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

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.

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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 ◽

10.1182/blood-2018-99-112500 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1944-1944 ◽

Cited By ~ 8

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.

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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 ◽

10.1182/blood-2021-151880 ◽

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.

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Transduction of Malignant Plasma Cells with Three Costimulatory Molecules (TRICOM) Elicits Myeloma-Specific Immune Response in Vitro – a Promising Strategy for Immunotherapy

Blood ◽

10.1182/blood.v120.21.1908.1908 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1908-1908

Author(s):

Katarina Luptakova ◽

Heidi Mills ◽

Jacalyn Rosenblatt ◽

Dina Stroopinsky ◽

Turner Kufe ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

Mononuclear Cells ◽

Plasma Cells ◽

Costimulatory Molecules ◽

Cell Populations ◽

Autologous Tumor

Abstract Abstract 1908 Introduction: Tumor vaccines hold promise as a means of eliciting anti-myeloma immunity and controlling disease that may be resistant to chemotherapy and biologic therapy. We have developed a whole cell tumor vaccine, whereby patient derived plasma cells are transduced with an attenuated vaccinia vector that contains transgenes for the costimulatory molecules B7.1 (CD80), ICAM-1 (CD54), and LFA-3 (CD58), designated TRIad of COstimulatory Molecules (TRICOM). In this manner, a broad array of tumor antigens, including those which may be specific to a given patient, are presented in the context of costimulatory molecules that have been shown to be synergistic in the stimulation of the effector T-cells. In the present study, we evaluated the phenotype and functional characteristics of TRICOM transduced primary myeloma cells. Methods and results: Plasma cells were isolated from bone marrow aspirates obtained from patients with multiple myeloma following Ficoll density centrifugation. Bone marrow derived mononuclear cells were infected with a replication-defective poxviral vector, the modified vaccinia Ankara strain (MVA), encoding TRICOM, or a control empty MVA vector. The expression of costimulatory molecules was assessed using flow cytometric analysis 3 hrs following viral infection. Viral transduction using the TRICOM vector at the dose of 20 MOI (multiplicity of infection) increased the mean percentage of CD38+ cells expressing CD80, CD54 and CD58 from a minimal baseline level (below 5%) to 70%, 56% and 47%, respectively (n=4). Transduction with control MVA vector did not augment expression of costimulatory molecules on plasma cells (mean percent expression of CD80, CD54 and CD58 of 2.6%, 2.7% and 3.8%, respectively, n=4). Of note, compared to CD38+ plasma cells, the CD38 negative fraction of bone marrow derived mononuclear cells demonstrated a significantly lower TRICOM transduction efficiency (mean percent expression of CD80, CD54 and CD58 of 16%, 17% and 16%, respectively, n=4, p<0.05 compared to CD38+ plasma cells). The ability of MVA-TRICOM transduced plasma cells to stimulate autologous T cell populations in vitro was assessed. Patient derived T-cells were purified from the non-adherent portion of PBMC by magnetic bead separation. MVA-TRICOM or empty MVA vector infected plasma cells were irradiated with 20Gy and co-cultured with autologous T cells at a 10:1 ratio of effector cells to vaccine for 7 days. MVA-TRICOM transduced plasma cells potently stimulated activated T cell responses, as assessed by the percentage of CD4+/CD25+/CD69+ T-cells (mean 7.8% of activated T-cells with TRICOM vaccine vs. 2.7% with control vaccine, n=3, p<0.05). In contrast, vaccine stimulation did not result in regulatory T-cell expansion, assessed as the percentage of cells co-expressing CD4,CD25 and FoxP3 (2.4% vs. 2.3%, for TRICOM and control vaccine, respectively, n=3). In concert with these findings, vaccine stimulation resulted in a polarization towards Th1 cytokine secretion, with 7.9% of CD4+ T-cells expressing intracellular IFN-γ after stimulation with TRICOM vaccine as compared to 5.4% after stimulation with the control vaccine (n=3, p<0.05). To further assess the expansion of tumor specific T cell populations, the ability of vaccine stimulated T cells to kill autologous tumor was assessed in a cell-based fluorogenic cytotoxicity assay. MVA-TRICOM transduced plasma cells potently stimulate the expansion of myeloma specific CTLs with the capacity to lyse autologous tumor targets. Mean CTL lysis was 20% and 8% for vaccine stimulated and unstimulated T cells respectively (n=2). Conclusions: Malignant plasma cells transduced with MVA-TRICOM strongly express costimulatory molecules, and potently stimulate activated, tumor reactive T cell populations. This preclinical data serves as a platform for developing a phase 1 clinical trial evaluating the use of MVA-TRICOM transduced autologous plasma cells in patients with multiple myeloma. Disclosures: No relevant conflicts of interest to declare.

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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 ◽

10.1182/blood.v126.23.2999.2999 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2999-2999 ◽

Cited By ~ 8

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.

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Wilms´Tumor 1 Protein Is Highly Expressed on Malignant Plasma Cells and Provides a Novel Target for Immunotherapeutic Approaches

Blood ◽

10.1182/blood.v126.23.98.98 ◽

2015 ◽

Vol 126 (23) ◽

pp. 98-98 ◽

Cited By ~ 2

Author(s):

Guenther Koehne ◽

Satyajit Kosuri ◽

Ekaterina Doubrovina ◽

Tao Dao ◽

Andrew Scott ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Plasma Cell ◽

Plasma Cells ◽

Therapeutic Potential ◽

Wilms Tumor ◽

Human Igg1 ◽

Wilms Tumor 1

Abstract Introduction: The Wilms' tumor 1 (WT1) protein is a tumor associated antigen that is potentially targetable by immunotherapeutic approaches. We have demonstrated the overexpression of WT1 in myeloma cells by IHC and in HLA-A*0201+ pts by staining with a high-affinity fully human IgG1 mAb (ESK1) specific to the RMFPNAPYL/HLA-A*0201 complex on malignant plasma cells. We report initial results from pts with plasma cell leukemia (PCL) or relapsed/refractory multiple myeloma (rMM) who have been treated with CD34-selected allo transplants followed by the administration of donor-derived WT1-specific T-cell infusions to induce an immunotherapeutic effect. Methods: In situ expression of WT1 was assessed by IHC analyses using a sequential double staining technique of MoAbs specific for CD138 and WT1.For staining with the RMFPNAPYL/HLA-A*0201 complex, BM samples were blocked with human FcR Blocking Reagent and then directly stained with MoAbs specific for CD38, CD56, CD45 and ESK1 or its isotype control human IgG1 and were analyzed by flow cytometry. WT1-specific T cells were generated from the original stem cell donors by sensitization of CD3+ enriched T-cell fractions with autologous APCs loaded with the pool of overlapping pentadecapeptides of WT1 (Invitrogen, Boston, MA). Cells were propagated in vitro with weekly restimulation and supplementation with IL-2 beginning at day 10-16. After 35-49 days, T-cells were harvested, counted and tested for antigen specific cytotoxicity, HLA-restriction, lack of alloreactivity and sterility. Pts received CD34-selected PBSC allografts after myeloablative cytoreduction with busulfan, melphalan and fludarabine. Pts were treated with 3 infusions of donor-derived WT1-specific T-cell infusions (5x10e6 cells/kg) starting 6 weeks post allo HSCT and at 4 weekly thereafter. Results: Marrow from all pts with immunohistochemical documented plasma cell involvement stained positive for WT1 IHC while WT1 staining remained negative in pts in CR. Only pts expressing HLA-A*0201 that stained positively for WT1 by IHC also demonstrated expression of WT1 by the RMFPNAPYL/HLA-A*0201 complex, whereas pts lacking HLA-A*0201 but with active disease stained positive for WT1 IHC but not ESK1 staining. Of 7 pts, 3 PCL and 4 rMM, treated with WT1-specific T cells, 4 pts had persistent disease post CD34-selected allotransplant. Of these 4 pts 2 pts developed a striking rise of WT1-specific T-cell frequencies and developed a complete remission post WT1 CTL infusions lasting for >2years. Conclusion: WT1 is overexpressed on malignant plasma cells and serves as a target for potential immunotherapeutic approaches in pts with multiple myeloma. Pts with persistent PCL following CD34-selected allografts treated with adoptive transfer of donor-derived WT1-specific cytotoxic T cells can achieve long lasting remission underscoring the therapeutic potential of T-cells specific for immunogenic WT1 peptides expressed on malignant plasma cells. Disclosures O'Reilly: Atara Biotherapeutics: Research Funding.

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Generation of T-cells reactive against CT-7 and BCMA peptides: Potential targets for T-cell immunotherapy of multiple myeloma

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.7615 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 7615-7615

Author(s):

L. D. Anderson ◽

D. G. Maloney ◽

S. R. Riddell

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Cell Therapy ◽

Plasma Cell ◽

Target Cells ◽

T Cell Repertoire ◽

Cell Repertoire ◽

T Cell Therapy

7615 Background: Multiple myeloma is a malignant plasma cell disorder that is incurable with chemotherapy or autologous stem cell transplantation (SCT), and novel therapies with lower toxicity are needed. There is evidence that T-cells can recognize myeloma and mediate anti-tumor effects, but the lack of defined target antigens other than idiotype has hindered the development of myeloma-specific T-cell therapy. We are investigating cancer-testis antigens and overexpressed “self”-proteins as candidate myeloma antigens, including MAGE-C1 (CT-7), which is expressed by >80% of myelomas, and B-Cell Maturation Antigen (BCMA), a plasma cell differentiation antigen commonly over-expressed in myeloma. Methods: To identify potential T-cell epitopes from CT-7 and BCMA, we scanned the protein sequences with computer algorithms and synthesized peptides predicted to bind to HLA-A2 and A3. CT-7 and BCMA are both “self” proteins to which the T-cell repertoire may be relatively tolerant, so we have utilized culture conditions that facilitate the expansion of rare myeloma-reactive T-cells. CD8+ T cells were stimulated in vitro with autologous dendritic cells pulsed with CT-7 or BCMA peptides in the presence of cytokines that avoid excessive nonspecific expansion of T-cells. Wells were screened for reactivity against peptide-pulsed target cells and myeloma cell lines. Results: A specific CD8+ T-cell response by both ELISPOT and cytotoxicity assays to at least one HLA-A2 peptide from each of the CT-7 and BCMA proteins has been identified in normal donors. CT-7 and BCMA-specific T-cells are being cloned in order to determine their ability to recognize primary myeloma cells. Experiments are also in progress to elicit responses to these peptides in myeloma patient samples and to screen HLA A3-binding epitopes. Conclusions: T-cells recognizing CT-7 and BCMA are detectable in the normal T-cell repertoire and can be isolated and expanded in vitro. We are currently pursuing the identification of additional antigenic epitopes in these proteins to define their potential utility as targets for vaccination or adoptive T-cell therapy. No significant financial relationships to disclose.

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Development of a fully human T cell engaging bispecific antibody for the treatment of multiple myeloma.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.8017 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 8017-8017 ◽

Cited By ~ 1

Author(s):

Ben Buelow ◽

Duy Pham ◽

Starlynn Clarke ◽

Shelley Force Aldred ◽

Kevin Dang ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Bispecific Antibody ◽

Plasma Cells ◽

Bispecific Antibodies ◽

Human Pbmcs

8017 Background: Although BCMA is a plasma cell specific surface molecule attractive as an antibody target in multiple myeloma, its scarcity on the cell surface may limit the efficacy of a conventional antibody. T-cell engaging bispecific antibody approaches are highly efficacious and are particularly well suited for a membrane target with limited expression, such as BCMA. Teneobio has developed a multivalent antibody platform based on modular human VH domains, which allowed us to build T cell engaging bispecific antibodies with low and high T cell agonistic activities. Methods: UniRats were immunized with either CD3 or BCMA antigens and antigen-specific UniAbs were identified by antibody repertoire sequencing and high-throughput gene assembly, expression, and screening. High affinity binding VH sequences were selected using recombinant proteins and cells. In vitro efficacy studies included T-cell activation by cytokine- and tumor cell kill by calcein-release assays. In vivo efficacy of the molecules was evaluated in NSG mice harboring myeloma cells and human PBMCs. Results: BCMA-specific UniAbs bound plasma cells with high affinities (100-700pM) and cross-reacted with cynomolgus plasma cells. Strong and weak T cell agonists were identified that bound human T cells with high and low affinities respectively and cross-reacted with cynomolgus T cells. T cell engaging bispecifics with a strong (H929 cytotoxicity:EC50=27pM) and a weak T cell activating arm (H929 cytotoxicity: EC50=1170pM) demonstrated T-cell activation and tumor-cell cytotoxicity in vitro; bispecifics with a weak CD3 engaging arm showed markedly reduced cytokine production even at doses saturating for cytotoxicity. In viv o, BCMAxCD3 bispecific antibodies reduced tumor load and increased survival when co-administered with human PBMCs as compared to controls. Conclusions: Our results suggest that T cell engaging bispecifics with low-affinity anti-CD3 arms could be preferred for the treatment of Multiple Myeloma.

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B-Cell Maturation Antigen Is a Promising Target for Genetically-Modified T-Cell Therapy of Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.937.937 ◽

2012 ◽

Vol 120 (21) ◽

pp. 937-937

Author(s):

Robert O. Carpenter ◽

Moses O. Evbuomwan ◽

Stefania Pittaluga ◽

Jeremy J. Rose ◽

Mark Raffeld ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

National Cancer Institute ◽

Plasma Cells ◽

Patent Application ◽

Target Cells ◽

Human Organs ◽

Specific Manner

Abstract Abstract 937 Multiple myeloma (MM) is a usually incurable malignancy of plasma cells. New therapies are urgently needed for MM. Development of immunotherapies targeting antigens expressed by MM cells could improve the outcomes of patients with multiple myeloma (MM). Chimeric antigen receptors (CARs) are fusion proteins containing antigen-recognition moieties and T-cell activation domains. T cells genetically modified to express CARs can specifically recognize tumor antigens, and CAR-expressing T cells have potent in vivo activity against some types of lymphoma and leukemia. Unfortunately, development of CAR-expressing T-cell therapies for MM has been hindered because most proteins expressed on MM cells are also expressed on essential normal cells. We report here our work developing CARs that target B-cell maturation antigen (BCMA). BCMA is a protein that has been reported to be selectively expressed by B-lineage cells including MM cells. We hypothesized that BCMA would be a suitable target for CAR-expressing T cells. We evaluated BCMA expression in all major human organs, and we designed and tested the first anti-BCMA CARs. We assessed BCMA RNA expression in all major human organs by quantitative PCR. Although BCMA RNA expression was mainly limited to hematologic tissues, low levels of BCMA RNA were detected in gastrointestinal organs, trachea, and testes. Next, we assessed BCMA protein expression in samples of 33 normal human organs by immunohistochemistry (IHC). BCMA-expressing plasma cells were detected in the lamina propria of several gastrointestinal organs by IHC, but BCMA protein was not detected in other gastrointestinal tissues. BCMA-expressing gastrointestinal plasma cells were probably the source of the low levels of BCMA RNA detected in normal gastrointestinal organs. Except for plasma cells, BCMA expression was not detected in any normal human tissues by IHC. Furthermore, BCMA was not detected on primary human CD34+hematopoietic cells by flow cytometry. In contrast, we detected uniform BCMA cell-surface expression by either flow cytometry or IHC on primary MM cells from 5 of 5 patients. Because BCMA is expressed by MM cells but not by normal essential tissues, we reasoned that BCMA would be an appropriate target for CAR-expressing T cells. We designed anti-BCMA CARs that contained antigen receptor domains derived from anti-BCMA monoclonal antibodies. The CARs also contained the signaling moiety of the CD28 molecule and the signaling domains of the CD3-zeta molecule. DNA encoding these CARs was ligated into a self-inactivating lentiviral vector, and human T cells were transduced with the anti-BCMA CARs. After transduction, anti-BCMA CAR expression was routinely detected on more that 60% of the transduced T cells by flow cytometry. Notably, we were able to successfully culture and transduce T cells from heavily treated patients with advanced MM. T cells expressing anti-BCMA CARs produced IFN-gamma, IL-2, and tumor necrosis factor in a BCMA-specific manner. T cell degranulation, which is required for perforin-mediated cytotoxicity, is associated with upregulation of CD107a. Anti-BCMA-CAR-transduced T cells upregulated CD107a when stimulated with BCMA-expressing target cells but not when stimulated with BCMA-negative target cells. Anti-BCMA-CAR-transduced T cells proliferated in a BCMA-specific manner. Anti-BCMA-CAR-transduced T cells killed BCMA-expressing multiple myeloma cells lines but not BCMA-negative cell lines. Importantly, anti-BCMA-CAR-transduced T cells produced interferon-gamma when cultured with BCMA-expressing primary MM cells but not when cultured with BCMA-negative peripheral blood mononuclear cells. Anti-BCMA-CAR-transduced T cells killed 66% of autologous primary MM cells in a BCMA-specific manner at a T cell to MM cell ratio of 7:1 in a 4-hour cytotoxicity assay. In summary, BCMA is expressed on MM cells of many patients, and BCMA is not expressed by normal essential tissues; therefore, BCMA is a suitable target for CAR-expressing T cells. We constructed and tested the first reported anti-BCMA CARs, and we showed that these T cells exhibited BCMA-specific functions including killing of primary MM cells. Adoptive transfer of anti-BCMA-CAR-transduced T cells is a promising new strategy for treating MM. These findings are the first steps toward clinical trials of anti-BCMA CAR-expressing T cells. Disclosures: Kochenderfer: National Cancer Institute: Inventor on patent application by National Cancer Institute., Inventor on patent application by National Cancer Institute. Patents & Royalties.

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AMG 701 induces cytotoxicity of multiple myeloma cells and depletes plasma cells in cynomolgus monkeys

Blood Advances ◽

10.1182/bloodadvances.2020002565 ◽

2020 ◽

Vol 4 (17) ◽

pp. 4180-4194 ◽

Cited By ~ 3

Author(s):

Rebecca L. Goldstein ◽

Ana Goyos ◽

Chi-Ming Li ◽

Petra Deegen ◽

Pamela Bogner ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cell ◽

T Cell Activation ◽

Half Life ◽

Plasma Cells ◽

Xenograft Model ◽

Assay Sensitivity ◽

Cynomolgus Monkeys ◽

Mouse Xenograft Model

Abstract 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.

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Analysis of the Multiple Myeloma HLA Peptidome Identifies a Naturally Presented Bcma-Derived Peptide As an Immunogenic T-Cell Epitope for Immunotherapeutic Approaches

Blood ◽

10.1182/blood-2018-99-113473 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3173-3173

Author(s):

Tatjana Bilich ◽

Simon D. Walz ◽

Annika Nelde ◽

Helmut R. Salih ◽

Ana Marcu ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Cell Lines ◽

Cd8 T Cells ◽

Specific Cell ◽

Phase I Clinical Trials ◽

Promising Target ◽

Hla Ligands

Abstract The B-cell maturation antigen (BCMA) is selectively expressed by cells of the B-lineage, including multiple myeloma (MM) cells, and constitutes a promising target for immunotherapeutic approaches. At present, BCMA is being evaluated as target for immunotherapeutic approaches, such as CAR T cells and bispecific antibodies, which have demonstrated promising results in phase I clinical trials. The utilization of cytotoxic T cells bearing T-cell receptors against BCMA constitutes an alternative promising approach to target MM cells. Therefore, the identification of BCMA-derived peptides that are naturally presented by human leukocyte antigens (HLA) and thus can serve as target structures for CD8+ T cells, is indispensable. In a previous study, we characterized the immunopeptidomic landscape of MM by mass spectrometry-based analysis of naturally presented HLA ligands from primary MM samples and MM cell lines (Walz et al., Blood, 2015). Comparative HLA peptidome profiling of the MM-derived HLA ligands versus the immunopeptidome of numerous benign samples from different tissues identified several strictly MM-associated antigens. Here, we evaluated this dataset for the presence of BCMA-derived MM-exclusive antigens and identified two HLA class I-restricted, BCMA-derived peptides in the immunopeptidome of our cohort comprising 15 primary MM samples and MM cell lines. Notably, one of these peptides showes strictly MM-associated presentation and was never detected on any benign tissues according to our extensive immunopeptidome database (135,354 HLA ligands originating from 16,626 source proteins detected in 337 samples from various benign tissues including blood, bone marrow, lung, kidney, liver, and spleen). This HLA-B*18-restricted ligand P(BCMA)B*18 is represented in 20% (3/15) of the analyzed MM immunopeptidomes. For immunological characterization of the P(BCMA)B*18 peptide, we performed in vitro artificial antigen-presenting cell-based priming experiments engaging naïve CD8+ T cells obtained from healthy volunteers (HV). Induction of tetramer-positive T-cell populations with frequencies ranging from 0.1-2.9% of viable CD8+ T cells was observed for all analyzed healthy whole blood donors, which demonstrates the immunogenicity of P(BCMA)B*18. Subsequently, we functionally characterized the induced P(BCMA)B*18-specific CD8+ T cells using intracellular cytokine staining. Upon stimulation with P(BCMA)B*18, we observed an increased IFNγ and TNF production specifically in the peptide-specific CD8+ T cells. In addition, the degranulation marker CD107a was found to be upregulated in the analyzed tetramer-positive T cells, confirming the activity of CD8+ T cells upon peptide-stimulation. Priming experiments using naïve CD8+ T cells obtained from MM patients as well as in vitro cytotoxicity assays with polyclonal peptide-specific effector T cells are presently ongoing in order to assess the capacity of P(BCMA)B*18-specific CD8+ T cells to induce antigen-specific cell lysis. Taken together, we identified a naturally presented and MM-associated, BCMA-derived peptide, which constitutes a promising target for tailored T cell-based immunotherapeutic approaches. Disclosures Salih: Several patent applications: Patents & Royalties: e.g. EP3064507A1. Kowalewski:Immatics Biotechnologies GmbH: Employment. Weisel:Amgen, BMS, Celgene, Janssen, and Takeda: Honoraria; Amgen, BMS, Celgene, Janssen, Juno, Sanofi, and Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen, Celgene, Janssen, and Sanofi: Research Funding.

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