INTRODUCTION
With three approvals and another ~100 in clinical development, bispecific antibodies (BsAbs) represent an important class of therapeutic modalities.1, 2 The intent of BsAb therapy is for a single molecule to interfere with multiple disease pathways by recognizing two different epitopes or antigens. These interactions can expand and prolong the efficacy of these modalities in complex disease indications. Another attractive quality of BsAbs is their potential to provide novel functionalities that do not exist in mixtures of the parental antibodies leading to synergistic biological effects. Given it has become clear that many disorders including cancer, metabolic diseases (including diabetes and cardiovascular illnesses), and autoimmune diseases display multiple and/or redundant mechanisms that fuel their progression, BsAbs have the potential to provide increasingly effective therapeutic options to patients compared with antibodies and other therapeutic entities that interact or modulate the activity of a single target.3-5 Innovation in the field of protein engineering and advancements in technology have led to the design enablement of over 100 BsAb formats.6 While some BsAbs are simply smaller proteins comprised of two linked antigen-binding fragments, a number of other BsAbs formats leverage the basic modular nature of the IgG structure. The IgG-like BsAb molecules consist of subunits on individual antibodies attached to an agonistic/antagonistic mAb that impart the ability to bind dual soluble or membrane bound ligands or a combination of both. These formats include DVD-Ig, cross-mAbs, IgG-extracellular domain (ECD) and IgG-scFv constructs.
Despite their exceptional therapeutic promise and structural tractability, the translation of BsAbs as medicines has been relatively slow compared to mAbs.2 For example, the dual activity of T cell redirection and engagement was described approximately >30 years prior to the 2009 launch of catumaxomab (withdrawn in 2017 for commercial reasons) and more recently blinatumomab (approved 2017) and amivantamab (approved 2021) both for treatment of cancer. The first BsAb approved outside of oncology is emicizumab for the treatment of haemophilia which also occurred more recently in 2017. Similar to most antibody therapeutics, the causalities of the slow clinical success for BsAbs can be generally related to several factors, including an incomplete understanding of the biological mechanism of action, poorly defined exposure-response profiles, insufficient safety margins, strategic industry decisions and immunogenicity. The increased inherent structural diversity and tractability BsAbs afford relative to mAbs also leads to potentially greater uncertainty in their pharmacokinetic and disposition profiles. Thus, in addition to the aforementioned challenges, unpredicted aberrant pharmacokinetic profiles requiring increased empirical protein engineering can also limit the potential advantages BsAbs offer pharmacologically relative to classical monospecific mAbs.
As a means to mitigate poor pharmacokinetics for mAbs, several studies have reported leveraging preclinical in vivo and in vitrophysiochemical characterization-based PK developability strategies during the discovery process.7-9 These approaches have been used to improve the probability of success by selecting or engineering mAbs with increased stability (physical, chemical and thermal stabilities) and lower non-specific or unintended interactions.10, 11 Improving the stability and lowering the risk of unintended interactions, in turn provides enhanced human exposure profiles to support the intended dose and frequency of administration. Indeed, we and other groups have reported connecting preclinical pharmacokinetics with various physiochemical characterization along with FcRn interaction analyses in an integrated manner to inform the selection and engineering of mAbs with optimized pharmacokinetic profiles.10-16 Our laboratories have extended these approaches to some BsAbs that utilize IgG-ECD and IgG-scFv formats.10, 13 These studies revealed that poor physiochemical properties in some BsAb formats contributed to increased clearance rate, driven by endothelial cell-based association/clearance mechanisms in the liver; moreover, the studies showed that engineering the structural configuration of the ECD mitigated aberrant pharmacokinetic behavior of the BsAbs. While these initial studies lay an important foundation for understanding non-target related factors influencing the disposition and pharmacokinetics of BsAbs, there remains a paucity of data and an incomplete understanding of the balance between the in vitro physiochemical factors andin vivo physiological mechanisms that influence the peripheral clearance and disposition of BsAbs. Moreover, while previous studies were able to connect physiochemical properties to IgG-ECD BsAb pharmacokinetics in a post-hoc analysis, there remains considerable opportunity to define the relative contribution of the various non-target related factors influencing the non-specific clearance of another BsAb format in an a priori manner. With these points in mind, we designed the present study to evaluate the physiochemical properties and connectivity of these with in vivo mechanism(s) involved in the clearance of two IgG-scFv constructs (deemed BsAb-1 and BsAb-2; Figure 1A) using preclinical models.
The IgG-scFv constructs were made with scFv units and mAbs targeting two distinct soluble ligands having minimal peripheral concentrations in normal animals, so that the in vivo kinetics and disposition could be evaluated in the absence of target mediated drug disposition (TMDD). The Fab (Fab-1) region of BsAb-1 binds to the same ligand as the scFv (scFv-1) component of BsAb-2 and relatedly, the Fab-2 region of BsAb-2 binds to the same target as scFv-2 in BsAb-1 (Figure 1A). While both BsAbs orientations were imparted with the same antigen binding properties, we observed rapid clearance (~2 mL/hr/kg) of BsAb-1 and acceptable clearance (~0.2 mL/hr/kg) of BsAb-2 in cynomolgus monkeys. Characterization of the two BsAbs revealed differences in physical and thermal stability profiles, as well as, in FcRn based interactions. The evaluation of the biodistribution of the two molecules in cynomolgus monkeys indicated distribution to the same organs to the same quantitative extent, but BsAb-1 was more rapidly cleared from tissue. Taken together, the in vitro and in vivo data indicate that the inferior physical stability properties and the poor release of BsAb-1 from FcRn at neutral pH are likely linked to its aberrant clearance in cynomolgus monkeys. The observation is mechanistically distinct than the proposed increased hydrophobic interaction findings alone that led to aberrant kinetics observed for other BsAb formats in the earlier studies, highlighting the complexity of the issue.10, 13 The findings in this report confirm the need for continued evaluation and delineation of the balance between factors influencing the disposition and pharmacokinetics of various BsAbs and the interplay of the BsAb format on these parameters.