The Synthesis of antiCD3-F(ab’)2 and Fab, and the Biodistribution of an antiCD3-F(ab’)2 Radiotracer in C57BL/6J and BALB/c mice

Background: The activation of tumor-infiltrating lymphocytes (TILs) has become a prominent immunotherapy technique for the treatment of various cancers. The binding of the antiCD3 immunoglobulin (IgG) to the CD3 T-cell receptor (TCR) produces the cytotoxic T-cell, thereby eliciting an immune response against biological malignancies. In addition to the therapeutic action that these T-cells induce, imaging of these cells serves as a foundation to monitor immunotherapy treatment efficacy. Antigen binding fragments, derived from full-sized therapeutic monoclonal antibodies (mAbs), have been shown to bind identically to their parent mAbs while also exhibiting more rapid clearance from the bloodstream. We developed novel protocols for the production of antiCD3 antigen binding fragments, characterized the fragments, and preliminarily evaluated their capabilities to rapidly and non-invasively analyze the efficacy of TIL therapy. Methods: A monoclonal antiCD3 antibody (mAb) was enzymatically digested into Fab and F(ab’)2 fragments by ficin and pepsin respectively. The antiCD3-F(ab’)2 fragment was isolated by size-exclusion high-performance liquid chromatography (SE-HPLC), and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), dynamic light scattering (DLS), and SE-HPLC. The fragment was then conjugated with the metal chelating agent p-SCN-Bn-NOTA (NOTA), radiolabeled with copper-64 (Cu-64 or 64Cu), and injected into immunocompetent (C57BL/6J) and immunocompromised (BALB/c) mice. 64Cu labeled antiCD3-F(ab’)2 uptake was evaluated ex vivo by measurement of radioactivity in target tissues at 6 h and 24 h posttail vein injection. Result: Protocols for the digestion of antiCD3 to crude Fab and F(ab’)2 were developed and optimized. SE-HPLC purification was successfully performed to isolate the antiCD3- F(ab’)2 fragment. The percent injected dose per gram of tissue (%ID/g) of 64Cu-NOTAantiCD3- F(ab’)2 between black and nude mice was statistically significant for the spleen at 6 h, and for the kidney, spleen, cervical lymph node (CLN), and axillary lymph node (ALN) at 24 h. Conclusion: Novel protocols for the fragmentation and purification of an antiCD3 mAb to Fab and F(ab’)2 were developed. The conjugated, radiolabeled antiCD3-F(ab’)2 fragment showed rapid clearance via the kidney, significant uptake in several lymphoid organs, and further potential for development as an imaging agent for cancer immunotherapy efficacy.