Final Results of a Phase 1 Study of AntiCD19 CAR-T Cells with TNFRSF19 Transmembrane Domain
Authors: Caimi Paolo F. , Ghobadi Armin , Reese Jane , Tomlinson Benjamin , Otegbeye Folashade , Ritchey Julie K , Trummer Tabatha , Schiavone Jennifer , Zamborsky Kayla , Eissenberg Linda G , Hollaway Julia , Schneider Dina , Bakalarz Kristen , Boughan Kirsten M , Metheny Leland , Patel Seema , Galloway Erin , Gallogly Molly , Krueger Winfried , Kadan Michael , Pacheco Sanchez Gabriela , Sharma Ashish , Wald David , Cooper Brenda W. , DiPersio John F. , Sekaly Rafick , Orentas Rimas J , Dropulic Boro , de Lima Marcos J.G. 
Published: November 2021

Abstract
Background: AntiCD19 CAR-T cells are effective against chemorefractory B cell lymphoma. Patients (pts) with rapidly progressive disease and urgent need for therapy have very poor prognosis and may not be able to receive CAR-T cells in time. Decreasing the apheresis to infusion time can make CAR-T cells rapidly available. We conducted a dual-center phase I trial using on-site manufacture of CAR-T cells for treatment of relapsed and refractory (r/r) B cell lymphoma.
Methods: Adult pts with r/r CD19+ B cell lymphomas who failed ≥ 2 lines of therapy were enrolled. Autologous T cells were transduced with a lentiviral vector (Lentigen Technology, Inc, LTG1563) encoding an antiCD19 binding motif, CD8 linker, TNFRS19 transmembrane region, and 4-lBB/CD3z intracellular signaling domains. GMP-compliant manufacture was done using CliniMACS Prodigy in a 12-day culture, subsequently shortened to 8 days. Dose escalation was done using 3+3 design. Lymphodepletion included cyclophosphamide (60mg/kg x 1) and fludarabine (25mg/m2/d x 3). Cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS) were graded using the Lee and CARTOX criteria, respectively. CAR-T persistence was measured with qPCR and flow cytometry. Plasma cytokine concentrations were measured using electrochemiluminescence (MesoScale Diagnostics, Inc).
Results: Thirty-one pts were enrolled and treated. Baseline patient and disease characteristics are listed in table 1. Twenty-nine (94%) pts were refractory to the prior line of therapy and 21 (68%) had symptomatic disease at the time of lymphocyte collection. CAR-T cell product manufacture was successful in all pts. Median transduction rate was 45% [range 15-66], median culture expansion was 36-fold [range 3-79]. CAR-T cell doses were 0.5 x 10 6/kg (n = 4), 1 x 10 6/kg (n = 16), and 2 x 10 6/kg (n = 11). Median time from apheresis to lymphodepletion was 7 days (range 2 - 15) and median time from apheresis to CAR-T cell infusion time was 13 days (range 9 - 20). Twenty-eight pts were infused fresh product.
Seventeen pts (55%) experienced CRS. Grade 1-2 CRS was observed in 15 pts (48%), grade ≥ 3 was observed in 3 pts (10%). One patient had grade 4 CRS that was later complicated by hemophagocytic syndrome and died on day 21; a second patient had grade 5 CRS in the context of bulky disease and died on day 8. Ten pts (32%) had ICANS and 4 pts had grade 3-4 ICANS. Treatment for CRS / ICANS included tocilizumab (n = 12), siltuximab (n = 4), anakinra (n = 3) and corticosteroids (n = 10). The most common all grade non - hematologic toxicity was fatigue, observed in 19 pts, all grade 1. Hematologic toxicity was common, with grade ≥ 3 neutropenia observed in all subjects.
Twenty-five (81%) presented disease response and twenty-two pts (71%) achieved complete response (CR). There were no statistically significant differences in the overall and complete response rates between dose levels. After a median follow up of 18 months (range 1 - 32), 5 pts relapsed, and 7 pts have died. Causes of death include progressive disease (n=5), CRS (n=1) and CRS/HLH (n=1). Two-year estimates of PFS and OS for the whole cohort were 67% (95%CI 52-88%) and 75% (95%CI 60-93%)(fig1), respectively. Two-year estimates for patients achieving disease response (CR or PR) were 82% (95%CI 67-99%) and 90% (95%CI 78-100%), respectively. The median duration of response has not been reached (95% CI 74-100). Among pts achieving CR, 94% (95% CI 61-100%) had sustained remission at 12 months.
Median time to peak CAR-T expansion, measured by PCR, was 14 days (IQR 14-19), without differences between dose levels, culture duration or fresh vs. cryopreserved infusion. All evaluable subjects had persistent CAR-Ts on PCR measurements done on days 30, 60 and 90. CAR-T cell dose did not have an impact in the time to peak in vivo CAR-T cell expansion or in the rate of CAR-T cell persistence (fig 2). Cytokine measurements have been conducted in 19 pts, with area under the curve (AUC) analyses showing pts with CRS had higher plasma concentrations of multiple cytokines (fig 3). Patients achieving CR had higher plasma concentrations of MIP3B.
Conclusions: Second generation antiCD19 CAR-T cells with TNFRS19 transmembrane domain have potent clinical activity. On-site manufacture was successful in all pts. This strategy, in combination with fresh product infusion, can make CAR-T cell therapy rapidly available for pts with high-risk r/r B cell lymphoma.
Figure 1 Figure 1.

Disclosures
Caimi: Amgen Therapeutics.: Consultancy; TG Therapeutics: Honoraria; XaTek: Patents & Royalties: Royalties from patents (wife); Kite Pharmaceuticals: Consultancy; Genentech: Research Funding; ADC Theraputics: Consultancy, Research Funding; Seattle Genetics: Consultancy; Verastem: Consultancy. Ghobadi: Wugen: Consultancy; Atara: Consultancy; Amgen: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Celgene: Consultancy. Schneider: Lentigen Technology: Current Employment. Boughan: Beigene: Speakers Bureau. Metheny: Incyte: Speakers Bureau; Pharmacosmos: Honoraria. Krueger: Lentigen: Current Employment. Kadan: Lentigen: Current Employment. Orentas: Lentigen: Patents & Royalties. Dropulic: Lentigen: Ended employment in the past 24 months, Patents & Royalties. de Lima: Miltenyi Biotec: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

OffLabel Disclosure:
AntiCD19 CAR-T cells with TNFRSF19 transmembrane domain for treatment of relapsed and refractory B cell lymphomas.