells.

Figure 2

Experimental plan of studies A, B and C sets with N: number of mice per group. mGM-CSF: mouse granulocyte macrophage colony stimulating factor. IS, immunostimulant; 15 mg/kg CP and 5 µg mGM-CSF; 3CL, three cell lines; -S, stressed cells; -H, haptenated cells; -SH, stressed and haptenated cells.

Figure 3

Effect of stimulated and/or haptenated CT26 vaccine. (A) Tumor growth curves of tumor-bearing mice (n = 10 per group). Administration of the CT26-SH (G4) vaccine significantly retarded tumor growth compared to that in the control group (G1) (p-value=0.003, Student t test at day 20). The CT26-SH group showed a better effect than the other two treatment groups (G2: CT26-S and G3: CT26-H) on day 20. ***: 0.001 ≥ p-value > 0.0001. (B) Kaplan-Meier survival analysis of tumor-bearing mice (n = 10 per group). With the best survival time for CT26-SH group (G4) compared to the control group (G1) (p-value=0.0748, log-rank test), CT26-S vaccinated group (G2), and CT26-H vaccinated group (G3). Log-rank test comparing all groups with each other, p-value =0.0044. NE: not evaluable; the bounds of confidence indices (CI) cannot be calculated. At Risk table shown in Supplementary Figure 1A .

Figure 4

Immunostimulant associated to CT26-SH vaccine. (A) Tumor growth curves of the tumor-bearing mice (n = 10 in the control group: G1, n = 10 in the CT26-SH group: G4, n = 10 in IS group: G5 and n = 9 in the IS + CT26-SH group: G6). Administration of the CT26-SH vaccine + IS (G6) significantly retarded tumor growth compared to control group (G1) (p<0.0003, Student’s t-test at day 20). ns: no significant. **: 0.01 ≥ p-value > 0.001; ***: 0.001 ≥ p-value > 0.0001; ****: 0.0001 ≥ p-value. (B) Kaplan-Meier survival analysis of tumor-bearing mice (n = 10 in the control group: G1, n = 10 in the CT26-SH group: G4, n = 10 in IS group: G5 and n = 9 in the IS + CT26-SH group: G6). Survival analyses indicated the best survival time in the IS + CT26-SH group compared to the control group (p-value=0.0046, log-rank test), IS group (p-value=0.0220, log-rank test) and CT26-SH vaccinated group (p-value=0.0748, log-rank test). Log-rank test comparing all the groups with each other, which shows the significance of one of the groups on this parameter log-rank test p-value=0,0044. NE, not evaluable, the bounds of confidence indices (CI) cannot be calculated. IS, immunostimulant: 15 mg/kg CP and 5 µg mGM-CSF. At Risk table shown in Supplementary Figure 1B .

Figure 5

Effect of three cell lines-based vaccine, stimulated and haptenated. (A) Tumor growth curves of tumor-bearing mice (n = 20 in the control group: G’1, n = 18 in the IS + CT26-SH group: G’2 and n = 18 in the IS + 3CL-SH group: G’3). Administration of the IS + 3CL-SH vaccine significantly retarded tumor growth compared to that in the control group (p-value =0.0067, Student’s t-test at day 22). **: 0.01 ≥ p-value > 0.001; ***: 0.001 ≥ p-value > 0.0001. (B) Kaplan-Meier analysis of the survival of tumor-bearing mice (n = 20 in the control group, n = 18 in the IS + CT26-SH group and n = 18 in the IS + 3CL-SH group). Survival analyses indicated that the IS + 3CL-SH group had the best survival time compared to the control group (p<0,0001, log-rank test) and the IS + CT26-SH vaccinated group (p-value=0,0023, log-rank test). Log-rank test comparing all the groups with each other, which shows the significance of one of the groups on this parameter log-rank test p-value < 0,0001. NE, not evaluable, the bounds of confidence indices (CI) cannot be calculated. 3CL-SH: three cell lines-based vaccine, stimulated and haptenated. IS, immunostimulant: 15 mg/kg CP and 5 µg mGM-CSF. At Risk table shown in Supplementary Figure 1C .

Figure 6

STC vaccine at proteomic level. (A) Venn diagram comparing the identified proteins in each cell models for untreated samples. (B) Venn diagram showing percentage of specific proteins of each cell line before and after simulation and haptenation. (C) Up-regulated proteins after simulation and haptenation, Venn diagram showing the percentage of specific and shared proteins for each cell line. (D) Venn diagram showing the overlapped proteins between CT26 cell line and the 3CL-SH vaccine. UP, up-regulated; NT, untreated.

Figure 7

Effect of the 3 cell lines-STC based vaccine on MC38 PD1-R. (A) Tumor growth curves of tumor-bearing mice (n = 15 per group, G’’1: control group, G’’2: anti-PD-1 group, G’’3: IS + anti-PD1 group and G’’4: IS + 3 CL-SH group. No statistical difference between the groups was observed at D12. (B) Individual curves for the tumor growth of mice of the G’’4: IS + 3 CL-SH group. (C) Kaplan-Meier analysis of the survival of tumor-bearing mice (n = 15 per group G’’1: control group, G’’2: anti-PD-1 group, G’’3: IS + anti-PD1 group and G’’4: IS + 3 CL-SH group). Significant improvement of the survival of mice bearing MC38- PD-1R tumor treated by 3 CL-SH+IS vaccine vs control (Log-rank Mantel-Cox test, p-value=0,0368). (D) CD8+ infiltration in the TME (Tumor Micro-environment) at D14 (n=5/group). Group treated with IS + 3 CL-SH vaccine displays a significant increase in the infiltration of cytotoxic CD8+ T cells when compared with the control group (p-value =0.045, Student’s t-test). ns, non-significant. (E) TAM 1 infiltration in the TME (Tumor Micro-environment) at D14 (n=5/group) showing increase in anti-tumoral M1 macrophages in the group treated with IS + 3 CL-SH vaccine vs control (p-value =0.02, Student’s t-test). 3CL-SH, three cell lines-based vaccine, stimulated and haptenated. ns, non-significant. IS, immunostimulant: 15 mg/kg CP and 5 µg mGM-CSF. *: 0.05 ≥ p-value.