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Fungal methionine synthase, Met6p, transfers a methyl group from 5-methyl-tetrahydrofolate to homocysteine to generate methionine. The enzyme is essential to fungal growth and is a potential anti-fungal drug design target. We have characterized the enzyme from the pathogen Candida albicans but were unable to crystallize it in native form. We converted Lys103, Lys104, and Glu107 all to Tyr (Met6pY), Thr (Met6pT) and Ala (Met6pA). All variants showed wild-type kinetic activity and formed useful crystals, each with unique crystal packing. In each case the mutated residues participated in beneficial crystal contacts. We have solved the three structures at 2.0–2.8 Å resolution and analyzed crystal packing, active-site residues, and similarity to other known methionine synthase structures. C. albicans Met6p has a two domain structure with each of the domains having a (βα)8-barrel fold. The barrels are arranged face-to-face and the active site is located in a cleft between the two domains. Met6p utilizes a zinc ion for catalysis that is bound in the C-terminal domain and ligated by four conserved residues: His657, Cys659, Glu679 and Cys739.

The cobalamin-independent methionine synthase enzyme catalyzes a challenging reaction: the direct transfer of a methyl from 5-methyl-tetrahydrofolate-glutamate3 to the l-homocysteine thiol. The enzyme has a dual (βα)8 TIM barrel structure that binds, activates and brings the reactants into reaction proximity by conformational movements. In the previously observed open structures, the substrates bind too far apart to react, but we have captured a ternary complex with both substrates bound in a closed form of the enzyme. The closing is described in terms of a hinge between the N- and C-terminal TIM barrels and a rearrangement of key loops within the C domain. The substrate specificity can now be rationalized and the structure reveals His707 as the acid that protonates the THF leaving group through a water molecule trapped in the closed active site. The substrates are correctly oriented for an in-line attack by l-homocysteine on the N(5)-methyl.

Death receptor 5 (DR5) is a tumor necrosis factor receptor (TNF) superfamily member that requires multimerization to activate the extrinsic apoptotic pathway and is broadly expressed on solid and hematologic cancers. IGM-8444 is a multivalent IgM DR5 agonist that efficiently multimerizes DR5 to induce tumor cell apoptosis while maintaining a favorable in vitro and in vivo safety profile. The universal mechanism of apoptotic cell death and the safety profile of IGM-8444 makes it an attractive combination partner with standard of care treatment regimens. Here we describe the characterization of single agent and combinatorial cytotoxicity with different classes of chemotherapeutic agents. IGM-8444 was first evaluated as a monotherapy across a panel of human solid tumor cell lines in vitro and xenograft tumor models in vivo. IGM-8444 responses ranged from highly sensitive to resistant. In a sensitive Colo205 model IGM-8444 additionally showed rapid intratumoral pharmacodynamic (PD) activity, inducing maximal caspase-3 cleavage at 6 hours post-dose. Next a panel of solid tumor (including colorectal, gastric, non-small cell lung cancer, and pancreatic) cell lines were selected to evaluate IGM-8444 in combination with standard of care chemotherapeutic agents. Synergistic cytotoxicity was observed when IGM-8444 was used in combination with certain classes of chemotherapeutic agents including topoisomerase inhibitors, microtubule inhibitors, nucleoside analogs, and platinum-based agents. Enhanced anti-tumor activity was also observed in xenograft mouse models where IGM-8444 was dosed in combination with these classes of chemotherapeutic compounds. Mechanistically, this synergistic combinatorial cytotoxicity may be explained by the reported increase in DR5 expression on tumor cells following treatment with many of these classes of chemotherapeutic agents. In summary, IGM-8444 shows in vitro cytotoxicity and in vivo PD and anti-tumor efficacy responses in preclinical models, with enhanced activity in combination with several classes of chemotherapies reported to upregulate DR5 expression. IGM-8444 combination with FOLFIRI standard of care is currently under evaluation in a Phase 1 study in patients with metastatic colorectal cancer (NCT04553692). Citation Format: Beatrice T. Wang, Thomas J. Matthew, Poonam Yakkundi, Miho Oyasu, Mélanie Desbois, Susan E. Calhoun, Ling Wang, Tasnim Kothambawala, Devinder K. Ubhi, Marvin S. Peterson, Eric W. Humke, Maya F. Kotturi, Bruce A. Keyt, Angus M. Sinclair. Characterization of the synergistic tumor cytotoxicity of agonistic DR5 IgM antibody IGM-8444 with chemotherapeutic agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6123.

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