Abstract 2719: Papiliximab, a bispecific nanobody targeting CD47 and PDL1 retards tumor growth without hemolysis

Abstract Cancer cells are known to express immune checkpoint proteins, enabling them to evade immune surveillance. In response, anti-cancer therapies targeting these proteins have been developed. While immune checkpoint inhibitors targeting PD-L1 have been clinically approved, their effectiveness as standalone therapies is limited, with only a 20% response rate. To improve responsiveness, bispecific monoclonal antibodies (MoAbs) targeting both CD47 and PD-L1 have been developed. These bispecific antibodies activate both the innate (by blocking CD47) and adaptive (by blocking PD-L1) immune systems simultaneously. However, conventional antibodies targeting CD47 have been dropped during development due to the risk of hemolysis. In this study, we developed a bispecific single-domain antibody (nanobody, Nb) that targets both CD47 and PD-L1 with a minimal hemagglutination risk. Using phage display libraries from alpacas immunized with recombinant antigens, we screened for an anti-CD47 Nb and an anti-PD-L1 Nb. The affinities (Kd) of these Nbs for their antigens were 7.0 × 10−9 and 9.7 × 10−9, respectively. To maximize affinity for both antigens, a bispecific antibody (Papiliximab) was designed to tandemly array anti-CD47 and PD-L1 Nbs with the Fc region of IgG4. Papiliximab has a lower affinity for RBC (up to 3 μM) than conventional anti-CD47 MoAbs reported earlier. Papiliximab successfully inhibited interactions between CD47/SIRP-α and PD-L1/PD-1, with IC50 values of 7.09 nM and 2.67 nM, respectively. Papiliximab induced the expression of IFN-γ by inhibiting the PD-L1/PD-1 interaction in the PBMC-based MLR (Mixed Lymphocyte reaction) assay. In addition, Papiliximab demonstrated the induction of a phagocytosis effect through CD47 blocking and its IgG4 Fc domain. Therefore, Papiliximab is a multifunctional hybrid bispecific antibody that modulates both innate and acquired immunity. Papiliximab exhibited no cross-reactivity with mouse antigens, but did with cynomolgus antigens. We tested its efficacy on the growth of human B cell lymphoma (Raji cells) and human breast cancer (MDA-MB-231 cells) in humanized NSG mice. Papiliximab was more effective in inhibiting tumor growth than mono-specific Abs and Nbs for PD-L1 or CD47, and their combination. These observations suggest that Papiliximab might be able to improve clinical remission more effectively than the combination of monotherapies without the risk of anemia associated with anti-CD47 MoAbs. We are now working to elucidate the mechanisms behind Papiliximab's superior efficacy and safety compared to the combination of monotherapies. Citation Format: Bum Seo Baek, Min Geun Kim, Jong Hyun Kim, In Young Jang, Sung Min Kim, Jeong Hwan Kim, Sang Beum Lee, Hyoung Tae Kim, Seung-Yong Seong. Papiliximab, a bispecific nanobody targeting CD47 and PDL1 retards tumor growth without hemolysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2719.