Abstract
Chimeric antigen receptor T (CAR T) cell therapies are a standard of care for the treatment of certain B-cell malignancies and have exhibited promising results in early-phase solid cancer trials. Clinical responses to CAR T-cell therapies are associated with robust CAR T-cell expansion. However, CAR T-cell pharmacokinetic analyses are not available in most treating centres, CAR T-cells can result in serious on- and off-target toxicities, and rare cases of CAR T-cell transformation have been reported. There is a need for tags that both permit the detection of CAR T-cells in clinical laboratories, and provide a ‘safety switch’ for the rapid depletion of CAR T-cells using a clinically-licensed medicine in the event of severe CAR T-cell-related toxicity or transformation.
To meet both requirements, we designed and developed a new protein tag (modCD20), comprising the transmembrane protein CD20 bearing mutations at selected intracellular phosphorylation sites. To minimise immunogenicity, extracellular domains of modCD20 are of wild type human sequence. We found that modCD20 is incorporated into the membrane of genetically-modified cells, and allows for flow cytometric detection of transduced cells, including with antibodies that are routinely available in clinical laboratories (Fig. 1). Unlike wild-type CD20, modCD20 does not induce calcium influx or cellular activation upon binding by the clinically-licensed anti-CD20 antibody, Rituximab. Moreover, parenteral Rituximab administration resulted in depletion of modCD20-expressing cells in vivo (Fig. 2).
Finally, we also designed an alternative modCD20 (modCD20R) that incorporates a single extracellular mutation to abrogate binding by Rituximab but retain binding of the alternate anti-CD20 monoclonal antibody Obinutuzumab; this may be of use as a CAR T-cell safety and detection marker for patients who have recently received Rituximab.
In conclusion, we have developed a new protein tag, which permits the detection of gene-transduced cells in routine clinical laboratories, and enables depletion of genetically modified cells using a clinically-licensed and widely available monoclonal antibody. In the rare event of a CAR T-cell-derived lymphoma, Rituximab can be readily incorporated into standard lymphoma treatment regimens. This modCD20 tag will facilitate the safe development of new CAR-T cell therapies and other adoptive cell therapies.