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The dramatic success of CAR T cell therapies in the treatment of certain B cell malignancies has led to a wave of novel cell therapies for cancer. All approved engineered cell therapies are derived from autologous T cells. This has posed barriers for patient access to qualified treatments that include a lengthy production time, inconsistent product characteristics, uncertain manufacturability, and a high cost of manufacturing. To address these obstacles, allogeneic “universal” cell therapies, derived from healthy donors or differentiated pluripotent cells are engineered and expanded in large quantities for off-the-shelf treatment. In allogeneic CAR T cell therapy, T cell receptor (TCR) knockout is required to prevent graft-versus-host disease of alloreactive T cells targeting against host tissue. Allogeneic cells may have a very limited half-life following infusion, however, as they are rapidly targeted by the patient’s own immune system causing host-versus-graft disease. Gene editing is used to increase the persistence of allogeneic T cells by enabling them to evade host T cell or natural killer (NK) cell surveillance. A conventional method of preventing host T cell rejection is to knockout (KO) the β-2 microglobulin (β2M) to diminish the expression of MHC class I protein. To further minimize rejection, overexpression of nonclassical MHC class I protein, HLA-E, is engineered into allogeneic T cells to evade host NK cell rejection. Strategies to improve allogenic CAR T cell persistence through immune evasion have implications for developing a therapeutic product consisting of both CAR T and CAR NK cells. Here we show the use of different methods to investigate the effectiveness of the immune evasion strategies. We found that the effectiveness of HLA-E expression in the suppression of NK cell rejection is highly correlated with the expression of CD94/NKG2A on the host NK cells. Host NK cells with low CD94/NKG2A expression will rapidly recognize and kill allogeneic CAR-T cells even when HLA-E is overexpressed. As it is not possible to control CD94/NKG2A expression in patient NK cells, we sought to identify alternative factors to allow allogeneic cells to evade rapid immune recognition without requiring long term suppression of immune response. To mitigate the donor-to-donor variation on alloreactivity, we developed a strategy to identify NK ligands that can effectively diminish host NK activation in the presence of allogeneic CAR T cells with TCR and B2M gene KOs. A platform using K562 cell line was developed to screen a wide variety of NK inhibitory peptides and synthetic ligands to identify their inhibitory activities. To date, we have identified several synthetic proteins that demonstrate enhanced NK inhibitory function. These synthetic proteins can be used to complement, or to replace HLA-E and enable more extended persistence of an allogeneic cell product in a broader patient population. Citation Format: Meriam Vejiga, Don Wang, Guangnan Li, Kyle Pratt, Phung Gip, James Trager, Ivan Chan. Immune masking strategies to extend the pharmacokinetics of allogeneic cell therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5511.

CD47 is a cell surface molecule that inhibits phagocytosis of cells that express it by binding to its receptor, SIRPα, on macrophages and other immune cells. CD47 is expressed at different levels by neoplastic and normal cells. Here, to reveal mechanisms by which different neoplastic cells generate this dominant ‘don’t eat me’ signal, we analyse the CD47 regulatory genomic landscape. We identify two distinct super-enhancers (SEs) associated with CD47 in certain cancer cell types. We show that a set of active constituent enhancers, located within the two CD47 SEs, regulate CD47 expression in different cancer cell types and that disruption of CD47 SEs reduces CD47 gene expression. Finally we report that the TNF-NFKB1 signalling pathway directly regulates CD47 by interacting with a constituent enhancer located within a CD47-associated SE specific to breast cancer. These results suggest that cancers can evolve SE to drive CD47 overexpression to escape immune surveillance.

Significance Macrophage-mediated programmed cell removal (PrCR) plays an essential role in tumor surveillance and elimination. Blockade of the don't-eat-me signal CD47 on tumor cells allows already expressed eat-me signals to induce PrCR to eliminate tumor cells. To date the molecular mechanism by which macrophages recognize and phagocytose tumor cells remains unclear. This paper demonstrates that the activation of Toll-like receptor (TLR) pathways in macrophages induces the phosphorylation of Bruton's tyrosine kinase (Btk), which catalyzes cell-surface exposure of calreticulin. Calreticulin on or secreted by macrophages plays a critical role in mediating adjacent tumor cell recognition and phagocytosis. These findings reveal a strategy to enhance the efficacy of PrCR through a combination of TLR/Btk activation and CD47 blockade, and advance our understanding of the underlying mechanism of macrophage-mediated PrCR of tumor cells.