Abstract 5761: Identification of Fc mutations that optimize agonistic activity of human anti-CD40 monoclonal antibodies by enhancing appropriate Fcg receptor interactions
Authors: Ye Shiming , Cohen Diane , Choi Donghee , Tan Siu sze , Xiong Joanna , Akamatsu Yoshiko , Chao Debra , Hollenbaugh Diane , Harding Fiona 
Published: August 2018

Abstract
Several agonistic antibodies against human CD40 are currently being explored as immune therapy for cancer patients. Previously, anti-CD40 antibody therapy showed some clinical efficacy, but significant toxicity at the efficacious dose limited further development. To develop an anti-CD40 agonistic antibody with an improved clinical therapeutic index, we focused on modifications to the heavy chain isotype that would impact interactions with specific Fc receptors.
In mouse models, anti-CD40 murine IgG1 demonstrated stronger immune stimulatory activities than murine IgG2a. The CD40 murine IgG2a antibody induced acute cytokine release within hours of dosing and led to mortality weeks later. To generate human antibodies with an Fc structure that functions similarly to murine IgG1, human IgG1 Fc mutations with reduced binding on human CD16, enhanced binding on CD32A/B, and maintained binding on CD64 were identified through a proprietary mammalian cell surface display technology. A panel of anti-human CD40 antibodies carrying different Fc regions were created. In a CD40 transfected HEK293 NFκB reporter cell assay, where agonistic activity was not related to Fcγ receptor interactions, anti-CD40 human IgG2 demonstrated stronger agonistic activity than human IgG1 antibodies with either wild type or mutated Fc. This result confirmed the reported role of human IgG2 hinge in impacting the agonistic activity of anti-CD40. In assays using primary B cells or monocyte-derived dendritic cells, which constitutively express Fcγ receptors, a subset of anti-CD40 human IgG1 antibodies showed stronger potency than the corresponding human IgG2, due to the better Fcγ receptor crosslinking for IgG1 antibodies. Swapping the human IgG2 hinge into IgG1 Fc did not further enhance the agonistic activity of anti-CD40 antibodies in the primary cell assays, suggesting that the Fc-crosslinking plays a dominant role in mediating the agonistic activity of these anti-CD40 antibodies in biological systems with cells expressing Fcγ receptor. Engineering the proprietary human IgG1 Fc mutations to these anti-CD40 antibodies further enhanced agonistic activity compared to wild type IgG1 in primary cell assays. In addition to the potent agonistic activity, anti-CD40 with the IgG1 Fc mutations demonstrated lower ADCC and reduced cytokine production driven by Fc receptor activation in a co-culture system. Finally, an Fc modified anti-CD40 agonistic antibody demonstrated an acceptable safety profile in cynomolgous monkey toxicity studies. These data suggest anti-CD40 with the proprietary IgG1 Fc mutations represents a newer generation of CD40 agonists for oncology immune therapy.
Citation Format: Shiming Ye, Diane Cohen, Donghee Choi, Siu sze Tan, Joanna Xiong, Yoshiko Akamatsu, Debra Chao, Diane Hollenbaugh, Fiona Harding. Identification of Fc mutations that optimize agonistic activity of human anti-CD40 monoclonal antibodies by enhancing appropriate Fcg receptor interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5761.