Cancer Immunology, Immunotherapy

, Volume 57, Issue 12, pp 1879–1890 | Cite as

Kinetics of anti-carcinoembryonic antigen antibody internalization: effects of affinity, bivalency, and stability

  • Michael M. Schmidt
  • Greg M. Thurber
  • K. Dane WittrupEmail author
Original Article


Theoretical analyses suggest that the cellular internalization and catabolism of bound antibodies contribute significantly to poor penetration into tumors. Here we quantitatively assess the internalization of antibodies and antibody fragments against the commonly targeted antigen carcinoembryonic antigen (CEA). Although CEA is often referred to as a non-internalizing or shed antigen, anti-CEA antibodies and antibody fragments are shown to be slowly endocytosed by LS174T cells with a half-time of 10–16 h, a time scale consistent with the metabolic turnover rate of CEA in the absence of antibody. Anti-CEA single chain variable fragments (scFvs) with significant differences in affinity, stability against protease digestion, and valency exhibit similar uptake rates of bound antibody. In contrast, one anti-CEA IgG exhibits unique binding and trafficking properties with twice as many molecules bound per cell at saturation and significantly faster cellular internalization after binding. The internalization rates measured herein can be used in simple computational models to predict the microdistribution of these antibodies in tumor spheroids.


Tumor targeting CEA Endocytosis Antibody fragments Affinity 



This work was supported by CA101830 and the NIGMS/MIT Biotechnology Training Program. The authors also thank the Ludwig Institute and Dr. Gerald Prud’homme for cell lines and plasmids.

Supplementary material

262_2008_518_MOESM1_ESM.pdf (1.2 mb)
MOESM1 (PDF 96 kb)


  1. 1.
    Ackerman M, Chalouni C, Raman V, Schmidt M, Ritter G, Mellman I, Wittrup KD (2008) A33 antigen displays persistent surface expression. Cancer Immunol Immunother (in press)Google Scholar
  2. 2.
    Adams GP et al (2001) High affinity restricts the localization and tumor penetration of single-chain fv antibody molecules. Cancer Res 61:4750–4755PubMedGoogle Scholar
  3. 3.
    Adams GP, Weiner LM (2005) Monoclonal antibody therapy of cancer. Nat Biotechnol 23:1147–1157PubMedCrossRefGoogle Scholar
  4. 4.
    Austin CD et al (2004) Endocytosis and sorting of ErbB2 and the site of action of cancer therapeutics trastuzumab and geldanamycin. Mol Biol Cell 15:5268–5282PubMedCrossRefGoogle Scholar
  5. 5.
    Baxter LT, Jain RK (1991) Transport of fluid and macromolecules in tumors. III. Role of binding and metabolism. Microvasc Res 41:5–23PubMedCrossRefGoogle Scholar
  6. 6.
    Beckman RA, Weiner LM, Davis HM (2007) Antibody constructs in cancer therapy: protein engineering strategies to improve exposure in solid tumors. Cancer 109:170–179PubMedCrossRefGoogle Scholar
  7. 7.
    Behr TM et al (2000) Therapeutic advantages of Auger electron- over beta-emitting radiometals or radioiodine when conjugated to internalizing antibodies. Eur J Nucl Med 27:753–765PubMedCrossRefGoogle Scholar
  8. 8.
    Bryan JN et al (2005) Comparative uptakes and biodistributions of internalizing versus noninternalizing copper-64 radioimmunoconjugates in cell and animal models of colon cancer. Nucl Med Biol 32:851–858PubMedCrossRefGoogle Scholar
  9. 9.
    Cai W et al (2007) PET imaging of colorectal cancer in xenograft-bearing mice by use of an 18F-labeled T84.66 anti-carcinoembryonic antigen diabody. J Nucl Med 48:304–310PubMedCrossRefGoogle Scholar
  10. 10.
    Casalini P et al (1997) Tumor pretargeting: role of avidin/streptavidin on monoclonal antibody internalization. J Nucl Med 38:1378–1381PubMedGoogle Scholar
  11. 11.
    Dooley H et al (1998) Stabilization of antibody fragments in adverse environments. Biotechnol Appl Biochem 28(Pt 1):77–83PubMedGoogle Scholar
  12. 12.
    Fallon EM, Lauffenburger DA (2000) Computational model for effects of ligand/receptor binding properties on interleukin-2 trafficking dynamics and T cell proliferation response. Biotechnol Prog 16:905–916PubMedCrossRefGoogle Scholar
  13. 13.
    Fan Z et al (1994) Antibody-induced epidermal growth factor receptor dimerization mediates inhibition of autocrine proliferation of A431 squamous carcinoma cells. J Biol Chem 269:27595–27602PubMedGoogle Scholar
  14. 14.
    Ford CH et al (1996) Novel flow cytometric analysis of the progress and route of internalization of a monoclonal anti-carcinoembryonic antigen (CEA) antibody. Cytometry 23:228–240PubMedCrossRefGoogle Scholar
  15. 15.
    Friedman LM et al (2005) Synergistic down-regulation of receptor tyrosine kinases by combinations of mAbs: implications for cancer immunotherapy. Proc Natl Acad Sci USA 102:1915–1920PubMedCrossRefGoogle Scholar
  16. 16.
    Fujimori K et al (1990) A modeling analysis of monoclonal antibody percolation through tumors: a binding-site barrier. J Nucl Med 31:1191–1198PubMedGoogle Scholar
  17. 17.
    Graff CP et al (2004) Directed evolution of an anti-carcinoembryonic antigen scFv with a 4-day monovalent dissociation half-time at 37 °C. Protein Eng Des Sel 17:293–304PubMedCrossRefGoogle Scholar
  18. 18.
    Graff CP, Wittrup KD (2003) Theoretical analysis of antibody targeting of tumor spheroids: importance of dosage for penetration, and affinity for retention. Cancer Res 63:1288–1296PubMedGoogle Scholar
  19. 19.
    Hammarstrom S (1999) The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Semin Cancer Biol 9:67–81PubMedCrossRefGoogle Scholar
  20. 20.
    Hedin A, Hammarstrom S, Larsson A (1982) Specificities and binding properties of eight monoclonal antibodies against carcinoembryonic antigen. Mol Immunol 19:1641–1648PubMedCrossRefGoogle Scholar
  21. 21.
    Jain RK (2001) Delivery of molecular and cellular medicine to solid tumors. Adv Drug Deliv Rev 46:149–168PubMedCrossRefGoogle Scholar
  22. 22.
    Kraeber-Bodere F et al (2006) Targeting, toxicity, and efficacy of 2-Step, pretargeted radioimmunotherapy using a chimeric bispecific antibody and 131I-labeled bivalent hapten in a phase I optimization clinical trial. J Nucl Med 47:247–255PubMedGoogle Scholar
  23. 23.
    Kyriakos RJ et al (1992) The fate of antibodies bound to the surface of tumor cells in vitro. Cancer Res 52:835–842PubMedGoogle Scholar
  24. 24.
    Lee YC et al (2002) Reversible dimer formation and stability of the anti-tumour single-chain Fv antibody MFE-23 by neutron scattering, analytical ultracentrifugation, and NMR and FT-IR spectroscopy. J Mol Biol 320:107–127PubMedCrossRefGoogle Scholar
  25. 25.
    Liu G et al (2005) Further investigations of morpholino pretargeting in mice–establishing quantitative relations in tumor. Eur J Nucl Med Mol Imaging 32:1115–1123PubMedCrossRefGoogle Scholar
  26. 26.
    Lund KA et al (1990) Quantitative analysis of the endocytic system involved in hormone-induced receptor internalization. J Biol Chem 265:15713–15723PubMedGoogle Scholar
  27. 27.
    Mattes MJ (2005) Binding parameters of antibodies: pseudo-affinity and other misconceptions. Cancer Immunol Immunother 54:513–516PubMedCrossRefGoogle Scholar
  28. 28.
    Mayor S, Rothberg KG, Maxfield FR (1994) Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking. Science 264:1948–1951PubMedCrossRefGoogle Scholar
  29. 29.
    Paxton RJ et al (1987) Sequence analysis of carcinoembryonic antigen: identification of glycosylation sites and homology with the immunoglobulin supergene family. Proc Natl Acad Sci USA 84:920–924PubMedCrossRefGoogle Scholar
  30. 30.
    Rao BM, Lauffenburger DA, Wittrup KD (2005) Integrating cell-level kinetic modeling into the design of engineered protein therapeutics. Nat Biotechnol 23:191–194PubMedCrossRefGoogle Scholar
  31. 31.
    Reiter Y et al (1996) Engineering antibody Fv fragments for cancer detection and therapy: disulfide-stabilized Fv fragments. Nat Biotechnol 14:1239–1245PubMedCrossRefGoogle Scholar
  32. 32.
    Sharma SK et al (2005) Sustained tumor regression of human colorectal cancer xenografts using a multifunctional mannosylated fusion protein in antibody-directed enzyme prodrug therapy. Clin Cancer Res 11:814–825PubMedGoogle Scholar
  33. 33.
    Shih LB et al (1994) Internalization of an intact doxorubicin immunoconjugate. Cancer Immunol Immunother 38:92–98PubMedCrossRefGoogle Scholar
  34. 34.
    Stein R et al (1999) Carcinoembryonic antigen as a target for radioimmunotherapy of human medullary thyroid carcinoma: antibody processing, targeting, and experimental therapy with 131I and 90Y labeled MAbs. Cancer Biother Radiopharm 14:37–47PubMedCrossRefGoogle Scholar
  35. 35.
    Sung C, van Osdol WW (1995) Pharmacokinetic comparison of direct antibody targeting with pretargeting protocols based on streptavidin–biotin binding. J Nucl Med 36:867–876PubMedGoogle Scholar
  36. 36.
    Thurber G, Wittrup KD (2008) Quantitative spatiotemporal analysis of antibody diffusion and endocytic consumption in tumor spheroids. Cancer Res (in press)Google Scholar
  37. 37.
    Thurber G, Zajik SC, Wittrup KD (2007) Theoretical criteria for antibody saturation of tumors and micrometastases. J Nucl Med 48:995–999PubMedCrossRefGoogle Scholar
  38. 38.
    Wegener WA et al (2000) Safety and efficacy of arcitumomab imaging in colorectal cancer after repeated administration. J Nucl Med 41:1016–1020PubMedGoogle Scholar
  39. 39.
    Yazdi PT, Wenning LA, Murphy RM (1995) Influence of cellular trafficking on protein synthesis inhibition of immunotoxins directed against the transferrin receptor. Cancer Res 55:3763–3771PubMedGoogle Scholar
  40. 40.
    Zimmermann W et al (1987) Isolation and characterization of cDNA clones encoding the human carcinoembryonic antigen reveal a highly conserved repeating structure. Proc Natl Acad Sci USA 84:2960–2964PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Michael M. Schmidt
    • 1
  • Greg M. Thurber
    • 2
  • K. Dane Wittrup
    • 1
    • 2
    Email author
  1. 1.Department of Biological EngineeringMassachusetts Institute of TechnologyCambridgeUSA
  2. 2.Department of Chemical EngineeringMassachusetts Institute of TechnologyCambridgeUSA

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