Combination treatment of radiofrequency ablation andpeptide neoantigen vaccination:Promising modality for futurecancer immunotherapy Jiawei Shou1†, Fan Mo2,3,4,5 †, Shanshan Zhang2,6 †, Lantian Lu2,7, Ning Han2,8, Liang Liu2, Min Qiu2, Hongseng Li1, Weidong Han1, Dongying Ma2, Xiaojie Guo2, Qianpeng Guo2, Qinxue Huang2, Xiaomeng Zhang2, Shengli Ye9, Hongming Pan1*, Shuqing Chen2,3,6,10*and Yong Fang1* 1Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China,2Hangzhou Neoantigen Therapeutics Co., Ltd., Hangzhou, China,3College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China,4Vancouver Prostate Centre, University of British Columbia, Vancouver, BC, Canada,5Hangzhou AI-Force Therapeutics Co., Ltd., Hangzhou, China,6Zhejiang California International Nanosystems Institute, Zhejiang University, Hangzhou, China,7School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, Australia,8Hangzhou AI-Nano Therapeutics Co., Ltd., Hangzhou, China,9Shulan (Hangzhou) Hospital, Hangzhou, China, 10ZJU-Hangzhou Global Scienti ﬁc and Technological Innovation Center, Hangzhou, China 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 t reatment 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 ef ﬁcacy 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- gELISpot assay showed that patients in Cohort 1 had stronger neoantigen-speci ﬁc immune responses at baseline and post vaccination. Mice receiving combination treatm ent of RFA and neoantigen vaccines displayed higher antitumor immune responses than mice receiving single modality. The combination of PD-1 b lockage with RFA and neoantigen vaccines further enhanced the antitumor response in mice. Frontiers in Immunology frontiersin.org 01 OPEN ACCESS EDITED BY Jai Rudra, Washington University in St. Louis, United States REVIEWED BY Rupsa Basu, Humane Genomics, United States Philippe Rochigneux, Institut Paoli-Calmettes (IPC), France Wayne Robert Thomas, University of Western Australia, Australia *CORRESPONDENCE Hongming Pan panhongming@zju.edu.cn Shuqing Chen chenshuqing@zju.edu.cn Yong Fang fangyong@zju.edu.cn †These authors share ﬁrst authorship SPECIALTY SECTION This article was submitted to Vaccines and Molecular Therapeutics,a section of the journalFrontiers in Immunology RECEIVED 22 July 2022 ACCEPTED 07 September 2022 PUBLISHED 29 September 2022 CITATION Shou J, Mo F, Zhang S, Lu L, Han N, Liu L, Qiu M, Li H, Han W, Ma D, Guo X, Guo Q, Huang Q, Zhang X, Ye S, Pan H, Chen S and Fang Y (2022) Combination treatment ofradiofrequency ablation and peptide neoantigen vaccination: Promising modality for futurecancer immunotherapy.Front. Immunol. 13:1000681. doi: 10.3389/fimmu.2022.1000681TYPE Original Research PUBLISHED 29 September 2022 DOI10.3389/fimmu.2022.1000681 Conclusion: Neoantigen vaccination after local RFA treatment could improve the clinical and immune response among patients of different cancer types. The synergistic antitumor potential s 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/ , identi ﬁer NCT03662815. KEYWORDS neoantigen vaccine, radiofrequency abl ation, immune checkpoint inhibition, cancer, immunotherapy Introduction Neoantigens are a type of tumor-speci ﬁca n t i g e n st h a ta r e generated by non-synonymous mutations ( 1). During the past ﬁve years, neoantigen-based personalized cancer vaccines have thrived as a result of recent advancement in sequencing technologies and immunoinformatic algorithms. H owever, neoantigen vaccination alone might be inef ﬁcient to elicit robust responses to eliminate tumors, due to the complicated tumor immunosuppressive microenvironment ( 2). Thus, a variety of different strategies have been explored in both preclinical and clinical settings to improve the ef ﬁcacy of neoantigen vaccines ( 3). For example, vaccine adjuvants such as toll-like receptors (TLRs) have been administered with neoantig en vaccines to enhance their anticancer ef ﬁcacy. Besides, various delivery approaches are being investigated to impr ove the unfavorable in vivo pharmacokinetics of neoantigens, and boost the insuf ﬁcient immune responses resulting from the discrepant physicochemical properties of neoantigens ( 2,4–8). Currently, it seems promising to couple neoantigen vaccines with other modalities that can modify or revert the immunosuppressive TME, such as immune checkpoint inhibition (ICI) ( 3–7,9,10). Up to date, a variety of trials have been launched to investigate the ef ﬁcacy and underlying mechanisms of the combination of ICIs with neoantigen vaccines (NCT03715985, NCT03422094, NCT02287428, NCT02950766, NCT03380871 and NCT02897765). Radiofrequency ablation (RFA) has been commonly utilized in clinics as a minimally invasive treatment, increasing local temperature to induce coagulative necrosis of tumor. Besides, it has been demonstrated that RFA can activate the host ’si m m u n e system through different mechanisms ( 11). Firstly, cell death resulted from RFA-induced hyperthermic injury and coagulativenecrosis usually leads to the release of both intracellular antigens and damage-associated molecular patterns (DAMPs) such as heat shock proteins, mobility group protein B1, as well as nucleic acids (DNA and RNA), which can be endocytosed and processed by dendritic cells (DCs) to elicit a systemic immune response ( 12– 14). Secondly, RFA can modulate tumor microenvironment (TMB) by upregulating the expression of immunostimulating factors including interferon- g(IFN- g), tumor necrosis factor alpha (TNF- a), interleukin (IL)-1 b, IL-8 and IL-2, and inhibiting the expression of immunosuppressive soluble IL-2 receptor and hepatocyte growth factor ( 12). Moreover, after RFA treatment, it is common to observe the accumulation of local tumor-in ﬁltrating lymphocytes and peripheral circulation lymphocytes ( 12), together with the decease of the proportions of regulatory T cells, tumor-associated macrophages andneutrophiles ( 14), mounting systemic antitumor responses. However, despite the above anticancer advantages, RFA alone may still be insuf ﬁcient to prevent cancer progression or inhibit cancer recurrence due to many factors such as the inef ﬁcient antigen presentation by DCs ( 13,15). Recently, the synergistic antitumor activity of RFA and ICI has been reported in both preclinical and clinical settings ( 11,15,16). Also, PD-L1, PD-1 and LAG3 were found to be upregulated in distant non-RFA tumors after RFA treatment, indicating the synergy between RFA and ICI ( 14,15). Several clinical trials have been launched to explore the ef ﬁcacy and mechanisms of the combination of RFA with ICI (NCT01853618; NCT02821754; NCT03939975) ( 12). As described above, both neoantigen vaccines and RFA require synergetic therapeut ic modality to further enhance their ef ﬁcacy to prevent tumor progression or inhibit tumor recurrence. The fact that RFA could elicit pre-vaccination response ( 10,17), encourages the combination of RFA with neoantigen vaccines to translate into clinical bene ﬁts. Since both RFA and neoantigen vaccination could synergize with ICIs; it would be worthwhile to investigate the synergies between RFA, neoantigen vaccines and ICIs in combating tumors. We report here the promising results of RFA treatment prior to neoantigenAbbreviations: AE, Adverse effect; DMSO, dimethyl sulfoxide; ICI, Immune checkpoint inhibition; OS, overall survival; PBMCs, Peripheral blood mononuclear cells; PFS, progression-free survival; RFA, Radiofrequency ablation; TCR, T cell receptor; TLR, toll-like receptor.Shou et al. 10.3389/ ﬁmmu.2022.1000681 Frontiers in Immunology frontiersin.org 02 vaccination in patients of different cancer types, as well as the synergistic antitumor effects of combinational RFA, neoantigen vaccination and ICIs in a mouse colorectal tumor model. Methods Clinical trial design and patient characteristics Patients aged ≥18 with different cancer types including colon cancer, melanoma, lung cancer, pancreatic cancer, biliary tract cancer, ovarian cancer, b reast cancer, gastric cancer, parotid cancer, and adrenal sebaceous adenocarcinoma were enrolled in a single-arm, open-label and investigator-initiated phase I study at Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University in China (NCT03662815). Eligible patients had a baseline Eastern Cooperative Oncology Group (ECOG) performance status of 0-1. The primary endpoints of this study were safety and feasibility, and the secondary endpoint was efﬁcacy based on progression-free survival (PFS), overall survival (OS), and neoantigen-speci ﬁc immune response. For all enrolled patients, the treatment scheme and clinical response of personalized vaccines were summarized in Supplementary Table S1 . Colorectal patients receiving RFA and neoantigen vaccines in our study were retrospectively reviewed and compared to colorectal patients (RFA colorectal group) who received only RFA at Sir Run Run Shaw Hospital af ﬁliated with Zhejiang University School of Medicine between 2017 and 2020, for their OS. Patients with similar characteristics to our patients including age, sex distribution, tumor metastases, prior systemic therapies and RFA treatments were selected for comparison. Clinical assessment, monitoring, and follow-up including physical examination, ECOG performance, vital sign, blood test as well as urinalysis were conducted on a regular basis according to the follow-up plan. Ima ging tests at baseline and approximately every 8 weeks post-vaccination were arranged for each patient to assess the clinical ef ﬁcacy. Enzyme-linked immunospots (ELISpot) assa y, T-cell receptor (TCR) sequencing, and ﬂow cytometry analyses on cytokines and T- cell subsets were performed pre- and post-vaccination to evaluate the speci ﬁc immune response ( Supplementary Methods ). All tumors were assessed by investigators according to RECIST v1.1 criterion at baseline and approximately every 8 weeks thereafter. Each patient was monitored during vaccine treatment and followed up every 3 months after the discontinuation of treatment. Treatment-related adverse events (AEs) were recorded and graded for safety evaluation according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.03) throughout the whole treatment. This study was approved by Institutional Review Board and Independent Ethics Committee, and conducted in accordance with the Declaration of Helsinki and theInternational Conference on H armonization Guidelines for Good Clinical Practice. All patients had given written informed consent before the initiation of any study procedures. Signed permission to disclose their personal health information (PHI) was collected. Vaccines and immunization schedule Whole-exome sequencing was performed for each patient (Supplementary Methods ;Supplementary Table S2 ). Personalized peptide vaccines based on identi ﬁed neoepitopes as well as HLA typing of each patient, were designed using our in-house pipeline iNeo-Suite ( Supplementary Methods ; Supplementary Table S3 and Supplementary Table S4 ). All peptides were manufactured through solid-phase peptide synthesis at GMP-like standard (bacteria-free, less than 10 EU/ mg endotoxin, > 95.0% purity) ( Supplementary Table S5 ). Water insoluble peptides were removed from the formulation of iNeo- Vac-P01, as auxiliary solvents such as dimethyl sulfoxide (DMSO) were not applicable due to ethical concerns. iNeo-Vac-P01 comprises of 5~20 peptides with the length ranging from 15 to 35 amino acids. The customized peptides were pooled into 3~4 groups according to their HLA typing, afﬁnity, and allele frequency, and delivered subcutaneously (s.c.) to both sides of upper arms and para-umbilical area at a dose of 100 or 300 mg/peptide per 1mL injection ( Supplementary Table S1). For vaccination, iNeo-Vac-P01 was administered during the ﬁrst month on day 1, 4, 8, 15 and 22 as prime. Boosts were given to each patient on day 78 and 162, and every 2 to 3 months afterwards until disease progression was found. Another batch of vaccines was designed and administered for several patients, as they had an extra puncture biopsy on their new lesions during the treatment and had good treatment compliance. Prior to each vaccination, 800 mL granulocyte-macrophage colony- stimulating factor (GM-CSF) was injected (s.c.) as adjuvant at a concentration of 50 mg/mL nearby each injection site of iNeo- Vac-P01. Animal study Seventy BALB/c female mice aged 6-8 weeks were purchased from Shanghai Slac Laboratory Animal Co. Ltd., and implanted with either bilateral or unilateral tumors. The mouse colon cancer cell line CT26 and breast cancer cell line 4T1 were obtained from Center for New Drug Safety Evaluation and Research, China Pharmaceutical University. Adherent tumor cells were cultured as described in Supplementary Methods . To obtain bilateral tumors, CT26 or 4T1 tumors were subcutaneously injected at the density of 1×106/100 mL on the leftﬂank, while CT26 tumor was injected at the density of 7.5×104/100 mL on the right ﬂank. Unilateral tumor model wasShou et al. 10.3389/ ﬁmmu.2022.1000681 Frontiers in Immunology frontiersin.org 03 established by subcutaneous injection of CT26 cells at a density of 7.5×104/100 mL on the right ﬂank. Twenty mice bearing CT26 unilateral tumor were randomly and evenly divided into 2 groups, and then treated with PBS (PBS group), and iNeo-Vac-P01 (Vac group), respectively. Similarly, another 4 groups of mice bearing CT26 bilateral tumors were treated with RFA, RFA in combination with anti- PD-1 ( InVivo MAb anti-mouse PD-1, Bio X Cell, Lebanon, USA) (RFA+anti-PD-1), RFA in combination with Vac (RFA+Vac), and RFA in combination with both Vac and anti-PD-1 (RFA +Vac+anti-PD-1), respectively. Only tumors on the left ﬂank were ablated. In addition, another group of mice (n=10) were implanted with bilateral heterogenous tumors (CT26 tumor on the right ﬂank, and 4T1 tumor on the left ﬂank), and received RFA on the left ﬂank to study the abscopal effect as a control group. Mice bearing bilateral tumors were scheduled to receive RFA treatment 6 days post tumor inoculation, where the size of the left ﬂank tumors reached around 6 mm in diameter. RFA was performed using a clinically available ablation device consisted of a radiofrequency energy generator (STARmed, RF generator VRS01) and a 17-gauge single ablation electrode with an active tip of 1 cm (STARmed, 18-07s07f). The tip was inserted orthogonally into the center of the tumor, with the energy titrated to ensure a tip temperature around 70°C for 3.5 to 4.5 minutes depending on the actual tumor size. CT26-speci ﬁc neoantigens were identi ﬁed, prioritized, and then designed into peptide vaccines ( Supplementary Methods , Supplementary Table S12 ). iNeo-Vac-P01 (100 mL, 0.1 mg/ peptide) was administered s.c. to mice on day 8, 9, 12, 16, 21 and 28 after tumor inoculation. Anti-PD-1 antibody (100 mL, 200 mg/ mL) was administered to mice intraperitoneally on day 14, 17, 20 and 23. Potential biomarkers were investigated using peripheral blood samples for ﬂow cytometry. Bothspleen and peripheral blood samples were tested using ELISpot assay toevaluate the neoantigen- speciﬁci m m u n er e s p o n s e( Supplementary Methods ). All experiments were approved by Laboratory Animal Management and Ethics Committee of ZCMU (approval number: IACUC-20210301-02) and performed in accordance with the guidelines of Animal Ethical and Welfare Committee of ZCMU. Anesthesia was performed by iso ﬂurane inhalation. All tumors were implanted into the mice ’sﬂanks to minimize distress and negative effects on normal body functions. When the tumor size reached a maximum diameter of 20 mm or micestarted to show signs of suffering such as reluctance to move, hunched posture, ≥20% weight loss, or body condition scored 2/ 5 or less, euthanasia was performed. At the end of this study, all mice were euthanized by CO 2inhalation. Statistical analysis The data used in the analyses of safety and clinical effects were collected from patients who received at least one dose ofiNeo-Vac-P01. Descriptive statistics were applied to determine the characteristics of baseline and assess the safety pro ﬁle of vaccine. Disease control rate (DCR) was de ﬁned as the proportion of complete response (CR), partial response (PR) and stable disease (SD) for best clinical response. Standard RECISTv1.1 guideline was applied for the analysis of clinical response. The Kaplan-Meier curve of patients was plotted with GraphPad Prism 5 (v5.01) using logrank test. Results Patient demographics A total of 28 patients with different tumor types, including colon cancer, melanoma, lung cancer, pancreatic cancer, biliary tract cancer, ovarian cancer, breast cancer and others were enrolled in this study from February 7th, 2018. All data in this study were collected before May 1st, 2021. Although patients were not initially randomized into separate cohorts, distinct differences in clinical response and immune response were found between Cohort 1 (RFA+Vac group, n=10) and Cohort 2 (Vac group, n=18) after retrospective analysis. Patients in Cohort 1, referred as RFA+Vac patients, had received single or multiple RFA treatments within 6 months before they received their ﬁrst dose of personalized vaccine; while patients in Cohort 2, referred as Vac patients, had not. The patientdemographics,baseline disease characteristics,and previous treatments were summarized in Table 1 ,i n d i c a t i n gt h a ta m o n g s t RFA+Vac patients, 4 (40%), 5 (50%) and 1 (10%) patients had liver metastases, both liver and lung metastases, and bone metastases, respectively. Personalized peptide vaccine, iNeo-Vac-P01, was successfully manufactured and scheduled for all patients. The treatment scheme and clinical response of each patient were presented in Supplementary Table S1 .N os i g n i ﬁcant difference was found for the lines of prior systematic therapy between RFA+Vac and Vac groups. Safety and tolerability of iNeo-Vac-P01 vaccination All patients received at least 5 d oses of vaccines as prime doses, and 1 - 6 boost doses, except for Patient P014 who dropped out ofstudy before the 5 thprime vaccination. A second batch of iNeo-Vac- P01 was scheduled for patients P013, P015 and P019 (Figure 1A ), and clinical evaluation on PFS of these patients was based on the ef ﬁcacy of vaccination during the ﬁrst batch only. The treatment was generally well tolerated, with m ost AEs being graded grade 1-2 (Table 2 ). The most commonly reported treatment-related toxicities were fatigue, fever and muscle soreness. No treatment- r e l a t e ds e r i o u sa d v e r s ee v e n t s( S A E s )o rd e a t hw a sr e p o r t e d .A l lA E s were reversible without special nursing. The types of treatment- related AEs were considered unrelated to the types of cancer.Shou et al. 10.3389/ ﬁmmu.2022.1000681 Frontiers in Immunology frontiersin.org 04 Neoantigen vaccination showed better clinical response in RFA+Vac patients In this study, at least one target lesion was evaluated for each patient. For the 9 evaluable RFA+Vac patients, 11.11% (1/9) displayed PR, 77.78% (7/9) displayed SD and 11.11% (1/9) d i s p l a y e dp r o g r e s s i o nd i s e a s e( P D ) ;w h i l ef o rt h e1 6e v a l u a b l eV a c patients, 68.75% (11/16) displayed SD and 31.25% (5/16) displayed PD (Figure 1 andSupplementary Table S11 ). Three patients (P002, P003 and P014) were excluded from due to the development of new lesions before their ﬁrst post-treatment follow-up assessment. Overall, tumor reduction was observed in 55.56% (5/9) RFA+Vac patients and only 25.00% (4/16) Vac patients ( Figures 1B ,C). Nine RFA+Vac patients and eighteen Vac patients were followed up for progression free survival (PFS) and overall survival (OS) (Figure 2 ). One patient (P014) in RFA+Vac group was excluded from this assessment due to study drop-out before the completion of primevaccination. The rest patients all received ≥5 doses of vaccines. The median PFS (mPFS) of RFA+Vac patients was 4.42 months (1.67 – 11.50 months); while Vac patients 2.82 months (1.53 –12.13 months) (Figure 2A ). The median OS (mOS) of RFA+Vac patients was 20.18 months (7.43 –36.73 months), longer than 10.94 months (2.27 – 38.17 months) for Vac patients ( Figure 2B ). Differences in mPFS and mOS between RFA+Vac and Vac groups were both statistically signiﬁcant ( p<0 . 0 5 ) . Combination treatment bene ﬁts colorectal patients in survival To further evaluate whether the application of personalized vaccine iNeo-Vac-P01 could improve the clinical response of patients after RFA treatment, the OS of colorectal cancer patients receiving combination treatment of RFA and iNeo-Vac-P01 in thisTABLE 1 Demographics and characteristics at baseline. Characteristics RFA+Vac Patients (N = 10) Vac Patients (N = 18) Age category-no. (%) <65 yrs.