ΔPK oncolytic activity includes modulation of the tumour cell milieu.

Oncolytic virotherapy is a unique cancer therapeutic that encompasses tumour cell lysis through both virus replication and programmed cell death (PCD) pathways. Nonetheless, clinical efficacy is relatively modest, likely related to the immunosuppressive tumour milieu. Our studies use the herpes simplex virus type 2 (HSV-2)-based oncolytic virus ΔPK that has documented anti-tumour activity associated with virus replication, PCD and cancer stem cell lysis. They are designed to examine whether ΔPK-mediated oncolysis includes the ability to reverse the immunosuppressive tumour microenvironment by altering the balance of cytokines directly secreted by the melanoma cells and to define its mechanism. Here, we show that melanoma cells secreted the immunosuppressive cytokine IL-10, and that secretion was inhibited by ΔPK through virus replication and c-Jun N-terminal kinase/c-Jun activation. ΔPK-induced IL-10 inhibition upregulated surface expression of MHC class I chain-related protein A, the ligand for the activating NKG2D receptor expressed on NK- and cytotoxic T-cells. Concomitantly, ΔPK also upregulated the secretion of inflammatory cytokines TNF-α, granulocyte macrophage colony-stimulating factor and IL-1ß through autophagy-mediated activation of Toll-like receptor 2 pathways and pyroptosis, and it inhibited the expression of the negative immune checkpoint regulator cytotoxic T-lymphocyte antigen 4. Pharmacologic inhibition of these processes significantly reduces the oncolytic activity of ΔPK.

Prevalent and Diverse Intratumoral Oncoprotein-Specific CD8+ T Cells within Polyomavirus-Driven Merkel Cell Carcinomas.

Merkel cell carcinoma (MCC) is often caused by persistent expression of Merkel cell polyomavirus (MCPyV) T-antigen (T-Ag). These non-self proteins comprise about 400 amino acids (AA). Clinical responses to immune checkpoint inhibitors, seen in about half of patients, may relate to T-Ag-specific T cells. Strategies to increase CD8+ T-cell number, breadth, or function could augment checkpoint inhibition, but vaccines to augment immunity must avoid delivery of oncogenic T-antigen domains. We probed MCC tumor-infiltrating lymphocytes (TIL) with an artificial antigen-presenting cell (aAPC) system and confirmed T-Ag recognition with synthetic peptides, HLA-peptide tetramers, and dendritic cells (DC). TILs from 9 of 12 (75%) subjects contained CD8+ T cells recognizing 1-8 MCPyV epitopes per person. Analysis of 16 MCPyV CD8+ TIL epitopes and prior TIL data indicated that 97% of patients with MCPyV+ MCC had HLA alleles with the genetic potential that restrict CD8+ T-cell responses to MCPyV T-Ag. The LT AA 70-110 region was epitope rich, whereas the oncogenic domains of T-Ag were not commonly recognized. Specific recognition of T-Ag-expressing DCs was documented. Recovery of MCPyV oncoprotein-specific CD8+ TILs from most tumors indicated that antigen indifference was unlikely to be a major cause of checkpoint inhibition failure. The myriad of epitopes restricted by diverse HLA alleles indicates that vaccination can be a rational component of immunotherapy if tumor immune suppression can be overcome, and the oncogenic regions of T-Ag can be modified without impacting immunogenicity.

Pulsed high-intensity focused ultrasound enhances apoptosis and growth inhibition of squamous cell carcinoma xenografts with proteasome inhibitor bortezomib.

PURPOSE: To investigate whether combining pulsed high-intensity focused ultrasound (HIFU) with the chemotherapeutic drug bortezomib could improve antitumor activity against murine squamous cell carcinoma (SCC) tumors. MATERIALS AND METHODS: All experiments were conducted with animal care and use committee approval. Murine SCC cells were implanted subcutaneously in C3H mice. When tumors reached 100 mm(3), mice were randomized to one of three groups for twice weekly intraperitoneal injections of 1.5 mg of bortezomib per kilogram of body weight, a proteasome inhibitor (n = 10); 1.0 mg/kg bortezomib (n = 11); or a control vehicle (n = 12). Within each group, half of the mice received pulsed HIFU exposure to their tumors immediately prior to each injection. The time for tumors to reach 650 mm(3) was compared among groups. Additional tumors were stained with terminal deoxynucledotidyl transferase-mediated dUTP nick end labeling and CD31 to assess apoptotic index and blood vessel density, respectively. RESULTS: Tumors in the control group, pulsed HIFU and control group, and 1.0 mg/kg of bortezomib alone group reached the size end point in 5.2 days +/- 0.8 (standard deviation), 5.3 days +/- 0.8, and 5.6 days +/- 1.1, respectively. However, pulsed HIFU and 1.0 mg/kg bortezomib increased the time to end point to 9.8 days +/- 2.9 (P < .02), not significantly different from the 8.8 days +/- 2.1 in tumors treated with 1.5 mg/kg bortezomib alone (P > .05). Combination therapy was also associated with a significantly higher apoptotic index (P < .05). CONCLUSION: Treatment of tumors with pulsed HIFU lowered the threshold level for efficacy of bortezomib, resulting in significant tumor cytotoxicity and growth inhibition at lower dose levels.

Merkel Cell Carcinoma in the Age of Immunotherapy: Facts and Hopes.

Merkel cell carcinoma (MCC) is a rare (∼2,000 U.S. cases/year) but aggressive neuroendocrine tumor of the skin. For advanced MCC, cytotoxic chemotherapy only infrequently (<10% of cases) offers durable clinical responses (>1 year), suggesting a great need for improved therapeutic options. In 2008, the Merkel cell polyomavirus (MCPyV) was discovered and is clonally integrated in approximately 80% of MCC tumors. The remaining 20% of MCC tumors have large numbers of UV-associated mutations. Importantly, both the UV-induced neoantigens in virus-negative tumors and the MCPyV T antigen oncogenes that are required for virus-positive tumor growth are immunogenic. Indeed, antigen-specific T cells detected in patients are frequently dysfunctional/"exhausted," and the inhibitory ligand, PD-L1, is often present in MCC tumors. These findings led to recent clinical trials involving PD-1 pathway blockade in advanced MCC. The combined data from these trials involving three PD-1 pathway blocking agents-avelumab, pembrolizumab, and nivolumab-indicated a high frequency of durable responses in treated patients. Of note, prior treatment with chemotherapy was associated with decreased response rates to PD-1 checkpoint blockade. Over the past year, these striking data led to major changes in advanced MCC therapy, including the first-ever FDA drug approval for this disease. Despite these successes, approximately 50% of patients with MCC do not persistently benefit from PD-1 pathway blockade, underscoring the need for novel strategies to broaden antitumor immune responses in these patients. Here, we highlight recent progress in MCC including the underlying mechanisms of immune evasion and emerging approaches to augment the efficacy of PD-1 pathway blockade. Clin Cancer Res; 24(9); 2035-43. ©2017 AACR.

HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells.

Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this 'missing-self' response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8+ T cells, do not bind anti-HLA antibodies and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression.

The oncolytic virus ΔPK has multimodal anti-tumor activity.

Oncolytic viruses (OVs) are an emerging cancer therapeutic, with a near complete absence of serious adverse effects. However, clinical efficacy is relatively modest, related to poor tumor penetration, failure to lyse cancer stem cells (CSCs) and blockade of immunogenic cell death by the immunosuppressive tumor microenvironment. To overcome such limitations, we developed an OV (known as ΔPK) with multimodal anti-tumor activity. ΔPK has potent anti-tumor activity both in melanoma cell lines and xenograft animal models, associated with virus replication and the induction of multiple independent programmed cell death pathways. It lyses CSCs through autophagy modulation and it reverses the immunosuppressive tumor microenvironment by altering the balance of cytokines secreted by the tumor cells. This includes decreased tumor cell secretion of the immunosuppressive and procancerous cytokines IL-10 and IL-18 and concomitant increased secretion of the proinflammatory cytokines TNF-α, GM-CSF, IL-6 and IL-1ß. ΔPK also upregulates the NKG2D ligand, MICA expressed by cytotoxic NK and T cells, and downregulates the negative immune checkpoint regulator cytotoxic T-lymphocyte antigen-4 (CTLA-4). ΔPK is well tolerated in human patients in whom it also alters the Th1/Th2 balance. Further studies are designed to elucidate the role of these contributions in different tumor types.