immuno-PET Agents Research Articles

Imaging the L-Type Amino Acid Transporter-1 (LAT1) with Zr-89 ImmunoPET

The L-type amino acid transporter-1 (LAT1, SLC7A5) is upregulated in a wide range of human cancers, positively correlated with the biological aggressiveness of tumors, and a promising target for both imaging and therapy. Radiolabeled amino acids such as O-(2-[18F]fluoroethyl)-L-tyrosine (FET) that are transport substrates for system L amino acid transporters including LAT1 have met limited success for oncologic imaging outside of the brain, and thus new strategies are needed for imaging LAT1 in systemic cancers. Here, we describe the development and biological evaluation of a novel zirconium-89 labeled antibody, [89Zr]DFO-Ab2, targeting the extracellular domain of LAT1 in a preclinical model of colorectal cancer. This tracer demonstrated specificity for LAT1 in vitro and in vivo with excellent tumor imaging properties in mice with xenograft tumors. PET imaging studies showed high tumor uptake, with optimal tumor-to-non target contrast achieved at 7 days post administration. Biodistribution studies demonstrated tumor uptake of 10.5 ± 1.8 percent injected dose per gram (%ID/g) at 7 days with a tumor to muscle ratio of 13 to 1. In contrast, the peak tumor uptake of the radiolabeled amino acid [18F]FET was 4.4 ± 0.5 %ID/g at 30 min after injection with a tumor to muscle ratio of 1.4 to 1. Blocking studies with unlabeled anti-LAT1 antibody demonstrated a 55% reduction of [89Zr]DFO-Ab2 accumulation in the tumor at 7 days. These results are the first report of direct PET imaging of LAT1 and demonstrate the potential of immunoPET agents for imaging specific amino acid transporters.

Abstract LB-351: Evaluation of two engineered antibody fragments derived from a human CD20 antibody as tracers for immunoPET .

Abstract Background: CD20 is a cell surface antigen that is expressed on the majority of B-cell lymphomas and B-cell leukemias. Several monoclonal antibodies (mAbs), including the fully human IgG1, Ofatumumab, have been used successfully to target human CD20. Ofatumumab is FDA-approved for the treatment of patients with chronic lymphocytic leukemia (CLL) and under ongoing clinical development for other hematological malignancies and autoimmune diseases. In this study Ofatumumab was engineered into smaller fragments including a minibody (scFv-CH3; 80kDa) and a cys-diabody (scFv dimer; 50kDa) to enhance pharmacokinetic parameters and optimize possible application for immunoPET imaging. Methods: The variable (V) genes of Ofatumumab were reformatted into two minibodies (Mb) and four cys-diabodies (Cys-Db), differing in orientation of the V genes and linker lengths. Following in vitro characterization of each variant, a lead candidate from each format was produced at larger scale for in vivo evaluation. Ofatumumab, Mb and Cys-Db were radioiodinated with I-131 (t1/2 8d) for biodistribution and I-124 (t1/2 4.2d) for PET imaging studies using the Iodogen method. Female SCID mice bearing Ramos (CD20+) and BC-1 (CD20−) tumors were used. Blocking studies were performed by pre-injecting 0.2-1mg of unlabeled Ofatumumab 24h prior to imaging. The percent injected dose per gram (%ID/g) was determined in harvested tumor, blood and organs. Results: The lead Cys-Db and Mb were shown to have similar binding affinity (0.8 nM) to that of the parental Ofatumumab. Receptor specific binding was confirmed by competitive cell binding assays using both fluorophore-conjugated and radiolabeled antibody fragments. Blood clearances resulted in MRTs of 3.8h, 8.3h and 24.9h for Cys-Db, Mb and Ofatumumab, respectively. Due to its slower initial distribution, the Mb had a higher blood AUC (306%ID/g*h) than that of Ofatumumab (234% ID/g*h) and Cys-Db (102% ID/g*h). Biodistribution studies of Ofatumumab revealed significant uptake in spleen, bone and liver, which was attributed to Fc-mediated clearance as this was not observed with the antibody fragments. Imaging studies with Mb and Cys-Db showed clear delineation of Ramos xenografts; 1.3% ID/g Cys-Db at 8h and 2.7% ID/g Mb at 24h. This resulted in a tumor to blood ratio of 1.7 for the Cys-Db at 8h and 1.1 for the Mb at 24h. Prior administration of excess unlabeled Ofatumumab reduced tumor uptake of the Cys-Db by 50% at 8h and decreased uptake of the Mb by 17% at 24h. Conclusion: Our preliminary results show that antibody fragments derived from Ofatumumab can be used to target CD20 positive cells in vitro and in vivo. The favorable pharmacokinetics of the Cys-Db resulted in higher tumor to blood ratios at 8h and 24h compared to the Mb or Ofatumumab and support the further development of this fragment as a CD20-targeted immunoPET agent. Citation Format: Jean Gudas, Tove Olafsen, David Ho, Derek Bartlett, Arye Lipman, Jean Gudas, Katy Sorenson, Jennifer Keppler, Christian Behrenbruch, Anna Wu, Sharon Ashworth, Jan Passchier, Roger Gunn, Paul Matthews. Evaluation of two engineered antibody fragments derived from a human CD20 antibody as tracers for immunoPET . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-351. doi:10.1158/1538-7445.AM2013-LB-351

Recombinant Anti-CD20 Antibody Fragments for Small-Animal PET Imaging of B-Cell Lymphomas

The CD20 cell surface antigen is expressed at high levels by over 90% of B-cell non-Hodgkin lymphomas (NHL) and is the target of the anti-CD20 monoclonal antibody rituximab. To provide more sensitive, tumor-specific PET imaging of NHL, we sought to develop PET agents targeting CD20. Two recombinant anti-CD20 rituximab fragments, a minibody (scFv-C(H)3 dimer; 80 kDa) and a modified scFv-Fc fragment (105 kDa), designed to clear rapidly, were generated. Both fragments were radiolabeled with (124)I, and the minibody was additionally labeled with (64)Cu (radiometal) after conjugation to 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA). The radioiodinated fragments and the radiometal-labeled minibody were evaluated in mice as small-animal PET imaging agents for the in vivo imaging of human CD20-expressing lymphomas. Rapid and specific localization to CD20-positive tumors was observed with the radioiodinated fragments. However, the tumor uptake levels and blood activities differed, resulting in different levels of contrast in the images. The better candidate was the minibody, with superior uptake (2-fold higher than that obtained with scFv-Fc) in CD20-positive tumors and low uptake in CD20-negative tumors. Ratios of CD20-positive tumors to CD20-negative tumors at 21 h were 7.0 +/- 3.1 (mean +/- SD) and 3.9 +/- 0.7 for the minibody and scFv-Fc, respectively. The ratio achieved with the (64)Cu-DOTA-minibody at 19 h was about 5-fold lower because of higher residual background activity in CD20-negative tumors. A radioiodinated minibody and a radioiodinated scFv-Fc fragment produced excellent, high-contrast images in vivo. These new immunoPET agents may prove useful for imaging CD20-positive lymphomas in preclinical models and in humans with NHL.