scholarly journals Allogeneic Anti-Bcma CAR-T Cells Show Tumour Specific Killing Against Primary Multiple Myeloma Cells from Different Genomic Sub-Groups

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1834-1834 ◽  
Author(s):  
Ana M Metelo ◽  
Ieuan Walker ◽  
Agnieszka Jozwik ◽  
Charlotte Graham ◽  
Charlotte Attwood ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Employment Equity ◽  
Relevant Model ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Introduction: Autologous anti-BCMA CAR-T cells have been successfully used in clinical trials for the treatment of relapsed refractory Multiple Myeloma (rrMM), achieving high initial response rates (>80%). However, in some patients these therapeutic responses were not sustained long-term and patients relapsed within 12-18 months1,2. Poor T cell fitness leading to early CAR-T cell exhaustion as well as BCMA negative tumour escape are thought to be factors contributing to treatment failure. In this study we describe for the first time the activity of an allogeneic anti-BCMA CAR-T cell product derived from young healthy donors (HD) against primary MM cells using patient bone marrow (BM) biopsies. In addition, we compare the performance of HD and MM patient-derived anti-BCMA CAR-T cells. Results: We have developed a clinically relevant model to test the efficacy of allogeneic anti-BCMA CAR-T cells against primary MM cells. This ex vivo platform uses bulk BM biopsies from MM patients to represent the heterogeneity seen in MM tumours in vivo, including their complex genomic background and unique immunosuppressive microenvironment. Newly diagnosed patients and rrMM patients with high risk genetics are included in the cohort. Using this model we show that allogeneic anti-BCMA CAR-T cells efficiently eliminate primary MM cells after 4 hours of co-culture, in a dose-dependent manner (n=9). These allogeneic anti-BCMA CAR-T cells specifically target BCMA-expressing primary MM cells (including samples with low BCMA levels and high risk genomic abnormalities, with specific anti-BCMA CAR-T cell killing of 13-73%), whilst not affecting non-tumour cells in the BM microenvironment. Moreover, we show that anti-BCMA CAR-T cells become significantly activated after exposure to CD138+ MM cells (>50% CD25+ T cells versus <10% CD25+ T cells against negative controls) and release a range of cytokines detected in the cell culture media by Luminex (including IFNγ, TNFα, IL8, GMCSF, IL-13, IL-12, MIP-1α, MIP-1β, RANTES, IL-5, IFN-α and IL-7). Finally, we compare the T cell profile of rrMM-derived anti-BCMA CAR-T cells (n=6) versus HD-derived anti-BCMA CAR-T cells (n=6), showing that HD-derived anti-BCMA CAR-T cells have a higher CD4/CD8 ratio (0.684 vs. 0.334, p<0.05), increased percentage of naïve CD4 T cells (13.6% vs. 5.05%, p<0.05) and naïve CD8 T cells (34.13% vs. 4.43%, p<0.05) and generate an expanded population of activated CD25+ T cells after exposure to MM cells. In contrast, MM-derived anti-BCMA CAR-T cells express increased levels of TIGIT (a checkpoint inhibitory molecule involved in MM relapse) and have a large percentage of permanently dysfunctional T cells (CD101+CD38+CD8+), which might affect their T cell fitness and persistence in vivo. Conclusion: To our knowledge, this is the first study showing that allogeneic anti-BCMA CAR-T cells are therapeutically active against primary MM cells, in a clinically relevant model that includes the BM microenvironment and different MM genomic subgroups. HD-derived anti-BCMA CAR-T cells were shown to have distinct phenotypic and functional characteristics compared to MM-derived anti-BCMA CAR-T cells. This work lends further support to the development of a first-in-human Phase 1 clinical trial for the treatment of rrMM patients using this allogeneic anti-BCMA CAR-T cell therapy. 1 Raje N et al. N Engl J Med. 2019; 380(18):1726-1737. 2 Zhao WH et al. J Hematol Oncol. 2018; 11(1):141. Disclosures Metelo: Pfizer: Research Funding; Allogene: Research Funding. Jozwik:Servier: Research Funding. Graham:Servier: Research Funding; Gillead: Other: Funding to attend educational meeting. Cuthill:Amgen: Other: Conference support; Takeda: Other: Conference support; Janssen: Speakers Bureau. Bentley:Allogene Therapeutics: Employment, Equity Ownership. Boldajipour:Pfizer: Employment. Sommer:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Sasu:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Benjamin:Takeda: Honoraria; Pfizer: Research Funding; Servier: Research Funding; Allogene: Research Funding; Gilead: Honoraria; Amgen: Honoraria; Eusapharm: Consultancy; Novartis: Honoraria.

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 Prophylactic Itacitinib (INCB039110) for the Prevention of Cytokine Release Syndrome Induced By Chimeric Antigen Receptor T-Cells (CAR-T-cells) Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1934-1934 ◽  
Author(s):  
Eduardo Huarte ◽  
Roddy S O'Connor ◽  
Melissa Parker ◽  
Taisheng Huang ◽  
Michael C. Milone ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytokine Release ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Background: T-cells engineered to express a chimeric antigen receptor (CAR-T-cells) are a promising cancer immunotherapy. Such targeted therapies have shown long-term relapse survival in patients with B cell leukemia and lymphoma. However, cytokine release syndrome (CRS) represents a serious, potentially life-threatening, side effect often associated with CAR-T cells therapy. The Janus kinase (JAK) tyrosine kinase family is pivotal for the downstream signaling of inflammatory cytokines, including interleukins (ILs), interferons (IFNs), and multiple growth factors. CRS manifests as a rapid (hyper)immune reaction driven by excessive inflammatory cytokine release, including IFN-g and IL-6. Itacitinib is a potent, selective JAK1 inhibitor which is being clinically evaluated in several inflammatory diseases. Aims: To evaluate in vitro and in vivo the potential of itacitinib to modulate CRS without impairing CAR-T cell anti-tumor activity. Materials and Methods: In vitro proliferation and cytotoxic activity of T cells and CAR-T cells was measured in the presence of increasing concentrations of itacitinib or tocilizumab (anti-IL-6R). To evaluate itacitinib effects in vivo, we conducted experiments involving adoptive transfer of human CD19-CAR-T-cells in immunodeficient animals (NSG) bearing CD19 expressing NAMALWA human lymphoma cells. The effect of itacitinib on cytokine production was studied on CD19-CAR-T-cells expanded in the presence of itacitinib or tocilizumab. Finally, to study whether itacitinib was able to reduce CRS symptoms in an in vivo setting, naïve mice were stimulated with Concanavalin-A (ConA), a potent T-cell mitogen capable of inducing broad inflammatory cytokine releases and proliferation. Results: In vitro, itacitinib at IC50 relevant concentrations did not significantly inhibit proliferation or anti-tumor killing capacity of human CAR-T-cells. Itacitinib and tocilizumab (anti-IL-6R) demonstrated a similar effect on CAR T-cell cytotoxic activity profile. In vivo, CD19-CAR-T-cells adoptively transferred into CD19+ tumor bearing immunodeficient animals were unaffected by oral itacitinib treatment. In an in vitro model, itacitinib was more effective than tocilizumab in reducing CRS-related cytokines produced by CD19-CAR-T-cells. Furthermore, in the in vivo immune hyperactivity (ConA) model, itacitinib reduced serum levels of CRS-related cytokines in a dose-dependent manner. Conclusion: Itacitinib at IC50 and clinically relevant concentrations did not adversely impair the in vitro or in vivo anti-tumor activity of CAR-T cells. Using CAR-T and T cell in vitro and in vivo systems, we demonstrate that itacitinib significantly reduces CRS-associated cytokines in a dose dependent manner. Together, the data suggest that itacitinib may have potential as a prophylactic agent for the prevention of CAR-T cell induced CRS. Disclosures Huarte: Incyte corporation: Employment, Equity Ownership. Parker:Incyte corporation: Employment, Equity Ownership. Huang:Incyte corporation: Employment, Equity Ownership. Milone:Novartis: Patents & Royalties: patents related to tisagenlecleucel (CTL019) and CART-BCMA; Novartis: Research Funding. Smith:Incyte corporation: Employment, Equity Ownership.

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.

Manufacturing an Enhanced CAR T Cell Product By Inhibition of the PI3K/Akt Pathway During T Cell Expansion Results in Improved In Vivo Efficacy of Anti-BCMA CAR T Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1893-1893 ◽  
Author(s):  
Molly R. Perkins ◽  
Shannon Grande ◽  
Amanda Hamel ◽  
Holly M. Horton ◽  
Tracy E. Garrett ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership ◽  
Cells Cultured

Abstract Patients treated with chimeric antigen receptor (CAR) T cells targeting CD19 for B cell malignancies have experienced rapid and durable tumor regressions. Manufacture of CAR T cells is challenged by the necessity to produce a unique drug product for each patient. Each treatment requires ex vivo culture of patient T cells to facilitate CAR gene transfer and to achieve therapeutic amounts of T cells. Paradoxically, ex vivo culture with IL-2 also decreases CAR T cell activity. Some investigators have proposed isolating central memory T cells (thought to be enriched for therapeutic T cells), yet isolation techniques are cumbersome and costly to scale commercially. Culture of T cells in IL-7 and IL-15 has also been shown by several investigators to improve therapeutic activity. Here we explored the potential for culture modifications to improve the therapeutic potential of CAR T cells without adding complexity to manufacturing. We tested this hypothesis using CAR T cells specific to B cell maturation antigen (BCMA) manufactured using standard IL-2 culture with an inhibitor of PI3K added to the media, or with IL-7 and IL-15 in place of IL-2. The in vivo activity was studied in NSG mouse models of human Burkitt's lymphoma (Daudi), and multiple myeloma (RPMI-8226), both of which express BCMA. In the lymphoma model, NSG mice were injected intravenously (IV) with 2 x 106 Daudi cells and allowed to accumulate a large tumor burden before being treated with 4 x 106 CAR+ T cells on day 18 post-tumor injection. At this late time point post implantation, mice had highly disseminated Daudi tumor (our goal was to model late stage disease observed in relapsed and refractory lymphoma). In this model of advanced disease, IL-2 cultured anti-BCMA CAR T cells had no effect on tumor growth (p = 0.22) and all mice succumbed to the tumors within two weeks after treatment. Anti-BCMA CAR T cells grown in IL-7 and IL-15 also failed to control tumor growth (p = 0.23). In sharp contrast, all animals treated with anti-BCMA CAR T cells cultured with the PI3K inhibitor survived and experienced complete long-term tumor regression (p=0.003). The same anti-BCMA CAR T cells were used in a model of multiple myeloma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells, and at 22 days post-implantation mice received a single IV administration of anti-BCMA CAR T cells (4 x 105 CAR+ T cells/mouse) cultured under various conditions. In this model, all treatment groups demonstrated tumor regression, regardless of the in vitro culture conditions. To evaluate CAR T cell durability, two weeks after initial tumor clearance, surviving animals were then re-challenged with RPMI-8226 cells on the opposite flank to model tumor relapse. We found that only animals that had been treated with anti-BCMA CAR T cells cultured with PI3K inhibition were immune to subsequent tumor challenge (p=0.005). Given the superior in vivo efficacy of anti-BCMA CAR T cells cultured with PI3K inhibition, we sought to identify phenotypic characteristics associated with the improved therapeutic activity. Anti-BCMA CAR T cells cultured with PI3K inhibition contained an increased frequency of CD62L+ CD8 T cells in the final product (p < 0.001) suggesting improved expansion of a distinct CD8 T cell subset. These data suggest that inhibition of PI3K during ex vivo expansion with IL-2 may generate a superior anti-BCMA CAR T cell product for clinical use. Furthermore, this approach could potentially be used in the manufacture of other T cell therapies. Disclosures Perkins: bluebird bio: Employment, Equity Ownership. Grande:bluebird bio: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Miller:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.

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 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 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 Knockout of CBLB Greatly Enhances Anti-Tumor Activity of CAR T Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 338-338
Author(s):  
Kathryn Hooper ◽  
Kyle Havens ◽  
Anne-Rachel Krostag ◽  
Michael S Magee ◽  
Unja Martin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Target Cells ◽  
Employment Equity ◽  
Car T Cells ◽  
Highly Efficient ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Abstract Chimeric antigen receptor (CAR) T cell therapies continue to show excellent outcomes in hematological cancers. Achieving success in additional tumor indications, however, will likely require modulating inhibitory pathways that limit CAR T cell potency. We developed a megaTAL nuclease targeting the gene encoding Casitas B-lineage lymphoma proto-oncogene-b (CBLB), a ubiquitin ligase that serves as an intracellular checkpoint that negatively regulates T cell activation. The megaTAL nuclease platform has been previously shown to drive highly efficient genome editing in primary T cells. Electroporation of primary T cells with mRNA encoding the CBLB megaTAL resulted in >90% indels at the target locus and a concomitant reduction of Cbl-b protein levels. Specificity characterization studies revealed three detectable non-exonic off-target sites with near negligible indel frequencies. We next assessed the functional impact of CBLB disruption in CAR T cells engineered to target the epidermal growth factor receptor (EGFR). When co-cultured with EGFR+ target cells, CAR T cells with megaTAL-mediated CBLB gene knockout had a 2-fold increase in pro-inflammatory cytokine production compared with mock-treated CAR T cells. We developed an A549 tumor xenograft model to test the activity of CBLB megaTAL-treated CAR T cells in vivo. While mock-treated CAR T cells had a transient impact on tumor growth, we observed complete and durable tumor elimination in mice infused with the CBLB megaTAL-treated CAR T cells. Improved responses in the megaTAL treated animals were particularly pronounced at lower CAR T cell doses, suggesting that CBLB knockout enhances the potency of CAR T cells. In summary, the CBLB megaTAL is a highly efficient and specific gene editing nuclease that enhances CAR T cell anti-tumor responses in vitro and in vivo, and thus could potentially improve the efficacy of CAR T therapy. Disclosures Hooper: bluebird bio: Employment, Equity Ownership. Havens:bluebird bio: Employment, Equity Ownership. Krostag:bluebird bio: Employment, Equity Ownership. Magee:bluebird bio: Employment, Equity Ownership. Martin:bluebird bio: Employment, Equity Ownership. Gupta:bluebird bio: Employment, Equity Ownership. Smurnyy:bluebird bio: Employment, Equity Ownership. Pechilis:bluebird bio: Employment, Equity Ownership. Rode:bluebird bio: Employment, Equity Ownership. Chavkin:bluebird bio: Employment, Equity Ownership. Grande:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership. Jarjour:bluebird bio: Employment, Equity Ownership. Astrakhan:bluebird bio: Employment, Equity Ownership.

Phase 1 Biomarker Analysis of the ZUMA-1 (KTE-C19-101) Study: A Phase 1-2 Multi-Center Study Evaluating the Safety and Efficacy of Anti-CD19 CAR T Cells (KTE-C19) in Subjects with Refractory Aggressive Non-Hodgkin Lymphoma (NHL)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2730-2730 ◽  
Author(s):  
John M. Rossi ◽  
Sattva S. Neelapu ◽  
William Y. Go ◽  
Yueh-wei Shen ◽  
Marika Sherman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Multiparameter Flow Cytometry ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T Cell ◽  
Biomarker Analysis ◽  
Car T ◽  
Equity Ownership

Abstract Introduction: A single institution study conducted at the National Cancer Institute (NCI) using anti-CD19 CAR T cells with CD28 and CD3-zeta signaling domains showed durable remissions in subjects with relapsed/refractory advanced B-cell malignancies (Kochenderfer et al. Blood 2012, JCO 2014, ASH 2014). In the NCI study, anti-CD19 CAR T cells expanded within 1 week of administration and generally cleared the peripheral blood within 2 months. Anti-CD19 CAR T cell infusion resulted in rapid elevation and subsequent resolution of circulating cytokines and chemokines within 3 weeks (Bot et al. ASCO 2015). KTE-C19 utilizes the same anti-CD19 CAR construct investigated at the NCI, manufactured using an optimized 6-8 day process (Better et al. ASCO 2014). Preliminary characterization of KTE-C19 expansion and serum pharmacodynamic (PD) markers from ZUMA-1 are presented. Methods: Subjects received KTE-C19 at a target dose of 2 x 106 (1 x 106 minimum) CAR-positive T cells/kg body weight after undergoing a fixed dose conditioning chemotherapy regimen of cyclophosphamide and fludarabine. KTE-C19 was characterized pre- and post-infusion by multiparameter flow cytometry for CAR expression and major phenotypic markers. KTE-C19 expansion and clearance was also monitored by multiparameter flow cytometry and quantitative PCR (qPCR) at weeks 1, 2, 4 and months 3 and 6 respectively. Forty one analytes from serum samples were evaluated by EMD Millipore Luminex® xMAP® multiplex assays at Days -5 (prior to conditioning chemotherapy), 0, 1, 3, 5, 7, 14 and 28 for a broad panel of cytokines, chemokines and immune effector related markers. Maximum fold increase (MFI) was defined as the maximum fold change of measured analytes above baseline (Day -5). Time to maximum value and time to resolution were calculated among subjects with increases after commencement of conditioning chemotherapy. Comparisons of fold increase were not adjusted for multiplicity. Results: As of 28 July 2015, 6 subjects have received KTE-C19. Three subjects had post-infusion samples evaluable for CAR T-cell expansion by qPCR. Peak levels occurred within 7 days and KTE-C19 was detectable at 1 month. Additional qPCR monitoring is ongoing. Circulating cytokines, chemokines and immune effector markers have been evaluated in 4 subjects. Samples post conditioning and pre-KTE-C19 infusion showed significant increases in the levels of IL-15 (p<0.005) and MCP-1 (p<0.030). Cytokine and chemokine levels achieved their peak levels 1-10 days post KTE-C19 infusion and returned to baseline generally within 3 weeks. Key pro-inflammatory markers were substantially upregulated as indicated in the table. Additional monitoring of KTE-C19 persistence (by qPCR), phenotype and activation status of CAR T cells is ongoing. Updated results will be presented. Pre-infusion characterization of KTE-C19 with respect to CAR expression and T-cell subsets including CD4, CD8, naïve, central memory and effector memory are presented in a separate ASH abstract. Conclusion: Six subjects with refractory aggressive NHL have received KTE-C19. CAR T cells expanded substantially and persisted through at least 1 month in 3 subjects evaluated to date. Pro-inflammatory, immune-homeostatic cytokines and chemokines peaked within 2 weeks. Timing of peak KTE-C19 expansion and serum cytokine profiles and clearance in this multicenter study with an optimized, shortened manufacturing process were consistent with results of the NCI study. This first biomarker analysis of ZUMA-1 demonstrates the PD activity of KTE-C19 and will help optimize future CAR T cell therapy. Clinical trial: NCT02348216.Table 1.MFI (range)Time to Maximum Median Days (range)Time to ResolutionaMedian Days (range)IL-6141 (17, 1010)5 (3, 9)18.5 (9, 28)IFN-gamma167 (8, 4387)4 (3, 5)9 (7, 10)CRP27 (1, 57)5 (3, 5)7 (7, 13)IL-1522 (12, 55)2 (1, 7)9 (9, 9)IL-27 (1, 31)1 (1, 3)5 (3, 5)IL-10132 (3, 561)5 (5, 5)9 (7, 11)MCP-116 (2, 25)2 (1, 5)7 (3, 10)Granzyme B42 (27, 773)5 (5, 7)8.5 (7, 14)a Time to resolution is the first post-KTE-C19 infusion day at which the level was ≤ 2x baseline. Disclosures Rossi: Kite Pharma: Employment, Equity Ownership; Amgen: Equity Ownership. Off Label Use: Tocilizumab for the management of CRS . Go:Kite Pharma: Employment, Equity Ownership; Amgen: Equity Ownership. Shen:Kite Pharma: Employment, Equity Ownership. Sherman:Amgen: Equity Ownership; Kite Pharma: Employment, Equity Ownership. Locke:Kite Pharma: Other: Scientific Advisory Boards. Bartlett:Insight: Research Funding; Janssen: Research Funding; Kite: Research Funding; Medimmune: Research Funding; Genentech: Research Funding; Pharmacyclics: Research Funding; Colgene: Research Funding; Millennium: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Gilead: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; MERC: Research Funding; Dynavax: Research Funding; Idera: Research Funding; Portola: Research Funding; Bristol Meyers Squibb: Research Funding; Infinity: Research Funding; LAM Theapeutics: Research Funding. Siddiqi:Pharmacyclics/Jannsen: Speakers Bureau; Seattle Genetics: Speakers Bureau; Kite pharma: Other: attended advisory board meeting. Navale:Amgen: Equity Ownership; Kite Pharma: Employment, Equity Ownership. Elias:Kite Pharma: Employment, Equity Ownership; Amgen: Equity Ownership. Wiezorek:Kite Pharma: Employment, Equity Ownership, Other: Officer of Kite Pharma. Bot:Kite Pharma: Employment, Equity Ownership.

scholarly journals Fresh Versus Cryopreserved/Thawed Bispecific Anti-CD19/CD20 CAR-T Cells for Relapsed, Refractory Non-Hodgkin Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4465-4465 ◽  
Author(s):  
Nirav N. Shah ◽  
Fenlu Zhu ◽  
Dina Schneider ◽  
Winfried Krueger ◽  
Andrew Worden ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Viability ◽  
Research Funding ◽  
Phase 1 ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Introduction Chimeric Antigen Receptor modified T (CAR-T) cell therapies have revolutionized the relapsed, refractory B cell malignancy landscape. Due to the complex steps involved with cell production, some third-party companies require T cells to be cryopreserved prior to shipping, while most manufacturers deliver modified CAR-T cells to the treating center in a cryopreserved state. This is vastly different to the approach taken with traditional cell based therapies, specifically allogeneic transplant (allo-HCT), an immunological treatment that relies on a graft-versus-tumor (GVT) effect to prevent disease relapse. Historically, "fresh" stem cells were felt to be superior to cryopreserved products due to concerns that cryopreservation may damage T cells and other mononuclear cells delaying engraftment and limiting GVT reactivity. As a result, in clinical practice most allo-HCT products are still given as fresh infusions without cryopreservation. In a Phase 1 clinical trial evaluating the safety of a bispecific anti-CD19, anti-CD20 CAR (LV20.19CAR), CAR-T cells were produced in a point-of-care fashion utilizing the CliniMACS Prodigy device. Local manufacturing allowed flexibility to administer either fresh LV20.19CAR-T cells without cryopreservation, or if indicated, thawed CAR-T cells post-cryopreservation. Methods Patients (pts) were treated on a Phase 1 dose escalation + expansion trial (NCT03019055) to demonstrate safety of 41BB/CD3z LV20.19CAR-modified T cells for adults with relapsed, refractory B cell NHL including DLBCL, MCL, FL, and CLL. The starting dose was 2.5x10^5 cells/kg with a target dose of 2.5x10^6 cells/kg. All pts received low dose fludarabine (30 mg/m2) x 3 days +cyclophosphamide (500 mg/m2) x 1 day for lymphodepletion. In the Phase 1 dose-escalation cohorts, pts received fractionated CAR-T cells over two days (30% on Day 0 and 70% on Day+1), while expansion cohort pts received CAR-T cells as a single infusion. The goal for all pts was to infuse fresh CAR-T cell prior to cryopreservation, however, CAR-T cell could be cryopreserved and infused at a later date for clinical / logistical reasons. Results A total of 20 pts received LV20.19CAR T cell therapy (Table 1). Fourteen pts received fresh CAR-T cells immediately post-harvest, 5 pts received post-thaw CAR-T cells, and 1 patient received a mixed fresh/cryopreserved product and was not included in this analysis. Reasons for cryopreserved administration was delay due to active infection (N=3), patient preference (N=1), and unexplained neutropenia (N=1). Among 19 evaluable pts, the CR rate (79% vs 40%), mean ferritin, mean CRP, and incidence of CRS and neurotoxicity were all higher in the fresh infusion group (Table 1), but not statistically significant. In terms of LV20.19 CAR-T product characteristics, mean cell viability at infusion was 93% for the fresh infusion group versus 63% for cryopreserved pts (p<0.01). Point-of-care administration allowed final cell doses to be adjusted for diminished viability among pts receiving cryopreserved product. Figure 1 demonstrates the in-vivo expansion and persistence of LV20.19CART cells among fresh versus post-thaw pts. The peak percentage of CAR-T cells within the CD3 compartment was higher in pts given fresh cell infusions (Figure 2), but was not statistically significant (p=0.08). Conclusions Cryopreservation is known to diminish cell viability and increase clinical costs associated with freezing and storage. To date, there is limited clinical data evaluating outcomes of pts receiving fresh CAR-T cells compared to thawed CAR-T cells post-cryopreservation. Although it is presumed that in-vivo CAR-T cell activity is comparable in both scenarios, among our pts, both cell viability and in-vivo expansion favored pts who received a fresh infusion. Unlike third-party CAR-T cell products where viability is unknown at the time of infusion, we adjusted the final dose to accommodate decreased cell viability. CR rates and incidence of CRS and NTX were higher among fresh infused pts suggesting greater in-vivo activity, although findings were not statistically significant, partially a result of the small sample size. While our findings are limited by small numbers in each cohort and variability in cell dose and diagnosis, these data suggest that cryopreservation of CAR-T cells may impact clinical responses and is a logistical step that needs further investigation. Disclosures Shah: Cell Vault: Consultancy, Equity Ownership; Oncosec: Equity Ownership; Lentigen: Honoraria, Research Funding; Exelexis: Equity Ownership; Geron: Equity Ownership; Celgene: Other: Advisory Board; Incyte: Consultancy; Oncosec: Equity Ownership; Kite Pharma: Other: Advisory Board. Zhu:Miltenyi Biotec: Research Funding. Schneider:Lentigen Technology, A Miltenyi Biotec Company: Employment. Krueger:Lentigen Technology, A Miltenyi Biotec Company: Employment. Worden:Lentigen Technology, A Miltenyi Biotec Company: Employment. Hamadani:Sanofi Genzyme: Research Funding, Speakers Bureau; Otsuka: Research Funding; ADC Therapeutics: Consultancy, Research Funding; Takeda: Research Funding; Celgene: Consultancy; Janssen: Consultancy; Pharmacyclics: Consultancy; Merck: Research Funding; Medimmune: Consultancy, Research Funding. Dropulic:Lentigen Technology, A Miltenyi Biotec Company: Employment. Hari:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Research Funding; Janssen: Consultancy, Honoraria; Kite: Consultancy, Honoraria; Amgen: Research Funding; Spectrum: Consultancy, Research Funding; Sanofi: Honoraria, Research Funding; Cell Vault: Equity Ownership; AbbVie: Consultancy, Honoraria. Johnson:Miltenyi Biotec: Research Funding; Cell Vault: Equity Ownership.

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