Biodistribution and tumor imaging of an anti-CEA single-chain antibody-albumin fusion protein.

Albumin fusion proteins have demonstrated the ability to prolong the in vivo half-life of small therapeutic proteins/peptides in the circulation and thereby potentially increase their therapeutic efficacy. To evaluate if this format can be employed for antibody-based imaging, an anticarcinoembryonic antigen (CEA) single-chain antibody(scFv)-albumin fusion protein was designed, expressed and radiolabeled for biodistribution and imaging studies in athymic mice bearing human colorectal carcinoma LS-174T xenografts. The [125 I]-T84.66 fusion protein demonstrated rapid tumor uptake of 12.3% injected dose per gram (ID/g) at 4 h that reached a plateau of 22.7% ID/g by 18 h. This was a dramatic increase in tumor uptake compared to 4.9% ID/g for the scFv alone. The radiometal [111 In]-labeled version resulted in higher tumor uptake, 37.2% ID/g at 18 h, which persisted at the tumor site with tumor: blood ratios reaching 18:1 and with normal tissues showing limited uptake. Based on these favorable imaging properties, a pilot [64 Cu]-positron emission tomography imaging study was performed with promising results. The anti-CEA T84.66 scFv-albumin fusion protein demonstrates highly specific tumor uptake that is comparable to cognate recombinant antibody fragments. The radiometal-labeled version, which shows lower normal tissue accumulation than these recombinant antibodies, provides a promising and novel platform for antibody-based imaging agents.

Combined radioimmunotherapy and chemotherapy of breast tumors with Y-90-labeled anti-Her2 and anti-CEA antibodies with taxol.

Because breast cancer cells often express either Her2/neu or carcinoembryonic antigen (CEA) or both, these tumor markers are good targets for radioimmunotherapy using Y-90-labeled antibodies. We performed studies on nude mice bearing xenografts from MCF7, a cell line that has low Her2 and CEA expression, to more accurately reflect the more usual situation in breast cancer. Although uptake of In-111 anti-CEA into tumors was lower than that for In-111-labeled anti-Her2, radioimmunotherapy (RIT) with Y-90 anti-CEA was equivalent to that of Y-90 anti-Her2. When either Y-90 antibody was combined with a split-dose treatment with Taxol, the antitumor effect was greater than with either agent alone. When Y-90 anti-CEA was combined with a single dose of Taxol, the results were equivalent to the split-dose regimen. RIT plus cold Herceptin had no additional effects on tumor size reduction over RIT alone. When animals were first treated with Y-90 anti-Her2 and imaged 1-2 weeks later with In-111 anti-CEA or anti-Her2, tumor uptake was higher for anti-CEA and improved over tumor uptake with no prior RIT. These results suggest that a split dose of RIT with anti-Her2 antibody followed by anti-CEA antibody would be more effective than a single dose of either. This prediction was partially confirmed in a controlled study comparing single- vs split-dose anti-Her2 RIT followed by either anti-Her2 or anti-CEA RIT. These studies suggest that combined RIT and Taxol therapy are suitable in breast cancers expressing either low amounts of Her2 or CEA, thus expanding the number of eligible patients for combined therapies. They further suggest that split-dose RIT using different combinations of Y-90-labeled antibodies is effective in antitumor therapy.

Tailoring the pharmacokinetics and positron emission tomography imaging properties of anti-carcinoembryonic antigen single-chain Fv-Fc antibody fragments.

Antibody fragments are recognized as promising vehicles for delivery of imaging and therapeutic agents to tumor sites in vivo. The serum persistence of IgG1 and fragments with intact Fc region is controlled by the protective neonatal Fc receptor (FcRn) receptor. To modulate the half-life of engineered antibodies, we have mutated the Fc-FcRn binding site of chimeric anti-carcinoembryonic antigen (CEA) antibodies produced in a single-chain Fv-Fc format. The anti-CEA T84.66 single-chain Fv-Fc format wild-type and five mutants (I253A, H310A, H435Q, H435R, and H310A/H435Q, Kabat numbering system) expressed well in mammalian cell culture. After purification and characterization, effective in vitro antigen binding was shown by competition ELISA. Biodistribution studies in BALB/c mice using (125)I- and (131)I-labeled fragments revealed blood clearance rates from slowest to fastest as follows: wild-type > H435R > H435Q > I253A > H310A > H310A/H435Q. The terminal half-lives of the mutants ranged from 83.4 to 7.96 hours, whereas that of the wild-type was approximately 12 days. Additionally, (124)I-labeled wild-type, H435Q, I253A, H310A, and H310A/H435Q variants were evaluated in LS174T xenografted athymic mice by small animal positron emission tomography imaging, revealing localization to the CEA-positive xenografts. The slow clearing wild-type and H435Q constructs required longer to localize to the tumor and clear from the circulation. The I253A and H310A fragments showed intermediate behavior, whereas the H310A/H435Q variant quickly localized to the tumor site, rapidly cleared from the animal circulation and produced clear images. Thus, attenuating the Fc-FcRn interaction provides a way of controlling the antibody fragment serum half-life without compromising expression and tumor targeting.