The first reports of bacteria as a cancer therapy date back to the pioneering work of Dr. William Coley–now widely regarded as the father of immunotherapy. As far back as 1891, Coley demonstrated that the intratumoral injection of live and later heat-killed isolates of Streptococcus pyogenes and Serratia marcescens could induce durable remission in patients with bone and soft tissue sarcoma. While this therapy was deemed to unsafe at the time, Coley’s findings have formed the basis for our modern understanding of immunology and cancer immunotherapy. Over the past two decades, the advent of synthetic biology is driving a new era of medicine through the genetic programming of living cells. This transformative approach enables the creation of engineered systems that sense and respond to diverse environments, permitting safe and effective targeted delivery of therapeutic payloads within disease sites. In this thesis, I seek to utilize principles from synthetic biology and immunology to engineer bacteria for immunotherapeutic delivery. I have developed multiple strains of non-pathogenic E. coli capable of colonizing solid tumors and delivering diverse immunotherapeutic payloads specifically within the tumor. This local delivery approach enables the utilization of therapeutic agents that may be otherwise systemically toxic. In one instance, we engineered an encoded nanobody antagonist of CD47 (CD47nb), an anti-phagocytic receptor commonly overexpressed in several human cancers. We show that delivery of CD47nb by tumor-colonizing bacteria increases activation of tumor-infiltrating T cells, stimulates rapid tumor regression, prevents metastasis, and leads to long-term survival in a syngeneic tumor model. Thus, engineered bacteria may be used for safe and local delivery of diverseimmunotherapeutic payloads leading to systemic antitumor immunity.

In 1867, Dr. Wilhelm Busch decisively exposed a cancer patient to erysipelas and notedtumor regression. The practice of inoculating tumors with bacteria became more widespread with the work of Dr. William Coley, beginning in 1891, who inoculated inoperable tumors and observed complete regressions though at times notable toxicity. These microbial manipulations of immunity now form the roots of cancer immunotherapy in modern history. Alongside the blossoming of cancer immunology since, the development of techniques to alter biological systems has given rise to synthetic biology. Together, these fields allow the programming of biological systems to precisely guide the cancer-immune interplay. As mammalian immunity targets bacterially-derived antigens due to the immunostimulatory nature of microbes, and tumors express various antigens, synthetic alteration of microbes to function as safe and effective anti-tumor vaccines is a natural proposition. In this work, I describe my development of such a microbial system comprised of a synthetic tumor-antigen construct optimized for expression in bacteria, the immunotoxin Listerolysin O, and a genetically edited probiotic E. coli chassis with multi-functional protease deletions. This platform encodes and delivers high levels of diverse tumor antigens sets, remodels the tumor microenvironment, and stimulates productive and durable anti-tumor immunity to control and eliminate primary and metastatic tumors. We show that this system induces tumor antigen-specific CD4+ and CD8+ T cells, activates NK cells, recruits and activates dendritic cells, and reduces immunosuppressive regulatory T cells, B cells, and myeloid cells within the tumor microenvironment. This work thus establishes a new class of anti-tumor vaccine which modulates all arms of immunity to achieve robust anti-tumor efficacy.