In CAR T?cell therapy targeting CD19 for lymphoma, host immune anti-murine CAR responses limited the efficacy of repeat dosing and possibly long-term persistence. BCMA-specific clones from which human CARs were engineered. Despite a narrow range of affinity for BCMA, dramatic differences in CAR T?cell SNT-207858 expansion were observed between unique scFvs in a repeat antigen stimulation assay. These results were confirmed by screening in a MM xenograft model, where only the top preforming CARs from the repeat antigen stimulation assay eradicated disease and prolonged survival. The results of this screening identified a highly effective CAR T?cell therapy with properties, including rapid expansion ( 10,000-fold, day 6), eradication of large tumor burden, and durable protection to tumor re-challenge. We generated a bicistronic construct including a second-generation CAR and a truncated-epithelial growth factor receptor marker. CAR T?cell vectors stemming from this work are under clinical investigation. expansion and accumulation of T? cells genetically modified SNT-207858 with our lead CAR. Highly active human BCMA-targeted CAR constructs stemming from this work are currently under clinical evaluation. Results Identification of Human Anti-BCMA scFvs for Incorporation into CAR Vectors We screened a human B cell-derived scFv phage display library (Figure?S1) containing 6? 1010 scFvs with recombinant human BCMA extracellular domain-immunoglobulin G1 (IgG1) Fc fusion (BCMA-Fc) protein to identify BCMA-specific human scFvs. After DNA sequencing, 57 unique and diverse BCMA-specific clones were identified containing light- and heavy-chain CDRs, each covering six subfamilies with HCDR3 length ranging from 5 to 18 amino acids. The binding specificity of the unique clones against full-length human BCMA expressing NIH 3T3 murine fibroblast artificial antigen-presenting cells (BCMA-aAPCs) was confirmed by flow cytometric analysis. 17 clones were further confirmed to bind to human MM cell lines by flow cytometry, and a subset of these scFvs were directly cloned into second-generation CAR vectors in a retroviral plasmid. Flow cytometric analysis after staining with BCMA-Fc confirmed CAR expression on the cell surface of donor T?cells; we consistently achieve Rabbit polyclonal to AFP (Biotin) similar retroviral transduction efficiencies (50%C60%) across scFvs investigated (Figure?1A). The majority of BCMA-targeted scFvs investigated had similar, single-digit nanomolar affinity for BCMA (Table S1). Open in a separate window Figure?1 Superior Expansion of BCMA(171) and BCMA(125) scFv Containing CAR T Cells Demonstrated by Repeat Antigen Stimulation Assay (A) Retroviral transduction efficiency; cell surface staining of human T?cells is?consistent regardless of scFv. Results from representative single donor. (B) Repeat antigen stimulation assay; CAR T?cells containing scFvs indicated (CD28 co-stimulatory domain) were placed on BCMA-aAPC or CD19-aAPC monolayers. Every 4?days, SNT-207858 CAR T?cells are counted, and the same number of CAR+ T?cells are re-plated on a new aAPC monolayer (arrows). CARs containing human anti-BCMA scFvs 171 and 125 convey superior expansion when plated on BCMA-aAPCs; mean? SEM; three independent experiments/donors; BCMA(125) CAR T?cells, plated on CD19 aAPCs do exhibit expansion at day 4. (C) Cytotoxicity analysis; CAR+ T?cells transduced with the same constructs as 1b are co-cultured at increasing E:T ratios with OPM2 human myeloma cell line. All scFvs lyse OPM2 cells in a dose-dependent manner. CAR T?cells incorporating scFvs 183, 171, 130, and 125 are superior to 137; mean? SEM; representative experiment in triplicate (*p? 0.005, two-way ANOVA). aAPC, NIH 3T3 artificial antigen presenting cell. CAR T Cell Expansion after Repeat Antigen Stimulation Distinguishes between scFv Clones As in B cell ALL,1 in CAR T?cell clinical trials of MM,10 CAR T?cell expansion in patients appears to correlate with clinical efficacy. expansion potential of CAR T?cells over multiple cycles of antigen stimulation. This repeat antigen stimulation assay revealed substantial differences in the expansion between novel scFvs incorporated into our CD28 containing CAR constructs, identifying scFv?clones 125 and 171 [BCMA(125), and BCMA(171), respectively] as superior expanders compared to those incorporating other scFvs. For example, BCMA(171) and BCMA(125) CAR T?cells uniquely continued to expand after four stimulations and expanded between a mean of 115- and 158-fold, respectively, across experiments using three independent donors (p? 0.005 compared to any other CAR). None of the other SNT-207858 scFvs studies continued to expand past the fourth stimulation, a group where?peak expansion was limited to between 2- and 30-fold (Figure?1B). Comparing cytotoxicity between these same CARs containing unique scFvs does not yield the same magnitude of differences as?the repeat antigen stimulation assay. To analyze cytotoxicity, we quantified ATP-dependent bioluminescence of an OMP2 MM cell line target transduced with firefly luciferase (ffLuc) 4?hr after co-culture with CAR T?cells.18 Cytotoxicity against OPM2-ffLuc cells demonstrate that CAR T?cells incorporating any of the anti-BCMA scFv studies lyse MM cell lines in a dose-dependent manner. However, it could not differentiate between the majority of CARs, with BCMA(183), BCMA(171), BCMA(130), and BCMA(125) all generating statistically equivalent cytotoxicity; only BCMA(137) was inferior to the rest (p? 0.005; Figure?1C). Assessment of Human Anti-BCMA CARs Confirms Results of Repeat Antigen Stimulation Assay in Distinguishing between Anti-BCMA scFvs NOD gamma (NSG) mice were engrafted with the bone marrow tropic human MM cell line OPM2.19 A large burden.