Generation and functional comparison of anti-CD20 chimeric antigen receptors

Abstract

Chimeric antigen receptor (CAR) T-cell therapy has proven effective, particularly in targeting relapsed, refractory haematological malignancies. However, optimal CAR design is not fully understood. It is known that the antigen epitope position, affinity of interaction and the length of the CAR construct can all impact function, but these factors often depend on the individual antigen context. CD20 is a validated immunotherapy target for monoclonal antibody (mAb) and CAR therapies. This thesis investigated the impact of incorporating single chain variable fragments (scFv) with different properties into an anti-CD20 CAR. Anti-CD20 scFv were generated from a panel of 5 parental mAb; rituximab, BHH2, B1-WG, 2F2 and Leu16. These mAb each possess different affinities, targeted epitopes and binding angles to CD20. Using molecular biology techniques, the scFv were each combined into a CAR construct comprising spacer, transmembrane and intracellular T-cell signalling domains.
It was found that 3 of the 5 CARs, rituximab, BHH2 and Leu16, could successfully be expressed on the cell surface of mammalian cell lines and bound in a specific manner to CD20. Stable cell lines expressing each of these CARs were generated using BWZ.36 and CTLL-2 cells to assess in vitro functional activity. The BWZ.36 IL-2 reporter cell line revealed a hierarchy of activation with the BHH2 CAR eliciting the highest response, followed by the rituximab and Leu16 CARs. CTLL-2 cells were shown to be non-cytotoxic, but capable of producing IFNγ, with the BHH2 CAR inducing the highest level of the cytokine.
CAR expression could not be established on primary mouse T cells despite high expression on other primary murine cells. However, CAR expression was achieved on primary human T cells. The Leu16 and BHH2 CARs were compared in in vitro cytotoxicity assays using transduced human T cells. BHH2 CAR consistently demonstrated a superior level of target-specific cytotoxicity. Cytokine analysis revealed that BHH2 CAR also induced a higher level of IFNγ production in the human T cells, although both constructs elicited similar levels of IL-2. Together, the findings of this thesis demonstrate that the inclusion of anti-CD20 scFvs with different binding epitopes and properties can impact CAR function, with the BHH2 CAR proving to have higher overall T-cell activation and cytotoxic abilities.

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ORCID for Mark Cragg: orcid.org/0000-0003-2077-089X

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