Abstract B178: Development and preclinical efficacy characterization of a systemically administered multiple Toll-like receptor (TLR) agonist for antitumor immunotherapy
Authors: Newman Michael J. 
Published: February 2019

Abstract
Background: The first cancer immunotherapy was developed in the 1890s by Dr. William Coley, who observed tumor regressions after injecting cancer patients with a heat-killed mixture of Gram-positive and negative pathogenic bacteria. Dr. Coley determined that the Gram-negative bacteria were the principal contributor to antitumor activity and that the product would likely be most effective when administered intravenously (i.v.), but was too toxic in this setting, limiting use to intramuscular and intratumoral administration. Coley’s toxins, as it was known, was credited with curing hundreds of late-stage cancer patients over 70 years. However, lack of knowledge regarding mechanism of action made it difficult to optimize or standardize, producing high variability. The use of multiple, local administration routes also likely contributed to variability in response, leading the FDA to refuse to grandfather in the product in 1963. We now know the mechanism of action of Coley’s toxins and the source of the i.v. toxicity. Gram-negative bacteria contain immune system danger signals, including multiple TLR agonists (activating TLRs 2, 4, 5 and 9), which directly and indirectly, via induction of cytokine and chemokine secretion, participate in the activation of most of the cellular mediators of innate and adaptive immune responses. Lipopolysaccharide (LPS), which activates TLR4, has been identified as a major contributor to both the antitumor activity and i.v. toxicity of Gram-negative bacteria. Decoy’s hypothesis is that significant reduction without complete elimination of LPS activity, in conjunction with killing and stabilization of nonpathogenic, Gram-negative bacteria, may produce a multiple TLR product that can safely and effectively induce antitumor immune responses via i.v. administration. Methods and Results: Nonpathogenic, Gram-negative E. coli were treated with polymyxin B and glutaraldehyde under conditions to kill and stabilize the cells, producing &gt;90% reduction in LPS endotoxin activity and pyrogenicity. Endotoxin activity and pyrogenicity were quantified using Limulus amebocyte lysate (LAL) and in vivo rabbit assays. Bacterial integrity was assessed by electron and light microscopy. Antitumor activity was determined using standard syngeneic and xenograft tumor models. Decoy-treated bacteria exhibited a 3-fold reduction in acute in vivo toxicity relative to untreated bacteria. Surprisingly, induction of antitumor cytokine secretion by murine and human peripheral blood mononuclear cells (PBMCs) was not compromised, relative to untreated bacteria. Treatment with Decoy bacteria (i.v.) produced significant single-agent antitumor activity against orthotopic murine colorectal carcinoma and metastatic murine pancreatic carcinoma. Synergistic combination activity, including eradication of established tumors, with a therapeutic index of up to 10-fold, was observed in combination with IL-2 or low-dose cyclophosphamide (LDC) in murine colorectal carcinoma models, with LDC in a subcutaneous (s.c.), murine non-Hodgkin’s lymphoma (NHL) model and with LDC plus rituximab in a s.c., human NHL model. Synergistic antitumor activity was also observed in combination with a low-dose, nonsteroidal anti-inflammatory drug (NSAID) in a metastatic, murine pancreatic carcinoma model. In addition, tumor eradications were observed in combination with NSAID and were enhanced by addition of anti-PD-1 therapy in a s.c., murine hepatocellular carcinoma model. Optimal (80-100%) tumor eradication was shown to be mediated by natural killer (NK), CD4+ and CD8+ T-cells. Immunologic memory (80-100% and partial), determined by rejection of subsequent tumor challenge, was demonstrated in both immune competent and innate only settings, respectively. Conclusion: We have developed an i.v. administered multiple TLR agonist, based on Coley’s toxins, that can be combined with a wide variety of existing agents to induce curative, antitumor immune responses.
Citation Format: Michael J. Newman. Development and preclinical efficacy characterization of a systemically administered multiple Toll-like receptor (TLR) agonist for antitumor immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B178.