A novel generation 1928zT2 CAR T cells induce remission in extramedullary relapse of acute lymphoblastic leukemia
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Anti-CD19 chimeric antigen receptor (CAR) T cells have shown promise in the treatment of B cell acute lymphocytic leukemia (B-ALL). However, its efficacy in B-ALL patients with extramedullary involvement is limited due to poor responses and neurotoxicity. Here, we utilized a third generation of CAR T cell vector, which contains the Toll/interleukin-1 receptor (ITR) domain of Toll-like receptor 2 (TLR2), to generate 1928zT2 T cells targeting CD19, and evaluated the efficacy of 1928zT2 T cells in relapse or refractory B-ALL patients with extramedullary involvement.
1928zT2 T cells were generated by 19-28z-TLR2 lentiviral vector transfection into primary human T lymphocytes. The anti-leukemia effect of 1928zT2 T cells were determined by killing assays and in xenografts. Three patients diagnosed as relapse or refractory ALL with extramedullary involvement were infused with 1928zT2 T cells, and the clinical responses were evaluated by BM smear, B-ultrasonography, PET/CT, histology, flow cytometry, qPCR, ELISA, and luminex assay.
1928zT2 T cells exhibited enhanced effector function against CD19+ leukemic cells in vitro and in a xenograft model of human extramedullary leukemia. Notably, the 1928zT2 T cells eradicated extramedullary leukemia and induced complete remission in the three relapse and refractory ALL patients without serious adverse effects. 1928zT2 T cells expanded robustly in the circulation of these three patients and were detected in the cerebrospinal fluid of patient 3. These three patients experienced cytokine release syndrome (CRS) with grade 2 or 3, which remitted spontaneously or after tocilizumab treatment. None of the three patients suffered neurotoxicity or needed further intensive care.
Our results demonstrate that 1928zT2 T cells with TLR2 incorporation augment anti-leukemic effects, particularly for eradicating extramedullary leukemia cells, and suggest that the infusion of 1928zT2 T cells is an encouraging treatment for relapsed/refractory ALL patients with extramedullary involvement.
ClinicalTrials.gov identifier NCT02822326. Date of registration: July 4, 2016.
KeywordsChimeric antigen receptor (CAR) T cells Acute lymphocytic leukemia (ALL) Extramedullary ALL Relapsed and refractory TLR2
Acute lymphoblastic leukemia
Allogeneic hematopoietic stem cell transplantation
- CAR-T cells
Chimeric antigen receptor T cells
Central nervous system
Cytokine releasing syndrome
Toll-like receptor 2
Despite great advances, relapsed and refractory B cell acute lymphoblastic leukemia (B-ALL) shows unfavorable prognosis, especially for adult patients [1, 2]. Although newly diagnosed ALL patients co-occurring with extramedullary involvement are rare, ALL with extramedullary tissues (EM-ALL) occurs in about 15 to 20% of all the relapse patients . The central nervous system (CNS) is a favorable site for ALL relapse, accounting for two thirds of extramedullary relapse . Besides, the extramedullary leukemia appears in the skin, breasts, bones, muscle, abdominal organs, and eyes. The presence of extramedullary leukemia is often associated with significantly shorter progression-free survival and overall survival . Worse yet, there is no standard management for these patients and they have to confront dismal outcomes.
Chimeric antigen receptor (CAR) T cells are emerging as a powerful therapy for relapse or refractory ALL [6, 7]; however, the conventional CAR-T cells had shown little evidence of antitumor activity against extramedullary leukemia . Moreover, the efficacy of CAR T cells against solid tumors is substantially poorer than that against leukemia [9, 10], impelling us to improve the design of CAR T vectors. The leukemia infiltrated into extramedullary tissues is often associated with poor response to therapy . To our knowledge, most previous clinical trials of CAR T cells treatment did not enroll the patients with extramedullary leukemia .
Recently, we generated 1928zT2 T cells targeting CD19 by introducing the Toll/interleukin-1 receptor (TIR) domain of Toll-like receptor 2 (TLR2) to 1928z . The activation of TLR2, a canonical costimulatory receptor, promotes the regression of established leukemia . In addition, incorporation with TLR2 domain improves expansion of CAR-T cells and potentiates the killing capacity of CAR T cells against CD19+ leukemic cells . Actually, the leukemia cells with potent abilities for cell migration, proliferation, adhesion, and protease activity facilitated themselves extramedullary infiltration [3, 4]. In this context, it is highly warranted to find tumor-specific T cells with enhanced activity against the leukemic cells of high affinity for the extramedullary relapse. The advent of novel 1928zT2 T cells with improved expansion and enhanced anti-tumor function causes it hopeful to treat such a complicated and aggressive disorder.
In this study, we report the remarkable ability of 1928zT2 T cells to induce complete remission (CR) in extramedullary tissues of three ALL patients, who had a relapse with resistant to available therapies. Our results provide evidences of extensive usage of CAR T cells in these adults, who have aggressive and often fatal relapsed ALL with extramedullary involvement.
Patient enrollment and characteristics
The characteristics of enrolled ALL patients with extramedullary involvement
CR on day 46
CR on day 10
CNS, pancreas, pleuroperitoneum, pelvic fascia, and LNs
CR on day 18
Patient 1 was a 34-year-old female diagnosed as B-ALL (CD19+, BCR/ABL-) in April, 2015 (Fig. 3a). Although she had no response to chemotherapy regimen of VDLCP at first, the patient achieved CR after Hyper CVAD A therapy. She received four cycles of chemotherapy and underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) from her 10/10 HLA allele-matched sister in November, 2015. However, 9 months later, she had a relapse in extramedullary tissues including her left breast and multiple lymph nodes identified by Positron emission tomography-computed tomography (PET/CT) (Fig. 3b). The extramedullary leukemia in breast was confirmed histologically (Fig. 3c), and leukemia blast cells were detected as positive for TdT, CD19, CD20, CD79a, CD34, CD99, CD10, PAX5, and Ki67 (15%), and negative for CD3 and Cyclin D1. B-mode ultrasound was used to monitor the tumor mass in the left breast, and about 2.8 × 1.6 cm size of an inhomogeneous hypo-echoic mass was identified (Fig. 3d). No evidence of relapse in BM and CNS was observed with persisted complete donor chimerism or negative minimal residual disease.
Patient 2 was a 15-year-old male also diagnosed as B-ALL (CD19+, BCR/ABL-) in October, 2014 (Fig. 4a). He underwent allo-HSCT from his 10/10 HLA allele-matched sibling in June, 2015, and unfortunately had a relapse in CNS 6 months later. Then, he achieved a second CR after intrathecal chemotherapy (IT), irradiation, and donor lymphocyte infusion (DLI). However, the leukemia recurred again 10 months later with BM and extramedullary involvement. The BM smear showed typical leukemic blasts account for 15% (Fig. 4b). The PET/CT revealed that an abnormal intense high metabolic region beneath the kidney capsule, indicating the extramedullary relapse in kidney (Fig. 4c). Although he received chemotherapy regimen of VDLCP, he had no response at all.
Patient 3 was a 20-year-old male with a third recurrence of ALL with CNS-3 status  and extramedullary involvement. He received a diagnosis of ALL (CD19+, BCR/ABL-) in 2000 when he was 3 years old (Fig. 5a). During the following 13 years, he had received more than 20 courses of intensive systemic chemotherapy (SC) and IT and remained being in CR. However, he had a relapse in BM and CNS in 2013. Then, he underwent allo-HSCT from a 10/10 HLA allele-matched unrelated female donor in 2014 and the second CR was achieved. Despite he received IT for more than six courses, the patient suffered from second recurrence with CNS-ALL. He had a good response to IT and irradiation and achieved CR for a third time. However, the cancer recurred again 10 months later with CNS and extramedullary involvement in June, 2017. The bone marrow examination still showed signs of complete remission. The whole-body PET/CT showed that extensive extramedullary relapse involving almost the entire pleura, peritoneum, pelvic fascia, pancreas, and multiple lymph nodes (Fig. 5b). A biopsy was performed on right-sided supraclavicular node mass, and characteristic megakaryocytes, erythroblasts and myeloid cells were observed microscopically. The markers were identified as CD3−, CD20−, CD19+, TdT+++, CD10+++, CD34−, and Ki67+ (90%), revealing the leukemic involvement (Fig. 5c). A palpable mass below xiphoid was identified by B-mode ultrasound, and inhomogeneous hypo-echoic mass about 3.0 × 1.9 cm in diameter was seen (Fig. 5d). In addition, aberrant blast cells were detected histologically (Fig. 5e) and examined CD19+CD10+ phenotype (Fig. 5f) in the CSF obtained by lumbar puncture. X/Y fluorescent in situ hybridization (FISH) showed that XY-type recipient cells accounted for 50% of the cerebrospinal cells, further indicating a relapse in the CSF (Fig. 5g).
Patients underwent an apheresis to obtain sufficient PBMCs for producing 1928zT2 T cells. The manufacture of 1928zT2 was completed in about 14 days. The cells were released for infusion after safety check. All patients received a lymphodepletion conditioning regimen which contained with fludarabine 25 mg/m2, cyclophosphamide 300 mg/m2, and/or plus other drugs. After 1-day rest, they received a split-dose infusion of 1928zT2 T cells (a total of 5 × 104–1 × 106 cells/kg and 10% on day 1, 30% on day 2, and 60% on day 3). No post-infusion cytokines such as IL-2 were administered. These patients were evaluated for responses and toxic effects. The expansion and persistence of circulating 1928zT2 T cells were also monitored. Adverse events such as CRS after 1928zT2 T cell infusion were graded according to National Institutes of Health criteria (Common terminology Criteria for Adverse Events, version 4) , and clinical responses were evaluated and defined as complete remission (CR), CR with incomplete count recovery (CRi), partial response (PR), stable disease (SD), or progressive disease (PD) according with the National Comprehensive Cancer Network (NCCN) guidelines. MRD negative was defined as the absence of leukemia cells in BM determined by flow cytometry, and the absence of fusion gene in BM determined by qPCR.
Generation and expansion of 1928zT2 T cells
The 1928zT2 T cells were generated as previously described . Briefly, the 19-28z-TLR2 lentiviral vector was established with 19-28z-TLR2, a CAR-linking FMC63-scFv, CD28 transmembrane and endodomain, the CD3ζ signaling domain, and the TIR domain (amino acid 639-784) of TLR2. Primary human T lymphocytes were isolated and collected, then stimulated with magnetic beads coated with anti-CD3/anti-CD28 antibodies (Miltenyi Biotec) at cell to bead ratio of 1 to 2. Approximately 72 h after activation, T cells were transfected with 19-28z-TLR2 lentiviral vector. These T cells were fed every 2 days with fresh media and used within 14 days of expansion.
Flow cytometry of CD19-CAR T cells detection
The percentages of transduced GFP-positive T cells, CD19 on leukemia cells, T cell phenotypes of the patient were determined by flow cytometry. The antibodies for CD3 (APC), CD4 (Percp-cy5.5), CD8 (PE), CD19 (APC), CD20 (PE), and CD22 (Percp-cy5.5) were used. The flow cytometry was performed with a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo software.
The target cells K562, K562-CD19, and NALM6 were tagged both GFP and luciferase (GL), specifically incubated with GFP T, 1928z T or 1928zT2 T cells at the indicated ratios in triplicate wells in U-bottomed 96-well plates. Viability of target cells was monitored 18 h later by adding 100 dl/well substrate D-luciferin firefly (Life Sciences) resolved at 150 μg/ml. Background luminescence was negligible (< 1% than the signal from the wells with only target cells). The viability percentage was calculated as experimental signal/maximal signal × 100%, and killing percentage was equal to 100 − viability percentage.
Xenograft models and in vivo assessment
To develop the mouse subcutaneous tumor xenograft model of human extramedullary ALL leukemia , NSI mice of ages 6 to 10 weeks were used. All procedures were performed under approval of the Institutional Animal Care and Use Ethics Committee. NALM6-GL (2 × 105 cells in 200 μl PBS) was injected subcutaneously, and 2 × 106 lentiviral transduced human T cells in 200 μl PBS were adoptively transferred to these mice systemically by tail vein injection 2 days later. In vivo whole body imaging of luciferase-labeled cells was performed by cooled CCD camera system (IVIS 100 Series Imaging System, Xenogen, Alameda, CA). D-Luciferin firefly, potassium salt (75 mg/kg) was injected, and the mice were imaged. Quantification of total and average emissions was performed using the Living Image software (Xenogen). The mice were killed at day 34, and their subcutaneous tumor masses were extracted and weighted.
Cytokine measurements and luminex technology
IL-6 levels in plasma were assessed using enzyme-linked immunosorbent assay (ELISA kit, eBioscience, USA) according to the manufacturer’s instructions. The immune-related cytokines were analyzed using Luminex MAGPIX system (Luminex Corp., Austin, TX) according to the manufacturers’ specifications. Samples were detected in triplicate relative to standards supplied by the manufacturer and analyzed for significant differences between different groups.
Quantitative real-time PCR
mRNA was extracted from cells with Trizol (Qiagen) and reverse transcribed into cDNA using the PrimeScript™ RT reagent Kit (Takara). All reactions were performed with TransStart Tip Green qPCR SuperMix (Transgene) on a Bio-Rad CFX96 real-time PCR machine. Delta CT calculations were relative to β-actin and were corrected for PCR efficiencies.
Statistical analysis was performed with SPSS software version 19.0 (Inc., Chicago, IL, USA). Student’s t test (two-tailed) or one-way analysis of variance was used, and data were presented as means ± SEM. A P value < 0.05 was considered significant.
1928zT2 T cells exhibited potent anti-leukemia effect in vitro
TLR2 incorporation promoted the anti-leukemia effect of 1928zT2 T cells ex vivo
Clinical responses to infusion of 1928zT2 T cells
In vivo expansion of 1928zT2 T cells
Toxicity of 1928zT2 T cells
Several cell adhesion and inflammation genes of patient 1 were also monitored by qPCR, and the increased expression pattern is shown in Fig. 7d. The patient was in a relatively stable state without any additional support. The serum cytokines of patient 3 were monitored using luminex assays, and the levels of IL-22, MCP-1, IL-6, and HGF increased prominently from day 6 to day 17 (Fig. 7e), coincident with the expansion of 1928zT2 T cells in the blood. The concentrations of other 34 tested cytokines including IFN-γ, IL-2, TNF-a, and IL-10 did not change much (Fig. 7e). None of these three patients presented neurotoxicity evidence.
Extramedullary involvement in ALL remains a therapeutic challenge . Although chemotherapy, radiation, surgical resection, and allo-HSCT are all modalities that can be incorporated into the therapy of EM-ALL, the optimal treatment remains unclear and these regimens would induce substantial short-term and long-term toxicities [19, 20]. Until recently, there has been no define therapy for these patients who probably have poor outcomes . We report three extramedullary relapse/refractory B-ALL patients achieved a complete remission by 1928zT2 T cells infusion, which implied the CAR T cells possess a potentially efficacy on EM-ALL relapsed or refractory. The three patients in this study had multiple relapses even though they had received chemotherapy, allo-HSCT, and/or radiation prior to CAR T cells infusion. Our results raise the prospect that 1928zT2 T cells infusion is able to rapidly eradicate extramedullary leukemia without any uncontrolled toxicities.
Notably, 1928zT2 T cells used in this study were incorporated with TLR2 domain, which showed improved anti-leukemia efficacy of CARs [12, 22]. These 1928zT2 T cells not only could eradicate the CD19-expressing NALM6 cells and K562 cells specifically and potently in vitro, they could significantly promote the remission of NALM-6 subcutaneous tumors ex vivo, indicating the TLR2 incorporation enhanced the anti-leukemia effects of CAR T cells. Of note, the patient 1 was infused with a dose of 1928zT2 T cells as low as 5 × 104 cells/kg and still achieved CR. As we know, the receptor of TLR2, an essential bridge of innate and adaptive immunity, has the potential to mobilize antigen-presentation response and promote specific activation and expansion of T cells, exhibiting critical regulation of overall immune responses . Previous studies have reported that TLR2 costimulation would promote adhesion and immune synaptic signaling between CAR T cells and target cells . In this study, we also found the obvious elevations of MCP-1  and HGF , the critical chemokines for directing migration of lymphocytes, in the patient 2 after 1928zT2 T cells infusion, indicating the possible involvement of TLR2 in adhesive signaling. In addition, the elevations of MCP-1 and HGF were coincident with the expansion and trafficking of 1928zT2 T cells, suggesting possible roles of MCP-1 and HGF in regulating the trafficking of 1928zT2 T cells. Further studies are required to investigate the mechanisms how TLR2 incorporation promote CAR T cells to infiltrate into tumors and disrupt tumor microenvironment, which are the major challenges for treating CNS and extramedullary leukemia.
1928zT2 T cells infusion may overcome many limitations of conventional therapies. For instance, CNS is regarded as a pharmacological sanctuary for leukemia cells . However, 1928zT2 T cells could traffic to the CNS and eradicate leukemia here without any overt CNS toxicities. Although some studies reported that CAR T cells emerging in CSF are associated with development of neurotoxicity , we did not find any evidence of neurotoxicity in patient 3. It was reported that the Phase II ROCKET trial of JCAR015 (19-28z CAR T cells) was halted for ALL due to the development of sever cerebral edema and subsequent death of several patients, which was associated with rapid expansion and exhaustion of CAR T-cells with CD28. Until now, there was no direct evidence of possible causal relationships of CD28 incorporation and neurotoxicity of CAR T-cells . It is speculated that CAR T cells incorporating 4-1BB are more persistent and less likely to undergo exhaustion than those incorporating CD28, contributing to improved tolerability [30, 31]. However, from the primary data of nine patients using 1928zT2 T cells with CD28 in our phase I clinical trial, no patients suffered from cerebral edema during the follow-up. Overall, the well tolerance and efficacy of 1928zT2 T cells in this study provides a promise for CNS-3 status leukemia patients, who were ineligible for CAR T cells before, though more patients are needed to be recruited in the clinical trial.
In addition, the incorporation of CD28 and TLR2 costimulatory domains in this study was different from the other third-generation CARs, which incorporate two costimulatory domains such as CD28 and 4-1BB or OX-40. These additional costimulatory signals could improve the expansion of CAR T cells. Most third-generation CAR T cells would improve cytokine secretion due to the productive T cell-stimulation and probably induce the severe CRS in patients. At present, most available CAR T cells usage in clinical were the second-generation ones and no direct comparison between these third generation CARs was reported. In this study, it was of note that the 1928zT2 T cells infusion just induced grade 2 or 3 CRS and these patients recovered spontaneously or after treatment with tocilizumab. Although we observed an onset of several cytokines elevations in parallel with robust T cell expansion in these patients, which was consistent with previous studies suggesting some degree of CRS is probably necessary for CAR T cells efficacy , the rest 34 tested cytokines did not fluctuate, indicating that the limited cytokine secretion was possibly related with the low toxicity of 1928zT2 T cells.
Our study demonstrated that the 1928zT2 T cells rapidly and reliably eradicated extramedullary and CNS leukemia and were well tolerated in these three ALL patients. Our results therefore suggested that adoptive transfer of 1928zT2 T cells has great potential to be used to treated ALL patients with extramedullary and CNS leukemia in the future.
This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 81671585, 81300446, 81370665, and 81522002); the Science and Technology Planning Project of Guangdong Province, China (2013B021800201, 2014B020212009, 2014B020226002, 2015B020227003, and 2017B020230004); Guangdong Provincial Applied Science and Technology Research & Development Program (2016B020237006 and 2016B030229006); and the Science and Technology Planning Project of Guangzhou, China (201400000003-4). CAR-T cells in this study were provided by Guangdong Zhaotai InVivo Biomedicine Co., Ltd. The clinical trial in this study was sponsored by Hunan Zhaotai Yongren Medical Innovation Co., Ltd.
Availability of data and materials
The material supporting the conclusion of this study has been included within the article.
JYW, PLL, ZYT, PL, and XD conceived and designed the study; JYW, PLL, and QL analyzed and interpreted the data; JYW, PLL, CH, WR, and XD wrote the manuscript; CWL, XH, SJW, DS, CXD, LSH, ZSL, MHZ, LJZ, YLW, XMC, and SXG provided the study materials or patients; QL, YXL, ZWJ, and DMC performed the experiments. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was approved by the Human Ethics Committees of the Guangdong General Hospital. The patient and donor gave their written informed consent in accordance with the Declaration of Helsinki. This study is registered at www.ClinicalTrials.gov as NCT02822326. All animal experimental protocols were performed in accordance with instruction guidelines from the China Council on Animal Care and approved by the guidelines of the Ethics Committee of Animal Experiments at Guangzhou Institutes of Biomedicine and Health (GIBH).
Consent for publication
The authors have obtained consent to publish from the participant to report individual patient data.
The authors declare that they have no competing interests.
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