Combination treatment of radiofrequency ablation and peptide neoantigen vaccination: Promising modality for future cancer immunotherapy

Abstract

Background: The safety and immunogenicity of a personalized neoantigen-based peptide vaccine, iNeo-Vac-P01, was reported previously in patients with a variety of cancer types. The current study investigated the synergistic effects of radiofrequency ablation (RFA) and neoantigen vaccination in cancer patients and tumor-bearing mice.

Methods: Twenty-eight cancer patients were enrolled in this study, including 10 patients who had received RFA treatment within 6 months before vaccination (Cohort 1), and 18 patients who had not (Cohort 2). Individualized neoantigen peptide vaccines were designed, manufactured, and subcutaneously administrated with GM-CSF as an adjuvant for all patients. Mouse models were employed to validate the synergistic efficacy of combination treatment of RFA and neoantigen vaccination.

Results: Longer median progression free survival (mPFS) and median overall survival (mOS) were observed in patients in Cohort 1 compared to patients in Cohort 2 (4.42 and 20.18 months vs. 2.82 and 10.94 months). The results of ex vivo IFN-γ ELISpot assay showed that patients in Cohort 1 had stronger neoantigen-specific immune responses at baseline and post vaccination. Mice receiving combination treatment of RFA and neoantigen vaccines displayed higher antitumor immune responses than mice receiving single modality. The combination of PD-1 blockage with RFA and neoantigen vaccines further enhanced the antitumor response in mice.

Conclusion: Neoantigen vaccination after local RFA treatment could improve the clinical and immune response among patients of different cancer types. The synergistic antitumor potentials of these two modalities were also validated in mice, and might be further enhanced by immune checkpoint inhibition. The mechanisms of their synergies require further investigation.

Clinical trial registration: https://clinicaltrials.gov/, identifier NCT03662815.

Keywords: cancer; immune checkpoint inhibition; immunotherapy; neoantigen vaccine; radiofrequency ablation.

Figure 1

Better clinical response in RFA+Vac patients upon receiving iNeo-Vac. (A) Swimmer plots of RFA+Vac patients (with yellow background) and Vac patients (with blue background); (B) Waterfall plots of RFA+Vac patients (shown in yellow) and Vac patients (shown in blue). Dashed line above 20% or below -30% indicate 20% increase (≥ 20% is considered as progressive disease) or 30% reduction (≤ -30% is considered as partial response) of the sum of the longest diameters of the tumors, respectively. # suggested that new lesion(s) was found; (C) Spider plots of RFA+Vac patients and Vac patients. The “best clinical response” in panel b was defined as the largest reduction of target lesion in size during treatment.

Figure 2

RFA+Vac patients showed better progression-free survival (PFS) and overall survival (OS). (A) Progression-free survival of Vac patients (shown in blue) and RFA+Vac patients (shown in yellow). (B) Overall survival of Vac patients (blue) and RFA+Vac patients (yellow). (C) Characteristics of patients in RFA+Vac colorectal group, in comparison to that of patients in RFA colorectal group. (D) Overall survival of RFA-pretreated colorectal cancer patients (RFA+Vac colorectal) (yellow) in this study, in comparison to RFA-treated colorectal cancer patients (RFA colorectal) (green) during the same period at Sir Run Run Shaw Hospital. Longer OS was observed for patients receiving additional neoantigen vaccination (p = 0.037). Survival data were compared by logrank test.

Figure 3

RFA+Vac patients showed better immune responses upon receiving iNeo-Vac-P01. (A) Ex vivo ELISpot assay results of each patient. Green triangle and red diamond represent the relative response rate, calculated per the ratios of peptides with positive ELISpot reads to all peptides immunized before and after vaccination, respectively. The bar chart shows the spot numbers of the peptide or peptide pool with best reads per 10⁵ PBMCs. # Indicated that peptide pools were used for ELISpot assay for this patient; + indicated that relatively high abundant novel clones of peripheral T cells were detected for this patient after vaccination. (B, C) Data shown in Box and Whiskers (with minimum to maximum, showing all points and median). (B) Baseline and post-vaccination ELISpot spot counts per 10⁵ PBMCs of RFA+Vac (yellow) and Vac (blue) patients. c) ELISpot spot counts per 10⁵ PBMCs at baseline and post vaccination of the peak response for RFA+Vac patients with t_int < 3 months, or 3 ≤ t_int < 6 months. No significant difference was found between the two groups at baseline (blue and yellow); however, the difference was intensified post vaccination, as patients with t_int < 3 months showed stronger peak response (red). Baseline response and peak response of all RFA+Vac patients showed statistically significant difference (p < 0.0051). (D) Flow cytometric analyses of RFA+Vac and Vac patients on T cell subsets. RFA+Vac and Vac patients were further divided into two subgroups: patients with good response (OS ≥ 12 months) and poor response (OS < 12 months); number of active CD4⁺ T cells (# per μL), and proportion of CD4⁺ or CD8⁺ T cells that expressed CTLA4 (%) at baseline, prime phase and boost phase were analyzed, respectively. (E) Flow cytometric analyses of RFA+Vac and Vac patients on cytokines including IFN-γ, IL-6 and TNF-α at baseline, prime phase, and boost phase. A t-test analysis was applied to indicate the significance in data difference with a two-tailed p value. ns, not significant (p > 0.05); *p ≤ 0.05; **p ≤ 0.01.

Figure 4

Case report of patient P015, who had multiple RFA treatments prior to vaccination. (A) The treatment scheme of patient P015. Two batches of iNeo-Vac-P01 were scheduled for this patient. (B) CT scan images of the tumor at different time points during the treatment. The red arrows indicated the corresponding tumor lesion. SLDs: sum of longest diameters. SLDs: sum of the longest diameters. (C, D) Ex vivo ELISpot assay of IFN-γ⁺ PBMCs during vaccination. (E) Increased abundance of peripheral neoantigen-specific T cell clones post vaccination as detected by TCR sequencing. (F) Multiplexed IF images of FFPE samples obtained from patient P015 pre- and post-vaccination. Signals of CD8, CD4 and Granzyme B are shown in fuchsias, green and yellow. Double stranded DNA is shown in blue. Shown on the right are the proportion (%) and density (counts per mm²) of CD4⁺ Granzyme B⁺ and CD8⁺ Granzyme B⁺ cells.

Figure 5

Combination treatment of RFA and neoantigen vaccination inhibited tumor growth and elicited robust local and systemic antitumor immune responses in mice. (A) Schematic illustration of the treatment mice received. Mouse models with unilateral or bilateral tumor(s) were established; mice with bilateral tumors received RFA treatment on the left flank tumor. (B, C) Tumor growth of mice receiving different treatments. Ablation of an irrelevant tumor (4T1) did not help inhibit the growth of CT26 tumor. Combinational use of RFA and iNeo-Vac-P01 (RFA+Vac) significantly slowed down the growth of CT26 tumor, and the inclusion of anti-PD-1 further enhanced this antitumor effect. (D) Flow cytometric analyses of PD-1 expression in tumor tissues (n=3). (E) Flow cytometric analyses of multifunctional T cells, gating on CD8⁺ perforin⁺ cells (n=3). (F) Flow cytometric analyses of cytotoxic T cells, gating on CD8⁺ IFN-γ⁺ cells (n=3). (G) Ex vivo ELISpot assay. Spleen cells were collected and re-stimulated by neoantigen peptides. Data shown in Mean ± SD; two-tailed p value was obtained through unpaired t test. ns, not significant (p > 0.05); **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.