Connexin Hemichannel Activation by S-Nitrosoglutathione Synergizes Strongly with Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing

In this study, we used B16-F10 cells grown in the dorsal skinfold chamber (DSC) preparation that allowed us to gain optical access to the processes triggered by photodynamic therapy (PDT). Partial irradiation of a photosensitized melanoma triggered cell death in non-irradiated tumor cells. Multiphoton intravital microscopy with genetically encoded fluorescence indicators revealed that bystander cell death was mediated by paracrine signaling due to adenosine triphosphate (ATP) release from connexin (Cx) hemichannels (HCs). Intercellular calcium (Ca2+) waves propagated from irradiated to bystander cells promoted intracellular Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria and rapid activation of apoptotic pathways. Combination treatment with S-nitrosoglutathione (GSNO), an endogenous nitric oxide (NO) donor that biases HCs towards the open state, greatly potentiated anti-tumor bystander killing via enhanced Ca2+ signaling, leading to a significant reduction of post-irradiation tumor mass. Our results demonstrate that HCs can be exploited to dramatically increase cytotoxic bystander effects and reveal a previously unappreciated role for HCs in tumor eradication promoted by PDT.

Uncialamycin-based antibody–drug conjugates: Unique enediyne ADCs exhibiting bystander killing effect

Antibody–drug conjugates (ADCs) have emerged as valuable targeted anticancer therapeutics with at least 11 approved therapies and over 80 advancing through clinical trials. Enediyne DNA-damaging payloads represented by the flagship of this family of antitumor agents, N-acetyl calicheamicin γ1I, have a proven success track record. However, they pose a significant synthetic challenge in the development and optimization of linker drugs. We have recently reported a streamlined total synthesis of uncialamycin, another representative of the enediyne class of compounds, with compelling synthetic accessibility. Here we report the synthesis and evaluation of uncialamycin ADCs featuring a variety of cleavable and noncleavable linkers. We have discovered that uncialamycin ADCs display a strong bystander killing effect and are highly selective and cytotoxic in vitro and in vivo.

90 Potentiation of T-cell mediated tumor killing via modulation of the fas/fasL pathway

BackgroundT-cell based immunotherapies such as CAR-T, bispecific mAb, transgenic T cells and checkpoint blockade have profound efficacy in multiple tumor types but share a common limitation – target antigen (Ag) escape.1 2 One approach to address this limitation has been therapy directed at a ‘parallel’ target (e.g. CD22 after CD19 loss), however, these lineage markers are frequently lost together.3 Here, we describe an alternate, broadly applicable, approach: potentiating fasL/fas-signaling to increase localized bystander killing of Ag-tumor cells and thereby prevent Ag escape.MethodsWe used a CRISPR/Cas9 library to screen for tumor expressed molecules that inhibit or facilitate T-cell killing. We then evaluated one candidate -fas- using murine transgenic T cells, murine and human CAR-T cells, bispecific mAb redirected PBMC, and tumoral RNAseq data from a large CAR-T clinical trial.ResultsGFP-specific (JEDI) CD8 T cells were co-cultured with on-target (GFP+) and bystander (mCherry+) lymphoma cells that had been transfected with a CRISPR/Cas9 library; this screen revealed several tumor-expressed candidate molecules inhibiting or facilitating T-cell killing. Notably, we observed a marked dependence on fas for on-target tumor killing and then, surprisingly, an exquisite dependence on fas for localized bystander tumor killing. (figure 1).Because bystander tumor killing appeared critically fas-dependent, we hypothesized that potentiating fas-signaling might increase bystander killing. An in vitro screen of small molecules that modulate fas-pathway revealed several candidates, including inhibitors of histone deacetylases (HDAC), inhibitors of apoptosis proteins (IAP) and Bcl-2 family members in murine and human systems (figure 2). To validate these candidates, we demonstrated that HDACi increased GFP-specific T cell killing of both on-target and bystander lymphoma cells, in a completely fas-dependent manner (figure 3). Similarly, using a bispecific antibody-based system, we demonstrated increased, fas-dependent, T cell killing of both on-target and bystander human lymphoma cells with inhibitors of IAP and bcl-2 family members (e.g. MCL1).Abstract 90 Figure 1See text for descriptionAbstract 90 Figure 2See text for descriptionAbstract 90 Figure 3See text for descriptionConclusionsT-cell mediated tumor killing can be potentiated with fas pathway modulators. This augmentation improves both fas-dependent Ag+ and Ag-tumor cell death. Further studies of modulating the fas pathway alongside T-cell based immunotherapies are needed as potential treatments to prevent antigen escape and improve patient outcomes.AcknowledgementsWe thank the flow cytometry core facility, microscopy core facility, and the CCMS animal facility at ISMMS.Ethics ApprovalThe studies were approved by The Mount Sinai Institutional Review Board.ReferencesZaretsky J, Garcia-Diaz A, Shin D, et al. Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma. N Engl J Med 2016: 375(9); 819–20.Majzner R, Mackall C. Tumor antigen escape from CAR T-cell therapy. Cancer Discov 2018;8(10):1219–1226.Jacoby E, Nguyen S, Fountaine T, et al. CD19 CAR immune pressure induces B-precusor acute lymphoblastic leukaemia lineage switch exposing inherent leukaemic plasticity. Nat Commun 2016; 7:12320.

Tumor neoantigen heterogeneity impacts bystander immune inhibition of pancreatic cancer growth

The threshold for immunogenic clonal fraction in a heterogeneous solid tumor required to induce effective bystander killing of non-immunogenic subclones is unknown. Pancreatic cancer poses crucial challenges for immune therapeutic interventions due to low mutational burden and consequent lack of neoantigens. Here, we designed a model to incorporate artificial neoantigens into genes of interest in cancer cells and to test the potential of said antigens to actuate bystander killing. By precisely controlling the abundance of a neoantigen in the tumor, we studied the impact of neoantigen frequency on immune response and immune escape. Our results showed that a single, strong, widely expressed neoantigen could lead to a robust antitumor response when at least 80% of cancer cells express the neoantigen. Further, immunological assays revealed induction of T-cell responses against a non-target self-antigen on KRAS oncoprotein, when we inoculated animals with a high frequency of tumor cells expressing a test neoantigen. Using nanoparticle-based gene therapy, we successfully altered the tumor microenvironment by perturbing interleukin-12 and interleukin-10 gene expression. The subsequent remodeling of the microenvironment reduced the threshold of neoantigen frequency at which bioluminescent signal intensity for tumor burden decreased 1.5-logfold, marking a robust tumor growth inhibition, from 83% to as low as 29%. Our results thus suggest that bystander killing is rather inefficient in immunologically cold tumors like pancreatic cancer and requires an extremely high abundance of neoantigens. However, the bystander killing mediated antitumor response can be rescued, when supported by adjuvant immune therapy.