Abstract 5318: Optimizing pharmacokinetics and targeting properties of recombinant anti-Lewis Y antibody hu3S193 in A431 tumor-bearing mice

Abstract

Abstract Aim: The humanized monoclonal antibody 3S193 (hu3S193) specifically binds the Lewis Y (Ley) antigen and has shown a long half-life in Phase I first-in-man trials. Previous results have shown that mutations in residues I253, H310 and H435 of the Fc region interfere with the neonatal receptor (FcRn) binding. We have generated a hu3S193 antibody with an alanine mutation in residue I253 (I253A) that shows a shorter half-life than parental hu3S193. The objective of this pilot study was to evaluate the radiolabeling properties of 89Zr-labelled hu3S193_I253A compared to its parent hu3S193 and evaluate them as small-animal positron emission tomography (PET) imaging agents. Methods: Site-directed mutagenesis was performed using a QuickChange XL II site-directed mutagenesis kit (Stratagene) to generate the hu3S193_I253A. The LONZA Glutamine Synthetase expression system was used for eukaryotic expression of antibody heavy and kappa light chain vectors. Mutants were produced in transient (Freestyle 293, Invitrogen) cells and tested for Ley binding using ELISA, FACS and BIAcore analysis. Parental hu3S193 and hu3S193_I253A were radiolabelled with 89Zr after conjugation to deferroxamine-p-SCN (Df). The 89Zr-Df-labelled antibodies were evaluated in small-animal PET studies at 4, 24, 48, and 72 hours post injection. At 72 hours after injection, animals were sacrificed. Blood samples were taken via cardiac puncture and organs were removed. Blood and organ samples were counted for radioactivity. Results: Analysis of hu3S193_I253A mutant by ELISA, BIAcore and FACS showed retention of Ley antigen binding ability. PET imaging demonstrated specific tumor uptake of both 89Zr-Df-labeled parental and hu3S193_I253A at 24 hours post injection and this uptake was maintained for 6 days after injection. Lesions as small as 16 mm3 were clearly detected with both agents. Higher uptake in the liver was detected when using 89Zr-Df-labeled hu3S193_I253A. The 72 hour biodistribution data displayed a higher non-specific uptake in the liver with hu3S193_I253A versus parental (i.e. 15 %ID/g versus 6 %ID/g respectively). For comparison, using 111In-CHX-A”-hu3S193_I253A liver uptake was 7 ± 0.3 %ID/g. This observation indicates that improvement to the 89Zr-labelling chemistry could reduce the liver uptake of faster clearing hu3S193 constructs. Conclusion: The pilot study of 89Zr-labelled hu3S193 and hu3S193_I253A demonstrated excellent tumor uptake of both agents, with high-contrast images generated of lesions as small as 16mm3. Improvements in 89Zr radiolabelling chemistry may be required to optimize imaging of fast clearing recombinant antibodies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5318. doi:10.1158/1538-7445.AM2011-5318