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.

Altering Antibody-Drug Conjugate Binding to the Neonatal Fc Receptor Impacts Efficacy and Tolerability.

Antibody-drug conjugates (ADC) rely on the target-binding specificity of an antibody to selectively deliver potent drugs to cancer cells. IgG antibody half-life is regulated by neonatal Fc receptor (FcRn) binding. Histidine 435 of human IgG was mutated to alanine (H435A) to explore the effect of FcRn binding on the pharmacokinetics, efficacy, and tolerability of two separate maytansine-based ADC pairs with noncleavable linkers, (c-DM1 and c-H435A-DM1) and (7v-Cys-may and 7v-H435A-Cys-may). The in vitro cell-killing potency of each pair of ADCs was similar, demonstrating that H435A showed no measurable impact on ADC bioactivity. The H435A mutant antibodies showed no detectable binding to human or mouse FcRn in vitro, whereas their counterpart wild-type IgG ADCs were found to bind to FcRn at pH = 6.0. In xenograft bearing SCID mice expressing mouse FcRn, the AUC of 7v-Cys-may was 1.6-fold higher than that of 7v-H435A-may, yet the observed efficacy was similar. More severe thrombocytopenia was observed with 7v-H435A-Cys-may as compared to 7v-Cys-may at multiple dose levels. The AUC of c-DM1 was approximately 3-fold higher than that of c-H435A-DM1 in 786-0 xenograft bearing SCID mice, which led to a 3-fold difference in efficacy by dose. Murine FcRn knockout, human FcRn transgenic line 32 SCID animals bearing 786-0 xenografts showed an amplified exposure difference between c-DM1 and c-H435A-DM1 as compared to murine FcRn expressing SCID mice, leading to a 10-fold higher dose required for efficacy despite a 6-fold higher AUC of the c-H435A-DM1. The accelerated clearance observed for the noncleavable maytansine ADCs with the H435A FcRn mutation led to reduced efficacy at equivalent doses and exacerbation of clinical pathology parameters (decreased tolerability) at equivalent doses. The results show that reduced ADC clearance mediated by FcRn modulation can improve therapeutic index.

Expression of anti-apoptotic factors modulates Apo2L/TRAIL resistance in colon carcinoma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) selectively induces apoptosis in transformed cells. Normal cells and certain tumor cells can evade Apo2L/TRAIL induced cell death, but the determinants of Apo2L/TRAIL sensitivity are poorly understood. To better understand the factors that contribute to Apo2L/TRAIL resistance, we characterized two colon carcinoma lines with pronounced differences in Apo2L/TRAIL sensitivity. Colo205 cells are highly sensitive to Apo2L/TRAIL whereas Colo320 cells are unresponsive. Components of the DISC (death inducing signaling complex) could be immunoprecipitated from both cell lines in response to Apo2L/TRAIL. Sensitizing agents including a proteasome inhibitor conferred Apo2L/TRAIL sensitivity in Colo320 cells, indicating that the apoptotic machinery was intact and functional. We specifically suppressed the expression of Bcl-2, FLIP or XIAP in Colo320 cells. Downregulation of either FLIP or XIAP but not Bcl-2 restored sensitivity of Colo320 cells to Apo2L/TRAIL. Moreover, stable knockdown of XIAP expression in Colo320 subcutaneous tumors resulted in suppression of tumor growth and sensitivity to Apo2L/TRAIL in vivo. Our results indicate that only a specific subset of anti-apoptotic proteins can confer resistance to Apo2L/TRAIL in Colo320 cells. Elucidation of the factors that contribute to Apo2L/TRAIL resistance in tumor cells may provide insight into combination therapies with Apo2L/TRAIL in a clinical setting.

Interleukin-15 interactions with interleukin-15 receptor complexes: characterization and species specificity.

Interleukin (IL-) 2 and IL-15 share the IL-2 receptor betagamma c subunits (IL-2Rbetagamma c) but have specific, unique alpha receptor subunits. We studied species specificity of human (hu), simian (si), and mouse (mu) IL-15 and found that hu and si IL-15 behaved similarly in all systems investigated. Hu and mu IL-15 bound hu or mu IL-15Ralpha with equal high affinity in the presence or absence of IL-2Rbetagamma c and exhibited similar proliferative activities on cells containing all three subunits. However, quantitative differences were noted in the specific activity of hu and mu IL-15 in both in vitro and in vivo systems utilizing IL-2Rbetagamma c in the absence of IL-15Ralpha. These data show that hu IL-15 may be used in mouse model systems, however care must be taken when comparing the efficacy and toxicity of cytokines across species.

Characterization of the in vivo function of TNF-alpha-related apoptosis-inducing ligand, TRAIL/Apo2L, using TRAIL/Apo2L gene-deficient mice.

To define the normal physiological role for the TRAIL/Apo2L in vivo, we generated TRAIL/Apo2L gene-targeted mice. These mice develop normally and show no defects in lymphoid or myeloid cell homeostasis or function. Although TRAIL/Apo2L kills transformed cells in vitro, TRAIL/Apo2L(-/-) mice do not spontaneously develop overt tumors at an early age. However, in the A20 B cell lymphoma-transferred tumor model, TRAIL/Apo2L(-/-) mice are clearly more susceptible to death from overwhelming tumor burden, due to increased lymphoma load in the liver. A20 tumors are susceptible to TRAIL/Apo2L killing in vitro, indicating that TRAIL/Apo2L may act directly to control A20 cells in vivo. Despite the fact that TRAIL binds osteoprotegerin and osteoprotegerin-transgenic mice are osteopetrotic, TRAIL/Apo2L(-/-) mice show no evidence of altered gross bone density, and no alterations in frequency or in vitro differentiation of bone marrow precursor osteoclasts. Moreover, leucine zipper TRAIL has no toxicity when repeatedly administered to osteoprotegerin(-/-) mice. Thus, TRAIL/Apo2L is important in controlling tumors in vivo, but is not an essential regulator of osteoprotegerin-mediated biology, under normal physiological conditions.

4-1BB-specific monoclonal antibody promotes the generation of tumor-specific immune responses by direct activation of CD8 T cells in a CD40-dependent manner.

4-1BB (CD137) is a member of the TNFR superfamily (TNFRSF9). T cell expression of 4-1BB is restricted to activated cells, and cross-linking has been shown to deliver a costimulatory signal. Here we have shown that treatment of tumor-bearing mice with agonistic 4-1BB-specific Abs can lead to T cell-mediated tumor rejection. In vivo mAb depletion experiments demonstrated that this rejection requires CD8(+) cells but not CD4(+) or NK cells. Both IFN-gamma- and CD40-mediated signals were also required, because no benefit was observed on treatment with 4-1BB mAb in mice in which the genes for these molecules had been knocked out. Interestingly, 4-1BB-mediated stimulation of immune responses in CD40L(-/-) mice is effective (although at a reduced level), and may suggest the existence of an alternative ligand for CD40. Additional experiments in IL-15(-/-) mice indicate that IL-15 is not required for either the generation of the primary tumor-specific immune response or the maintenance of the memory immune response. In contrast, the presence of CD4 cells during the primary immune response appears to play a significant role in the maintenance of effective antitumor memory. Finally, in mice in which the number of dendritic cells had been expanded by Fms-like tyrosine kinase3 ligand treatment, the antitumor effects of 4-1BB ligation were enhanced.