To the best of our knowledge, this is the first thorough examination of the effects of affinity on in vivo tumor targeting of intact IgG molecules. In the studies described above, we have determined that the intrinsic affinity of intact IgG molecules is a critical determinant is the accumulation and penetration into solid tumors in vivo. Working with a series of IgG molecules spanning a wide range of intrinsic affinity for the same target epitope, we found that as the affinity of the antibody for its target antigen increased, the distribution became more perivascular in nature. Furthermore, as the higher affinity antibodies exhibited a greater degree of internalization and catabolism in both our in vitro and in vivo studies, we postulate that the limited penetration of the higher affinity antibodies was due to their more efficient catabolism and degradation by the perivascular tumor cells. Antibodies that exhibit efficient penetration into solid tumors would likely target a greater proportion of the tumor, potentially exhibiting improved therapeutic efficacy. Accordingly, the data presented here provide a context for understanding how to design antibodies for specific applications that require either perivascular targeting or thorough tumor penetration.
The role of affinity in tumor targeting with antibody-based molecules has been the subject of significant study and interest. However, until now, these studies have been performed with antibodies that target different epitope on the tumor antigen (24
) or with antibody fragments or targeting molecules based on alternate scaffolds (8
). These studies offered important insights into the impact of affinity. For example, we observed that scFv with high affinities exhibit distinct perivascular accumulation while low affinity scFv demonstrate a broad, almost homogenous, distribution throughout the vascularized regions of the tumor (8
). However, this prior work did not provide direct evidence for the role of affinity in tumor targeting by intact IgG molecules. Targeting different epitopes with different affinity IgGs could bias the outcome of the studies by altering biological processes such as the rates of internalization and recycling. The use of fragments such as scFv molecules or alternate scaffolds such as affibodies or darpins would be subject to different rates of systemic elimination due to differences in size, lack of interaction with the immunoglobulin salvage receptor FcRn and varied sites of catabolism. The studies described in this paper were specifically designed to definitively test the impact of intrinsic affinity for a targeted tumor antigen on the ability of IgG molecules to target tumors in the in vivo
setting. By using a panel of antibodies that exhibited specificity for identical epitopes of HER2 ECD, and were nearly identical in their sequences and structure we were able to isolate and assess the influence of affinity.
In the current study, analyzing the behavior of bivalent IgG's offered both familiar and new insights into how antibodies target and distribute throughout tumors. First, we observed that, using the same in vivo model as was used for scFv studies described above, increasing affinity again did not enhance tumor uptake. In fact, 125I-labeled H3B1, the highest affinity IgG that was tested, had less tumor accumulation than the other affinity variants, and this difference grew between 24hr and 120hr (). Next, as with the scFv in previous studies, it was observed that high affinity IgG have less penetration than do their lower affinity counterparts. Interestingly, there seems to be a threshold between the C6.5 (intrinsic affinity = 23nM) and ML39 (intrinsic affinity = 7.3nM) IgGs in our model that distinguishes more tumor-penetrative from the more perivascularly-retained MAbs (). This observation clearly implies the existence of a binding site barrier in this system, but does not explain the mechanistic basis for the observation.
To address this issue, in vitro
internalization assays were performed (). These studies demonstrated that G98A, and to a lesser extent C6.5, dissociate from the surface of the cells more completely than their higher affinity counterparts. This likely explains why these antibodies distributed more widely throughout the tumors as assessed by IHC. In contrast, ML39, H3B1 and trastuzumab were extensively degraded in vitro
by tumor cells and exhibited limited in vivo
tumor penetration. Furthermore, these higher affinity antibodies possessed rates of monovalent dissociation that were slower than the reported internalization rate of HER2 () While the binding of antibodies to shed antigen within the tumor interstitial space has been shown to block the function of the antibodies in some tumors (27
), the differences we observed in the tumor uptake of the antibodies labeled with residualizing and non-residualizing radioisotopes suggests that this was not a defining limitation in the current study. These findings suggest that irreversible binding of antigen due to internalization and degradation by tumor cells contributes to the “binding site barrier” that limits penetration into the tumor.
Antibody internalization and degradation led to the impressive in vivo
consumption of MAb by tumors (). We found that trastuzumab was more efficiently degraded than C6.5, even though the two antibodies have similar measured functional affinities (12
). While the antibodies bind to distinct HER2 epitopes, they also differ in intrinsic binding site affinity by 255-fold, either property may be responsible for trastuzumab's more complete internalization and degradation. These observations, coupled with trastuzumab's limited perivascular tumor penetration, are consistent with models illustrated by others (see in (16
)). However, there is a lag in the tumor clearance of C6.5 after MAb levels decrease in circulation (). We speculate that this could be due to the rapid rate of C6.5 dissociation relative to the rate of HER2 internalization by the targeted tumor cells. As shown in , it is evident that as affinity increases, so does in vivo
consumption of the antibody by the tumor. Future studies should consider the total exposure of a MAb to cancer cells as a true area under the (concentration vs time) curve for the tumor itself and not rely on a single time point.
While the ultimate correlation of affinity and penetration with efficacy requires additional studies, we have demonstrated a clear structure: function relationship between a MAb and its target wherein the antibody's intrinsic binding affinity has a tangible impact on distribution, and therefore in vivo exposure of tumor to drug. The ultimate applicability of these observations depends on numerous factors including the relationship of targeting to efficacy, the internalization rate of the targeted antigen, and the targeted antigen's expression density. Despite these caveats, the studies presented here describe principles that should be considered when designing MAb for imaging or therapeutic applications.