Engineering Metabolically Armored T Cell Therapies for Solid Tumor Translation

Altered metabolism is a hallmark of cancer that directly inhibits antitumor immunity and the translation of engineered T cell therapies to solid tumor cancers. In particular, the immunosuppressive metabolite adenosine accumulates in the tumor microenvironment (TME) at concentrations 100-fold that of healthy tissues. Adenosine accumulation strongly inhibits T cell activation, proliferation, and effector function. Interestingly, adenosine deaminase (ADA) enzymes are capable of degrading adenosine into benign and readily cleared metabolites. We hypothesize that T cells that have been engineered to express and secrete ADA enzymes would be ‘armored’ to resist adenosine-mediated immunosuppression. Therefore, we sought to complete proof-of-concept studies that demonstrate that it is feasible to engineer T cell therapies capable of delivering ADA protein to the tumor microenvironment.

There exist two ADA isoforms, one that is intracellularly restricted (ADA1) and one that is secreted (ADA2). We’ve engineered HEK293T and Jurkat T cells with ADA enzymes and mutants thereof to assess their level protein production and enzyme activity in lysates and supernatants. Using kinetic assays, we confirmed that ADA1 is largely intracellularly restricted, making ADA2 the pertinent protein for T cell therapy applications. We’ve tested promoter and other variations in genetic circuitry to increase the secretion of ADA2. We further mutated active site residues in the protein that improved the Km, which resulted in a 10-fold activity increase of the enzyme. Collectively, this work interfaces cellular and protein engineering simultaneously to demonstrate the first ever attempt at engineering improved T cell therapies that degrade immunosuppressive metabolites. These improvements will serve to facilitate the translation of T cell therapies to the solid tumor microenvironment.

Finally, we’ll discuss ongoing efforts to identify even further improved variants of ADA2, through structure-based alignments with homologous enzymes and the construction and screening of a large protein library, as well as construction of an IL-2 promoter-driven luciferase construct that we can use to demonstrate the capability immune cells and the Jurkat cell line to resist adenosine-mediated immune suppression in vitro.