Point-of-care Anti-CD19 Chimeric Antigen Receptor T-cell Therapy for Relapsed/refractory Follicular Lymphoma

Abstract

Patients with relapsed/refractory follicular lymphoma (R/R-FL) often require multiple treatment lines. We performed a phase 1b/2 single-center clinical trial of autologous point-of-care anti-CD19 chimeric antigen receptor (CAR) T-cells in R/R-FL patients treated patients with ≥ 2 treatment lines.

All 26 patients enrolled received CAR T-cell infusion at a median of 11 days after leukapheresis. Seventy-seven percent of patients had POD24. At enrollment, disease stage was III-IV in 85% of the patients, 77% had high-risk FLIPI score, and 77% had progressive disease.

Grade III-IV cytokine release and immune effector cell-associated neurotoxicity syndromes occurred in 12% and 16% of the patients, respectively. Overall response rate at 1-month was 88%. The median follow-up was 15.4 months. One-year overall and progression-free survival were 100% and 63%, respectively.

In conclusion, point-of-care CAR T-cell, manufactured within 11 days, induced a high response rate with an acceptable safety profile in patients with high-risk R/R-FL.

INTRODUCTION

Follicular lymphoma (FL) is the most common indolent lymphoma type. Although the disease is largely responsive to first-line chemoimmunotherapy, in many patients it is characterized by multiple relapses and a slow-indolent pattern of progression, hence it is considered incurable in most situations.[1] Nevertheless, a subset of patients has an aggressive form of the disease, distinguished by refractoriness to, or relapse during the first 24 months after initial chemoimmunotherapy (POD24). This group of patients has a dismal prognosis and typically requires various lines of therapy.[2–4]

There is no defined management sequence for FL patients who relapsed after or were refractory (R/R) to two treatment lines. Numerous regimens were described with various response rates and intensities.[5–10] However, median progression-free survival (PFS) achieved with those therapies ranged between 8–14 months. Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy exhibits unprecedented efficacy in patients with aggressive lymphoma.[11,12] Among patients with R/R FL, the ELARA[13] trial, followed by the ZUMA-5[14] trial, demonstrated high response rates and an acceptable safety profile (overall response rate [ORR] of 86.2% and 94%, respectively), leading to the United States Food and Drug Administration (FDA) approval of tisagenlecleucel and axicabtagene ciloleucel for this indication.[15,16]

Since November 2016 we offer point-of-care (POC), locally produced, academic anti-CD19 CAR T-cell therapy for various B-cell malignancies. We previously reported a median vein-to-vein production time of 10 days.[17–21] Here, we describe the outcome of 26 adult patients with R/R FL without histological evidence of transformation to aggressive lymphoma, treated in a single center using this product. During the study period, no registered CAR T-cell products for FL were locally available.

MATERIALS AND METHODS

Study design

This is a phase 1b/2 clinical trial (NCT02772198), approved by the institutional review board and the Israeli Ministry of Health. Informed consent has been obtained from all patients. Inclusion criteria were age ≥18 years, failure of at least 2 prior anti-lymphoma therapies, adequate CD3 count (above 250 CD3+ cells per microliter blood), no immunosuppressive treatment, and preserved organ function. Patients with uncontrolled rapidly progressing disease, or active central nervous system involvement were excluded, as well as patients with active hepatitis B or C, HIV, and pregnant women.[17] Primary endpoints were disease response at one month, best response, and safety. Secondary endpoints were overall survival (OS), PFS, and production feasibility.

CAR T-cell production and Administration

Fresh peripheral blood mononuclear cells were isolated. T-cells were activated and transduced with a gammaretrovirus encoding the CD19 CAR (based on an FMC63-derived single-chain fragment variable, a CD28 costimulatory domain, and a CD3-ζ signaling domain). CAR T-cells quality was controlled throughout the manufacturing process and included cell identity, transduction efficacy, cell count, viability, potency, purity, the replication competent of the retrovirus polymerase chain reaction, and sterility (tested on day +8, except for mycoplasma contamination tested on day +9 using polymerase chain reaction). The fresh cell product was delivered for immediate infusion after being washed and counted. Bridging chemoimmunotherapy was allowed. Lymphodepletion included fludarabine 25 mg/m² over 3 days (days −4 to −2) and cyclophosphamide 900 mg/m² once (day −2), followed by infusion of 1×10⁶ CAR T cells per kg recipient in the inpatient setting.[17,19,20] Antibiotic and antiviral prophylaxis included trimethoprim-sulfamethoxazole and acyclovir, respectively, given for 12 months starting on day 0.

Definitions

Response was assessed using positron emission tomography-computed tomography (PET-CT) and interpreted according to the Lugano criteria[22], relative to the assessment performed before leukapheresis. The ORR was defined as the proportion of patients who achieved a complete response (CR) or partial response (PR). One month response was defined as the disease response on the 28^th day following infusion, or the earliest possible assessment if PET-CT could not be performed due to the patient’s clinical condition. OS was defined as the time from cell infusion to the date of death from any cause. PFS was defined as the time from cell infusion to the date of either first documented progression or death due to any cause. Duration of response (DOR) was defined as the time from the first response assessment to disease progression, relapse, or death from any cause, among patients who achieved a CR or PR. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were graded according to the American Society for Transplantation and Cellular Therapy Consensus Grading.[23] Adverse events were graded according to the Common Terminology Criteria for Adverse Events (CTCAE v5.0).

Statistical analysis

Categorical variables were described by frequency and percentage. Continuous variables were summarized by median and range. The Kaplan-Meier method was used for survival description and the Log-Rank test was used for survival comparison. The median follow-up was calculated by the reverse Kaplan-Meier method. All p-values were 2-sided, and p < 0.05 was considered statistically significant. Data were analyzed using R (version 4.1.2).

RESULTS

Population characteristics

Twenty-six patients (median age, 62 [range 34–78]) with R/R FL enrolled between March 2017 and September 2022. Patients and disease characteristics are described in Table 1. Most patients had a good performance status (KPS ≥ 90, 81%), although with high-risk disease features: Follicular Lymphoma International Prognostic Index (FLIPI) score at leukapheresis ≥ 3 in 20 (77%) patients; POD24 in 20 (77%) patients; disease stage III-IV at leukapheresis in 22 (85%) patients; and a median number of 3 (range 2–6) prior treatment lines. Twenty-two (85%) patients had an exposure to bendamustine prior to leukapheresis. Production feasibility was 100%. CAR T-cells were manufactured in 25 patients from fresh leukapheresis products with a median vein-to-vein time of 11 days (range 9–22). CAR T-cells were manufactured from cryopreserved leukapheresis product in one patient to allow bendamustine administration as a bridging to CAR T-cell therapy while preventing T-cell function impairment. Only two patients (8%) received bridging therapy (rituximab, n=1; bendamustine, n=1). The last follow-up was as of November 2022.

Table 1:

Patient and disease characteristics

Characteristic	Overall, N = 26 *
Age at CAR T	62 (34 – 78)
Age ≥ 65 years	10 (38%)
Gender (male)	16 (62%)
Karnofsky performance status at admission
≥ 90	21 (81%)
< 90	5 (19%)
POD24	20 (77%)
Time from follicular lymphoma diagnosis (years)	4 (0.6 – 19.6)
Previous treatment lines
2–3	19 (73%)
≥ 4	7 (27%)
Time from previous anti-lymphoma therapy (months)	4 (1 – 52)
Primary refractory disease ¥	6 (23%)
Previous autologous stem-cell transplantation	5 (19%)
Disease response at CAR T
Partial response	3 (12%)
Stable disease	3 (12%)
Progressive disease	20 (77%)
Bulky disease at CAR T	3 (12%)
Disease stage at CAR T
I-II	4 (15%)
III-IV	22 (85%)
Disease grade at most recent biopsy
1	4 (15%)
2	14 (54%)
3a	8 (31%)
FLIPI score at CAR T
Low risk (0–1)	3 (12%)
Intermediate risk (2)	3 (12%)
High risk (≥3)	20 (77%)
Elevated LDH at leukapheresis	9 (35)

^*Median (range); n (%)

^¥Primary refractory disease was specified if complete response was never achieved in a prior therapy.

Abbreviations: CAR, chimeric antigen receptor; POD24, progression of disease within 24 months of first chemoimmunotherapy; FLIPI, follicular lymphoma international prognostic index; LDH, lactate dehydrogenase.

Toxicity

The toxicity profile and therapy administered are presented in Table 2. CAR T-cell therapy was well tolerated in most of the patients. CRS of any grade occurred in 22 (85%) patients, although only 3 (12%) patients had grade 3 CRS. Most patients with CRS did not require therapy. ICANS of any grade occurred in 6 patients (24%), and grade 3–4 ICANS was noted in 4 (16%) patients. Tocilizumab was administered to 4 (15%) patients. Corticosteroids were administered to 6 (23%) patients. Adverse events were captured during the first 28 days and included two patients (8%) with a cardiovascular event (one with grade 1 chest pain and one with grade 3 atrial fibrillation). One patient (4%) had grade 3 vascular access complication. Cellular therapy-related mortality was not observed.

Table 2.

Toxicity and treatment

Characteristic	Overall, N = 26 *
CRS (maximal grade)
No CRS	4 (15%)
1	13 (50%)
2	6 (23%)
3	3 (12%)
4	0 (0%)
Time from infusion to CRS (days)	5 (2 – 9)
CRS duration	4 (1 – 13)
Tocilizumab administration	4 (15%)
ICANS (maximal grade)
No ICANS	20 (77%)
1	0 (0%)
2	2 (8%)
3	3 (12%)
4	1 (4%)
Time from infusion to ICANS (days)	9 (4 – 15)
ICANS duration	3 (1 – 6)
ICANS treatment
Antiepileptics	12 (46%)
Corticosteroids	6 (23%)
Cardiovascular adverse event	2 (8%)
Vascular access complication	1 (4%)
Hematological toxicity
Severe neutropenia (<0.5k/μl)	20 (77%)
Neutropenic fever	16 (80%)
Severe thrombocytopenia (≤50 K/μl)	6 (23%)
Anemia requiring PRBC	10 (48%)

^*Median (range); n (%)

Abbreviations: CRS, cytokine release syndrome; ICANS, Immune effector cell-associated neurotoxicity syndrome; PRBC, packed red blood cells.

Severe neutropenia (<0.5k/μl) occurred in 20 patients (77%) at a median of 6 days following cell infusion (range [−4]-91) and with a median duration of 8 days (range 2–136). Neutropenic fever was noted in 16 patients (80%). A second wave of severe neutropenia was captured in 6 (32%) patients. Resolution of severe neutropenia was recorded in all patients before censoring. Severe thrombocytopenia (≤50 K/μl) occurred in 6 (23%) patients. Bleeding events were not captured. Anemia requiring packed red blood cells occurred in 10 (48%) patients during the first 30 days following cell infusion. Hypogammaglobulinemia was observed in 17/18 (94%) patients who had immunoglobulins measurement after CAR T-cell therapy. Notably, all those patients (except two without an immunoglobulin measurement at enrollment) had hypogammaglobulinemia also at study enrollment. Two patients received intravenous immunoglobulins for hypogammaglobulinemia and recurrent infections.

Infectious complications were noted from lymphodepletion and during the first 100 days following cell infusion. One patient had E. coli sepsis during lymphodepletion, and cell infusion was deferred. One patient had suspected pseudomonas pneumonia. One patient was infected with COVID-19 on the 5^th day following cell infusion and was admitted to the intensive care unit. Two patients had cytomegalovirus reactivation, and one patient had an uncomplicated viral infection.

Response and survival

Disease response was evaluated in all patients. One-month ORR was 88% (n=23; CR, n=22 [85%]; PR, n=1 [3%]). Progressive disease was reported in 3 (12%) patients. The one patient with PR achieved a CR after one year of follow-up without additional treatment, thus the best response was CR in 23 (88%) patients and progressive disease in 3 (12%) patients (Table 3.). Figure 1. presents the disease status pre-CAR T-cell therapy and at one-month post-infusion.

Table 3.

Response after CAR T-cell therapy

Characteristic	Overall, N = 26
1-month response assessment
Overall response rate	23 (88%)
Complete response	22 (85%)
Partial response	1 (4%)
Progressive disease	3 (12%)
Best response
Overall response rate	23 (88%)
Complete response	23 (88%)
Partial response	0 (0%)
Progressive disease	3 (12%)

Figure 1. Alluvial plot showing 1-month disease response rate in follicular lymphoma patients treated with point-of-care CAR-T cell therapy.

Abbreviations: CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease.

The median follow-up was 15.4 months (IQR 8.3–24.4). The median OS was not reached. One- and 2-year OS were 100% (95% CI: 100%–100%) and 94% (95% CI: 83%–100%), respectively. The median PFS was not reached (95% CI: 12- not-reached). One- and 2-year PFS were 63% (95% CI: 45%–88%) and 51% (95% CI: 33%–79%), respectively (Figure 2A-B). The median PFS for patients with POD24 was 14 months (95% CI: 14- not-reached), though no comparison between patients with or without POD24 could have been made due to the low number of patients without POD24. One-year PFS was not significantly different in patients who received CAR T as their third line vs. patients who received CAR T as a more advanced treatment line (67% [95% CI 47–97] vs. 51% [24–100], p=0.8); in patients with elevated lactate dehydrogenase at leukapheresis (67% [95% CI 45–100] vs. 53% [28–100], p=0.2); or in patients with thrombocytopenia at admission (73% [95% CI 51–100] vs. 61% [39–95], p=0.8). The median DOR was not reached. One and 2-year DOR were 71% (95% CI: 52%–96%) and 57% (95% CI: 37%–88%), respectively (Figure 2C).

Figure 2. Kaplan-Meier curves of patients who received point-of-care CAR T-cell therapy.

A, overall survival; B, progression-free survival; and C, duration of response.

DISCUSSION

Follicular lymphoma is often characterized by multiple relapses and decreasing PFS after each succeeding therapy.[1] The AUGMENT[24] trial reported an ORR of 78% and a median PFS of 39.4 months with lenalidomide and rituximab in patients with R/R FL. This therapy has become the standard of care as the second line in many institutions.

Third-line treatment regimens emerged during the last decade. However, most of those treatments offer limited ORR and PFS, and require prolonged treatment courses. Clinical trials involving PI3K inhibitors such as idelalisib, duvelisib, copanlisib, and umbralisib reported an ORR ranging between 42%–57% and a median PFS ranging between 8.3–11 months. Notably, those therapies had a considerable toxicity profile.[7–10] Recently, idelalisib, duvelisib, and umbralisib, that had been approved for use as monotherapy in R/R FL by the FDA were voluntarily withdrawn. The withdrawals were due either to the inability to complete confirmatory studies in the required period or because of emerging data from confirmatory phase 3 combination studies that suggest an imbalance in OS in favor of the comparator.[25–27] The EZH2 inhibitor, tazemetostat, was approved in R/R FL patients with or without EZH2 mutation. It is well tolerated and achieves an ORR of 69% and a median PFS of 13.8 months.[5] Mosunetuzumab, a bispecific antibody, was approved by the FDA and the European Medicines Agency (EHA) after results from the phase 2 study showed an ORR of 80% and a median PFS of 17.9 months.[6]

In contrast to the treatments mentioned above, CAR T-cell therapy has the benefit of a single administration without the need for an ongoing therapy. The ELARA trial, evaluating tisagenlecleucel in patients with FL refractory to ≥ 2 treatment lines, reported an ORR of 86% (69% CR).[13] Recent long-term follow-up report of this trial, with a median follow-up of 28.9 months, described 2-year PFS and DOR of 57% and 64%, respectively.[28] CRS was reported in 49% of the patients (without grade ≥ 3 CRS) and ICANS was reported in 4% (3% grade ≥ 3 neurological events). The updated analyses of the ZUMA-5 study [29,30], recently presented, evaluating axicabtagene ciloleucel in patients with indolent non-Hodgkin lymphoma, reported an ORR of 94% (79% CR) among the group of patients with R/R FL. With a median follow-up of 41.7 months, the median PFS and DOR were 40.2 and 38.6 months, respectively. The median PFS was 40.2 months in patients with POD24, whereas median PFS was not reached in patients without POD24. The median OS was not reached, and the estimated OS at 3 years was 75%. Grade ≥ 3 CRS and neurologic events were reported in 6% and 15% of the patients with FL, respectively.

The current study demonstrated that POC academic anti-CD19 CAR T-cell therapy with CD28 costimulatory domain is highly effective in a heavily pre-treated population of R/R FL with high-risk disease features: 77% of the patients with POD24, 77% with high-risk FLIPI score, and 85% with stage III-IV disease, all at leukapheresis. POC production enables very short production time, abrogates the need for cryopreservation and shipment of the cells, and allows the administration of CAR T-cell therapy without the need for bridging chemotherapy in most patients.[17] Indeed, we report here a production feasibility of 100%. All patients screened received the cells in a short vein-to-vein time of 11 days, with only 2 patients (8%) receiving bridging therapy. Furthermore, the in-house production enables us to provide rapid service to patients from our center and from centers nearby, who need urgent treatment and cannot wait for a commercial slot or in cases production failure of a commercial product.[31]

Point-of-care production of CAR T-cell therapy is wide-spreading in recent years. The Barcelona group reported the use of ARI-0001, a 4–1BB CAR T-cell product, in 47 patients with CD19+ malignancies. They reported production feasibility of 87%; cell production time of 7–10 days, though with a median vein-to-vein period of 42 days; and an ORR of 75% in the non-Hodgkin’s lymphoma (NHL) cohort. [32] Maschan et al. reported the use of multiple-site POC production of a 4–1BB product in Moscow and Cleveland in a cohort of 54 patients with NHL and B-ALL. They report production feasibility of 96–100%; median vein-to-vein period of 13 days; and an ORR of 83% in the NHL cohort. [33] Martinez-Cibrian et al. recently presented their experience with an automated POC platform for the production of anti-CD19 CAR T-cells in patients with chronic lymphocytic leukemia and reported 100% production feasibility among 7 patients, and median vein-to-vein period of 7 days.[34] In addition, our group also recently presented encouraging results of POC anti-BCMA CAR T-cell therapy in heavily pre-treated R/R multiple myeloma patients with 100% production feasibility and median vein-to-vein period of 11 days.[35]

The efficacy results presented here are comparable to the results reported in the pivotal trials of the approved products in R/R FL: ORR of 88% along with a 2-year PFS of 51%, similar to the results reported in the ELARA trial.[13] The safety profile was similar to that reported for axi-cel, which also contains a CD28 co-stimulatory domain. Interestingly, it appears that the rate of severe ICANS noted for tisa-cel, axi-cel and our POC constract were lower in R/R FL patients compared with those reported in patients with aggressive B-cell Lymphoma. Here, we report grade 3–4 ICANS rate of 16%, compared with 22% in patients with aggressive B-cell lymphoma treated with the same POC product,[17] and a rate of 28% grade 3–4 neurological events that are reported in the ZUMA-1 trial.[36] The reason for this difference is unclear and might be attributed to the disease features.

Early and late hematological toxicities were reported following CAR T-cell therapy in large B-cell lymphoma patients.[37] In R/R FL patients, The ELARA and ZUMA-5 trials reported grade ≥3 neutropenia in 32% and 60% of the patients, respectively.[13,14] The current study reports a higher rate of neutropenia (severe neutropenia [grade 4] in 77% of the patients), with neutropenic fever that occurred in 80% of the patients, suggesting that our construct might produce a more severe hematological toxicity, or that this phenomenon can be attributed to the patient characteristics of our cohort. This was also consistent with grade ≥3 thrombocytopenia, occurring in 23% of the patients in this study, compared with 9.3% and 23% in the ELARA and ZUMA-5 trials, respectively.

Our findings are limited by the relatively low number of patients treated with our POC CAR T-cell therapy, restricting our ability to detect risk factors for inferior survival rates. We also did not evaluate CAR T persistence and expansion. This study is unique in that it is the first to depict outcomes of POC CAR T-cell product in R/R FL patients, to the best of our knowledge.

In summary, we report the outcome of 26 R/R follicular lymphoma patients who received point-of-care CAR T-cell therapy in a single center. Point-of-care production was rapid and efficient, and abrogated the need for cryopreservation, bridging therapy, and product shipment. CAR T-cell therapy was well tolerated in most of the patients and resulted in high ORR and encouraging survival. The outcome presented here is similar to the outcome reported in the clinical trials of the approved products, with a similar safety profile compared to the ZUMA-5 trial.