Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme.

The Trp metabolite kynurenine (KYN) accumulates in numerous solid tumours and mediates potent immunosuppression. Bacterial kynureninases (KYNases), which preferentially degrade kynurenine, can relieve immunosuppression in multiple cancer models, but immunogenicity concerns preclude their clinical use, while the human enzyme (HsKYNase) has very low activity for kynurenine and shows no therapeutic effect. Using fitness selections, we evolved a HsKYNase variant with 27-fold higher activity, beyond which exploration of >30 evolutionary trajectories involving the interrogation of >10 variants led to no further improvements.

Engineering of the Recombinant Expression and PEGylation Efficiency of the Therapeutic Enzyme Human Thymidine Phosphorylase.

Human thymidine phosphorylase (HsTP) is an enzyme with important implications in the field of rare metabolic diseases. Defective mutations of HsTP lead to mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a disease with a high unmet medical need that is associated with severe neurological and gastrointestinal complications. Current efforts focus on the development of an enzyme replacement therapy (ERT) using the ortholog (EcTP).