Influence of Affinity and Antigen Internalization on the Uptake and Penetration of Anti-HER2 Antibodies in Solid Tumors

Developmental Therapeutics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania.
Cancer Research (Impact Factor: 9.33). 03/2011; 71(6):2250-9. DOI: 10.1158/0008-5472.CAN-10-2277
Source: PubMed


Antibody drugs are widely used in cancer therapy, but conditions to maximize tumor penetration and efficacy have yet to be fully elucidated. In this study, we investigated the impact of antibody binding affinity on tumor targeting and penetration with affinity variants that recognize the same epitope. Specifically, we compared four derivatives of the C6.5 monoclonal antibody (mAb), which recognizes the same HER2 epitope (monovalent K(D) values ranging from 270 to 0.56 nmol/L). Moderate affinity was associated with the highest tumor accumulation at 24 and 120 hours after intravenous injection, whereas high affinity was found to produce the lowest tumor accumulation. Highest affinity mAbs were confined to the perivascular space of tumors with an average penetration of 20.4 ± 7.5 μm from tumor blood vessels. Conversely, lowest affinity mAbs exhibited a broader distribution pattern with an average penetration of 84.8 ± 12.8 μm. In vitro internalization assays revealed that antibody internalization and catabolism generally increased with affinity, plateauing once the rate of HER2 internalization exceeded the rate of antibody dissociation. Effects of internalization and catabolism on tumor targeting were further examined using antibodies of moderate (C6.5) or high-affinity (trastuzumab), labeled with residualizing ((111)In-labeled) or nonresidualizing ((125)I-labeled) radioisotopes. Significant amounts of antibody of both affinities were degraded by tumors in vivo. Furthermore, moderate- to high-affinity mAbs targeting the same HER2 epitope with monovalent affinity above 23 nmol/L had equal tumor accumulation of residualizing radiolabel over 120 hours. Results indicated equal tumor exposure, suggesting that mAb penetration and retention in tumors reflected affinity-based differences in tumor catabolism. Together, these results suggest that high-density, rapidly internalizing antigens subject high-affinity antibodies to greater internalization and degradation, thereby limiting their penetration of tumors. In contrast, lower-affinity antibodies penetrate tumors more effectively when rates of antibody-antigen dissociation are higher than those of antigen internalization. Together, our findings offer insights into how to optimize the ability of therapeutic antibodies to penetrate tumors.

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    • "This is because very high affinity interactions between antibodies and tumour antigens are predicted to impair efficient tumour penetration of the monoclonal antibodies and thus diminish effective in vivo targeting [28-32]. Similar findings were recently reported by Rudnick et al. [27], who studied the impact of affinity on the in vivo tumour-targeting properties of anti-HER2 antibodies. They found that the highest affinity mAbs exhibited an average penetration of 20.4 ± 7.5 μm from tumour blood vessels. "
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    • "To ascertain the purity and biological integrity of the radioconjugate, an immunoreactivity assay was conducted and the immunoreactive fraction at infinite LAT1 antigen excess was calculated as 0.79 ± 0.11. We examined the binding kinetics of [89Zr]DFO-Ab2 where surface bound [89Zr]DFO-Ab2 was internalized at 37 °C for various intervals up to 24 h as shown in Figure 2 [31]. Over-time, the surface-bound activity decreased as the internalized fraction increased. "
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    • "Most studies examining affinity have been performed using antibodies but are expected to hold true for other targeting ligands. Both mathematical modeling and experimental studies have demonstrated that lower affinity antibodies experience better tissue distribution [67–69]. This “binding site barrier” was first described in a modeling study by Fujimori et al. and is based on the successful binding of a ligand to tumor cells near the site of entry into the tumor; this binding then impedes ligand distribution throughout the tumor [67]. "
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