Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Mar 15;71(6):2250-9.
doi: 10.1158/0008-5472.CAN-10-2277.

Influence of affinity and antigen internalization on the uptake and penetration of Anti-HER2 antibodies in solid tumors

Stephen I Rudnick  1 , Jianlong Lou, Calvin C Shaller, Yong Tang, Andres J P Klein-Szanto, Louis M Weiner, James D Marks, Gregory P Adams
Affiliations

Influence of affinity and antigen internalization on the uptake and penetration of Anti-HER2 antibodies in solid tumors

Stephen I Rudnick et al. Cancer Res. .

Abstract

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Affinity limits the tumor uptake of antibodies. For each anti-HER2 affinity variant, 20μg 125I labeled IgG was injected into scid mice bearing s.c. SK-OV-3 tumors. The amount of antibody in tumors was determined 24hr (left) and 120hr later (right). The monovalent affinity, as previously determined by SPR, is given for each antibody in the graph key. The statistical significance (P) in comparing C6.5 to H3B1 is given above each timepoint. The average of 5 mice ± standard error of the mean is graphed per point.
Figure 2
Figure 2
Affinity limits antibody penetration into tumors. Representative IHC of s.c. SK-OV-3 tumors 120hr after i.p. injection of 500μg of anti-HER2 IgG. Adjacent sections of tumors from untreated mice were stained with hematoxylin and eosin (A) and for human HER2 (B). Dual staining for blood vessels (brown) and human IgG (purple) was performed on tumors from untreated mice (C) and mice treated with G98A (D), C6.5 (E), ML39 (F), H3B1 (G), and trastuzumab (H). The bar in panel C represents 100microns with 10× magnification. The average distance (±sem) of IgG penetration from randomly selected blood vessels is plotted in panel I.
Figure 3
Figure 3
Affinity, relative to the rate of antigen internalization, determines the extent of antibody catabolism in vitro. Internalization assays were conducted by incubating 450ng 125I labeled anti-HER2 IgGs with 5×105 SK-OV-3 cells. Samples were taken immediately following or 1, 2, 4, 8, 24hr after unbound IgG was washed from cells. The percent of total antibody is shown with respect to time for surface bound (A), dissociated (B), internalized (C), and degraded (D) antibody. The average ±1 standard deviation is shown from triplicate samples. The correlation of degradation with antigen internalization (E) was made by plotting the 24hr timepoint in D against the SPR determined rate of dissociation of the parental scFv from each affinity clone. The monovalent scFv affinity used for trastuzumab (unfilled square) was determined for the 4D5 Fab elsewhere (28). The region shaded in gray represents the range of internalization rates (ke) as determined by others (18).
Figure 4
Figure 4
Comparison of radiolabel reveals that tumors consume significant amounts of antibody in vivo. Biodistribution experiments were conducted as described in figure 1. 20μg of 125I (triangles) or 111In (circles) labeled C6.5 IgG (left) and trastuzumab (right) were injected into scid mice bearing s.c. SK-OV-3 tumor xenografts. The amount of antibody in tumors (filled symbols and black line) and in circulation (empty symbols and grey line) was determined after 8, 24, 48, 72, and 120hr. Each point is the average of 5 mice ± the standard error of the mean.
Figure 5
Figure 5
Use of residualizing isotope reveals that affinity does not limit the amount of antibody delivered to the tumor. For each anti-HER2 affinity variant, 20μg 111In labeled IgG was injected into scid mice bearing s.c. SK-OV-3 tumors. The amount of antibody in tumors was determined 24hr (left) and 120hr later (right). The average of 5 mice ± standard error of the mean is graphed per point.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Fujimori K, Covell DG, Fletcher JE, Weinstein JN. Modeling analysis of the global and microscopic distribution of immunoglobulin G, F(ab')2, and Fab in tumors. Cancer research. 1989;49:5656–63. - PubMed
    1. Fujimori K, Covell DG, Fletcher JE, Weinstein JN. A modeling analysis of monoclonal antibody percolation through tumors: a binding-site barrier. J Nucl Med. 1990;31:1191–8. - PubMed
    1. Jain RK. Physiological barriers to delivery of monoclonal antibodies and other macromolecules in tumors. Cancer research. 1990;50:814s–9s. - PubMed
    1. Ackerman ME, Pawlowski D, Wittrup KD. Effect of antigen turnover rate and expression level on antibody penetration into tumor spheroids. Molecular cancer therapeutics. 2008;7:2233–40. - PMC - PubMed
    1. Thurber GM, Zajic SC, Wittrup KD. Theoretic criteria for antibody penetration into solid tumors and micrometastases. J Nucl Med. 2007;48:995–9. - PubMed

Publication types

MeSH terms