Comparative analysis of in vitro activity of a CD20-specific CART cells based on the human monoclonal antibody ofatumumab

Summary

Despite the armamentarium of highly efficacious therapeutic approaches to treat patients with haemotological malignancies, the choice of therapies to help those with relapsing forms of leukemia and lymphoma has been limited. The goal of the present study was to design a novel CD20-specific CAR and perform a side-by-side comparison of its in vitro activity with that of the published CD20-specific CARs.

Materials and methods

To this end, we produced lentiviral constructs encoding two reference CARs based on the murine mAbs 1F5 and Leu16, as well as the CAR based on the sequence of a fully human mAb ofatumumab (2F2). In these CARs, structure of the hinge region was additionally varied, and alternative designs encompassing a hinge derived from human IgG or lacking a spacer altogether were tested. T cells from a healthy donor were isolated, activated, and transduced with the psedotyped lentiviral particles obtained with the above constructs.

Results

Positive control CAR T cells transduced with a “gold standard” CD19-specific CAR identical to the one used in Kymriah CAR T cell product were also produced (Fig. 1). Next, we asked whether the CAR T cells obtained displayed any differences in cytotoxicity against CD20-positive Raji cells. FACS-based cytotoxicity data shown in Figure 2 indicate that regardless of the CAR design, all CAR T cells were highly active. Nevertheless, this type of analysis merely represents a snapshot of cytotoxic activity at a selected time point (4 hrs), so we proceeded to measure the CAR T cell cytotoxicity in real time by running impedance-based cell proliferation assays (RTCA, iCelligence). Notably, RTCA platform typically requires that target cells be adherent, so we turned to HEK 293T cells overexpressing CD20 as a model. RTCA data collected over 24 hrs indicate that T cells expressing a hingeless version of the 2F2-based CAR outperform the cells expressing other CAR designs at time points past 4 hr, consistent with the FACS data.

Figure 1. Primary human T cells transduced with the indicated CAR constructs display robust surface expression of the CAR

Figure 2. CAR T cells show a significant cytotoxicity against target Raji cells upon 4-hour co-incubation. As negative controls, non-transduced T cells from healthy donors were used

Conclusion

Thus, our second-generation hingeless 2F2-based CAR is a promising candidate for in vivo experiments, given its pronounced CD20-specific in vitro activity and the important advantage of a fully human sequence used as the antigen-recognition module.

This study was supported by the RFBR grant № 19-415-543015 р_мол_а.

Keywords

Т cells, chimeric antigen receptor, CAR T cell therapy, ofatumumab.