Introduction: Monoclonal antibodies blocking CTLA-4 or PD-1 ligand interaction have been shown to induce durable clinical responses in a subset of melanoma and NSCLC patients. The clinical efficacy of these antibodies has been linked to their ability to restore robust anti-tumor immune responses. Based on clinical and preclinical data it is expected that combinations of antibodies blocking inhibitory and/or costimulatory molecules could benefit patients that do not respond to existing immunotherapies. However dual blockade of immuno-modulatory receptors (iMODs) has also been shown to increase immune-related toxicity as has been shown for a combination of CTLA-4 and PD-1 antibodies. Bispecific antibodies are a novel therapeutic modality capable of specifically binding two different targets. As such they may be ideally suited to address dual blockade of iMODs. Previous work on other cell surface targets has shown that bispecific antibodies can exert functional activities that cannot be reproduced by monoclonal antibody combinations. In addition, bispecific antibodies that more selectively target specific cell populations, based on the co-expression of the two targets, could have reduced safety liabilities in patients. We report here the generation and characterization of common light chain (cLC) Fab panels against PD-1, PD-L1, LAG-3, CD137, OX40 and TIM-3 for combination in a full length IgG1 bispecific antibody format and present the functional evaluation of some of these combinations in human in vitro functional assays.

Methods: Humanized cLC mice (MeMo®) were immunized with recombinant protein or DNA, and materials harvested from these mice were used to generate Fab regions against these antigens using phage display. Furthermore, large and diverse synthetic cLC Fab-phagemid libraries were used to select iMOD specific Fabs. The resulting cLC Fabs were cloned into bispecific antibody expression constructs for further testing. Recombinant proteins (both wild type and domain swapped orthologs) were generated for each target and used for binding and competition in both ELISA and FACS. Reporter assays using NFAT or NFkB-luc in Jurkat cells were used for each target to measure functional activity of antibodies. Furthermore, primary human assays using CD3 or SEB activation with cytokine release were used as a read-out of T cell activity.

Results: Two parallel strategies were applied to generate panels of cLC Fabs against PD-1, PD-L1, LAG-3, CD137, OX40 and TIM-3; mouse immunization and phage selection yielded >1350 specific Fab arms whereas synthetic library selections yielded >850 Fabs. Initial characterization and binning of representative clones were carried out in bispecific IgG format (iMOD specific Fabs combined with a Tetanus toxoid-specific control Fab arm) allowing for stringent ranking of the iMOD panels in a monovalent format for (domain) specificity, cynomolgus cross-reactivity, affinity, stability, and ligand blocking potency. The resulting characteristics of the identified bins were used to generate diverse bispecific antibody panels that combined different iMOD Fabs. These were first tested in reporter assays to select functional clones, that were then confirmed in a SEB driven T cell proliferation assay. Several bispecific candidates have been identified that show significantly increased cytokine production and other functional parameters compared to benchmark antibodies.

Conclusion: Large diverse Fab panels binding six different iMODs were generated for use in bispecific antibody combinations. Characterization and screening of large panels of bispecific IgG has been carried out in relevant functional assays. Results from the screening of bispecific antibodies targeting iMOD combinations support the concept of empirical screening to identify potent immunomodulatory combinations.

Citation Format: Rinse Klooster, Cecile Geuijen, Mark Throsby. Generation of immuno-modulatory receptor binding bispecific antibodies to modulate tumor immunity [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B088.