Oncovir (United States)

Abstract Purpose To evaluate safety and immunogenicity of intramuscularly delivered personalized neoantigen synthetic long peptide (SLP) vaccines in patients with advanced solid tumors. Patients and Methods In this Phase I trial, 12 patients with advanced melanoma (n=9) or renal cell carcinoma (n=3) who could no longer access further standard treatments received intramuscular neoantigen SLP vaccines with poly-ICLC. Each vaccine contained ∼20 predicted neoantigen peptides. Adverse events were monitored throughout vaccination and follow-up. Immune profiling was performed at baseline and predefined post-vaccination time points. Results Intramuscular neoantigen vaccination was well tolerated, with only grade 1–2 local pain or fever and no immune-mediated toxicities. All participants developed de novo T-cell responses, detectable within one week. On average, 46% of peptides per patient were immunogenic, inducing both CD8⁺ and CD4⁺ neoantigen-specific responses. Patients previously treated with immune checkpoint inhibitors (ICIs) had higher baseline immunity but achieved comparable post-vaccination responses to ICI-naïve patients. IFN-γ–dominant CD8⁺ and TNF-α–dominant CD4⁺ responses were observed, along with increased effector memory differentiation. Two patients with higher CD8⁺ TEMRA proportions were the longest survivors. Tumor biopsies revealed enhanced CD8⁺ infiltration, and epitope spreading occurred in one of two evaluable cases. Analysis of 239 peptides showed greater immunogenicity for dual MHC I/II–binding, cysteine-containing, and in-frame indel- or low-VAF–derived mutations, while proline substitutions reduced responses. Conclusions Intramuscular neoantigen SLP vaccination with poly-ICLC is safe and induces rapid, mutation-specific T-cell immunity with robust CD8⁺ effector responses. These findings support intramuscular administration as a promising strategy for peptide-based cancer vaccines. Translational relevance Personalized neoantigen vaccines offer a promising strategy to enhance tumor-specific immunity, but most prior studies using intradermal or subcutaneous delivery have shown limited induction of cytotoxic CD8⁺ T cells. This study demonstrates that intramuscular administration of personalized neoantigen synthetic long peptide vaccines with poly-ICLC is safe, feasible, and capable of eliciting rapid, mutation-specific CD4⁺ and CD8⁺ T-cell responses in patients with advanced melanoma and renal cell carcinoma. Vaccine-induced immunity was dominated by IFN-γ–producing cells and accompanied by a shift toward effector memory phenotypes. In selected cases, post-treatment tumor biopsies revealed increased CD8⁺ infiltration. These findings support intramuscular delivery as a practical and effective platform for neoantigen-based cancer vaccine.

BackgroundDistinct subsets of CD4+ T cells can either promote tumor immunity by facilitating priming and expansion of cytolytic CD8+ T cells or, as Tregs, inhibit anti-tumor activity. Recently, other immunosuppressive CD4+T cell populations have been reported in mice and humans. However, the roles, origins and physiologic targets of the latter remain poorly defined. Our previous studies showed that, under defined conditions, synthetic long peptide (SLP) neoantigen (neoAg) cancer vaccines induced therapeutic rejection of established tumors, but efficacy was variable and depended on the quantity and quality of MHC-II neoAgs in the vaccine.1 Herein we identify a population of tumor-specific inhibitory non-Treg cytolytic CD4+ T cells induced by high MHC-II neoAg doses in vaccines, define their biology and mechanism of action, and present strategies to circumvent their formation/inhibitory function during immunotherapy.MethodsDifferent methylcholanthrene-induced mouse sarcoma cell lines that never express MHC-II were implanted into syngeneic mice that were then treated with tumor specific SLP neoAg vaccines containing varying amounts of MHC-II neoAgs.ResultsVaccines containing tumor specific MHC-I neoAgs plus low doses of MHC-II neoAg (LDVax) promote tumor rejection. In contrast, similar vaccines containing high doses of MHC-II neoAg (HDVax) were ineffective. HDVax inhibited antitumor efficacy of anti-PD-1 and anti-4–1BB in an antigen-specific manner but not anti-CTLA4 indicating that HDVax induced an inhibitory immune cell population. Inhibition could also be achieved by adoptive transfer of antigen-specific HDVax-induced CD4+ T cells into vaccine-naïve, tumor-bearing mice. Single-cell and bulk RNA sequencing and multicolor flow cytometry revealed that HDVax-induced CD4+ T cells express high levels of LILRB4, Granzyme-B (GZMB), Perforin and CCL5. TCRSeq and scRNAseq showed that the HDVax-induced inhibitory CD4+T cells were distinct from Foxp3+ Tregs. HDVax-induced CD4+ T cells inhibited tumor immunity by killing type 1 conventional dendritic cells (cDC1), as evidenced by in vitro killing assays and by low recovery of cDC1 from tumors in HDVax-treated mice compared to LDVax-treated or control mice. Killing was dependent on GZMB protein expression in the CD4+ T cells. Treatment of these cells with blocking anti-LILRB4 reduced cellular expression of GZMB and circumvented HDVax-induced inhibition. A similar reprogramming occurred following genetic depletion of cDC2 or treatment with a novel cis-targeted IL-2 mutein (CD8-IL2) that selectively activates CD8+ T cells.2ConclusionsThese results provide a roadmap to improve efficacy of cancer vaccines and potentially other immunotherapies by circumventing the generation/function of a heretofore unrecognized inhibitory CD4+ T cell population.AcknowledgementsWe thank all members of the Schreiber laboratory for discussions and technical support. We thank Likui Yang of the Immunomonitoring Laboratory (IML) at Washington University in St.Louis, who provided tetramers for MHC-I and MHC-II neoAgs. This work was supported by grants to Robert Schreiber from the National Cancer Institute of the National Institutes of Health (R01CA190700), the Parker Institute for Cancer Immunotherapy, and a Stand Up to Cancer-Lustgarten Foundation Pancreatic Cancer Foundation Convergence Dream Team Translational Research Grant and postdoctoral training grant (T32CA009547) from the National Cancer Institute to Hussein Sultan.ReferencesGubin MM. et al. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature 2014;515:577–581.Moynihan K, et al. Selective activation of CD8+ T cells by a CD8-targeted IL-2 results in enhanced anti-tumor efficacy and safety. JITC 2021.Ethics ApprovalAll in vivo experiments were performed in our specific-pathogen-free facility using procedures approved by the AAALAC-accredited Animal Studies Committee of Washington University in St Louis and followed all relevant ethical regulations.

Effective anti-tumour immunity in humans has been associated with the presence of T cells directed at cancer neoantigens, a class of HLA-bound peptides that arise from tumour-specific mutations. They are highly immunogenic because they are not present in normal tissues and hence bypass central thymic tolerance. Although neoantigens were long-envisioned as optimal targets for an anti-tumour immune response, their systematic discovery and evaluation only became feasible with the recent availability of massively parallel sequencing for detection of all coding mutations within tumours, and of machine learning approaches to reliably predict those mutated peptides with high-affinity binding of autologous human leukocyte antigen (HLA) molecules. We hypothesized that vaccination with neoantigens can both expand pre-existing neoantigen-specific T-cell populations and induce a broader repertoire of new T-cell specificities in cancer patients, tipping the intra-tumoural balance in favour of enhanced tumour control. Here we demonstrate the feasibility, safety, and immunogenicity of a vaccine that targets up to 20 predicted personal tumour neoantigens. Vaccine-induced polyfunctional CD4+ and CD8+ T cells targeted 58 (60%) and 15 (16%) of the 97 unique neoantigens used across patients, respectively. These T cells discriminated mutated from wild-type antigens, and in some cases directly recognized autologous tumour. Of six vaccinated patients, four had no recurrence at 25 months after vaccination, while two with recurrent disease were subsequently treated with anti-PD-1 (anti-programmed cell death-1) therapy and experienced complete tumour regression, with expansion of the repertoire of neoantigen-specific T cells. These data provide a strong rationale for further development of this approach, alone and in combination with checkpoint blockade or other immunotherapies.

The design of efficacious and cost-effective therapeutic vaccines against cancer remains both a research priority and a challenge. For more than a decade, our laboratory has been involved in the development of synthetic peptide-based anti-cancer therapeutic vaccines. We first dedicated our efforts in the identification and validation of peptide epitopes for both CD8 and CD4 T cells from tumor-associated antigens (TAAs). Because of suboptimal immune responses and lack of therapeutic benefit of peptide vaccines containing these epitopes, we have focused our recent efforts in optimizing peptide vaccinations in mouse tumor models using numerous TAA epitopes. In this focused research review, we describe how after taking lessons from the immune system’s way of dealing with acute viral infections, we have designed peptide vaccination strategies capable of generating very high numbers of therapeutically effective CD8 T cells. We also discuss some of the remaining challenges to translate these findings into the clinical setting.

Long peptides are efficiently presented to both CD4(+) and CD8(+) T cells after intracellular processing by antigen-presenting cells. To investigate the safety and in vivo immunogenicity of synthetic overlapping long peptides (OLP) from a human tumor self-antigen, we conducted a phase I clinical trial with OLP from cancer-testis antigen NY-ESO-1 in various adjuvant combinations. Twenty-eight patients with advanced ovarian cancer in second or third remission were enrolled sequentially in three cohorts and received at least one vaccination. Patients in Cohort 1 (n = 4) received 1.0 mg OLP, Cohort 2 (n = 13) received OLP in Montanide-ISA-51, and Cohort 3 (n = 11) received OLP + 1.4 mg Poly-ICLC in Montanide-ISA-51 on weeks 1, 4, 7, 10, and 13. Humoral and cellular responses were evaluated by standardized immunomonitoring techniques (ELISA, ELISPOT assay, intracellular cytokine staining, and tetramer staining). The vaccine was generally well tolerated with injection site reactions and fatigue that resolved. NY-ESO-1-specific antibody and CD8(+) T cells were undetectable after vaccination with OLP alone, but were found in 6 of 13 (46%) and 8 of 13 (62%) patients, respectively, after vaccination with OLP+Montanide, and in 10 of 11 (91%) and 10 of 11 (91%) patients, respectively, after vaccination with OLP+Montanide+Poly-ICLC. NY-ESO-1-specific CD4(+) T cells were detected in all patients with greater frequency and polyclonality when Montanide-ISA-51 was used for vaccination. Inclusion of Poly-ICLC as an adjuvant further accelerated the induction of NY-ESO-1-specific immune responses. The current study shows that NY-ESO-1 OLP vaccine is safe and rapidly induces consistent integrated immune responses (antibody, CD8(+) and CD4(+)) in nearly all vaccinated patients when given with appropriate adjuvants.