1109 Engineering T cells with CD40-based chimeric receptors to enhance adoptive T cell therapy

<h3>Background</h3> We recently provided proof-of-concept that T-cell receptor (TCR)-gene therapy using T cells genetically engineered to express TCRs targeting mutant KRAS G12D could mediate regression of metastatic disease in a patient with pancreatic cancer.¹ However, TCR-gene therapy hasn’t been effective in other patients and thus strategies to enhance activity of the T cells are required. In some patients treated with TCR-gene therapy, the engineered T cells often persist in the patients, suggesting that the T cells ultimately lose the potency required to mediate durable tumor regression. Signals derived from costimulatory receptors can drive effective T-cell responses but these signals may be missing or insufficient in the tumor microenvironment (TME). CD40 is a potent costimulatory protein found on the surface of antigen-presenting cells such as B cells, DCs, and macrophages and plays a major role in activating these cell types, but T cells generally do not express CD40 except transiently upon activation. Thus, we hypothesize that overexpression of CD40 or CD40 chimeric receptors can increase antitumor T-cell function when CD40 is engaged by an agonistic anti-CD40 antibody (CDX-1140, Celldex Therapeutics). <h3>Methods</h3> To test this, we designed CD40 non-antigen chimeric receptors (NACRs), which were co-expressed with a KRAS G12D-reactive TCR. Our CD40 NACRs contained the extracellular domain of CD40 fused to the transmembrane and cytoplasmic domains derived from 10 different families of receptors (e.g., IL-2R, TLR, TNF, etc.). <h3>Results</h3> To evaluate the function of our CD40 NACRs, we stimulated the T cells with anti-CD40 antibody and performed intracellular staining for molecules such as phospho-STAT5 or effector cytokines like IFN-g and TNF. We evaluated the in vitro killing ability of our CD40 NACRs by co-culturing the T cells with pancreatic and colorectal cancer cell lines expressing HLA-C*08:02 and KRAS G12D. Despite evidence of biochemical activity of some of the receptors, most CD40 NACRs did not significantly enhance the in vitro killing ability of the T cells against the tested cancer cell lines. However, a few CD40-based receptors, including wild type CD40, potently enhanced killing of tumor cell lines in vitro. Surprisingly, the enhanced potency of some of these receptors was seen in the absence of anti-CD40 antibody, suggesting that endogenous CD40L expression may be contributing to increased T-cell effector function. Overall, our study highlights the potential of CD40 gene engineering to enhance adoptive cell therapy. Additional characterization of our lead CD40-based receptors is ongoing. <h3>Reference</h3> Rom Leidner, Nelson Sanjuan Silva, Huayu Huang, David Sprott, Chunhong Zheng, Yi-Ping Shih, Amy Leung, Roxanne Payne, Kim Sutcliffe, Julie Cramer, Steven A Rosenberg, Bernard A Fox, Walter J Urba, Eric Tran. Neoantigen T-cell receptor gene therapy in pancreatic cancer. <i>N Engl J Med</i>. 2022 Jun 2;<b>386</b>(22):2112–2119.