Pre-Emptive Donor Lymphocyte Infusion with CD19-Directed, CAR-Modified T Cells Infused after Allogeneic Hematopoietic Cell Transplantation for Patients with Advanced CD19+ Malignancies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 862-862 ◽  
Author(s):  
Partow Kebriaei ◽  
Stefan O. Ciurea ◽  
Mary Helen Huls ◽  
Harjeet Singh ◽  
Simon Olivares ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hematopoietic Cell Transplantation ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Lymphoid Malignancies ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Background: Allogeneic hematopoietic cell transplantation (HCT) can be curative in a subset of patients with advanced lymphoid malignancies but relapse remains a major reason for treatment failure. Donor-derived, non-specific lymphocyte infusions (DLI) can confer an immune anti-malignancy effect but can be complicated by graft-versus-host-disease (GVHD). Chimeric antigen receptor (CAR)-modified T cells directed toward CD19 have demonstrated dramatic efficacy in patients with refractory ALL and NHL. However, responses are often associated with life-threatening cytokine release syndrome. Aim: We hypothesized that infusing CAR-modified, CD19-specific T-cells after HCT as a directed DLI would be associated with a low rate of GVHD, better disease control, and a less severe cytokine release syndrome since administered in a minimal disease state. Methods: We employed a non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a CD19-specific CAR (designated CD19RCD28 that activates via CD3z & CD28) in donor-derived T cells for patients with advanced CD19+ lymphoid malignancies. T-cells were electroporated using a Nucleofector device to synchronously introduce two DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T-cells stably expressing the CAR were retrieved over 28 days of co-culture by recursive additions of g-irradiated activating and propagating cells (AaPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The AaPC were derived from K562 cells and genetically modified to co-express CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound version of IL-15. Results: To date, we have successfully treated 21 patients with median age 36 years (range 21-62) with advanced CD19+ ALL (n=18) or NHL (n=3); 10 patients had active disease at time of HCT. Donor-derived CAR+ T cells (HLA-matched sibling n=10; 1 Ag mismatched sibling n=1; haplo family n=8; cord blood n=2) were infused at a median 64 days (range 42-91 days) following HCT to prevent disease progression. Transplant preparative regimens were myeloablative, busulfan-based (n=10) or reduced intensity, fludarabine-based (n=11). All patients were maintained on GVHD prophylaxis at time of CAR T-cell infusion with tacrolimus, plus mycophenolate mofeteil for cord, plus post-HCT cyclophosphamide for haplo donors. The starting CAR+ T-cell dose was 106 (n=7), escalated to 107 (n=6), 5x107 (n=5), and currently at 108 (n=3) modified T cells/m2 (based on recipient body surface area). Patients have not demonstrated any acute or late toxicity to CAR+ T cell infusions. Three patients developed acute grades 2-4 GVHD (liver n=1, upper GI n=1, skin=1) which was within the expected range after allogeneic HCT alone. Of note, the rate of CMV reactivation after CAR T cell infusion was 24% vs. 41 % previously reported for our patients without CAR T cell infusion (Wilhelm et al. J Oncol Parm Practice, 2014, 20:257). Nineteen patients have had at least 30 days follow-up post CAR T-cell infusion and are evaluable for disease progression. Forty-eight percent of patients (n=10) remain alive and in complete remission (CR) at median 5.2 months (range 0-21.3 months) following CAR T cell infusion. Importantly, among 8 patients who received haplo-HCT and CAR, 7 remain in remission at median 4.2 months. Conclusion: We demonstrate that infusing donor-derived CD19-specific CAR+ T cells, using the SB and AaPC platform, in the adjuvant HCT setting as pre-emptive DLI may provide an effective and safe approach for maintaining remission in patients at high risk for relapse. Graft-vs-host disease did not appear increased by administration of the donor derived CAR-T cells. Furthermore, the add-back of allogeneic T cells appears to have contributed to immune reconstitution and control of opportunistic viral infection. Disclosures Huls: Intrexon and Ziopharm: Employment, Equity Ownership. Singh:Intrexon and Ziopharm: Equity Ownership, Patents & Royalties. Olivares:Intrexon and Ziopharm: Equity Ownership, Patents & Royalties. Su:Ziopharm and Intrexon: Employment. Figliola:Intrexon and Ziopharm: Equity Ownership, Patents & Royalties. Kumar:Ziopharm and Intrexon: Equity Ownership. Jena:Ziopharm Oncology: Equity Ownership, Patents & Royalties: Potential roylaties (Patent submitted); Intrexon: Equity Ownership, Patents & Royalties: Potential royalties (Patent submitted). Ang:Intrexon and Ziopharm: Equity Ownership. Lee:Intrexon: Equity Ownership; Cyto-Sen: Equity Ownership; Ziopharm: Equity Ownership.

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 Development of molecular and pharmacological switches for chimeric antigen receptor T cells

2019 ◽  
Vol 8 (1) ◽  
Author(s):  
Bill X. Wu ◽  
No-Joon Song ◽  
Brian P. Riesenberg ◽  
Zihai Li
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Intervention Strategy ◽  
Antigen Receptor ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract The use of chimeric antigen receptor (CAR) T cell technology as a therapeutic strategy for the treatment blood-born human cancers has delivered outstanding clinical efficacy. However, this treatment modality can also be associated with serious adverse events in the form of cytokine release syndrome. While several avenues are being pursued to limit the off-target effects, it is critically important that any intervention strategy has minimal consequences on long term efficacy. A recent study published in Science Translational Medicine by Dr. Hudecek’s group proved that dasatinib, a tyrosine kinase inhibitor, can serve as an on/off switch for CD19-CAR-T cells in preclinical models by limiting toxicities while maintaining therapeutic efficacy. In this editorial, we discuss the recent strategies for generating safer CAR-T cells, and also important questions surrounding the use of dasatinib for emergency intervention of CAR-T cell mediated cytokine release syndrome.

Chimeric Antigen Receptor T-Cell Therapies for Aggressive B-Cell Lymphomas: Current and Future State of the Art

2019 ◽  
pp. 446-453 ◽  
Author(s):  
Jeremy S. Abramson ◽  
Matthew Lunning ◽  
M. Lia Palomba
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Chimeric Antigen Receptor ◽  
Refractory Disease ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Aggressive B-cell lymphomas that are primary refractory to, or relapse after, frontline chemoimmunotherapy have a low cure rate with conventional therapies. Although high-dose chemotherapy remains the standard of care at first relapse for sufficiently young and fit patients, fewer than one-quarter of patients with relapsed/refractory disease are cured with this approach. Anti-CD19 chimeric antigen receptor (CAR) T cells have emerged as an effective therapy in patients with multiple relapsed/refractory disease, capable of inducing durable remissions in patients with chemotherapy-refractory disease. Three anti-CD19 CAR T cells for aggressive B-cell lymphoma (axicabtagene ciloleucel, tisagenlecleucel, and lisocabtagene ciloleucel) are either U.S. Food and Drug Administration approved or in late-stage development. All three CAR T cells produce durable remissions in 33%–40% of treated patients. Differences among these products include the specific CAR constructs, costimulatory domains, manufacturing process, dose, and eligibility criteria for their pivotal trials. Notable toxicities include cytokine release syndrome and neurologic toxicities, which are usually treatable and reversible, as well as cytopenias and hypogammaglobulinemia. Incidences of cytokine release syndrome and neurotoxicity differ across CAR T-cell products, related in part to the type of costimulatory domain. Potential mechanisms of resistance include CAR T-cell exhaustion and immune evasion, CD19 antigen loss, and a lack of persistence. Rational combination strategies with CAR T cells are under evaluation, including immune checkpoint inhibitors, immunomodulators, and tyrosine kinase inhibitors. Novel cell products are also being developed and include CAR T cells that target multiple tumor antigens, cytokine-secreting CAR T cells, and gene-edited CAR T cells, among others.

CAR-T Therapy for Lymphoma with Prophylactic Tocilizumab: Decreased Rates of Severe Cytokine Release Syndrome without Excessive Neurologic Toxicity

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-31 ◽  
Author(s):  
Paolo F Caimi ◽  
Ashish Sharma ◽  
Patricio Rojas ◽  
Seema Patel ◽  
Jane Reese ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Advisory Board ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Car T Cells ◽  
Car T Cell ◽  
Significant Difference ◽  
Car T

INTRODUCTION: Anti-CD19 chimeric antigen receptor T (CAR-T) cells have demonstrated activity against relapsed/refractory lymphomas. Cytokine release syndrome (CRS) and CAR-T related encephalopathy syndrome (CRES/ICANS) are well-known complications of CAR-T cell therapy. Tocilizumab, a humanized monoclonal antibody targeting the interleukin 6 (IL-6) receptor, is approved for treatment of CRS. Our institutional standard was modified to administer prophylactic tocilizumab before infusion CAR-T cell products. We present the outcomes of subjects treated with locally manufactured antiCD19 CAR-T cells (TNFRSF19 transmembrane domain, CD3Zeta/4-1BB costimulatory signaling) with and without prophylactic tocilizumab. METHODS: Relapsed / refractory (r/r) lymphoma patients (pts) treated with anti-CD19 CAR-T cells at our institution were included. Baseline demographic and clinical characteristics, as well as laboratory results were obtained from our Hematologic Malignancies and Stem Cell Therapy Database. Prior to institution of prophylactic tocilizumab, pts received this agent only if they presented evidence of CRS grade 2 or higher. In May 2019, our institutional practice changed to provide tocilizumab 8mg/kg, 1 hour prior to infusion of CAR-T cell product. CRS was measured according to the ASTCT Consensus Grading, whereas CRES was measured using the CARTOX-10 criteria. Comparisons between groups were done with the Mann-Whitney U test for continuous variables and Fisher's exact test for categorical variables. RESULTS: Twenty-three relapsed / refractory lymphoma pts were treated with antiCD19 CAR-T cells; 15 pts received prophylactic tocilizumab. Median follow up was 312 days (range 64 - 679) days. Baseline characteristics are listed in table 1. Both groups were similar: There were no statistically differences in the rate of bulky, refractory disease, prior ASCT or number or prior lines of therapy. Baseline lymphocyte counts, C - reactive protein (CRP) and were also comparable between groups (Table 2). We did not observe immune adverse reactions to tocilizumab infusion. There were no differences in the incidence of cytopenias or infectious complications between groups. CRS of any grade was observed in 6/8 (75%) of pts without prophylactic tocilizumab vs. 6/15 (40%) in pts treated with prophylactic tocilizumab (p = 0.23), whereas CRS grade >1 was observed in 5 pts (62.5%) without prophylactic tocilizumab and in 3 pts (20%) treated with prophylactic tocilizumab (p = 0.02). There was no significant difference in the incidence of all grade CRES (no prophylaxis, 3/8 [38%] pts; prophylaxis 5/15 [30%] pts, p = 0.2969). There was a statistically significant difference in the peak CRP and peak ferritin without difference in the peak lymphocyte count after CAR-T infusion (Table 2, Figure 1). Patients given prophylactic tocilizumab had higher IL-6 plasma concentrations on day 2 after infusion (Figure 2). Complete response was observed in 4/8 (50%) pts without prophylactic tocilizumab vs. 12/15 (80%) pts with prophylactic tocilizumab (p = 0.18). All pts had detectable Anti-CD19 CAR-T cells on day 30, both groups had peak CAR-T expansion on day 14, with no statistically significant differences in expansion rates between groups. All evaluable subjects have had CAR-T persistence on days 60, 90, 180, and 365. CONCLUSIONS: Use of prophylactic tocilizumab prior to infusion of antiCD19 CAR-T cells is associated with reduced incidence of severe CRS and decreased levels of clinical laboratory markers of inflammation, despite increases in plasma concentration of IL-6. This decreased rate of grade ≥2 CRS is not associated with impaired disease control and did not result in increased rates of neurologic toxicity. Prophylactic tocilizumab does not appear to affect CAR-T cell expansion or persistence. Figure 1 Disclosures Caimi: ADC therapeutics: Other: Advisory Board, Research Funding; Celgene: Speakers Bureau; Amgen: Other: Advisory Board; Bayer: Other: Advisory Board; Verastem: Other: Advisory Board; Kite pharmaceuticals: Other: Advisory Board. Worden:Lentigen, a Miltenyi biotec company: Current Employment. Kadan:Lentigen, a Miltenyi biotec company: Current Employment. Orentas:Lentigen Technology, a Miltenyi Biotec Company: Research Funding. Dropulic:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. de Lima:Celgene: Research Funding; Pfizer: Other: Personal fees, advisory board, Research Funding; Kadmon: Other: Personal Fees, Advisory board; Incyte: Other: Personal Fees, advisory board; BMS: Other: Personal Fees, advisory board. OffLabel Disclosure: Use of tocilizumab as prophylaxis for CRS is not approved, whereas use for treatment is approved and on label.

scholarly journals Development of CAR-T Cell Constructs with Broad Anti-Tumor Tropism

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 24-24
Author(s):  
Ameet K. Mishra ◽  
Iris Kemler ◽  
David Dingli
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Car T Cells ◽  
Car T Cell ◽  
Nsg Mice ◽  
Human T Cells ◽  
Car T

Chimeric antigen receptor T (CAR-T) cell therapy is a transformative approach to cancer eradication. CAR-T is expensive in part due to the restricted use of each CAR construct for a specific set of tumors such as B cell lymphoma targeted with CD19 and multiple myeloma targeted with BCMA. A CAR construct with broad anti-tumor activity can be advantageous due to wide applicability and scalability of production. We show that CD126, the IL-6 receptor alpha, is an antigen that is expressed by many hematologic and solid malignancies including multiple myeloma, non-Hodgkin lymphoma, acute myeloid leukemia, pancreatic and prostate adenocarcinoma, non-small cell lung cancer and malignant melanoma amongst others. High CD126 expression is a negative prognostic marker in many malignancies. The two CD126 targeting CAR-T cell constructs contain the CD28 anchoring domain followed by 4-1BB and CD3 zeta signaling domain. Lentiviral vectors were generated with triple plasmid (CAR, psPAX2 and pMD2.G) transfection of 293T cells and the vector concentrated by ultracentrifugation and used to transduce human T cells. T cells were isolated from leuko-reduction cones using negative selection with magnetic beads. The transduction efficiency was around 60%. The T cells were activated with anti-CD3/CD28 beads and expanded for two weeks before using for downstream experiments. CD126 CAR-T cells are able to kill many tumor cells in an antigen specific manner and with an efficiency that is directly proportional to the cell surface expression of CD126 expression (rho = 0.6, p = 0.0019). The presence of soluble CD126 in the culture media did not interfere with CAR-T cell killing. The CAR-T constructs bind murine CD126. However, injection of CD126 targeting CAR-T cells in NSG mice did not lead to any evidence of hepatotoxicity and weight loss despite possible expression of this antigen on hepatocytes. In vivo studies in NSG mice with multiple myeloma (RPMI-8226) and prostate adenocarcinoma (DU-145) xenograft models (n=10 tumors per group) showed that the intravenously injected CD126 targeted CAR-T cells (107) infiltrated the tumors, expanded, produced human interferon gamma and killed the tumor cells (p<0.001). Bioluminescence imaging showed control of tumor growth in the actively treated tumors compared to the controls (p<0.05). At post mortem, mice injected with CD126 targeted CAR-T cells had smaller residual tumors compared to controls injected with non-engineered human T cells from the same donor. Binding of sIL-6R by CAR-T cells could mitigate cytokine release syndrome. In support of this, murine SAA-3 levels (the equivalent of human CRP) were lower in mice injected with CD126 CAR-T compared to controls (p<0.05), suggesting that binding of sIL-6R by CAR-T cells could mitigate cytokine release syndrome. CD126 provides a novel therapeutic for CAR-T cells in a broad variety of tumors with low risk of toxicity. Disclosures Dingli: Apellis: Consultancy; Millenium: Consultancy; Janssen: Consultancy; Bristol Myers Squibb: Research Funding; Sanofi-Genzyme: Consultancy; Alexion: Consultancy; Rigel: Consultancy; Karyopharm Therapeutics: Research Funding.

Remissions of Multiple Myeloma during a First-in-Humans Clinical Trial of T Cells Expressing an Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. LBA-1-LBA-1 ◽  
Author(s):  
Syed Abbas Ali ◽  
Victoria Shi ◽  
Michael Wang ◽  
David Stroncek ◽  
Irina Maric ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Plasma Cells ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Bone Marrow Plasma

Abstract B-cell maturation antigen (BCMA) is a protein expressed by normal and malignant plasma cells. We are conducting a phase I clinical trial of an anti-BCMA chimeric antigen receptor (CAR-BCMA) that incorporates an anti-BCMA single-chain variable fragment, a CD28 domain, and a CD3-zeta T-cell activation domain (Carpenter et al. Clinical Cancer Research 2013). Autologous T cells are genetically modified to express the CAR with a gamma-retroviral vector. Patients receive a single infusion of CAR-BCMA T cells. Before the CAR T-cell infusions, patients receive a chemotherapy regimen of 300 mg/m2 of cyclophosphamide and 30 mg/m2 of fludarabine with each chemotherapy agent given daily for 3 days. The purpose of the chemotherapy is to enhance activity of the CAR T cells by depleting endogenous leukocytes. Twelve patients have been enrolled, and 11 patients have been treated on one of 4 dose levels, 0.3x106, 1x106, 3x106, and 9x106CAR+ T cells/kg of bodyweight. Patients had advanced multiple myeloma (MM) with a median of 7 prior lines of therapy. Of the 6 patients treated on the lowest 2 dose levels, one patient had a transient partial remission (PR) of 2 weeks duration; the other 5 patients had responses of stable disease (SD). On the 3rddose level, 2 patients obtained responses of stable disease, and one patient obtained a response of very good PR (VGPR) with complete elimination of MM bone disease on positron emission tomography (PET) scan, normalization of serum free light chains, and clearance of bone marrow plasma cells. Toxicity among patients on the first 3 dose levels was mild and included cytopenias attributable to chemotherapy, fever in 3 patients, and signs of cytokine release syndrome including tachycardia and hypotension in Patient 8 who had a VGPR. Two patients have been treated on the highest dose level of 9x106CAR+ T cells/kg. The first patient on this dose level, Patient 10, had MM making up 90% of total bone marrow cells before treatment. Starting 4 hours after infusion of CAR T cells, Patient 10 exhibited signs of cytokine release syndrome including fever, tachycardia, dyspnea, acute kidney injury, coagulopathy, hypotension requiring vasopressor support, and muscle damage manifesting as an elevated creatine kinase level and weakness. His neutrophil count was less than 500/µL before the CAR-BCMA T-cell infusion and remained below 500/µL for 40 days after the CAR T-cell infusion before recovering. He also experienced prolonged thrombocytopenia. Patient 10’s myeloma was rapidly eliminated after CAR-BCMA T-cell infusion. By immunohistochemistry staining for CD138, bone marrow plasma cells decreased from 90% before treatment to 0% one month after the CAR T-cell infusion. The serum M-protein decreased from 1.6 g/dL before treatment to undetectable 2 months after treatment. The serum and urine immunofixation electrophoresis tests were negative 2 months after the CAR T-cell infusion. Patient 10’s current myeloma response is stringent complete remission. The second patient treated on the 9x106CAR+ T cells/kg dose level, Patient 11, had IgG lambda MM with 80% bone marrow plasma cells before treatment. Patient 11 experienced signs of cytokine release syndrome with toxicities including fever, tachycardia, hypotension, delirium, hypoxia, and coagulopathy. Patient 11’s M-protein decreased from 3.6 g/dL before treatment to 0.8 g/dL 4 weeks after treatment. His serum lambda free light chain decreased from 95.9 mg/dL before treatment to 0.15 mg/dL 4 weeks after treatment. Four weeks after CAR T-cell infusion, bone marrow plasma cells were undetectable. T cells containing the CAR-BCMA gene were detected in the blood of all 10 patients evaluated with peak levels of 0.04 to 18.2% of blood mononuclear cells. Patient 10 had the highest peak absolute number of blood CAR T cells with 51 CAR+ T cells/µL. Blood levels of IL-6 and other inflammatory cytokines were highest in patients with clinical signs of cytokine release syndrome, and the 3 patients with the highest serum IL-6 levels also had the most impressive anti-myeloma responses. Before treatment, the mean serum BCMA level of treated patients was 243 ng/mL. In responding patients, serum BCMA levels decreased after treatment. Toxicities in patients receiving CAR-BCMA T cells were similar to toxicities in leukemia patients treated with anti-CD19 CAR T cells. Our findings demonstrate strong anti-myeloma activity in the first clinical trial of a CAR targeting BCMA. Disclosures: Wang: Celgene: Research Funding. Kochenderfer:bluebird bio Inc.: Research Funding. Off Label Use: Use of cyclophosphamide and fludarabine as a conditioning regimen for adoptively-transferred T cells will be part of the presentation.

scholarly journals Monocytes Are Required for Both Optimal Anti-Leukemic Efficacy and the Cytokine Release Syndrome By CAR-T Cells: Lessons from an Innovative Xenotolerant Mouse Model

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 997-997
Author(s):  
Margherita Norelli ◽  
Monica Casucci ◽  
Barbara Camisa ◽  
Laura Falcone ◽  
Catia Traversari ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Secondary Transfer

Abstract Background: Chimeric antigen-receptor (CAR)-engineered T cells promise to cure chronic and acute leukemias refractory to standard treatments. Before this promise is fulfilled, however, two crucial issues need to be solved: i) how to circumvent the emergence of secondary resistance (e.g. due totarget-antigen loss; leukemic lineage switch); ii) how to manage associated toxicities (e.g. the cytokine release syndrome, CRS; lineage aplasias). Unfortunately, all these issues cannot be addressed pre-clinically in currently available NSG mouse models, because they lack human hematopoiesis and, furthermore, ultimately develop xenograft-versus-host disease (X-GVHD), preventing the evaluation of long-term effects. Methods: We have developed an innovative xenotolerant model by transplanting human hematopoietic stem cells (HSCs) intraliver in newborn NSG mice triple transgenic for human SCF, GM-SCF and IL-3 (SGM3). Differently from "classical" NSG, SGM3 mice reconstituted high levels of human T cells (>1000 cells per microL at week 8), which, once transferred in secondary recipients, persisted up to 200d without causing X-GVHD, even after irradiation. Robust and specific xenotolerance was confirmed by in vitrohyporesponsiveness to NSG, bot not to C57/Bl6 antigens (irradiated splenocytes) or human HLAs (PBMCs). Secondary transfer experiments in leukemic and/or HSC-humanized SGM-3 mice have been then designed for studying the determinants of CAR-T cell efficacy and associated toxicities in the absence of confounding xenoreactivity. Results: SGM3-derived T cells were transduced ex vivo with either a CD19 or a CD44v6 CAR (both having a CD28 2G design) after activation with CD3/CD28-beads and IL-7/IL-15, resulting in a preferential and functional CD45RA+/CD62L+/CD95+ stem memory T cell (TSCM) phenotype. Once transferred in secondary recipients previously engrafted with a CD19+/CD44v6 leukemic cell line, CD19 or CD44v6 CAR-T cells equally mediated rapid tumor clearance both in low and high tumor-burden settings, in the absence of malaise or elevated human IL-6 levels in vivo. At later time points (after 100d), however, approximately 50% of responding mice relapsed despite significant CAR-T cell persistence in vivo (>50 cells per microL). A significant fraction of leukemia relapses were characterized by post-transcriptional down-regulation of CD44v6 expression or CD19 loss, respectively. Conversely, secondary transfer of SGM3-derived CAR-T cells in leukemic SGM3 mice that had been previously humanized with HSCs resulted in the development of a clinical syndrome similar to the CRS observed in clinical trials (high fevers, elevated IL-6, TNF-alpha and serum amyloid A levels - mouse analog of C-reactive protein in humans), resulting in 30% lethality. This CRS was anticipated and shortened for CD44v6 compared with CD19 CAR-T cells and worse in the case of 4-1BB compared with the original CD28 2G CAR designs. Strikingly, mice recovering from the CRS benefited from durable leukemic remissions, yet experienced long-lasting CD19+ B-cell or CD44v6+ monocyte aplasias. Deepness of remission was confirmed in "tertiary" recipients, which did not develop leukemia after the infusion of bone-marrow cells from mice in remission 150d since CAR-T cell infusion. Interestingly, in this model, tocilizumab administration at the time of either CD19 or CD44v6 CAR-T cell infusion efficiently prevented the CRS, but did not interfere with their comparable and long-term anti-leukemic effects. Conversely, depleting monocytes/macrophages before therapeutic CAR-T cell infusion by either lyposomal clodronate or by the prophylactic CD44v6 CAR-T cells inhibited CRS development, but also resulted in significantly worse leukemia-free survival (at 250d, 0% vs 80%, P<0.0001). Conclusions: A number of lessons can be learned from this innovative xenotolerant mouse model of CAR-T cell immunotherapy: monocytes are required for both i) optimal anti-leukemic efficacy, and ii) the occurrence of CRS; iii) tocilizumab prevents the CRS without interfering with efficacy; iv) monocyte aplasia induced by CD44v6 CAR-T cells does not impact on their efficacy, at least in the theraeputic setting, and may ameliorate CRS toxicity. As for CD44v6 CAR-T cells, this model could be used for effectively predicting the efficacy and associated toxicities of new CAR-T cell therapies, speeding up their clinical development. Disclosures Traversari: MolMed SpA: Employment. Bordignon:MolMed SpA: Employment. Ciceri:MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy. Bondanza:Formula Pharmaceuticals: Honoraria; TxCell: Research Funding; MolMed SpA: Research Funding.

scholarly journals Preclinical Evaluation of ALLO-819, an Allogeneic CAR T Cell Therapy Targeting FLT3 for the Treatment of Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3921-3921 ◽  
Author(s):  
Cesar Sommer ◽  
Hsin-Yuan Cheng ◽  
Yik Andy Yeung ◽  
Duy Nguyen ◽  
Janette Sutton ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Target Cells ◽  
Employment Equity ◽  
T Cell Therapy ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Autologous chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B-cell leukemias, lymphomas and multiple myeloma, raising interest in using CAR T cell therapies in AML. These therapies are produced using a patient's own T cells, an approach that has inherent challenges, including requiring significant time for production, complex supply chain logistics, separate GMP manufacturing for each patient, and variability in performance of patient-derived cells. Given the rapid pace of disease progression combined with limitations associated with the autologous approach and treatment-induced lymphopenia, many patients with AML may not receive treatment. Allogeneic CAR T (AlloCAR T) cell therapies, which utilize cells from healthy donors, may provide greater convenience with readily available off-the-shelf CAR T cells on-demand, reliable product consistency, and accessibility at greater scale for more patients. To create an allogeneic product, the TRAC and CD52 genes are inactivated in CAR T cells using Transcription Activator-Like Effector Nuclease (TALEN®) technology. These genetic modifications are intended to minimize the risk of graft-versus-host disease and to confer resistance to ALLO-647, an anti-CD52 antibody that can be used as part of the conditioning regimen to deplete host alloreactive immune cells potentially leading to increased persistence and efficacy of the infused allogeneic cells. We have previously described the functional screening of a library of anti-FLT3 single-chain variable fragments (scFvs) and the identification of a lead FLT3 CAR with optimal activity against AML cells and featuring an off-switch activated by rituximab. Here we characterize ALLO-819, an allogeneic FLT3 CAR T cell product, for its antitumor efficacy and expansion in orthotopic models of human AML, cytotoxicity in the presence of soluble FLT3 (sFLT3), performance compared with previously described anti-FLT3 CARs and potential for off-target binding of the scFv to normal human tissues. To produce ALLO-819, T cells derived from healthy donors were activated and transduced with a lentiviral construct for expression of the lead anti-FLT3 CAR followed by efficient knockout of TRAC and CD52. ALLO-819 manufactured from multiple donors was insensitive to ALLO-647 (100 µg/mL) in in vitro assays, suggesting that it would avoid elimination by the lymphodepletion regimen. In orthotopic models of AML (MV4-11 and EOL-1), ALLO-819 exhibited dose-dependent expansion and cytotoxic activity, with peak CAR T cell levels corresponding to maximal antitumor efficacy. Intriguingly, ALLO-819 showed earlier and more robust peak expansion in mice engrafted with MV4-11 target cells, which express lower levels of the antigen relative to EOL-1 cells (n=2 donors). To further assess the potency of ALLO-819, multiple anti-FLT3 scFvs that had been described in previous reports were cloned into lentiviral constructs that were used to generate CAR T cells following the standard protocol. In these comparative studies, the ALLO-819 CAR displayed high transduction efficiency and superior performance across different donors. Furthermore, the effector function of ALLO-819 was equivalent to that observed in FLT3 CAR T cells with normal expression of TCR and CD52, indicating no effects of TALEN® treatment on CAR T cell activity. Plasma levels of sFLT3 are frequently increased in patients with AML and correlate with tumor burden, raising the possibility that sFLT3 may act as a decoy for FLT3 CAR T cells. To rule out an inhibitory effect of sFLT3 on ALLO-819, effector and target cells were cultured overnight in the presence of increasing concentrations of recombinant sFLT3. We found that ALLO-819 retained its killing properties even in the presence of supraphysiological concentrations of sFLT3 (1 µg/mL). To investigate the potential for off-target binding of the ALLO-819 CAR to human tissues, tissue cross-reactivity studies were conducted using a recombinant protein consisting of the extracellular domain of the CAR fused to human IgG Fc. Consistent with the limited expression pattern of FLT3 and indicative of the high specificity of the lead scFv, no appreciable membrane staining was detected in any of the 36 normal tissues tested (n=3 donors). Taken together, our results support clinical development of ALLO-819 as a novel and effective CAR T cell therapy for the treatment of AML. Disclosures Sommer: Allogene Therapeutics, Inc.: Employment, Equity Ownership. Cheng:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Yeung:Pfizer Inc.: Employment, Equity Ownership. Nguyen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Sutton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Melton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Valton:Cellectis, Inc.: Employment, Equity Ownership. Poulsen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Djuretic:Pfizer, Inc.: Employment, Equity Ownership. Van Blarcom:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Chaparro-Riggers:Pfizer, Inc.: Employment, Equity Ownership. Sasu:Allogene Therapeutics, Inc.: Employment, Equity Ownership.

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

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

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

scholarly journals A New Era in Endothelial Injury Syndromes: Toxicity of CAR-T Cells and the Role of Immunity

2020 ◽  
Vol 21 (11) ◽  
pp. 3886
Author(s):  
Eleni Gavriilaki ◽  
Ioanna Sakellari ◽  
Maria Gavriilaki ◽  
Achilles Anagnostopoulos
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hematopoietic Cell Transplantation ◽  
Endothelial Injury ◽  
Common Denominator ◽  
Cell Toxicity ◽  
New Era ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Immunotherapy with chimeric antigen receptor T (CAR-T cells) has been recently approved for patients with relapsed/refractory B-lymphoproliferative neoplasms. Along with great efficacy in patients with poor prognosis, CAR-T cells have been also linked with novel toxicities in a significant portion of patients. Cytokine release syndrome (CRS) and neurotoxicity present with unique clinical phenotypes that have not been previously observed. Nevertheless, they share similar characteristics with endothelial injury syndromes developing post hematopoietic cell transplantation (HCT). Evolution in complement therapeutics has attracted renewed interest in these life-threatening syndromes, primarily concerning transplant-associated thrombotic microangiopathy (TA-TMA). The immune system emerges as a key player not only mediating cytokine responses but potentially contributing to endothelial injury in CAR-T cell toxicity. The interplay between complement, endothelial dysfunction, hypercoagulability, and inflammation seems to be a common denominator in these syndromes. As the indications for CAR-T cells and patient populations expand, there in an unmet clinical need of better understanding of the pathophysiology of CAR-T cell toxicity. Therefore, this review aims to provide state-of-the-art knowledge on cellular therapies in clinical practice (indications and toxicities), endothelial injury syndromes and immunity, as well as potential therapeutic targets.

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