Microbial-inspired immune-stimulating antibody-toxin conjugate (ATC) for cancer immunotherapy - PROJECT SUMMARY Generating antitumor immune responses requires the phagocytosis of tumor cells and subsequent cross-presentation of tumor-derived antigens by antigen-presenting cells. However, these processes are impeded by phagocytosis checkpoints and inefficient cytosolic transport of antigenic peptides from phagolysosomes. CD47, an integrin family member, is highly expressed in multiple human cancer cells and plays an important role in helping cancer cells evade phagocytosis by professional antigen-presenting cells (APCs). Even in the cases where tumor cells are phagocytosed, escape of cancer cell-derived proteins and peptides from phagolysosomes into the cytosol where they can be further processed by proteasomes and loaded onto MHC molecules for cross-presentation to T cells is limited. Therefore, strategies to promote tumor cell phagocytosis and subsequent phagolysosomal escape of tumor cell-derived cellular components in antigen-presenting cells, are critical for activating innate immune functionalities of APCs and their ability to prime effector T cell responses. Here, we propose to engineer a novel antibody-drug/toxin conjugate (ATC) inspired by the phagolysosomal escape mechanisms of intracellular infectious pathogens including Listeria monocytogenes. We linked the anti-CD47 antibody with the L. monocytogenes toxin LLO via a cleavable linker (CD47-LLO). The antibody binds to CD47-expressing tumor cells, promoting their phagocytosis by phagocytes. Once exposed to the reducing conditions within the phagolysosomes, the linker between CD47-LLO is cleaved, and the released LLO is activated in the acidic environment to form pores on the phagosome membrane. This creates a channel for digested tumor and protein fragments to leak into the cytosol to promote the cross-presentation of tumor antigens. We hypothesize that CD47-LLO, as a novel cancer immunotherapeutic, promotes innate and adaptive immune cell interactions to produce potent and durable antitumor responses against localized and metastatic cancers. To test this central hypothesis, we will first in Aim 1 determine the mechanisms of action of CD47-LLO, including the impact of the ATC treatment on antigen presentation in APCs and the diversity of T cell clones. In Aim 2, we will study the impact of CD47- LLO treatment on immune cell populations and their interactions with tumor cells and one another within the tumor microenvironment. Finally, in Aim 3, we will test a humanized version of CD47-LLO against patient tumors established in mice reconstituted with a functional human immune system, as a single agent or in combination with checkpoint blockade. If successful, our proposed research would pave the way for developing a first-in-class, non-cytotoxic ATC that promotes immune activation to generate potent antitumor responses.
