EpoxySEAKER and onSEAKER: CAR T-Cell Systems for Targeted Local Biosynthesis of Therapeutic Warheads

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EPOXYSEAKER AND ONSEAKER: CAR T-CELL SYSTEMS FOR TARGETED LOCAL BIOSYNTHESIS OF THERAPEUTIC WARHEADS Our labs have developed novel SEAKER (Synthetic Enzyme-Armed KillER) CAR T-cells that express enzymes that cleave masking groups from systemically administered non-toxic prodrugs. The localization of SEAKER cells to tumors allows for specific conversion of the prodrug to anticancer agent at the site of interest. These SEAKER cells overcome limitations of CAR T-cell therapy such as T-cell exhaustion, immunosuppression and antigen variance by creating a cascade effect through proliferation of T-cells, constitutive secretion of enzymes and catalytic generation of the active drugs. The enzymes continue to produce cytotoxic compounds at the tumor site even after the T-cells functionally exhaust. We will engineer this system to produce novel “epoxySEAKER” and “onSEAKER” cells that express bacterial biosynthetic enzymes and convert non- toxic prodrug substrates into cytotoxic compounds through an enzyme-mediated therapeutic warhead installation. Limiting the bulkiness of the prodrug by eliminating the need for masking moieties we expect greater pharmacological properties of the prodrugs. Installing the warhead moiety onto the prodrug rather than cleaving a masking group will eliminate the non-specific activation of cytotoxic agents and reduce the on-target, off-site toxicity associated with the target compounds. We plan to use our two novel SEAKER systems to target the tumor environment, elicit immunologic responses, and subsequently secrete EpnF or TsnB9 which will enzymatically activate prodrug variants of FDA approved drugs in situ. We will generate multiple prodrugs for the two SEAKER systems and evaluate their conversion to the active compounds using recombinant enzymes in vitro. Compounds such as carfilzomib and panobinostat are associated with on-target off-site toxicity when administered for the treatment of multiple myeloma. Tumor targeting “epoxySEAKER” or “onSEAKER” cells will localize the secretion of synthetic enzyme to the tumor site, allowing systemic administration of non-toxic prodrug, subsequent in situ compound synthesis and reduced off-site toxicity demonstrated by the target compounds. The additive therapeutic effect of the CAR-T cells and small-molecules will increase the effectiveness of treatment and may expand the target scope of CAR-T cell therapy.