CAR-Engineered T Cells Specific for the Elotuzumab Target SLAMF7 Eliminate Primary Myeloma Cells and Confer Selective Fratricide of SLAMF7+ Normal Lymphocyte Subsets
Authors: Danhof Sophia , Gogishvili Tea , Koch Silvia , Schreder Martin , Knop Stefan , Einsele Hermann , Hudecek Michael 
Published: October 2019

Abstract
Background: SLAMF7 (CS1, CD319) is uniformly and highly expressed in multiple myeloma (MM) where it promotes adhesion and survival of malignant plasma cells (mPCs) in the bone marrow niche. It is absent on normal solid organ tissues but known to be expressed on lymphocyte subsets (T, B and NK cells). Clinical evaluation of the anti-SLAMF7 monoclonal antibody (mAb) Elotuzumab (huLuc63) has resulted in marked reversible lymphodepletion and conferred potent anti-MM efficacy in combination therapy. Here, we evaluated the potential to generate SLAMF7-directed chimeric antigen receptor (CAR) modified T cells from previously treated MM patients and analyzed their potency against autologous mPCs as well as fratricidal activity against normal lymphocyte subsets.
Methods: Flow cytometric analyses for SLAMF7 expression on mPCs and normal lymphocyte subsets of MM patients (n=67) and healthy donors (n=20) was performed using specific mAbs and matched isotype controls. A SLAMF7-specific CAR was constructed using the VH/VL targeting domains of mAb huLuc63, fused to an Ig-Fc spacer and a signaling module of CD3ζ and CD28. Lentiviral gene transfer was performed into CD3/CD28-bead stimulated bulk CD4+ and CD8+ T cells of MM patients (n=7). CAR transgene positive T cells were enriched using an EGFRt transduction marker and expanded for functional analyses.
Results: We confirmed high SLAMF7 expression levels on mPCs in all analyzed samples and detected SLAMF7 expression on a fraction of normal lymphocytes obtained from peripheral blood of MM patients, including naïve and memory CD4+ (95% CI: 33-59%) and CD8+ T cells (75-95%), B cells (25-35%) and NK cells (94-98%). Remarkably, the proportion of SLAMF7+ cells was significantly higher in MM patients compared to healthy donors in all corresponding lymphocyte subsets (p<0.05). Despite high level SLAMF7 expression on the input T cell population, functional CD4+ and CD8+ T cells expressing the SLAMF7-CAR could be readily generated in all 7 MM patients, and expanded to therapeutically relevant doses in a single expansion cycle following enrichment (>107 cells). We analyzed the kinetics of SLAMF7 expression on CD4+ and CD8+ CAR T cells during the manufacturing process and detected rapid disappearance of SLAMF7+ T cells in T cell lines modified with the SLAMF7-CAR. By contrast unmodified T cells and T cell lines expressing a CD19-CAR retained a significant proportion of SLAMF7+ T cells, suggesting that expression of the SLAMF7-CAR induced killing of SLAMF7+ T cells. In vitro functional testing of SLAMF7-CAR CD4+ and CD8+ T-cell lines confirmed potent specific lysis of SLAMF7+ MM cell lines including MM1.S and OPM-2 and stable SLAMF7-transfectants of K562, as well as antigen specific IFNγ secretion and productive proliferation. In a flow cytometry based cytotoxicity assay, co-incubation of mPCs with autologous (or allogeneic) SLAMF7-CAR T cells resulted in elimination of >90% of mPCs after a 4-hour incubation period, whereas CD19-CAR or unmodified T cells had no discernible effects. Moreover, in an in vivo xenograft MM model (NSG/MM1.S) a single administration of SLAMF7-CAR T cells resulted in complete MM clearance and long-term survival, whereas mice treated with CD19-CAR or unmodified T cells rapidly expired from progressive disease. Finally, we analyzed the fratricidal potential of SLAMF7-CAR T cells to predict hematologic toxicities that might occur in a clinical setting. Co-incubation of purified CD4+ and CD8+ primary T cells, B cells and NK cells with SLAMF7-CAR T cells resulted in rapid and specific elimination of only SLAMF7+ subsets, whereas SLAMF7- cells remained viable and functional as confirmed for CD4+ and CD8+ T cells by inducible IFNγ secretion.
Conclusion: Our data demonstrate that SLAMF7-specific CAR T cells can be reproducibly generated from MM patients and exert remarkable anti-myeloma efficacy in pre-clinical models in vitro and in vivo. Lymphocytic fratricide does not preclude the manufacture of SLAMF7-CAR T cells but might be associated with acute (cytokine storm) or chronic (viral infections) side effects in a clinical setting. However, such toxicities may be prevented e.g. by preparative lymphodepletion and antiviral prophylaxis and enable the implementation of SLAMF7-CAR T cell therapy as a safe and effective modality in the treatment of MM.

Disclosures
Knop: Celgene Corporation: Consultancy. Einsele:Novartis: Consultancy, Honoraria, Speakers Bureau; Amgen/Onyx: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau.