Diverse immunotherapies can effectively treat syngeneic brainstem tumors in the absence of overt toxicity

Background: Immunotherapy has shown remarkable clinical promise in the treatment of various types of cancers. However, clinical benefits derive from a highly inflammatory mechanism of action. This presents unique challenges for use in pediatric brainstem tumors including diffuse intrinsic pontine glioma (DIPG), since treatment-related inflammation could cause catastrophic toxicity. Therefore, the goal of this study was to investigate whether inflammatory, immune-based therapies are likely to be too dangerous to pursue for the treatment of pediatric brainstem tumors. Methods: To complement previous immunotherapy studies using patient-derived xenografts in immunodeficient mice, we developed fully immunocompetent models of immunotherapy using transplantable, syngeneic tumors. These four models – HSVtk/GCV suicide gene immunotherapy, oncolytic viroimmunotherapy, adoptive T cell transfer, and CAR T cell therapy – have been optimized to treat tumors outside of the CNS and induce a broad spectrum of inflammatory profiles, maximizing the chances of observing brainstem toxicity. Results: All four models achieved anti-tumor efficacy in the absence of toxicity, with the exception of recombinant vaccinia virus expressing GMCSF, which demonstrated inflammatory toxicity. Histology, imaging, and flow cytometry confirmed the presence of brainstem inflammation in all models. Where used, the addition of immune checkpoint blockade did not introduce toxicity. Conclusions: It remains imperative to regard the brainstem with caution for immunotherapeutic intervention. Nonetheless, we show that further careful development of immunotherapies for pediatric brainstem tumors is warranted to harness the potential potency of anti-tumor immune responses, despite their possible toxicity within this anatomically sensitive location

APOBEC3B-mediated Corruption of the Tumor Cell Immunopeptidome Induces Heteroclitic Neoepitopes for Cancer Immunotherapy

APOBEC3B, an anti-viral cytidine deaminase which induces DNA mutations, has been implicated as a mediator of cancer evolution and therapeutic resistance. Mutational plasticity also drives generation of neoepitopes, which prime anti-tumor T cells. Here, we show that overexpression of APOBEC3B in tumors increases resistance to chemotherapy, but simultaneously heightens sensitivity to immune checkpoint blockade in a murine model of melanoma. However, in the vaccine setting, APOBEC3B-mediated mutations reproducibly generate heteroclitic neoepitopes in vaccine cells which activate de novo T cell responses. These cross react against parental, unmodified tumors and lead to a high rate of cures in both subcutaneous and intra-cranial tumor models. Heteroclitic Epitope Activated Therapy (HEAT) dispenses with the need to identify patient specific neoepitopes and tumor reactive T cells ex vivo. Thus, actively driving a high mutational load in tumor cell vaccines increases their immunogenicity to drive anti-tumor therapy in combination with immune checkpoint blockade

Subversion of NK-cell and TNFα Immune Surveillance Drives Tumor Recurrence

Understanding how incompletely cleared primary tumors transition from minimal residual disease (MRD) into treatment resistant, immune-invisible recurrences has major clinical significance. We show here that this transition is mediated through the subversion of two key elements of innate immune surveillance. In the first, the role of TNFalpha changes from an antitumor effector against primary tumors into a growth promoter for MRD. Second, whereas primary tumors induced a natural killer (NK)-mediated cytokine response characterized by low IL6 and elevated IFNgamma, PD-L1hi MRD cells promoted the secretion of IL6 but minimal IFNgamma, inhibiting both NK cell and T-cell surveillance. Tumor recurrence was promoted by trauma- or infection-like stimuli inducing VEGF and TNFalpha which stimulated the growth of MRD tumors. Finally, therapies which blocked PD1, TNFalpha or NK cells delayed or prevented recurrence. These data show how innate immune surveillance mechanisms, which control infection and growth of primary tumors, are exploited by recurrent, competent tumors and identifies therapeutic targets in patients with MRD known to be at high risk of relapse

Ad5NULL-A20 - a tropism-modified, αvβ6 integrin-selective oncolytic adenovirus for epithelial ovarian cancer therapies

Purpose: Virotherapies are maturing in the clinical setting. Adenoviruses (Ad) are excellent vectors for manipulability and tolerance of transgenes. Poor tumour-selectivity, off-target sequestration and immune inactivation hamper clinical efficacy. We sought to completely redesign Ad5 into a refined, tumour selective virotherapy targeted to αvβ6 integrin, which is expressed in a range of aggressively transformed epithelial cancers but non-detectable in healthy tissues. Experimental Design: Ad5NULL-A20 harbours mutations in each major capsid protein to preclude uptake via all native pathways. Tumour-tropism via αvβ6-targeting was achieved by genetic insertion of A20 peptide (NAVPNLRGDLQVLAQKVART) within the fiber knob protein. The vector's selectivity in vitro and in vivo was assessed. Results: The tropism-ablating triple mutation completely blocked all native cell entry pathways of Ad5NULL-A20 via coxsackie and adenovirus receptor (CAR), αvβ3/5 integrins and coagulation factor 10 (FX). Ad5NULL-A20 efficiently and selectively transduced αvβ6+ cell lines and primary clinical ascites-derived EOC ex vivo, including in the presence of pre-existing anti-Ad5 immunity. In vivo biodistribution of Ad5NULL-A20 following systemic delivery in non-tumour-bearing mice was significantly reduced in all off-target organs, including a remarkable 107-fold reduced genome accumulation in the liver compared to Ad5. Tumour uptake, transgene expression and efficacy were confirmed in a peritoneal SKOV3 xenograft model of human EOC, where oncolytic Ad5NULL-A20-treated animals demonstrated significantly improved survival compared to those treated with oncolytic Ad5. Conclusions: Oncolytic Ad5NULL-A20 virotherapies represent an excellent vector for local and systemic targeting of αvβ6-over-expressing cancers, and exciting platforms for tumour selective over-expression of therapeutic anti-cancer modalities, including immune checkpoint inhibitors

Oncolytic virotherapy induced CSDE1 neo-antigenesis restricts VSV replication but can be targeted by immunotherapy

In our clinical trials of oncolytic vesicular stomatitis virus expressing interferon beta (VSV-IFNβ), several patients achieved initial responses followed by aggressive relapse. We show here that VSV-IFNβ-escape tumors predictably express a point-mutated CSDE1P5S form of the RNA-binding Cold Shock Domain-containing E1 protein, which promotes escape as an inhibitor of VSV replication by disrupting viral transcription. Given time, VSV-IFNβ evolves a compensatory mutation in the P/M Inter-Genic Region which rescues replication in CSDE1P5S cells. These data show that CSDE1 is a major cellular co-factor for VSV replication. However, CSDE1P5S also generates a neo-epitope recognized by non-tolerized T cells. We exploit this predictable neo-antigenesis to drive, and trap, tumors into an escape phenotype, which can be ambushed by vaccination against CSDE1P5S, preventing tumor escape. Combining frontline therapy with escape-targeting immunotherapy will be applicable across multiple therapies which drive tumor mutation/evolution and simultaneously generate novel, targetable immunopeptidomes associated with acquired treatment resistance

Size effects of graphene nanoplatelets on the properties of high-density polyethylene nanocomposites: Morphological, thermal, electrical, and mechanical characterization

High-density polyethylene (HDPE)-based nanocomposites incorporating three different types of graphene nanoplatelets (GnPs) were fabricated to investigate the size effects of GnPs in terms of both lateral size and thickness on the morphological, thermal, electrical, and mechanical properties. The results show that the inclusion of GnPs enhance the thermal, electrical, and mechanical properties of HDPE-based nanocomposites regardless of GnP size. Nevertheless, the most significant enhancement of the thermal and electrical conductivities and the lowest electrical percolation threshold were achieved with GnPs of a larger lateral size. This could have been attributed to the fact that the GnPs of larger lateral size exhibited a better dispersion in HDPE and formed conductive pathways easily observable in scanning electron microscope (SEM) images. Our results show that the lateral size of GnPs was a more regulating factor for the above-mentioned nanocomposite properties compared to their thickness. For a given lateral size, thinner GnPs showed significantly higher electrical conductivity and a lower percolation threshold than thicker ones. On the other hand, in terms of thermal conductivity, a remarkable amount of enhancement was observed only above a certain filler concentration. The results demonstrate that GnPs with smaller lateral size and larger thickness lead to lower enhancement of the samples' mechanical properties due to poorer dispersion compared to the others. In addition, the size of the GnPs had no considerable effect on the melting and crystallization properties of the HDPE/GnP nanocomposites. © 2020 Evgin et al.This work was partially supported by the Science Grant Agency VEGA, project no. 2/0010/18, and 2/0093/16 (Slovakia).2/0010/18, 2/0093/1

Inhibitory Receptors Induced by VSV Viroimmunotherapy Are Not Necessarily Targets for Improving Treatment Efficacy

Systemic viroimmunotherapy activates endogenous innate and adaptive immune responses against both viral and tumor antigens. We have shown that therapy with vesicular stomatitis virus (VSV) engineered to express a tumor-associated antigen activates antigen-specific adoptively transferred T cells (adoptive cell therapy, ACT) in vivo to generate effective therapy. The overall goal of this study was to phenotypically characterize the immune response to VSV+ACT therapy and use the information gained to rationally improve combination therapy. We observed rapid expansion of blood CD8+ effector cells acutely following VSV therapy with markedly high expression of the immune checkpoint molecules PD-1 and TIM-3. Using these data, we tested a treatment schedule incorporating mAb immune checkpoint inhibitors with VSV+ACT treatment. Unlike clinical scenarios, we delivered therapy at early time points following tumor establishment and treatment. Our goal was to potentiate the immune response generated by VSV therapy to achieve durable control of metastatic disease. Despite the high frequency of endogenous PD-1+ TIM-3+ CD8+ T cells following virus administration, antibody blockade did not improve survival. These findings provide highly significant information about response kinetics to viroimmunotherapy and juxtapose the clinical use of checkpoint inhibitors against chronically dysfunctional T cells and the acute T cell response to oncolytic viruses