scholarly journals Cord Blood T Regulatory Cells Can Dampen Cytokine Release Syndrome and Improve on Target Efficacy of CD19 CAR T Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 15-16
Author(s):  
Ke Zeng ◽  
Li Li ◽  
Meixian Huang ◽  
Mi-Ae Lyu ◽  
Mitsutaka Nishimoto ◽  
...  
Keyword(s):  
T Cells ◽  
Inflammatory Cytokines ◽  
Research Funding ◽  
Treg Cells ◽  
Tumor Burden ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Raji Cells ◽  
Car T Cells ◽  
Car T

Background.As a T cell driven process where release of inflammatory cytokines as a result of the proliferation and on target cell kill by chimeric antigen receptor (CAR)-T cells, cytokine release syndrome (CRS) can be potentially targeted by adoptive therapy with T regulatory (Treg) cells. Specifically, allogeneic cord blood (CB) derived Treg cells have now shown safety and efficacy in graft vs host disease (GVHD), we hypothesized that CB Treg cell therapy can be exploited for treatment of CRS. Method.Xenogenic lymphoma model was created using NSG mice where 0.3x106 GFP-labeled Raji cells were injected on day 0 in all mice followed by 0.3x106 cells of i) mock-CAR T, ii) no CART, ii) CD19-CAR T cells on day +5. Additional injections of 1x107 CB Treg cells on day +11, +18, +25 were added to the no CAR T arm and the CD19-CAR T arm such that there were 3 mice per arm. Mice were followed for weight, GVHD score and survival. Non-invasive bioluminescence was used to perform serial imaging to evaluate the tumor burden. Serial blood was drawn for cell analysis and cytokine assay. Result. As shown in figure A, in vivo proliferation of GFP-labeled Raji cells was evident in all mice day by day +4. CD19-CAR T but not the mock-CAR T cells decreased the tumor burden at day+11. However, at day +14 all mice including CD19-CAR T cell recipients showed progression whereas CD19-CAR T+CB Treg cell recipient showed no evidence of bioluminescence. A superior survival in the CD19-CAR T+CB Treg cells recipients was evident when compared to other treatment arms (Table A). At the time of euthanasia, different organs were evaluated for the detection of the CD19-CART cells and were recovered only in the CD19-CART+CB Treg cells recipients (Table B) . The CD19-CAR T recipients showed an increase in the inflammatory cytokines on day +16 PB samples including IFN-gamma (Figure B) and TNF-alpha (Figure C) which were decreased in the CD19-CAR T + CB Treg arm. Furthermore, a reciprocal increase of the anti-inflammatory cytokine IL-1RA was observed in the CD19-CAR T + CB Treg arm compared to the CD19-CAR T alone (Figure D). Conclusion. The addition of CB Treg cells to CD19-CAR T cells in a xenogenic lymphoma model led to dampening of the cytokine storm and improved on target efficacy of CAR T cells. This combination should be examined in clinical setting. Disclosures Sadeghi: Cellenkos Inc.:Current Employment.Nastoupil:Genentech, Inc.:Honoraria, Research Funding;Karus Therapeutics:Research Funding;Bayer:Honoraria;Gamida Cell:Honoraria;Gilead/KITE:Honoraria;Novartis:Honoraria, Research Funding;Merck:Research Funding;TG Therapeutics:Honoraria, Research Funding;LAM Therapeutics:Research Funding;Janssen:Honoraria, Research Funding;Pfizer:Honoraria, Research Funding;Celgene:Honoraria, Research Funding.Patel:Oncopeptides:Consultancy;Janssen:Consultancy, Research Funding;Precision Biosciences:Research Funding;Takeda:Consultancy, Research Funding;Nektar:Consultancy, Research Funding;Celgene:Consultancy, Research Funding;Bristol Myers Squibb:Consultancy, Research Funding;Poseida:Research Funding;Cellectis:Research Funding.Parmar:Cellenkos Inc.:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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 In vivo generation of human CD 19‐ CAR T cells results in B‐cell depletion and signs of cytokine release syndrome

2018 ◽  
Vol 10 (11) ◽  
Author(s):  
Anett Pfeiffer ◽  
Frederic B Thalheimer ◽  
Sylvia Hartmann ◽  
Annika M Frank ◽  
Ruben R Bender ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Cell Depletion ◽  
B Cell Depletion ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Car T Cells ◽  
Car T

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

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

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

scholarly journals Novel Chimeric Antigen Receptor T Cells for the Treatment of Hodgkin Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 806-806 ◽  
Author(s):  
Marco Ruella ◽  
Saad S Kenderian ◽  
Olga Shestova ◽  
Taylor Chen ◽  
John Scholler ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
B Cell ◽  
Cell Line ◽  
Immune Cells ◽  
Research Funding ◽  
Tumor Burden ◽  
Car T Cells ◽  
Car T

Abstract Hodgkin lymphoma (HL) generally carries a good prognosis. However, 10-15% of patients relapse or are refractory to first-line therapy. These patients have a poor prognosis and would benefit from innovative approaches. Our group and others have demonstrated the clinical efficacy of anti-CD19 chimeric antigen receptor redirected T cells (CART19, CTL019) for refractory B cell malignancies. Despite the B-cell origin of the malignant Hodgkin Reed-Sternberg (HRS) cells, B-cell antigens, in particular CD19, are typically not expressed in HL. We sought to define a HL-associated cell membrane antigen that could be targeted by CAR T cells. Given the relative paucity of the malignant cells and the importance of the immunosuppressive tumor microenvironment in HL, the ideal target would be expressed on neoplastic cells as well as on infiltrating immune cells in order to provide robust stimulation of the CAR T cells. Immunohistochemistry for novel HL targets on 10 patient samples revealed that 5/10 patients expressed CD123 on the HRS cells. CD123 was also seen on immune cells of the microenvironment in most samples. CD123 is the α chain of the receptor for interleukin-3 (IL-3), an important cytokine in hematopoietic growth and differentiation that has been previously shown to promote HL cell line growth (Aldinucci et al, Leuk & Lymph, 2005). As primary HL is non-engraftable in mice we turned to immortalized HL cell lines and confirmed that CD123 is expressed by flow cytometry and Q-PCR in four different HL cell lines (HDLM-2, KMH2, SUPHD1, and L428). To determine the role of IL-3 signaling in HL we engrafted NOD-SCID-γ-chain KO mice that overexpress human cytokines including IL-3 (NSG-S mice) with the luciferase-expressing HDLM-2 cell line. After i.v. injection, the neoplastic cells progressively formed disseminated soft tissue masses. Serial injections of a neutralizing anti-IL3 antibody slowed the growth of tumor, suggesting that CD123 may be a particularly relevant target in HL. We therefore sought to investigate the utility of anti-CD123 CAR T cells (CART123) for the treatment of HL. We have recently described the activity of CART123 in human acute myeloid leukemia (Gill et al, Blood, 2014). Our construct is a 2nd generation CAR, comprising 4-1BB co-stimulatory and CD3-ζ chain signaling domains with an anti-CD123 scFv. In vitro, CART123 specifically degranulate, proliferate, produce cytokines and kill HL cells (Table 1). Moreover, long-term co-culture (20 days) of CART123 with HDLM-2 cells at a 1:1 ratio led to T cell proliferation and complete elimination of HL cells by day 4. To confirm these in vitro data, we developed a rigorous in vivo model injecting 1 million luciferase+ HDLM-2 cells i.v. on day 0. Serial bioluminescent imaging (BLI) demonstrated low level of tumor on day 7, which was followed by gradual increase in tumor burden over approximately 6 weeks, reproducing the indolent nature of the human disease. At day 43 when the tumor burden was 20-fold higher than baseline, mice were treated with 1.5 million CART123 cells or control T cells. CART123 induced complete and durable eradication of disseminated tumor within 14 days, leading to 100% relapse-free and 100% overall survival at 6 months (Figure 1 and 2). Tumor elimination was associated with extensive CAR T cell expansion as detected by flow cytometry in serial peripheral blood bleedings. In summary, we show for the first time that human CD123-redirected T cells display potent therapeutic activity against disseminated HL. We have previously demonstrated that CART123 lead to myelosuppression, suggesting that our findings could be translated to treat patients with refractory HL with a combined CART123 and rescue autologous bone marrow transplantation. Table 1 In vitro activity of CART123 compared to untransduced control T cells (UTD) against a HL cell line (HDLM-2). IN VITRO EXPERIMENT CART123* UTD CD107a Degranulation (4 hrs, E:T = 1:5) 59.3% 2.69% Specific Killing (24 hrs) E:T = 2:1 57% 5% E:T = 0.25:1 27% 1% Proliferating cells (CFSE based) (5 days, E:T = 1:1) 96.4% 20% Cytokine production (24 hrs, E:T = 1:1) (Luminex, MFI) INF-γ 38,265 42 IL-2 85,604 0 TNF-α 10,684 55 MIP-1β 40,038 111 IL-6 16,425 110 GM-CSF 99,915 285 *All P values are <0.05 when compared to UTD Figure 2 Figure 2. Disclosures Ruella: Novartis: Research Funding. Kenderian:Novartis: Research Funding. Shestova:Novartis: Research Funding. Chen:Novartis: Research Funding. Scholler:Novartis: Research Funding. June:Novartis: Patents & Royalties, Research Funding. Gill:Novartis: Research Funding.

scholarly journals Therapeutic Targeting of Monokine Production Is a Promising Strategy to Attenuate Cytokine-Release Syndrome in CAR-T Cell Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2067-2067
Author(s):  
Muneyoshi Futami ◽  
Keisuke Suzuki ◽  
Satomi Kato ◽  
Yoshio Tahara ◽  
Yoichi Imai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Cytokine Production ◽  
Cytokine Release ◽  
Killing Effect ◽  
Car T Cells ◽  
Car T Cell ◽  
Tnf Α ◽  
Car T

Cancer immunotherapy using chimeric antigen receptor-armed T cells (CAR-T cells) have shown excellent outcomes in hematological malignancies. However, cytokine release syndrome (CRS), characterized by excessive activation of CAR-T cells and macrophages remains to be overcome. Steroid administration usually resolves signs and symptoms of CRS but abrogates CAR-T cell expansion and persistence. Tocilizumab, a humanized monoclonal antibody against interleukin-6 receptor (IL-6R), attenuates CRS without significant loss of CAR-T cell activities, while perfect rescue of CRS symptoms cannot be achieved by IL-6/IL-6R blockade. There is actual need for novel strategies to prevent or cure CRS. TO-207, an N-benzoyl-L-phenylalanine derivative compound, significantly inhibits inflammatory cytokine production in a human monocyte/macrophage-specific manner. Here we tested TO-207 for its ability to inhibit cytokine production without impaired CAR-T cell function in a CRS-simulating co-culture system consisting of CAR-T cells, target leukemic cells and monocytes. To observe a precise pattern of cytokine release from CAR-T cells and monocytes, we first established a co-culture system that mimics CRS using K562/CD19 cells, 19-28z CAR-T cells, and peripheral blood CD14+ cells. IFN-γ was produced exclusively from CAR-T cells, and TNF-α, MIP-1α, M-CSF, and IL-6 were produced from both CAR-T cells and monocytes, but monocytes were the major source of these cytokine production. MCP-1, IL-1β, IL-8, and IL-10 were released exclusively from monocytes. To observe the effect of drugs on cytokine production, prednisolone (PSL), TO-207, tocilizumab, and anakinra (an IL-1R antagonist) were added to the co-culture. PSL exhibited suppressive effects on TNF-α and MCP-1 production. Tocilizumab did not suppress these cytokines. Anakinra up-regulated IL-6 and IL-1β production, probably due to activation of negative feedback loops. Interestingly, TO-207 widely suppressed all of these monocyte-derived cytokines including TNF-α, IL-6, IL-1β, MCP-1, IL-8, and GM-CSF. Next, we observed whether the cytokine inhibition by TO-207 attenuates killing effect of CAR-T cells. PSL attenuated killing effect of CD4+ CAR-T cells and CD8+ CAR-T cells toward K562/CD19 cells. In contrast, TO-207 did not exhibit any change in cytotoxicity of CD4+ CAR-T cells and CD8+ CAR-T cells. To determine whether the effect of PSL and TO-207 on cytotoxicity changes in the presence of CD14+ monocytes, CD14+ cells were added to the co-culture. In the absence of CAR-T cells, PSL induced a modest attenuation of cytotoxicity, whereas to the CAR-T cells, PSL exhibited a significant attenuation of cytotoxicity. TO-207 exhibited a minimal effect on cytotoxicity in the absence or presence of CAR-T cells. These results suggested that CAR-T cells play a major role in the cytotoxicity toward leukemia cells, and drugs that do not affect CAR-T cell functions, such as TO-207, maintain their cytotoxic effects on leukemia cells. In conclusion, our present co-culture model with K562/CD19 cells, 19-28z CAR-T cells, and CD14+ monocytes accurately recapitulate killing effect and cytokine release profiles. IFN-γ was produced exclusively by CAR-T cells, but majority of other cytokines such as TNF-α, MIP-1α, M-CSF, IL-6, MCP-1, IL-1β, IL-8, and IL-10 were from CD14+ monocytes/macrophages. Because killing effect was largely dependent on CAR-T cells while cytokine production was dependent on monocytes/macrophages, selective inhibition of pro-inflammatory cytokines from monocytes by TO-207 would be ideal for treatment of CAR-T-related CRS. These results encourage us to consider a clinical application for CRS. Figure Disclosures Futami: Torii Pharmaceutical: Research Funding. Suzuki:Torii Pharmaceutical: Employment. Kato:Torii Pharmmaceutical: Research Funding. Tahara:Torii Pharmaceutical: Employment. Imai:Celgene: Honoraria, Research Funding; Janssen Pharmaceutical K.K: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Mimura:Torii Pharmaceutical: Employment. Watanabe:Torii Pharmaceutical: Employment. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding.

scholarly journals Anti-CD19 ARTEMISTM Therapy Drastically Reduces Cytokine Release without Compromising Efficacy Against Preclinical Lymphoma Models

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3354-3354
Author(s):  
Hong Liu ◽  
Li Long ◽  
Shon Green ◽  
Lucas H Horan ◽  
Bryan Zimdahl ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Cytokine Release ◽  
Raji Cells ◽  
Car T Cells ◽  
Nsg Mice ◽  
Car T ◽  
Equity Ownership

Abstract Anti-CD19 chimeric antigen receptor (CAR) T cell therapies for B cell malignancies have demonstrated the remarkable curative potential of T cell immunotherapies. However, in clinical trials anti-CD19-CAR T cells continue to trigger life threatening adverse events that are often associated with excessive cytokine release and excessive T-cell proliferation. We reasoned that the activation pathway of current CAR T cells could be altered to better regulate proliferation and cytokine secretion, and thus disentangle the correlation between cytokine release syndrome (CRS) and efficacy of T cell-based therapies. Through protein engineering, we developed the ARTEMISTM (1) signaling platform which when expressed on primary T-cells results in a dramatic reduction of cytokine release during tumor cell lysis, without sacrificing efficacy. Using a human phage display library, we also identified several human CD19 antibodies with improved specificity and affinity that will be less immunogenic as compared to the murine-derived anti-CD19 antibodies that are currently used in most trials. Our lead antibody clone CD19(7) was then engineered into both CD28z-CAR and ARTEMISTM platforms for comparison. When tested in vitro, both CD19(7)-ARTEMISTM T cells and CD19(7)-CD28z-CAR T cells specifically lysed multiple CD19+ leukemia and lymphoma cell lines with similar potencies. However, during the 16 hour killing assays, ARTEMIS™ T cells secreted over 1000-fold less IL-2 and dramatically lower levels of IFN-γ, GM-CSF, IL-10 and IL-6. ARTEMISTM T cells also accumulated less PD-1, LAG3, and TIM3 on their surface during culturing and following in vitro killing, indicating a diminished propensity for exhaustion. Furthermore, during in vitro T cell expansion, ARTEMISTM cells were enriched for naïve/central memory subpopulations, had lower expression of granzyme B, a marker of terminal differentiation, and had reduced rates of receptor internalization upon antigen engagement. These characteristics suggest that T-cells activated through the ARTEMISTM receptor will have improved persistence and long-term proliferation potential, as well as a safer, more controlled cytokine release when used for T-cell therapies. When tested in vivo against CD19+ Raji systematic lymphoma xenografts, intravenous administration of CD19(7)-ARTEMISTM T cells caused rapid, complete, and lasting tumor regression that was better than that achieved with an equal dose of CD19(7)-CD28z-CAR T cells (Figure 1). In agreement with our in vitro data, mice treated with ARTEMISTM T cells had nearly undetectable levels of cytokines in their blood at 24 hours post dosing, a time in which CD19(7)-CAR-treated mice had markedly elevated levels of human IFN-γ, IL-2, TNFα, and IL-10. While flow cytometry analysis of the peripheral blood showed that CD19(7)-CAR T cells expanded more rapidly in mice, CD19(7)-ARTEMISTM T cells better controlled Raji tumor growth and were negative for PD-1 expression which was high on circulating CAR T cells. At 7 weeks post dosing, a time when all ARTEMISTM T cell-treated mice had no detectable tumors, they were re-challenged with Raji lymphoma. While tumors grew rapidly in control mice, ARTEMISTM T cell-treated mice resisted the Raji lymphoma re-challenge, indicating that ARTEMISTM T cells persisted in these mice despite the absence of tumors and remained antigen-responsive (Figure 2). Our data demonstrates that CD19(7)-ARTEMISTM T cells are highly potent against lymphoma preclinical models while releasing drastically lower levels of cytokines. Thus we have developed and pre-clinically validated a novel fully human anti-CD19 T cell therapy that has the potential to persist longer in patients and, importantly, presents a lower risk of cytokine-related toxicities without compromising efficacy. A clinical trial testing CD19(7)-ARTEMISTM T cell therapy in humans is expected to begin in 2017. Figure 1 Raji lymphoma tumor growth in NSG mice treated with either donor-matched untransduced T cells (Mock), CD19(7)-CAR, or CD19(7)-ARTEMISTM T cells (5x106 receptor-positive cells per mouse) Figure 1. Raji lymphoma tumor growth in NSG mice treated with either donor-matched untransduced T cells (Mock), CD19(7)-CAR, or CD19(7)-ARTEMISTM T cells (5x106 receptor-positive cells per mouse) Figure 2 Raji lymphoma tumor growth in NSG mice previously treated with CD19(7)-ARTEMISTM T cells who had complete regression (0.5x106 Raji cells/mouse). As controls, Raji-naïve mice were implanted with Raji cells following an injection of Mock T cells. (1)ARTEMISTM is trademarked by Eureka Therapeutics, Inc. Figure 2. Raji lymphoma tumor growth in NSG mice previously treated with CD19(7)-ARTEMISTM T cells who had complete regression (0.5x106 Raji cells/mouse). As controls, Raji-naïve mice were implanted with Raji cells following an injection of Mock T cells. / (1)ARTEMISTM is trademarked by Eureka Therapeutics, Inc. Disclosures Liu: Eureka Therapeutics: Employment, Equity Ownership, Patents & Royalties. Long:Eureka Therapeutics: Employment, Equity Ownership. Green:Eureka Therapeutics: Employment. Horan:Eureka Therapeutics: Employment. Zimdahl:Eureka Therapeutics: Employment. Liu:Eureka Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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.

Cytokine Release Syndrome (CRS) after Chimeric Antigen Receptor (CAR) T Cell Therapy for Relapsed/Refractory (R/R) CLL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1983-1983 ◽  
Author(s):  
David L. Porter ◽  
Simon F. Lacey ◽  
Wei-Ting Hwang ◽  
Pamela Shaw ◽  
Noelle V. Frey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Research Funding ◽  
Grading System ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Car T ◽  
Life Threatening ◽  
Guide Intervention

Abstract CTL019 are autologous T cells genetically modified to express a chimeric antigen receptor (CAR) consisting of an external anti-CD19 domain with the CD3z and 4-1BB signaling domains, and mediate potent anti-tumor effects in patients (pts) with advanced, R/R CLL, ALL and NHL. CRS is the most serious toxicity of CTL019 therapy; symptoms can include fevers, nausea, myalgias, capillary leak, hypoxia, and hypotension. Standard CRS grading criteria are not applicable to CAR T cell therapies. To better capture clinical manifestations of CRS and guide intervention after CTL019, we devised a novel CRS grading scale. that was applied to 40 pts treated with CTL019 for R/R CLL; 14 pts on an initial pilot and 26 pts on an ongoing dose-optimization trial (reported separately). Our new CRS grading system is shown below. Pts were 80% male, a median age of 65 (range 51-78) and received a median of 4 prior therapies (range 1-10). 41% had known mutation at p53. 83% of 24 pts tested had unmutated IgVH. Response rate to CTL019 (CR+PR) was 42%. CRS was the major toxicity and occurred in 57% (23/40) of pts. CRS was gr 1 in 10%, gr 2 in 17%, gr 3 in 15% and gr 4 in 15%. Development of CRS correlated with response; 13/23 (57%) pts with CRS responded versus 4/17 (24%) pts without CRS responded (p=0.05). CRS was associated with elevations in IL-6, IFN-g, and other cytokines; details for 33 pts will be presented. Peak fold-increase over baseline for IL-6 was a median of 10.6x (range 0.28–649) and for IFN- g a median of 32.9x (1–7243x). For pts with CRS, this increase in IL-6 was a median of 23.5x compared to 1.86x in pts without CRS (p=0.001); and in IFN- g was a median of 97.2xin pts with CRS compared to 24.2x without (p=0.018). Increasing CRS severity was associated with peak fold change in IL-6 (p< 0.0001) and IFN- g (p=0.015). Notably, unlike cytokine changes associated with sepsis, TNF-a did not markedly increase during CRS. CRS occurred with a consistent and often dramatic increase in ferritin, C reactive protein (CRP), and hemophagocytosis, suggesting concurrent macrophage activation syndrome (MAS). Increasing CRS severity was associated with an increasing trend for peak ferritin (log scale, p<0.001) and peak CRP (p<0.001). The median peak ferritin was 13,463 ng/ml in pts with CRS compared to 378 in pts without (p<0.001). Median peak CRP was 16 mg/dl in pts with CRS compared to 3.86 in pts without (p=0.002). CRS required intervention in 8 pts. 1 pt was successfully treated with corticosteroids. Given marked increases in IL-6, 7 patients received the IL6-receptor antagonist tocilizumab with or without corticosteroids with resolution of CRS. Tocilizumab was given to 1/7 pts with gr 2 CRS, 1/6 pts with gr 3 and 5/6 pts with gr 4. Several pts also received corticosteroids and/or etanercept. All pts had resolution of CRS signs with no TRM from CRS. CRS is the most significant complication of CTL019 and can be life threatening. A novel CRS grading system was needed to identify CRS severity more accurately guide intervention timing. CTL019-related CRS is associated with a unique cytokine profile and has been manageable with anti-cytokine therapy in pts with R/R CLL. CRS appears to correlate with response of CLL to CTL019. Further study is needed to develop reliable methods to predict severity and minimize CRS toxicity without inhibiting anti-leukemia activity of CTL019. New CRS Grading System for CTL019 Abstract 1983. Table Grade 1 Grade 2 Grade 3 Grade 4 Mild: Treated with supportive care such as anti-pyretics, anti-emetics Moderate: Requiring IV therapies or parenteral nutrition; some signs of organ dysfunction (i.e. gr 2 Cr or gr 3 LFTs) related to CRS and not attributable to any other condition. Hospitalization for management of CRS related symptoms including fevers with associated neutropenia. More severe: Hospitalization required for management of symptoms related to organ dysfunction including gr 4 LFTs or gr 3 Cr related to CRS and not attributable to any other conditions; this excludes management of fever or myalgias. Includes hypotension treated with IV fluids or low-dose pressors, coagulopathy requiring FFP or cryoprecipitate, and hypoxia requiring supplemental O2 (nasal cannula oxygen, high flow 02, CPAP or BiPAP). Pts admitted for management of suspected infection due to fevers and/or neutropenia may have gr 2 CRS. Life-threatening complications such as hypotension requiring “high dose pressors”, hypoxia requiring mechanical ventilation. Disclosures Porter: Novartis: Patents & Royalties, Research Funding; Genentech (spouse employment): Employment. Off Label Use: Use of genetically modified T cells (CTL019) to treat CLL and use of tocilizumab to treat cytokine release syndrome.. Lacey:Novartis: Research Funding. Hwang:NVS: Research Funding. Frey:Novartis: Research Funding. Chew:Novartis: Patents & Royalties, Research Funding. Chen:Novartis: Research Funding. Kalos:Novartis: Patents & Royalties, Research Funding. Gonzalez:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. Litchman:Novartis: Employment. Shen:Novartis: Employment. Quintas-Cardamas:Novartis: Employment. Wood:Novartis Pharma: Employment. Levine:Novartis: Patents & Royalties, Research Funding. June:Novartis: Patents & Royalties, Research Funding. Grupp:Novartis: Research Funding.

Latest in Clinical Application of CAR Cell Therapy for B-cell Malignancy and Transplantation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. SCI-24-SCI-24
Author(s):  
Crystal L. Mackall
Keyword(s):  
Clinical Trials ◽  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
B Cell ◽  
Graft Failure ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Car T Cells ◽  
Car T

Unparalleled remission rates in patients with chemorefractory B-ALL treated with CD19-CAR T cells illustrate the potential for immunotherapy to eradicate chemoresistant cancer. CD19-CAR therapy is poised to fundamentally alter the clinical approach to relapsed B-ALL and ultimately may be incorporated into frontline therapy. Despite these successes, as clinical experience with this novel modality has increased, so has understanding of factors that limit success of CD19-CAR T cells for leukemia. These insights have implications for the future of cell based immunotherapy for leukemia and provide a glimpse of more global challenges likely to face the emerging field of cancer immunotherapy. Five challenges limiting the overall effectiveness of CD19-CAR therapy will be discussed: 1) T cell exhaustion is a differentiation pathway that occurs in T cells subjected to excessive T cell receptor signaling. A progressive functional decline occurs, manifest first by diminished proliferative potential and cytokine production, following by diminished cytolytic function and ultimately cell death. High leukemic burdens predispose CD19-CAR T cells to exhaustion as does the presence of a CD28 costimulatory signal, while a 4-1BB costimulatory signal diminishes the susceptibility to exhaustion. This biology is likely responsible for limited CD19-CAR persistence observed in clinical trials using a CD19-zeta-28 CAR compared to that observed using a CD19-zeta-BB CAR. 2) Leukemia resistance occurs in approximately 20% of patients treated with CD19-CAR and is associated with selection of B-ALL cells lacking CD19 targeted by the chimeric receptor. Emerging data demonstrates two distinct biologies associated with CD19-epitope loss. Isoform switch is characterized by an increase in CD19 isoforms specifically lacking exon 2, which binds the scFvs incorporated into CD19-CARs currently in clinical trials. Lineage switch is characterized by a global change in leukemia cell phenotype, and is associated with dedifferentiation toward a more stem-like, or myeloid leukemia in the setting of CD19-CAR for B-ALL. These insights raise the prospect that effectiveness of immunotherapy for leukemia may be significantly enhanced by targeting of more than one leukemia antigen. 3) CAR immunogenicity describes immune responses induced in the host that can lead to rejection of the CD19-CAR transduced T cells. Anti-CAR immune responses have been observed by several groups, and mapping is underway to identify the most immunogenic regions of the CAR, as a first step toward preventing this complication. 4) The most common toxicities associated with CD19-CAR therapy are cytokine release syndrome, neurotoxicity and B cell aplasia. Cytokine release syndrome is primarily observed in the setting of high disease burdens and efforts are underway to standardize grading and treatment algorithms to diminish morbidity. Increased information is needed to better understand the neurotoxicity observed in the context of this therapy. Although clinical data is limited, B cell aplasia appears to be adequately treated with IVIG replacement therapy. 5) Technical graft failure (e.g. inadequate expansion/transduction) is a challenge that has received limited attention, primarily since many trials have not reported the percentage of patients in whom adequate products could not be generated. We have observed that technical graft failure is often associated with a high frequency of contaminating myeloid populations in the lymphocyte product and selection approaches designed to eradicate myeloid populations have resulted in improved T cell expansion and transduction. These results suggest that optimization of lymphocyte selection may diminish the incidence of technical graft failure. Disclosures Mackall: Juno: Patents & Royalties: CD22-CAR. Off Label Use: cyclophosphamide.

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