PET imaging of human T cells using a high specific activity Cu-64-labeled anti-CD3 antibody

224 Objectives Population based studies of host immune responses to cancers have demonstrated the importance of immunologic biomarkers for determining patient prognosis and response to therapies. ImmunoPET with radiolabeled monoclonal antibodies (mAbs) targeting human T-cells provides a sensitive modality to evaluate T cell populations in the tumor microenvironment. In this study we demonstrate that the protein dose can be decreased by generating a high specific activity radiolabeled probe. Methods Teplizumab, a humanized anti-CD3 IgG1 mAb, was reduced and conjugated with NODAGA-maleimide to hinge disulfide bonds and the biologic activity assessed using a panel of functional and biochemical assays. Pharmacokinetic, biodistribution and PET imaging studies were performed in nude mice bearing CD3 expressing tumors after radiolabeling with residualizing (111In- and 64Cu) and non-residualizing (124I) radionuclides. Results Conjugation of Teplizumab with 7-8 NODAGA chelators on inter-chain hinge cysteine residues did not impact structural integrity or binding to CD3 (EC50 0.3 nM). In vitro functional assays demonstrated that conjugation to NODAGA further attenuated the functional activity of Teplizumab. Test radiolabeling with 64Cu produced specific activities >100 µCi/µg. Tumor bearing mice micro-dosed with 1 µg of 64Cu-NODAGA-antibody with a specific activity of 100 µCi/µg, showed high contrast PET images as early as 24h. The tumor and blood uptakes at 48h were 38.6±7.7% ID/g and 6.8±4.0% ID/g, respectively. In vivo studies demonstrated higher tumor targeting with residualizing radionuclides compared to 124I. The 111In-NODAGA-mAb exhibited faster blood clearance than the 124I-mAb: 48.6h vs. 88.4h. Conclusions Modification of hinge inter-chain cysteine residues with NODAGA enabled radiolabeling of a mAb to high specific activity without perturbing critical properties or compromising binding affinity. Properties of conjugated and radiolabeled Teplizumab in vitro and in vivo suggest that this radiotracer can provide a means for safely monitoring T cell distribution in patients.