Abstract 2374: Bispecific and trispecific GPRC5D antibodies with potent cell-killing activity against multiple myeloma
Authors: Roth Hayley , Rogers Daniel , Sanchez Ileine , Tyrell Breanna , Barnes Trevor , Snyder Ami , Nowgu Nenna , Doolan Kyle , Doranz Benjamin J. , Chambers Ross , Rucker Joseph B. 
Published: March 2024

Background: GPRC5D is a G protein-coupled receptor that is expressed on multiple myeloma cells but absent from most healthy tissues except for hair follicles. It is an attractive target for anti-tumor modalities including T cell-engaging bispecific antibodies. Recent clinical data demonstrate that combination therapy of T cell-engaging molecules individually targeting GPRC5D and BCMA on tumor cells offers unprecedented therapeutic benefits in relapsed/refractory multiple myeloma patients. A trispecific antibody that simultaneously engages these molecules while activating T cells is expected to provide comparable and additional benefits as a monotherapy.

Methods: Multipass membrane proteins are valuable therapeutic targets but are largely inaccessible for antibody discovery due to structural complexity and high conservation. We developed an antibody discovery platform (MPS) tailored for membrane proteins that utilizes advanced immunization techniques including DNA, mRNA, and Lipoparticles (virus-like particles) and evolutionarily divergent host species.

From the parental antibodies isolated, we engineered panels of GPRC5DxCD3 bispecific and GPRC5DxBCMAxCD3 trispecific antibodies using multiple formats and CD3 arms that encompass different geometries and binding stoichiometries. GPRC5D multispecific antibodies were tested for target specificity using Integral Molecular’s Membrane Proteome Array (MPA), and epitope residues were defined using alanine scanning mutagenesis. Both bispecific and trispecific antibodies were tested for in vitro potency and cytokine release using MM.1R and RPMI 8226 multiple myeloma cell lines. In vivo efficacy for a subset of antibodies was determined using xenograft models in PBMC-engrafted mice.

Results: We immunized chickens with GPRC5D to obtain high-titer immune responses and configured resulting antibodies as T cell-engaging bispecific molecules with a CD3 binding arm. A subset of GPRC5DxCD3 bispecific antibodies displayed potent T cell-mediated cytotoxicity with picomolar potency on multiple myeloma cell lines. Antibody specificity profiling showed high specificity binding, and epitope mapping helped to explain why only some GPRC5D antibodies were active as multispecific antibodies.

Bispecific molecules were then re-engineered as trispecific antibodies with additional binding to BCMA, a well-validated multiple myeloma target. GPRC5DxBCMAxCD3 trispecifics showed improved cytotoxic activity against multiple myeloma cells. Both bispecific and trispecific antibodies demonstrated a large window between cell killing and cytokine release. We will present in vivo efficacy data for a panel of bi- and trispecific molecules against MM.1R xenografts in PBMC-engrafted mice.

Citation Format: Hayley Roth, Daniel Rogers, Ileine Sanchez, Breanna Tyrell, Trevor Barnes, Ami Snyder, Nenna Nowgu, Kyle Doolan, Benjamin J. Doranz, Ross Chambers, Joseph B. Rucker. Bispecific and trispecific GPRC5D antibodies with potent cell-killing activity against multiple myeloma [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 2374.