Actomyosin dynamics and T cell receptor signaling are tightly coupled to ensure proper dynamics and function of signaling microclusters within the immunological synapse.

The T cell actin network generates mechanical forces that regulate LFA-1 activity at the immunological synapse.

Summary Ca2+ mobilization and cytoskeletal reorganization are key hallmarks of T-cell activation, and their interdependence has long been recognized. Recent advances in the field have elucidated the molecular pathways that underlie these events and have revealed several points of intersection. Ca2+ signaling can be divided into two phases: initial events leading to release of Ca2+ from endoplasmic reticulum stores, and a second phase involving STIM 1 (stromal interaction molecule 1) clustering and CRAC (calcium-release activation calcium) channel activation. Cytoskeletal dynamics promote both phases. During the first phase, the actin cytoskeleton promotes T-cell receptor mechanotransduction and serves as a dynamic scaffold for microcluster assembly. Proteins that drive actin polymerization such as WASp (Wiskott-Aldrich syndrome protein) and HS1 (hematopoietic lineage cell-specific protein 1) promote signaling through PLCγ1 (phospholipase Cγ1) and release of Ca2+ from endoplasmic reticulum stores. During the second phase, the WAVE (WASP-family verprolin homologous protein) complex and the microtubule cytoskeleton promote STIM 1 clustering at sites of plasma membrane apposition, opening Orai channels. In addition, gross cell shape changes and organelle movements buffer local Ca2+ levels, leading to sustained Ca2+ mobilization. Conversely, elevated intracellular Ca2+ activates cytoskeletal remodeling. This can occur indirectly, via calpain activity, and directly, via Ca2+-dependent cytoskeletal regulatory proteins such as myosin II and L-plastin. While it is true that the cytoskeleton regulates Ca2+ responses and vice versa, interdependence between Ca2+ and the cytoskeleton also encompasses signaling events that occur in parallel, downstream of shared intermediates. Inositol cleavage by PLCγ1 simultaneously triggers both endoplasmic reticulum store release and diacylglycerol-dependent microtubule organizing center reorientation, while depleting the pool of phosphatidylinositol-4,5-bisphosphate, an activator of multiple actin regulatory proteins. The close interdependence of Ca2+ signaling and cytoskeletal dynamics in T cells provides positive feedback mechanisms for T-cell activation and allows for finely tuned responses to extracellular cues.

T-cell–mediated approaches have shown promise in myeloma treatment. However, there are currently a limited number of specific myeloma antigens that can be targeted, and multiple myeloma (MM) remains an incurable disease. G-protein–coupled receptor class 5 member D (GPRC5D) is expressed in MM and smoldering MM patient plasma cells. Here, we demonstrate that GPRC5D protein is present on the surface of MM cells and describe JNJ-64407564, a GPRC5DxCD3 bispecific antibody that recruits CD3+ T cells to GPRC5D+ MM cells and induces killing of GPRC5D+ cells. In vitro, JNJ-64407564 induced specific cytotoxicity of GPRC5D+ cells with concomitant T-cell activation and also killed plasma cells in MM patient samples ex vivo. JNJ-64407564 can recruit T cells and induce tumor regression in GPRC5D+ MM murine models, which coincide with T-cell infiltration at the tumor site. This antibody is also able to induce cytotoxicity of patient primary MM cells from bone marrow, which is the natural site of this disease. GPRC5D is a promising surface antigen for MM immunotherapy, and JNJ-64407564 is currently being evaluated in a phase 1 clinical trial in patients with relapsed or refractory MM (NCT03399799).

The Ras GTPase-activating-like protein IQGAP1 is a multi-modular scaffold that controls signaling and cytoskeletal regulation in fibroblasts and epithelial cells. However, the functional role of IQGAP1 in T cell development, activation and cytoskeletal regulation has not been investigated. Herein, we show that IQGAP1 is dispensable for thymocyte development, as well as microtubule organizing center polarization and cytolytic function in CD8+ T cells. However, IQGAP1-deficient CD8+ T cells, as well as Jurkat T cells suppressed for IQGAP1 were hyper-responsive, displaying increased IL-2 and IFN-γ production, heightened LCK activation, and augmented global phosphorylation kinetics following TCR ligation. Additionally, IQGAP1-deficient T cells exhibited increased TCR-mediated F-actin assembly and amplified F-actin velocities during spreading. Moreover, we found that discrete regions of IQGAP1 regulated cellular activation and F-actin accumulation. Taken together, our data suggest that IQGAP1 acts as a dual negative regulator in T cells, limiting both TCR-mediated activation kinetics and F-actin dynamics via distinct mechanisms.

B-cell maturation antigen (BCMA), a member of the tumor necrosis factor family of receptors, is predominantly expressed on the surface of terminally differentiated B cells. BCMA is highly expressed on plasmablasts and plasma cells from multiple myeloma (MM) patient samples. We developed a BCMAxCD3 bispecific antibody (teclistamab [JNJ-64007957]) to recruit and activate T cells to kill BCMA-expressing MM cells. Teclistamab induced cytotoxicity of BCMA+ MM cell lines in vitro (H929 cells, 50% effective concentration [EC50] = 0.15 nM; MM.1R cells, EC50 = 0.06 nM; RPMI 8226 cells, EC50 = 0.45 nM) with concomitant T-cell activation (H929 cells, EC50 = 0.21 nM; MM.1R cells, EC50 = 0.1 nM; RPMI 8226 cells, EC50 = 0.28 nM) and cytokine release. This activity was further increased in the presence of a γ-secretase inhibitor (LY-411575). Teclistamab also depleted BCMA+ cells in bone marrow samples from MM patients in an ex vivo assay with an average EC50 value of 1.7 nM. Under more physiological conditions using healthy human whole blood, teclistamab mediated dose-dependent lysis of H929 cells and activation of T cells. Antitumor activity of teclistamab was also observed in 2 BCMA+ MM murine xenograft models inoculated with human T cells (tumor inhibition with H929 model and tumor regression with the RPMI 8226 model) compared with vehicle and antibody controls. The specific and potent activity of teclistamab against BCMA-expressing cells from MM cell lines, patient samples, and MM xenograft models warrant further evaluation of this bispecific antibody for the treatment of MM. Phase 1 clinical trials (monotherapy, #NCT03145181; combination therapy, #NCT04108195) are ongoing for patients with relapsed/refractory MM.

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B-cell maturation antigen (BCMA) is a tumor necrosis factor (TNF) family surface protein predominantly expressed on terminally differentiated B-cells. BCMA signals through P38/NF-κB pathway upon binding to its ligands; a proliferation inducing ligand (APRIL) and B-cell activator of the TNF family (BAFF) and promote anti-apoptotic gene expression. BCMA expression is elevated in plasma blasts, plasma cells from spleen and bone marrow and correlates with disease progression in multiple myeloma (MM). BCMA expression in premalignant MM settings such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) also gives an opportunity for early cancer interception. To target cancer cells expressing BCMA, we developed a BCMAxCD3 bispecific antibody using the Genmab DuoBody® technology (Ab-957) to recruit T cells to BCMA-expressing MM cells so that T cells could be activated and induced to kill BCMA+ cancer cells. This antibody can induce cytotoxicity of BCMA+ MM cell lines in vitro (H929 cells: EC50=0.15nM, MM1.R cells: EC50=0.06nM, RPMI8226 cells: EC50=0.45nM) with a concomitant T cell activation (H929 cells: EC50=0.21nM, MM1.R cells: EC50=0.1nM, RPMI8226 cells: EC50=0.28nM). In contrast, this antibody was unable to kill BCMA- cancer cell line (MV4-11), demonstrating the specificity of the cytotoxicity. Ab-957 also inhibited tumor development or growth in two BCMA+ MM murine xenograft models inoculated with human T cells. Furthermore, this antibody could deplete BCMA+ cells in bone marrow samples from MM patient's in an ex-vivo assay with an average EC50 value of 2.5 nM. Lastly, Ab-957 is well-tolerated in cynomolgus monkey and is being developed for Phase I clinical trial in patients with multiple myeloma. Disclosures Pillarisetti: Janssen: Employment. Baldwin:Janssen: Employment. Babich:Janssen: Employment. Majewski:Janssen: Employment. Barone:Janssen: Employment. Li:Janssen: Employment. Zhang:Janssen: Employment. Chin:Janssen: Employment. Luistro:Janssen: Employment. Mendonça:Janssen: Employment. Nanjunda:Janssen: Employment. Rudnick:Janssen Pharmaceuticals R&D: Employment. Bellew:Janssen: Employment. Elsayed:Janssen: Employment, Other: stock options. Attar:Janssen: Employment. Gaudet:Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued.