GPRC5D multispecific antibodies: Engineering for affinity, cross-species reactivity, and bi/trispecific engagement in multiple myeloma

Background: GPRC5D is a G protein-coupled receptor expressed on multiple myeloma cells but largely absent from healthy tissues, making it an attractive target for T cell-engaging multispecific antibodies. Antibodies that simultaneously engage GPRC5D and other molecules (e.g. BCMA and CD3) to activate T cells are anticipated to provide excellent efficacy as monotherapies. Multipass membrane proteins are valuable therapeutic targets but often inaccessible for antibody discovery due to structural complexity and high conservation. Using our membrane protein-optimized discovery platform (MPS), we generated a panel of GPRC5D antibodies and engineered them as bi- and trispecific molecules. We also developed a platform for comprehensive CDR-Scanning of our lead antibody for affinity maturation, developability, and cross-species reactivity for preclinical testing. Methods: Antibodies against GPRC5D were isolated and formatted as GPRC5D×CD3 and GPRC5D×BCMA×CD3. These molecules were tested for in vitro and in vivo potency, cytokine release, and binding specificity. To further optimize our lead, we developed and optimized method for comprehensive CDR-Scanning where each residue across 6 CDR regions was substituted to all 19 other amino acid variants, generating 986 single residue variants. Variants were individually evaluated for expression and binding using ELISA and assessed for expression and reactivity against both human and cynomolgus GPRC5D. Beneficial substitutions (>200% wild-type binding) were combined using structure-guided and AI/ML-informed design strategies for further validation. Results: We identified multispecific antibodies that exhibited potent T cell-mediated cytotoxicity with picomolar potency on multiple myeloma cell lines, a large window between cell killing and cytokine release and robust tumor control against multiple myeloma xenografts in a humanized mouse model. Antibody specificity profiling confirmed high specificity binding to the intended targets, without detectable off-target interactions across the membrane proteome. Saturating mutagenesis of the CDR regions of the GPRC5D-binding moiety of the lead antibody identified 14 variants with elevated binding to the human GPRC5D >200% WT, and 10 variants with elevated binding to cynomolgus GPRC5D. Combination yielded more than 20 variants with binding >4,000% WT. Conclusions: GPRC5D multispecific antibodies hold promise as potent and safe therapeutics for multiple myeloma. Through CDR-Scanning, we are advancing a new generation of GPRC5D antibodies with enhanced attributes, including affinity improvements, pH sensitivity, reduced polyreactivity, improved half-life, and cross-reactivity in cynomolgus for preclinical development.