Therapeutic strategies utilizing multi-functional and bispecific antibodies (BsAb) that engage multiple pathways are a promising way to improve and prolong efficacy of biologics in complex disease indications. In the early stages of discovery, BsAbs often exhibit a broad range of pharmacokinetic (PK) behavior. Optimization of the neonatal Fc receptor (FcRn) interactions and removal of undesirable physiochemical properties have been used to improve the ‘pharmacokinetic developability’ for various monoclonal antibody (mAb) therapeutics, yet there is a sparsity of such information for BsAbs. The present work evaluated the influence of FcRn interactions and inherent physiochemical properties on the pharmacokinetics of two related single chain variable fragment (scFv) based BsAbs. Despite their close relation, the two BsAbs exhibited disparate PK in cynomolgus monkeys with BsAb-1 having aberrant clearance of ~2 mL/hr/kg and BsAb-2 displaying a typical PK with a clearance of ~0.2 mL/hr/kg. Evaluation of the biophysical characteristics of the molecules, such as charge, non-specific binding, thermal stability, and hydrophobic properties, as well as FcRn interaction characterization showed some differences. In-depth drug disposition results revealed that poor physical stability and incomplete release from FcRn are the primary factors these contributed to the rapid clearance of the BsAb with aberrant PK.

• Background And Purpose: Many oncology ADCs have failed to demonstrate efficacy in clinic because of dose-limiting toxicity. We developed an approach that harnesses ADC affinity to broaden the therapeutic index (TI). • Experimental Approach: Two anti-mesenchymal-epithelial transition factor (MET) monoclonal antibodies (mAbs) with high affinity (HAV) or low affinity (LAV) were studied. IVM studies were conducted in rats to visualize and identify the cellular disposition of the mAbs within the liver and kidney. A rat HT-29 xenograft model was used to evaluate the in vivo anti-tumor efficacy of the two mAbs conjugated to monomethyl auristatin E (MMAE). The pharmacokinetics (PK) and toxicity profiles were also evaluated in rats. Single photon emission computed tomography (SPECT/CT) studies revealed the ADC tissue biodistribution, while mass spectroscopy measured free payload concentration in different organs. • Key Results: The estimated TI for LAV-ADC was at least 3 times greater than the HAV-ADC. The LAV- and HAV-ADCs showed similar levels of anti-tumor activity, while the 111In-DTPA studies showed simlar amounts of the ADCs in HT29 tumors. Although the LAV-ADC has slower blood clearance, higher liver toxicity was observed with HAV-ADC. While the SPECT/CT 111In- and 124I- DTPA findings showed HAV-ADC has higher accumulation and rapid clearance in normal tissues, the IVM studies confirmed HAV mAb accumulates within hepatic sinusoidal endothelial cells while the LAV mAb does not. • Conclusion And Implications: Lowering the MET binding affinity provides a larger TI for MET-ADC. Decreasing the affinity of the ADC reduces the TMDD to MET expressed in normal tissues while maintaining uptake/delivery to the tumor.