scholarly journals Efficient Gene Transduction and Reprogramming of Hematopoietic Cells Including T-Cells By Using a Non-Integrating Measles Virus Vector

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3494-3494
Author(s):  
Jiyuan Liao ◽  
Yasushi Soda ◽  
Ai Sugawara ◽  
Yoshie Miura ◽  
Takafumi Hiramoto ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Gene Transfer ◽  
Research Funding ◽  
Measles Virus ◽  
Gene Transduction ◽  
Efficient Gene ◽  
Car T ◽  
Ipsc Generation ◽  
Equity Ownership

Abstract By the ectopic expression of reprogramming genes OCT, KLF4, SOX2 and MYC (OKSM), somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs). Human iPSCs are considered a promising cell source to provide an import tool for the basic investigation and the advanced medicine including gene therapy and regenerative medicine. To establish iPSCs, integration-free Sendai virus (SV) vectors have been most widely used do far, but transduction and reprogramming of T cells without stimulation is still very challenging. On the other hand, a great success of chimeric antigen receptor T cell (CAR-T) therapies highlighted the importance of anti-cancer immunity for the cancer treatment. Particularly, many refractory patients with acute lymphoblastic leukemia and B-cell lymphoma were successfully treated with CD19-CAR-T therapies, however, some patients died before receiving the treatment due to long preparation time of CAR-Ts. Therefore, rapid production systems of CAR-Ts are desired, and for this purpose, efficient and safe gene transduction systems to T cells should be developed. In this study, we developed a new non-integrating measles virus (MV) vector-based delivery system with F deletion to eliminate cell membrane fusion-associated cytotoxicity. MV vectors transduced genes through MV receptors including CD46 and signaling lymphocyte activation molecule (CD150/SLAM). First, we examined transduction efficiencies of MV vectors and SV vectors in hematopoietic cells by using GFP expression vectors (MV-Gs and SV-Gs). Compared to SV-Gs, our MV-Gs allowed more efficient gene transfer into most hematopoietic cell type including T (3-fold) and B cells (7-fold) (Fig. 1). Furthermore, at the same multiplicity of infection (MOI) of viral transduction, MV-Gs induced less apoptosis in T cell subset compared to SV-Gs (Fig. 2) due to the slower kinetics of viral RNA amplification in the transduced cells 24 h ,48 h and 72 h post transduction. Those results encouraged us to examined if MV vectors are more potent than SV vectors in iPSC generation from unstimulated T cells. To address this question, we developed MV vectors harboring four reprogramming genes (MV-OKSMGs) and compared with SV vectors harboring these genes (SV-OKSMGs). As expected, with the MV-OKSMGs, we could generate high-quality iPSCs with the similar morphology, pluripotency markers, karyotype and differentiation capacity as human embryonic stem cells. Upon the less cytotoxicity, iPSC generation efficiency of MV-OKSMGs was much higher than that of SV-OKSMGs for unstimulated T cells (0.47 ± 0.25% vs 0.008 ± 0.009%). Considering the safe history of MV vaccine, carrying capabilities of multiple genes, more flexible receptors and higher transduction efficiency for resting T cells, our exclusive MV vector would be a potential gene transfer system for iPSC generation and lymphocyte-based-immunotherapies such as CAR-T therapies. Disclosures Liao: neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding. Soda:Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Sugawara:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Miura:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Tahara:TAKARA BIO, INC.: Research Funding. Takishima:neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding. Hirose:TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding. Hijikata:Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Miyamoto:Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; neopharma Japan Co. Ltd: Research Funding. Takeda:TAKARA BIO, INC.: Research Funding. Tani:neopharma Japan Co. Ltd: Research Funding; Oncolys BioPharma Inc.: Equity Ownership; SymBio Pharmaceuticals Limited: Equity Ownership; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding.

scholarly journals F-Deletion Non-Integrating Measles Virus Vector: A Promising Tool for T-Cell Engineering and Naive iPSCs Generation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5750-5750
Author(s):  
Jiyuan Liao ◽  
Yasushi Soda ◽  
Ai Sugawara ◽  
Yoshie Miura ◽  
Takafumi Hiramoto ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Measles Virus ◽  
Transduction Efficiency ◽  
Promising Tool ◽  
Primed State ◽  
Equity Ownership ◽  
T Cell Engineering

Although great successes of chimeric antigen receptor T-cell (CAR-T) therapy highlighted the importance of anti-cancer immunity for cancer treatment, there are still some problems remained, i.e., long preparation time, extremely high cost and potential risk by insertional mutagenesis. To developmore rapid and safer T-cell engineering systems than current procedures using retrovirus or lentiviral vectors, we have long been focusing on measles virus as a new vector because of its high infectivity to T cells including resting state and rapid gene expression without chromosome integration. Presently, Sendai virus vectors (SVs), which is also a Paramyxoviridaevirus-based vector, are widely used for gene delivery and induced pluripotent stem cells (iPSCs), but the transduction to undifferentiated T cells (UTs) is a big challenge for SVs. We, therefore, compared the gene transduction efficiency between our measles vector (MVs) and SVs. We also compared iPSCs generation efficiencies between MVs and SVs. We engineered our non-replicating and non-integrating measles virus vectors (MV)with F deletion to eliminate cell membrane fusion-associated cytotoxicity.Based on the original property of measles virus,our recombinant MVsallowed more efficient gene transduction to various hematopoietic cells including UTs and B cells than SVs. Importantly, MVs induced less apoptosis compared withSVs due to their slower amplification of viral RNA in transduced cells. Moreover, we could establish iPSCs from UTs with MVs harboring reprogramming genes 50 times more efficiently than SVs harboring the same reprogramming genes. MV-induced iPSCs derived from CD3+T cells (MV-TiPSCs) were similar to regular human pluripotent stem cells (hPSCs: embryonic stem cells and iPSCs), which are in primed state, in morphology, the expressions of pluripotent markers and the ability to differentiate into three germ layers. On the other hand, without using naive induction culture condition, MV-induced iPSCs derived from CD34+hematopoietic progenitor cells (MV-HPC-iPSCs) presented a dome shape and showed a transcriptome profile close to naive iPSCs. To further confirm naive-like properties of MV-HPC-iPSCs, we evaluated gene expression patterns of these cells for 22 common genes most differently expressed in naive and primed hPSCs reported in previous reports (Fig.1).As expected, MV-HPC-iPSCs were clustered in naive hPSCs group while MV-HPC-iPSCs after culturing in primed induction condition showed primed-like features (Fig.2). Moreover, whole genome bisulfite sequencing analysis showed that MV-HPC-iPSCs had lower methylation than primed MV-HPC-iPSCs. These results strongly suggested that MV could induce naive-like iPSCs directly, and primed induction culture changed the cells to primed state with increasing genomic methylation. Considering the very safe history of MV vaccine, the capabilities of simultaneous expressions of multiple genes and the high transduction efficiency for hematopoietic cells including UTs, our MVs will be useful to directly induce naive state iPSCs, and be a promising tool for developing new T-cell immunotherapies. Disclosures Liao: TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding. Soda:TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding. Miura:Neoprecision therapeutics: Research Funding. Tahara:TAKARA BIO, INC.: Research Funding. Miyamoto:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Takeda:TAKARA BIO, INC.: Research Funding. Tani:Oncolys BioPharma Inc.: Equity Ownership; SymBio Pharmaceuticals Limited: Equity Ownership; TAKARA BIO, INC.: Research Funding; Neoprecision therapeutics: Equity Ownership, Research Funding; neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Equity Ownership.

scholarly journals Combination of NKTR-255, a Polymer Conjugated Human IL-15, with CD19 CAR T Cell Immunotherapy in a Preclinical Lymphoma Model

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2866-2866 ◽  
Author(s):  
Cassie Chou ◽  
Simon Fraessle ◽  
Rachel Steinmetz ◽  
Reed M. Hawkins ◽  
Tinh-Doan Phi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Board Member ◽  
Ad Hoc ◽  
Advisory Board ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Background CD19 CAR T immunotherapy has been successful in achieving durable remissions in some patients with relapsed/refractory B cell lymphomas, but disease progression and loss of CAR T cell persistence remains problematic. Interleukin 15 (IL-15) is known to support T cell proliferation and survival, and therefore may enhance CAR T cell efficacy, however, utilizing native IL-15 is challenging due to its short half-life and poor tolerability in the clinical setting. NKTR-255 is a polymer-conjugated IL-15 that retains binding affinity to IL15Rα and exhibits reduced clearance, providing sustained pharmacodynamic responses. We investigated the effects of NKTR-255 on human CD19 CAR T cells both in vitro and in an in vivo xenogeneic B cell lymphoma model and found improved survival of lymphoma bearing mice receiving NKTR-255 and CAR T cells compared to CAR T cells alone. Here, we extend upon these findings to further characterize CAR T cells in vivo and examine potential mechanisms underlying improved anti-tumor efficacy. Methods CD19 CAR T cells incorporating 4-1BB co-stimulation were generated from CD8 and CD4 T cells isolated from healthy donors. For in vitro studies, CAR T cells were incubated with NKTR-255 or native IL-15 with and without CD19 antigen. STAT5 phosphorylation, CAR T cell phenotype and CFSE dilution were assessed by flow cytometry and cytokine production by Luminex. For in vivo studies, NSG mice received 5x105 Raji lymphoma cells IV on day (D)-7 and a subtherapeutic dose (0.8x106) of CAR T cells (1:1 CD4:CD8) on D0. To determine optimal start date of NKTR-255, mice were treated weekly starting on D-1, 7, or 14 post CAR T cell infusion. Tumors were assessed by bioluminescence imaging. Tumor-free mice were re-challenged with Raji cells. For necropsy studies mice received NKTR-255 every 7 days following CAR T cell infusion and were euthanized at various timepoints post CAR T cell infusion. Results Treatment of CD8 and CD4 CAR T cells in vitro with NKTR-255 resulted in dose dependent STAT5 phosphorylation and antigen independent proliferation. Co-culture of CD8 CAR T cells with CD19 positive targets and NKTR-255 led to enhanced proliferation, expansion and TNFα and IFNγ production, particularly at lower effector to target ratios. Further studies showed that treatment of CD8 CAR T cells with NKTR-255 led to decreased expression of activated caspase 3 and increased expression of bcl-2. In Raji lymphoma bearing NSG mice, administration of NKTR-255 in combination with CAR T cells increased peak CAR T cell numbers, Ki-67 expression and persistence in the bone marrow compared to mice receiving CAR T cells alone. There was a higher percentage of EMRA like (CD45RA+CCR7-) CD4 and CD8 CAR T cells in NKTR-255 treated mice compared to mice treated with CAR T cells alone and persistent CAR T cells in mice treated with NKTR-255 were able to reject re-challenge of Raji tumor cells. Additionally, starting NKTR-255 on D7 post T cell infusion resulted in superior tumor control and survival compared to starting NKTR-255 on D-1 or D14. Conclusion Administration of NKTR-255 in combination with CD19 CAR T cells leads to improved anti-tumor efficacy making NKTR-255 an attractive candidate for enhancing CAR T cell therapy in the clinic. Disclosures Chou: Nektar Therapeutics: Other: Travel grant. Fraessle:Technical University of Munich: Patents & Royalties. Busch:Juno Therapeutics/Celgene: Consultancy, Equity Ownership, Research Funding; Kite Pharma: Equity Ownership; Technical University of Munich: Patents & Royalties. Miyazaki:Nektar Therapeutics: Employment, Equity Ownership. Marcondes:Nektar Therapeutics: Employment, Equity Ownership. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy; Lyell Immunopharma: Equity Ownership, Patents & Royalties, Research Funding. Turtle:Allogene: Other: Ad hoc advisory board member; Novartis: Other: Ad hoc advisory board member; Humanigen: Other: Ad hoc advisory board member; Nektar Therapeutics: Other: Ad hoc advisory board member, Research Funding; Caribou Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; T-CURX: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Patents & Royalties: Co-inventor with staff from Juno Therapeutics; pending, Research Funding; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Other: Ad hoc advisory board member.

scholarly journals NOTCH and CAR Signaling Control T Cell Lineage Commitment from Pluripotent Stem Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-30
Author(s):  
Sjoukje van der Stegen ◽  
Pieter Lindenbergh ◽  
Roseanna Petrovic ◽  
Benjamin Whitlock ◽  
Raedun Clarke ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Cell Lineage ◽  
Lineage Commitment ◽  
Publicly Traded ◽  
Car T Cell ◽  
Car T ◽  
Single Positive

Chimeric Antigen Receptor (CAR) T cells are a new treatment paradigm for relapsed/refractory hematopoietic malignancies. However, their autologous nature imposes manufacturing constraints that can delay CAR T cell availability and increase their cost. We previously established proof of principle that αβ T cell-derived induced pluripotent stem cells (TiPSCs) can provide a self-renewing source for in vitro CAR T cell production (Themeli, Nat Biotechnol, 2013). The use of cloned TiPSC further enhances the feasibility of verifying genome integrity of the genetically engineered stem cells and should in principle yield highly homogenous cell products. Using αβ T cell-derived TiPSCs transduced with a well-defined CD19-specific CAR (1928z; Park, NEJM, 2018), we previously demonstrated that TiPSCs can be differentiated into CAR T cells. These T cells retained their endogenous T cell receptor (TCR) and also displayed characteristics of innate lymphoid cells. We have now examined how the timing of CAR expression as well as the CAR signaling strength influence T cell lineage commitment, enabling better control towards αβ T cell lineage commitment. αβ T cell lineage development depends in part on a precisely orchestrated interactions between NOTCH and (pre)TCR signaling, the timing and strength of which are crucial for αβ lineage commitment. Because TiPSCs harbor rearranged TCRα and TCRβ genes, mature TCR expression occurs earlier than if it required VDJ recombination, skewing differentiation towards acquiring innate features including CD4-CD8- double-negative or CD8αα single-positive phenotypes. We show that providing strong NOTCH stimulation counteracts the effects of early antigen receptor expression, facilitating CD4+CD8αβ+ double positive (DP) formation. We hypothesized that CAR signaling in the absence of ligand binding (tonic signaling) may mimic a TCR signal, the strength and timing of which could re-direct lineage commitment. We therefore investigated CARs providing different levels of signaling strength and the impact of delaying the onset of CAR expression. Tonic CAR signaling was measured in peripheral blood T cells expressing 1928z or 1928z-1XX, a construct in which the second and third ITAM in the CD3ζ domain have been mutated to be non-functional (Feucht, Nat Med, 2019), following either retroviral transduction (SFG vector) orTRAC-targeted cDNA integration, placing CAR expression under the transcriptional control of the TCRα promoter (Eyquem, Nature, 2017). CAR signaling in the absence of antigen exposure, measured by phosphorylation of ITAM3, ERK1/2 and ZAP70, was reduced by bothTRAC-targeting and reduction of functional ITAMs, with additive effects when combined inTRAC-1928z-1XX. Three of these engineering strategies (virally expressed 1928z,TRAC-1928z andTRAC-1928z-1XX) were evaluated in the context of TiPSC-derived T cell differentiation. Virally expressed 1928z (resulting in constitutive CAR expression throughout differentiation) resulted in the predominant generation of innate-like CD8αα T cells, associated with the absence of early T cell lineage markers such as CD5, CD2 and CD1a. Delayed expression of 1928z throughTRACtargeting resulted in increased CD5, CD2 and CD1a, but did not yield any more CD4+CD8αβ+ DP cells. In TiPSC expressingTRAClocus-encoded 1928z-1XX, a greater DP population emerged, from which CD8αβ single-positive T cells could be induced. Phenotypic analyses of clonal TRAC-1928z-1XX TiPSC lines further establish the interplay between CAR and NOTCH1 in determining αβ lineage commitment. Together these data show that early TCR and CAR expression skew T cell lineage commitment towards an innate-like T cell fate, which can be overcome by controlling the strength and timing of NOTCH, TCR and CAR signaling. These studies pave the way for the predetermined generation of a variety of CAR T cell types endowed with different functional attributes. Disclosures Whitlock: Fate Therapeutics Inc.:Current Employment, Current equity holder in publicly-traded company.Clarke:Fate Therapeutics Inc.:Current Employment, Current equity holder in publicly-traded company.Valamehr:Fate Therapeutics, Inc:Current Employment, Current equity holder in publicly-traded company.Riviere:Juno Therapeutics:Other: Ownership interest, Research Funding;Takeda:Research Funding;Fate Therapeutics Inc.:Consultancy, Other: Ownership interest , Research Funding;FloDesign Sonics:Consultancy, Other: Ownership interest;Atara:Research Funding.Sadelain:Atara:Patents & Royalties, Research Funding;Fate Therapeutics:Patents & Royalties, Research Funding;Mnemo:Patents & Royalties;Takeda:Patents & Royalties, Research Funding;Minerva:Other: Biotechnologies , Patents & Royalties.

scholarly journals Durable Clinical Responses in Heavily Pretreated Patients with Relapsed/Refractory Multiple Myeloma: Updated Results from a Multicenter Study of bb2121 Anti-Bcma CAR T Cell Therapy

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 740-740 ◽  
Author(s):  
Jesus G. Berdeja ◽  
Yi Lin ◽  
Noopur Raje ◽  
Nikhil Munshi ◽  
David Siegel ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T ◽  
Clinical Responses ◽  
Equity Ownership ◽  
Dose Levels

Abstract Introduction: Chimeric antigen receptor (CAR) T cell therapies have demonstrated robust and sustained clinical responses in several hematologic malignancies. Data suggest that achieving acceptable benefit:risk profiles depends on several factors, including the specificity of the antigen target and characteristics of the CAR itself, including on-target, off-tumor activity.To test the safety and efficacy of CAR T cells in relapsed and/or refractory multiple myeloma (RRMM), we have designed a second-generation CAR construct targeting B cell maturation antigen (BCMA) to redirect T cells to MM cells. BCMA is a member of the tumor necrosis factor superfamily that is expressed primarily by malignant myeloma cells, plasma cells, and some mature B cells. bb2121 consists of autologous T cells transduced with a lentiviral vector encoding a novel CAR incorporating an anti-BCMA scFv, a 4-1BB costimulatory motif and a CD3-zeta T cell activation domain. Methods: CRB-401 (NCT02658929) is a multi-center phase 1 dose escalation trial of bb2121 in patients with RRMM who have received ≥ 3 prior regimens, including a proteasome inhibitor and an immunomodulatory agent, or are double-refractory, and have ≥ 50% BCMA expression on malignant cells. Peripheral blood mononuclear cells are collected via leukapheresis and shipped to a central facility for transduction, expansion, and release testing prior to being returned to the site for infusion. Patients undergo lymphodepletion with fludarabine (30 mg/m2) and cyclophosphamide (300 mg/m2) daily for 3 days then receive 1 infusion of bb2121. The study follows a standard 3+3 design with planned dose levels of 50, 150, 450, 800, and 1,200 x 106 CAR+ T cells. The primary outcome measure is incidence of adverse events (AEs), including dose-limiting toxicities (DLTs). Additional outcome measures were quality and duration of clinical response assessed according to the IMWG Uniform Response Criteria for Multiple Myeloma, evaluation of minimal residual disease (MRD), overall and progression-free survival, quantification of bb2121 in blood, and quantification of circulating soluble BCMA over time. Results: Asof May 4, 2017, 21 patients (median 58 [37 to 74] years old) with a median of 5 (1 to 16) years since MM diagnosis, had been infused with bb2121, and 18 patients were evaluable for initial (1-month) clinical response. Patients had a median of 7 prior lines of therapy (range 3 to 14), all with prior autologous stem cell transplant; 67% had high-risk cytogenetics. Fifteen of 21 (71%) had prior exposure to, and 6 of 21 (29%) were refractory to 5 prior therapies (Bort/Len/Car/Pom/Dara). Median follow-up after bb2121 infusion was 15.4 weeks (range 1.4 to 54.4 weeks). As of data cut-off, no DLTs and no treatment-emergent Grade 3 or higher neurotoxicities similar to those reported in other CAR T clinical studies had been observed. Cytokine release syndrome (CRS), primarily Grade 1 or 2, was reported in 15 of 21 (71%) patients: 2 patients had Grade 3 CRS that resolved in 24 hours and 4 patients received tocilizumab, 1 with steroids, to manage CRS. CRS was more common in the higher dose groups but did not appear related to tumor burden. One death on study, due to cardiopulmonary arrest more than 4 months after bb2121 infusion in a patient with an extensive cardiac history, was observed while the patient was in sCR and was assessed as unrelated to bb2121. The overall response rate (ORR) was 89% and increased to 100% for patients treated with doses of 150 x 106 CAR+ T cells or higher. No patients treated with doses of 150 x 106 CAR+ T cells or higher had disease progression, with time since bb2121 between 8 and 54 weeks (Table 1). MRD negative results were obtained in all 4 patients evaluable for analysis. CAR+ T cell expansion has been demonstrated consistently and 3 of 5 patients evaluable for CAR+ cells at 6 months had detectable vector copies. A further 5 months of follow up on reported results and initial data from additional patients will be presented. Conclusions: bb2121 shows promising efficacy at dose levels above 50 x 106 CAR+ T cells, with manageable CRS and no DLTs to date. ORR was 100% at these dose levels with 8 ongoing clinical responses at 6 months and 1 patient demonstrating a sustained response beyond one year. These initial data support the potential of CAR T therapy with bb2121 as a new treatment paradigm in RRMM. CT.gov study NCT02658929, sponsored by bluebird bio and Celgene Disclosures Berdeja: Teva: Research Funding; Janssen: Research Funding; Novartis: Research Funding; Abbvie: Research Funding; Celgene: Research Funding; BMS: Research Funding; Takeda: Research Funding; Vivolux: Research Funding; Amgen: Research Funding; Constellation: Research Funding; Bluebird: Research Funding; Curis: Research Funding. Siegel: Celgene, Takeda, Amgen Inc, Novartis and BMS: Consultancy, Speakers Bureau; Merck: Consultancy. Jagannath: MMRF: Speakers Bureau; Bristol-Meyers Squibb: Consultancy; Merck: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Medicom: Speakers Bureau. Turka: bluebird bio: Employment, Equity Ownership. Lam: bluebird bio: Employment, Equity Ownership. Hege: Celgene Corporation: Employment, Equity Ownership. Morgan: bluebird bio: Employment, Equity Ownership, Patents & Royalties. Quigley: bluebird bio: Employment, Equity Ownership, Patents & Royalties. Kochenderfer: Bluebird bio: Research Funding; N/A: Patents & Royalties: I have multiple patents in the CAR field.; Kite Pharma: Research Funding.

scholarly journals A Phase 1 Study with Point-of-Care Manufacturing of Dual Targeted, Tandem Anti-CD19, Anti-CD20 Chimeric Antigen Receptor Modified T (CAR-T) Cells for Relapsed, Refractory, Non-Hodgkin Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4193-4193 ◽  
Author(s):  
Nirav N Shah ◽  
Fenlu Zhu ◽  
Carolyn Taylor ◽  
Dina Schneider ◽  
Winfried Krueger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Research Funding ◽  
Phase 1 ◽  
Third Party ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Abstract Background: CAR-T cell therapy directed against the CD19 antigen is a breakthrough treatment for patients (pts) with relapsed/refractory (R/R) B-cell NHL. Despite impressive outcomes, not all pts respond and many that respond still relapse. Affordability and accessibility are further considerations that limit current commercial models of CAR-T products. Commercial CAR-T manufacturing is complex, time consuming, and expensive with a supply chain starting at the treating center with apheresis of mononuclear cells, cryopreservation, and shipping to and from a centralized third-party manufacturing site. We addressed these limitations in a Phase 1 clinical trial evaluating a first-in-human bispecific tandem CAR-T cell directed against both CD19 and CD20 (CAR-20.19-T) antigens for pts with R/R B-cell NHL. Through dual targeting we hope to improve response rates and durability of response while limiting antigen escape. We eliminated third party shipping logistics utilizing the CliniMACS Prodigy, a compact tabletop device that allows for automated manufacturing of CAR-T cells within a GMP compliant environment within the hospital. Most materials and reagents used to produce the CAR-T cell product were single-sourced from the device manufacturer. Methods: Phase 1 (NCT03019055), single center, dose escalation + expansion study to demonstrate feasibility and safety of locally manufactured second generation 41BB + CD3z CAR-20.19-T cells via the CliniMACS Prodigy. Feasibility was measured by ability to generate a target CAR-20.19-T cell dose for a minimum of 75% of subjects. Safety was assessed by the presence of dose limiting toxicities (DLTs) through 28 days post-infusion. Dose was escalated in a 3+3 fashion with a starting dose of 2.5 x 10^5 cells/kg, a target DLT rate <33%, and a goal treatment dose of 2.5 x 10^6 cells/kg. Adults with R/R Diffuse Large B-cell Lymphoma (DLBCL), Follicular Lymphoma (FL), Mantle Cell Lymphoma (MCL) or Chronic Lymphocytic Leukemia (CLL) were eligible. CAR-T production was set for a 14-day manufacturing process. Day 8 in-process testing was performed to ensure quality and suitability of CAR-T cells for a potential fresh infusion. On Day 10, pts eligible for a fresh CAR-T infusion initiated lymphodepletion (LDP) chemotherapy with fludarabine 30 mg/m2 x 3 days and cyclophosphamide 500 mg/m2 x 1 day, and cells were administered after harvest on Day 14. Pts ineligible for fresh infusion received cryopreserved product and LDP was delayed accordingly. Results: 6 pts have been enrolled and treated with CAR-20.19-T cells: 3 pts at 2.5 x 10^5 cells/kg and 3 pts at 7.5 x 10^5 cells/kg. Median age was 53 years (48-62). Underlying disease was MCL in 3 pts, DLBCL in 2 pts, and CLL in 1 patient. Baseline data and prior treatments are listed in Table 1. CAR-T production was successful in all runs and all pts received their target dose. Three pts received fresh CAR-T cells and 3 pts received CAR-T cells after cryopreservation. To date there are no DLTs to report. No cases of Grade 3/4 cytokine release syndrome (CRS) or neurotoxicity (NTX) were observed. One patient had Grade 2 CRS and Grade 2 NTX requiring intervention. The other had self-limited Grade 1 CRS and Grade 1 NTX. Median time to development of CRS was Day +11 post-infusion. All pts had neutrophil recovery (ANC>0.5 K/µL) by Day 28. Response at Day 28 (Table 2) is as follows: 2/6 pts achieved a complete response (CR), 2/6 achieved a partial response (PR), and 2/6 had progressive disease (PD). One subject with a PR subsequently progressed at Day 90. The 3 pts who did progress all underwent a repeat biopsy, and all retained either CD19 or CD20 positivity. Pts are currently being enrolled at the target dose (2.5 x 10^6 cells/kg) and updated results will be provided at ASH. Conclusions: Dual targeted anti-CD19 and anti-CD20 CAR-T cells were successfully produced for all pts demonstrating the feasibility of a point-of-care manufacturing process via the CliniMACS Prodigy device. With no DLTs or Grade 3-4 CRS or NTX to report, and 2/6 heavily pre-treated pts remaining in CR at 3 and 9 months respectively our approach represents a feasible and promising alternative to existing CAR-T models and costs. Down-regulation of both target antigens was not identified in any patient following CAR-T infusion, and in-process studies suggest that a shorter manufacturing timeline is appropriate for future trials (10 days). Disclosures Shah: Juno Pharmaceuticals: Honoraria; Lentigen Technology: Research Funding; Oncosec: Equity Ownership; Miltenyi: Other: Travel funding, Research Funding; Geron: Equity Ownership; Exelexis: Equity Ownership. Zhu:Lentigen Technology Inc., A Miltenyi Biotec Company: Research Funding. Schneider:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Krueger:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Worden:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Hamadani:Sanofi Genzyme: Research Funding, Speakers Bureau; Merck: Research Funding; Janssen: Consultancy; MedImmune: Consultancy, Research Funding; Cellerant: Consultancy; Celgene Corporation: Consultancy; Takeda: Research Funding; Ostuka: Research Funding; ADC Therapeutics: Research Funding. Johnson:Miltenyi: Research Funding. Dropulic:Lentigen, A Miltenyi Biotec company: Employment. Orentas:Lentigen Technology Inc., A Miltenyi Biotec Company: Other: Prior Employment. Hari:Takeda: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Kite Pharma: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Spectrum: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Amgen Inc.: Research Funding; Sanofi: Honoraria, Research Funding.

scholarly journals Development and Evaluation of a Human Single Chain Variable Fragment (scFv) Derived Bcma Targeted CAR T Cell Vector Leads to a High Objective Response Rate in Patients with Advanced MM

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 742-742 ◽  
Author(s):  
Eric L Smith ◽  
Sham Mailankody ◽  
Arnab Ghosh ◽  
Reed Masakayan ◽  
Mette Staehr ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Employment Equity ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Abstract Patients with relapsed/refractory MM (RRMM) rarely obtain durable remissions with available therapies. Clinical use of BCMA targeted CAR T cell therapy was first reported in 12/2015 for RRMM, and based on small numbers, preliminary results appear promising. Given that host immune anti-murine CAR responses have limited the efficacy of repeat dosing (Turtle C. Sci Trans Med 2016), our goal was to develop a human BCMA targeted CAR T cell vector for clinical translation. We screened a human B cell derived scFv phage display library containing 6x1010 scFvs with BCMA expressing NIH 3T3 cells, and validated results on human MM cell lines. 57 unique and diverse BCMA specific scFvs were identified containing light and heavy chain CDR's each covering 6 subfamilies, with HCDR3 length ranges from 5-18 amino acids. 17 scFvs met stringent specificity criteria, and a diverse set was cloned into CAR vectors with either a CD28 or a 4-1BB co-stimulatory domain. Donor T cells transduced with BCMA targeted CAR vectors that conveyed particularly desirable properties over multiple in vitro assays, including: cytotoxicity on human MM cell lines at low E:T ratios (&gt;90% lysis, 1:1, 16h), robust proliferation after repeat antigen stimulation (up to 700 fold, stimulation q3-4d for 14d), and active cytokine profiling, were selected for in vivo studies using a marrow predominant human MM cell line model in NSG mice. A single IV injection of CAR T cells, either early (4d) or late (21d) after MM engraftment was evaluated. In both cases survival was increased when treated with BCMA targeted CAR T cells vs CD19 targeted CAR T cells (median OS at 60d NR vs 35d p&lt;0.05). Tumor and CAR T cells were imaged in vivo by taking advantage of luciferase constructs with different substrates. Results show rapid tumor clearance, peak (&gt;10,000 fold) CAR T expansion at day 6, followed by contraction of CAR T cells after MM clearance, confirming the efficacy of the anti-BCMA scFv/4-1BB containing construct. Co-culture with primary cells from a range of normal tissues did not activate CAR T cells as noted by a lack of IFN release. Co-culture of 293 cells expressing this scFv with those expressing a library of other TNFRSF or Ig receptor members demonstrated specific binding to BCMA. GLP toxicity studies in mice showed no unexpected adverse events. We generated a retroviral construct for clinical use including a truncated epithelial growth factor receptor (EGFRt) elimination gene: EGFRt/hBCMA-41BBz. Clinical investigation of this construct is underway in a dose escalation, single institution trial. Enrollment is completed on 2/4 planned dose levels (DL). On DL1 pts received cyclophosphamide conditioning (3g/m2 x1) and 72x106 mean CAR+ T cells. On DL2 pts received lower dose cyclophosphamide/fludarabine (300/30 mg/m2 x3) and 137x106 mean CAR+ T cells. All pts screened for BCMA expression by IHC were eligible. High risk cytogenetics were present in 4/6 pts. Median prior lines of therapy was 7; all pts had IMiD, PI, high dose melphalan, and CD38 directed therapies. With a data cut off of 7/20/17, 6 pts are evaluable for safety. There were no DLT's. At DL1, grade 1 CRS, not requiring intervention, occurred in 1/3 pts. At DL2, grade 1/2 CRS occurred in 2/3 pts; both received IL6R directed Tocilizumab (Toci) with near immediate resolution. In these 2 pts time to onset of fever was a mean 2d, Tmax was 39.4-41.1 C, peak CRP was 25-27mg/dl, peak IL6 level pre and post Toci were 558-632 and 3375-9071 pg/ml, respectively. Additional serum cytokines increased &gt;10 fold from baseline in both pts include: IFNg, GM CSF, Fractalkine, IL5, IL8, and IP10. Increases in ferritin were limited, and there were no cases of hypofibrinogenemia. There were no grade 3-5 CRS and no neurotoxicities or cerebral edema. No pts received steroids or Cetuximab. Median time to count recovery after neutropenia was 10d (range 6-15d). Objective responses by IMWG criteria after a single dose of CAR T cells were observed across both DLs. At DL1, of 3 pts, responses were 1 VGPR, 1 SD, and 1 pt treated with baseline Mspike 0.46, thus not evaluable by IMWG criteria, had &gt;50% reduction in Mspike, and normalization of K/L ratio. At DL2, 2/2 pts had objective responses with 1 PR and 1 VGPR (baseline 95% marrow involvement); 1 pt is too early to evaluate. As we are employing a human CAR, the study was designed to allow for an optional second dose in pts that do not reach CR. We have treated 2 pts with a second dose, and longer follow up data is pending. Figure 1 Figure 1. Disclosures Smith: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: BCMA targeted CAR T cells, Research Funding. Almo: Cue Biopharma: Other: Founder, head of SABequity holder; Institute for Protein Innovation: Consultancy; AKIN GUMP STRAUSS HAUER & FELD LLP: Consultancy. Wang: Eureka Therapeutics Inc.: Employment, Equity Ownership. Xu: Eureka Therapeutics, Inc: Employment, Equity Ownership. Park: Amgen: Consultancy. Curran: Juno Therapeutics: Research Funding; Novartis: Consultancy. Dogan: Celgene: Consultancy; Peer Review Institute: Consultancy; Roche Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Liu: Eureka Therpeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

scholarly journals In Vitro Generation of Human Pluripotent Stem Cell-Derived T Cells for Immunotherapy

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 691-691
Author(s):  
Amélie Montel-Hagen ◽  
Christopher S. Seet ◽  
Suwen Li ◽  
Brent Chick ◽  
Patrick Chang ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Pluripotent Stem Cells ◽  
Research Funding ◽  
Cytokine Release ◽  
Highly Efficient ◽  
Car T ◽  
Engineered T Cells

Abstract Adoptive cell therapy using T cells engineered to express antigen-specific T cell receptors (TCR-T) or chimeric antigen receptors (CAR-T) offer targeted and potentially curative treatments for malignancy. Current approaches rely on the genetic modification and expansion of mature circulating T-cells. Such processes are limited to autologous T cells due to the risk of graft-versus-host (GvHD) disease from allogeneic T cells through endogenous TCR expression as well as rejection through MHC incompatibility. Furthermore, prolonged ex-vivo expansion of T cells may reduce in vivo efficacy and harvesting sufficient T cells from lymphopenic patients is challenging. Direct in vitro differentiation of engineered T cells from human pluripotent stem cells (HSPCs) may overcome these problems by providing an unlimited source of cells that can be genetically edited, permitting the suppression of endogenous TCR expression through allelic exclusion, and the de novo generation of naïve antigen-specific T cells. We have developed an in vitro Artificial Thymic Organoid (ATO) system that induces highly efficient and reproducible production of mature naïve T cells from human hematopoietic stem cells and progenitor cells (HSPC). Here, we report the preclinical development of a modified ATO system that supports highly efficient in vitro differentiation and positive selection of naive human T cells from at least 5 different lines of human pluripotent stem cells (PSC), including Embryonic stem cells (ESC) and induced Pluripotent Stem Cells (iPSC). T cell differentiation from PSC was very similar phenotypically to that from HSPC. As in normal human thymopoiesis, the first evidence for T cell commitment was expression of CD7 and CD5, followed by the CD3-CD8lo "ISP8" stage, then CD4+CD8+ "DP" stage and finally production of CD3+CD8+CD4- "CD8SP" and Cd3+CD4+CD8- "CD4SP". As is typical with both monolayer cultures and ATOs (and opposite to normal thymus), CD8SP predominated over CD4SP. Surprisingly, differentiation occurred more rapidly from PSC than with HSPC. As with HSPC-ATOs, CD8SP from PSC ATOs showed a mature naïve conventional T cell phenotype i.e. CD3+TCRab+CD4- CD45RA+CD62L+CD27+ and exhibited a diverse, thymic-like TCR repertoire, and robust TCR-dependent cytokine release and proliferation. The differentiation in ATOs of an ESC line that expresses an HLA-A*02:01-restricted αβ TCR specific for NY-ESO-1 resulted in a markedly increased cell yield with an enhanced generation of naïve CD3+TCRαβ+CD8αβ+ conventional T cells, the majority of which were antigen-specific by tetramer staining. TCR-engineered T cells produced from PSC in ATOs displayed a near complete lack of endogenous TCR Vβ expression, consistent with induction of allelic exclusion by the exogenous TCR during T cell development. The TCR engineered T cells underwent polyfunctional cytokine release, and proliferation in response to artificial APCs. Moreover, the differentiation in ATOs of an ESC line that expresses a CD19-specific 2nd generation (CD28/CD3zeta) CAR construct resulted in the production of CD5+CD7+ CD45RA+ CAR T cells. As reported previously, the ESC-derived CAR T cells did not express CD4, CD8 or CD3; however, they responded to PMA/ionomycin and underwent specific cytokine release and degranulation in response to target cells expressing CD19. PSC-derivedATOs thus present a highly efficient platform for the generation of clinically relevant mature naïve and potentially non-alloreactive TCR and CAR engineered T cells for adoptive immunotherapy. Disclosures Montel-Hagen: Kite Pharma: Research Funding. Seet: Kite Pharma: Research Funding. Crooks: Kite Pharma: Research Funding.

scholarly journals NKG2D-CAR Transduced Primary Natural Killer Cells Efficiently Target Multiple Myeloma Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 590-590 ◽  
Author(s):  
Alejandra Leivas ◽  
Paula Rio ◽  
Rebeca Mateos ◽  
Mari Liz Paciello ◽  
Almudena Garcia-Ortiz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Cell Therapy ◽  
Nk Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Car T ◽  
Equity Ownership

Abstract Introduction Immunotherapy represents a new weapon in the fight against multiple myeloma. Current clinical outcomes using CAR-T cell therapy against multiple myeloma show promise in the eradication of the disease. However, these CARs observe relapse as a common phenomenon after treatment due to the reemergence of neoantigens or negative cells. CARs can also be targeted using non-antibody approaches, including the use of receptors, as NKG2D with a wider range of ligands, and ligands to provide target specificity. Different cell types have been used to improve CAR cell therapy. CAR-T cells are the most commonly used. However, despite its effectiveness, there are still problems to face. The toxicity of the cytokine release syndrome is well known, that is why memory CD45RA- T cells are used to avoid collateral effects, although having lower efficacy. However, CAR-NK cells may have less toxicity and provide a method to redirect these cells specifically to refractory cancer. The objective of this work was to compare the anti-tumor activity of CAR-T, NKAEs and CAR-NK cells from multiple myeloma patients. Methods The activated and expanded NK cells (NKAE) were generated by coculture of peripheral blood mononuclear cells with the previously irradiated CSTX002 cell line. The CD45RA- T cells were obtained by depletion with CD45RA magnetic beads and subsequent culture. The NKAE and T were transduced with an NKG2D-CAR with signaling domains of 4-1BB and CD3z. The expansion of NKAE and the expression of NKG2D-CAR were evaluated by flow cytometry based on the percentage of NK cell population and transduction efficiency by the expression of NKG2D. Europium-TDA release assays (2-4 hours) were performed to evaluate in vitro cytotoxic activity. The antitumor activity of the NKAE (n=4) and CD45RA- (n=4) cells against MM U-266 cells was studied. Methylcellulose cultures were performed to assess the activity against the clonogenic tumor cell. In vivo studies were carried out in NSG mice receiving 5.106 of U266-luc MM cells i.v. injected at day 1. At day 4, mice received 15.106 i.v. injected of either CAR-NKAE or untransduced NKAE cells. Results In vitro. The killing activity of primary NKAE cells (n=4) was 86.6% (± 13.9%), considerably higher than that of CD45RA- lymphocytes (16.7% ± 13.6%) from the same patient (n=4). Even CD45RA- T cells from healthy donors (n=4) exhibit lower anti tumoral capacity (28.2% ± 9.7%) than NKAE cells. The transduction with an NKG2D CAR (MOI=5) improved the activity of autologous NKAE cells by 10% (96.4% ± 19%) leading to a nearly complete destruction of U-266 MM cells, and that of CD45RA- allogenic healthy cells in 19% (47.4% ± 12.6%). Nevertheless, CD45RA- autologous T cells transduced with NKG2D-CAR minimally improved their activity by 5.8% (22.5% ± 10.6%). Additionally, the CAR-NKAE cells were able to destroy the clonogenic tumor cell responsible for the progression of the MM from RPMI-8226 cell line. At an 8:1 ratio the CAR-NKAE cells were able to destroy 71.2% ± 2.5% of the clonogenic tumor cells, while the NKAE reached 56.5% ± 2.6% at a maximum ratio of 32: 1. The toxicity of the CAR-NKAE cells on healthy tissue from the same patient was assessed, and no activity against autologous PBMCs was observed, 1,8% at a maximun ratio of 32:1 (effector:target). In vivo. NKAE cells and CAR-NKAE cells were efficient in abrogating MM growth. However, CAR-NKAE cells treatment showed higher efficiency 14 days after tumor cells injection. Forty-two days after tumor cells injection, only animals receiving CAR-NKAE cells treatment remain free of disease (Figure 1). Conclusions It is feasible to modify primary NKAE cells and CD45RA- T cells from primary MM cells to safely express an NKG2D-CAR. Our data show that CD45RA- T cells from patients are not effective in vitro against MM even once transduced with our CAR. The resulting CAR-NKG2D NKAE cells are the most appropriate strategy for the destruction of MM in vitro and in vivo in our model. These results form the basis for the development of an NKG2D-CAR NK cell therapy in MM. Disclosures Rio: Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Lee:Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Martinez-Lopez:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Vivia: Honoraria; Pfizer: Research Funding; BMS: Research Funding; Novartis: Research Funding.

scholarly journals B-Cell Maturation Antigen (BCMA)-Specific Chimeric Antigen Receptor T Cells (CART-BCMA) for Multiple Myeloma (MM): Initial Safety and Efficacy from a Phase I Study

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1147-1147 ◽  
Author(s):  
Adam D. Cohen ◽  
Alfred L. Garfall ◽  
Edward A Stadtmauer ◽  
Simon Francis Lacey ◽  
Eric Lancaster ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Depth Of Response ◽  
Car T ◽  
Equity Ownership ◽  
Grade 3

Abstract Background : BCMA is expressed on MM cells, and CAR T cells targeting BCMA have pre-clinical anti-MM activity. CART-BCMA is an autologous T cell product engineered by lentiviral transduction to express a fully human BCMA-specific CAR with CD3ζ and 4-1BB signaling domains, and then expanded ex vivo using CD3/CD28 beads. Methods: In this ongoing, 3+3 dose-escalation study, relapsed/refractory MM patients (pts) receive CART-BCMA cells as split-dose infusions (10% on day 0, 30% on day 1, and 60% on day 2). Three cohorts are planned: 1) 1-5 x 108 CART cells alone; 2) cyclophosphamide (CTX) 1.5 g/m2 + 1-5 x 107 CART cells; and 3) CTX 1.5 g/m2 + 1-5 x 108 CART cells. Pts need serum creatinine (Cr) <2.5 mg/dL or Cr clearance≥30 ml/min, and adequate hepatic, cardiac, and pulmonary function. BCMA expression on MM cells is analyzed by flow cytometry, though no pre-specified level is required for eligibility. CART-BCMA frequency and activation status are assessed in blood and marrow by flow cytometry. Levels of CAR-transduced cells are also measured by qPCR using a transgene-specific primer/probe pair. Soluble BCMA, BAFF and APRIL levels in serum are assessed by ELISA. Bioactivity of the infusion product and CART-related cytokine release syndrome are analyzed by Luminex. Responses are assessed by IMWG criteria. Results: To date, 11 pts have been screened, and 6 treated in cohort 1. Reasons for not receiving treatment were screen fail (n=2), rapid MM progression/renal failure (n=2), and pt/MD choice (n=1). The 6 treated pts were all IMID/PI-refractory with high risk cytogenetics and median 9 lines of therapy (Table). All expressed BCMA on MM cells, and achieved the minimum target dose of 1x108 CART-BCMA cells. All but 2 received 100% of planned dose, with 2 (pts 01and 03) receiving 40% (3rd infusions held for fever). Cytokine release syndrome (CRS) occurred in 5 patients: 2 grade 3 requiring tocilizumab (pts 01 and 03), 1 grade 2, and 2 grade 1. High-grade CRS was associated with elevated levels of IL-6, IFNg, MCP1, MIG, IL2Ra, and IL-10, as seen in our acute lymphoblastic leukemia CTL019 trial (Teachey et al, 2016). There was 1 DLT: grade 4 PRES (posterior reversible encephalopathy syndrome) in pt 03, with severe delirium, recurrent seizures, obtundation, and cerebral edema on MRI. This resolved after anti-epileptics, high-dose methylprednisolone and cyclophosphamide, without long-term neurologic sequelae. Other grade 3/4 toxicities to date include hypophosphatemia (n=3 pts), hypocalcemia (n=2), and anemia, neutropenia, lymphopenia, thrombocytopenia, hypofibrinogenemia, fatigue, pneumonia, UTI, elevated Alk phos and AST, hypokalemia, hypertension, and pleural effusion (n=1 each). CART-BCMA cells were detected in blood and marrow by CAR-specific PCR in all 6 pts, and in 4/6 by flow cytometry, with 2 pts, 01 and 03, having massive CART expansion peaking at 90% and 76% of peripheral CD3+ T cells, respectively. CART-BCMA cells during peak expansion were predominantly CD8+ and highly activated. Pt 01 has ongoing CART-BCMA persistence, with ongoing stringent CR at 7 months and MRD-negative bone marrow by flow cytometry. Pt 03, who had pleural and possible dural MM involvement, had CART-BCMA cells found in pleural fluid and CSF, and achieved VGPR (IF+ only) with resolution of extramedullary disease on PET/CT scan. She progressed at 5 months, associated with significant reduction of CART-BCMA cells and loss of BCMA expression on her MM cells by flow cytometry, suggestive of antigen escape. Two pts (02, 11) had modest CART-BCMA expansion, with 1 minimal response (MR) lasting 2 months, and 1 ongoing MR 1 month post-infusion. Two pts (09, 10) had minimal expansion and no response. Soluble BCMA levels, which were elevated in all pts at baseline, declined in parallel with CART-BCMA expansion and correlated with depth of response, with an accompanying increase in previously suppressed BAFF and APRIL levels in serum. Conclusions: CART-BCMA cells can be manufactured from heavily-pretreated MM pts, and demonstrate promising in vivo expansion and clinical activity, even without lymphodepleting conditioning. Depth of response correlates with degree of CART-BCMA expansion and CRS. Toxicities to date include CRS and in 1 pt, severe reversible neurotoxicity, as described in other CAR T cell studies. Expanded accrual in cohort 1, as well as in cohorts with CTX conditioning, is ongoing, with updated data to be presented at the meeting. Table Table. Disclosures Cohen: Bristol-Meyers Squibb: Consultancy, Research Funding; Janssen: Consultancy. Garfall:Bioinvent: Research Funding; Novartis: Consultancy, Research Funding; Medimmune: Consultancy. Stadtmauer:Novartis: Consultancy; Takada: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Teva: Consultancy; Janssen: Consultancy. Lacey:Novartis: Research Funding. Lancaster:Janssen: Consultancy; Medimmune, Inc.: Consultancy; Grifols, Inc.: Other: Teaching courses. Vogl:Millennium: Consultancy, Research Funding; Celgene: Consultancy; Karyopharm: Consultancy; Teva: Consultancy; Acetylon: Research Funding; Glaxo Smith Kline: Research Funding; Calithera: Research Funding; Constellation: Research Funding. Ambrose:Novartis: Research Funding. Plesa:Novartis: Patents & Royalties, Research Funding. Kulikovskaya:Novartis: Research Funding. Weiss:Prothena: Other: Travel, accommodations, Research Funding; Novartis: Consultancy; GlaxoSmithKline: Consultancy; Janssen: Consultancy, Other: Travel, accommodations, Research Funding; Millennium: Consultancy, Other: Travel, accommodations. Richardson:Novartis: Employment, Patents & Royalties, Research Funding. Isaacs:Novartis: Employment. Melenhorst:Novartis: Patents & Royalties, Research Funding. Levine:Novartis: Patents & Royalties, Research Funding. June:Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; University of Pennsylvania: Patents & Royalties; Tmunity: Equity Ownership, Other: Founder, stockholder ; Johnson & Johnson: Research Funding; Celldex: Consultancy, Equity Ownership; Immune Design: Consultancy, Equity Ownership; Pfizer: Honoraria. Milone:Novartis: Patents & Royalties, Research Funding.

scholarly journals Overcoming the Immunosuppressive Tumor Microenvironment of Hodgkin Lymphoma Using Chimeric Antigen Receptor T Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 43-43 ◽  
Author(s):  
Marco Ruella ◽  
Michael Klichinsky ◽  
Saad S Kenderian ◽  
Olga Shestova ◽  
Amy Ziober ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hodgkin Lymphoma ◽  
Research Funding ◽  
Poor Prognosis ◽  
University Of Pennsylvania ◽  
Car T Cell ◽  
Car T ◽  
Immunosuppressive Tumor Microenvironment ◽  
Equity Ownership

Abstract Patients with Hodgkin lymphoma (HL) who relapse or are refractory to first-line therapy have a poor prognosis and some of these patients require innovative therapeutic approaches. Our group and others have demonstrated the clinical efficacy of anti-CD19 chimeric antigen receptor T cells (CART19) for B cell malignancies. However, B cell antigens such as CD19 are typically absent from Hodgkin Reed-Sternberg (HRS) cells. Targeting the CD30 antigen using CART cells has met with limited clinical success to date. In HL the neoplastic HRS cells often represent ~1% of the tumor mass and the remainder is comprised of a highly immunosuppressive tumor microenvironment (TME). Within the TME, tumor-associated macrophages (TAM) have a key role in promoting tumor growth and inhibiting the anti-tumor immune response, and the presence of TAM in HL is associated with a poor prognosis. HL therefore represents an excellent model of tumor-induced immunosuppression that can potentially represent a barrier to successful CART therapy. The objective of this study was therefore to develop an anti-HL CAR T cell immunotherapy that would also overcome the immunosuppression of the HL microenvironment. We sought to define a tumor antigen that was co-expressed on HRS and on TAM. Using immunohistochemistry and flow cytometry we confirmed previous reports that CD123 (the IL-3Rα chain) is expressed on HRS in 50-60% of HL patients and in 100% HL cell lines (HDLM-2, KMH2, SUPHD1, and L428). Importantly, CD123 was also seen on several HL-associated immune cells of the TME, including TAM. Exposure of macrophages to HDLM-2 cells polarized them to an M2-like phenotype (Figure 1 A) with expression of CD123. Luminex analysis of HDLM-2/TAM co-culture supernatants revealed high levels of IL-13, and blockade of IL-13 partially reversed the immunosuppressive phenotype. We then developed a model of TAM-mediated CAR T cell inhibition, using immunosuppressive M2-like macrophages derived by exposure to IL-4 or to HDLM-2 cells. These macrophages could suppress “third party” CART19 proliferation in response to B-acute lymphoblastic leukemia. We then used anti-CD123 CART cells (CART123) to target TAM. Importantly, CART123 actively recognized and killed TAM, thereby resisting TAM-mediated inhibition (as opposed to control CART19 cells) (Figure 1 B). We therefore sought to investigate the utility of anti-CD123 CART for the treatment of HL (initial results for this part presented at ASH 2014). In vitro, CART123 specifically degranulate, proliferate, produce cytokines and kill HL cells. A clinically-relevant xenograft model of HL was generated using luciferase+ HDLM-2 cells in NSG mice. In this model mice showed progressive increase in tumor burden over approximately 6 weeks, reproducing the indolent nature of the human disease. At day ~43 mice were randomized to receive 1.5 million CART123 cells or control T cells. CART123 induced complete and durable eradication of disseminated tumor within 14 days, leading to 100% relapse-free and 100% overall survival at 8 months while mice treated with control T cells had a median survival of 128 days (Figure 2 A). Tumor elimination was associated with extensive CAR T cell expansion as detected by flow cytometry in serial peripheral blood bleedings. In order to prove the formation of an immunological memory, at day 250 CART123 treated mice were re-challenged with the HDLM-2 cell line. The tumor was promptly rejected, associated with a re-expansion of CART123 cells in the peripheral blood (~10 months after T cell injection) leading to an overall survival advantage as compared to controls (Figure 2 B). In summary, we showed that human CD123-redirected T cells display potent therapeutic activity against disseminated HL. In Hodgkin Lymphoma (HL) CD123 is expressed on malignant Reed-Sternberg cells as well as in M2-polarized tumor associated macrophages (TAMs): importantly CART123 can target simultaneously both the HRS and the TAM without being resistant to immunosuppression. We have previously demonstrated that CART123 can lead to myelosuppression in preclinical models, suggesting that our findings could be translated to treat patients with refractory HL in the context of stem cell transplantation using “short-acting” RNA-CAR123 T cells or depletable lentivirally-transduced CART123. Disclosures Ruella: Novartis: Patents & Royalties. Kenderian:Novartis: Patents & Royalties, Research Funding. Wasik:Gilead Sciences: Equity Ownership; Seattle Genetics: Honoraria; Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: NPM-ALK as an omncogene; University of Pennsylvania: Patents & Royalties: CAR T-cells; Gilead Sciences: Research Funding; Pharmacyclics: Research Funding. June:Novartis: Honoraria, Patents & Royalties, Research Funding; Johnson & Johnson: Honoraria; Pfizer: Honoraria; Celldex: Consultancy, Equity Ownership; Immune Design: Consultancy, Equity Ownership; Tmunity Therapeutics: Equity Ownership; Novartis: Honoraria, Patents & Royalties, Research Funding. Gill:Novartis: Patents & Royalties, Research Funding.

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