BI 836909, a Novel Bispecific T Cell Engager for the Treatment of Multiple Myeloma Induces Highly Specific and Efficacious Lysis of Multiple Myeloma Cells in Vitro and Shows Anti-Tumor Activity in Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2999-2999 ◽  
Author(s):  
Susanne Hipp ◽  
Petra Deegen ◽  
Joachim Wahl ◽  
Diann Blanset ◽  
Oliver Thomas ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Xenograft Model ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell ◽  
Anti Tumor Activity ◽  
Dose Dependent

Abstract BI 836909 is a Bispecific T cell Engager (BiTE®), designed to redirect the body's endogenous T cells towards cells expressing B cell maturation antigen (BCMA) on the cell surface. BCMA is a highly plasma cell specific antigen and shows homogeneous expression on the cell surface of multiple myeloma, plasma cell leukemia and plasmacytoma cells. In normal tissues, BCMA expression is restricted to plasma cells, while other normal tissues do not express BCMA. This highly selective expression pattern makes BCMA an ideal target for T cell redirecting therapy. The pharmacological effect of BI 836909 depends on its simultaneous binding to both the CD3 epsilon subunit of the T cell receptor complex on T cells as well as to BCMA on multiple myeloma cells, resulting in the lysis of the BCMA-expressing cells. In vitro, unstimulated peripheral blood mononuclear cells (PBMCs) were co-cultured with several multiple myeloma cell lines and increasing concentrations of BI 836909, and tumor cell lysis, T cell activation, and induction of cytokine release were assessed. BI 836909 induced dose-dependent redirected lysis of human multiple myeloma cell lines with EC90 values ranging from 16 to 810 pg/mL. Viability of BCMA-negative cells was not affected, demonstrating the specificity of BI 836909 for BCMA. The expression of the activation markers CD69 and CD25 on T cells and the release of cytokines by T cells were target-dependent and occurred only in the presence of BCMA-positive cells. In vivo anti-tumor activity of BI 836909 was assessed in NOD/SCID mice reconstituted with human T cells and bearing subcutaneous or orthotopic xenografts derived from human multiple myeloma cell lines. In the subcutaneous NCI-H929 xenograft model, animals were treated with BI 836909 by daily intravenous or subcutaneous bolus injections. Statistically significant dose-dependent anti-tumor activity was observed at doses of 50 µg/kg/day and higher. The efficacy of BI 836909 was comparable after intravenous and subcutaneous administration, when the difference in bioavailability of the different routes was considered. In an orthotopic L-363 xenograft model, treatment with BI 836909 resulted in a statistically significant prolonged survival at doses of 5 µg/kg/day and higher. BI 836909 shows comparable cross-reactive binding to both BCMA and CD3 epsilon of human and macaque origin at picomolar and low nanomolar affinities respectively, thus allowing the assessment of pharmacodynamics, pharmacokinetics, and safety in non-human primates. In toxicity studies, cynomolgus monkeys were administered doses of up to 135 µg/kg/day of BI 836909 via continuous intravenous infusion, and up to 405 µg/kg/day via daily subcutaneous injection for up to 28 days. A dose- dependent decrease in plasma cells was observed in the bone marrow of treated animals compared to the vehicle control group, consistent with BCMA expression on cynomolgus monkey plasma cells, this demonstrated the pharmacological activity of BI 836909. These pre-clinical data demonstrate that BI 836909 is a highly potent, efficacious and BCMA-selective T cell redirecting agent and support clinical testing of BI 836909 in multiple myeloma patients. Disclosures Hipp: Boehringer Ingelheim RCV GmbH & Co KG, Dr. Boehringer-Gasse 5-11, 1121 Vienna, Austria: Employment. Deegen:Amgen Research (Munich) GmbH, Staffelseestrasse 2, 81477 Munich, Germany: Employment. Wahl:Amgen Research (Munich) GmbH, Staffelseestrasse 2, 81477 Munich, Germany: Employment. Blanset:Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, CT 06877, USA: Employment. Thomas:Amgen Research (Munich) GmbH, Staffelseestrasse 2, 81477 Munich, Germany: Employment. Rattel:Amgen Research (Munich) GmbH, Staffelseestrasse 2, 81477 Munich, Germany: Employment. Adam:Boehringer Ingelheim RCV GmbH & Co KG, Dr. Boehringer-Gasse 5-11, 1121 Vienna, Austria: Employment. Friedrich:Amgen Research (Munich) GmbH: Employment.

scholarly journals REGN5458, a Bispecific BCMAxCD3 T Cell Engaging Antibody, Demonstrates Robust In Vitro and In Vivo Anti-Tumor Efficacy in Multiple Myeloma Models, Comparable to That of BCMA CAR T Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1944-1944 ◽  
Author(s):  
David J Dilillo ◽  
Kara Olson ◽  
Katja Mohrs ◽  
T. Craig Meagher ◽  
Kevin Bray ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Car T Cells ◽  
Nsg Mice ◽  
Car T

Abstract Improving therapies for multiple myeloma (MM) remains a high medical need because of the significant morbidity and mortality of the disease. Targeted immunotherapies represent a promising opportunity to fill this clinical need. B cell maturation antigen (BCMA) is an attractive cell-surface target for MM due to its consistent expression on MM patient malignant plasma cells and expression limited in normal tissue primarily to plasma cells. Redirection of a patient's T cells to recognize tumors by CD3-binding bispecific molecules or through the generation of chimeric antigen receptor (CAR) T cells, has shown preliminary evidence of clinical activity. Bispecific antibodies concurrently engage a tumor antigen on cancer cells and the CD3 signaling machinery on T cells, bringing the tumor cell and T cell into proximity and facilitating T cell activation and tumor cell killing. By contrast, CAR T cell therapy involves re-infusion of the patient's own T cells after ex vivo engineering to express CARs targeting tumor antigens and triggering T cell signaling. Here we describe the generation of REGN5458, a human bispecific antibody that binds to BCMA and CD3. In vitro, REGN5458 efficiently activates T cells and induces polyclonal T cell killing of myeloma cell lines with a range of BCMA cell-surface densities, and also induces cytotoxicity of primary human plasma cells. Similar to gamma-sectretase inhibitors, incubation of myeloma cell lines with REGN5458 increased surface levels of BCMA. In xenogenic studies, after BCMAhigh NCI-H929 and BCMAlow MOLP-8 MM cells were co-implanted with PBMC and grown subcutaneously in immunodeficient NOD/SCID/L2Rgamma-deficient (NSG) mice, REGN5458 doses as low as 0.4 mg/kg significantly suppressed the growth of both tumors. Using aggressive, systemic xenogenic tumor models, in which NSG mice were engrafted with PBMC and intravenously injected with BCMAhigh OPM-2 cells or BCMAlow MOLP-8 cells expressing luciferase, REGN5458 reduced tumor burden and suppressed tumor growth at doses as low as 0.4 mg/kg. In immunocompetent mice genetically engineered to express human CD3, REGN5458 inhibited the growth of syngeneic murine tumors expressing human BCMA at doses as low as 0.04 mg/kg. Finally, as REGN5458 binds to cynomolgus CD3 and BCMA and mediates cytotoxicity of primary cynomolgus plasma cells, the pharmacology of REGN5458 was evaluated in cynomolgus monkeys. REGN5458 administration was well-tolerated, resulting in a mild inflammatory response characterized by transiently increased CRP and serum cytokines. Importantly, REGN5458 treatment led to the depletion of BCMA+ plasma cells in the bone marrow, demonstrating cytotoxic activity in non-human primates. The anti-tumor efficacy of REGN5458 was compared to BCMA-specific CAR T cells using 2nd generation CAR lentiviral constructs containing a single-chain variable fragment binding domain from REGN5458's BCMA binding arm and 4-1BB and CD3z signaling domains. Human PBMC-derived T cells were transduced to express this CAR and expanded. Both REGN5458 and the BCMA CAR T cells demonstrated similar targeted cytotoxicity of myeloma cell lines and primary patient blasts in vitro, and were capable of clearing established systemic OPM-2-luciferase myeloma tumors in NSG mice, but with different kinetics: treatment with REGN5458 resulted in rapid clearance of tumors within 4 days, whereas treatment with BCMA CAR T cells allowed tumors to continue to grow for 10-14 days following injection before rapidly inducing tumor clearance. Thus, REGN5458 exerts its therapeutic effect rapidly after injection, using effector T cells that are already in place. In contrast, BCMA CAR T cells require time to traffic to the tumor site and expand, before exerting anti-tumor effects. Collectively, these data demonstrate the potent pre-clinical anti-tumor activity of REGN5458 that is comparable to that of CAR T cells, and provide a strong rationale for clinical testing of REGN5458 in patients with MM. Disclosures Dilillo: Regeneron Pharmaceuticals: Employment. Olson:Regeneron Pharmaceuticals: Employment. Mohrs:Regeneron Pharmaceuticals: Employment. Meagher:Regeneron Pharmaceuticals: Employment. Bray:Regeneron Pharmaceuticals: Employment. Sineshchekova:Regeneron Pharmaceuticals: Employment. Startz:Regeneron Pharmaceuticals: Employment. Retter:Regeneron Pharmaceuticals: Employment. Godin:Regeneron Pharmaceuticals: Employment. Delfino:Regeneron Pharmaceuticals: Employment. Lin:Regeneron Pharmaceuticals: Employment. Smith:Regeneron Pharmaceuticals: Employment. Thurston:Regeneron Pharmaceuticals: Employment. Kirshner:Regeneron Pharmaceuticals: Employment.

Constitutive and Immunoproteasome Inhibitors Increase IκB and Decrease NF-κB Expression In Dendritic Cell

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3493-3493
Author(s):  
Ahmad-Samer Samer Al-Homsi ◽  
Zhongbin Lai ◽  
Tara Sabrina Roy ◽  
Niholas Kouttab
Keyword(s):  
Multiple Myeloma ◽  
T Cell ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Peripheral Blood ◽  
Research Funding ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell

Abstract Introduction Constitutive and immunoproteasome inhibitors (C&IPI) were thought to suppress nuclear factor-κB (NF-κB) pathway by preventing IκB degradation, which prevents NF-κB translocation into the nucleus. This mechanism of action has since been questioned by a number of studies. First, bortezomib promoted constitutive NF-κB activity in endothelial cell carcinoma. Second, NF-κB constitutive activity was resistant to bortezomib in multiple myeloma cell lines. Third, bortezomib increased IκB mRNA but post-transcriptionally downregulated IκB in normal cells and in multiple myeloma cell lines resulting in induced canonical NF-κB activation. Lastly, bortezomib increased nuclear levels of IκB as opposed to lowering cytoplasmic levels in cutaneous T cell lymphoma cell line suggesting that nuclear translocation of IκB was possibly responsible for NF-κB inhibition. The inhibitory activity of C&IPI on dendritic cells (DC) is of interest in the prevention of graft versus host disease (GvHD). It has been shown that different C&IPI impede DC maturation and T cell priming both in vitro and in vivo. Herein we sought to understand the mechanism of action of proteasome and immunoproteasome inhibitors on DC and to test their effect on IκB and NF-IκB expression. Materials and Methods We first performed RT PCR on lysates of DC obtained from the peripheral blood of 7 patients who received post-transplant cyclophosphamide and bortezomib as prevention of GvHD on a phase I clinical trial. Patients received allogeneic transplantation from matched-related or unrelated donors. Patients received no other immunosuppressive therapy except for rabbit anti-thymocyte globulin for those receiving graft from unrelated donor. Steroids were not allowed on the study. Samples were obtained on days +1, +4, and +7. The results were analyzed in comparison to samples obtained on day 0 before stem cell infusion. We then performed the same experiment on lysates of DC obtained from the peripheral blood of healthy volunteer donors. DC were untreated or incubated with bortezomib (10 nM for 4 h), carfilzomib (30 nM for 1 h), oprozomib (100 nM and 300 nM for 4 h), ONX 0914 (200 nM for 1 h), PR-825 (125 nM for 1 h), or PR-924 (1000 nM for 1 h). The drug concentration and duration of exposure were chosen based on the IC50 on proteasome activity and to reproduce in vivo conditions. We also performed IκB western blot on DC isolated from peripheral blood of healthy volunteers, untreated or incubated with bortezomib (10 nM for 4 h) or oprozomib (300 nM for 4 h). Each experiment was performed at least in triplicate. Results We found that the combination of cyclophosphamide and bortezomib significantly and progressively increased IκB mRNA while decreasing NF-κB mRNA in DC studied ex vivo. We also found that all studied C&IPI increased IκB mRNA to a variable degree while only oprozomib (300 nM) decreased NF-κB mRNA in DC in vitro. Finally, both bortezomib and oprozomib increased IκB protein level in DC in vitro (figure). Conclusion Our data suggest that C&IPI increase IκB expression in DC. As opposed to the previously reported data in other cell types, the effect is not associated with post-transcriptional downregulation. Cyclophosphamide and bortezomib also decrease NF-κB expression in DC in vivo while only oprozomib had the same effect in vitro. The effect of C&IPI on IκB and NF-κB expression may represent a new mechanism of action and suggests their effect may be cell-type dependent. Disclosures: Al-Homsi: Millennium Pharmaceuticals: Research Funding. Off Label Use: The use of cyclophosphamide and bortezomib for GvHD prevention. Lai:Millennium Pharmaceuticals: Research Funding.

scholarly journals Dominant Expression of PVRIG and TIGIT Inhibitory Pathways in Bone Marrow of Multiple Myeloma Patients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4717-4717
Author(s):  
Masha Frenkel ◽  
Zoya Alteber ◽  
Ning Xu ◽  
Mingjie Li ◽  
Haiming Chen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Inhibitory Receptor ◽  
Preclinical Models ◽  
Cell Numbers

Abstract Introduction Blocking inhibitory immune checkpoints holds promise to treat multiple myeloma (MM) patients. However, currently available checkpoint inhibitors have not shown significant clinical benefits for MM patients, warranting the need for alternative checkpoint blockers. The immune checkpoint TIGIT was recently shown to be the most upregulated immune inhibitory receptor on CD8+ T cells in MM patients' bone marrow (BM), compared to other checkpoints (Guillerey C., Blood. 2018). Preclinical models demonstrated the dominant effects of TIGIT blockade or depletion, by significantly improving mice survival, reducing myeloma cell numbers and exhausted T cell hallmarks (Minnie S., Blood. 2018). As a result, several clinical trials using anti-TIGIT monoclonal antibodies have been recently initiated in MM patients. The DNAM-1 family, in addition to TIGIT, also includes the inhibitory receptor PVRIG, that competes with the co-activating receptor DNAM-1 for the binding to the shared ligand PVRL2, similarly to the TIGIT/PVR/DNAM-1 interaction. Accordingly, TIGIT and PVRIG co-blockade were shown to synergize in enhancing T cell activity and anti-tumor activity in preclinical models (Whelan S., Cancer Immunol. Res. 2019). PVRL2 together with PVR (ligand of TIGIT) were shown to be highly expressed on plasma cells and on CD14+ cells in BM of MM patients (Lozano E., Clin. Cancer Res. 2020). This study aimed at evaluating DNAM-1 axis receptors expression in MM patients' BM. Methods Fresh BM aspirates were collected from 21 MM patients with progressive disease (PD) or in complete response (CR) after obtaining IRB approval. BM mononuclear cells were isolated and single cell suspensions were obtained followed by staining with anti-human antibodies to evaluate DNAM-1 axis members and PD-1 expression. BM biopsies from 6 MM patients (each patient had 4 core on the Tissue Micro-Array T291 USBiomax) were stained for PVRL2 expression by immuno-histochemistry (IHC). Results Flow cytometry analysis of PD-1 and DNAM-1 axis receptors revealed a significant lower fraction of PD1+ cells among cell populations examined compared with other receptors. TIGIT expression was the highest on NK, CD8+ and NKT cells compared to CD4+ T cells, which is in line with previous published data (Lozano E. Clin. Cancer Res. 2020). In contrast, DNAM-1 was expressed on CD8+ T, NK and NKT cells with prominent high expression on CD4+ T cells (Fig 1A). The highest expression among the receptors was of PVRIG on all lymphoid populations, except CD4+ where DNAM-1 was more highly expressed. Importantly, 50% of CD8+ T cells co-expressed TIGIT and PVRIG, supporting a combinatorial therapeutic approach (Fig. 1B). Additionally, the expression of the PVRL2 ligand on MM plasma and endothelial cells was demonstrated by IHC. FACS analysis further supported PVRL2 expression on plasma cells in MM BM (Fig 2). A higher expression of PVRIG, TIGIT and PD-1 was present on DNAM-1 negative CD8+ T cells (Fig 3A, B), suggesting accumulation of exhausted cells in MM tumor microenvironment (TME) as previously described (Minnie S., Blood. 2018). PVRIG had significantly higher expression on DNAM+ cells, compared to PD-1 and TIGIT (Fig 3C), suggesting the potential of its blockade to enhance DNAM-1 activation and subsequent proliferation of earlier differentiated memory cells in MM TME. Finally, CR patients had a trend for higher DNAM-1 expression on CD8+ T cells compared to those with PD (Fig 3D). This is consistent with other reports in mice showing that the expression of DNAM-1 negatively correlates with BM myeloma cell numbers (Minnie S., Blood. 2018). Conclusions DNAM-1 axis receptors are dominantly expressed on lymphocytes in BM of MM patients, with PVRIG exhibiting the most prominent expression. The reduced expression of DNAM-1 in PD patients' TME, compared to CR patients, suggests a link between DNAM-1 axis and clinical outcomes. Recent data suggest TIGIT is an attractive target for blockade in MM. Our new findings highlight for the first time the dominant expression of PVRIG, as well as TIGIT, and suggest that combined blockade of TIGIT with PVRIG may potentially benefit MM patients, placing the DNAM-1 axis as a dominant pathway in MM therapy. Figure 1 Figure 1. Disclosures Frenkel: Compugen Ltd.: Current Employment, Other: in the event of frontal participation, I will be reimbursed for my travel expenses by Compugen Ltd.. Alteber: Compugen Ltd.: Current Employment. Cojocaru: Compugen Ltd.: Current Employment. Ophir: Compugen Ltd.: Current Employment.

scholarly journals The Effects of BCMA Expression, Soluble BCMA, and Combination Therapeutics on the Anti-Tumor Activity of HPN217, a BCMA-Targeting Tri-Specific T Cell Engager Against Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1185-1185
Author(s):  
Patrick P Ng ◽  
Wade Aaron ◽  
Evan Callihan ◽  
Golzar Hemmati ◽  
Che-Leung Law ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Half Life ◽  
Plasma Cells ◽  
Target Cells ◽  
Publicly Traded ◽  
Anti Tumor Activity

Abstract Introduction B-cell maturation antigen (BCMA) is a cell surface receptor highly and selectively expressed on normal plasma cells and transformed plasma cells in multiple myeloma (MM) patients. Upon ligand binding, BCMA initiates signals that promote the survival of MM cells and the production of immunosuppressive factors. Therapeutics that target BCMA are being investigated in the clinic, with encouraging preliminary results. HPN217 is a Tri-specific T Cell-Activating Construct (TriTAC) specific to BCMA, to serum albumin for half-life extension, and to CD3ε for redirecting T cells against MM cells. It is currently being evaluated in a phase 1 /2 clinical trial for relapsed or refractory MM (NCT04184050). Herein, we describe translational studies to examine factors that may impact the therapeutic efficacy of HPN217, including the target BCMA, in membrane-bound or soluble form, and concomitant or combination therapeutics such as γ-secretase inhibitor (GSI) and dexamethasone. Results To evaluate the effects of HPN217 against primary MM cells, we used a patient-derived 3D-culture system (3DTEBM) designed to recapitulate the biology within the bone marrow microenvironment. 3DTEBM seeded with bone marrow accessory cells and autologous plasma recreate niches along an oxygen gradient that enable the survival and expansion of autologous MM cells without additional nutrient supplements. 3DTEBM's were established from 5 MM patients with varying ratios of autologous CD3+ T cells to MM cells (0.15-0.6). Although the functional competence of the T cells was unknown, HPN217 was able to mediate MM cell killing in 80% of the cultures with up to 71% of MM cells eliminated at a T cell/MM cell ratio of 0.45. The anti-tumor efficacy of HPN217 correlated strongly (R 2 = 0.99) with BCMA expression on the MM cells as measured by flow cytometry, suggesting the number of target receptors can be a limiting factor in efficacy. Consistent with this result, pre-incubation of target cells with 1 or 10 μg/mL anti-BCMA reduced the activity of HPN217 in T cell-dependent cellular cytotoxicity (TDCC) assays using healthy donor T cells and MM cell lines. Soluble BCMA (sBCMA) is produced when the extracellular domain of BCMA is cleaved by γ-secretase. It may act as a sink for HPN217. There was no correlation between the activity of HPN217 and the quantity of sBCMA in 3DTEBM. However, in TDCC assays, the addition of 6.25, 25 and 100 nM recombinant BCMA respectively led to 4-, 9- and 28-fold increases in the EC 50 of HPN217. Taken together, these data underscore the importance of preserving BCMA on MM cells and reducing sBCMA in circulation. Interestingly, treatment of MM cell line RPMI8226 with the GSI LY-3039478 for 24 hours increased the cell surface expression of BCMA by 3.6 folds. Using RPMI8226 as target cells in the 3DTEBM system, LY-3039478 increased the killing efficacy of HPN217-redirected primary T cells by 1.9 folds. Dexamethasone (Dex) is used with other therapeutics for treating MM. It is also commonly given to manage cytokine release syndrome (CRS) caused by T cell engagers. We conducted TDCC assays in the presence of 0.07-300 nM Dex to simulate plasma concentrations relevant to dose levels of Dex premedication for CRS. The highest Dex concentrations caused ≤3-fold increases in the EC 50 of HPN217. Considering this and the plasma half-life of i.v. injected Dex at <5 h, the suppressive effect of Dex on the anti-tumor activity of HPN217-redirected T cells may be limited. We then evaluated if MM.1S-Luc cell line xenografts in NCG mice would be a suitable model to extend the above in vitro findings to an in vivo setting. Lesions in the spine, skull and femur in NCG mice treated with vehicle could be detected by bioluminescent imaging. All mice succumbed to the disease within 40 days. By contrast, animals treated with HPN217 were protected in a dose-dependent manner. Mice that received the highest dose remained 100% disease-free at the end of the study (Figure 1). Conclusions We demonstrated HPN217 mediated BCMA-dependent primary MM cell killing by autologous T cells, and that the density of BCMA target on the surface of MM cells and sBCMA affected the efficacy of HPN217 in cultures. GSI, which increased the expression of BCMA on MM cells, enhanced the efficacy of HPN217. On the other hand, Dex had limited negative effect. HPN217 in combination with approved and experimental MM therapeutics is being evaluated in the 3DTEBM and MM.1S-Luc models. Figure 1 Figure 1. Disclosures Ng: Harpoon Therapeutics: Current Employment, Current equity holder in publicly-traded company. Aaron: Harpoon Therapeutics: Current Employment, Current equity holder in publicly-traded company. Callihan: Harpoon Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hemmati: Harpoon Therapeutics: Current Employment, Current equity holder in publicly-traded company. Law: Harpoon Therapeutics: Current Employment, Current equity holder in publicly-traded company. Azab: Cellatrix, LLC: Current Employment, Current holder of individual stocks in a privately-held company. Sun: Harpoon Therapeutics: Consultancy, Current equity holder in publicly-traded company, Ended employment in the past 24 months.

Preclinical Evaluation of a Potent Anti-Bcma CD3 Bispecific Molecule for the Treatment of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 383-383 ◽  
Author(s):  
Siler H Panowski ◽  
Tracy Kuo ◽  
Amy Chen ◽  
Tao Geng ◽  
Thomas J Van Blarcom ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Half Life ◽  
Plasma Cells ◽  
Target Cells ◽  
Dependent Manner ◽  
Human Igg

Abstract Multiple myeloma (MM) is a debilitating disease characterized by the abnormal accumulation of malignant plasma cells in the bone marrow. Despite recent advances in myeloma therapy, including proteasome inhibitors, immunomodulatory drugs, and targeted antibody therapies, patients relapse and the disease remains incurable and one of high unmet need. T cell redirecting therapies are a new and exciting class of therapeutics that harness the potent cytotoxic activity of T cells and redirect it to target tumor cells. T cell redirecting therapies are only as good as their targeted tumor associated antigen (TAA) and the potent nature of the therapy requires a lack of TAA expression in essential normal tissue. B-cell Maturation Antigen, BCMA, is a tumor necrosis factor superfamily member highly expressed on the surface of myeloma cells. Detectable normal BCMA tissue expression appears limited to plasmablasts and mature plasma cells, making it an ideal T cell redirecting target for the treatment of MM. Other groups have developed T cell redirecting therapies against BCMA, including CAR T and BiTE therapy (a short half-life CD3 bispecific). Here we present preclinical studies on a fully-human IgG CD3 bispecific molecule targeting BCMA (half-life in mice of ~3 days). This molecule utilizes anti-BCMA and anti-CD3 targeting arms paired through hinge mutation technology and placed in an IgG2A backbone. The molecule binds to BCMA-expressing myeloma cell lines and to T cells with affinities of 20pM and ~40nM, respectively. T cells co-cultured with MM cell lines were activated and de-granulated in the presence of BCMA bispecific. In vitro cytotoxicity assays revealed the high potency of the molecule, as it was able to drive lysis of MM target cells with an EC50 of 6± 8 pM (mean ± SD). We also observed strong in vitro potency with the BCMA bispecific in four different MM primary patient samples, EC50 =0.093±0.1 nM (mean ± SD). When the same four samples were targeted with a BCMA antibody drug conjugate (ADC), 3 of the samples gave EC50 values of 1.25±0.7 nM (mean ± SD) - i.e. a 43 fold decrease in potency compared to the CD3 bispecific. The fourth patient did not respond to the ADC. Together, these results illustrate the potential advantages of a CD3 bispecific over an ADC for targeting BCMA. In orthotopic, established, tumor mouse models utilizing three different MM cell lines, (OPM2, MM.1S and MOLP8), a single injection of BCMA bispecific effectively treated tumors in a dose-dependent manner. Re-dosing the bispecific was able to provide additional and prolonged efficacy. The extreme potency of T cell redirecting therapies results in outstanding efficacy, but can also lead to lysis of normal cells expressing even minute levels of target. The species cross-reactivity of the BCMA bispecific allowed for exploratory toxicity studies in cynomologus monkeys. The molecule was able to effectively deplete normal plasma B cells expressing low levels of BCMA, providing evidence of activity. Activity was accompanied by a cytokine spike following initial dosing. No cytokine release was observed following a second bispecific dose. Encouragingly, animals experienced no additional adverse events (AEs), confirming the favorable safety profile of BCMA as a target for MM. In summary, we report on a fully human IgG CD3 bispecific molecule targeting BCMA for the treatment of multiple myeloma. Our BCMA bispecific is expected to have an antibody-like half-life in humans and, taken together, our findings support that the molecule has the potential to be both a potent and safe therapeutic. Disclosures Panowski: Pfizer Inc.: Employment. Kuo:Alexo Therapeutics: Employment. Chen:Alexo Therapeutics: Employment. Geng:Kodiak Sciences: Employment. Van Blarcom:Pfizer Inc.: Employment. Lindquist:Pfizer Inc.: Employment. Chen:Pfizer Inc.: Employment. Chaparro-Riggers:Pfizer Inc.: Employment. Sasu:Pfizer Inc.: Employment.

Fully Human CD38 Antibodies Efficiently Trigger ADCC of Multiple Myeloma Cell Lines and Primary Tumor Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3377-3377 ◽  
Author(s):  
Matthias Peipp ◽  
Michel de Weers ◽  
Thomas Beyer ◽  
Roland Repp ◽  
Paul Parren ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Cells ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Treatment Modalities ◽  
Potential Candidate ◽  
Isolated Tumor Cells ◽  
Myeloma Cells ◽  
Multiple Myeloma Cell

Abstract Although new treatment modalities have recently been added to the standard regimens for multiple myeloma, the clinical outcome for patients with advanced disease is often limited. Monoclonal antibodies are increasingly used for tumor therapy, and may also represent interesting options for multiple myeloma patients. CD38 is one of the most promising target antigens on malignant plasma cells, which are evaluated in preclinical and early clinical studies as targets for antibody therapy. CD38 is a type II transmembrane protein with ectoenzymatic activity, which is involved in calcium mobilization. Human CD38 is predominantly expressed by bone marrow precursor cells and by terminally differentiated plasma cells. Multiple myeloma cells show moderate to high expression levels - making CD38 a potential candidate as target for immunotherapy. A panel of 42 fully human CD38 antibodies was generated by immunizing human Ig transgenic mice. Immunofluorescence studies with CD38 transfected cells demonstrated antigen-specific, high affinity binding, and cross-blocking experiments revealed four distinct epitope groups. Seven antibodies, representing each of the four groups, were selected for further analyses. ADCC and CDC activity against CD38-positive myeloma cell lines (AMO-1 and JK6), and against freshly-isolated primary multiple myeloma cells was investigated. Human whole blood served as effector source, which was then fractionated into plasma (containing human complement), mononuclear (MNC) or granulocytic (PMN) effector cells. All antibodies mediated concentration-dependent killing of both multiple myeloma cell lines - using human mononuclear cells as effector source. Also complement-dependent killing of freshly isolated myeloma cells was observed. However, none of the antibodies recruited PMN for tumor cell lysis. Importantly, CD38 antibodies also killed freshly isolated tumor cells from a rare patient with a CD38/138- positive plasma cell leukemia, which was chemotherapy- refractory at the time of analysis. Furthermore, CD38 antibodies effectively prevented outgrowth of CD38-positive tumor cells in SCID mouse xenograft models. Antibody 005 was significantly more effective in these assays compared to the remaining panel of CD38 antibodies. In conclusion, CD38 antibodies efficiently mediated killing of multiple myeloma cell lines as well as freshly isolated tumor cells and prevented tumor outgrowth in xenografted SCID mice. Antibody 005 was superior in mediating CDC and ADCC via MNC - particularly at low antibody concentrations.

Lenalidomide Promotes CRBN-Mediated Ubiquitination and Degradation of IKZF1 and IKZF3

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. LBA-5-LBA-5
Author(s):  
Jan Krönke ◽  
Namrata Udeshi ◽  
Anupama Narla ◽  
Peter Grauman ◽  
Slater Hurst ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Transcription Factors ◽  
B Cell ◽  
Cell Lines ◽  
Ubiquitin Ligase ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell ◽  
Protein Levels ◽  
Human T Cells

Abstract Lenalidomide is a highly effective drug for the treatment of multiple myeloma and has activity in additional B cell lymphomas. Lenalidomide has been shown to bind the CRBN-DDB1 E3 ubiquitin ligase, but it is unknown how lenalidomide alters the activity of this enzyme complex, and how this leads to therapeutic efficacy.  We used a combination of quantitative proteomic approaches to demonstrate that lenalidomide acts by a novel mechanism of action for a therapeutic agent: in multiple myeloma cells, lenalidomide increases the binding of two substrates, IKZF1 (Ikaros) and IKZF3 (Aiolos), to the CRBN substrate adaptor; increases the ubiquitination of these substrates; and causes the targeted degradation of these transcription factors that are essential for the differentiation and survival of plasma cells including multiple myeloma cells. To identify targets of the CRBN-DDB1 ubiquitin ligase that are altered by lenalidomide, we applied SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative mass spectrometry studies to globally assess changes in ubiquitination and proteome levels in the multiple myeloma cell line MM1S. Two members of the Ikaros transcription factor family, IKZF1 and IKZF3, were differentially ubiquitinated and decreased after lenalidomide treatment.  Subsequent validation experiments in various cell lines demonstrated that lenalidomide, thalidomide, and pomalidomide cause a decrease of endogenous and ectopically expressed IKZF1 and IKZF3 protein levels but not mRNA levels. Furthermore, we confirmed that IKZF1 and IKZF3 bind CRBN in the presence of lenalidomide, supporting CRBN’s role as a substrate adaptor. Consistent with this, shRNA mediated knockdown or overexpression of a CRBN mutant (CRBNYWAA) that does not bind lenalidomide abrogated lenalidomide’s effect on IKZF1 and IKZF3. Moreover, CRBN promoted IKZF3 ubiquitination in vitro in the presence of lenalidomide, demonstrating that it is an enzymatic substrate. Using deletion mutants of IKZF3 we identified a 58-amino-acid degron in the N-terminal zinc finger domain that is sufficient for lenalidomide-induced degradation. Based on sequence alignment of that region between lenalidomide responding Ikaros proteins IKZF1 and IKZF3 vs. non-responding IKZF2, IKZF4 and IKZF5 we substituted a single amino acid (IKZF3Q147H) that prevented binding of IKZF3 to CRBN and conferred resistance to lenalidomide induced degradation. IKZF1 and IKZF3 are essential transcription factors for terminal B cell differentiation. We evaluated the biological effects of IKZF1 and IKZF3 loss using shRNAs in a variety of cell lines. IKZF1 and IKZF3 specific shRNAs inhibited the growth of multiple myeloma cell lines while lenalidomide insensitive cell lines derived from other hematopoietic neoplasms were unaffected. Similarly, a dominant negative IKZF3 mutant resulted in growth inhibition of MM1S cells. In contrast, expression of IKZF3Q147Hconferred lenalidomide resistance to MM1S cells. Lenalidomide induces IL-2 expression and release in T cells. We found that lenalidomide induced a dose-dependent decrease of IKZF1 and IKZF3 protein levels in primary human T cells. Previous studies have shown that IKZF3 is a transcriptional repressor of IL-2. To further evaluate the effect of IKZF3 loss, we transduced primary human T cells with shRNAs targeting either IKZF3 or control. IL2 RNA levels increased 3.3 fold after lenalidomide treatment in T cells expressing control shRNAs. In contrast, the baseline IL2 RNA level in T cells transduced with IKZF3 specific shRNAs was 3.7 fold higher compared to controls and this effect could not be further stimulated by lenalidomide. In conclusion, selective targeting of two lymphoid transcription factors, IKZF1 and IKZF3, explains lenalidomide’s selective growth inhibition in multiple myeloma and likely other B cell lymphomas as well as its immunomodulatory effects in T cells. Furthermore, selective ubiquitination and degradation of specific targets provides a novel mechanism of therapeutic activity for proteins that are not otherwise amenable to small-molecule inhibition. Disclosures: Ebert: Celgene: Membership on an entity’s Board of Directors or advisory committees.

scholarly journals The Mucolipin TRPML2 Channel Enhances the Sensitivity of Multiple Myeloma Cell Lines to Ibrutinib and/or Bortezomib Treatment

Biomolecules ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 107
Author(s):  
Giorgio Santoni ◽  
Consuelo Amantini ◽  
Federica Maggi ◽  
Oliviero Marinelli ◽  
Matteo Santoni ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Plasma Cells ◽  
Biological Effects ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell ◽  
Bortezomib Treatment

Multiple myeloma (MM) is a haematological B cell malignancy characterised by clonal proliferation of plasma cells and their accumulation in the bone marrow. The aim of the present study is the evaluation of biological effects of Ibrutinib in human MM cell lines alone or in combination with different doses of Bortezomib. In addition, the relationship between the expression of TRPML2 channels and chemosensitivity of different MM cell lines to Ibrutinib administered alone or in combination with Bortezomib has been evaluated. By RT-PCR and Western blot analysis, we found that the Ibrutinib-resistant U266 cells showed lower TRPML2 expression, whereas higher TRPML2 mRNA and protein levels were evidenced in RPMI cells. Moreover, TRPML2 gene silencing in RPMI cells markedly reverted the effects induced by Ibrutinib alone or in combination with Bortezomib suggesting that the sensitivity to Ibrutinib is TRPML2 mediated. In conclusion, this study suggests that the expression of TRPML2 in MM cells increases the sensitivity to Ibrutinib treatment, suggesting for a potential stratification of Ibrutinib sensitivity of MM patients on the basis of the TRPML2 expression. Furthermore, studies in vitro and in vivo should still be necessary to completely address the molecular mechanisms and the potential role of TRPML2 channels in therapy and prognosis of MM patients.

scholarly journals Expression of the bcl-2 gene in human multiple myeloma cell lines and normal plasma cells

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 495-502 ◽  
Author(s):  
M Pettersson ◽  
H Jernberg-Wiklund ◽  
LG Larsson ◽  
C Sundstrom ◽  
I Givol ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
B Cell Differentiation ◽  
Multiple Myeloma Cell ◽  
Normal Plasma ◽  
Human Multiple Myeloma

Abstract The bcl-2 gene, encoding a mitochondrial membrane protein suggested to play an important role in cell survival, is translocated into the Ig loci in about 80% of human follicular lymphomas, which results in a high level of expression. This report shows that bcl-2 was expressed in eight of eight human multiple myeloma cell lines and in normal lymph node and bone marrow plasma cells. In the majority of the myeloma lines, the level of expression was comparable with that observed in Karpas 422, a follicular lymphoma cell line carrying a 14;18 translocation of the bcl-2 gene. DNA rearrangements of the bcl-2 locus were evident in only one of the myeloma cell lines, U-266–1970. In this cell line, which exhibited the highest bcl-2 expression, a fourfold increased copy number of the bcl-2 gene was estimated by Southern analysis. This amplification was lost in cells of later passages (U-266– 1984), suggesting that bcl-2 might possibly have played a role in the tumor development in vivo. Our results are in contrast to previous observations in murine plasmacytoma, in which bcl-2 was shown to be silent. The results also contradict the published observation that bcl- 2 is not expressed at terminal stages of B-cell differentiation. It is at present unclear whether the high expression of bcl-2 in human myeloma is the result of a deregulated expression associated with the malignant phenotype or a mere reflection of the bcl-2 expression typical of normal plasma cells.

Molecular Characterization of Human Multiple Myeloma Cell Lines by Genome-Wide Profiling.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1793-1793
Author(s):  
Carolina Elosua ◽  
Purificacion Catalina ◽  
Cristina Palma ◽  
Brian A Walker ◽  
Nicholas J Dickens ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Uniparental Disomy ◽  
Myeloma Cell ◽  
Molecular Pathogenesis ◽  
Gene Localization ◽  
Multiple Myeloma Cell

Abstract Abstract 1793 Poster Board I-819 Multiple Myeloma (MM) is a malignancy depicted by clonal expansion of plasma cells in the bone marrow. There are two broad genetic subtypes of multiple myeloma as defined as hyperdiploid multiple myeloma (H-MM), characterized by trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19, and 21, and nonhyperdiploid multiple myeloma (NH-MM) associated with primary translocations involving the immunoglobulin heavy chain (IgH). These two subtypes of multiple myeloma have two different molecular pathogenesis and characteristic changes of each have been already observed. In our study, we previously described the patterns of genetic lesions and molecular pathogenesis of 12 HMCLs with Affymetrix 500K Single Nucleotide Polymorphism-based mapping arrays, now we re-depict those patterns using the Illumina 1M array set and compare the two studies. These techniques allow the examination and identification of copy number changes, bi-allelic deletions and the identification of loss of heterozygosity (LOH) due to loss and uniparental disomy (UPD), as well as gene localization and identification. The 12 HMCLs utilized are characterized for their structural alterations and not by hyperdiploidy. In addition, so as to fulfill the selection criteria, a minimum of 3 cell lines must present the alterations cited below. Previously described gains were observed in 1q, 7q, 8, 11q, 18, 19, and 20q; but also found at 4q. The bi-allelic deletions were ascertained on 3p. Similarly, we identified the regions of mono-allelic deletions on 1, 2q, 6q, 8q, 9p, 11q, 12, 13q, 14q, 17p, and 20p. In addition, described regions of bi-allelic deletions were detected on 1p, 6q, 8p, 13q, 16q, and 22q, and furthermore located on 2q, 3, 4q, 9, 10q, 12p, and 20p. Finally, the UPD obtained were traced on 1q, 4q, 8q, 10q, and 22q. The use of the new platform has allowed us to re-identify, significantly increase in information above the original set and finely delimit the regions previously described. Taken together, the dysregulated genes from the myeloma genome indicate that the crucial pathways in myeloma include NF-kB, apoptosis, cell-cycle and critical intracellular signaling pathways including the JAK/STAT, Wnt signaling, RAS/RAF/MAPK and PI3k/AKT. Disclosures No relevant conflicts of interest to declare.

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