Harnessing the innate immune system: a novel bispecific antibody targeting CD47 and CD24 for selective tumor clearance

Background Tumor-associated macrophages (TAMs) abundantly infiltrate tumors and possess potent antitumor capabilities. “Don‘t eat me” signals like CD47 allow tumors to evade macrophages and proliferate unchecked. CD47 is upregulated in many tumors and interacts with the SIRPα expressed on macrophages to restrict effector function. Similarly, CD24 interacts with the Siglec-10 on TAMs to inhibit engulfment. Despite their potential, there is still a lack of effective therapeutics targeting macrophages. Recent clinical trials targeting CD47 have demonstrated limited efficacy and significant side effects in solid tumors, primarily due to the expression of CD47 on healthy cells such as red blood cells (RBCs). We therefore developed novel anti-CD47 variable domain of heavy chain of heavy-chain antibodies (vHHs) with strong ligand-blocking activity while demonstrating minimal binding to RBCs and incorporated these vHHs to generate an anti-CD47/CD24 bispecific antibody that preserves Fc-effector function and achieves improved tumor targeting while maintaining the blockade of antiphagocytic signals elicited by both CD47 and CD24. Methods Yeast display was employed to generate vHHs targeting CD47 and fully human monoclonal antibodies against CD24, respectively. The antigen binding epitopes of the vHHs to CD47 were predicted using AlphaFold3. Bispecific antibodies were designed, constructed, and characterized in vitro. Antitumor efficacy was evaluated in a human immune cell reconstitution mouse model, while safety was evaluated using a humanized syngeneic mouse model. Furthermore, the underlying mechanisms and alterations in tumor microenvironment were explored ex vivo. Results VHHs targeting CD47 and a fully human antibody against CD24 were identified, all exhibiting potent ligand-blocking activity. The bispecific antibody BiAb-103C, engineered on a human IgG1 scaffold, had strong binding to CD47 + CD24 + tumor cells and could effectively inhibit the CD47-SIRPα interaction. Fc-effector activity was observed towards CD24 (but not CD47) single-positive cells to promote phagocytosis and antibody-dependent cellular cytotoxicity of CD47 + CD24 + tumor cells. In mice, antibody candidates demonstrated notable antitumor activity alongside favorable safety observations. Conclusions Our study presents the discovery of an anti-CD47/CD24 bispecific antibody that offers a promising therapeutic strategy to address the challenges associated with both the efficacy and safety of CD47-targeting agents, offers insight into macrophage-driven cancer immunotherapy, and could potentially provide a therapeutic option for patients non-responsive to immunotherapy.