Abstract 5741: Harnessing macrophages, while protecting T cells, enhances anti-tumor efficacy
Authors: Yamada-Hunter Sean A. , Theruvath Johanna , Radosevich Molly T. , McIntosh Brianna J. , Freitas Katherine A. , Martinez-Velez Naiara , Sotillo Elena , Leruste Amaury , Xu Peng , Desai Moksha H. , Sahaf Bita , Banuelos Allison , Wasserman Savannah L. , Weissman Irving L. , Cochran Jennifer R. , Mackall Crystal L. 
Published: April 2023

Abstract
Chimeric antigen receptor T (CAR T) cells are synthetically engineered to target specific tumor antigens. CD47 is a ubiquitous receptor that serves as a “don’t eat me” signal by binding to SIRPɑ on macrophages and is often over-expressed by cancer cells. Despite individual promise of CAR T and anti-CD47 therapies, neither has demonstrated clear efficacy in treating solid tumors in the clinic and there is thus an urgent need to develop novel approaches that enhance the potency of these therapies. We interrogated the potential pairing of CAR T with anti-CD47 therapy to overcome tumor resistance mechanisms inherent to each therapy alone, by engaging non-redundant properties of two arms of the immune system. However, upon coadministration of anti-CD47 therapy and CAR T cells across multiple tumor models in vivo, we observed potent and consistent macrophage-mediated clearance of CAR-T cells via on-target, off-tumor binding of anti-CD47 therapies to CAR T cells. This anti-CD47 mediated CAR T cell depletion blunts the therapeutic benefits of treatment and renders the pairing of the current versions of the two agents impractical.To overcome this challenge, we used directed evolution and yeast surface display to engineer a novel variant of CD47 (eCD47) with selective binding, identifying mutations that resulted in loss of binding to the anti-CD47 antibody B6H12, while maintaining the CD47 “don’t eat me” function through binding to SIRPɑ, which is essential for T cell persistence in vivo. T cells engineered to express eCD47, but not native, wild-type CD47, were resistant to targeting by multiple anti-CD47 antibodies but maintained binding to SIRPɑ. These T cells were no longer susceptible to anti-CD47 mediated macrophage phagocytosis in vitro, nor were they depleted in vivo after B6H12 administration. We demonstrated a striking improvement in therapeutic efficacy upon treatment of multiple solid tumor models when anti-CD47 therapy was combined with CAR T cells expressing eCD47, compared to combination with CAR T cells expressing wild-type CD47. We interrogated a mechanistic basis for this improved efficacy in an osteosarcoma model through single-cell RNA-sequencing of isolated tumors. We discovered that CAR T treatment led to a large influx of unique populations of macrophages into the tumor, which were lost upon CAR T depletion after anti-CD47 treatment. These T cell recruited-macrophages were maintained after anti-CD47 treatment in the presence of CAR T cells expressing eCD47, harnessing macrophage mediated anti-tumor activity after combination treatment. Thus, for the first time, eCD47 allows for effective pairing of CAR T therapy and anti-CD47 therapy for cancer treatment, by sparing T cells from macrophage mediated depletion, and revealing impressive synergy when adoptive T cell therapy is combined with macrophage activation.
Citation Format: Sean A. Yamada-Hunter, Johanna Theruvath, Molly T. Radosevich, Brianna J. McIntosh, Katherine A. Freitas, Naiara Martinez-Velez, Elena Sotillo, Amaury Leruste, Peng Xu, Moksha H. Desai, Bita Sahaf, Allison Banuelos, Savannah L. Wasserman, Irving L. Weissman, Jennifer R. Cochran, Crystal L. Mackall. Harnessing macrophages, while protecting T cells, enhances anti-tumor efficacy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5741.