The first Bispecific T-cell Engager (BiTE®) targeting B-cell maturation antigen (BCMA) on multiple myeloma (MM) cells and CD3 on T cells is currently in clinical development (NCT02514239). This first-generation BiTE® has a short serum half-life and is delivered by continuous IV infusion. We here investigated the T cell-redirected cytotoxicity and immunomodulatory effects of AMG 701, a BCMA-targeting BiTE® with extended half-life, alone and in combination with lenalidomide (len), in MM cell lines and patient-derived samples. AMG 701 has a plasma half-life of 112 hr. in non-human primates (Cancer Res 2018;78(13 Suppl):Abstract nr LB-299) and is currently being evaluated clinically (NCT03287908). We here show that AMG 701 significantly induced T cell-mediated lysis of BCMA-positive MM cells resistant or sensitive to current anti-MM agents including bortezomib and lenalidomide (len). EC₅₀ values ranged from 0.64-2.54 ng/ml following overnight treatment with AMG 701 at effector to target (E/T) ratio of 10 to 1. Moreover, following overnight treatment AMG 701-induced MM cell lysis remained robust even at low concentrations (< 2 ng/ml) and low E/T ratios (2/1, 1/2). Importantly, the presence of myeloma-supporting osteoclasts or bone marrow stromal cells did not significantly alter the ability of AMG 701 to lyse MM cells. In the presence of BCMA-positive target cells, AMG 701 rapidly induced degranulation of CD4+ and CD8+ T cells in a dose-dependent manner, evidenced by upregulated surface CD107a expression. AMG 701 triggered lysis also induced secretion of IFNγ and TNFα, to a greater extent in CD8+ than CD4+ T cell subsets. Importantly, AMG 701 (1d treatment) induced lysis of autologous patient tumor cells from relapsed and refractory MM. Combined effects of AMG 701 with len were next investigated using effector cells (PBMC or CD3+ T) pretreated with len. Len enhanced AMG 701-mediated T cell lysis of MM cells even in the presence of osteoclasts. In the presence of BCMA-positive target cells, len pretreatment also further enhanced AMG 701-induced secretion of IFNγ and TNFα from T cells, to a greater extent in CD8+ than CD4+ T cell subsets. In the absence of AMG 701, len-specific lysis of MM cells was observed, confirming len-enhanced cytotoxic potential of T cells against MM cells. We next studied the potential immunomodulatory effects of AMG 701 by flow cytometry analysis. AMG 701 was added to co-cultures of MM cells and CellTrace^TM violet-labeled effector cells and T cells were analyzed at various time points. After 4d-co-incubation, AMG 701 induced more proliferation of CD8+ than CD4+ T cells (9.94% vs 0.8% at 1 ng/ml; and 47.5% vs 16.7% at 10 ng/ml, respectively). Higher levels of CD25 and CD69 were also observed in CD8+ than CD4+ T cells. Transient but not persistent upregulation of immune checkpoint molecules PD-1, TIM-3, and LAG-3 were seen on CD4+ and CD8+ T cells after coincubation with AMG 701 and BCMA-positive target cells. In parallel, the CD8+/CD4+ T cell ratios were increased (1.21 to 3.48-fold at d1 to d5, p<0.001); and further increased by 7d (1.97 to 8.64 folds at d1 to d8, p<0.001). AMG 701 induced differentiation of CD4+ and CD8+ T cells from naïve T cells to central memory (CM) and effector memory (EM) T cells, as demonstrated by changes in expression of CD62L and CD45RA. Moreover, CM and EM T cells were increased consistently after AMG 701 treatment (median % of CM+EM on CD4+ T cells: 74.8% (d1), 82.5% (d5), and 91.5% (d8), p<0.01; median % of CM+EM on CD8+ T cells: 54.4% (d1), 83.6% (d5), and 91.1% (d8), p<0.001). Furthermore, the proliferating T cells derived …