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

Department of Biological Engineering, Massachusetts Institute of Technology, Building E19-551, 50 Ames Street, Cambridge, MA, 02139, USA.
Cancer Immunology and Immunotherapy (Impact Factor: 3.94). 05/2008; 57(12):1879-90. DOI: 10.1007/s00262-008-0518-1
Source: PubMed


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.

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Available from: Greg M Thurber, Mar 27, 2014
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    • "We chose a carcinoembryonic antigen (CEA) specific single-chain Fv fragment (scFv) shMFE20, in which the inter domain linker (Gly4Ser)3 had been changed to (Gly4Ser)4 to increase the monomer to dimer ratio in the production process (Supplementary Fig. S1). A disulfide bond had been engineered to further stabilize the monomeric form of the construct21 by performing two amino acid substitutions: Gly to Cys and Ala to Cys at Kabat positions H44 and L100 respectively, resulting in a unique cystine-stabilized single-chain Fv fragment (sscFv). The location of the artificial cystine corresponds to that identified as a general site within the scFv framework to stabilize Fvs by disulfide bonds2223. "
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