Head-to-head comparison of in-house produced CD19 CAR-T cell in ALL and NHL patients

Abstract

BackgroundCD19 chimeric antigen receptor T (CAR-T) cells demonstrate remarkable remission rates in pediatric and adult patients with refractory or relapsed (r/r) acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). In 2016, we initiated a clinical trial with in-house produced CD19 CAR-T cells with a CD28 co-stimulatory domain. We analyzed, for the first time, differences in production features and phenotype between ALL and NHL patients.MethodsNon-cryopreserved CAR-T cells were produced from patients’ peripheral blood mononuclear cells within 9 to 10 days. 93 patients with r/r ALL and NHL were enrolled under the same study. CAR-T cells of ALL and NHL patients were produced simultaneously, allowing the head-to-head comparison.ResultsAll patients were heavily pretreated. Three patients dropped out from the study due to clinical deterioration (n=2) or production failure (n=1). Cells of ALL patients (n=37) expanded significantly better and contained more CAR-T cells than of NHL patients (n=53). Young age had a positive impact on the proliferation capacity. The infusion products from ALL patients contained significantly more naïve CAR-T cells and a significantly higher expression of the chemokine receptor CXCR3. PD-1, LAG-3, TIM-3, and CD28 were equally expressed. 100% of ALL patients and 94% of NHL patients received the target dose of 1×10e6 CAR-T/kg. The overall response rate was 84% (30/36) in ALL and 62% (32/52) in NHL. We further compared CAR-T cell infusion products to tumor infiltrating lymphocytes (TIL), another common type of T cell therapy, mainly clinically effective in solid tumors. CAR-T cells contained significantly more naïve T cells and central memory T cells and significantly less CCR5 compared to TIL infusion products.ConclusionsThe in-house production of CAR-T cells is highly efficient and fast. Clinical response rate is high. CAR-T cells can be successfully produced for 99% of patients in just 9 to 10 days. Cells derived from ALL patients demonstrate a higher proliferation rate and contain higher frequencies of CAR-T cells and naïve T cells than of NHL patients. In addition, understanding the differences between CAR-T and TIL infusion products, may provide an angle to develop CAR-T cells for the treatment of solid tumors in the future.Trial registration numberClinicalTrials.gov; CAR-T:NCT02772198, First posted: May 13, 2016; TIL:NCT00287131, First posted: February 6, 2006.

Gamma-Delta CAR-T Cells Show CAR-Directed and Independent Activity Against Leukemia

“…Final products had an average of 1.15% γδT and 0.77% γδCAR-T ( Figure 1G). The, percentage of γδCAR-T in the final product was not associated with response (17). To summarize, using this protocol we are able to produce a pure fraction of γδT cells lacking αβ T cells, with high expression of the CD19 CAR, and this within a course of 2 weeks.…”

“…The median fold change of the γδT cells was 185 (range, 29–1,376) for transduced cells compared with a median of 363 (range, 81–2,350) for un-transduced γδT cells, with a variable range between different donors ( Figure 1E , p = 0.2 paired T -test). As controls, we ran in parallel a standard CAR-T cell (sCAR-T) production for each donor, using a protocol as for the clinical setting ( 17 ), just in a smaller scale and transduction performed on day 5 instead of 2. CAR transduction efficacy ranged between 40 and 80% ( Figure 1F ), and did not differ between sCAR-T and γδCAR-T (60.5% ± 13.2 and 65.3% ± 18.3, respectively).…”

“…Patient material was obtained as part of a clinical trial previously reported (NCT02772198) (17,18) and approved by the Sheba Medical Center IRB and the Israeli Ministry of Health. All animal experiments were approved by Institutional Ethical Review Process Committees and were performed under Israel Institutional Animal care and use committee approval (1131/17/ANIM).…”

“…Peripheral-blood mononuclear cells (PBMCs) were isolated using centrifugation on LymphoprepTM density gradients (Alere technologies) and were activated in T cell medium with 100 IU/ml IL2 and either OKT3 50 ng/ml (Invitrogen) for standard T cell expansion (previously published) (17), or zoledronic acid 2.94 uM (Novartis) for γδT cell expansion. On day 5 of culture, activated cells were transduced with the CD19 CAR retrovirus, based on an MSGV backbone transduced with an FMC63-CD28-CD3zeta plasmid, kindly provided by Steve Feldman.…”

“…Standard CAR T cells for the clinical setting were produced the same way as standard CAR T described here, with the only differences that clinical CAR T production utilizes 300 IU/ml IL-2 (instead of 100 IU/ml), transduction is performed on day 2 (instead of day 5) and human AB serum is supplemented to the culture medium (instead of FBS) (17).…”