A Fit-for-purpose Strategy for Clinical Immunogenicity Assessment of Multivalent Bispecific Antibodies.

Bispecific antibodies (BsAbs) have emerged as a promising class of therapeutics to treat complex diseases, offering advantages in dual targeting simultaneously compared to monospecific antibodies. However, BsAbs often require advanced engineering, and the novel formats present challenges for the development of clinical anti-drug antibody (ADA) assays. Immunogenicity evaluation is a required study endpoint during the clinical development of biotherapeutics, and bridging immunoassay is a common method for developing clinical ADA assays. However, in two of our BsAb programs, the traditional bridging enzyme-linked immunosorbent assay (ELISA) was unable to detect surrogate ADAs directed against the arm containing multivalent domains. Further investigations revealed that the surrogate ADAs to the multivalent binding domain of the two BsAbs predominantly form 1:1 complexes with the drug, even in the presence of a significant excess of the BsAbs. To overcome the limitations of traditional bridging ELISA, we explored alternative assay approaches and developed fit-for-purpose ADA assays tailored to supporting multivalent BsAbs. Here, we present two case studies of multivalent BsAb analyzed using a stepwise ELISA format, where the drug is used for capture and a recombinant human high affinity Fc gamma receptor 1A (FcγRIa) is used for detection of the ADAs, leveraging the LALAPG attenuated effector function mutations present in both BsAbs. This work highlights the complexity of bioanalytical challenges in developing advanced therapeutic modalities and showcases the innovative solutions required to support the rapidly evolving field of BsAb therapeutics.