A durable 4-1BB-based CD19 CAR-T cell for treatment of relapsed or refractory non-Hodgkin lymphoma

Eligibility criteria

Patients with CD19-positive r/r B-NHL who previously received at least one line of treatment were recruited (Supplementary Table S1 ). The inclusion criteria include: 1) patients with DLBCL, follicular lymphoma (FL), or primary mediastinal B-cell lymphoma (PMBCL) who are refractory or have relapsed after at least two previous lines of treatment; or who have relapsed after transplantation; 2) patients with mantle cell lymphoma who have received at least one line of treatment; 3) patients with evaluable disease lesions; 4) age ≥18 years; 5) life expectancy ≥90 d; 5) Eastern Cooperative Oncology Group (ECOG) performance status of 0−2; 6) females of childbearing potential with a negative blood pregnancy test; and 7) those who voluntarily participate in the trial and sign the informed consent form (ICF). Exclusion criteria are as follows: 1) patients with high-risk organ involvement: tumors invade one of the following organs: central nervous system, gastrointestinal tract, lungs, pericardium and large vessels; 2) those who have a graft-versus-host disease and need to use immunosuppressive agents; or who have an autoimmune system disease; 3) those who received chemotherapy or radiotherapy within 3 d before peripheral blood mononuclear cell (PBMC) collection; 4) those who have used systemic steroids within 5 d prior to PBMC collection (except for recent or current use of inhaled steroids); or 5) patients with active Hepatitis B or Hepatitis C virus, human immunodeficiency virus (HIV) or other untreated active infections.

Table S1. Baseline characteristics of all 22 patients.

Features	Dose level
	5×105/kg (N=3)	1×106/kg (N=13)	3×106/kg (N=6)
BMI, body mass index; ECOG, Eastern Cooperative Oncology Group; Chemo, chemotherapy; Radio, radiotherapy; BTK, Bruton’s tyrosine kinase; PD-1, programmed death-1.
Age (year)
	47.33±12.58	56.31±9.91	46.33±15.10
Min, Max	34, 59	36, 69	27, 62
Median (Q3−Q1)	49 (25)	61 (33)	51 (35)
Gender [n (%)]
Male	3 (100)	6 (46.2)	5 (83.3)
Female	0	7 (53.8)	1 (16.7)
Height (cm)
	174.0±10.82	164.7±7.30	171.7±11.18
Min, Max	165, 186	153, 179	150, 180
Median (Q3−Q1)	171 (21)	165 (26)	176 (30)
Weight (kg)
	80.67±10.60	67.54±10.47	76.17±14.08
Min, Max	71, 92	48, 87	54, 97
Median (Q3−Q1)	79 (21)	68 (39)	77 (43)
BMI
	26.79±4.36	24.79±2.58	25.69±2.99
Min, Max	23, 31	20, 28	24, 32
Median (Q3−Q1)	26 (9)	24 (8)	24 (8)
Diagnosis [n (%)]
Indolent	2 (66.7)	3 (23.1)	1 (16.7)
Aggressive	1 (33.3)	10 (76.9)	5 (83.3)
ECOG [n (%)]
0	3 (100)	10 (76.9)	6 (100)
1	0	3 (23.1)	0
Prior therapies [n (%)]
Chemo	1 (33.3)	10 (76.9)	4 (66.7)
Chemo + Radio	2 (66.7)	0	1 (16.7)
BTK inhibitor + Chemo	0	2 (15.4)	0
PD-1 inhibitor + Chemo	0	0	1 (16.6)
PD-1 inhibitor + Chemo+ Radio	0	1 (9.1)	0
Prior lines of therapies [n (%)]
1	1 (33.3)	0	1 (16.7)
2	1 (33.3)	6 (46.2)	5 (83.3)
>2	1 (33.4)	7 (53.8)	0

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Study design

This was a single center phase I/II clinical study (ClinicalTrials.gov identifier: NCT03528421) to evaluate the safety and efficacy of IM19 in patients with r/r B-NHL. To assess the dose-limiting toxicity (DLT) of IM19, three dose levels (5×10⁵/kg, 1×10⁶/kg and 3×10⁶/kg) were assessed. Once DLT occurred in more than 2 patients, the recruitment should be terminated at this dose level. This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Peking University Cancer Hospital (IRBPUCH No. 2018YJZ03). All participants were informed of the possible risks and side effects of the therapy and provided the signed informed consent.

A schematic design of the clinical trial is shown in Figure 1 . Apheresis was performed in all eligible patients. After pretreatment with fludarabine [25 mg/(m²·d)] and cyclophosphamide [250 mg/(m²·d] for 3 consecutive days, patients were infused with IM19 on d 0. The primary endpoints were 3-month ORR and the incidence of DLT. The secondary objectives included CR, partial response (PR) and ORR on d 28 and at month 6. The endpoints such as hematology, blood biochemistry, C reactive protein (CRP), CRS and NT were closely monitored especially within one month. On d 1, 4, 7, 10, 14, 21, 28, 90 and 180, the peripheral blood samples were collected to test the CAR-T and cytokines using flow cytometry.

Vector construction and lentivirus production

IM19 CAR was composed of FMC63-derived scFv, hinge and transmembrane domains of CD8α, and intracellular domains of 4-1BB and CD3ζ. The CAR gene was ligated to an EF1α promoter-based lentiviral transfer plasmid pLenti6.3/V5 (ThermoFisher, Waltham, MA, USA). The transfer plasmid, packaging plasmids (pLP1 and pLP2, ThermoFisher, Waltham, MA, USA) and envelope plasmid (pLP/VSVG, ThermoFisher, Waltham, MA, USA) were co-transfected into suspension 293 cells using polyethyleneimine (Polysciences, Warrington, PA, USA). After purification from the culture medium using Core 700 chromatography (GE Healthcare, Chicago, IL, USA), the IM19 CAR lentiviral vectors were formulated, cryopreserved, and sent for quality control in Immunochina Pharmaceuticals Co., Ltd. (Beijing, China).

Manufacture of IM19 CAR-T cells

IM19 CAR-T cells were manufactured by Immunochina Pharmaceuticals Co., Ltd. (Beijing, China). Briefly, PBMCs were isolated from the apheresis using a density gradient medium Ficoll (GE Healthcare, Chicago, IL, USA). T cells were purified and activated by CD3/CD28 Dynabeads (ThermoFisher, Waltham, MA, USA) at a T cell/bead ratio of 1:1. One day later, T cells were transduced with IM19 lentiviral vector and cultured in X-VIVO 15 medium (Lonza Group, Basel, Switzerland) containing 100 U/mL of IL-2 at a density of 1.5×10⁶/mL for 7−11 d. Then CAR-T cells were formulated, cryopreserved, and sent for quality control in Immunochina Pharmaceuticals Co., Ltd.

Flow cytometry analysis of CAR-T cells

Flow cytometry analysis was performed using NovoCyte 2060R (ACEA Biosciences, San Diego, CA, USA) according to previous report (17). The transduction efficiency of IM19 CAR gene into T cells was detected using a phycoerythrin (PE)-labeled anti-CAR antibody which was developed by Immunochina Pharmaceuticals for recognizing the scFv of IM19. PE-anti-CD4 and FITC-anti-CD8 antibodies (BD Biosciences, San Jose, CA, USA) were used to detect the CD4/CD8 ratio. The APC-anti-CD45RA and FITC-anti-CD62L antibodies (Biolegend, San Diego, CA, USA) were used to evaluate the ratio of memory T cells.

Cytometric bead array assay of cytokines

The blood was sampled at the follow-up time points. The protein levels of interleukins (IL-2, IL-4, IL-6, IL-10 and IL-17A), interferon (IFN)-γ and tumor necrosis factor (TNF) were evaluated using the Cytometric Bead Array (CBA) Human Th1/Th2/Th17 Cytokine Kit (BD Biosciences, San Jose, CA, USA) and flow cytometry analysis. All assays were carried out according to the manufacturer’s protocol. Flow cytometry was used to detect the cytokine levels in serum samples. The serum data were expressed in picograms per milliliter.

Evaluation of clinical outcomes

The patients were followed up, with adverse events evaluated according to the clinical symptoms, hematology, blood biochemistry and immune indexes. NT was graded according to National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) (18). CRS was assessed in line with the Consensus Criteria of American Society for Transplantation and Cellular Therapy (ASTCT) (19). The tumor burden was assessed using positron emission tomography-computed tomography (PET-CT) for response evaluation based on the revised criteria for response assessment (20).

Statistical analysis

Statistical analyses were performed with GraphPad Prism (Version 8.0, GraphPad Software, Inc., La Jolla, CA, USA) and IBM SPSS (Version 22.0, IBM Corp., New York, USA). A two-sided Wilcoxon or Mann-Whitney U test was conducted for a comparison of two groups. D’Agostino-Pearson test and Anderson-Darling test were used for normality tests. For three or more groups, statistical analysis was based on one-way ANOVA and Kruskal Wallis test. Kaplan-Meier curve was used to analyze the survival of patients. A threshold of P<0.05 was considered statistically significant for all analyses.

Characteristics of patients and IM19 CAR-T cells

From June 2018 to October 2019, a total of 26 patients with r/r B-NHL were recruited, and 22 eligible patients aged 28−69 years were screened. A dose-escalation study was first performed to evaluate DLT and the optimum dose in 15 patients at the dose levels of 5×10⁵/kg (3 patients), 1×10⁶/kg (6 patients) and 3×10⁶/kg (6 patients). Then an extensive investigation of the optimum dose was carried out in 7 patients at the dose level of 1×10⁶/kg (Figure 1A , Table 1 ). The baseline characteristics of patients are summarized in Table 1 , and features of patients are analyzed in Supplementary Table S1 . Three lymphoma subtypes were present in this study, including DLBCL (13/22), FL (7/22) and marginal zone B-cell lymphoma (MZL) (2/22). And 90.9% (20/22) of patients received at least 2 lines of therapies before enrollment.

After PBMCs were isolated from the leukapheresis, T cells were purified, activated and transduced with the IM19 lentiviral vectors. The manufacturing time was 6−18 d. When expanding to the dose level, cells were evaluated for the CAR expression ratio, CD4/CD8 ratio and the percentage of memory T cells (Supplementary Table S2 ). Under our manufacturing process, the percentage of naïve, stem cell memory, central memory and effector memory T cells in almost all final IM19 products remained at high levels (Supplementary Table S2 ). On d 0, patients were intravenously infused with IM19 CAR-T cells, and were closely followed up from d 4 to d 28 for safety evaluation. The response was assessed on d 28, 90 and 180 (Figure 1B ).

Safety of IM19 CAR-T cells

As of March 1st, 2020, the cut-off date, 22 patients were infused with IM19 CAR-T cells. The adverse events associated with CAR-T cells were monitored. CRS was observed in 59.1% (13/22, 95% CI: 38.7%−76.8%) of patients among whom one patient (4.5%) showed both grade 3 CRS and grade 3 NT at the dose level of 3×10⁶/kg (Figure 2A , Supplementary Table S3 ). Tocilizumab and dexamethasone were used for the toxicity management. All the adverse events resolved within 2 months. All patients showed tolerability to IM19 at three dose levels, and the DLT was not reached.

To further evaluate the adverse events, we assessed the hematology, blood biochemistry and blood inflammatory cytokines at the follow-up time point. Almost all patients experienced cytopenia and 8 patients showed decreased platelet count after treatment with IM19. Multiple cytokines, especially IL-6, IL-10 and IFN-γ, increased in 12 patients from d 4 to d 28 (Figure 2B −D ). The patient who experienced severe CRS and NT showed much higher levels of IL-6, IL-10 and IFN-γ (Figure 2 ).

Clinical responses of IM19 CAR-T cells

One month and three months after treatment with IM19, patients underwent PET-CT scanning for response evaluation. Decrease in sum of the products of diameters (SPD) was observed in 16 patients, and the ORR was 72.7% (16/22, 95% CI: 51.5%−87.1%) and CR was 40.9% (9/22, 95% CI: 23.2%−61.3%) on d 28 ((Figure 3A ,B ), Table 2 ). The 3-month ORR and CR were 59.1% (13/22, 95% CI: 38.7%−76.8%) and 50.0% (11/22, 95% CI: 30.7%−69.3%), and the 6-month ORR and CR were 50.0% (11/22, 95% CI: 30.7%−69.3%) and 45.5% (10/22, 95% CI: 26.9%−65.4%), respectively (Table 2 ). Nine patients (40.9%) showed ongoing response even after 1 year (Figure 3A ). Evident eradication of tumors and durable effectiveness were observed in CR patients, and representative PET-CT images are shown in Figure 3C . Six patients were evaluated as progressive disease (PD) on d 28 (Figure 3A ,B ). For patients with aggressive NHL, the ORR was 62.5% (10/16, 95% CI: 38.5%−81.6%) and CR was 31.3% (5/16, 95% CI: 13.9%−55.9%) on d 28 (Table 2 ). The median PFS was 6 months after IM19 infusion (Figure 3D ), and 1-year overall survival (OS) was 77.8% (Figure 3E ).

Correlation of IM19 CAR-T characteristics with clinical outcomes

We further evaluated the potential factors which were associated with the safety and clinical responses. The levels of IM19 CAR-T cells in peripheral blood were analyzed at the follow-up time point. In vivo expansion and high levels of IM19 from d 4 to d 14 were observed in all patients, and then decreased and persisted at a low level. The IM19 CAR-T could be detected in some of the patients even beyond 8 months (Figure 4A ). The ratio of CD4/CD8 in final IM19 products was not significantly correlated with the CRS grades. The number of CAR-T cells was proportional to the severity of CRS (Figure 4B ). Importantly, the clinical responses (CR vs. PR; PR vs. PD) at month 3 were apparently associated with the peak level of CAR-T cells in peripheral blood, although there were only 2 patients in the PR group (Figure 4C ). Particularly, the median area under the curve of IM19 levels within 28 d (AUC-28) in patients of CR and PR groups was more than 10-fold higher than that in PD group (Figure 4D ). In addition, the transduction efficiency of CAR and the CD4/CD8 ratio in final IM19 products showed no distinct correlation with clinical responses at month 1 or month 3 (Figure 4C ), and they were consistently not correlated with the peak level of CAR-T cells in peripheral blood (Figure 4E ).