This study describes the development of highly specific and effective CAR T-cells expressing novel Chimeric Antigen Receptors targeting the Kappa Myeloma Antigen. Multiple myeloma (MM) is an incurable malignancy of differentiated plasma cells accompanied by severe bone lesions, cytopenia and hypercalcemia. The majority of MM patients produce excess monoclonal immunoglobulin and/or isotype-restricted free light chains (FLC). The monoclonal antibody KappaMab (kMAb-previously-MDX-1097) binds to a unique conformational epitope on the Kappa Myeloma Antigen (KMA). KMA consists of kFLC that is not associated with Ig heavy chain but is non-covalently associated with sphingomyelin on the cell membrane. KMA is present on k-restricted MM, some k-type lymphomas and B cells associated with Waldenstroms macroglobulinemia and not present on normal B cells, immune cells or normal human tissue. The restricted expression of KMA makes it an ideal target and the unique specificity of KMAb suggests that this antibody is an excellent candidate for the development of a novel treatment for myeloma patients.The scFv gene from the kMAb was fused with the co-stimulatory domains from either CD28 or CD137 (41BB), along with the activation domain of the CD3z; subunit to construct several second generation CARs in the piggyBac transposon vector. In addition, a novel chimeric construct of KMA fused to mCherry was designed, to enable the creation of stable antigen presenting cell lines. Transfected CD3+Tcells were enriched and expanded using irradiated autologous PBMCs and the irradiated KMA-mCherry cell line with the addition of 200IU of IL-15 every 48 hours. After a three week expansion, the CAR T-cells were phenotyped and analysed for functional specificity. Release of IFN-γ upon co-culture with KMA+ or KMA- cells indicated target specific activation. The potency of the CAR T-cells was confirmed in cytotoxicity assays, using 51Cr labelled cells. Pre-clinical data indicated that the KM.CAR T-cell containing the CD8 spacer was the most efficacious among all the KM.CAR T-cells generated, even when there were moderate levels of antigen presentation. The CD28 co-stimulatory domain containing KM.CAR T-cells expressed and expanded better than the ones containing the 41BB co-stimulatory domain. All the KM.CAR T-cells were effective in clearing target cells that expressed KMA. There was a mix of central and effector memory cells at the end of culture which indicated a potential for long term survival and function in vivo. Most of the KM.CAR T-cells showed negligible expression of the exhaustion marker PD-1. Myeloma is becoming the next haematological malignancy after leukaemia to demonstrate phenomenal response rates to CAR therapy. Our in vitro data indicates that the KM.CAR T-cell is likely to be effective in kappa restricted myeloma patients. In vivo testing of these CAR T-cells in a Rag2-/- myeloma mouse model system is currently underway.

Citation Format: Kavitha Gowrishankar, Rosanne Dunn, David Gottlieb, Kenneth Micklethwaite. CAR T-cells targeting the kappa myeloma antigen for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3572.