Natalia N Steevens

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.

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.

Abstract BACKGROUND: Chemotherapy followed by autologous T cells that are genetically modified to express a CD19-specific chimeric antigen receptor (CAR) has shown promise as a novel therapy for patients with relapsed or refractory B cell acute lymphoblastic leukemia (B-ALL); however, the risk of severe cytokine release syndrome (sCRS) and neurotoxicity has tempered enthusiasm for widespread application of this approach. 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. METHODS: We are conducting the first clinical trial that administers CD19 CAR-T cells manufactured from a defined composition of T cell subsets to adults with relapsed or refractory B-ALL. CD8+ and CD4+ T cells 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 2x105, 2x106 or 2x107 CAR-T cells/kg. Prior to CAR-T cell infusion, patients underwent lymphodepletion with a high-dose cyclophosphamide (Cy)-based regimen with or without fludarabine (Flu). RESULTS: Twenty-nine adults with B-ALL (median age 40, range 22 - 73 years; median 17% marrow blasts, range 0 - 97%), including 10 patients who had relapsed after allogeneic transplantation, received at least one CAR-T cell infusion. Twenty-four of 26 restaged patients (92%) achieved bone marrow (BM) complete remission (CR) by flow cytometry. CD4+ and CD8+ CAR-T cells expanded in vivo after infusion and their number in blood correlated with the infused CAR-T cell dose. Thirteen patients received lymphodepletion with Cy-based regimens without Flu. Ten of 12 restaged patients (83%) achieved BM CR by flow cytometry; however, 7 of these (70%) relapsed a median of 66 days after CAR-T cell infusion. Disease relapse correlated with a loss of CAR-T cell persistence in blood. We observed a CD8 cytotoxic T cell response to the murine scFv component of the CAR transgene that contributed to CAR-T cell rejection, and resulted in lack of CAR-T cell expansion after a second CAR-T cell infusion in 5 patients treated for persistent or relapsed disease. To minimize immune-mediated CAR-T cell rejection 14 patients were treated with Cy followed by Flu lymphodepletion (Cy/Flu, Cy 60 mg/kg x 1 and Flu 25 mg/m2 x 3-5) before CAR-T cell infusion. All patients (100%) who received Cy/Flu lymphodepletion achieved BM CR after CAR-T cell infusion. CAR-T cell expansion and persistence in blood was higher in Cy/Flu-lymphodepleted patients compared to their counterparts who received Cy alone (Day 28 after 2x106 CAR-T cells/kg: CD8+ CAR-T cells, mean 55.8/μL vs 0.10/μL, p<0.01; CD4+ CAR-T cells, 2.1/μL vs 0.02/μL, p<0.01), enabling reduction in CAR-T cell dose for Cy/Flu-treated patients. Patients who received Cy/Flu lymphodepletion appear to have longer disease-free survival (DFS) than those who received Cy alone (Cy/Flu, median, not reached; Cy alone, 150 days, p=0.09). CAR-T cell infusion was associated with sCRS, characterized by fever and hypotension requiring intensive care in 7 of 27 patients (26%) and neurotoxicity (≥ grade 3 CTCAE v4.03) in 13 of 27 patients (48%). Two patients died following complications of sCRS. Patients with sCRS or neurotoxicity had higher peak serum levels of IL-6, IFN-γ, ferritin and C-reactive protein compared to those without serious toxicity. Importantly IL-6, IFN-γ and TNF-α levels in serum collected on day 1 after CAR-T cell infusion from those who subsequently developed neurotoxicity were higher than those collected from their counterparts who did not develop neurotoxicity (IL-6, p<0.01; IFN-γ, p=0.05; TNF-α, p=0.04), providing potential biomarkers to test early intervention strategies to prevent neurotoxicity. The risks of sCRS and neurotoxicity correlated with higher leukemic marrow infiltration and increasing CAR-T cell dose. We have now adopted a risk-stratified approach to CAR-T cell dosing in which the CAR-T cell dose inversely correlates to the patient's bone marrow tumor burden. CONCLUSION: Risk-stratified dosing of CD19 CAR-T cells of defined subset composition is feasible and safe in a majority of patients with refractory B-ALL, and results in a CR rate of 92%. Addition of Flu to Cy-based lymphodepletion improves CAR-T cell expansion, persistence and DFS. Disclosures Turtle: Juno Therapeutics: Patents & Royalties, Research Funding. Berger:Juno Therapeutics: Patents & Royalties. Jensen:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Riddell:Adaptive Biotechnologies: Consultancy; Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Membership on an entity's Board of Directors or advisory committees. Maloney:Seattle Genetics: Honoraria; Janssen Scientific Affairs: Honoraria; Roche/Genentech: Honoraria; Juno Therapeutics: Research Funding.

Abstract BACKGROUND: The adoptive transfer of CD19-specific chimeric antigen receptor-modified (CD19 CAR) T cells is a promising strategy for treating patients with CD19+ B cell acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (NHL). Dramatic responses have been observed in a subset of patients receiving CD19 CAR T cell therapy, and prior studies suggest that persistence of transferred T cells may correlate with the extent of tumor regression. The use of unselected T cells to prepare CAR T cells results in variation in the phenotypic composition of the infused product in individual patients, making it difficult to determine whether particular T cell subsets contribute to efficacy and/or toxicity. Studies in our lab demonstrated that genetically modified effector T cells derived from purified T cell subsets differ in the capacity to persist in vivo after adoptive transfer, and that a combination of CAR-modified CD8+ central memory (TCM) and CD4+ T cells provides optimal antitumor activity in tumor xenograft models. Based on these data, we designed the first clinical trial in which patients with CD19+ B cell malignancies receive CD19 CAR T cells comprised of a defined composition of CD8+ TCM and CD4+T cells engineered to express a CD19 CAR. METHODS: Patients with relapsed or refractory CD19+ ALL, CLL or NHL are eligible for this phase I/II study. CD8+ TCM and CD4+ T cells were separately enriched by immunomagnetic selection from a leukapheresis product from each patient, and cryopreserved. The CD8+ TCM and CD4+ T cells were stimulated in independent cultures with anti-CD3/anti-CD28 paramagnetic beads, and transduced with a lentivirus encoding the murine FMC63 anti-CD19 scFv, 4-1BB and CD3 zeta signaling domains. After in vitro expansion, the cell product for infusion was formulated in a 1:1 ratio of CD4+:CD8+ CAR+ T cells. A truncated non-functional human epidermal growth factor receptor (EGFRt) encoded in the transgene cassette allowed identification of transgene-expressing T cells by flow cytometry. Lymphodepleting chemotherapy was administered followed by infusion of EGFRt+ CAR T cells at one of three dose levels (2 x 105 EGFRt+ cells/kg, 2 x 106 EGFRt+ cells/kg, 2 x 107 EGFRt+cells/kg). RESULTS: Twenty patients with relapsed or refractory ALL (n = 9), NHL (n = 10) or CLL (n = 1), including those who failed prior autologous (n = 4) or allogeneic (n = 4) stem cell transplant have been treated on the trial. Fifteen of 20 treated patients received a product that conformed to the prescribed CD8+ T­CM:CD4 composition. Five patients received a product manufactured using a modified strategy either due to low blood lymphocyte counts (n = 3) or due to failure to propagate T cells in culture (n = 2). CD8+ TCM and CD4+ T cells have been isolated from 12 additional patients and cryopreserved for therapy. Patients have been treated at all three dose levels without acute infusional toxicity. Severe cytokine release syndrome (sCRS) consisting of fever, hypotension, and reversible neurotoxicity associated with elevated serum IFN-γ and IL-6 was only observed in ALL patients with a high tumor burden. One ALL patient treated at the highest cell dose died of complications associated with sCRS. None of the NHL patients had sCRS. Of patients who are >6 weeks after CD19 CAR T cell therapy, best responses included complete (n=1) or partial (n=5) remission in 6/9 patients with NHL and complete remission in 5/7 patients with ALL. Both CD4+ and CD8+ CAR-T cells expanded in vivo and could be detected in blood, marrow and CSF. The peak level and duration of persistence of both CD4+ and CD8+ EGFRt+ T cells were associated with clinical response. TCRBV gene sequencing of flow sorted CD4+ and CD8+ EGFRt+CAR T cells from 2 patients showed that proliferating CAR T cells were polyclonal. A subset of NHL patients in whom CAR T cells became undetectable developed a T cell immune response to sequences in the murine CD19-specific scFv component of the CAR transgene. CONCLUSION: Adoptive immunotherapy with CD19 CAR T cells of defined subset composition is feasible and safe in a majority of heavily pretreated patients with refractory B cell malignancies and has potent anti-tumor activity. Persistence of CAR-T cells may be limited in some patients by transgene product immunogenicity. Data from this ongoing clinical trial will be updated at the meeting. Disclosures Turtle: Juno Therapeutics: Research Funding. Berger:Juno Therapeutics: Patents & Royalties. Hudecek:Juno Therapeutics: Patents & Royalties. Jensen:Juno: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Riddell:Juno Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Maloney:Juno Therapeutics: Research Funding.

3006 Background: Genetically modified T cells derived from distinct T cell subsets differ in the capacity to persist after adoptive transfer. We are conducting the first phase I/II clinical trial in which patients (pts) with CD19+ B cell malignancies receive T cells comprised of a defined composition of CD8+ TCM and CD4+ T cells engineered to express a CD19 CAR. Methods: CD8+ TCM and CD4+ T cells were separately enriched from each patient, transduced with a CD19 CAR lentivirus and expanded in vitro. The cell product for infusion was formulated in a 1:1 ratio of CD8+:CD4+ CAR+ T cells and infused at one of three dose levels (2x105 – 2x107 CAR-T cells/kg) after lymphodepleting chemotherapy. Results: Thirty-seven pts with ALL (n = 20), NHL (n = 14) or CLL (n = 3) have been treated and 33/37 received a product that conformed to a prescribed CD8+:CD4+ composition. There was no serious acute infusional toxicity. Severe cytokine release syndrome (sCRS) consisting of fever, hypotension, coagulopathy and neurotoxicity associated with elevated serum IFN-γ and IL-6 was only observed in ALL pts with high tumor burden. One ALL patient treated at the highest cell dose died of complications associated with sCRS. No NHL or CLL pts had sCRS. Eighteen of 20 ALL pts were evaluated for response, with 15 (83%) achieving complete marrow remission by high resolution flow cytometry. Clinical responses in NHL included complete (n = 1) or partial (n = 6) remission in 7/13 pts. Two of 3 CLL pts achieved marrow remission by flow cytometry. The peak level and duration of persistence of both CD4+ and CD8+ CAR-T cells were associated with clinical response. We are investigating the impact of distinct lymphodepletion regimens on CAR-T cell proliferation and persistence in vivo. A T cell immune response to the murine CD19-specific scFv component of the CAR transgene was detected in a subset of pts with limited CAR-T cell persistence. Conclusions: Adoptive immunotherapy with CD19 CAR-T cells of defined subset composition is feasible and safe in a majority of heavily pretreated pts with refractory B cell malignancies and has potent anti-tumor activity at low cell doses. CAR-T cell doses for phase II studies in ALL and NHL cohorts have been determined. Clinical trial information: NCT01865617.