Universal SLAMF7-Specific CAR T-Cells As Treatment for Multiple Myeloma

Introduction : Signaling lymphocytic activation molecule F7 (SLAMF7, also called CS1) is highly expressed on multiple myeloma (MM) tumor cells but has limited expression in a subset of hematopoietic cells among normal tissues, making it a rational target for chimeric antigen receptor (CAR) T-cell therapy in MM. Currently, most CAR-T cell therapy products are generated from autologous T-cells, which is a major limitation logistically and likely difficult to do for lymphopenic and most critically ill patients. Therefore, we have designed allogeneic "off-the-shelf" engineered CAR T-cells (derived from normal healthy donor peripheral blood mononuclear cells), which contain an inactivation of the TCRα constant (TRAC) gene using TALEN^® gene-editing technology to prevent graft-versus-host disease. The SLAMF7 gene is also inactivated using TALEN^® in order to minimize the risk of fratricide of SLAMF7-specific CAR⁺ T-cells (Galetto et al, ASH 2015, Abstract 116). Here, we report in vitro and in vivo efficacy results from double knock-out (KO) (TRAC and SLAMF7) SLAMF7-specific universal CAR T-cells (UCARTCS1) against MM cell lines and primary MM tumor cells.

Methods : The function of UCARTCS1 cells was tested in vitro against MM cell line (MM.1S) and primary MM tumor cells using flow-cytometry-based cytotoxicity, degranulation, CFSE proliferation, and multiplex cytokine induction assays. The efficacy of UCARTCS1 was also tested in vivo against primary MM xenografts in human fetal bone implanted into NOD/SCID/IL2rg^null mice (NSG-hu). Double KO (TRAC and SLAMF7) T-cells lacking SLAMF7 CAR served as controls. The potential toxicity of UCARTCS1 against hematopoietic cells has also been assessed in vitro using the cytotoxicity assay.

Results : UCARTCS1, but not control double KO T-cells, specifically lysed MM.1S (median, 93% lysis; range, 78-98% with UCARTCS1 vs. median, 17%; range, 15-47% with control T-cells; n=10 experiments) and primary MM tumor cells (n=10 samples) (median 59%; range, 20-90% with UCARTCS1 vs. median 9%; range, 0-36% with control T cells). In agreement with this, we observed specific degranulation in both CD4⁺ and CD8⁺ UCARTCS1 cells but not control T-cells in presence of MM.1S cells and primary MM tumor cells. In addition, significant and specific proliferation of both CD4⁺ and CD8⁺ UCARTCS1 cells but not control T-cells was observed when they were co-cultured with MM.1S or primary MM tumor samples (n=8). Analysis of culture supernatants for ten cytokines showed that UCARTCS1 cells primarily produced IFN-γ and GM-CSF in presence of primary MM tumor cells (n=6) suggesting a Th1/Tc1 response. On the other hand, UCARTCS1 did not induce any significant lysis of normal donor peripheral blood mononuclear cells or CD34⁺ hematopoietic cells from bone marrow aspirates of healthy donors.

To test the efficacy of UCARTCS1 cells in vivo we injected 1x10⁶ CD138-purified primary MM tumor cells into the NSG-hu mouse model. After the tumor had been established (6-8 weeks with a serum M-protein level >15 µg/ml), mice were treated intravenously with a single injection of either 10x10⁶ total cells/mouse ofUCARTCS1 or control T-cells. Mice treated with control T-cells developed gradual increase in M-protein levels (median, 339 µg/ml; range, 100-700 µg/mL) whereas the M-protein levels rapidly became undetectable in the mice treated with UCARTCS1 cells and remained undetectable until they were euthanized at approximately 50 days after adoptive transfer.

Conclusion : Our studies revealed that UCARTCS1 cells specifically target and lyse primary MM tumor cells both in vitro and in vivo . Further, UCARTCS1 cells specifically degranulated, produced Th1/Tc1 cytokines, and proliferated in response to primary MM tumor cells. Our results support further development and testing of this universal "off-the-shelf" allogeneic SLAMF7-specific CAR-T product in patients with MM.