Monoclonal antibody internalization rates as diagnostic indicators for the immunotherapy of cancer
Recent fundamental discoveries within immunology and cancer biology have led to monoclonal antibody (mAb) immunotherapies; transforming the treatment of cancer. Currently, there are more than 25 approved mAb products for use in oncology and more in the late stages of clinical trials. mAbs generally exhibit desirable pharmacokinetic (PK) characteristics such as slow clearance and long biological half-lives although significant interindividual variability (IIV) in PK is often observed. Anthropometric variables (e.g. weight), demographic variables (age, gender, and race), dose, co-administered drugs, and co-morbidities are often considered as variables in computational modeling of antibody PK; however, much of the variability remains to be explained. Few studies have been performed examining determinants of mAb PK, and little effort has been placed on the identification of diagnostic indicators of IIV.
The high affinity of mAbs for their targets, which is desirable concerning pharmacodynamics, often leads to non-linear, target-mediated pharmacokinetics. In situations where target-drug binding affinity is high, the interaction plays a significant role in drug pharmacokinetics a phenomenon known as target-mediated drug disposition (TMDD). In many cases, after mAbs bind to a specific target, they are internalized by endocytosis and destroyed through lysosomal degradation as an antibody-antigen complex. Multiple intra- and inter-patient factors including binding affinity of mAbs, antigen density, expression of endocytic machinery, and the target turnover kinetics, affect the extent of receptor-mediated destruction. Our overarching hypothesis is that the characterization of tissue-specific (T cell) mAb-target protein internalization rates will help explain the variability seen in patients treated with mAb-based therapies. This project aims to 1) implement a robust and reliable flow cytometry-based method for determining mAb-target internalization 2) determine the coefficient of variation (CV) of mAb-target internalization in healthy donors.
This is a new avenue of research in the lab that is focused on implementing our findings in a more translational setting. Before using protein internalization rates for patient immune monitoring, rigorous validation studies must be performed to establish proper experimental and analytical workflows. However, the feasibility of our approach is supported by robust literature in the fields of endocytosis and protein turnover. For implementation of the methodology, we will focus on established targets of mAb therapies (CTLA4, PD1, LAG-3, TIM-3, TIGIT). Target proteins will be expanded as time allows. For these experiments, healthy donor buffy coats will be purchased from an external source under an IRB-exempt protocol. Primary human T cell subsets will be isolated, activated, stained with target fluorescent antibodies, and put back into culture. Time points over 24 hours will be taken to determine target protein turnover rate. The intensity of fluorescent-tagged antibodies at each time point will be determined by flow cytometry and used to calculate the mAb-target internalization rate. We are looking for new partners with clinical flow cytometry and T cell biology expertise in order to collect data that will be leveraged for team-based extramural grant submissions.
- Translational Research