Christian Behrenbruch

Three-Dimensional Nanostructured Substrates toward Efficient Capture of Circulating Tumor Cells

During the progression of metastasis, cancer cells detach from the solid primary tumor, enter the blood stream, and travel to different tissues of the body. These breakaway cancer cells in the peripheral blood are known as circulating tumor cells (CTCs).[1] In addition to conventional diagnostic imaging and serum marker detection, quantification of CTCs in patient blood provides new and valuable information about managing cancer.[2–5] Over the past decade, CTC counting has been used for examining cancer metastasis, predicting patient prognosis, and monitoring the therapeutic outcomes of cancer.[6] However, isolation of CTCs has been technically challenging due to the extremely low abundance (a few to hundreds per milliliter) of CTCs among a large number of hematologic cells in the blood (109 mL−1).[4, 7, 8] Several technology platforms for isolating/counting CTCs have been developed with strategies that involve immunomagnetic beads or microfluidic devices.[3, 4,9, 10] The former utilizes capture-agent-coated magnetic beads to immunologically recognize CTCs in the blood, followed by magnetic isolation. However, these bead-based approaches are limited by their low CTC-capture yield and purity. Recently, a number of microfluidic technologies[9, 10] has been established for capturing viable CTCs from whole-blood samples with improved efficiency and selectivity compared to the bead-based approach.[3, 7] While different device architectures were applied in these CTC-sorting microchips, the improved CTC-capture efficiencies were achieved by increasing CTC/substrate contact frequency and duration.

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

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-C H 3; 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 (t 1/2 8d) for biodistribution and I-124 (t 1/2 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