(160l) Engineering Glycans into Antibody Fab Domains for Improved Therapeutic Efficacy

Glycosylation of therapeutic proteins has been shown to influence their safety, bioactivity, and pharmacokinetic (PK) properties. As a result, the emergence of therapeutic monoclonal antibodies (mAbs) has prompted the mandated restriction of their glycosylation. Current therapeutic mAbs are immunoglobulin G (IgG) type, with glycosylation limited to a single Fc domain glycan. We hypothesized that these restrictions on atypical mAb glycosylation limit therapeutic benefits based on consolidating evidence that Fab domain glycans improve biological activity and PK properties. To test this hypothesis, we glycoengineered variants of two functionally divergent human interleukin-2 (IL-2) binding mAbs: the mAb 602, which biases IL-2 activity toward immune effector cells; and the mAb F5111, which biases IL-2 activity toward regulatory T cells. These “test case” mAbs demonstrate a route to clinical translation for glycoengineered mAbs with atypical glycosylation. Several glycovariants of both 602 and F5111 were designed to include one or two new glycosylation sites in the Fab region and showed successful expression and glycosylation. To ensure that our glycoengineering efforts did not compromise mAb function, we characterized the IL-2 affinity and competitive binding activities of these variants and demonstrated that their function was indistinguishable from the parent mAbs. We further confirmed that the immune-biasing activity of the variants was also identical to that of the parent mAbs. Overall, our results demonstrate how mAbs can be engineered to include atypical glycosylations for potentially improved pharmacological properties, allowing them to act as superior therapeutics.