Strategies for Cancer Immunotherapy Development: Preclinical Studies

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Combination of HDAC inhibition and cytokine enhances therapeutic HPV vaccine therapy: Human papillomavirus (HPV)-associated malignancies continue to present a major health concern despite the development of prophylactic vaccines. Standard therapies offer limited benefit to patients with advanced-stage disease. Despite improved outcomes with programmed cell death protein-1 (PD-1) targeted therapies, treatment resistance and modest response rates highlight a significant unmet need to develop novel therapies for these patients. PDS0101 (designated HPV vaccine) is a liposomal nanoparticle HPV16-specific therapeutic vaccine that has been shown to generate strong HPV-specific responses in preclinical and clinical studies. The current study assessed the efficacy of this HPV vaccine in combination with the tumor-targeting immunocytokine NHS-IL12 (PDS01ADC), plus either anti-PD-1 or the class I histone deacetylase inhibitor Entinostat. Mice bearing HPV16+, anti-PD-1 refractory TC-1 and mEER tumors were treated with HPV vaccine, NHS-IL12, and either anti-PD-1 or Entinostat to determine antitumor efficacy and survival benefits. A comprehensive analysis of the tumor microenvironment was performed using flow cytometry, multiplex immunofluorescence, chemokine and cytokine assessment, and single-cell RNA sequencing with T-cell receptor (TCR) enrichment. The combination of HPV vaccine and NHS-IL12 with either Entinostat or anti-PD-1 yielded significant antitumor activity and prolonged survival in antiPD-1 refractory models of HPV16+ cancer, with superior activity employing Entinostat versus anti-PD-1 combination. Entinostat triple therapy increased overall and HPV16-specific tumor CD8+ T-cell infiltration with heightened cytotoxicity. TCR sequencing revealed a CD8+ T-cell clone unique to vaccine-treated cohorts, which displayed an enriched cytotoxic transcriptional profile with triple therapy. These effects were paralleled by strong differentiation of tumor-associated macrophages (TAMs) towards pro-inflammatory, antitumor M1-like cell states. Single-cell transcriptomic analysis indicated all three agents were required for highest modulation of both CD8+ T cells and TAMs conducive to tumor control. A biomarker signature reflecting the preclinical findings was found to be associated with improved survival in patients with HPV-associated malignancies. Together, these findings provide a rationale for the combination of HPV vaccine, NHS-IL12, and Entinostat in the clinical setting for patients with HPV16-associated malignancies. Combination of a therapeutic cancer vaccine targeting the endogenous retroviral envelope protein ERVMER34-1 with immune-oncology agents facilitates expansion of neoepitope-specific T cells and promotes tumor control: Endogenous retroviruses (ERVs) are remnants of retrovirus germline infections that occurred over the course of evolution and constitute between 5% and 8% of the human genome. While ERVs tend to be epigenetically silenced in normal adult human tissues, they are often overexpressed in carcinomas and may represent novel immunotherapeutic targets. This study characterizes the ERV envelope protein ERVMER34-1 as a target for a therapeutic cancer vaccine. The expression of ERVMER34-1 in multiple healthy adult and cancer tissues was assessed, as was its immunogenicity, to ascertain whether specific T cells could lyse human carcinoma cell lines expressing ERVMER34-1. Furthermore, the ability of a rationally designed ERVMER34-1-targeted therapeutic vaccine to induce tumor clearance in two murine carcinoma models expressing ERVMER34-1 was examined either as a monotherapy or in combination with anti-programmed cell death protein-1/programmed death-ligand 1 monoclonal antibody (mAb) or the interleukin-15 superagonist N-803. The ERVMER34-1 protein was shown to be overexpressed in 232/376 of human carcinomas analyzed while being absent in most healthy adult tissues. High levels of ERVMER34-1 RNA expression associate with decreased survival in uveal melanoma, adenoid cystic, and head and neck carcinomas. ERVMER34-1-specific T cells were detected in peripheral blood mononuclear cells (PBMCs) of patients with cancer but not healthy donors following an overnight stimulation. However, reactive T cells are readily expanded from both healthy donor and patient with cancer PBMCs following a 7-day in vitro stimulation. Furthermore, ERVMER34-1-specific T cells selectively kill human carcinoma cell lines expressing ERVMER34-1. A novel, rationally designed, therapeutic cancer vaccine targeting ERVMER34-1 mediated tumor control in established syngeneic murine tumors expressing the full-length ERVMER34-1 protein. When combined with checkpoint blockade, the vaccine promoted expansion of neoepitope-reactive T cells whose function was further enhanced when combined with N-803. This expansion of neoepitope-reactive T cells was associated with tumor control. This study reveals the potential of a vaccine that targets the retroviral envelope protein ERVMER34-1 and supports its continued development toward clinical testing as a new class of therapeutic cancer vaccine. Generation of murine tumor models refractory to anti-PD-1/-L1 therapies due to defects in antigen processing/presentation or IFN? signaling using CRISPR/Cas9: Immune checkpoint blockade (ICB) targeting the programmed cell death protein 1 (PD-1) and its ligand 1 (PD-L1) fails to provide clinical benefit for most cancer patients due to primary or acquired resistance. Drivers of ICB resistance include tumor antigen processing/presentation machinery (APM) and IFNgamma signaling mutations. Thus, there is an unmet clinical need to develop alternative therapies for these patients. To this end, we developed a CRISPR/Cas9 approach to generate murine tumor models refractory to PD-1/-L1 inhibition due to APM/IFNgamma signaling mutations. Guide RNAs were employed to delete B2m, Jak1, or Psmb9 genes in ICB-responsive EMT6 murine tumor cells. B2m was deleted in ICB-responsive MC38 murine colon cancer cells. We report a detailed development and validation workflow including whole exome and Sanger sequencing, western blotting, and flow cytometry to assess target gene deletion. Tumor response to ICB and immune effects of gene deletion were assessed in syngeneic mice. This workflow can help accelerate the discovery and development of alternative therapies and a deeper understanding of the immune consequences of tumor mutations, with potential clinical implications.