The first chimeric antigen receptor (CAR) T-cell therapies have been approved for treatment of B-cell malignancies. This is mainly due to the success of CAR T-cells targeting B-lymphocyte antigen CD19, which has led to astonishing results in clinical trials. Since CD19 is a general B-cell antigen, CAR-T-cells eliminate all B-lineage cells, including nonmalignant B cells. Therefore, the patients suffer from the impaired humoral immune response, increasing susceptibility to severe infections. Since B-cell lymphoma and chronic lymphocytic leukemia cells have a clonally restricted expression of Immunoglobulin (Ig) light chains, either Ig-kappa or Ig-lambda, Ig-kappa positive tumor cells can be targeted while sparing normal Ig-lambda positive B-cells. In this respect, we isolated the sequence encoding the antigen-binding parts of an anti-Ig kappa antibody and designed a second-generation CAR construct (IGK CAR). Expression of IGK CAR in expanded peripheral blood T-cells and subsequent testing of the CAR T-cells in various in vitro assay with targeT-cells demonstrated cytokine production and potent killing of Ig-kappa expressing B-cell lines such as BL-41, whereas no response was observed against Ig lambda positive B-cell lines such as Granta-519. We compared IGK CAR with a clinical CD19 CAR (fmc63) and observed similar potency in targeT-cell killing. Previous reports have shown that the presence of free immunoglobulins present in human serum could inhibit IGK CAR T-cells, and our tests confirmed this. To improve IGK CAR T-cells in the presence of IgGs while maintaining the specificity, we utilized a combinatorial CAR system, where the signaling domains were split. Our design demonstrated efficient killing of Ig-kappa positive cells and was less sensitive to free IgG as compared to IGK CAR T-cells. Additionally, we observed a trade-off between specificity and cytotoxic potential. Increasing one individual component of the combinatorial system made the cells less prone to serum IgG inhibition but demonstrated somewhat higher cytotoxic activity against Ig-kappa negative targets. Our fully adjustable design, therefore, brings another perspective to the field by regulating the individual expression levels according to the treatment needs, hence enabling T-cells to be either more aggressive or specific depending on the treatment efficiency and on the on-target toxicity in patients. Taken together, our in vitro data demonstrate that IGK-CD19 CAR combination is as potent as IGK or CD19 CAR T-cells, and provides an alternative by combining their benefits into one design and thus reduces on-target toxicity.

Citation Format: Hakan Köksal, Pierre Dillard, Sólrún Melkorka Maggadóttir, Gunnar Kvalheim, Erlend Bremertun Smeland, June Helen Myklebust, Else Marit Inderberg, Sébastien Wälchli. Combinatorial IGK-CD19 CAR primarily targets IgK+ malignant B-cells and is less prone to serum IgG inhibition [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A035.