Abstract 2052: Screening novel format antibodies to design bispecific ADCs that address target heterogeneity
Authors: Barnscher Stuart D. , Urosev Dunja , Yin Kevin , Rojas Andrea Hernández , Lawn Sam , Fung Vincent , Wong Jodi , Sagoe-Wagner Araba , Degefie Lemlem , Livernois Ali , Kim Catrina , Moore Paul A. , Rich Jamie R. 
Published: March 2024

Abstract
Addressing inter-patient and intra-tumoral target heterogeneity is a challenge for antibody drug conjugates (ADCs). The most common approach to mitigate intra-tumoral ADC target heterogeneity is to employ a bystander active payload. Once the ADC is internalized and metabolized, the payload can diffuse into tumor cells, independent of target expression. This strategy has proven effective, as evidenced by all but one of the eleven FDA-approved ADCs incorporating a bystander active payload. Nevertheless, it is important to note that in most cases there is clear evidence of an expression-response relationship, and the bystander approach does not specifically address inter-patient heterogeneity in target expression. Bispecific ADCs that can target two different tumor associated antigens (TAAs) are a promising approach to overcoming challenges associated with spatial and temporal target heterogeneity. A traditional bispecific ADC design employs a bivalent IgG where one paratope interacts with target A, and the other paratope interacts with target B. This design ensures that the molecule is maximally active when both targets A and B are present. However, this enhanced specificity approach has limitations, as it requires cellular co-expression of both targets to be effective. In contrast, a novel-format bispecific ADC targeting two different TAAs independently could increase the addressable patient population relative to a monospecific ADC. For example, a bispecific ADC with the potential for both independent and dual targeting of folate receptor alpha (FRα) and NaPi2b, established targets in ovarian cancers, could significantly expand the number of patients who could benefit relative to an ADC against either target alone. Here we describe a novel approach to the design and screening of a FRα x NaPi2b bispecific ADC library with the aim of targeting tumors that express either FRα, NaPi2b, or both targets. A library of 48 bispecific molecules was designed, employing multiple paratopes and variable antibody formats. The library comprised three valency bins, 1+1, 2+1, and 2+2, corresponding to the number of binding arms to each target. Bispecific antibodies were generated from 31 half antibodies containing Azymetric™ Het_Fc mutations. Complementary half-antibodies were combined and oxidized to generate interchain disulfide bonds, with bispecific antibody purity in the range of 90%. Antibodies and their corresponding ADCs, prepared through direct conjugation to a cytotoxic payload, were evaluated on a panel of FRα-expressing and Napi2b-expressing cancer cell lines. Key ADC parameters, including binding, internalization, and in vitro cytotoxicity, highlighted paratope combinations, valencies, and geometric formats that offer the unique potential to overcome target heterogeneity in ovarian cancer.
Citation Format: Stuart D. Barnscher, Dunja Urosev, Kevin Yin, Andrea Hernández Rojas, Sam Lawn, Vincent Fung, Jodi Wong, Araba Sagoe-Wagner, Lemlem Degefie, Ali Livernois, Catrina Kim, Paul A. Moore, Jamie R. Rich. Screening novel format antibodies to design bispecific ADCs that address target heterogeneity [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 2052.