CART Cell Therapy Targeting CD84 Alone or in Combination with CD19 for the Treatment of B-Cell Malignancies

Most B-cell acute lymphoblastic leukemia (B-ALL) patients (>70%) and B-cell lymphoma patients (>50%) achieve complete remission with CD19-directed CART cell therapy (CART19). However, leukemic cells that either lack or have low levels of CD19 expression may lead to relapse. In large B-cell lymphoma (LBCL), a low or negative CD19 expression has been correlated with resistance to CART19 therapy. Moreover, it has been reported that patients with lower pre-therapy median CD19 density had increased risk of relapse after axicabtagene ciloleucel. On the other hand, loss of cell surface CD19 has been observed in more than 30% of relapses after CART19 therapy in B-ALL. Consequently, new strategies are needed in this scenario. The CD84 (SLAMF5) immunoreceptor is overexpressed in B-cell malignancies and presents a promising novel target for CART cell therapy, either alone or in combination with the CD19 antigen.

We developed a second-generation CART cell targeting CD84 (CART84) (anti-CD84scFv-CD8αH-TM-4-1BB-CD3z). Its in vitro and in vivo effectiveness was evaluated against B-cell malignancies using NALM-6, a B-ALL cell line, and Ramos, a Burkitt lymphoma cell line. CART84 cells were cytotoxic towards B-ALL and aggressive B-cell lymphoma, both in vitro and in vivo. Cytokine production and proliferation assays demonstrated that CART84 specifically proliferated and secreted cytokines when co-cultured with CD84⁺ tumor cells. CART84 controlled disease progression and increased survival of CART-treated mice in a NSG immunodeficient B-ALL mouse model.

Second, we developed a dual CART cell targeting both CD19 and CD84 antigens (CD19/CD84-DUAL1) using an “AND” strategy. The CD19/CD84 DUAL1 CART cells were cytotoxic towards CD19+/CD84+ NALM-6 cells, but also exhibited non-specific killing of CD19-/CD84+ MOLM-13 cells (a cell line derived from acute myeloid leukemia). We identified the dimerization of their hinge domains as the potential cause of this non-specific killing. To prevent dimerization, we incorporated C164S and C181S mutations in the hinge domains, resulting in the CD19/CD84 DUAL1m CART cell. The CD19/CD84 DUAL1m CART cells specifically killed NALM-6 cells expressing both antigens (CD19⁺/CD84⁺), without affecting MOLM-13 cells (CD19^-/CD84⁺).

Next, we designed two versions of CD19 and CD84 dual CART cells using an “IF BETTER” strategy while maintaining the mutations: CD19/CD84-DUAL2m (anti-CD19scFv-4-1BB-CD3z/anti-CD84scFv-4-1BB) and CD19/CD84-DUAL3m (anti-CD19scFv-4-1BB-CD3z-anti-CD84scFv-CD28). Both “IF-BETTER” dual CART cells exhibited specific in vitro cytotoxicity towards CD19⁺/CD84⁺ NALM-6 cells, but not towards CD19^-/CD84⁺ MOLM-13 cells.

In parallel, we developed a dual CART cell targeting both CD19 and CD71 (transferrin receptor protein 1) (CD19/CD71 DUAL4m). Equivalent cytotoxicity assays were conducted with DUAL4m CART cells, which specifically killed CD19⁺/CD71⁺ NALM-6 cells in vitro, but not CD19^-/CD71⁺ MOLM-13 cells. Notably, CD19/CD84-directed CART cells (DUAL2m and DUAL3m) displayed slightly higher in vitro cytotoxicity than the CD19/CD71-directed dual CART cells and controlled disease progression in a NSG immunodeficient B-ALL mouse model.

Finally, a NALM-6 cell line with low CD19 expression was generated to simulate the B-ALL relapses with diminished CD19 expression. Using CRISPR-Cas9 technology, we knocked out the CD19 gene. Subsequently, the CD19-negative cell line was transduced with the CD19 transgene under the control of the PGK100 promoter. In vitro cytotoxicity assays were performed, and all dual CART cells effectively killed this CD19-low expressing NALM-6 B-ALL cell line.

In summary, CART84 cells demonstrated cytotoxicity towards B-ALL and aggressive B-cell lymphoma, both in vitro and in vivo, highlighting their potential as a therapeutic option for relapsed CD19^negative B-cell malignancies. Furthermore, the “IF-BETTER” CD19/CD84-targeted dual CART cells, enhanced with their co-receptor, offer a promising approach for addressing CD19^low B-ALL and B-cell lymphomas, while maintaining the safety profile of CD19-directed CART cell therapy.