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.

scholarly journals Chemically Controlled, Immunosuppression-Resistant, Anti-Bcma CAR-T Cells for Treatment of Antibody-Mediated Autoimmunity

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2203-2203 ◽  
Author(s):  
Sowndharya Rajavel ◽  
Cade E. Ito ◽  
Keith Abe ◽  
Valerie Guerrero ◽  
Gene I. Uenishi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Side Effect ◽  
Plasma Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Nsg Mice ◽  
Car T ◽  
Equity Ownership

Abstract Auto-reactive antibody production by plasma cells is the direct cause of many auto-immune diseases. In such cases elimination of plasma cells would ameliorate the disease. Chimeric antigen receptor T (CAR-T) cells with cytotoxicity toward cells expressing B-cell maturation antigen (BCMA) have shown remarkable promise for the treatment of multiple myeloma, a plasma cell neoplasm. Elimination of non-malignant plasma cells is a side-effect of anti-BCMA CAR-T treatment of multiple myeloma, suggesting the use of these anti-BCMA CAR T cells for auto-immune indications. Unfortunately, CAR-T administration requires use of lymphodepletion to achieve efficient cell engraftment, and is often accompanied by cytokine release syndrome (CRS), a potentially life-threatening side-effect. As lymphodepletion and CRS pose morbidity/mortality risks that are unacceptable for therapy of many auto-immune diseases, we have utilized CRISPR-Cas9 gene editing to develop a controllable CAR-T cell platform that provides for (1) engraftment with non-cytotoxic transient immunosuppression; and (2) small-molecule dependent CAR T-cell expansion. We have implemented this platform using a unique dual targeting approach in which a BCMA CAR transgene is integrated into the TRAC locus, and additional payloads are integrated into a second locus, thus also enabling an allogeneic manufacturing process. Transgene integration occurred in >50% of cells individually with several percent of cells targeted at two loci. TRAC-targeted, anti-BCMA CAR T cells demonstrated CAR-dependent, target-cell-BCMA-dependent cytotoxicity towards both high-BCMA- and low-BCMA-expressing cell lines and in multiple myeloma cells xenografted into NSG mice. Drug-regulation properties and immunosuppression resistance are the subject of ongoing experiments. Anti-BCMA CAR T cells that are chemically controlled, incapable of graft-versus-host disease, and insensitive to immunosuppression may be an attractive treatment option a variety of antibody-mediated auto-immune conditions. Disclosures Rajavel: Casebia Therapeutics: Employment. Ito:Casebia Therapeutics: Employment. Abe:Casebia Therapeutics: Employment. Guerrero:Casebia Therapeutics: Employment. Uenishi:Casebia Therapeutics: Employment. Scharenberg:Casebia Therapeutics: Employment; Generation Bio: Equity Ownership; Alpine Immune Sciences: Equity Ownership. Cost:Casebia Therapeutics: Employment.

scholarly journals CD171- and GD2-specific CAR-T cells potently target retinoblastoma cells in preclinical in vitro testing

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Lena Andersch ◽  
Josefine Radke ◽  
Anika Klaus ◽  
Silke Schwiebert ◽  
Annika Winkler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Cell Lines ◽  
T Cell Therapy ◽  
Retinoblastoma Cell ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Background Chimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects. Methods CD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3+) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs. Results All retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing. Conclusion Both CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.

scholarly journals Clinical Development of a Non-Gene-Edited Allogeneic Bcma-Targeting CAR T-Cell Product in Relapsed or Refractory Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 27-28
Author(s):  
A. Samer Al-Homsi ◽  
Sebastien Anguille ◽  
Jason Brayer ◽  
Dries Deeren ◽  
Nathalie Meuleman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Gene Editing ◽  
Cell Product ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Background Autologous CAR T-cell therapy targeting the B-cell maturation antigen (BCMA) has shown impressive objective response rates in patients with advanced multiple myeloma (MM). Clinical grade manufacturing of autologous CAR T-cells has limitations including vein-to-vein delivery time delay and potentially sub-optimal immunological capability of T-cells isolated from patients with advanced disease. Allogeneic CAR T-cell products, whereby cells from healthy third-party donors are used to generate an "off-the-shelf" CAR T-cell product, have the potential to overcome some of these issues. To circumvent the primary potential risk of graft-versus-host disease (GvHD) associated with the use of allogeneic T-cells, abrogation of the T-cell receptor (TCR) expression in the CAR T-cells, via gene editing, is being actively pursued. To avoid the potential safety risks and manufacturing challenges associated with gene editing, the allogeneic CYAD-211 CAR T-cell product exploits short hairpin RNA (shRNA) interference technology to down-regulate TCR expression thus avoiding the risk of life-threatening GvHD. Aim The aim is to generate a BCMA-specific allogeneic CAR T-cell product using a non-gene editing approach and study its activity both in vitro and in vivo. CYAD-211 combines a BCMA-specific CAR with a single optimized shRNA targeting the TCR CD3ζ subunit. Downregulation of CD3ζ impairs the TCR expression on the surface of the donor T-cells, preventing their reactivity with the normal host tissue cells and potential GvHD induction. Maintaining all the elements required for the therapy within a single vector (all-in-one vector) provides some significant manufacturing advantages, as a solitary selection step will isolate cells expressing all the desired traits. Results CYAD-211 cells produce high amounts of interferon-gamma (IFN-γ) during in vitro co-cultures with various BCMA-expressing MM cell lines (i.e., RPMI-8226, OPM-2, U266, and KMS-11). Cytotoxicity experiments confirmed that CYAD-211 efficiently kills MM cell lines in a BCMA-specific manner. The anti-tumor efficacy of CYAD-211 was further confirmed in vivo, in xenograft MM models using the RPMI-8226 and KMS-11 cell lines. Preclinical data also showed no demonstrable evidence of GvHD when CYAD-211 was infused in NSG mice confirming efficient inhibition of TCR-induced activation. Following FDA acceptance of the IND application, IMMUNICY-1, a first-in-human, open-label dose-escalation phase I clinical study evaluating the safety and clinical activity of CYAD-211 for the treatment of relapsed or refractory MM patients to at least two prior MM treatment regimens, is scheduled to begin recruitment. IMMUNICY-1 will evaluate three dose-levels of CYAD-211 (3x107, 1x108 and 3x108 cells/infusion) administered as a single infusion after a non-myeloablative conditioning (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, daily for 3 days) according to a classical Fibonacci 3+3 design. Description of the study design and preliminary safety and clinical data from the first cohort will be presented at ASH 2020. Conclusion CYAD-211 is the first generation of non-gene edited allogeneic CAR T-cell product based on shRNA technology. The IMMUNICY-1 clinical study seeks to provide proof of principle that single shRNA-mediated knockdown can generate fully functional allogeneic CAR T-cells in humans without GvHD-inducing potential. We anticipate that subsequent generations of this technology will incorporate multiple shRNA hairpins within a single vector system. This will enable the production of allogeneic CAR T-cells in which multiple genes of interest are modulated simultaneously thereby providing a platform approach that can underpin the future of this therapeutic modality. Figure 1 Disclosures Al-Homsi: Celyad: Membership on an entity's Board of Directors or advisory committees. Brayer:Janssen: Consultancy; Bristol-Myers Squibb, WindMIL Therapeutics: Research Funding; Bristol-Myers Squibb, Janssen, Amgen: Speakers Bureau. Nishihori:Novartis: Other: Research support to institution; Karyopharm: Other: Research support to institution. Sotiropoulou:Celyad Oncology: Current Employment. Twyffels:Celyad Oncology: Current Employment. Bolsee:Celyad Oncology: Current Employment. Braun:Celyad Oncology: Current Employment. Lonez:Celyad Oncology: Current Employment. Gilham:Celyad Oncology: Current Employment. Flament:Celyad Oncology: Current Employment. Lehmann:Celyad Oncology: Current Employment.

scholarly journals Anti-CD19 ARTEMISTM Therapy Drastically Reduces Cytokine Release without Compromising Efficacy Against Preclinical Lymphoma Models

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3354-3354
Author(s):  
Hong Liu ◽  
Li Long ◽  
Shon Green ◽  
Lucas H Horan ◽  
Bryan Zimdahl ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Cytokine Release ◽  
Raji Cells ◽  
Car T Cells ◽  
Nsg Mice ◽  
Car T ◽  
Equity Ownership

Abstract Anti-CD19 chimeric antigen receptor (CAR) T cell therapies for B cell malignancies have demonstrated the remarkable curative potential of T cell immunotherapies. However, in clinical trials anti-CD19-CAR T cells continue to trigger life threatening adverse events that are often associated with excessive cytokine release and excessive T-cell proliferation. We reasoned that the activation pathway of current CAR T cells could be altered to better regulate proliferation and cytokine secretion, and thus disentangle the correlation between cytokine release syndrome (CRS) and efficacy of T cell-based therapies. Through protein engineering, we developed the ARTEMISTM (1) signaling platform which when expressed on primary T-cells results in a dramatic reduction of cytokine release during tumor cell lysis, without sacrificing efficacy. Using a human phage display library, we also identified several human CD19 antibodies with improved specificity and affinity that will be less immunogenic as compared to the murine-derived anti-CD19 antibodies that are currently used in most trials. Our lead antibody clone CD19(7) was then engineered into both CD28z-CAR and ARTEMISTM platforms for comparison. When tested in vitro, both CD19(7)-ARTEMISTM T cells and CD19(7)-CD28z-CAR T cells specifically lysed multiple CD19+ leukemia and lymphoma cell lines with similar potencies. However, during the 16 hour killing assays, ARTEMIS™ T cells secreted over 1000-fold less IL-2 and dramatically lower levels of IFN-γ, GM-CSF, IL-10 and IL-6. ARTEMISTM T cells also accumulated less PD-1, LAG3, and TIM3 on their surface during culturing and following in vitro killing, indicating a diminished propensity for exhaustion. Furthermore, during in vitro T cell expansion, ARTEMISTM cells were enriched for naïve/central memory subpopulations, had lower expression of granzyme B, a marker of terminal differentiation, and had reduced rates of receptor internalization upon antigen engagement. These characteristics suggest that T-cells activated through the ARTEMISTM receptor will have improved persistence and long-term proliferation potential, as well as a safer, more controlled cytokine release when used for T-cell therapies. When tested in vivo against CD19+ Raji systematic lymphoma xenografts, intravenous administration of CD19(7)-ARTEMISTM T cells caused rapid, complete, and lasting tumor regression that was better than that achieved with an equal dose of CD19(7)-CD28z-CAR T cells (Figure 1). In agreement with our in vitro data, mice treated with ARTEMISTM T cells had nearly undetectable levels of cytokines in their blood at 24 hours post dosing, a time in which CD19(7)-CAR-treated mice had markedly elevated levels of human IFN-γ, IL-2, TNFα, and IL-10. While flow cytometry analysis of the peripheral blood showed that CD19(7)-CAR T cells expanded more rapidly in mice, CD19(7)-ARTEMISTM T cells better controlled Raji tumor growth and were negative for PD-1 expression which was high on circulating CAR T cells. At 7 weeks post dosing, a time when all ARTEMISTM T cell-treated mice had no detectable tumors, they were re-challenged with Raji lymphoma. While tumors grew rapidly in control mice, ARTEMISTM T cell-treated mice resisted the Raji lymphoma re-challenge, indicating that ARTEMISTM T cells persisted in these mice despite the absence of tumors and remained antigen-responsive (Figure 2). Our data demonstrates that CD19(7)-ARTEMISTM T cells are highly potent against lymphoma preclinical models while releasing drastically lower levels of cytokines. Thus we have developed and pre-clinically validated a novel fully human anti-CD19 T cell therapy that has the potential to persist longer in patients and, importantly, presents a lower risk of cytokine-related toxicities without compromising efficacy. A clinical trial testing CD19(7)-ARTEMISTM T cell therapy in humans is expected to begin in 2017. Figure 1 Raji lymphoma tumor growth in NSG mice treated with either donor-matched untransduced T cells (Mock), CD19(7)-CAR, or CD19(7)-ARTEMISTM T cells (5x106 receptor-positive cells per mouse) Figure 1. Raji lymphoma tumor growth in NSG mice treated with either donor-matched untransduced T cells (Mock), CD19(7)-CAR, or CD19(7)-ARTEMISTM T cells (5x106 receptor-positive cells per mouse) Figure 2 Raji lymphoma tumor growth in NSG mice previously treated with CD19(7)-ARTEMISTM T cells who had complete regression (0.5x106 Raji cells/mouse). As controls, Raji-naïve mice were implanted with Raji cells following an injection of Mock T cells. (1)ARTEMISTM is trademarked by Eureka Therapeutics, Inc. Figure 2. Raji lymphoma tumor growth in NSG mice previously treated with CD19(7)-ARTEMISTM T cells who had complete regression (0.5x106 Raji cells/mouse). As controls, Raji-naïve mice were implanted with Raji cells following an injection of Mock T cells. / (1)ARTEMISTM is trademarked by Eureka Therapeutics, Inc. Disclosures Liu: Eureka Therapeutics: Employment, Equity Ownership, Patents & Royalties. Long:Eureka Therapeutics: Employment, Equity Ownership. Green:Eureka Therapeutics: Employment. Horan:Eureka Therapeutics: Employment. Zimdahl:Eureka Therapeutics: Employment. Liu:Eureka Therapeutics: Employment, Equity Ownership, Patents & Royalties.

CD123-Specific Chimeric Antigen Receptor Redirected T Cells Exhibit Potent Cytolytic Activity and Multiple Effector Functions Against Acute Myeloid Leukemia without Altering Normal Hematopoietic Colony Formation in Vitro

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 950-950
Author(s):  
Armen Mardiros ◽  
Cedric Dos Santos ◽  
Tinisha McDonald ◽  
Christine Brown ◽  
Xiuli Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Colony Formation ◽  
Cytolytic Activity ◽  
Car T Cells ◽  
Tnf Α ◽  
Donor T Cells ◽  
Car T

Abstract Abstract 950 Current treatment regimes for acute myeloid leukemia (AML) achieve complete remissions in only a subset of individuals and most adult patients will relapse within 5-years, emphasizing the need for novel treatment alternatives. One such therapy may be the administration of T cells engineered to express chimeric antigen receptors (CARs) specific for AML-associated antigens. CARs are typically composed of a single chain variable fragment (scFv) from a monoclonal antibody fused to the CD3ζ signaling domain and may contain one or more costimulatory endodomains. When expressed in T cells, CARs redirect T cell specificity to surface antigens on target cells in an MHC-independent manner. The interleukin 3 receptor alpha chain (IL3Rα, CD123) is a cell surface receptor which is aberrantly over-expressed on multiple hematologic malignancies including AML. Previous work has demonstrated that CD123 is not expressed on all CD34+/CD38− hematopoietic stem cells and is restricted to cells of the myeloid lineage, making CD123 an attractive target for CAR T cell therapy. We have therefore generated two novel CD123-specific (CD123R) CARs using scFvs from previously characterized antibodies, designated 26292 and 32716, which bind two distinct epitopes on CD123. Here we demonstrate that T cells expressing CARs derived from either 26292 or 32716 effectively redirect T cell specificity against CD123+ cells. Healthy donor T cells (n=3) engineered to express the CD123R CARs efficiently lysed CD123+ cell lines LCL and KG1a while sparing the CD123− cell line K562 as demonstrated by a 4 hour chromium-51 (51Cr) release assay. Additionally, both of the CD123R CAR T cells produced similar levels of IFN-γ and TNF-α and displayed comparable levels of antigen-dependent proliferation following co-culture with CD123+ cell lines. The potent cytolytic activity and activation of our CD123-targeting T cells was not limited to tumor cell lines. Indeed, CD123R CAR T cells, but not donor-matched CD19-specific (CD19R) CAR T cells, robustly lysed panel of primary AML samples (n=6, 3 – persistent, 1 – relapsed, 2 - untreated) (* p<0.05, ** p<0.001 using the unpaired students' t-test comparing 26292 or 32716 CAR T cells to donor-matched CD19R CAR T cells), and exhibited multiple effector functions for both CD4 and CD8 T cell subsets (ie CD107a degranulation, IFN-γ and TNF-α production, and antigen specific proliferation) when co-cultured with primary AML samples (n=3, 2 – relapsed, 1-persistent). To examine the effect our CD123-specific T cells have on normal and leukemic progenitor cells, we co-cultured CD123R CAR T cells, or donor-matched CD19-targeting T cells, with either CD34-enriched cord blood (CB, n=3) or primary AML samples (n=3, 2 – relapsed, 1 - untreated) for 4 hours (E:T 25:1) prior to plating in semisolid methylcellulose progenitor culture. CD123-targeting T cells did not significantly reduce the number of colony-forming unit granulocyte-macrophage (CFU-GM) or burst-forming unit erythroid (BFU-E) colonies from CB when compared to CD19R CAR T cells. Finally, while CD19-specific T cells had little impact on leukemic colony formation of primary AML samples, CD123-targeting T cells significantly reduced leukemic colony formation in vitro. Collectively, our data demonstrate that CD123-specifc CARs can be expressed in primary healthy donor T cells, distinguish between CD123+ and CD123− cells, and mediate robust anti-leukemic activity against a panel of poor-risk primary AML patient samples. Importantly, we demonstrate that CD123R CAR T cells have little impact on normal progenitor colony formation while significantly reducing the growth of clonogenic myeloid leukemic progentiors in vitro. Thus, CD123R CAR T cells are a promising candidate for future immunotherapy of AML. Disclosures: Bhatia: Novartis: Consultancy, Honoraria. Jensen:ZetaRx: Equity Ownership, Patents & Royalties.

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.

scholarly journals A TCR Mimic Antibody-Directed CAR T Cell Specific for Intracellular NDC80 Is Broadly Cancer Reactive and Displays High Activity Against Hematological Malignancies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 20-21
Author(s):  
Martin G. Klatt ◽  
Zhiyuan Yang ◽  
Jianying Liu ◽  
Tatyana Korontsvit ◽  
Sung Soo Mun ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Private Company ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Hla Ligand ◽  
Car T

Chimeric antigen receptor (CAR) T cells represent a novel class of FDA-approved drugs with high efficacy against refractory B cell derived malignancies and potentially other cancer types. However, target selection for CAR T cell therapy remains challenging as cell surface proteins are not cancer-specific and therefore often not adaptable for CAR T cell therapy. In contrast, many intracellular proteins can be highly tumor specific and are targetable after proteasomal degradation and presentation on human leukocyte antigen (HLA) complexes recognized by T cell receptor mimic antibodies. This class of antibodies recognizes peptide:HLA complexes with a similar mode of recognition as a TCR, but with the clinical versatility and applicability of an antibody. To identify a tumor specific target that is presented as a peptide in conjunction with the highly prevalent HLA allele A*02:01, we immunopurified peptide:HLA complexes from various cancer cell lines of different origins, separated HLA ligands from complexes and identified their peptide sequences via mass spectrometry. Network analysis of the resulting HLA ligand datasets identified shared biological processes among the tumor cell lines that were not present in network analyses of published datasets of healthy human tissue HLA ligandomes. Through this filtering process several potential targets were identified and an HLA ligand derived from kinetochore NDC80 protein homolog (NDC80) was selected as a target. The NDC80 derived peptide was detected in over 90% of the A*02 positive cell lines tested and never reported to be present in HLA ligand datasets of healthy human tissues. Furthermore, NDC80 has been shown to be differentially expressed in malignant compared to adjacent non-malignant tissues and is associated with poor prognosis in many cancer types. After utilizing E-ALPHA®phage library screening, one clone (NDC80-L1) was selected as the lead TCR mimic antibody. Overall, NDC80-L1 showed high specificity for the target HLA:peptide complex in both antibody and CAR T cell format in vitro and demonstrated binding primarily to the central region of the HLA ligand as determined by alanine screening assays. The exquisite specificity of NDC80-L1 was further illustrated by NDC80 knockdown experiments as well as successful immunopurification of the target peptide together with no relevant off-targets from BV173 ALL cells in mass spectrometry assays. Given the high specificity, sensitivity was assessed primarily in a potent CAR T cell format: Multiple tumor cell lines of different origin (e.g. ALL, AML, lymphoma, melanoma, mesothelioma, pancreatic and thyroid cancer) were successfully killed in vitro by NDC80-L1 CAR T cells, but no toxicity towards A*02:01 positive CAR T cells, healthy PBMCs or NDC80 target negative cell lines was observed. Interestingly, NDC80-L1 CAR T cells demonstrated highest efficacy in hematological malignancies most likely correlating with elevated expression of antigen presentation machinery and rapid cell division which leads to strong surface expression of NDC80 peptides. In summary, CAR T cells directed against peptide/HLA-A*02 derived from the NDC80 protein effectively kill multiple cancer cell lines in vitro without evidence of relevant off-target killing. However, the improved killing especially against ALL, AML and lymphomas highlights the potential of these CAR T cells to preferentially eliminate cancer cells with high proliferative capacity. Future in vivo studies with CAR T cell and antibody format will further investigate this TCR mimic antibody's potential as a tumor-agnostic therapeutic agent. Disclosures Klatt: MSKCC/EUREKA: Patents & Royalties: MSKCC AND EUREKA THERAPUETICS HAVE FILED A PATENT FOR THIS ANTIBODY/SCFV. Yang:Eureka Therapuetics: Current Employment, Current equity holder in private company, Patents & Royalties: MSKCC and Eureka have filed patent for this TCRm and ScvF. Liu:Eureka Therapue: Current Employment, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm. Dao:Eureka Therapeutics: Consultancy. Liu:Eureka Therapeutics: Current Employment, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm. Scheinberg:Eureka Therapeutics: Consultancy, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm; Actinium: Consultancy, Current equity holder in private company; Sellas: Consultancy, Current equity holder in private company; Contrafect: Current equity holder in private company; Arvenas: Current equity holder in private company; Sapience: Consultancy, Current equity holder in private company; Iovance: Current equity holder in private company; Oncopep: Consultancy; Pfizer: Consultancy, Current equity holder in private company; Lantheus: Current equity holder in private company; Enscyse: Current equity holder in private company.

scholarly journals Superior Efficacy of CAR-T Cells Using Marrow-Infiltrating Lymphocytes (MILsTM) As Compared to Peripheral Blood Lymphocytes (PBLs)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4437-4437 ◽  
Author(s):  
Eric R. Lutz ◽  
Srikanta Jana ◽  
Lakshmi Rudraraju ◽  
Elizabeth DeOliveira ◽  
Jing Zhou ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership

Background The type of T cell used in generating chimeric antigen receptor (CAR) T cells is an important choice. Evidence suggests that T cells that are early in the effector/memory differentiation pathway with more stemness and greater potential to persist are better than more differentiated T cells with less stemness that are more readily exhausted and have less potential to persist. Marrow-infiltrating Lymphocytes (MILsTM) is a novel form of adoptive T cell therapy composed of patient-autologous, polyclonal CD4 and CD8 T cells that are activated and expanded from the bone marrow. Genetically unmodified MILsTM have demonstrated antitumor activity in patients with multiple myeloma and are being developed for several other tumor types, including non-small cell lung cancer and other solid tumors. Distinguishing features of bone marrow T cells used to produce MILsTM include their memory phenotype, inherent tumor antigen-specificity, higher CD8:CD4 ratio and ability to persist long-term when compared to peripheral blood lymphocytes (PBLs) which is the T cell source used to produce currently approved CAR-T therapies. Based on these differences, we hypothesize that MILsTM provide a more robust and better fit platform for CAR-T therapy compared to PBLs. Using a CD38-specific, 4-1BB/CD3z-signaling CAR as an initial model, we have demonstrated the feasibility of producing CAR-modified MILsTM (CAR-MILsTM) and showed that CAR-MILsTM demonstrate superior killing in vitro compared to CAR-T cells generated from patient-matched PBLs (CAR-PBLs). Herein, we build on our previous data and add a second BCMA-specific CAR model. We use the two multiple myeloma model systems to compare cytolytic potential, functionality, and expression of phenotypic markers of memory, stemness and exhaustion between patient-matched CAR-MILsTM and CAR-PBLs. Methods Matched pairs of CAR-MILsTM and CAR-PBLs were produced from the bone marrow and blood of multiple myeloma patients. Two different in vitro cytotoxicity assays, the RTCA xCelligence real-time impedance and FACS assays, were used to evaluate antigen-specific killing of target tumor cells. Functionality of CD4 and CD8 CAR-T cells, at the single-cell level, was evaluated by measuring the secretion of 32 cytokines and chemokines following in vitro antigen-specific stimulation using IsoPlexis IsoCode chips and analyzed using IsoPeak. Expression of markers of T cell memory (CD45RO & CCR7/CD62L), stemness (CD27) and exhaustion (PD1 & TIM3) on CAR-MILsTM and CAR-PBLs prior to and following antigen-specific stimulation was evaluated by flow-cytometry (FACS). Results CAR-MILsTM demonstrated superior killing of tumor target cells in vitro, regardless of the antigen specificity of the CAR, when compared to matched CAR-PBLs and this superiority persisted even upon repeated antigen encounter - a factor that may be critical in guaranteeing better anti-tumor efficacy and persistence. CAR-MILsTM demonstrated increased polyfunctionality (secretion of 2+ cytokines per cell) and an increased polyfunctional strength index (PSI) following antigen-stimulation compared to CAR-PBL in both CD4 and CD8 T cells. The enhanced PSI in CAR-MILsTM was predominately mediated by effector, stimulatory and chemoattractive proteins associated with antitumor activity including Granzyme B, IFNg, IL-8, MIP1a and MIP1b. Coincidentally, increased PSI and enhanced secretion of these same proteins was reported to be associated with improved clinical responses in patients with Non-Hodgkin lymphoma treated with CD19-specific CAR-T therapy. Expression of memory markers on CD4 and CD8 T cells were similar in CAR-MILsTM and CAR-PBLs both prior to and following antigen-stimulation. Although expression of CD27, PD1 and TIM3 were similar at baseline, CAR-MILs maintained higher levels of CD27 and lower levels of PD1 and TIM3 compared to CAR-PBLs following antigen-stimulation in both CD4 and CD8 T cells. Conclusions Collectively, our data suggest that CAR-MILsTM have several advantages over CAR-PBLs, including increased cytolytic potential, enhanced polyfunctionality, increased stemness and less exhaustion. Based on these differences and the inherent antitumor properties of MILsTM, we speculate that CAR-MILsTM would be more potent and effective than currently approved CAR-T products derived from PBLs. Disclosures Lutz: WindMIL Therapeutics: Employment, Equity Ownership. Jana:WindMIL Therapeutics: Employment, Equity Ownership. Rudraraju:WindMIL Therapeutics: Employment, Equity Ownership. DeOliveira:WindMIL Therapeutics: Employment, Equity Ownership. Zhou:Isoplexis: Employment, Equity Ownership. Mackay:Isoplexis: Employment, Equity Ownership. Borrello:Aduro: Patents & Royalties: intellectual property on allogeneic MM GVAX; BMS: Consultancy; WindMIL Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Celgene: Honoraria, Research Funding, Speakers Bureau. Noonan:WindMIL Therapeutics: Employment, Equity Ownership, Patents & Royalties; Aduro: Patents & Royalties: intellectual property on allogeneic MM GVAX.

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 Any closer to successful therapy of multiple myeloma? CAR-T cell is a good reason for optimism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Faroogh Marofi ◽  
Safa Tahmasebi ◽  
Heshu Sulaiman Rahman ◽  
Denis Kaigorodov ◽  
Alexander Markov ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Survival Rate ◽  
T Cell ◽  
Plasma Cells ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

AbstractDespite many recent advances on cancer novel therapies, researchers have yet a long way to cure cancer. They have to deal with tough challenges before they can reach success. Nonetheless, it seems that recently developed immunotherapy-based therapy approaches such as adoptive cell transfer (ACT) have emerged as a promising therapeutic strategy against various kinds of tumors even the cancers in the blood (liquid cancers). The hematological (liquid) cancers are hard to be targeted by usual cancer therapies, for they do not form localized solid tumors. Until recently, two types of ACTs have been developed and introduced; tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR)-T cells which the latter is the subject of our discussion. It is interesting about engineered CAR-T cells that they are genetically endowed with unique cancer-specific characteristics, so they can use the potency of the host immune system to fight against either solid or liquid cancers. Multiple myeloma (MM) or simply referred to as myeloma is a type of hematological malignancy that affects the plasma cells. The cancerous plasma cells produce immunoglobulins (antibodies) uncontrollably which consequently damage the tissues and organs and break the immune system function. Although the last few years have seen significant progressions in the treatment of MM, still a complete remission remains unconvincing. MM is a medically challenging and stubborn disease with a disappointingly low rate of survival rate. When comparing the three most occurring blood cancers (i.e., lymphoma, leukemia, and myeloma), myeloma has the lowest 5-year survival rate (around 40%). A low survival rate indicates a high mortality rate with difficulty in treatment. Therefore, novel CAR-T cell-based therapies or combination therapies along with CAT-T cells may bring new hope for multiple myeloma patients. CAR-T cell therapy has a high potential to improve the remission success rate in patients with MM. To date, many preclinical and clinical trial studies have been conducted to investigate the ability and capacity of CAR T cells in targeting the antigens on myeloma cells. Despite the problems and obstacles, CAR-T cell experiments in MM patients revealed a robust therapeutic potential. However, several factors might be considered during CAR-T cell therapy for better response and reduced side effects. Also, incorporating the CAT-T cell method into a combinational treatment schedule may be a promising approach. In this paper, with a greater emphasis on CAR-T cell application in the treatment of MM, we will discuss and introduce CAR-T cell’s history and functions, their limitations, and the solutions to defeat the limitations and different types of modifications on CAR-T cells.

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