Generation of FMC63 scFv specific anti-idiotype antibodies for quality control of anti-CD19 CAR T cell trials

Abstract

Chimeric antigen receptor (CAR) T cell therapy is an emerging form of immunotherapy that has revolutionised the treatment of some cancers. This is especially true for relapsed and refractory (r/r) lymphoma where it has provided previously terminal patients with a new, potentially curative, treatment option. While the therapeutic outcomes for CAR T cells have been impressive, the quality control during therapy manufacture remains an area in which a lot of progress is required. There is currently no reagent capable of directly detecting the expression of CAR on the surface of transduced T cells. This leads to a large degree of potential variability between CAR T cell preparations that cannot be quantified.

This research aimed to address this problem through the generation of an anti-idiotypic antibody capable of recognising the FMC63 derived CAR utilised for anti-CD19 CAR T cell therapy. This was done via the immunisation of mice with FMC63 scFv containing antigen and generation of hybridomas. Hybridomas were screened against FMC63 scFv via ELISA to determine those that were secreting scFv specific monoclonal antibodies (mAb). Clones secreting mAb identified as scFv specific were then screened against cell surface expressed anti-CD19 CAR via flow cytometry. None of the mAb that were seen to be specific for FMC63 scFv via ELISA were able to detect CAR expression on the surface of cells. Isotype analysis of FMC63 scFv specific mAb indicated a lack of T cell help, and thus a lack of many of the typical process mAb undergo to become high-affinity and antigen specific. As such, an alternative technique in which additional T cell epitopes were introduced to the antigen was developed.

These results identify that immunisation of mice with a weak FMC63 scFv containing antigen is not sufficient for the production of a high-affinity anti-idiotypic mAb for detection of FMC63 derived CAR.