DISCUSSION
During our pharmacokinetic characterization of two related scFv-based BsAb constructs, we observed an unusually rapid ~10-fold faster clearance of BsAb-1 relative to BsAb-2. Both BsAb-1 and BsAb-2 constructs were made with scFv domains fused to the HC C-terminal of IgG4 mAbs. The two BsAbs displayed comparable binding affinity to the same soluble ligands that had no/minimal peripheral concentrations in normal animals; moreover, the BsAbs had no specific interaction with cell surface receptors thus, eliminating both circulating ligand-mediated and cell surface target-mediated drug disposition as potential mechanisms for the observed clearance. In addition, since both BsAbs examined use an IgG4 parental Fc that has been engineered to eliminate interactions with Fcγ receptors, direct binding with blood cells is not expected to be a viable clearance mechanism either. Given the disparity in clearance was not related to target binding or Fcγ receptor interactions, the focus of the present effort became delineating the non-target related physiological mechanism(s) affecting the BsAb in vivo behavior.
Non-target related physiological factors influencing the disposition and pharmacokinetics of BsAbs are poorly understood. In previous reports we demonstrated that association with liver sinusoidal endothelial cells (LSECs) led to the accumulation of BsAbs in liver and was responsible for the unusually rapid clearance of several BsAbs including some IgG-scFv constructs.13 Given these findings, LSEC clearance became an initial plausible mechanism to explore for BsAb-1 and BsAb-2 that could readily explain the atypical elements of the BsAb clearance. Interestingly, IHC analyses of liver from cynomolgus monkey PK studies conducted with the BsAbs showed that LSECs were not the root cause of the rapid clearance of BsAb-1 relative to BsAb-2 (Figure 4). These negative IHC data were also consistent with radiolabel biodistribution studies in cynomolgus monkeys, which showed that BsAb-1 and BsAb-2 had a similar rate and extent of distribution to tissues (Figure 2). The similar rate and extent of accumulation in the tissues observed for BsAb-1 and BsAb-2 indicate comparable tissue disposition for the molecules and by extension, that the PK differences were not due to clearance via a specific organ. The key difference noted in the biodistribution studies of BsAb-1 and BsAb-2 was the rate of clearance of the molecules from the major organs of clearance. The increased clearance of BsAb-1 relative to BsAb-2 from all the major tissues of elimination along with the greater amount of catabolite recovery of BsAb-1 in urine, strongly suggests that following uptake into tissues BsAb-1 was not efficiently recycled back to the blood circulation and instead degraded. Given these data, we speculated other plausible mechanisms, such as physiochemical properties leading to differential molecular stability, non-specific binding profiles and/or FcRn interactions, may be causative and account for the rapid tissue elimination of BsAb-1.
Physiochemical properties including positive charge, poor thermal stability and hydrophobic-based interaction potential that can facilitate non-specific interactions have been linked to the pharmacokinetic developability of mAbs.7-9 While there are a paucity of such studies for BsAbs, in previous reports we found that increased global structural stabilization and reduced hydrophobicity were connnected with in vivo kinetics for BsAbs with an ECD format.10 In the case of BsAb-1 and BsAb-2 in this report, the molecules have largely comparable physiochemical profiles, with BsAb-1 exhibiting lower first melting temperature than BsAb-2 (Table 1). Taken together, the physiochemical differences are relatively small and we speculate that these factors alone are not the major contributors to the differential clearance of BsAb-1 and BsAb-2.
Another plausible mechanism that we and others have suggested which can negatively affect the pharmacokinetics of mAbs (and by extension BsAbs) is the FcRn interaction profile.16, 24, 25 The FcRn interaction profile includes the direct binding interactions of molecules to FcRn at both acidic (pH ~6) and neutral pH (pH ~7.4), as well as, characterization of the rate of the dissociation of the IgG:FcRn complex as the pH increases. The later parameter contextualizes interactions with FcRn which would impede mAb recycling and release within the endosomal compartment and into the peripheral circulation. 20, 24, 26 In vitroanalyses of BsAb-1 and BsAb-2 using previously published approaches showed that BsAb-1 and BsAb-2 bound to FcRn similarly at pH 6 and showed no binding to FcRn at pH 7.4 (Table 2). These data indicated that direct FcRn binding was unlikely related to the differential clearance observations between BsAb-1 and BsAb-2. However, additional characterization of the FcRn release profile for each BsAb did show striking differences. In the FcRn release assessment, complexes of each of the BsAb with FcRn at acidic pH (pH ~6) were formed and the amount of BsAb which remained bound to FcRn once the complex was exposed to neutral pH (pH ~7.4) was measured as a surrogate of FcRn intracellular binding and extracellular release activities. There is ~8 times larger amount of BsAb-1 that remained bound to FcRn once the complex was exposed to neutral pH compared to BsAb-2, indicating that BsAb-1 is less efficiently released from FcRn upon the pH change (Table 2). Taken together, the poor FcRn release profile may also contribute to the more rapid in vivoclearance of BsAb-1 and provide an additional and perhaps a major culprit mechanism for the PK difference observed relative to BsAb-2. Studies with mAbs have shown that altered release from FcRn can affect both the distribution phase (α phase) and the elimination phase (β phase) of the kinetic time course.27 The PK profile of BsAb-1 showed a rapid distribution and short half-life consistent with the hallmarks of altered FcRn release at neutral pH, further supporting that poor release from FcRn is a likely the perpetrator mechanism for the poor PK behavior of BsAb-1. Interestingly, analyses of the FcRn release profile of the parental mAb (mAb-1) used to construct BsAb-1 did not show any evidence of poor dissociation from the receptor at neutral pH (refer to Results section). This suggests that the fusion of scFv-2 to mAb-1 altered the FcRn interactions of BsAb-1, which was not the case when scFv-1 was fused to mAb-2 to construct BsAb-2.
While dysfunctional FcRn interactions have been noted to negatively impact mAb PK, previous studies of other BsAbs showed no connectivity to FcRn as causative in rapid clearance observations.10, 13, 15, 28-30 To the best of our knowledge, the data presented herein is the first report connecting altered FcRn release to BsAb PK. We speculate that there are likely differences in the intracellular trafficking of BsAb-1 and BsAb-2 linked mechanistically with FcRn-mediated recycling that connect to the in vivo catabolism, elimination and PK observations for the two molecules (Figure 5). We postulate that at the cellular level the molecules are largely comparably internalized via fluid phase endocytosis into endosomes which facilitates binding FcRn within the acidic environment of this compartment. The relative similarity in the BsAb-1 and BsAb-2 physiochemical and direct FcRn binding properties (at acidic and neutral pH) are consistent with this proposed non-specific intracellular internalization and endosomal FcRn binding mechanisms. Next, in the case of BsAb-2, the in vitro and in vivo data indicate the molecule likely undergoes ‘productive recycling’ which is connected to antibody-based biologics with acceptable PK developability. In this situation, intracellular BsAb-2 is mostly salvaged from lysosomal degradation by FcRn-mediated recycling. Indeed, the efficient release from FcRn at neutral pH in vitro supports the molecule’s PK profile. Additionally, the slowed blood clearance of BsAb-2 supports the molecule being productively recycled back into the blood circulation once the receptor:BsAb complex is exposed to the neutral pH outside cells. In contrast, for BsAb-1 which displayed poor PK, we hypothesize that the molecule undergoes ‘non-productive recycling’ whereby when the FcRn:BsAb complex is exposed to neutral pH there is inefficient release of BsAb-1 from FcRn. The inefficient release may shift the trafficking equilibrium such that BsAb-1 is not released from cells and eventually degraded. The ~54% of BsAb-1 that remained bound to FcRn at neutral pH in vitro is consistent with this speculation (Table 2). Along those lines, the greater rate and extent of BsAb-1 catabolites found in urine (relative to BsAb-2) is also supportive of the proposed mechanism (Figure 3). Additional interrogation of our BsAb constructs using other approaches including cell-based trafficking and imaging studies may provide further insight in future studies.
In summary, the findings in this report are an important demonstration that BsAb PK can be impacted by a variety of physiological and biochemical factors. There are multiple PK developability considerations including the nature of the BsAb targets (target/turnover/tissue distribution), the physiochemical properties of the BsAbs, and the BsAb structural configuration that can influence disposition and elimination differentially. Careful delineation of preponderance of these factors on a molecule-to-molecule basis can ultimately lead to the selection and design of BsAbs with increased therapeutic value for patients.