Courtney Beers

NKG2D splice variants: a reexamination of adaptor molecule associations

NKG2D is a homodimeric C-type lectin-related receptor expressed on natural killer (NK) cells and T cells. In mice, alternative deoxyribonucleic acid (DNA) splicing generates two isoforms of NKG2D that differ in the length of their cytoplasmic domains. Their ability to induce cellular activation is mediated via association with two membrane-bound, signaling adaptor molecules, DAP10 and DAP12. It has been reported that the long form of NKG2D associates exclusively with DAP10, whereas the short variant can interact with either adaptor. The short isoform was reported to be almost undetectable in naïve NK cells. Using two distinct cell types, we demonstrate that like the short isoform, the long variant of NKG2D also associates not only with DAP10 but also with DAP12. Using reporter cells (70Z/3), we demonstrate that DAP12 can compete equally with DAP10 for association with both variants of NKG2D when DAP10 and DAP12 are coexpressed. Cross-linking either isoform of NKG2D induces a calcium flux when associated exclusively with DAP10 or DAP12. Moreover, using quantitative polymerase chain reaction (PCR), we also show that the short isoform of NKG2D is expressed in naïve NK cells. Our data suggest that signaling via mouse NKG2D isoforms is more complex than originally presented.

Local Delivery of OncoVEXmGM-CSF Generates Systemic Antitumor Immune Responses Enhanced by Cytotoxic T-Lymphocyte–Associated Protein Blockade

Purpose: Talimogene laherparepvec, a new oncolytic immunotherapy, has been recently approved for the treatment of melanoma. Using a murine version of the virus, we characterized local and systemic antitumor immune responses driving efficacy in murine syngeneic models. Experimental Design: The activity of talimogene laherparepvec was characterized against melanoma cell lines using an in vitro viability assay. Efficacy of OncoVEXmGM-CSF (talimogene laherparepvec with the mouse granulocyte-macrophage colony-stimulating factor transgene) alone or in combination with checkpoint blockade was characterized in A20 and CT-26 contralateral murine tumor models. CD8+ depletion, adoptive T-cell transfers, and Enzyme-Linked ImmunoSpot assays were used to study the mechanism of action (MOA) of systemic immune responses. Results: Treatment with OncoVEXmGM-CSF cured all injected A20 tumors and half of contralateral tumors. Viral presence was limited to injected tumors and was not responsible for systemic efficacy. A significant increase in T cells (CD3+/CD8+) was observed in injected and contralateral tumors at 168 hours. Ex vivo analyses showed these cytotoxic T lymphocytes were tumor-specific. Increased neutrophils, monocytes, and chemokines were observed in injected tumors only. Importantly, depletion of CD8+ T cells abolished all systemic efficacy and significantly decreased local efficacy. In addition, immune cell transfer from OncoVEXmGM-CSF-cured mice significantly protected from tumor challenge. Finally, combination of OncoVEXmGM-CSF and checkpoint blockade resulted in increased tumor-specific CD8+ anti-AH1 T cells and systemic efficacy. Conclusions: The data support a dual MOA for OncoVEXmGM-CSF that involves direct oncolysis of injected tumors and activation of a CD8+-dependent systemic response that clears injected and contralateral tumors when combined with checkpoint inhibition. Clin Cancer Res; 23(20); 6190–202. ©2017 AACR.

Abstract 258: Talilmogene laherparepvec increases the anti-tumor efficacy of the anti-PD-1 immune checkpoint blockade

Talimogene laherparepvec is an investigational oncolytic immunotherapy based on a modified herpes simplex virus type-1 (HSV-1) designed to selectively replicate in tumors and to initiate a systemic immune response to target cancer cells that have metastasized. Intralesional administration of talimogene laherparepvec is intended to result in oncolysis within injected tumors. Iterative viral replication of virus within permissive tumor tissue results in lytic cell destruction and local release of progeny virus and tumor derived antigens. GM-CSF, a product of the viral transgene, is also produced locally such that it can recruit and stimulate antigen presenting cells which, in addition to relevant tumor-derived antigens, are required for the initiation of a systemic antitumor immune response. Recently, in a retrospective analysis of a Phase III melanoma trial investigators found that about two-thirds of the lesions injected with talimogene laherparepvec shrank 50% or more. And the same effect was seen in about a third of all uninjected tumors in the skin and lymph nodes and about a sixth of uninjected visceral lesions providing an indication that the treatment is triggering the desired immune system effect. Ongoing clinical trials are investigating T-VEC in combination with the immune checkpoint inhibiting antibodies ipilimumab and pembrolizumab in advanced melanoma. We sought to determine if the combination of intratumoral injection of talimogene laherparepvec and the systemic delivery of the checkpoint anti-PD1 antibody could increase the anti-tumor efficacy in a preclinical syngeneic contralateral tumor model. Using established MC-38 colon carcinoma tumors in C57Bl/6 mice, we delivered three intratumoral injections three days apart of the talimogene laherparepvec surrogate OncoVEXmuGM-CSF and twice weekly systemic injections of an antagonistic anti-PD-1 antibody. Either OncoVEXmuGM-CSF or anti-PD-1 alone induced modest tumor growth inhibition/tumor regressions of contralateral tumors. In combination, the OncoVEXmuGM-CSF injected tumor displayed 8/10 complete regressions and the distant tumors had 2/10 complete regressions. Peripheral blood was analyzed at 4 days and 10 days post the initial OncoVEXmuGM-CSF and anti-PD-1 antibody injections. OncoVEXmuGM-CSF increased the percent of PD-L1+ CD4 and CD8 T cells and the combination increased the percent of activated CD8+ T cells. Our findings demonstrate that localized therapy with talimogene laherparepvec augments the systemic anti-tumor immune response seen with anti-PD-1 therapy, providing a strong rationale for continued investigation of such combinations in the clinic. Citation Format: Julia Piasecki, Tiep le, Rafael Ponce, Courtney Beers. Talilmogene laherparepvec increases the anti-tumor efficacy of the anti-PD-1 immune checkpoint blockade. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 258. doi:10.1158/1538-7445.AM2015-258

Abstract 4287: Talimogene laherparepvec activates systemic T-cell-mediated anti-tumor immunity

Talimogene laherparepvec is an investigational oncolytic immunotherapy based on a modified herpes simplex virus type-1 (HSV-1) immunotherapy designed to selectively replicate in tumors and to initiate an anti-tumor immune response. Intralesional administration of talimogene laherparepvec is intended to result in oncolysis within injected tumors. Iterative replication of virus within permissive tumor tissue results in lytic cell destruction and local release of progeny virus and tumor derived antigens. GM-CSF, a product of the viral transgene, is also produced locally such that it can recruit and stimulate antigen presenting cells which, in addition to relevant tumor-derived antigens, are required for the initiation of a systemic antitumor immune response. The talimogene laherparepvec-induced immune mediated mechanism of action in both the virus-injected and distant tumors have yet to be fully understood therefore, we evaluated viral replication, tumor cell lysis, and the changes in immune cell populations in both the injected and distant tumors using syngeneic contralateral tumor models. Animals received a single intratumoral dose of the murine surrogate of talimogene laherparepvec (OncoVEXmuGM-CSF), which induced regression in the majority of virus-injected tumors and tumor growth inhibition/regression in the contralateral (uninjected) tumors. HSV-1 antigen was only detected by IHC in the virus-injected tumor and not the contralateral tumor (and no other tissues). Virally-mediated tumor destruction (oncolysis) was also localized to only the virus-injected tumor. In preliminary experiments, morphometric analysis of the tumor tissue revealed that the percent area of CD3+ and CD8+ lymphocytes were significantly increased in both the virus-injected and contralateral tumors compared to the formulation control mice. In addition, the percent area occupied by CD103+ cells (a marker found on potent cytotoxic T cell stimulating dendritic cells) was increased in the virus-injected tumor compared to the contralateral tumor and the tumors in the control animals. Although cellular infiltration was increased in both virus-injected and contralateral tumors and was inversely correlated with tumor volume, the decreased volume of injected tumors was attributed to viral oncolysis. Taken together these data supports that talimogene laheparepvec activates a systemic T cell mediated anti-tumor immune response. Citation Format: Julia Piasecki, Jim Rottman, Tiep Le, Rafael Ponce, Courtney Beers. Talimogene laherparepvec activates systemic T-cell-mediated anti-tumor immunity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4287. doi:10.1158/1538-7445.AM2015-4287

Talilmogene Iaherparepvec generates systemic T-cell-mediated anti-tumor immunity

Talimogene laherparepvec is an investigational oncolytic immunotherapy based on a modified herpes simplex virus type-1 (HSV-1) that is designed to selectively replicate in tumor tissue and to stimulate a systemic antitumor immune response. Intralesional administration of talimogene laherparepvec is intended to result in oncolysis within injected tumors. Iterative viral replication of virus within permissive tumor tissue results in lytic cell destruction and local release of progeny virus and cellular derived antigens. GM-CSF, a product of the viral transgene, is also produced locally such that it can recruit and stimulate antigen presenting cells which, in addition to relevant tumor-derived antigens, are required for the initiation of a systemic antitumor immune response. A phase 3 clinical trial of talimogene laherparepvec in regionally or distantly metastatic melanoma has recently reported a 26% ORR objective response rate (6% for GM-CSF alone){(J Clin Oncol 31, 2013 (suppl; abstr LBA9008)}. Since the immune mediated anti-tumor mechanisms have yet to be fully understood, we sought to identify immune-specific changes post talimogene laherparepvec administration in the A20 syngeneic contralateral tumor model. We analyzed immune populations in both injected and non-injected tumors and in peripheral hematopoietic tissues at 2, 5 and 10 days post injection. Animals received one 5x106 PFU intratumoral dose of talimogene laherparepvec which induces complete regression in approximately 70-100% of the injected tumors and approximately 50-60% of the contralateral non-injected tumors. Injection of virus resulted in marked increases in injected tumor draining lymph node and spleen size compared to control animals. Upon further flow cytometric analysis, changes in T-cell populations were observed in the injected and non-injected tumor draining lymph nodes, spleen and peripheral blood and T-cells expressed higher levels of cell surface activation markers as early as 2 days post-treatment in talimogene laherparepvec treated animals as compared to vehicle control animals. To functionally examine the T-cell-mediated anti-tumor immunity, we evaluated the memory response by re-challenging mice that completely regressed their primary A20 tumors with either A20 or three other Balb/c-derived tumor cell lines; 4T1, CT26 and RENCA. Interestingly, the mice displayed broad immunity to Balb/c tumor cells and rejected the A20, CT26 and RENCA but not 4T1 tumor cells whereas all the tumor cell lines grew robustly in control naive animals. In addition, splenic T-cells from talimogene laherparepvec-treated mice induced A20 tumor cell killing ex vivo while T-cells from vehicle control-treated mice did not. Intratumoral injection of talimogene laherparepvec systemically activates T cells and generates anti-tumor immunity in mice.