Ohad Ilovich1, Arutselvan Natarajan1 , Sharon Hori1 , Ataya Sathirachinda1 , Richard Kimura1 , Ananth Srinivasan1 , Mathias Gebauer3, Jochen Kruip3, Ingo Focken3, Christian Lange3, Chantal Carrez4, Ingrid Sassoon4, Veronique Blanc4, Susanta K. Sarkar5, and Sanjiv S. Gambhir1,2
- Department of Radiology, Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305
- Departments of Bioengineering and Materials Science & Engineering, Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305
- Sanofi R&D, BioInnovation Novel Protein Therapeutics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany
- Sanofi Oncology, Vitry, France
- Sanofi Oncology, Cambridge, Massachusetts
To develop and compare three copper 64 ((64)Cu)-labeled antibody fragments derived from a CA6-targeting antibody (huDS6) as immuno-positron emission tomography (immuno-PET)-based companion diagnostic agents for an antibody-drug conjugate by using huDS6.Three antibody fragments derived from huDS6 were produced, purified, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and evaluated in the following ways: (a) the affinity of the fragments and the DOTA conjugates was measured via flow cytometry, (b) the stability of the labeled fragments was determined ex vivo in human serum over 24 hours, and (c) comparison of the in vivo imaging potential of the fragments was evaluated in mice bearing subcutaneous CA6-positive and CA6-negative xenografts by using serial PET imaging and biodistribution. Isotype controls with antilysozyme and anti-DM4 B-Fabs and blocking experiments with an excess of either B-Fab or huDS6 were used to determine the extent of the antibody fragment (64)Cu-DOTA-B-Fab binding specificity. Immunoreactivity and tracer kinetics were evaluated by using cellular uptake and 48-hour imaging experiments, respectively. Statistical analyses were performed by using t tests, one-way analysis of variance, and Wilcoxon and Mann-Whitney tests.The antibody fragment (64)Cu-DOTA-B-Fab was more than 95% stable after 24 hours in human serum, had an immunoreactivity of more than 70%, and allowed differentiation between CA6-positive and CA6-negative tumors in vivo as early as 6 hours after injection, with a 1.7-fold uptake ratio between tumors. Isotype and blocking studies experiments showed tracer-specific uptake in antigen-positive tumors, despite some nonspecific uptake in both tumor models.Three antibody fragments were produced and examined as potential companion diagnostic agents. (64)Cu-DOTA-B-Fab is a stable and effective immuno-PET tracer for CA6 imaging in vivo.
CovalX Technology Used (Click each option to learn more)
DOTA conjugation to antibody fragments using metal free buffers and the diabody was reduced using dithiothreitol. Next, it was reacted with 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimido ethylacetamide. Using a mass spectrometer that had been modified with a CovalX high mass detection system, the mean DOTA-fragment ratio was determined. To find this, the change in mass was divided by the mass of a single DOTA substituent.