Phase 1/2 Study of AUTO3 the First Bicistronic Chimeric Antigen Receptor (CAR) Targeting CD19 and CD22 Followed By an Anti-PD1 in Patients with Relapsed/Refractory (r/r) Diffuse Large B Cell Lymphoma (DLBCL): Results of Cohort 1 and 2 of the Alexander Study
Authors: Ardeshna Kirit M. , Marzolini Maria A V , Norman Jane , Al-Hajj Muhammad , Thomas Simon , Faulkner Jim , Kotsopoulou Ekaterini , Pule Martin , Peddareddigari Vijay G R , Khokhar Nushmia Z , Jonnaert Maud , Chen Robert , Osborne Wendy 
Published: February 2020

Introduction: CD 19 CAR T cell therapies have shown significant activity in patients with r/r DLBCL, however relapses due to CD19 loss or PDL1 upregulation are common. In this study, we are evaluating the safety and efficacy of AUTO3, a CAR T cell therapy designed to target CD19 and CD22 simultaneously followed by 3 doses of anti-PD1 monoclonal antibody pembrolizumab (Pem) treatment. The incorporation of dual targeting and anti-PD1 to target relapses due to antigen loss and PDL1 upregulation is novel and first in its kind.
Methods & Patients: We constructed a novel bicistronic retroviral vector encoding both an anti-CD19 CAR and an anti-CD22 CAR. Antigen binding domains were humanized incorporating an OX40 co-stimulatory domain for the CD19 CAR and a 41BB co-stimulatory domain for the CD22 CAR. The CD22 CAR was enhanced by incorporating a novel pentameric spacer. The cell product was manufactured in a semi-automated and closed process.
Patients (≥ 18 years) with r/r DLBCL not otherwise specified (NOS), high grade, or transformed from indolent histology; Eastern Cooperative Oncology Group Performance Status &lt;2, adequate renal, hepatic, cardiac function and an absolute lymphocyte count ≥0.5 x 10e9/L are eligible. Patients with CNS disease, prior allogeneic stem cell transplant, prior CD19 or CD22 directed therapy are excluded, as well as patients with any contraindication to receiving Pem.
All patients received lymphodepletion with 30 mg/m2/day fludarabine and 300 mg/m2/day cyclophosphamide for 3 days prior to AUTO3 infusion. Patients in cohort 1 received 50 x 10e6 and patients in cohort 2 received 150 x 10e6 transduced CAR T-cells. The first 3 patients in lowest dose cohort received AUTO3 alone, subsequently all patients should receive AUTO3 followed by 3 doses of Pem 200 mg given every 3 weeks starting day 14. The primary endpoints of phase 1 are frequency of dose-limiting toxicities (DLTs) and grade (G) 3-5 toxicity. Key secondary endpoints include overall response rate, complete response rate, duration of response, disease free survival, overall survival, and biomarker endpoints such as the level of AUTO3 cells in blood and duration of B cell aplasia.
Results: As of the data cut-off date (05-July, 2019), 24 patients have been enrolled, 4 patients were screen failures, 1 patient is in screening, and 19 patients underwent leukapheresis. 16/16 patients had product successfully manufactured and 3 patient products in the manufacturing process. 5/16 patients discontinued before dosing and 11 patients were dosed with AUTO3 in cohort 1 and 2. Four with AUTO3 alone, and 7 with AUTO3 followed by Pem. Seven patients received 50x10e6 AUTO3, and 4 patients received 150 x 10e6 AUTO3. Median age was 49, median 3 prior lines of treatment, and 27% had prior autologous transplant. Seven patients had DLBCL NOS and 4 patients had transformed DLBCL from marginal zone or follicular lymphoma. Eleven patients in cohort 1 and 2 had a minimum of 4 week follow up and were evaluable for safety and efficacy analysis.
No AUTO3 related deaths and no DLTs were observed. G &gt; 3 treatment emergent adverse events &gt; 10 % were neutropenia (73%), thrombocytopenia (64%), anemia (64%), and infection (18%). Treatment emergent adverse events &gt; 25% regardless of grades were pyrexia (82%), thrombocytopenia (82%), neutropenia (73%), anemia (64%), nausea (36%), vomiting (36%), fatigue (36%), headache (36%), constipation (36%), dyspnea (27%), and cough (27%). 27% experienced cytokine release syndrome (CRS), all were grade 1, no ≥ G2 CRS were noted. Only one case (9%) of neurotoxicity was noted which was grade 3 and treated with steroids and tocilizumab. Dose levels 50 and 150 x 10e6 have been cleared without DLTs.
At the lowest dose level of 50 x 10e6 cells, the objective response rate (ORR) was 57% and complete remission rate (CRR) was 29%. The ORR and CRR at dose 150 x 10e6 cells was 50%. Updated follow up and additional patient data at higher dose levels as well as cellular kinetics, product characteristics and relevant biomarkers will be presented.
Conclusions: AUTO3, a novel bicistronic anti CD19/CD22 CAR T therapy, has a manageable safety profile in combination with Pem. Notable is the lack of severe CRS (0%). The efficacy data with 2/4 patients achieving CR at dose 150 x 10e6 cells is promising. The study continues to enroll patients at higher dose levels of AUTO3 followed by Pem.

Disclosures
Al-Hajj: Autolus Therapeutics: Employment, Equity Ownership. Thomas:Autolus: Employment, Equity Ownership. Faulkner:Autolus Therapeutics: Employment, Equity Ownership. Kotsopoulou:Autolus Therapeutics: Employment, Equity Ownership. Pule:Autolus: Employment, Equity Ownership, Patents & Royalties. Peddareddigari:Autolus Therapeutics: Employment, Equity Ownership. Khokhar:Autolus Therapeutics: Employment, Equity Ownership. Jonnaert:Autolus Therapeutics: Employment. Chen:Autolus Therapeutics: Employment. Osborne:Novartis: Other: Travel; Roche: Consultancy, Honoraria, Other: Travel, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; MSD: Consultancy; Servier: Consultancy; Gilead: Consultancy; Takeda: Consultancy, Honoraria, Other: Travel, Speakers Bureau.