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-?-lyase (MGL) could effectively inhibit human tumors xenografted in mice. However, bacterial-derived MGLs are unstable in serum (t1/2 = 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-?-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 kcat/KM of 5.6 � 102 M-1 s-1 and displayed a serum deactivation t1/2 = 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 kcat/KM 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.

Engineering a novel human enzyme for systemic L-methionine depletion cancer therapy

247th National Meeting (2014)

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