Tumor-targeted interleukin-12 synergizes with entinostat to overcome PD-1/PD-L1 blockade-resistant tumors harboring MHC-I and APM deficiencies

Abstract

BackgroundImmune checkpoint blockade (ICB) has achieved unprecedented success in treating multiple cancer types. However, clinical benefit remains modest for most patients with solid malignancies due to primary or acquired resistance. Tumor-intrinsic loss of major histocompatibility complex class I (MHC-I) and aberrations in antigen processing machinery (APM) and interferon gamma (IFN-γ) pathways have been shown to play an important role in ICB resistance. While a plethora of combination treatments are being investigated to overcome ICB resistance, there are few identified preclinical models of solid tumors harboring these deficiencies to explore therapeutic interventions that can bypass ICB resistance. Here, we investigated the combination of the epigenetic modulator entinostat and the tumor-targeted immunocytokine NHS-IL12 in three different murine tumor models resistant to αPD-1/αPD-L1 (anti-programmed cell death protein 1/anti-programmed death ligand 1) and harboring MHC-I, APM, and IFN-γ response deficiencies and differing tumor mutational burden (TMB).MethodsEntinostat and NHS-IL12 were administered to mice bearing TC-1/a9 (lung, HPV16 E6/E7+), CMT.64 lung, or RVP3 sarcoma tumors. Antitumor efficacy and survival were monitored. Comprehensive tumor microenvironment (TME) and spleen analysis of immune cells, cytokines, and chemokines was performed. Additionally, whole transcriptomic analysis was carried out on TC-1/a9 tumors. Cancer Genome Atlas (TCGA) datasets were analyzed for translational relevance.ResultsWe demonstrate that the combination of entinostat and NHS-IL12 therapy elicits potent antitumor activity and survival benefit through prolonged activation and tumor infiltration of cytotoxic CD8+T cells, across αPD-1/αPD-L1 refractory tumors irrespective of TMB, including in the IFN-γ signaling-impaired RVP3 tumor model. The combination therapy promoted M1-like macrophages and activated antigen-presenting cells while decreasing M2-like macrophages and regulatory T cells in a tumor-dependent manner. This was associated with increased levels of IFN-γ, IL-12, chemokine (C-X-C motif) ligand 9 (CXCL9), and CXCL13 in the TME. Further, the combination therapy synergized to promote MHC-I and APM upregulation, and enrichment of JAK/STAT (janus kinase/signal transducers and activators of transcription), IFN-γ-response and antigen processing-associated pathways. A biomarker signature of the mechanism involved in these studies is associated with patients’ overall survival across multiple tumor types.ConclusionsOur findings provide a rationale for combining the tumor-targeting NHS-IL12 with the histone deacetylase inhibitor entinostat in the clinical setting for patients unresponsive to αPD-1/αPD-L1 and/or with innate deficiencies in tumor MHC-I, APM expression, and IFN-γ signaling.

Preclinical and clinical studies of a tumor targeting IL-12 immunocytokine

“…Multiple preclinical and clinical studies have demonstrated the tumor suppressive ability of NHS-IL12 as a single agent ( 22 , 35 – 37 , 56 ). Mounting preclinical and clinical evidence supports the use of NHS-IL12 as an integral component of combination therapies for the treatment of solid cancers, including in combination with immune checkpoint blockade and other treatment modalities ( 35 , 40 , 43 , 45 , 46 , 52 , 53 , 58 , 60 , 61 ). A unique aspect of NHS-IL12 versus rIL-12 is its ability to localize to the tumor, thereby eliciting local IFNγ production via activation, maturation, and expansion of NK and CD8 + TILs.…”

“…Due to the sustained increases of IFNγ noted with the combination of entinostat and NHS-IL12, a follow-up study investigated this combination therapy in TC-1/a9 (HPV E6/E7 + ), CMT.64 and RVP3 tumor models resistant to PD-1/PD-L1 blockade due to varying MHC-I and APM deficiencies ( 53 ) ( Figures 3D, E ). While many of the findings here mirrored previous findings of the important role of tumor-infiltrating CD8 + T cells, M1 macrophages, and T cell trafficking chemokines, this investigation demonstrated that the sustained pro-inflammatory response of entinostat and NHS-IL12 may be utilized for patients harboring innate and acquired resistance to checkpoint blockade therapies targeting the PD-1/PD-L1 axis.…”

“…Additionally, this study demonstrated the role of NK cells and various antigen presenting cell populations, such as dendritic and B cells. Bulk RNAseq analysis of TC-1/a9 tumors showed enrichment of antigen processing and presentation, in addition to IFNγ and Jak/Stat signaling pathways, often dysregulated in checkpoint blockade resistance ( 53 , 54 ). Together, these studies demonstrated the translatability of this combination for the treatment of patients harboring solid malignancies, including those resistant to PD-1/PD-L1-targeted therapies.…”

A landscape of checkpoint blockade resistance in cancer: underlying mechanisms and current strategies to overcome resistance

“… 158 Moreover, in murine tumor models resistant to anti-PD-1/anti-PD-L1 therapy, tumor-targeted NHS-IL12 (IL-12 fused to an antibody that binds exposed DNA commonly found in necrotic tumors) when administered with a histone deacetylase inhibitor potentiated CD8 + T cell-dependent antitumor activity and provided survival benefit. 33 However, a phase 1b study evaluating the combination of NHS-IL12 and avelumab was discontinued due to lack of efficacy (NCT02994953). Several active studies that involve NHS-IL12 with anti-PD-L1/anti-TGF- fusion protein are ongoing but have yet to post results (NCT04287868, NCT04303117).…”

“… 29–33 Prevailing strategies are focused on transforming immunological “cold” tumors into “hot” tumors 30 , 34 , 35 by increasing tumoral T cell infiltration, enhancing cytotoxic T cell function, 30 , 32 , 36 repolarizing immunosuppressive population, 31–33 , 37 or overcoming tumor-intrinsic resistance mechanisms related to loss of major histocompatibility complex class I (MHC-I), aberrations in antigen processing machinery (APM), and interferon gamma (IFN-γ) pathways. 33 , 38–40 In this review, we will discuss the response of ICI in the real world and how this compares to the responses observed in clinical trials, as well as the underlying mechanisms promoting primary and acquired CBR. Finally, we aim to discuss current efforts to suppress these mechanisms that lead to CBR with the end goal of improving patient response to ICI therapy.…”