Theoretical analyses of antibody transport in solid tumors suggest that cellular internalization and catabolism of bound antibodies significantly retards penetration of these drugs into the tumor. In order to test these predictions using CEA-specific antibodies as a model system, the rates of antibody and antibody fragment internalization by CEA expressing tumor cells were measured. Fluorescence measurements using flow cytometry provided a quantitative and facile method for measuring trafficking kinetics that was higher throughput than imaging approaches and avoided artifacts of incomplete antibody stripping observed with acid washing protocols.
With the exception of IgG M85151a (discussed below), all anti-CEA antibodies and antibody fragments tested were internalized in LS174T cells slowly with uptake half-times of 10–16 h. This time scale is consistent with the metabolic turnover rate of CEA in the absence of antibody (t1/2
~ 15 h) suggesting that the antibodies are taken up passively with the antigen and do little to drive or modulate this uptake. The tested antibodies and antibody fragments have no effect on surface levels of CEA following binding, which is also consistent with a passive uptake mechanism. Since CEA is a GPI-linked protein with no cytoplasmic or transmembrane protein domains, the observed uptake is likely the result of bulk membrane turnover rather than specific protein-mediated pathways [19
]. Such non-specific trafficking of CEA may explain the results of immunofluorescent microscopy experiments in which internalized anti-CEA scFvs colocalized partially but incompletely with markers of multiple endocytic pathways. Similar slow metabolic turnover has been observed for other antibodies targeting non-receptor cell surface antigens [23
Slow cellular uptake of the high affinity anti-CEA scFv ds-sm3E-M on the order of 11–17 h was also observed in two additional colon carcinoma cell lines, LIM1215 and SW-1222. In contrast, uptake was significantly faster (t1/2 ~ 4 h) in a fibrosarcoma cell line HT-1080 transfected with a CEA expression plasmid. It is unclear if this faster uptake is due to greater overall cell surface turnover in this cell line or some function of the artificial CEA overexpression.
Cellular trafficking studies in other systems have suggested that the affinity and valency of soluble ligands or antibodies may alter cellular uptake rates by influencing the fraction of molecules that are recycled to the cell surface following endocytosis, or by altering antigen clustering dynamics on the cell surface [12
]. In the case of the anti-CEA scFvs examined here, however, no significant difference in the net uptake constant (ke
) was observed for molecules with a range of affinity, stability to protease digestion, and valency. The lack of an affinity dependency may suggest that CEA binding occupancy in the endosome has little effect on the fraction of antibodies that are recycled versus degraded, or alternatively, that both the high and low affinity scFvs are able to maintain antigen binding in the endosome due to the high concentration of CEA. Similarly, the equivalent uptake of the monovalent and bivalent scFvs suggests that crosslinking of two CEA molecules on the cell surface has little effect on the antigen’s distribution or trafficking. This result is consistent with previous observations that bivalent binding of IgGs against folate receptor, another GPI-linked protein, were insufficient to drive receptor clustering on the surface or increase uptake [28
Although affinity and valency have little impact on the uptake constant (ke
) of anti-CEA antibodies, they may still influence the total amount of internalized antibody which depends on both ke
and the amount of antibody bound (d[Ab]internal
). This distinction is clearly observed with the behavior of the low affinity scFv ds-shMFE-M in the continuous uptake and surface decay experiments. While ds-shMFE-M is internalized when continuously incubated with the cells at a 20 nM concentration, it dissociates from the cells prior to internalization in the surface decay assay. Both of these cases are relevant to in vivo tumor targeting. The continuous uptake experiments are similar to the loading phase of tumor targeting where a high plasma concentration maintains a sufficient antibody concentration in the tumor to drive binding and internalization, while the surface decay assay represents the retention phase when antibody clears from the plasma and tumor. In contrast to the low affinity case, the high affinity and bivalent antibodies have dissociation rates (koff
) slower than the internalization rate (ke
) such that binding will be essentially irreversible in both targeting regimes [23
Unlike the remainder of the tested antibodies, IgG M85151a exhibited a distinct trafficking profile with a significantly faster net uptake rate (t1/2
~ 5 h) and the ability to downregulate surface CEA. These properties were both valency dependent, as a monovalent Fab fragment of M85151a was internalized slowly and had no effect on surface CEA. Both the M85151a IgG and Fab also bind with approximately twice as many molecules per cell at saturation as compared to other antibodies of equivalent valency. One potential explanation for this twofold higher cell binding stoichiometry is the possibility that these antibodies bind to more than one epitope per CEA molecule. Monoclonal antibodies capable of binding multiple epitopes per CEA molecule have been reported previously, a phenonmenon attributed to the high sequence homology of repeat domains within the antigen [20
]. If M85151a does in fact bind two epitopes per CEA molecule, it may also provide a mechanism for the faster uptake. Bivalent antibodies that bind to a single epitope per antigen can only crosslink two molecules. In contrast, a bivalent molecule that binds more than one epitope per molecule may be able to crosslink larger clusters of antigens. Previous studies have demonstrated that the formation of large clusters of GPI-linked proteins can increase antigen localization in caveolae, as well as drive greater antigen internalization and downregulation [10
Although the experiments presented here indicate that the internalization of anti-CEA antibodies and antibody fragments in LS174T cells occurs slowly, this rate is sufficient to significantly impact antibody distribution and retention in the tumor. Thurber and Wittrup have shown that anti-CEA scFvs are able to penetrate significantly farther into LS174T spheroids when incubated at 20°C versus 37°C due to reduced cellular internalization [36
]. Similarly, Ackermann et al. have demonstrated that the slowly internalized IgG M111147 is able to penetrate significantly farther into LS174T spheroids than the rapidly internalized IgG M85151a (in preparation). The difference in penetration distance can be quantitatively predicted from the ke
values and cell surface binding stoichiometry measured here.
Based on these results and other computational predictions, we suggest that antibodies with slow cellular internalization rates should have advantages for most tumor targeting applications due to their improved penetration and retention in the tumor (with the exception of immunotoxins and antibody-drugs that must be internalized to be cytotoxic [39
]). In some cases, it may be possible to engineer more slowly internalized antibodies by either selecting for proteins that are efficiently recycled following endocytosis or by using monovalent antibodies that avoid faster uptake due to antigen clustering. Alternatively, antibody internalization may be reduced by targeting antigens with slower metabolic turnover. One promising target is the colorectal cancer marker A33 which has extended cell surface persistence due to interactions at the tight junction [1