scholarly journals A Novel Second Generation CD19 CAR for Therapy of High Risk/Relapsed Paediatric CD19+ Acute Lymphoblastic Leukaemia and Other Haematological Malignancies: Preliminary Results from the Carpall Study

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4026-4026
Author(s):  
Sara Ghorashian ◽  
Anne Marijn Kramer ◽  
Sarah Jayne Albon ◽  
Catherine Irving ◽  
Lucas Chan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Memory T Cells ◽  
Transduction Efficiency ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Post Infusion ◽  
Equity Ownership

Abstract Introduction: Recent clinical trials with T cells engineered to express 2nd generation CD19 chimeric antigen receptors (CARs) unprecedented anti-leukemic responses. We have developed a novel CD19CAR with a new scFv in the 41BBz format (CAT-41BBz CAR) which confers enhanced cytotoxicity and cytokine secretion in response to stimulation with CD19+ targets in vitro as well as equivalent in vivo anti-tumour efficacy to the FMC63 41BBZ CAR in use in clinical studies. We have designed, optimized and validated GMP-grade CAR T cell production using this novel CAR. Based on these data, we have recently initiated a Phase I clinical study (CARPALL) of this novel CAR in pediatric patients with relapsed ALL and other CD19+ hematological malignancies to determine the safety profile and durability of responses to CD19CART therapy. This will be critical in determining whether CD19CAR T cells are best used as a stand-alone therapy or as a bridge to stem cell transplant (SCT). Methods: We initially optimized our GMP production methodology in terms of activation method, cytokine milieu and expansion conditions on healthy donor peripheral blood mononuclear cells (PBMCs) to give optimal transduction efficiency and preserve early memory subsets within the CAR T cell product. We have subsequently validated this methodology using unstimulated leucaphereses from 5 lymphopenic patients with ALL. PBMCs were activated with anti-CD3/CD28 microbeads (Dynabeads CTS) and then lentivirally transduced with the CAT CAR vector. T cells were then expanded in the WAVE bioreactor before bead removal on a magnetic system and cryopreservation. Patients on study receive lymphodepletion with fludarabine and cyclophosphamide followed by a single dose of 106 CAR+ T cells/kg and are then monitored as an in-patient for 14 days post infusion for toxicities such as cytokine release syndrome or neurotoxicity. The primary end-points of the study are toxicity and the proportion of patients achieving molecular CR at 1 month post CD19CAR T cell infusion. Following this, patients undergo intensive monitoring of disease status for a total of 2 years post infusion. To determine the durability of responses, patients achieving a molecular CR will be monitored closely for the re-emergence of molecular level disease without additional consolidative therapy or SCT Results: We were able to generate the target dose of 1x106 CAR+ T cells/kg in 6 of 7 production runs (involving 2 healthy donors and 5 patients) to date, all of which met sterility release criteria. Transduction efficiency was on average 37% (range 7-84%, see table 1). Mean viral copy was 4.2 (range 1.2-5.8). Memory T cells of stem cell-like phenotype (CAR+ CCR7+ CD45RA+ CD95+ CD127+) formed a mean of 9% (range 0-31%), central memory T cells (CAR+ CCR7+ CD45RA-) formed a mean of 43% (range 16-70%) and effector memory T cells formed a mean of 31% (range 0-77%) of the final CAR T cell product. The percentage of CAR T cells expressing dual exhaustion markers (TIM3+ PD-1+) was on average 5% (range 2-8%). So far 2 patients have been treated. Conclusions We have optimized and successfully validated a robust GMP production method for CD19CAR T cells lentivirally transduced with a novel CD19CAR. Preliminary results of therapy with CAT-41BBz CAR T cells in initial patients on the clinical study will be presented. Disclosures Qasim: Autolus: Consultancy, Equity Ownership, Research Funding; Cellectis: Research Funding; Calimmune: Research Funding; Catapult: Research Funding. Pule:Autolus Ltd: Employment, Equity Ownership, Research Funding; UCL Business: Patents & Royalties; Amgen: Honoraria; Roche: Honoraria.

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 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 Initial Results from a Phase 1 Clinical Study of bb21217, a Next-Generation Anti Bcma CAR T Therapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 488-488 ◽  
Author(s):  
Nina Shah ◽  
Melissa Alsina ◽  
David S Siegel ◽  
Sundar Jagannath ◽  
Deepu Madduri ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Phase 1 ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Abstract Introduction: Immunomodulatory chimeric antigen receptor (CAR) T cell therapy directed against B-cell maturation antigen (BCMA) has shown promising results for the treatment of relapsed refractory multiple myeloma (RRMM) in several phase 1 clinical studies in patients with advanced disease. Persistence of CAR T cells post infusion may be one determinant of duration of response. bb21217 is a next-generation anti-BCMA CAR T cell therapy based on investigational therapy bb2121 (Friedman 2018, Hum Gene Ther 29:585). It uses the same scFv, 4-1BB costimulatory motif and CD3-zeta T cell activation domain as bb2121 with the addition of phosphoinositide 3 kinase inhibitor bb007 during ex vivo culture to enrich the drug product for T cells displaying a memory-like phenotype. Evidence suggests that CAR T cells with this phenotype may be more persistent and more potent than unselected CAR T cells. CRB-402 is a first-in-human clinical study of bb21217 in patients with RRMM designed to assess the safety, pharmacokinetics, efficacy and duration of effect of bb21217. Methods: CRB-402 (NCT03274219) is an ongoing, multi-center phase 1 dose escalation trial of bb21217 in approximately 50 patients with RRMM who have received ≥ 3 prior regimens, including a proteasome inhibitor and an immuno-modulatory agent, or are double-refractory. During dose escalation, enrollment is restricted to patients with ≥ 50% BCMA expression by IHC on malignant plasma cells. Peripheral blood mononuclear cells are collected via leukapheresis and sent 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 bb21217 as a single infusion. Planned dose levels are 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 are quality and duration of clinical response assessed according to the IMWG Uniform Response Criteria for MM, evaluation of minimal residual disease (MRD), progression-free and overall survival, and quantification of CAR+ cells in blood. Results: Asof June 15, 2018, 8 patients (median age 64 [min;max 54 to 70]) have received bb21217. All patients to date received a dose of 150 x 106 CAR+ T cells. Four had high tumor burden, defined as ≥ 50% bone marrow plasma cells pre-infusion. Patients had a median of 9 (min;max 4 to 17) prior lines of therapy and 7/8 had prior autologous stem cell transplant; 50% had high-risk cytogenetics. Four of 8 (50%) had previously received Bort/Len/Car/Pom/Dara. Median follow-up after bb21217 infusion was 16 weeks (2 to 27 weeks) and 7 patients were evaluable for initial (1-month) clinical response. As of data cut-off, 5 of 8 patients developed cytokine release syndrome (CRS; 1 Grade 1, 3 Grade 2, 1 Grade 3) and responded to supportive care or tocilizumab. This included 1 patient with high tumor burden who experienced DLTs consisting of grade 3 CRS and grade 4 encephalopathy with signs of posterior reversible encephalopathy syndrome on MRI. This patient received tocilizumab, corticosteroids and cyclophosphamide, improved neurologically and achieved a sCR. Following this event, the dose escalation cohort was divided into two groups based on tumor burden and dosing continued at 150x106 CAR+ T cells. No deaths occurred. With 1 to 6 months since treatment, 6 of 7 patients had demonstrated clinical response per IMWG criteria: currently 1 sCR, 3 VGPR, 2 PR. MRD negative results at 10-5 nucleated cells were obtained by next-generation sequencing in 3 of 3 evaluable responders. Robust CAR+ T cell expansion during the first 30 days was observed in 7 of 7 evaluable patients. Two of 2 patients evaluable at 6 months had detectable CAR vector copies. Conclusions: Early efficacy results with bb21217 CAR T therapy in RRMM at a dose of 150 x 106 CAR+ T cells are encouraging, with 6 of 7 patients demonstrating clinical responses. The adverse events observed to date are consistent with known toxicities of CAR T therapies. CAR+ T cells were measurable at 6 months post treatment in both evaluable patients. Enrollment in the study is ongoing; longer follow-up and data in more patients will establish whether treatment with bb21217 results in sustained CAR+ T cell persistence and responses. Disclosures Shah: Kite: Consultancy; Indapta Therapeutics: Consultancy; University of California San Francisco: Employment; Nekktar: Consultancy; Teneobio: Consultancy; Sanofi: Consultancy; Janssen: Research Funding; Indapta Therapeutics: Equity Ownership; Amgen: Consultancy; Bluebird: Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy; Takeda: Consultancy; Sutro Biopharma: Research Funding; Nkarta: Consultancy. Siegel:Takeda: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Karyopharm: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Merck: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau. Jagannath:Multiple Myeloma Research Foundation: Speakers Bureau; Merck: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Medicom: Speakers Bureau. Kaufman:Karyopharm: Other: data monitoring committee; BMS: Consultancy; Janssen: Consultancy; Abbvie: Consultancy; Roche: Consultancy. Turka:bluebird bio, Inc: Employment, Equity Ownership. Lam:bluebird bio, Inc: Employment, Equity Ownership. Massaro:bluebird bio, Inc: Employment, Equity Ownership. Hege:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties: multiple; Mersana: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; SITC: Membership on an entity's Board of Directors or advisory committees; Arcus Biosicences: Membership on an entity's Board of Directors or advisory committees. Petrocca:bluebird bio, Inc: Employment, Equity Ownership. Berdeja:Glenmark: Research Funding; Novartis: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Bristol-Myers Squibb: Research Funding; Bluebird: Research Funding; Amgen: Research Funding; Celgene: Research Funding; Poseida Therapeutics, Inc.: Research Funding; Takeda: Research Funding; Teva: Research Funding; Sanofi: Research Funding. Raje:AstraZeneca: Research Funding; Takeda: Consultancy; Merck: Consultancy; Janssen: Consultancy; Celgene: Consultancy; BMS: Consultancy; Amgen Inc.: Consultancy; Research to Practice: Honoraria; Medscape: Honoraria.

scholarly journals Response to Bcma CAR-T Cells Correlates with Pretreatment Target Antigen Density and Is Improved By Small Molecule Inhibition of Gamma Secretase

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1856-1856 ◽  
Author(s):  
Damian J. Green ◽  
Margot Pont ◽  
Andrew J. Cowan ◽  
Gabriel O Cole ◽  
Blythe Duke Sather ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Research Funding ◽  
Gamma Secretase ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Introduction: The adoptive transfer of B-Cell Maturation Antigen (BCMA) chimeric antigen receptor (CAR) T cells is demonstrating early promise in multiple myeloma [MM], however durable responses remain elusive and most studies report >50% of patients relapsing within 18 months of receiving CAR-T cell therapy. The mechanism of relapse is likely the consequence of multiple factors including the variable distribution of BCMA on tumor cells, allowing cells with low antigen density to escape. Initial target density, receptor downregulation and the emergence of antigen loss variants have all been implicated in relapse following CAR-T cells directed against CD22 and CD19. Reduced or absent BCMA expression may similarly be linked to relapse in MM. We have previously demonstrated that BCMA cleavage by the γ-secretase complex reduces ligand density for CAR-T cell recognition, and that a small molecule γ-secretase inhibitor (GSI) markedly increases surface BCMA levels in a dose-dependent fashion while improving CAR-T cell recognition in preclinical models. Methods and Results: In a phase I first-in-human study (NCT03338972) employing a CAR-T cell construct encoding a fully human BCMA scFv and 4-1BB/CD3z, rapid and deep objective responses at CAR-T cell doses starting at 5 x 107 have been observed. All patients had bone marrow (BM) involvement at baseline (mean 42.5 % CD138+ by IHC) and 14/15 had no detectable disease in the BM 28 days after therapy. One patient with comparatively very low BCMA expression (BCMA antibody binding capacity [ABC; QuantiBRITE] = 269; 16.9% of the malignant plasma cells (PCs) BCMA+ by flow cytometry) was the only subject with persistent tumor cells in the BM 28 days after therapy. Despite complete BM responses in all remaining patients, late relapses have occurred. Differences in the BCMA expression level on tumor cells prior to CAR-T cells between long term responders and those with relapse are evident. Among the 12 subjects with at least 3 months of follow up, those remaining in remission (median 12 months, range 3-16; data cut off 7/15/19) demonstrated a median pre-treatment BCMA ABC of 1761 (range 781-2922, n=5), in contrast patients with relapse (mean of 7.3 months, range 2-12) had a median pre-treatment BCMA ABC of 920 (range 260-1540, n=7). Six patients with a pretreatment mean ABC of 919 (range 260-1540) had BM evaluable for BCMA expression at relapse and the mean ABC decreased to 304 (range 121-519). The percent PCs expressing BCMA decreased from 77.5% (range 13 - 99.8) to 30% (range 10.4-60.4). The impact of gamma secretase inhibition on BCMA expression was assessed on BM cells obtained from a patient relapsing after BCMA CAR-T cells. At relapse a 9.5-fold decrease in ABC from baseline was observed. The cells were cultured for 5 hours in the presence of GSI (JSMD194) at a concentration of 1mM, which is readily achievable by oral administration. A significant increase in BCMA antigen expression was observed (ABC=917). The impact of modulating BCMA expression on tumor cells by concurrently administering an oral GSI with CAR-T cells is being explored in a phase one clinical trial (NCT03502577). In this setting, the GSI has increased BCMA expression when low level residual BCMA was observed following relapse after prior BCMA therapy failure. Two patients have been evaluated for response to an JSMD194 after failing other BCMA targeted agents. One received a prior BCMA CAR-T cell product and after relapse demonstrated a BCMA ABC of 769. Target expression increased in this patient almost nine-fold to 6828 (ABC) after three oral doses of JSMD194. A second patient had a BCMA ABC of 666 after failing a BCMA bispecific T cell engager. BCMA density increased over 14-fold to 9583 after GSI. Comprehensive data from the combination GSI and BCMA CAR-T cell trial are being reported separately. Conclusion: Pretreatment BCMA target density quantified with a uniform flow cytometry method of measurement and performed on all patients enrolled on a single center BCMA CAR-T cell clinical trial is associated with the durability of response. Further, BCMA expression can be significantly increased following GSI exposure in patients evidencing low BCMA ABC at baseline or when downregulation is the consequence of prior BCMA targeting therapy. The capacity for GSIs to increase BCMA target density and decrease soluble BCMA levels is a promising approach to be exploited in clinical trials. Disclosures Green: Juno Therapeutics: Consultancy, Patents & Royalties, Research Funding; Celgene: Consultancy; GSK: Consultancy; Seattle Genetics: Research Funding; Cellectar: Research Funding. Pont:Fred Hutchinson Cancer Research Center: Other: Inventor on a patent. Cowan:Sanofi: Consultancy; Juno: Research Funding; Abbvie: Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Cellectar: Consultancy. Sather:Lyell Immunopharma: Employment, Equity Ownership. Blake:Celgene: Employment, Equity Ownership. Works:Celgene: Employment, Equity Ownership. Maloney:Juno Therapeutics: Honoraria, Patents & Royalties: patients pending , Research Funding; A2 Biotherapeutics: Honoraria, Other: Stock options ; BioLine RX, Gilead,Genentech,Novartis: Honoraria; Celgene,Kite Pharma: Honoraria, Research Funding. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy; Lyell Immunopharma: Equity Ownership, Patents & Royalties, Research Funding. OffLabel Disclosure: Oral Gamma Secretase Inhibitor. Purpose is to increase expression of B Cell Maturation Antigen

scholarly journals T Cells Expressing an Anti-B-Cell Maturation Antigen (BCMA) Chimeric Antigen Receptor with a Fully-Human Heavy-Chain-Only Antigen Recognition Domain Induce Remissions in Patients with Relapsed Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3230-3230 ◽  
Author(s):  
Lekha Mikkilineni ◽  
Elisabet E. Manasanch ◽  
Norris Lam ◽  
Danielle Vanasse ◽  
Jennifer N. Brudno ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Car T Cells ◽  
B Cell Maturation ◽  
Car T ◽  
Post Infusion ◽  
Equity Ownership ◽  
Grade 3 ◽  
Dose Levels

Chimeric antigen receptor (CAR) T cells expressing B-cell maturation antigen (BCMA) can target and kill multiple myeloma (MM). BCMA was chosen as a target for MM because it is expressed by almost all cases of MM but has a restricted expression pattern on normal cells. CAR antigen-recognition domains made up of monoclonal antibody-derived, single-chain-variable fragments (scFv) are potentially immunogenic. To reduce the risk of recipient immune responses against CAR T cells, we used the sequence of a novel anti-BCMA, fully-human, heavy-chain-only binding domain designated FHVH33. The FHVH33 binding domain sequence was from TeneoBio, Inc. FHVH33 is smaller than a scFv. FHVH33 lacks the light chain, artificial linker sequence, and 2 associated junctions of a scFv, so it is predicted to be less immunogenic than a scFv, especially murine-derived scFvs. We constructed a CAR incorporating FHVH33, CD8α hinge and transmembrane domains, a 4-1BB costimulatory domain, and a CD3ζ T-cell activation domain. The CAR, FHVH33-CD8BBZ, is encoded by a γ-retroviral vector. FHVH33-CD8BBZ-expressing T cells (FHVH-BCMA-T) exhibited a full range of T-cell functions in vitro and eliminated tumors and disseminated malignancy in mice (Lam et al, Blood (ASH abstract) 2017 vol 130: 504). We are conducting the first clinical trial of FHVH-BCMA-T. Patients receive conditioning chemotherapy on days -5 to -3 with 300 mg/m2 of cyclophosphamide and 30 mg/m2 of fludarabine followed by infusion of FHVH-BCMA-T on day 0. This dose-escalation trial has 5 planned dose levels (DL). Twelve patients have received FHVH-BCMA-T on 3 DLs, 0.75x106, 1.5x106 and 3x106 CAR+ T cells/kg of bodyweight. Three patients were enrolled on the trial but not treated. The median age of patients enrolled was 63 (range 52-70); patients received a median of 6 lines of anti-myeloma therapy (range 3-10) prior to treatment with FHVH-BCMA-T. Ten patients out of 12 patients have achieved objective responses (OR). Five patients have obtained CRs or VGPRs to date. One patient achieved a partial remission (PR) 26 weeks after FHVH-BCMA-T infusion through a continued decrease in a measurable plasmacytoma. Five out of 7 patients who had myeloma with high-risk cytogenetics had an OR (Table). ORs occurred in patients with large soft-tissue plasmacytomas. Loss of BCMA expression on myeloma cells after treatment was documented in 2 patients. Two patients who developed progressive MM after CAR T-cell infusion had evidence of minimal residual disease in bone marrow 1-2 months post infusion of CAR T cells (patients 7,8). Eleven out of 12 patients had cytokine release syndrome (CRS); CRS grades ranged from 1-3 (Lee et al. Biol Blood Marrow Transplant 25 (2019) 625-638). The median peak C reactive protein (CRP) of the patients with CRS was 156.3 mg/L. Of 12 patients, 1 received the interleukin-6-receptor antagonist tocilizumab on day +6 to treat grade 3 CRS with hypotension requiring low-dose pressor therapy, grade 2 ejection fraction (EF) decrease and elevation of creatinine kinase (CK). All parameters returned to baseline by day +10. Patient 12 had a grade 3 decrease in EF which resolved by day +29. Two patients had grade 2 neurotoxicity that resolved without intervention: patient 3 had headaches, dysarthria and word-finding difficulties that resolved after 6 days while patient 6 had headaches on day +4. Patient 12 had grade 3 neurotoxicity with confusion on day +2; she was given dexamethasone with improvement in mental status the same day. After attaining a response, patient 6 died from influenza complications 6 weeks after FHVH-BCMA-T infusion. A median of 10.6% (range 1.1-46) of bone marrow T cells were CAR+ when assessed 14 days after FHVH-BCMA-T infusion. We assessed blood CAR+ cells by quantitative PCR. The median peak level of CAR+ cells was 76.5 cells/µl (range 3-347 cells/µl) and the median day post-infusion of peak blood CAR+ cell levels was 13 (range 9-14). The results from this phase 1 trial demonstrate that FHVH-BCMA-T cells can induce responses at low dose levels. Patients who had no CRS or low-grade CRS achieved objective responses. Toxicity was limited and reversible. Accrual to this trial continues. A maximum tolerated dose has not been determined yet. These results encourage further development of FHVH CAR-T. Table Disclosures Manasanch: Janssen: Honoraria; Sanofi: Honoraria; Takeda: Honoraria; Merck: Research Funding; Skyline Diagnostics: Research Funding; Sanofi: Research Funding; Quest Diagnostics: Research Funding; Celgene: Honoraria. Trinklein:Teneobio, Inc.: Employment, Equity Ownership. Buelow:Teneobio, Inc.: Employment, Equity Ownership. Kochenderfer:Kite and Celgene: Research Funding; Bluebird and CRISPR Therapeutics: Other: received royalties on licensing of his inventions. OffLabel Disclosure: Cyclophosphamide and fludarabine are used in combination for conditioning chemotherapy prior to CAR T-cell infusion

scholarly journals Transcend NHL 001: Immunotherapy with the CD19-Directed CAR T-Cell Product JCAR017 Results in High Complete Response Rates in Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4192-4192 ◽  
Author(s):  
Jeremy S. Abramson ◽  
Lia Palomba ◽  
Leo I Gordon ◽  
Matthew Lunning ◽  
Jon Arnason ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Research Funding ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T Cell ◽  
Non Hodgkin Lymphoma ◽  
Car T ◽  
Equity Ownership

Abstract Background: Based on promising results seen in patients treated with CD19-directed CAR-T cells in relapsed or refractory (R/R) pediatric B-cell acute lymphoblastic leukemia (Gardner, ASCO 2016) and adult B-cell non-Hodgkin lymphoma (Turtle, ASCO 2016), we are conducting a multicenter phase 1 trial of JCAR017 in R/R diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) (ClinicalTrials.gov Identifier: NCT02631044). JCAR017 is a second-generation, CD19-directed CAR-T cell product of defined cellular composition consisting of a 1:1 ratio of CD8+:CD4+ CAR+ T cells. Methods: Patients with R/R DLBCL (de novo or transformed from indolent lymphoma), follicular lymphoma grade 3B, or MCL and adequate organ function are eligible. There was no minimum absolute lymphocyte count (ALC) requirement for apheresis and no test expansion required. Treatment includes lymphodepletion (fludarabine 30 mg/m2 and cyclophosphamide 300 mg/m2 daily for 3 days) and JCAR017 given 2-7 days post-lymphodepletion at a starting dose of 5 x 107 CAR+ T cells (DL1). Single-dose and two-dose schedules are being evaluated. Primary objectives include safety and pharmacokinetics (PK) of JCAR017 measured by flow cytometry and quantitative PCR. Secondary objectives include complete and overall response (CR, OR) rates and duration of response (DOR). Response is assessed using the Lugano (2014) criteria. Results: As of August 1, 2016, 39 patients have been enrolled and 28 patients apheresed. Fourteen patients have been treated, all at DL1. Eight were male and 6 female. Thirteen patients had DLBCL and 1 had MCL. Median age was 61 years (range 37-79) and median number of prior therapies was 5 (range 2-9). Ten patients had undergone prior transplant (7 autologous; 3 allogeneic). Of the 14 patients, there were no cases of severe cytokine release syndrome (sCRS); 3 patients had low grade CRS (21%) (2 grade 1; 1 grade 2) and none required treatment with tocilizumab. Two of the 14 treated patients (14%) had neurotoxicity: 1 grade 4 encephalopathy and 1 grade 4 seizure. Both were in patients with DLBCL and were dose-limiting toxicities. Two deaths were seen in the DLBCL group and were due to disease progression. Twelve patients had at least 1 post-treatment response assessment; 11 patients with DLBCL and 1 with MCL. The patient with MCL had progressive disease at day 29 (D29). In the DLBCL group, response rates were: 82% (9/11) OR, 73% (8/11) CR, 9% (1/11) PR and 18% (2/11) PD at the time of post-treatment assessment on D29. All but one patient who achieved a CR were in remission at the time of this data cut. One DLBCL patient in CR had a parenchymal brain lesion in the right temporal lobe that also completely resolved. Of note, this patient had no CRS or neurotoxicity associated with JCAR017 treatment. The PK profile of JCAR017 in the peripheral blood and bone marrow show cellular expansion in all patients with persistence out to at least 3 months in patients with adequate follow up. Exploratory biomarker analyses will be presented at the meeting along with updated clinical data. Conclusions: Treatment with the defined cellular composition product JCAR017 following lymphodepletion with fludarabine and cyclophosphamide results in high CR rates in patients with heavily pretreated DLBCL, including the first report of a CR in a patient with secondary CNS lymphoma. Observed toxicities are manageable and compare favorably to other reported CAR T-cell products. Disclosures Abramson: Gilead: Consultancy; Kite Pharma: Consultancy; Abbvie: Consultancy; Seattle Genetics: Consultancy. Gordon:Northwestern University: Patents & Royalties: Patent for gold nanoparticles pending. Lunning:Celgene: Consultancy; Bristol-Myer-Squibb: Consultancy; Pharmacyclics: Consultancy; Genentech: Consultancy; Juno: Consultancy; AbbVie: Consultancy; Gilead: Consultancy; TG Therapeutics: Consultancy; Spectrum: Consultancy. Arnason:Gilead: Consultancy. Forero-Torres:Genentech/Roche: Research Funding; Seattle Genetics: Research Funding; Juno: Research Funding; Incyte: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Pfizer: Research Funding. Albertson:Juno Therapeutics: Employment, Equity Ownership. Sutherland:Juno therapeutics: Employment. Xie:Juno Therapeutics: Employment, Equity Ownership. Snodgrass:Juno therapeutics: Employment. Siddiqi:Pharmacyclics, LLC, an AbbVie Company: Speakers Bureau; Janssen: Speakers Bureau; Seattle Genetics: Speakers Bureau; Kite pharma: Other: Funded travel, 1 day registration, and 1 night hotel stay for EHA2016 so I could present trial data there.

scholarly journals Point-of-Care Manufacturing of CD20.19 Bi-Specific Chimeric Antigen Receptor T (CAR-T) Cells in a Standard Academic Cell Processing Facility for a Phase I Clinical Trial in Relapsed, Refractory NHL

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4553-4553 ◽  
Author(s):  
Fenlu Zhu ◽  
Nirav N Shah ◽  
Dina Schneider ◽  
Huiqing Xu ◽  
Katherine Chaney ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Research Funding ◽  
Target Cells ◽  
Cell Processing ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Abstract Adoptive cell therapy with autologous CAR-T cells has induced remarkable responses in patients with treatment-refractory hematologic malignancies, which has led to FDA approvals for two CAR-T products. However, limitations exist with commercial CAR-T centralized production: (1) off-site manufacturing can take several weeks and requires shipping from and to the treating facility; (2) off-site manufacturing limits treatment options for progressing patients; (3) high cost of the commercial products may limit their availability. To address these challenges, we used the fully automated Miltenyi CliniMACS Prodigy device, a GMP-compliant closed system, to manufacture autologous CAR-T cells for a Phase I trial (NCT03019055) evaluating a first-in-human bi-specific CAR that targets CD19 and CD20 (CD20.19 CAR). CAR-T manufacturing was performed exclusively using the CliniMACS Prodigy device and reagents obtained from Miltenyi Biotec. Production was performed within the Medical College of Wisconsin (MCW) Cell Therapy Laboratory, an ISO7 air handling environment. Manufacturing was set at 14 days, and production was as follows. First, peripheral blood mononuclear cells (MNC) were collected by apheresis, with a collection goal of 4 blood volumes to eliminate risk of a low CD3 yield in heavily pre-treated patients. Next, MNC were loaded onto the Prodigy, and CD4 and CD8 T cells enriched by positive immunomagnetic selection. To start the culture process, enriched T cells were suspended in TexMACS medium supplemented with 3% human AB serum and 200 U/mL IL-2, and TransACT reagent was added to stimulate the T cells in the Prodigy cell culture chamber. The following day (day 1), lentiviral vector expressing anti-CD19 and anti-CD20 (in tandem) with CD3ζ and 4-1BB stimulatory domains was added to the stimulated cells. Culture washes and feedings were done on days 5, 6, 8, 10 and 12 of manufacture, and final products harvested on Day 14. Protein L staining was used to detect expression of CD20.19 CAR on the T cells. On Day 14, eligible patients received fresh CAR-T cells, while for others the product was cryopreserved and administered on a later date. To date, the MCW Cell Therapy Laboratory has successfully generated CAR-T cell products for all 6 patients enrolled thus far on the Phase 1 clinical trial with no production failures (Table 1). Three patients received cryopreserved product and 3 patients received fresh product. The enriched T cells were 94.3% CD3+ (87.8-97.4%), and average T cell recovery from the apheresis cell products was 65.2% (54.2-80.0%). Protein L staining indicated 20.8% average CD20.19 CAR expression. Patient CAR-T cells were able to kill CD19+ and CD20+ target cells in vitro and produce IFN-gamma in response to the same target cells. An average yield of 5.9e+8 (4.3-7.9e+8) CAR T cells was obtained at harvest, which exceeded the required cell dose for all patients. The CAR-T cells were comprised of both CD4 and CD8 T cells, with higher expression on CD4 T cells; average CAR-T CD4:CD8 ratio on the final products was 2.8. The majority of T cells (average of 81.5%) had an effector-memory phenotype. In-process testing performed on Day 8 of manufacturing demonstrated sufficient numbers of CAR-T cells needed for patient infusions were already present, and that the CAR-T cells only expanded an additional 1.9 to 3.5-fold between Days 8 and 14. In conclusion, we have successfully demonstrated feasibility for point-of-care CAR-T cell production for clinical use from patient apheresis products utilizing the CliniMACS Prodigy device. Time to production was efficient (14 days), and patient-derived CAR-T cell products were reproducibly generated in a standard cell processing laboratory within an academic medical center. A major clinical advantage of CAR-T cells generated on-site is the flexibility in treatment. Patients can receive cells either immediately (i.e., fresh) or the cells can be cryopreserved for later infusion if the patient is not able to receive fresh cells. Based on our results, we intend to decrease the cell processing time to 10 days. Disclosures Zhu: Lentigen Technology Inc., A Miltenyi Biotec Company: Research Funding. Shah:Juno Pharmaceuticals: Honoraria; Oncosec: Equity Ownership; Geron: Equity Ownership; Exelexis: Equity Ownership; Miltenyi: Other: Travel funding, Research Funding; Lentigen Technology: Research Funding. Schneider:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Keever-Taylor:Medical College of Wisconsin: Research Funding. Dropulic:Lentigen, A Miltenyi Biotec company: Employment. Orentas:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Hari:Bristol-Myers Squibb: Consultancy, Research Funding; Amgen Inc.: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Kite Pharma: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Spectrum: Consultancy, Research Funding; Sanofi: Honoraria, Research Funding. Johnson:Miltenyi: Research Funding.

scholarly journals CAR T Cell Therapy Targeting G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D), a Novel Target for the Immunotherapy of Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 589-589 ◽  
Author(s):  
Eric L. Smith ◽  
Kimberly Harrington ◽  
Mette Staehr ◽  
Reed Masakayan ◽  
Jon Jones ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership ◽  
G Protein Coupled

Abstract Early clinical results using BCMA targeted CAR T cell therapies for advanced multiple myeloma (MM) have shown promise. However, BCMA expression can be variable, and BCMA downregulation has been correlated with relapse (Brudno J. JCO. 2018; Cohen A. ASH. 2017). Targeting multiple antigens may enhance response durability. We report that the orphan seven transmembrane G protein coupled receptor, GPRC5D, is an attractive additional target for CAR T cell therapy of MM. GPRC5D mRNA expression was previously identified in bone marrow cells from patients with MM; however its protein expression could not be detected with available FACS reagents (Frigyesi I. Blood. 2014). We evaluated 83 primary marrow samples by quantitative immunofluorescence (Q-IF) for CD138, BCMA, and GPRC5D. In 98% of the samples, CD138+ cells expressed surface GPRC5D. In most samples, the majority of CD138+ cells expressed both BCMA and GPRC5D, however, in several cases the dominant CD138+ population expressed only BCMA or GPRC5D, with GPRC5D expression independent of BCMA across samples (R2=0.156; Figure 1). The potential for "on target/off tumor" binding by targeting GPRC5D was evaluated by screening 30 essential normal tissue types by IHC (n=3 donors/type) followed by validation of any positive findings by RNA in situ hybridization and quantitative PCR. Results in non-plasma cell normal tissue were consistent with prior reports of GPRC5D off target expression restricted to cells from the hair follicle, a potentially immune privileged site. We developed GPRC5D-targeted CARs considering immunogenicity, spacer length, and tonic signaling. To minimize potential anti-CAR immunity, a human B cell-derived phage display library was screened. Seven diverse and highly specific human single chain variable fragments (scFvs) were identified. 42 CARs were derived from the 7 scFvs by modifying scFv orientation (VH/VL; VL/VH) and incorporating a short, medium, or long IgG4 based spacer. To monitor CAR-mediated signaling, we transduced each CAR into a Jurkat reporter line with RFP inserted in-frame, downstream of endogenous NR4A1 (Nur77). Nur77 expression is an immediate-early, specific marker of CD3z signaling (Ashouri J. J Immunol. 2017). Using this reporter, we observed that (1) a long spacer provided enhanced antigen-dependent activation across all anti-GPRC5D CARs; and (2) the use of different scFvs resulted in vastly different levels of tonic signaling. We selected potential lead and backup constructs and evaluated CAR activity on primary human T cells. When co-cultured specifically with MM cell lines, anti-GPRC5D CAR T cells secreted a polyfunctional cytokine profile, proliferated, and effectively lysed target cells. CD138+/CD38hi primary MM bone marrow aspirate cells were also specifically lysed. In vivo efficacy of GPRC5D-targeted CAR T cells was evaluated in NSG mice engrafted with a human MM cell line (ffLuc+) bone marrow xenograft. Donor T cells were gene-modified to express anti-GPRC5D CARs with either a 4-1BB or a CD28 co-stimulatory domain and membrane-anchored Gaussia luciferase (GLuc). Compared to control CAR T cells specific for an irrelevant target, anti-GPRC5D CAR T cells with either co-stimulatory domain proliferated and homed to the site of MM (Gluc imaging), eradicated MM xenograft (ffLuc imaging), and increased survival (Figure 2). One scFv that was highly functional in our GPRC5D CAR screen was evaluated for off-target binding against either >200 G protein-coupled receptors (cell based), or >4000 human transmembrane proteins (scFv-Fc based), and demonstrated binding only to GPRC5D. Studies with murine and cynomolgus cross-reactive GPRC5D targeting CARs did not show signs of alopecia or other unexpected toxicity in either species. In a murine model of post-BCMA CAR T cell treated antigen escape (CRISPR BCMA KO of a subpopulation of MM cells), anti-GPRC5D CAR T cells rescue BCMA- relapse. These results indicate that GPRC5D will be an important target for the immunotherapy of MM. We are translating this 4-1BB-containing, human-derived, GPRC5D-targeted CAR construct to the clinic. Disclosures Smith: Celgene: Consultancy, Patents & Royalties: CAR T cell therapies for MM, Research Funding. Harrington:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Masakayan:Agentus Inc: Employment. Jones:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Long:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Ghoddusi:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Do:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Pham:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Wang:Eureka Therapeutics: Employment, Equity Ownership. Liu:Eureka Therapeutics, Inc.: Employment, Equity Ownership. Xu:Eureka Therapeutics: Employment, Equity Ownership. Riviere:Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Fate Therapeutics Inc.: Research Funding. Liu:Eureka Therapeutics, Inc.: Employment, Equity Ownership. Sather:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Brentjens:Juno Therapeutics, a Celgene Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

scholarly journals CD19 CAR-T Cells Are Highly Effective in Ibrutinib-Refractory Chronic Lymphocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 56-56 ◽  
Author(s):  
Cameron J Turtle ◽  
Laila-Aicha Hanafi ◽  
Daniel Li ◽  
Colette Chaney ◽  
Shelly Heimfeld ◽  
...  
Keyword(s):  
T Cells ◽  
Lymph Node ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Abstract BACKGROUND: Ibrutinib, a Bruton Tyrosine Kinase (BTK) inhibitor causes partial responses (PR) in a majority of patients (pts) with chronic lymphocytic leukemia (CLL). However, complete responses (CR) are rare and high-risk pts who progress on ibrutinib have short survival. Lymphodepletion chemotherapy followed by infusion of CD19-specific chimeric antigen receptor (CAR)-modified T cells has produced encouraging responses in CLL in phase 1 clinical trials, but the majority of pts in those studies had not previously received or failed ibrutinib. METHODS: We treated 18 adults with CLL who had previously received ibrutinib with anti-CD19 CAR-T cells that were manufactured from defined CD4+ and CD8+ T cell subsets obtained by immunomagnetic selection of leukapheresis products, formulated in a final 1:1 ratio of CD8+:CD4+ CAR+ T cells, and infused at 1 of 3 dose levels (2x105, 2x106 or 2x107 CAR-T cells/kg) after lymphodepletion chemotherapy. RESULTS: Eighteen pts (median age 60; range 40-73) with a median of 5 previous therapies (range 3-9), including 3 pts that failed prior allogeneic stem cell transplant were enrolled and treated on the study. All pts were refractory to or had relapsed after receiving a regimen containing fludarabine and rituximab, and all pts had previously received ibrutinib; 11 were ibrutinib-refractory, 3 were ibrutinib-intolerant, and 4 were refractory to venetoclax. Twelve pts had a complex karyotype and 11 pts had 17p deletion. The median percentage of abnormal B cells in marrow was 77% (range 0.4 Ð 96). All pts had extramedullary disease and 2 had CNS disease. Lymphodepletion chemotherapy consisted of cyclophosphamide (Cy) 30-60 mg/kg x 1 and fludarabine (Flu) 25 mg/m2/day x 3 days (n=15); Flu 25 mg/m2/day x 3 days alone (n=2); and Cy 60 mg/kg alone (n=1). CAR-T cells were manufactured for all pts and 16/18 received CD4+ and CD8+ CAR-T cells in the defined 1:1 ratio. Four pts received 2x105, 13 received 2x106 and 1 received 2x107 CAR-T cells/kg. Seventeen pts have completed response and toxicity assessment. Analysis of all pts with B cell malignancies treated with Cy/Flu and CAR-T cells on our trial showed that the highest dose level (2x107 CAR-T cells/kg) was too toxic for an initial CAR-T cell infusion, and identified a maximum tolerated first dose of 2x106 CAR-T cells/kg. Cytokine release syndrome (CRS) was graded according to Lee et al (Blood, 2014). After a single cycle of lymphodepletion chemotherapy and CAR-T cell infusion, 8 pts developed grade (gr) 0-1, 5 had gr 2, 3 had gr 3, and 1 had gr 4 CRS. Four pts developed gr ³3 neurotoxicity (NT). No gr 5 events were observed, no pts were intubated, and only 1 pt required pressors. Three pts received tocilizumab and dexamethasone to treat CRS and/or NT. Four pts received a second cycle of lymphodepletion chemotherapy and CAR-T cells at a 10-fold higher dose than the 1st infusion for persistent disease. CRS and NT (gr3) was observed in 3 of 4 pts after the second cycle of therapy. Restaging was performed 4 weeks after the last CAR-T cell infusion. The ORR was 76% (8 PR and 5 CR). Two of the pts with PR by lymph node size criteria (IWCLL 2008) had negative PET scans after therapy. Among ibrutinib-refractory (n=10) or intolerant pts (n=3), the ORR was 77% (7 PR and 3 CR). In venetoclax refractory pts, 2 of 4 responded (PR). Only 1 of the 3 pts who did not receive Cy/Flu lymphodepletion responded. At day 28, 11 of 13 (85%) pts who received Cy/Flu lymphodepletion and a CAR-T cell infusion at ²2x106 CAR-T cells/kg had complete elimination of marrow disease by flow cytometry; 10/13 (77%) with nodal disease had a PR or CR at restaging, 1 had a mixed response, and 2 had progressive disease (PD). No malignant IGH sequences were detected in marrow of 4/4 pts in CR who underwent IGH deep sequencing. Pts with CR had a higher peak percentage of CD8+ (p=0.006), but not CD4+ CAR-T cells in blood. Robust CAR-T cell expansion was seen in some non-responders, which in conjunction with the lower response rate in nodal sites compared to blood, suggests that factors in the malignant lymph node environment may inhibit CAR-T cell activity. No pt in CR has relapsed or died with a median follow-up of 8.4 months. For the 13 pts that received Cy/Flu lymphodepletion and ≤ 2 x 106 CAR-T cells/kg, OS is 100% and PFS is shown in Fig. 1. CONCLUSION: CD19 CAR-T cells of defined CD4:CD8 ratio are highly active in CLL and can induce high response rates and durable CRs in poor prognosis pts who have previously failed ibrutinib. Disclosures Turtle: Juno Therapeutics: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria. Li:Juno Therapeutics: Employment, Equity Ownership. Riddell:Adaptive Biotechnologies: Consultancy, Honoraria; Cell Medica: Consultancy, Honoraria; Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Maloney:Juno Therapeutics: Research Funding; Genentech/Roche: Honoraria.

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