Lorinda Soma

BACKGROUND: T cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR-T cell products were prepared from unselected T cells. METHODS: We conducted a clinical trial to evaluate CD19 CAR-T cells that were manufactured from defined CD4+ and CD8+ T cell subsets and administered in a defined CD4+:CD8+ composition to adults with B cell acute lymphoblastic leukemia after lymphodepletion chemotherapy. RESULTS: The defined composition product was remarkably potent, as 27 of 29 patients (93%) achieved BM remission, as determined by flow cytometry. We established that high CAR-T cell doses and tumor burden increase the risks of severe cytokine release syndrome and neurotoxicity. Moreover, we identified serum biomarkers that allow testing of early intervention strategies in patients at the highest risk of toxicity. Risk-stratified CAR-T cell dosing based on BM disease burden decreased toxicity. CD8+ T cell-mediated anti-CAR transgene product immune responses developed after CAR-T cell infusion in some patients, limited CAR-T cell persistence, and increased relapse risk. Addition of fludarabine to the lymphodepletion regimen improved CAR-T cell persistence and disease-free survival. CONCLUSION: Immunotherapy with a CAR-T cell product of defined composition enabled identification of factors that correlated with CAR-T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR-T cell dosing strategies that mitigated toxicity and improved disease-free survival. TRIAL REGISTRATION: ClinicalTrials.gov NCT01865617. FUNDING: R01-CA136551; Life Science Development Fund; Juno Therapeutics; Bezos Family Foundation.

CD19-specific chimeric antigen receptor (CAR)-modified T cells have antitumor activity in B cell malignancies, but factors that affect toxicity and efficacy have been difficult to define because of differences in lymphodepletion and heterogeneity of CAR-T cells administered to individual patients. We conducted a clinical trial in which CD19 CAR-T cells were manufactured from defined T cell subsets and administered in a 1:1 CD4(+)/CD8(+) ratio of CAR-T cells to 32 adults with relapsed and/or refractory B cell non-Hodgkin's lymphoma after cyclophosphamide (Cy)-based lymphodepletion chemotherapy with or without fludarabine (Flu). Patients who received Cy/Flu lymphodepletion had increased CAR-T cell expansion and persistence, and higher response rates [50% complete remission (CR), 72% overall response rate (ORR)] than patients who received Cy-based lymphodepletion without Flu (8% CR, 50% ORR). The CR rate in patients treated with Cy/Flu at the maximally tolerated dose was 64% (82% ORR; n = 11). Cy/Flu minimized the effects of an immune response to the murine single-chain variable fragment component of the CAR, which limited CAR-T cell expansion and clinical efficacy in patients who received Cy-based lymphodepletion without Flu. Severe cytokine release syndrome (sCRS) and grade ≥3 neurotoxicity were observed in 13 and 28% of all patients, respectively. Serum biomarkers, one day after CAR-T cell infusion, correlated with subsequent sCRS and neurotoxicity. Immunotherapy with CD19 CAR-T cells in a defined CD4(+)/CD8(+) ratio allowed identification of correlative factors for CAR-T cell expansion, persistence, and toxicity, and facilitated optimization of lymphodepletion that improved disease response and overall and progression-free survival.

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Abstract BACKGROUND: Autologous T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) have demonstrated activity in patients with relapsed or refractory B cell NHL and CLL. The functional heterogeneity that is inherent in CAR-T cell products that are manufactured from undefined T cell subsets has hindered definition of dose-response relationships and identification of factors that may impact efficacy and toxicity, such as the lymphodepletion regimen and infused cell dose. We manufactured anti-CD19 CAR-T cells from a defined composition of CD4+ and CD8+ T cell subsets to treat adults with relapsed or refractory B cell NHL or CLL. T cell subsets were enriched from each patient, transduced with a CD19 CAR lentivirus and separately expanded in vitro before formulation for infusion in a 1:1 ratio of CD8+:CD4+ CAR+ T cells at one of three dose levels (2x105, 2x106 or 2x107 CAR-T cells/kg). CAR-T cells were administered 48-96 hours after lymphodepletion with either cyclophosphamide (Cy, 60 mg/kg)+/- etoposide or Cy (60 mg/kg) and fludarabine (25 mg/m2 daily for 3-5 days (Cy/Flu). RESULTS: Adult patients with relapsed/refractory CD19 expressing B cell NHL (n=28, median age 59 years, range 36-70) or CLL (n=6, median age 60 years, range 54-64) were treated with at least one CAR-T cell infusion. NHL histologies include diffuse large B cell or transformed NHL (DLBCL, n=18), follicular NHL (FL, n= 6) or mantle cell lymphoma (MCL, n=4). 15 patients had failed prior autologous (n=13) or allogeneic (n=3) transplants. Twelve of the 28 NHL patients received lymphodepletion with Cy-based regimens without fludarabine. Expansion of CAR-T cells and clinical responses were observed in 50% (CR=1 (DLBCL), PR=5 (2 FL, 2 DLBCL, 1 MCL), no response=6). Patients were treated at all three dose levels without dose limiting toxicity or severe cytokine release syndrome (sCRS). With this regimen, we observed short CAR-T cell persistence in most patients and demonstrated a CD8-mediated immune response to the murine scFv component of the CAR transgene that correlated with loss of CAR-T cells. Retreatment with CAR-T cells with or without chemotherapy in 5 patients led to no significant T cell expansion or clinical responses. To minimize transgene rejection fludarabine was added to the lymphodepletion regimen administered to the subsequent 16 NHL patients. Clinical responses were evaluated in 12 of 16 patients (2 not yet evaluable, 2 early deaths). Addition of Flu to the lymphodepletion regimen increased the CR rate to 42%, compared to 8% with Cy alone. Clinical responses were identified in 6 of 8 patients with DLBCL (3 CR, 3 PR) and 2 of 3 patients with FL (2 CR). The overall response rate was 67%. We noted higher peak CAR-T cell levels in blood in the Cy/Flu group (n=13) compared with the Cy only group (n=11) (CD8+ CAR-T cells, median 31.9 cells/ml vs 0.55 cells/ml, p = 0.009; CD4+ CAR-T cells, median 16.5 cells/ml vs 0.31 cells/ml, p= 0.007), and CAR-T cell persistence was longer in Flu-treated patients (see Figure 1 for patients treated at 2 x 107/kg). Surprisingly, 2 of 7 patients who received 2 x 107 CAR-T cells/kg experienced dose-limiting toxicity necessitating dose de-escalation. Markedly elevated IL-6 levels were observed within the first day after CAR-T cell infusion in patients who subsequently developed severe toxicity, which may provide an opportunity to test early interventional approaches to minimize toxicity. Six patients with relapsed and refractory CLL received CAR-T cells. Five of 6 restaged patients had complete clearance of blood and/or marrow disease by high-resolution flow cytometry 4 weeks following treatment. Overall clinical responses included 3 CR, 1 PR and 2 no response. One patient with a PR died from refractory pulmonary aspergillus infection. Patients with CR remain in remission at 1-10 months after therapy. CONCLUSION: Immunotherapy with CD19 CAR-T cells of defined subset composition is feasible in patients with NHL and CLL and has potent anti-tumor activity. Toxicity is related to cell dose. The addition of Flu to a Cy-based lymphodepletion regimen results in greater CAR-T cell expansion and persistence, and improves the CR rate after CD19 CAR-T cell therapy. Disclosures Turtle: Juno Therapeutics: Patents & Royalties, Research Funding. Berger:Juno Therapeutics: Patents & Royalties. Jensen:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy. Maloney:Juno Therapeutics: Research Funding; Janssen Scientific Affairs: Honoraria; Seattle Genetics: Honoraria; Roche/Genentech: Honoraria.