Engineering an Fc-inert feline IgG1 by targeted mutations: Application to anti-PD-1 antibody development

Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment in humans; however, research on ICIs in cats remains limited, and no clinical trials have been conducted for feline neoplastic diseases. Here, we developed a mouse monoclonal antibody (clone 1A1-2) targeting the feline PD-1 molecule and generated a mouse-feline chimeric antibody (1A1-2-fIgG₁) by replacing the constant region of 1A1-2 with that of feline IgG₁. However, administering 1A1-2-fIgG₁ to cats may deplete PD-1-expressing effector T-cells via complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis, as feline IgG₁ binds to CD64, CD16, and C1q. We engineered two 1A1-2-fIgG₁ mutants with amino acid substitutions in the constant region to reduce the interactions between the Fc fragment and C1q or FcγRs and mitigate these effector functions. These mutations successfully abolished the binding to CD64, CD32, and CD16 while preserving the affinity for FcRn, which is essential in maintaining the half-life of antibodies in the blood. Furthermore, the mutants exhibited impaired binding to C1q. Despite these modifications, the mutated antibodies effectively restored IFN-γ production, which had been suppressed by PD-1/PD-L1 signaling in stimulated lymphocytes, to levels comparable to those of the original antibody. These findings reveal that the engineered antibodies have potential for future clinical applications in feline oncology.