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Multiple myeloma (MM) is a hematologic malignancy that is characterized by the accumulation of abnormal plasma cells (PCs) in the bone marrow (BM). Patient outcome may be improved with BiTE (bispecific T-cell engager) molecules, which redirect T cells to lyse tumor cells. B-cell maturation antigen (BCMA) supports PC survival and is highly expressed on MM cells. A half-life extended anti-BCMA BiTE molecule (AMG 701) induced selective cytotoxicity against BCMA-expressing MM cells (average half-maximal effective concentration, 18.8 ± 14.8 pM), T-cell activation, and cytokine release in vitro. In a subcutaneous mouse xenograft model, at all doses tested, AMG 701 completely inhibited tumor formation (P < .001), as well as inhibited growth of established tumors (P ≤ .001) and extended survival in an orthotopic MM model (P ≤ .01). To evaluate AMG 701 bioactivity in cynomolgus monkeys, a PC surface phenotype and specific genes were defined to enable a quantitative digital droplet polymerase chain reaction assay (sensitivity, 0.1%). Dose-dependent pharmacokinetic and pharmacodynamic behavior was observed, with depletion of PC-specific genes reaching 93% in blood and 85% in BM. Combination with a programmed cell death protein 1 (PD-1)–blocking antibody significantly increased AMG 701 potency in vitro. A model of AMG 701 binding to BCMA and CD3 indicates that the distance between the T-cell and target cell membranes (ie, the immunological synapse) is similar to that of the major histocompatibility complex class I molecule binding to a T-cell receptor and suggests that the synapse would not be disrupted by the half-life extending Fc domain. These data support the clinical development of AMG 701.

Multiple myeloma (MM) is a cancer of the antibody-producing plasma cells (PC). MM is invariably fatal due to frequent disease relapse and/or treatment refractoriness, and therapies that provide deeper and more durable responses are needed. BiTE® (Bispecific T Cell Engager) molecules are immunotherapy agents that redirect a patient’s T cells to lyse tumor cells by simultaneously engaging a tumor associated antigen (TAA) on cancer cells and CD3ε on T cells. Clinical activity has been observed in MM using BiTE® molecules and other T cell engagers that target B-cell maturation antigen (BCMA). BCMA is an attractive MM TAA due to its broad prevalence and restricted normal tissue profile, mainly PCs and plasmablasts. However, heterogenous expression in MM cancer cells, potential antigen loss, and the presence of high levels of soluble BCMA in MM patient sera present challenges that may prevent BCMA-only-targeted therapies from achieving their full potential. To address these issues, we generated a BiTE® molecule capable of engaging BCMA and a second MM TAA, CS1 (also known as SLAMF7). This BiTE® molecule also contains an Fc-based domain to provide half-life extension (HLE). Here, we evaluated the in vitro and in vivo properties of this BCMA-CS1 HLE BiTE® molecule. The BCMA-CS1 HLE BiTE® molecule had nanomolar binding affinity for human BCMA, CS1, and CD3ε and the non-human primate (NHP) orthologues. In vitro, we observed picomolar activity against human MM cell lines with a range of BCMA and CS1 expression in a T cell dependent cellular cytotoxicity (TDCC) assay using human T cells or NHP peripheral blood mononuclear cells. The BCMA-CS1 HLE BiTE® molecule retained TDCC activity in the presence of soluble BCMA up to 2500 ng/mL as well as against human MM cells engineered to express only BCMA or CS1. In vivo, this BiTE® molecule inhibited tumor growth in a MM xenograft model. We also evaluated the BCMA-CS1 HLE BiTE® molecule in NHP over 15 days (IV dosing; intra-animal dose escalation from 60→240 μg/kg or 180→540 μg/kg on days 1 and 8). We observed hallmarks of BiTE® molecule activity in all groups, including transient decreases in circulating lymphocytes and moderate increases in cytokines like MCP-1. We measured the PC-specific transcripts BCMA and J-chain in NHP bone marrow and blood as surrogates for PC levels using ddPCR. These transcripts were reduced in both treatment groups (≥90%) with the strongest effects occurring in the 180→540 μg/kg group. Lastly, we showed that CS1 is highly and broadly expressed in MM patient samples and is restricted to a few normal hematopoietic cell types including PCs, NK cells, T cells, and some monocytes. These data suggest that the BCMA-CS1 HLE BiTE® molecule has potent in vitro and in vivo activity and may provide therapeutic benefit for MM patients by expanding the population of MM cancer cells that can be eliminated by a BiTE® molecule while overcoming common mechanisms that can impair BCMA-only-targeted MM therapies. Citation Format: Elizabeth T. Andrews, Stephanie C. Casey, Mohammad Farhad Amani, Grit Lorenczewski, Mozhgan Farshbaf, Lisa Winkel, Matthias Klinger, John M. Harrold, Famke Aeffner, Ana Goyos, Matthias Friedrich, Tara Arvedson, Matthew G. Chun. Evaluation of a dual-targeting BCMA-CS1 HLE BiTE® molecule for multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6251.

Notch signaling plays a crucial role in cell development and proliferation. The Notch receptor is an inducible transcription factor found on the cell surface. Upon interaction with its ligand, the Notch receptor is cleaved, releasing the Notch Intra-cellular Domain (NICD), which enters the nucleus and complexes with CBF1 and Mastermind (MAML1) to activate target genes. MAML1 is also involved in many other cell signaling pathways independently of Notch signaling. In this study, we focused on determining whether MAML family members follow the same path as the Notch intra-cellular domain, which undergoes ubiquitination and degradation via the ubiquitin-proteasome pathway. We also examined the regions of MAML1 required for ubiquitination and the kinase(s) that may be involved in phosphorylation of MAML1. We determined that MAML1 was ubiquitinated as the shortest-lived family member and that the (75-300) amino acid region of MAML1 is essential for ubiquitination and degradation of MAML1. We also found MAML2, which is not ubiquitinated, to be the most stable member of the family.

Early studies demonstrated the involvement of ubiquitination of the Notch intracellular domain for rapid turnover of the transcriptional complex at Notch target genes. It was shown that this ubiquitination was promoted by the co-activator Mastermind like 1 (MAML1). MAML1 also contains numerous lysine residues that may also be ubiquitinated and necessary for protein regulation. In this study, we show that over-expressed MAML1 is ubiquitinated and identify eight conserved lysine residues which are required for ubiquitination. We also show that p300 stimulates ubiquitination and that Notch inhibits ubiquitination. Furthermore, we show that a mutant MAML1 that has decreased ubiquitination shows increased output from a HES1 reporter gene assay. Therefore, we speculate that ubiquitination of MAML1 might be a mechanism to maintain low levels of the protein until needed for transcriptional activation. In summary, this study identifies that MAML1 is ubiquitinated in the absence of Notch signaling to maintain low levels of MAML1 in the cell. Our data supports the notion that a precise and tight regulation of the Notch pathway is required for this signaling pathway.