Combinatorial IL13Rα2 chimeric antigen receptor-T cells plus checkpoint blockade to treat solid tumors in murine and canine models.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 152-152
Author(s):  
Yibo Yin ◽  
Alina Boesteanu ◽  
Chong Xu ◽  
Avery Posey ◽  
Danielle Cook ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Combined Treatment ◽  
Checkpoint Blockade ◽  
Single Chain ◽  
Normal Tissues ◽  
Car T Cells ◽  
Car T ◽  
Canine Tumors

152 Background: Glioblastoma (GBM) is a deadly cancer, with a 5-year survival rate of less than 10%. T cells can be redirected to kill cancer cells using chimeric antigen receptors (CAR), a promising method to treat solid tumors. IL13Rα2 is expressed in many solid tumors but not normal tissues, and provides a target for CAR T cells as a powerful immunotherapy treatment. Methods: Human and canine tumors were screened for IL13Rα2 expression by flow cytometry and RT-PCR. Different IL13Rα2 single-chain variable fragments (scFv) were tested in CARs in vitro against human and canine recombinant IL13Rα2 protein to identify a cross-reactive clone. IL13Rα2 CARs expressed in human and canine T cells and showed antigen-specific stimulation by cytokine secretion and target cell lysis. Solid tumors were used to establish in vivo subcutaneous and orthotopic xenograft models in NSG mice. Mice received a single treatment of 2-5 million CAR T cells IV with or without anti PD-1, CTLA-4 or TIM3 mAb checkpoint blockade. Results: IL13Rα2 was detected on three human GBM (D270, U251 and U87), four canine osteosarcomas (BW-, CS-, MC- and SK-KOSA) and two canine lung cancer cell lines (Cacal3, Cacal5). Two different IL13Rα2 CAR were generated that recognized human IL13Rα2 and not IL13Rα1, one also recognized canine IL13Rα2. Five million IL13Rα2 CAR T-cells delivered IV were able to eliminate established GBM tumors. Use of fewer CAR T cells initially controlled tumor growth, followed by tumor outgrowth correlated with expression of T cell exhaustion markers. Combined treatment with CARs plus checkpoint blockade mAb delivered IP had synergistic effects, restoring anergic T-cell function and improving tumor treatment. Conclusions: IL13Rα2 is highly expressed on human and canine tumors, but not normal tissues. IL13Rα2 specific CAR T cells successfully respond to human and canine tumors and treat GBM in a xenograft mouse model, although this effect was transient at lower treatment doses. Addition of checkpoint blockade mAbs to CAR therapy was beneficial for the treatment of solid tumors. We plan to utilize this treatment in a canine spontaneous cancer preclinical model and translate into clinical trials for patients with GBM.

scholarly journals Fully human antibody VH domains to generate mono and bispecific CAR to target solid tumors

2021 ◽  
Vol 9 (4) ◽  
pp. e002173
Author(s):  
Guanmeng Wang ◽  
Xin Zhou ◽  
Giovanni Fucà ◽  
Elena Dukhovlinova ◽  
Peishun Shou ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Antigen Expression ◽  
Human Antibody ◽  
Single Chain ◽  
Car T Cells ◽  
Car T

BackgroundChimeric antigen receptor (CAR) T cells are effective in B-cell malignancies. However, heterogeneous antigen expression and antigen loss remain important limitations of targeted immunotherapy in solid tumors. Therefore, targeting multiple tumor-associated antigens simultaneously is expected to improve the outcome of CAR-T cell therapies. Due to the instability of single-chain variable fragments, it remains challenging to develop the simultaneous targeting of multiple antigens using traditional single-chain fragment variable (scFv)-based CARs.MethodsWe used Humabody VH domains derived from a transgenic mouse to obtain fully human prostate-specific membrane antigen (PSMA) VH and mesothelin (MSLN) VH sequences and redirect T cell with VH based-CAR. The antitumor activity and mode of action of PSMA VH and MSLN VH were evaluated in vitro and in vivo compared with the traditional scFv-based CARs.ResultsHuman VH domain-based CAR targeting PSMA and MSLN are stable and functional both in vitro and in vivo. VH modules in the bispecific format are capable of binding their specific target with similar affinity as their monovalent counterparts. Bispecific CARs generated by joining two human antibody VH domains can prevent tumor escape in tumor with heterogeneous antigen expression.ConclusionsFully human antibody VH domains can be used to generate functional CAR molecules, and redirected T cells elicit antitumoral responses in solid tumors at least as well as conventional scFv-based CARs. In addition, VH domains can be used to generate bispecific CAR-T cells to simultaneously target two different antigens expressed by tumor cells, and therefore, achieve better tumor control in solid tumors.

EXTH-59. GENERATION OF A THIRD GENERATION CAR T CELL THAT SIMULTANEOUSLY TARGETS WILDTYPE EGFR AND ITS MUTANT ISOFORM EGFRVIII FOR TREATMENT OF GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi176-vi176
Author(s):  
Daniel Wilkinson ◽  
Katherine Ryan ◽  
Vidyalakshmi Chandramohan ◽  
Daniel Landi ◽  
Darell Bigner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Solid Tumors ◽  
Third Generation ◽  
Single Chain ◽  
Car T Cells ◽  
Car T Cell ◽  
Hematological Cancers ◽  
Car T

Abstract Glioblastoma (GBM) is the most aggressive primary brain cancer with a median survival of less than 16 months. This dire prognosis signifies the urgent need for improved treatment options, such as immunotherapy. Chimeric antigen receptor (CAR) T cells have helped revolutionize immunotherapy, achieving considerable success in eliminating hematological cancers but generally failing to control solid tumors. One major hindrance to CAR T cell success in solid tumors is tumor heterogeneity. Tumor-associated or tumor-specific antigens (TAA or TSA, respectively) are rarely expressed by all malignant cells within a tumor. As a specific example in GBM, the most prevalent TSA, EGFRvIII, is present in just 30% of tumors, and then on only 30-50% of cells. Our pre-clinical and clinical experiences with CAR T cells reveal that tumors possessing as few as 5-10% EGFRvIII-negative cells will easily escape EGFRvIII-targeted CARs. Tumor cells that lack EGFRvIII expression often overexpress the wildtype isoform of EGFR (EGFRwt). Notably, EGFR is absent on normal brain. Therefore, a superior approach would be to simultaneously target EGFRvIII and EGFRwt, an approach that would bypass EGFR heterogeneity in EGFRwt/EGFRvIII-expressing tumors. Here, we generated a third generation CAR using the D2C7 single-chain variable fragment (scFv) targeting moiety that recognizes an epitope present in EGFRwt and EGFRvIII. Initial in vitro characterization of D2C7 CAR validated the specificity and function of D2C7 CAR, as it potently killed murine cell lines engineered to express either EGFRwt or EGFRvIII, but not a cell line expressing neither. Concomitant IFN-γ release supported these conclusions. Additionally, D2C7 CAR killed the human-derived GBM cell line U87 and vIII-transfected U87, U87vIII. Importantly, intracranially-administered D2C7 CAR significantly prolonged survival of mice bearing orthotopic U87vIII or U87/U87vIII heterogeneous tumors compared to mock-treated controls. Altogether, these data provide evidence that D2C7 CAR T cells represent a viable therapeutic option for EGFRwt/EGFRvIII heterogeneous tumors.

scholarly journals CAR T cells in solid tumors: challenges and opportunities

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Faroogh Marofi ◽  
Roza Motavalli ◽  
Vladimir A. Safonov ◽  
Lakshmi Thangavelu ◽  
Alexei Valerievich Yumashev ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Solid Tumor ◽  
Transmembrane Domain ◽  
Main Body ◽  
Single Chain ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Background CARs are simulated receptors containing an extracellular single-chain variable fragment (scFv), a transmembrane domain, as well as an intracellular region of immunoreceptor tyrosine-based activation motifs (ITAMs) in association with a co-stimulatory signal. Main body Chimeric antigen receptor (CAR) T cells are genetically engineered T cells to express a receptor for the recognition of the particular surface marker that has given rise to advances in the treatment of blood disorders. The CAR T cells obtain supra-physiological properties and conduct as “living drugs” presenting both immediate and steady effects after expression in T cells surface. But, their efficacy in solid tumor treatment has not yet been supported. The pivotal challenges in the field of solid tumor CAR T cell therapy can be summarized in three major parts: recognition, trafficking, and surviving in the tumor. On the other hand, the immunosuppressive tumor microenvironment (TME) interferes with T cell activity in terms of differentiation and exhaustion, and as a result of the combined use of CARs and checkpoint blockade, as well as the suppression of other inhibitor factors in the microenvironment, very promising results were obtained from the reduction of T cell exhaustion. Conclusion Nowadays, identifying and defeating the mechanisms associated with CAR T cell dysfunction is crucial to establish CAR T cells that can proliferate and lyse tumor cells severely. In this review, we discuss the CAR signaling and efficacy T in solid tumors and evaluate the most significant barriers in this process and describe the most novel therapeutic methods aiming to the acquirement of the promising therapeutic outcome in non-hematologic malignancies.

scholarly journals Adoptive Immunotherapy beyond CAR T-Cells

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 743
Author(s):  
Aleksei Titov ◽  
Ekaterina Zmievskaya ◽  
Irina Ganeeva ◽  
Aygul Valiullina ◽  
Alexey Petukhov ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Solid Tumors ◽  
Adoptive Immunotherapy ◽  
Γδ T Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Immunotherapy ◽  
Car T

Adoptive cell immunotherapy (ACT) is a vibrant field of cancer treatment that began progressive development in the 1980s. One of the most prominent and promising examples is chimeric antigen receptor (CAR) T-cell immunotherapy for the treatment of B-cell hematologic malignancies. Despite success in the treatment of B-cell lymphomas and leukemia, CAR T-cell therapy remains mostly ineffective for solid tumors. This is due to several reasons, such as the heterogeneity of the cellular composition in solid tumors, the need for directed migration and penetration of CAR T-cells against the pressure gradient in the tumor stroma, and the immunosuppressive microenvironment. To substantially improve the clinical efficacy of ACT against solid tumors, researchers might need to look closer into recent developments in the other branches of adoptive immunotherapy, both traditional and innovative. In this review, we describe the variety of adoptive cell therapies beyond CAR T-cell technology, i.e., exploitation of alternative cell sources with a high therapeutic potential against solid tumors (e.g., CAR M-cells) or aiming to be universal allogeneic (e.g., CAR NK-cells, γδ T-cells), tumor-infiltrating lymphocytes (TILs), and transgenic T-cell receptor (TCR) T-cell immunotherapies. In addition, we discuss the strategies for selection and validation of neoantigens to achieve efficiency and safety. We provide an overview of non-conventional TCRs and CARs, and address the problem of mispairing between the cognate and transgenic TCRs. Finally, we summarize existing and emerging approaches for manufacturing of the therapeutic cell products in traditional, semi-automated and fully automated Point-of-Care (PoC) systems.

scholarly journals 221 CRISPR screen identifies loss of IFNγR signaling and downstream adhesion as a resistance mechanism to CAR T-cell cytotoxicity in solid but not liquid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A234-A234
Author(s):  
Rebecca Larson ◽  
Michael Kann ◽  
Stefanie Bailey ◽  
Nicholas Haradhvala ◽  
Kai Stewart ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Solid Tumors ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundChimeric Antigen Receptor (CAR) therapy has had a transformative impact on the treatment of hematologic malignancies1–6 but success in solid tumors remains elusive. We hypothesized solid tumors have cell-intrinsic resistance mechanisms to CAR T-cell cytotoxicity.MethodsTo systematically identify resistance pathways, we conducted a genome-wide CRISPR knockout screen in glioblastoma cells, a disease where CAR T-cells have had limited efficacy.7 8 We utilized the glioblastoma cell line U87 and targeted endogenously expressed EGFR with CAR T-cells generated from 6 normal donors for the screen. We validated findings in vitro and in vivo across a variety of human tumors and CAR T-cell antigens.ResultsLoss of genes in the interferon gamma receptor (IFNγR) signaling pathway (IFNγR1, JAK1, JAK2) rendered U87 cells resistant to CAR T-cell killing in vitro. IFNγR1 knockout tumors also showed resistance to CAR T cell treatment in vivo in a second glioblastoma line U251 in an orthotopic model. This phenomenon was irrespective of CAR target as we also observed resistance with IL13Ralpha2 CAR T-cells. In addition, resistance to CAR T-cell cytotoxicity through loss of IFNγR1 applied more broadly to solid tumors as pancreatic cell lines targeted with either Mesothelin or EGFR CAR T-cells also showed resistance. However, loss of IFNγR signaling did not impact sensitivity of liquid tumor lines (leukemia, lymphoma or multiple myeloma) to CAR T-cells in vitro or in an orthotopic model of leukemia treated with CD19 CAR. We isolated the effects of decreased cytotoxicity of IFNγR1 knockout glioblastoma tumors to be cancer-cell intrinsic because CAR T-cells had no observable differences in proliferation, activation (CD69 and LFA-1), or degranulation (CD107a) when exposed to wildtype versus knockout tumors. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell adhesion pathways compared to wildtype glioblastoma cells after exposure to CAR T-cells. We found that loss of IFNγR1 reduced CAR T-cell binding avidity to glioblastoma.ConclusionsThe critical role of IFNγR signaling for susceptibility of solid tumors to CAR T-cells is surprising given that CAR T-cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumors, IFNγR signaling was required for sufficient adhesion of CAR T-cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumors differ in their interactions with CAR T-cells and suggests that enhancing T-cell/tumor interactions may yield improved responses in solid tumors.AcknowledgementsRCL was supported by T32 GM007306, T32 AI007529, and the Richard N. Cross Fund. ML was supported by T32 2T32CA071345-21A1. SRB was supported by T32CA009216-38. NJH was supported by the Landry Cancer Biology Fellowship. JJ is supported by a NIH F31 fellowship (1F31-MH117886). GG was partially funded by the Paul C. Zamecnik Chair in Oncology at the Massachusetts General Hospital Cancer Center and NIH R01CA 252940. MVM and this work is supported by the Damon Runyon Cancer Research Foundation, Stand Up to Cancer, NIH R01CA 252940, R01CA238268, and R01CA249062.ReferencesMaude SL, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378:439–448.Neelapu SS, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med 2017;377:2531–2544.Locke FL, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1–2 trial. The Lancet Oncology 2019;20:31–42.Schuster SJ, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med 2017;377:2545–2554.Wang M, et al. KTE-X19 CAR T-cell therapy in relapsed or refractory mantle-cell lymphoma. N Engl J Med 2020;382:1331–1342.Cohen AD, et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J Clin Invest 2019;129:2210–2221.Bagley SJ, et al. CAR T-cell therapy for glioblastoma: recent clinical advances and future challenges. Neuro-oncology 2018;20:1429–1438.Choi BD, et al. Engineering chimeric antigen receptor T cells to treat glioblastoma. J Target Ther Cancer 2017;6:22–25.Ethics ApprovalAll human samples were obtained with informed consent and following institutional guidelines under protocols approved by the Institutional Review Boards (IRBs) at the Massachusetts General Hospital (2016P001219). Animal work was performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) (2015N000218 and 2020N000114).

scholarly journals CAR T-cell therapy of solid tumors: promising approaches to modulating antitumor activity of CAR T cells

2019 ◽  
pp. 5-12 ◽  
Author(s):  
Ya.Yu. Kiseleva ◽  
A.M. Shishkin ◽  
A.V. Ivanov ◽  
T.M. Kulinich ◽  
V.K. Bozhenko
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Solid Tumors ◽  
Functional Activity ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Adoptive immunotherapy that makes use of genetically modified autologous T cells carrying a chimeric antigen receptor (CAR) with desired specificity is a promising approach to the treatment of advanced or relapsed solid tumors. However, there are a number of challenges facing the CAR T-cell therapy, including the ability of the tumor to silence the expression of target antigens in response to the selective pressure exerted by therapy and the dampening of the functional activity of CAR T cells by the immunosuppressive tumor microenvironment. This review discusses the existing gene-engineering approaches to the modification of CAR T-cell design for 1) creating universal “switchable” synthetic receptors capable of attacking a variety of target antigens; 2) enhancing the functional activity of CAR T cells in the immunosuppressive microenvironment of the tumor by silencing the expression of inhibiting receptors or by stimulating production of cytokines.

SynNotch CAR circuits enhance solid tumor recognition and promote persistent antitumor activity in mouse models

2021 ◽  
Vol 13 (591) ◽  
pp. eabd8836
Author(s):  
Axel Hyrenius-Wittsten ◽  
Yang Su ◽  
Minhee Park ◽  
Julie M. Garcia ◽  
Josef Alavi ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
T Cell ◽  
Antitumor Activity ◽  
Solid Tumors ◽  
Mouse Models ◽  
Solid Tumor ◽  
Car T Cells ◽  
Therapeutic Benefits ◽  
Car T

The first clinically approved engineered chimeric antigen receptor (CAR) T cell therapies are remarkably effective in a subset of hematological malignancies with few therapeutic options. Although these clinical successes have been exciting, CAR T cells have hit roadblocks in solid tumors that include the lack of highly tumor-specific antigens to target, opening up the possibility of life-threatening “on-target/off-tumor” toxicities, and problems with T cell entry into solid tumor and persistent activity in suppressive tumor microenvironments. Here, we improve the specificity and persistent antitumor activity of therapeutic T cells with synthetic Notch (synNotch) CAR circuits. We identify alkaline phosphatase placental-like 2 (ALPPL2) as a tumor-specific antigen expressed in a spectrum of solid tumors, including mesothelioma and ovarian cancer. ALPPL2 can act as a sole target for CAR therapy or be combined with tumor-associated antigens such as melanoma cell adhesion molecule (MCAM), mesothelin, or human epidermal growth factor receptor 2 (HER2) in synNotch CAR combinatorial antigen circuits. SynNotch CAR T cells display superior control of tumor burden when compared to T cells constitutively expressing a CAR targeting the same antigens in mouse models of human mesothelioma and ovarian cancer. This was achieved by preventing CAR-mediated tonic signaling through synNotch-controlled expression, allowing T cells to maintain a long-lived memory and non-exhausted phenotype. Collectively, we establish ALPPL2 as a clinically viable cell therapy target for multiple solid tumors and demonstrate the multifaceted therapeutic benefits of synNotch CAR T cells.

scholarly journals Combination Immunotherapy with CAR T Cells and Checkpoint Blockade for the Treatment of Solid Tumors

Cancer Cell ◽  
2019 ◽  
Vol 36 (5) ◽  
pp. 471-482 ◽  
Author(s):  
Rachel Grosser ◽  
Leonid Cherkassky ◽  
Navin Chintala ◽  
Prasad S. Adusumilli
Keyword(s):  
T Cells ◽  
Solid Tumors ◽  
Checkpoint Blockade ◽  
Combination Immunotherapy ◽  
Car T Cells ◽  
Car T

scholarly journals Advancing CAR T-Cell Therapy for Solid Tumors: Lessons Learned from Lymphoma Treatment

Cancers ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 125 ◽  
Author(s):  
Aleksei Titov ◽  
Aygul Valiullina ◽  
Ekaterina Zmievskaya ◽  
Ekaterina Zaikova ◽  
Alexey Petukhov ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Solid Tumors ◽  
B Cell Lymphoma ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Chimeric antigen receptor (CAR) immunotherapy is one of the most promising modern approaches for the treatment of cancer. To date only two CAR T-cell products, Kymriah® and Yescarta®, have been approved by the Food and Drug Administration (FDA) for the treatment of lymphoblastic leukemia and B-cell lymphoma. Administration of CAR T-cells to control solid tumors has long been envisaged as one of the most difficult therapeutic tasks. The first two clinical trials conducted in sarcoma and neuroblastoma patients showed clinical benefits of CAR T-cells, yet multiple obstacles still hold us back from having accessible and efficient therapy. Why did such an effective treatment for relapsed and refractory hematological malignancies demonstrate only relatively modest efficiency in the context of solid tumors? Is it due to the lucky selection of the “magic” CD19 antigen, which might be one of a kind? Or do lymphomas lack the immunosuppressive features of solid tumors? Here we review the existing knowledge in the field of CAR T-cell therapy and address the heterogeneity of solid tumors and their diverse strategies of immunoevasion. We also provide an insight into prospective developments of CAR T-cell technologies against solid tumors.

A Novel and Highly Potent CAR T Cell Drug Product for Treatment of BCMA-Expressing Hematological Malignances

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3094-3094 ◽  
Author(s):  
Alena A. Chekmasova ◽  
Holly M. Horton ◽  
Tracy E. Garrett ◽  
John W. Evans ◽  
Johanna Griecci ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Drug Product ◽  
Single Chain ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership

Abstract Recently, B cell maturation antigen (BCMA) expression has been proposed as a marker for identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM and some lymphoma tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Targeting BCMA maybe a therapeutic option for treatment of patients with MM and some lymphomas. We are developing a chimeric antigen receptor (CAR)-based therapy for the treatment of BCMA-expressing MM. Our anti-BCMA CAR consists of an extracellular single chain variable fragment (scFv) antigen recognition domain derived from an antibody specific to BCMA, fused to CD137 (4-1BB) co-stimulatory and CD3zeta chain signaling domains. Selection of our development candidate was based on the screening of four distinct anti-BCMA CARs (BCMA01-04) each comprised of unique single chain variable fragments. One candidate, BCMA02 (drug product name bb2121) was selected for further studies based on the robust frequency of CAR-positive cells, increased surface expression of the CAR molecule, and superior in vitro cytokine release and cytolytic activity against the MM cell lines. In addition to displaying specific activity against MM (U226-B1, RPMI-8226 and H929) and plasmacytoma (H929) cell lines, bb2121 was demonstrated to react to lymphoma cell lines, including Burkitt's (Raji, Daudi, Ramos), chronic lymphocytic leukemia (Mec-1), diffuse large B cell (Toledo), and a Mantle cell lymphoma (JeKo-1). Based on receptor density quantification, bb2121 can recognize tumor cells expressing less than 1000 BCMA molecules per cell. The in vivo pharmacology of bb2121 was studied in NSG mouse models of human MM and Burkitt's lymphoma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells. After 18 days, mice received a single intravenous (IV) administration of vehicle or anti-CD19Δ (negative control, anti-CD19 CAR lacking signaling domain) or anti-BCMA CAR T cells, or repeated IV administration of bortezomib (Velcade®; 1 mg/kg twice weekly for 4 weeks). Bortezomib, which is a standard of care for MM, induced only transient reductions in tumor size and was associated with toxicity, as indicated by substantial weight loss during dosing. The vehicle and anti-CD19Δ CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors, increased body weights, and 100% survival. Flow cytometry and immunohistochemical analysis of bb2121 T cells demonstrated trafficking of CAR+ T cells to the tumors (by Day 5) followed by significant expansion of anti-BCMA CAR+ T cells within the tumor and peripheral blood (Days 8-10), accompanied by tumor clearance and subsequent reductions in circulating CAR+ T cell numbers (Days 22-29). To further test the potency of bb2121, we used the CD19+ Daudi cell line, which has a low level of BCMA expression detectable by flow cytometry and receptor quantification analysis, but is negative by immunohistochemistry. NSG mice were injected IV with Daudi cells and allowed to accumulate a large systemic tumor burden before being treated with CAR+ T cells. Treatment with vehicle or anti-CD19Δ CAR T cells failed to prevent tumor growth. In contrast, anti-CD19 CAR T cells and anti-BCMA bb2121 demonstrated tumor clearance. Adoptive T cell immunotherapy approaches designed to modify a patient's own lymphocytes to target the BCMA antigen have clear indications as a possible therapy for MM and could be an alternative method for treatment of other chemotherapy-refractory B-cell malignancies. Based on these results, we will be initiating a phase I clinical trial of bb2121 for the treatment of patients with MM. Disclosures Chekmasova: bluebird bio, Inc: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Evans:bluebird bio, Inc: Employment, Equity Ownership. Griecci:bluebird bio, Inc: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Seidel:bluebird bio, Inc: Employment, Equity Ownership. Ryu:bluebird bio, Inc: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.

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