Melcher, Alan

Abstract T cell immune tolerance is established in part through the activity of the Auto-immune Regulator (AIRE) transcription factor in the medullary Thymic Epithelial Cells (mTEC) of the thymus. AIRE induces expression of SELF peripheral tissue-specific antigens for presentation to naïve T cells to promote activation/deletion of potentially autoreactive T cells. We show, for the first time to our knowledge, that tumors mimic the role of AIRE in mTEC to evade immune rejection. Thus, by expressing a broad range of SELF epitopes against which minimal functional T cell reactivities exist because of thymic deletion, AIRE acts as a master controller of SELFNESS, effectively cloaking the tumor from T cell attack. Moreover, we describe a completely novel immunotherapy in which engineered changes in AIRE expression in tumor cells alters their profile of SELFNESS, exposing both AIRE-modified, and parental unmodified, tumor cells to T cell attack. Consistent with our studies, patient RNAseq shows expression of AIRE predicts response to immune therapies with a strong correlation between AIRE expression and markers of TCR signaling. Therefore, by re-setting the immunological SELFNESS of cancer cells, this novel AIRE-mediated immunotherapy 1). converts a highly tolerized T cell compartment into a heteroclitic tumor-reactive T cell population; 2) confers de novo sensitivity to immune checkpoint blockade upon non-immunogenic tumors; 3). completely removes the need to identify potentially immunogenic tumor-associated antigens as targets for generation of de novo CD8+ and helper CD4+ T cell responses; and 4) leads to potent T cell-mediated rejection of aggressive, immunologically cold, non-immunogenic tumors.

2062 Background: While cancer therapeutics have made tremendous progress within the past several decades, this benefit has not been seen in many primary cancers of the brain. Particularly confounding have been high-grade gliomas (HGG), which retain a dismal prognosis. Currently, novel therapies are being explored to rise to this unmet, critical need. One such therapy are CAR T cells, immune cells which have been engineered to target malignancy-specific antigens. Unfortunately, the efficacy of CAR T cell therapies against solid tumors is significantly limited, in large part due to impaired expansion/persistence in the immune suppressive tumor microenvironment (TME). Here we show that in vivo reactivation of CAR T cells through their native T Cell Receptor (TCR) by an oncolytic virus (OV) has therapeutic benefit in HGG. Methods: An EGFRvIII third-generation MSGV1 retroviral CAR construct containing the CD28, 4-1BB, and CD3z moieties, in tandem with the scFv derived from the human monoclonal antibody 139 and the marker Thy1.1 (38) was used to generate our CAR T cells. C57Bl/6 mice were used for in vivo experiments and both B16- and CT2A-EGFRvIII murine glioma cell lines were injected in the brain to model HGG. OVs and CAR T cells were given systemically by tail vein. OVs used include reovirus, vesicular stomatitis virus, and adenovirus. Results: By using OV in combination with EGFRvIII CAR T cells, we were able to generate a CD8 CAR population with TCR specificity for both the EGFRvIII and OV epitopes. These dual-specific (DS) CAR T expressed a memory phenotype and persisted for much longer than conventional CAR T cells. Further, we showed that these DS CAR T cells are more cytotoxic and can respond more rapidly than their conventional counterparts. We created a novel delivery mechanism for this combination OV + CAR T therapy using virus-loaded CAR T cells to bypass initial antiviral clearance from the immune system. Treatment with these OV-loaded CAR T cells lead to significant benefit in mice with HGG tumors which could be further enhanced by a systemic boost with OV, which rapidly re-activated DS CAR T cells against tumor and resulted in long-term cures of greater than 80% of treated animals. Conclusions: These promising results show that DS CAR T cells can overcome the critical therapeutic challenge of CAR T as a treatment for solid tumors. Given these promising results, we will go on to develop a clinical trial in which CAR T cells will be pre-loaded with OV and administered intravenously to patients with HGG, followed by systemic boosting with virus to re-activate DS CAR T cells against their tumor.

Abstract Previously, we reported that killing tumor cells in vivo with the HSV thymidine kinase/ganciclovir system generates potent antitumor immunity, determined in part by the mechanism by which the cells die and by the levels of inducible heat shock protein (hsp) expression induced during the process of cell death. Here, we show that induction of hsp70 expression induces an infiltrate of T cells, macrophages, and predominantly dendritic cells (DCs) into the tumors as well as an intratumoral profile of Th1 cytokine expression (IFN-γ, TNF-α, and IL-12) and enhances immunogenicity via a T cell-mediated mechanism. In addition, the protection conferred by hsp70 is both tumor and cell specific. We also demonstrate that hsp70 targets immature APC to make them significantly more able to capture Ags. This is likely to optimize cross-priming of the infiltrating APC with tumor Ags, which are simultaneously being released by the dying cells. In addition, using an Myc epitope-tagged hsp70 expression vector, we present evidence that hsp70 released from dying tumor cells is taken up directly into DCs and may, therefore, be involved in direct chaperoning of Ags into DCs. Taken together, our data suggest that hsp70 induction serves to signal the immune system of the presence of an immunologically relevant (dangerous) situation against which an immune reaction should be raised.

Abstract Background Brain tumors are the leading cause of cancer death for pediatric patients. Pelareorep, an immunomodulatory oncolytic reovirus, has intravenous efficacy in preclinical glioma models when preconditioned with GM-CSF (sargramostim). We report a phase I trial with the primary goal of evaluating the safety of sargramostim/pelareorep in pediatric patients with recurrent or refractory high-grade brain tumors and a secondary goal of characterizing immunologic responses. Methods The trial was open to pediatric patients with recurrent or refractory high-grade brain tumors (3 + 3 cohort design). Each cycle included 3 days of subcutaneous sargramostim followed by 2 days of intravenous pelareorep. Laboratory studies and imaging were acquired upon recruitment and periodically thereafter. Results Six patients participated, including three glioblastoma, two diffuse intrinsic pontine glioma, and one medulloblastoma. Two pelareorep dose levels of 3 × 108 and 5 × 108 tissue culture infectious dose 50 (TCID50) were assessed. One patient experienced a dose limiting toxicity of persistent hyponatremia. Common low-grade (1 or 2) adverse events included transient fatigue, hypocalcemia, fever, flu-like symptoms, thrombocytopenia, and leukopenia. High-grade (3 or 4) adverse events included neutropenia, lymphopenia, leukopenia, hypophosphatemia, depressed level of consciousness, and confusion. All patients progressed on therapy after a median of 32.5 days and died a median of 108 days after recruitment. Imaging at progression did not show evidence of pseudoprogression or inflammation. Correlative assays revealed transient but consistent changes in immune cells across patients. Conclusions Sargramostim/pelareorep was administered to pediatric patients with recurrent or refractory high-grade brain tumors. Hyponatremia was the only dose limiting toxicity (DLT), though maximum tolerated dose (MTD) was not determined.

Abstract Improving the chances of curing patients with cancer who have had surgery to remove metastatic sites of disease is a priority area for cancer research. Pexa-Vec (Pexastimogene Devacirepvec; JX-594, TG6006) is a principally immunotherapeutic oncolytic virus that has reached late-phase clinical trials. We report the results of a single-center, nonrandomized biological end point study (trial registration: EudraCT number 2012-000704-15), which builds on the success of the presurgical intravenous delivery of oncolytic viruses to tumors. Nine patients with either colorectal cancer liver metastases or metastatic melanoma were treated with a single intravenous infusion of Pexa-Vec ahead of planned surgical resection of the metastases. Grade 3 and 4 Pexa-Vec–associated side effects were lymphopaenia and neutropaenia. Pexa-Vec was peripherally carried in plasma and was not associated with peripheral blood mononuclear cells. Upon surgical resection, Pexa-Vec was found in the majority of analyzed tumors. Pexa-Vec therapy associated with IFNα secretion, chemokine induction, and resulted in transient innate and long-lived adaptive anticancer immunity. In the 2 patients with significant and complete tumor necrosis, a reduction in the peripheral T-cell receptor diversity was observed at the time of surgery. These results support the development of presurgical oncolytic vaccinia virus-based therapies to stimulate anticancer immunity and increase the chances to cure patients with cancer.

Oncolytic viruses (OVs) encoding a variety of transgenes have been evaluated as therapeutic tools to increase the efficacy of chimeric antigen receptor (CAR)–modified T cells in the solid tumor microenvironment (TME). Here, using systemically delivered OVs and CAR T cells in immunocompetent mouse models, we have defined a mechanism by which OVs can potentiate CAR T cell efficacy against solid tumor models of melanoma and glioma. We show that stimulation of the native T cell receptor (TCR) with viral or virally encoded epitopes gives rise to enhanced proliferation, CAR-directed antitumor function, and distinct memory phenotypes. In vivo expansion of dual-specific (DS) CAR T cells was leveraged by in vitro preloading with oncolytic vesicular stomatitis virus (VSV) or reovirus, allowing for a further in vivo expansion and reactivation of T cells by homologous boosting. This treatment led to prolonged survival of mice with subcutaneous melanoma and intracranial glioma tumors. Human CD19 CAR T cells could also be expanded in vitro with TCR reactivity against viral or virally encoded antigens and was associated with greater CAR-directed cytokine production. Our data highlight the utility of combining OV and CAR T cell therapy and show that stimulation of the native TCR can be exploited to enhance CAR T cell activity and efficacy in mice.

Background Combination herpes simplex virus (HSV) oncolytic virotherapy and BRAF inhibitors (BRAFi) represent promising immunogenic treatments for BRAF mutant melanoma, but an improved understanding of the immunobiology of combinations is needed to improve on the benefit of immune checkpoint inhibitors (ICI). Methods Using a BRAFV600E-driven murine melanoma model, we tested the immunogenicity of HSV/BRAFi in immunocompetent C57BL mice. In addition to standard FACS analysis, we used the ‘Timer of Cell Kinetics and Activity’ system, which can analyze the temporal dynamics of different T cell subsets. This immune data was used to inform the selection of ICI for triple combination therapy, the effects of which were then further characterized using transcriptomics. Results Adding BRAFi treatment to HSV improved anti-tumor effects in vivo but not in vitro. Immune characterization showed HSV or dual therapy led to fewer intratumoral Treg, although with a more activated phenotype, together with more effector CD8 +T cells. Tocky analysis further showed that HSV/BRAFi dual treatment reduced the Tocky signal (reflecting engagement with cognate antigen), in both Treg and conventional subsets of CD4+, but not in CD8 +cells. However, a higher percentage of Treg than of conventional CD4 +maintained frequent engagement with antigens on treatment, reflecting a predominance of suppressive over effector function within the CD4 +compartment. The only T cell subset which correlated with a reduction in tumor growth was within Tocky signal positive conventional CD4+, supporting their therapeutic role. Targeting CD25 high, antigen-engaged Treg with a depleting anti-CD25 ICI, achieved complete cures in 100% of mice with triple therapy. Transcriptomic analysis confirmed reduction in Foxp3 on addition of anti-CD25 to HSV/BRAFi, as well as increases in expression of genes reflecting interferon signaling and cytotoxic activity. Conclusions Combination HSV/BRAFi is an immunogenic therapy for BRAF mutant melanoma, but cannot fully control tumors. Dual therapy results in changes in T cell dynamics within tumors, with relatively maintained antigen signaling in Treg compared with conv CD4+. Antigen-engaged CD4 +effectors correlate with tumor growth control, and depletion of Treg by addition of an anti-CD25 ICI, releasing suppression of conventional CD4 +effectors by Treg, enhances survival and activates immune signaling within tumors.

Recognizing immune responses to oncolytic virotherapy opens the way for new combinations

Background Oncolytic reovirus therapy for cancer induces a typical antiviral response to this RNA virus, including neutralizing antibodies. Concomitant treatment with cytotoxic chemotherapies has been hypothesized to improve the therapeutic potential of the virus. Chemotherapy side effects can include immunosuppression, which may slow the rate of the antiviral antibody response, as well as potentially make the patient more vulnerable to viral infection. Method Reovirus neutralizing antibody data were aggregated from separate phase I clinical trials of reovirus administered as a single agent or in combination with gemcitabine, docetaxel, carboplatin and paclitaxel doublet or cyclophosphamide. In addition, the kinetics of individual antibody isotypes were profiled in sera collected in these trials. Results These data demonstrate preserved antiviral antibody responses, with only moderately reduced kinetics with some drugs, most notably gemcitabine. All patients ultimately produced an effective neutralizing antibody response. Conclusion Patients’ responses to infection by reovirus are largely unaffected by the concomitant drug treatments tested, providing confidence that RNA viral treatment or infection is compatible with standard of care treatments.

AbstractThe apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) family protects against infection by degrading incoming viral genomes through cytosine deamination. Here, we review how the potential to unleash these potent DNA mutagens comes at a price as APOBEC DNA mutagenesis can contribute to development of multiple types of cancer. In addition, because viral infection induces its expression, APOBEC is seen as the enemy of oncolytic virotherapy through mutation of the viral genome and by generating virotherapy-resistant tumors. Therefore, overall APOBEC in cancer has received very poor press. However, we also speculate how there may be silver linings to the storm clouds (kataegis) associated with APOBEC activity. Thus, although mutagenic genomic chaos promotes emergence of ever more aggressive subclones, it also provides significant opportunity for cytotoxic and immune therapies. In particular, the superpower of cancer immunotherapy derives in part from mutation, wherein generation of tumor neoantigens—neoantigenesis—exposes tumor cells to functional T-cell repertoires, and susceptibility to immune checkpoint blockade. Moreover, APOBECs may be able to induce suprathreshold levels of cellular mutation leading to mitotic catastrophe and direct tumor cell killing. Finally, we discuss the possibility that linking predictable APOBEC-induced mutation with escape from specific frontline therapies could identify mutated molecules/pathways that can be targeted with small molecules and/or immunotherapies in a Trap and Ambush strategy. Together, these considerations lead to the counterintuitive hypothesis that, instead of attempting to expunge and excoriate APOBEC activity in cancer therapy, it might be exploited—and even, counterintuitively, encouraged.