CLIC-01: Manufacture and distribution of non-cryopreserved CAR-T cells for patients with CD19 positive hematologic malignancies

Abstract

Access to commercial CD19 CAR-T cells remains limited even in wealthy countries like Canada due to clinical, logistical, and financial barriers related to centrally manufactured products. We created a non-commercial academic platform for end-to-end manufacturing of CAR-T cells within Canada's publicly funded healthcare system. We report initial results from a single-arm, open-label study to determine the safety and efficacy of in-house manufactured CD19 CAR-T cells (entitled CLIC-1901) in participants with relapsed/refractory CD19 positive hematologic malignancies. Using a GMP compliant semi-automated, closed process on the Miltenyi Prodigy, T cells were transduced with lentiviral vector bearing a 4-1BB anti-CD19 CAR transgene and expanded. Participants underwent lymphodepletion with fludarabine and cyclophosphamide, followed by infusion of non-cryopreserved CAR-T cells. Thirty participants with non-Hodgkin's lymphoma (n=25) or acute lymphoblastic leukemia (n=5) were infused with CLIC-1901: 21 males (70%), median age 66 (range 18-75). Time from enrollment to CLIC-1901 infusion was a median of 20 days (range 15-48). The median CLIC-1901 dose infused was 2.3 × 10⁶ CAR-T cells/kg (range 0.13-3.6 × 10⁶/kg). Toxicity included ≥ grade 3 cytokine release syndrome (n=2) and neurotoxicity (n=1). Median follow-up was 6.5 months. Overall response rate at day 28 was 76.7%. Median progression-free and overall survival was 6 months (95%CI 3-not estimable) and 11 months (95% 6.6-not estimable), respectively. This is the first trial of in-house manufactured CAR-T cells in Canada and demonstrates that administering fresh CLIC-1901 product is fast, safe, and efficacious. Our experience may provide helpful guidance for other jurisdictions seeking to create feasible and sustainable CAR-T cell programs in research-oriented yet resource-constrained settings.

Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT03765177, identifier NCT03765177.

Keywords: CAR-T cells; hematologic malignancies; immune effector cells; in-house manufacturing; leukaemia; lymphoma; point-of-care manufacturing; prodigy.

Figure 1

Patient Flow Diagram.

Figure 2

Growth kinetics of patient CLIC-1901 CAR-T cell products (n=30) manufactured using the CliniMACS Prodigy platform. CD4/CD8 T cells were enriched from bulk patient apheresis and either 50 million (black lines) or 100 million (grey lines) cells were used to initiate a 12-day culture using serum-free TexMACS medium and the ‘enhanced feeding protocol’ version of the TcT process. Cells were activated with TransAct immediately upon seeding and were transduced with lentivirus the following day. (A) shows the total number of viable cells present (measured on days 0, 5, 8 and 12) and (B) shows the number of CAR T-expressing cells present (measured on days 8 and 12). Dotted lines on (B) indicate the upper (200 × 10⁶) and lower (1 × 10⁶/kg; average 70 kg) numbers of CAR-T cells required for standard dosage according to clinical protocol (10-fold lower doses are required for patients with high disease burden).

Figure 3

Memory markers in infusion product. CD4+ and CD8+ T cells in infusion products consisted mainly of cells with central memory or effector memory phenotypes. Cryopreserved cells were thawed, stained, and analyzed using an Aurora spectral cytometer as described in Materials and Methods. Graphs show the percentage of central memory (CCR7+ CD45RO+), effector memory (CCR7- CD45RO+), naïve/stem cell memory (CCR7+ CD45RO-), and effector (CCR7- CD45RO-) cells within the CAR+CD4+ (left), and CAR+CD8+ (right) T cell populations (gated on CD3+ CD45+ single viable cells).

Figure 4

(A) Progression-free survival for N=30 infused. (B) Progression free survival by disease.

Figure 5

Overall survival for N=30 infused.

Figure 6

Swimmer’s Plot of Patient-level Data. Each bar represents one CLIC-01 participant infused with CLIC-1901. Participants are grouped into disease type (ALL, DLBCL, MCL, and Other). X-axis denotes the days after CAR-T infusion. Vertical dashed lines mark expected disease assessment time points. Hollow diamonds indicate the days where a disease assessment was made for each participant. Bars are coloured according to the response at each disease assessment, with the time between 2 assessments being coloured by the earlier assessment time. For the time before the first assessment, bars are coloured by the response observed at the first assessment. Periods of CRS or neurotoxicity are denoted by yellow bars within the overall bar. An × marks the death of a participant, and → denotes the patient is still known to be alive at that time point. For this figure, follow up is truncated at 1 year.

Figure 7

CAR cell phenotypes in the infusion product for responders versus non-responders. Independent ANOVAs were run to test for differences in CAR-T cell phenotypes between responders (n = 12) and non-responders (n = 14) at day 180, with Benjamini & Hochberg (BH) multiple test correction. Increased frequencies of Naïve/SCM CD4+ CAR-T cells (ANOVA p = 0.010, BH-adjusted p = 0.077) and CD27+ CD4+ CAR-T cells (ANOVA p = 0.019, BH-adjusted p = 0.107) in the infusion product correlated with favourable responses at day 180, as did decreased frequencies of PD1+ CD4+ CAR-T cells (ANOVA p = 0.00041, BH-adjusted p = 0.009) and TIGIT+ CD4+ CAR-T cells (ANOVA p = 0.0057, BH-adjusted p = 0.066). For each cell subset, grey points show individual measurements for each subject, while the green (responders) or purple (non-responders) points show the mean with whiskers extending to the mean ± standard deviation for each group.