Antibody internalization after cell surface antigen binding is critical for immunotoxin development.

Immunotoxin potency is dependent on cell surface binding specificity as well as internalization efficiency. Current approaches for immunotoxin development are dependent on existing antibodies that were selected for high affinity and/or high production yield. However, these antibodies may demonstrate low internalization efficiency upon cell surface binding and thus are not necessarily the best candidates for immunotoxin design. Here, we have developed an assay with a novel protein, DTG3, to compare and evaluate the internalization efficiency of monoclonal antibodies in order to circumvent the possibility of low internalization. DTG3 is a fusion protein containing the N-terminus of diphtheria toxin (DT) and three copies of streptococci Protein G immunoglobulin binding domains. We show that antibody-DTG3 complexes formed in the test tube are able to bind their antigen on the target cell surface, resulting in cell internalization, DT-mediated protein synthesis inhibition, and host cell apoptosis. We tested this system with two well-studied antibodies, antihuman CD3ε, and anti-PSMA antibodies and were able to show efficiency of this assay. We further examined commercially available anti-CD123 antibodies for potential leukemia-targeting immunotoxin development. Finally, we applied this system in the early-stage screening of newly generated anti-CD123 hybridomas. Our data showed that this internalization assay system is sensitive, time efficient, and reproducible, and has provided a tool to compare monoclonal antibodies for the clinical development of effective immunotoxins for the treatment of a variety of neoplasms.