Engineered CD47 protects T cells for enhanced antitumor immunity

Adoptively transferred T cells and agents designed to block the CD47/SIRPalpha axis are promising cancer therapeutics that activate distinct arms of the immune system. We administered anti-CD47 with adoptively transferred T cells with the goal of enhancing antitumor efficacy but observed rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors, which abrogated therapeutic benefit. anti-CD47 mediated CAR T clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered a CD47 variant (47E) that engaged SIRPalpha and provided a “don’t-eat-me” signal that was not blocked by anti-CD47 antibodies. TCR or CAR T cells expressing 47E were resistant to clearance by macrophages following anti-CD47, and mediated significant, sustained macrophage recruitment into the TME. Although many of the recruited macrophages manifested an M2-like profile, the combined therapy synergistically enhanced antitumor efficacy. This work identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T cell directed therapeutics with those designed to activate macrophages. It further delivers a therapeutic approach capable of simultaneously harnessing the antitumor effects of T cells and macrophages that manifests enhanced potency against solid tumors. Orthotopic 143B osteosarcoma human tumors were established in NSG mice. Mice were treated with either no T cells, mock transduced T cells, CD47 WT (47WT-), or engineered CD47 (47E-) expressing Her2.BBz human CAR T cells, each ± the anti-humanCD47 antibody B6H12 (8 experimental samples total, pooled from 3 mice per treatment group; n = 24 mice). Dissociated tumors from this 143B experiment were sorted for live cells using a Live/Dead stain at the Stanford Shared FACS facility. Single-cell RNAseq libraries were prepared using the Chromium Next GEM Single Cell 5' v2 platform (10x GENOMICS). Libraries were sent to Novogene for sequencing on a NovoSeq S4 lane (PE150) with approximately 30,000 mean reads per cell.