Purpose:

Although chimeric antigen receptor T-cell (CAR-T) therapy development for B-cell malignancies has made significant progress in the last decade, broadening the success to treating T-cell acute lymphoblastic leukemia (T-ALL) has been limited. We conducted two clinical trials to verify the safety and efficacy of GC027, an “off-the-shelf” allogeneic CAR-T product targeting T-cell antigen, CD7. Here, we report 2 patients as case reports with relapsed/refractory T-ALL who were treated with GC027.

Patients and Methods:

Both the trials reported here were open-label and single-arm. A single infusion of GC027 was given to each patient after preconditioning therapy.

Result:

Robust expansion of CAR-T cells along with rapid eradication of CD7+ T lymphoblasts were observed in the peripheral blood, bone marrow, and cerebrospinal fluid. Both patients achieved complete remission with no detectable minimal residual disease. At data cutoff, 30 September 2020, 1 of the 2 patients remains in ongoing remission for over 1 year after CAR T-cell infusion. Grade 3 cytokine release syndrome (CRS) occurred in both patients and was managed by a novel approach with a ruxolitinib-based CRS management. Ruxolitinib showed promising activity in a preclinical study conducted at our center. No graft-versus-host disease was observed.

Conclusions:

The two case reports demonstrate that a standalone therapy with this novel CD7-targeted “off-the-shelf” allogeneic CAR-T therapy may provide deep and durable responses in select patients with relapsed/refractory T-ALL. GC027 might have a potential to be a promising new approach for treating refractory/relapsed T-ALL. Further studies are warranted.

Translational Relevance

This study describes a potential new therapeutic option for relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL).

CD19-targeted autologous chimeric antigen receptor T-cell (CAR-T) therapy for the treatment of B-cell malignancies has made great progress over the last decade, and two CD19-targeted CAR-T products have been approved by the FDA (1, 2) in the last 4 years. CD7 is expressed in 95% of T-cell acute lymphoblastic leukemia (T-ALL) cases, as well as in T- and natural killer (NK)-cell lineages, making it an ideal target for T-ALL treatment (3). However, because of a shared common surface antigen, fratricide is a key challenge for CAR-T therapies in T-cell malignancies (4). Lengthy production time, the potential leukemia cell contamination, as well as the high manufacturing cost are the main challenges for the production of autologous CAR-T products. GC027, a CD7-targeted “off-the-shelf” allogeneic CAR-T product, shortens the time to treatment and reduces the treatment cost. Currently, two investigator-initiated trials are ongoing, and here, we report the initial clinical safety and response of GC027 in 2 patients with T-ALL.

This report includes case report data of 2 patients from two ongoing trials, a trial registered at www.chictr.org.cn as ChiCTR1900025311 (patient 1) and a trial registered at www.isrctn.com as ISRCTN19144142 (patient 2). The data cut-off date was 30 September 2020. Both trials were approved by the Ethics Review Board of 920th Hospital of Joint Logistics Support Force of People's Liberation Army of China (Kunming, Yunnan, P.R. China). The studies were conducted in accordance with the Declaration of Helsinki. All participants were informed of the trial details and they voluntarily provided written informed consent for enrollment. A timeline of study interventions is depicted in Fig. 1B. CAR-T persistence was not expected to be longer than 1 month. Otherwise, CAR-T cells would need to be eliminated by lympho-depletion to avoid long-term lymphopenia (T and NK cell). Cytokine release syndrome (CRS) was graded by ASBMT consensus (5). Algorithms for managing CRS and neurotoxicity are listed in the Supplementary Materials and Methods. The study additionally investigated ruxolitinib for the management of CRS, with supporting preclinical study data in the Supplementary Materials and Methods. GC027 was manufactured using non-HLA matched healthy donor T cells which was described in our previous abstract in 2020 American Association for Cancer Research Annual Meeting (6). Characterization of the infused CAR-T cells is listed in the Supplementary Data (Supplementary Table S1). The CD7-targeted CAR includes a single-chain variable fragment, which binds to CD7, a CD8 hinge, transmembrane domain, a 4-1BB costimulation region, and a CD3 primary signaling domain. The expression of T-cell receptor α and CD7 was disrupted by CRISPR/Cas9 system to avoid graft-versus-host disease (GvHD) and mutual killing between CAR-T cells (fratricide). Methods for the detection of fusion genes were reported by Padella and colleagues (7). Methods for flow cytometry, minimal residual disease (MRD) detection, cytokine analysis, IHC analysis, qPCR analysis, and tumor killing assay are provided in the Supplementary Materials and Methods. Kruskal–Wallis test was used to analyze the cytokine release levels among different groups in the preclinical research of ruxolitinib.

Figure 1.

A, Pretreatment course of the 2 patients reported. (1) The patient received maintenance portion of BFM 2000 regimen when his disease relapsed; (2) reinduction regimen was VDLP: vincristine, daunorubicin, L-asparaginase, and prednisone; (3) induction regimen was VDLP: vincristine, daunorubicin, L-asparaginase, and prednisone; and (4) another chemotherapy was VDCP regimen: vincristine, daunorubicin, cyclophosphamide, and prednisone. B, Timeline of study interventions. C, Ruxolitinib reduces cytokine release from GC027 during in vitro killing of T-ALL cell line, CCRF-CEM. E:T, Effector:Target ratio. D, Changes of body temperature and systolic pressure after infusion, and the clinical interventions for each patient. Each red circle and black square indicates the maximum temperature and minimal systolic blood pressure on the day of evaluation. Each blue triangle and green triangle indicates 5 mg ruxolitinib and 50 mg etanercept, respectively. Serum cytokines levels and inflammatory markers were measured at indicated timepoints after CAR-T infusion. Values ≤1 pg/mL were marked as 1 pg/mL.

Figure 1.

A, Pretreatment course of the 2 patients reported. (1) The patient received maintenance portion of BFM 2000 regimen when his disease relapsed; (2) reinduction regimen was VDLP: vincristine, daunorubicin, L-asparaginase, and prednisone; (3) induction regimen was VDLP: vincristine, daunorubicin, L-asparaginase, and prednisone; and (4) another chemotherapy was VDCP regimen: vincristine, daunorubicin, cyclophosphamide, and prednisone. B, Timeline of study interventions. C, Ruxolitinib reduces cytokine release from GC027 during in vitro killing of T-ALL cell line, CCRF-CEM. E:T, Effector:Target ratio. D, Changes of body temperature and systolic pressure after infusion, and the clinical interventions for each patient. Each red circle and black square indicates the maximum temperature and minimal systolic blood pressure on the day of evaluation. Each blue triangle and green triangle indicates 5 mg ruxolitinib and 50 mg etanercept, respectively. Serum cytokines levels and inflammatory markers were measured at indicated timepoints after CAR-T infusion. Values ≤1 pg/mL were marked as 1 pg/mL.

Close modal

We found in preclinical studies that, CAR-T viability, specific killing, and expansion were not significantly affected by ruxolitinib under concentrations lower than 1 μmol/L (Supplementary Fig. S2), which were around the physiologic plasma concentration at regular clinical dosage (5–20 mg twice a day, 0.3–1.5 μmol/L). Our result also showed that even minimal concentration of 0.25 μmol/L could significantly reduce cytokine secretion by CAR-T cells, including IL4, IL10, TNFα and IFNγ (P < 0.01; Fig. 1C). On the basis of these findings, we decided to use ruxolitinib for the management of CRS in the clinic.

Patient 1 is a 24-year-old male with relapsed T-ALL which was refractory to reinduction therapy. He was diagnosed in 2017, and achieved complete remission with a negative test for MRD after induction therapy. The karyotype of his T-ALL at diagnosis was normal and no fusion gene was detected. He had relapsed disease 16 months after the original diagnosis and had no response to reinduction chemotherapy (Fig. 1A). After enrollment, the patient received a 6-day lymphodepleting regimen (fludarabine 37 mg/m2 for 6 days, cyclophosphamide 900 mg/m2 for 4 days, prednisone 60 mg/m2 for 4 days, and melphalan 37 mg/m2 for 2 days) before a single dose of 2.64 × 108 total CAR-T cells (6.44 × 106/kg). One day before CAR-T infusion, there were still 25% blast cells in his bone marrow.

Patient 2 is a 39-year-old male, diagnosed with T-ALL in 2019. His leukemia cells had a normal karyotype, and no fusion gene was detected. He had no sign of central nervous system (CNS) invasion, and his initial T-ALL phenotype was cCD3+CD2+CD5+CD7+CD4+CD8+. The patient received induction chemotherapy at diagnosis, but had no response and his disease progressed to 83.4% lymphoblasts in bone marrow and 270 blast cells/μL in cerebrospinal fluid (CSF) along with CNS symptoms (headache). Therefore, the patient received another intensive chemotherapy, including intrathecal chemotherapy administration. The patient had a partial response and bone marrow lymphoblasts dropped to 6.7%. He still had CNS infiltration in his CSF. Unfortunately, salvage regimens with nelarabine and clofarabine are not available in China (not approved yet), and in our professional opinion the patient would unlikely benefit from cytarabine-based regimens without the combination of chemotherapy drugs with proven efficacy, such as nelarabine or clofarabine. On the basis of these considerations, we decided to enroll the patient into the study. While he was evaluated for enrollment on study, his disease quickly progressed with increasing lymphoblasts in the bone marrow and enlarging adenopathy on the neck (Fig. 1A). Imaging evaluations, including CT scan and ultrasound analysis, showed no other extramedullary disease except for the enlarged adenopathy on his neck. At enrollment, donor-specific antibodies, including HLA-I, HLA-II, and MICA, were verified to be negative. The patient also received a 6-day lymphodepleting regimen with daily doses of fludarabine 30 mg/m2, cyclophosphamide 600 mg/m2, and methylprednisolone 60 mg/m2. After a lymphodepleting regimen, his adenopathy was resolved, but there were still 3.9% lymphoblasts in the bone marrow and 0.3% in the peripheral blood as detected by flow cytometry. The patient received a total of 6.02 × 108 total CAR-T cells (1.1 × 107/kg).

Grade 3 CRS occurred in both patients (Fig. 1C), and the major symptoms were pyrexia and hypotension. Pyrexia occurred within several hours after infusion. Ruxolitinib and etanercept were administered in an effort to control CRS once the temperature was more than 39°C. After the use of ruxolitinib and etanercept, pyrexia was quickly resolved, but fever recurred about 8 hours after ruxolitinib (plasma half-life, ∼3–6 hours) administration. As follow-up, ruxolitinib was given at 5 mg/dose every 8 hours until patient's body temperature remained stable below 39°C. The hypotension was sensitive to norepinephrine, and a low dose of 1–3.5 mg/hour norepinephrine was administered to support the systolic blood pressure to be over 90 mmHg. Serum cytokines levels were analyzed by cytometric bead array (CBA) assay, and inflammatory markers, such as C reaction protein (CRP), were monitored by IHC. Levels of IL1β, IL2, IL5, IL8, IL10, IL6, IFNγ, TNFα, and CRP were significantly elevated after CAR-T infusion (Fig. 1C; Supplementary Fig. S1). No signs of neurotoxicity were observed in both patients. A tolerable headache occurred in patient 2 at day 2. This symptom was judged to be fever related, given the absence of other neurologic symptoms, such as seizure and unconsciousness. Patient's cranial imaging assessment showed no signs of cerebral edema. The patient's headache resolved once his body temperature decreased. All other symptoms gradually resolved along with the decrease of cytokine levels. We observed no signs of GvHD, coagulopathy, tumor lysis syndrome, and hypoxia in these 2 patients.

Disease responses were evaluated for both patients. Bone marrow examinations were performed on days 21 and 30 for patient 1, and on days 14 and 28 for patient 2. No evidence of disease was observed in bone marrow by morphology and flow cytometry for both patients. Lumbar puncture biopsy was performed for patient 2 on days 8 and 14, with no evidence of disease observed by morphology and flow cytometry. None of the 2 patients had available donors for hematopoietic stem cell transplantation, so neither of them was considered for allogeneic transplant. As of data cutoff of 30 September 2020, patient 1 remains in ongoing remission for over 1 year, while patient 2 relapsed 48 days after infusion.

Robust expansion of CAR-T cells was observed in both patients. Frequent ruxolitinib use did not seem to impact CAR T-cell expansion and efficacy of the CAR-T therapy. This is a critical finding for future approaches for the potential use of ruxolitinib for CRS prevention and management. CAR-T cells were detected in peripheral blood, bone marrow, and CSF (Fig. 2; Supplementary Fig. S3). Eradication of CD7+ cells was observed along with CAR-T expansion. Peak of CAR T-cell expansion was observed around days 8–11. Peripheral blood was drawn from patient 2 at day 9, and his mononuclear cells were purified and proven to have CD7-specific cytotoxicity against ALL cell line as demonstrated by in vivo killing assays (Fig. 2A). No marked evidence of myelosuppression and CD7-related on-target, off-tumor toxicity was observed. No CAR-T cells were detected either by flow cytometry or qPCR beyond day 30, and gradual recovery of CD7+ cells was observed.

Figure 2.

Detection of blood cells and cytotoxicity assay. A, Changes of CAR transgene copies in peripheral blood (PB) and blood cell counts, including total lymphocyte, CAR-T cells, and CD7+ lymphocyte, of patient 1 (P1) and patient 2 (P2). Cell count ≤ 1 cell/μL and DNA ≤ 10 copies/μg were treated as 1 cell/μL and 10 copies/μg. Results of 6-hour cytotoxicity against T-ALL cell line, CCRF-CEM, and B-ALL cell line, Nalm-6, using patient 2′s day 9 peripheral blood mononuclear cells (PBMC). B, Flow cytometry analysis of CAR-T cells (CD3CD7) in peripheral blood, bone marrow (BM), and CSF of patient 2.

Figure 2.

Detection of blood cells and cytotoxicity assay. A, Changes of CAR transgene copies in peripheral blood (PB) and blood cell counts, including total lymphocyte, CAR-T cells, and CD7+ lymphocyte, of patient 1 (P1) and patient 2 (P2). Cell count ≤ 1 cell/μL and DNA ≤ 10 copies/μg were treated as 1 cell/μL and 10 copies/μg. Results of 6-hour cytotoxicity against T-ALL cell line, CCRF-CEM, and B-ALL cell line, Nalm-6, using patient 2′s day 9 peripheral blood mononuclear cells (PBMC). B, Flow cytometry analysis of CAR-T cells (CD3CD7) in peripheral blood, bone marrow (BM), and CSF of patient 2.

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The results from both case reports demonstrate that the allogeneic CD7-targeted CAR-T product, GC027, is effective in eliminating T-ALL, and by providing long-term duration of response, this may provide a future stand-alone treatment option for T-ALL in relapsed or refractory patients who represent a high unmet medical need patient population. More studies are needed to further evaluate the safety and efficacy of the product. To achieve successful allogeneic universal CAR T-cell implantation, the most critical step is to avoid graft rejection by patients' own allo-reactive killer cells, including T and NK cells. So far, the only reported allogeneic CAR-T approach with successful clinical data is dependent on administration of an anti-CD52 antibody (alemtuzumab) to suppress the host T and NK cells (both are CD52 positive) and create a therapeutic window for allogeneic CAR-T expansion (8). Because both T and NK cells express CD7, instead of using anti-CD52 antibody, GC027 was able to target patients' own alloreactive T and NK cells. We also applied an enhanced lymphodepletion regimen that is stronger than the conventional FLU/CTX regimen used by commercial CAR-T products and other investigational CAR-T therapies, to better reduce the “target burden” and create a therapeutic window for GC027.

For CRS management, the most commonly used medication is tocilizumab, which is approved by the FDA for the treatment of severe and life-threatening CRS (9). Steroids are also recommended if further support is needed in addition to tocilizumab. However, the application of steroids may influence CAR-T expansion and persistence (10). Tocilizumab only blocks IL6 signaling and its effect on CAR-T therapy–induced neurotoxicity has not been fully understood yet. Recently, dasatinib was reported to decrease secretion of several key cytokines in CRS by direct suppression of CAR-T functions (11). More options are needed to better manage the adverse events, such as CRS induced by CAR-T therapies. Ruxolitinib is a JAK/STAT pathway inhibitor and was reported to reduce CAR-T–induced CRS in murine models (12, 13). Ruxolitinib was also recently reported to be a promising CRS intervention for patients with COVID-19 experiencing severe symptoms and prevented multiorgan failure in patients with severe systemic hyperinflammation (14). In preclinical studies, we observed that at physiologic concentrations, ruxolitinib was able to reduce the production of proinflammatory cytokines and short-term applications of ruxolitinib did not influence the target-specific cytotoxicity and expansion of CAR-T cells. Theoretically, as a Jak1/2 inhibitor, ruxolitinib should suppress the downstream activation of receptors for IL2, IL6, and IFNγ in multiple types of immune cells that participate in CRS response. However, it lacks evidence to suppress TNFα receptor signaling (15). Therefore, a novel CRS management approach with a combination of ruxolitinib and etanercept (TNFα inhibitor) was tested to manage CRS in patients with T-ALL treated with CAR-T. We observed that the application of ruxolitinib did not significantly impact target-specific cytotoxicity and expansion of CAR-T cells as seen in the clinical responses observed in these 2 patients. More studies are needed to further show the efficacy and safety of this specific novel CRS management approach. The study is still ongoing and up to cut-off date, a total of 5 adult patients with T-ALL have been enrolled. We are planning on presenting updated data on all patients in an upcoming scientific meeting. The novel approach to treat CRS with ruxolitinib as a potential alternative option to standard-of-care approaches will be further evaluated in our ongoing study.

X. Wang reports a patent for WO2020088631A1 pending. J. Liu reports other from Gracell Bio outside the submitted work, and employment with Gracell Bio. C. Yang reports a patent for WO2020088631A1 issued. L. Shen reports other from Gracell Biotechnologies during the conduct of the study and outside the submitted work and employment with Gracell Biotechnologies. J. He reports other from Gracell Bio outside the submitted work and employment with Gracell Bio. M. Sersch reports other from Gracell Bio outside the submitted work and employment with Gracell Bio. W. Cao reports other from Gracell Bio outside the submitted work, as well as has a patent for WO2020088631A1 pending, and founder of Gracell Bio. No disclosures were reported by the other authors.

S. Li: Conceptualization, data curation, investigation, methodology, writing-original draft, project administration, writing-review and editing. X. Wang: Conceptualization, investigation, methodology, writing-review and editing. Z. Yuan: Data curation, investigation. L. Liu: Data curation, investigation. L. Luo: Data curation, investigation. Y. Li: Data curation, investigation. K. Wu: Data curation. J. Liu: Writing-original draft, project coordination. C. Yang: Data curation. Z. Li: Project coordination. D. Wang: Project coordination. L. Shen: Supervised blood sample analysis. X. Ye: Facilitated and supervised clinical blood sample analysis. J. He: Supervised CAR-T production and QC analysis. C. Han: Blood sample analysis. Y. Wang: Data curation. D. Zhang: Data curation. Y. Dong: Data curation. L. Fang: Patient care, patient data collection. Y. Chen: Patient care, patient data collection. M. Sersch: Writing-review and editing. W. Cao: Writing-review and editing, led and supervised the product design, preclinical study and the product manufacturing. S. Wang: Supervision, investigation, project administration, designed and led the trial.

The authors thank Kunrong Yang and Xiaoming Yang from 920th Hospital for morphologic examinations, and Zucong Chen from The People's Hospital of Dehong Prefecture for clinical assistance. This study was sponsored by Gracell Biotechnologies Co., Ltd.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data