Delivery of cell-killing doses of ionizing radiation to tumors is the objective of antibody-directed radioimmunotherapy (RIT). In practice, however, collateral damage to healthy bone marrow and kidneys limits the maximum delivered radiation dose. Pretargeted RIT (PRIT) aims to overcome this limitation by separating the pharmacokinetics of tumor targeting and radionuclide delivery. First-generation PRIT utilizes the streptavidin/biotin interaction for radionuclide capture, and exhibits promise in animal tumor xenograft models. However, problems with streptavidin immunogenicity, kidney localization, and endogenous biotin intrinsically limit this approach. The perspective of this proposal is that the principle of PRIT is sound, but that to reach its full potential the protein targeting agents must be optimized, and the pharmacokinetics of tumor penetration must be subjected to rigorous engineering analysis. This project brings together faculty from Biological Engineering and Radiation Oncology to collaboratively develop the essential reagents and dosing strategies to enable PRIT to be maximally effective. Protein engineering by directed evolution will be applied to: 1) construct human antibody fragments that capture radiometal chelates (DOTA with yttrium, gallium, or bismuth) effectively irreversibly; and 2) to develop anti-CEA antibody fragments that bind persistently through cycles of endocytic trafficking through acidic compartments. The processes of diffusion, binding, and metabolism of antibody fragments in micrometastases will be characterized by quantitative fluorescence microscopy of single LS174T human colon adenocarcinoma tumor cells and spheroid cultures, and analyzed within a mathematical modeling framework to determine the limiting kinetic processes and predict necessary concentrations and times for saturation binding. These predictions will be tested with quantitative biodistribution studies in LS174T xenografts in nude mice. Taken together, these studies will establish a firm foundation from which to optimize PRIT. In this second and final allowed revision of the proposal, we have eliminated radiation dosimetry and focused on endocytic trafficking of antibody fragments as a critical process limiting the permeation and retention of bispecific antibodies, based on direct evidence for rapid antibody uptake by LS174T cells. This hypothesis informs the planned efforts in protein engineering, cell culture, and in vivo biodistribution studies. ? ?

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
Project #
Application #
Study Section
Cancer Immunopathology and Immunotherapy Study Section (CII)
Program Officer
Wong, Rosemary S
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Massachusetts Institute of Technology
Engineering (All Types)
Schools of Engineering
United States
Zip Code
Chen, Tiffany F; Li, Kevin K; Zhu, Eric F et al. (2018) Artificial Anti-Tumor Opsonizing Proteins with Fibronectin Scaffolds Engineered for Specificity to Each of the Murine Fc?R Types. J Mol Biol 430:1786-1798
Cheal, Sarah M; Xu, Hong; Guo, Hong-Fen et al. (2016) Theranostic pretargeted radioimmunotherapy of colorectal cancer xenografts in mice using picomolar affinity ??Y- or ยน??Lu-DOTA-Bn binding scFv C825/GPA33 IgG bispecific immunoconjugates. Eur J Nucl Med Mol Imaging 43:925-37
Tzeng, Alice; Kauke, Monique J; Zhu, Eric F et al. (2016) Temporally Programmed CD8?+ DC Activation Enhances Combination Cancer Immunotherapy. Cell Rep 17:2503-2511
de Picciotto, Seymour; Dickson, Paige M; Traxlmayr, Michael W et al. (2016) Design Principles for SuCESsFul Biosensors: Specific Fluorophore/Analyte Binding and Minimization of Fluorophore/Scaffold Interactions. J Mol Biol 428:4228-4241
Maass, Katie F; Kulkarni, Chethana; Quadir, Mohiuddin A et al. (2015) A Flow Cytometric Clonogenic Assay Reveals the Single-Cell Potency of Doxorubicin. J Pharm Sci 104:4409-4416
Yang, Nicole J; Liu, David V; Sklaviadis, Demetra et al. (2015) Antibody-mediated neutralization of perfringolysin o for intracellular protein delivery. Mol Pharm 12:1992-2000
de Picciotto, Seymour; Imperiali, Barbara; Griffith, Linda G et al. (2014) Equilibrium and dynamic design principles for binding molecules engineered for reagentless biosensors. Anal Biochem 460:9-15
Van Deventer, James A; Wittrup, Karl Dane (2014) Yeast surface display for antibody isolation: library construction, library screening, and affinity maturation. Methods Mol Biol 1131:151-81
Pirie, Christopher M; Liu, David V; Wittrup, K Dane (2013) Targeted cytolysins synergistically potentiate cytoplasmic delivery of gelonin immunotoxin. Mol Cancer Ther 12:1774-82
Reuel, Nigel F; Grassbaugh, Brittany; Kruss, Sebastian et al. (2013) Emergent properties of nanosensor arrays: applications for monitoring IgG affinity distributions, weakly affined hypermannosylation, and colony selection for biomanufacturing. ACS Nano 7:7472-82

Showing the most recent 10 out of 39 publications