Advances in Engineering a Human Cystathionine-γ-Lyase for Systemic L‑Methionine Depletion Cancer Therapy

It has been known for nearly a half century that many human tumors are much more sensitive than normal tissues to L-methionine (L-Met) starvation. More recently, systemic L-Met depletion by administration of Pseudomonas putida methionine-γ-lyase (MGL) could effectively inhibit human tumors xenografted in mice. However, bacterial-derived MGLs are unstable in serum (t_1/2 = 1.9 ± 0.2 h) and are highly immunogenic in primates. Since the human genome does not encode a human MGL enzyme, we created de novo a methionine degrading enzyme by reengineering the structurally homologous pyridoxal phosphate-dependent human enzyme cystathionine-γ-lyase (hCGL). hCGL degrades L-cystathionine but displays no promiscuous activity toward L-Met. Rational design and scanning saturation mutagenesis led to the generation of a variant containing three amino acid substitutions (hCGL-NLV) that degraded L-Met with a k_cat/K_M of 5.6 × 10² M⁻¹ s⁻¹ and displayed a serum deactivation t_1/2 = 30 ± 3 h (PEGylated). In vitro, the cytotoxicity of hCGL-NLV toward neuroblastoma cell lines was essentially indistinguishable from that of the P. putida MGL. Furthermore, intravenous administration hCGL-NLV was able to sufficiently deplete serum L-Met to retard growth of neuroblastoma xenografts in a mouse model.  In order to minimize dose requirement, we further engineered hCGL-NLV to identify next generation variants with improved kinetics. The current best variant has a five-fold higher k_cat/K_M as compared to hCGL-NLV and an improved in vitro cytotoxicity profile, with no appreciable loss of stability. We are currently testing this new variant with a variety of cancer cell lines as well as determining its PK/PD and efficacy in a mouse model.