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Our previous clinical study achieved complete remission (CR) rates of >90% following chimeric antigen receptor T cells targeting CD19 (CART19) treatment of refractory/relapsed B cell acute lymphoblastic leukemia (r/r B-ALL); however, the influence of the leukemia burden in peripheral blood (PB) blasts remains unclear. Here, we retrospectively analyzed 143 patients treated with CART19 (including 36 patients with PB blasts) to evaluate the effect of peripheral leukemia burden at the time of apheresis. One hundred seventeen patients with high disease burdens achieved 91.5% CR or incomplete count recovery CR and 86.3% minimal residual disease-negative CR, and 26 patients with low disease burdens obtained 96.2% MRD− CR. Collectively, 9 of 36 (25%) patients with PB blasts and 2 of 107 (1.87%) patients without PB blasts did not respond to CART19 therapy. The leukemia burden in PB negatively influenced ex vivo cell characteristics, including the transduction efficiency of CD3+ T cells and their fold expansion, and in vivo cell dynamics, including peak CART19 proportion and absolute count, fold expansion, and persistence duration. Further studies showed that these patients had higher programmed death-1 expression in CART19 products. Our data imply that PB blasts negatively affected CART19 production and the clinical efficacy of CART19 therapy in patients with r/r B-ALL., Graphical abstract, Chunrong Tong and colleagues demonstrate for the first time the adverse effects of peripheral blood (PB) blasts in refractory/relapsed B cell acute lymphoblastic leukemia treated with chimeric antigen receptor T cells targeting CD19. Their data implied that PB blasts at the time of apheresis negatively affected ex vivo and in vivo CART19 characteristics and subsequent clinical efficacy.

Purpose: B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T-cell therapy results in high remission rates in patients with relapsed/refractory (R/R) multiple myeloma. However, the factors associated with prognosis following CAR T-cell therapy are unknown. Patients and Methods: Between July 1, 2018 and July 31, 2020, 61 patients with R/R multiple myeloma received anti-BCMA CAR T-cell therapy (Chictr.org number, ChiCTR1800017404). Step-wise multivariate Cox regression and competing risk analyses were conducted to identify poor prognosis–associated risk factors. Results: Sixty patients (98.4%) experienced cytokine release syndrome (CRS), including 33, 23, and 4 cases of CRS grades 1 to 2, 3, and 4, respectively. The objective response rate (ORR) was 98.3%, and the complete remission (CR) rate was 70.3%. With a median follow-up period of 21.1 months, the 1-year overall survival (OS) and progression-free survival (PFS) rates were 78.0% and 50.2%, respectively. The median PFS was 12.7 months. Cox modeling revealed that poor PFS was associated with extramedullary disease [HR = 2.59, 95% confidence interval (95% CI) = 1.29–5.21, P = 0.008], light chain multiple myeloma (HR = 2.53, 95% CI = 1.03–5.97, P = 0.035), high-risk cytogenetics (HR = 2.80, 95% CI = 1.27–6.14, P = 0.01), and prior treatment with more than 3 therapeutic lines (HR = 3.14, 95% CI = 1.34–7.34, P = 0.008). Among the 41 CR cases, competing risk analyses demonstrated higher relapse predispositions in those with extramedullary disease (HR = 4.51, 95% CI = 1.86–10.9, P = 0.001), light chain multiple myeloma (HR = 4.89, 95% CI = 1.52 – 15.7, P = 0.008), or high-risk cytogenetics (HR = 5.09, 95% CI = 1.63–15.9, P = 0.005). Conclusions: Anti-BCMA CAR T-cell therapy is safe and effective for R/R multiple myeloma. For patients with high-risk factors, improvements to extend remission and more specific individualized therapies are needed.

Chimeric antigen receptor T cell (CAR-T) therapy has revolutionized the clinical treatment of hematological malignancies due to the prominent anti-tumor effects. B cell maturation antigen (BCMA) CAR-T cells have demonstrated promising effects in patients with relapsed/refractory multiple myeloma. However, the dynamics of CAR-T cell proliferation and cytotoxicity in clinical patients remains unexplored. Here, we longitudinally profiled the transcriptomes of 55,488 T cells including CAR-T products, CAR-T cells, and endogenous T cells at the peak and remission phases in a plasma cell leukemia (PCL) patient treated with BCMA CAR-T cells by single-cell transcriptomic analysis. Our results showed distinct CAR-T and endogenous T cell subsets indicating stage-specific expression in proliferation, cytotoxicity, and intercellular signaling pathways. Furthermore, we found that CAR-T cells at peak phase gradually convert to a highly cytotoxic state from a highly proliferative state along a development trajectory. Moreover, re-analysis of a single cell study from CD8(+) CD19 CAR-T confirmed our findings. These commonalities suggest conserved mechanisms for CAR-T treatment across hematological malignancies. Taken together, our current study provides insight into CAR-T cell dynamics during CAR-T therapy and proves that both BCMA CAR-T and CD19 CAR-T have similar transcriptional characteristics, especially at the CAR-T peak phase.

Although recent clinical trials have demonstrated the efficacy of CD19-directed chimeric antigen receptor (CAR) T-cell therapy for refractory or relapsed B acute lymphoblastic leukemia (r/r B-ALL), most trials exclude patients with high-burden CNS leukemia (CNSL) to avoid the risk of severe neurotoxicity. There were only sparse cases describing the effect of CAR T cells on low-burden CNSL, and the safety and effectiveness of CAR T cells in high-burden CNSL remains unknown. Here, we retrospectively analyzed the results of CD19 CAR T-cell therapy in 12 pediatric patients that had low (Blasts 5/μL blasts in CSF or a solid mass before CAR T-cell expansion, four developed severe (grade 3–4) neurotoxicity featured by persistent cerebral edema and seizure, and they fully recovered after intensive managements. Sustained remission was achieved in 9 of the 12 patients, resulted in a 6-month leukemia-free survival rate of 81.8% (95% CI 59.0–100). Only one patient has CNS relapse again. Our study demonstrates that CAR T cells are effective in clearing both low- and high-burden CNSL, but a high CNSL burden before CAR T-cell expansion may cause severe neurotoxicity requiring intense intervention.

Cytokine release syndrome (CRS) and immune effector cell‐associated neurotoxicity are two major CAR T related toxicities. With the interventions of Tocilizumab and steroids, many patients can recover from severe CRS. However, some patients are refractory to steroids and develop life‐threatening consequences. Ruxolitinib is an oral JAKs inhibitor and promising drug in inflammatory diseases. In this pilot study, we evaluate the efficacy of Ruxolitinib in CRS. Of 14 r/r B‐ALL children who received CD19 or CD22 CAR T cell therapies, 4 patients developed severe (≥grade 3) CRS with symptoms that were not alleviated with high‐dose steroids and thus received ruxolitinib. Rapid resolution of CRS symptoms was observed in 4 patients after ruxolitinib treatment. Serum cytokines significantly decreased after ruxolitinib intervention. All patients achieved complete remission on day 30 after infusion, and we could still detect CAR T expansion in vivo despite usage of ruxolitinib. There were no obvious adverse events related to ruxolitinib. In vitro assays revealed that ruxolitinib could dampen CAR T expansion and cytotoxicity, suggesting that the timing and dosage of ruxolitinib should be carefully considered to avoid dampening anti‐leukaemia response. Our results suggest that ruxolitinib is active and well tolerated in steroid‐refractory and even life‐threatening CRS.

Pan and colleagues report one of the first prospective evaluations of planned sequential chimeric antigen receptor (CAR) T-cell therapy targeting CD19 and then CD22 in a phase 1 trial, indicating acceptable toxicity and encouraging durable efficacy in pediatric patients with relapsed/refractory (r/r) acute lymphoblastic leukemia (ALL).

Although chimeric antigen receptor T (CAR-T) cell therapy has proven to be effective in treating relapsed or refractory B-cell hematological malignancies, severe hematological toxicities remain an intractable issue. This retrospective study assessed the characteristics and risk factors of new-onset severe cytopenia following CAR-T cell infusion in 76 patients with r/r acute lymphoblastic leukemia. The rates of new-onset severe cytopenia were high, including severe neutropenia (SN) (39/56, 70%), severe anemia (SA) (35/66, 53%), and severe thrombocytopenia (ST) (31/64, 48%). Comparatively, cohorts with higher cytokine release syndrome (CRS) grades had higher incidence of severe cytopenia with prolonged duration. Multivariable analyses showed that elevated maximum (max) lg D-dimer and delayed peak time of CRS are independent risk factors for SN recovery; increased max lg IL-10 and delayed CRS recovery are risk factors for SA; high max lg ferritin is a risk factor for ST; and longer period to CRS onset or CRS recovery and higher grade of CRS are risk factors for prolonged hematological toxicities. These observations led to the conclusion that profiles of CRS, including its duration, severity and serum markers are correlated to the incidence and recovery of new-onset severe cytopenia, prompting clinical intervention for post-CAR-T severe cytopenia.

BackgroundIn clinical applications of CAR T-cell therapy, life-threatening adverse events including cytokine release syndrome and neurotoxicity can lead to treatment failure. Outcomes of patients treated with anti-CD30 CAR T- cell have been disappointing in relapsing/refractory (r/r) classical Hodgkin’s Lymphoma (cHL).MethodsIn order to understand the applicable population of multiple CAR T-cell therapy, we examined the expression of CD19, CD20, and CD30 by immunohistochemistry (IHC) in 38 paraffin-embedded specimens of cHL. In the past two years, we found only one patient with cHL who is eligible for combined anti-CD19 and CD30 CAR T-cell treatment. This patient’s baseline characteristics were prone to severe adverse events. We treated this patient with low doses and multiple infusions of anti-CD19 and CD30 CAR T-cell.ResultsThe positive expression of CD19+ + CD30+ in Reed-Sternberg (RS) cells is approximately 5.2% (2/38). The patient we treated with combined anti-CD19 and CD30 CAR T-cell did not experience severe adverse events related to CAR T-cell therapy and received long term progression-free survival (PFS).ConclusionFor high risk r/r cHL patients, low doses of CAR T-cell used over different days at different times might be safe and effective. More clinical trials are warranted for CD19 and CD30 CAR T-cell combination therapy.

Chimeric antigen receptor (CAR)-T cell therapy emerges as a new treatment for refractory or relapsed (r/r) B-cell non-Hodgkin lymphoma (B-NHL); however, the overall response rate (ORR) of which in the B-NHL patients is much lower compared to the patients with r/r B acute lymphoblastic leukemia (B-ALL). We previously confirmed that sequential infusions of CD20 and CD22 CAR-T cells significantly improved the prognosis of the B-NHL patients, while some advanced patients still progressed to death during these CAR-T cell treatments. In this study, we showed that timely sequential administration of the second CAR-T cells could enhance expansion of prior CAR-T cells with stronger tumor-killing capacity in vitro and in vivo. We further conducted compassionate treatments on two advanced B-NHL patients with short-interval sequential infusions of CD19/22/20 CAR-T cells. Disease progression was observed in both patients after primary CAR-T cell infusion but robust re-expansion of prior CAR-T cells and anti-tumor effects was induced by infusion of a secondary CAR-T cells. These results indicate sequential infusions of CAR-T cells with a short interval may improve therapeutic efficacy in the B-NHL patients by promoting expansion of prior CAR-T cells.

Chimeric antigen receptor (CAR) T-cell therapies that target either CD19 or CD22 alone have potent antilymphoma effects. However, antigen escape–mediated relapse often occurs. CAR T cells targeting both CD19 and CD22 may overcome this limitation. In this study, we developed bispecific CAR T cells simultaneously recognizing CD19- and CD22-expressing targets and assessed their safety and efficacy profiles in patients with relapsed/refractory aggressive B-cell lymphoma. Twenty-four patients were screened, and 16 were found eligible for the study. CAR T-cell–associated toxicities were recorded. Responses, overall survival (OS), and progression-free survival (PFS) were assessed. Of the 16 eligible patients, 14 (87.5%) achieved objective response and 10 (62.5%) achieved complete response (CR). The 2-year OS and PFS rates were 77.3% and 40.2%, respectively. Achieving CR (P = 0.046) and the number of prior chemotherapy lines (n = 2; P = 0.047) were independent prognostic factors associated with favorable PFS. The 2-year OS and PFS among patients who achieved CR were higher than among those who did not (P = 0.015 and P < 0.001, respectively). The 2-year PFS among patients who received two prior lines of chemotherapy was higher than that among patients who received more than two lines of chemotherapy (P = 0.049); OS did not differ between the groups. Severe grade 4 cytokine-release syndrome (CRS) was observed in 1 patient; 4 and 11 patients had grades 1 and 2 CRS, respectively. No patients developed neurotoxicity. CD19/CD22 dual-targeted CAR T cells may be a safe, potent antilymphoma cell-based targeted immunotherapy.

Erythropoietin-producing hepatocellular carcinoma A2 (EphA2) is overexpressed in more than 90% of non-small cell lung cancer (NSCLC) but not significantly in normal lung tissue. It is therefore an important tumor antigen target for chimeric antigen receptors (CAR)-T-based therapy in NSCLC. Here, we developed a specific CAR targeted to EphA2, and the anti-tumor effects of this CAR were investigated. A second generation CAR with co-stimulatory receptor 4-1BB targeted to EphA2 was developed. The functionality of EphA2-specific T cells in vitro was tested with flow cytometry and real-time cell electronic sensing system assays. The effect in vivo was evaluated in xenograft SCID Beige mouse model of EphA2 positive NSCLC. These EphA2-specifc T cells can cause tumor cell lysis by producing the cytokines IFN-γ when cocultured with EphA2-positive targets, and the cytotoxicity effects was specific in vitro. In vivo, the tumor signals of mice treated with EphA2-specifc T cells presented the tendency of decrease, and was much lower than the mice treated with non-transduced T cells. The anti-tumor effects of this CAR-T technology in vivo and vitro had been confirmed. Thus, EphA2-specific T-cell immunotherapy may be a promising approach for the treatment of EphA2-positive NSCLC.

Refractory or relapsed B lymphoblastic leukemia (B-ALL) patients have a dismal outcome with current therapy. We treated 42 primary refractory/hematological relapsed (R/R) and 9 refractory minimal residual disease by flow cytometry (FCM-MRD+) B-ALL patients with optimized second generation CD19-directed CAR-T cells. The CAR-T-cell infusion dosages were initially ranged from 0.05 to 14 × 105/kg and were eventually settled at 1 × 105/kg for the most recent 20 cases. 36/40 (90%) evaluated R/R patients achieved complete remission (CR) or CR with incomplete count recovery (CRi), and 9/9 (100%) FCM-MRD+ patients achieved MRD-. All of the most recent 20 patients achieved CR/CRi. Most cases only experienced mild to moderate CRS. 8/51 cases had seizures that were relieved by early intervention. Twenty three of twenty seven CR/CRi patients bridged to allogeneic hematopoietic stem cell transplantation (allo-HCT) remained in MRD- with a median follow-up time of 206 (45-427) days, whereas 9 of 18 CR/CRi patients without allo-HCT relapsed. Our results indicate that a low CAR-T-cell dosage of 1 × 105/kg, is effective and safe for treating refractory or relapsed B-ALL, and subsequent allo-HCT could further reduce the relapse rate.

Mantle cell lymphoma (MCL) is a distinct histological type of B-cell lymphoma with a poor prognosis. Several agents, such as proteasome inhibitors, immunomodulatory drugs, and inhibitors of B cell lymphoma-2 and Bruton's tyrosine kinase have shown efficacy for relapsed or refractory (r/r) MCL but often have short-term responses. Chimeric antigen receptor (CAR) T-cell therapy has emerged as a novel treatment modality for r/r non-Hodgkin's lymphoma. However, long-term safety and tolerability associated with CAR T-cell therapy are not defined well, especially in MCL. In this report, we described a 70-year-old patient with r/r MCL with 48-month duration of follow-up who achieved long-term remission after CAR T-cell therapy. CAR T-cell-related toxicities were also mild and tolerated well even in this elderly patient. This report suggested that CAR T-cell therapy is a promising treatment modality for patients with MCL, who are generally elderly and have comorbid conditions.

Introduction: The source of chimeric antigen receptor (CAR) T cells can be autologous or allogeneic, depending on the history of allogeneic hematopoietic stem cell transplantation (allo-HSCT). For patients with a history of allo-HSCT, CAR T cells are allogeneic, which can be manufactured from donors' peripheral blood mononuclear cells (PBMC) (donor-derived allogeneic CAR T cells, DD-alloCAR) or patients' own PBMC (recipient-derived allogeneic CAR T cells, RD-alloCAR). Unlike autoCAR T cells, which are activated by CAR only, alloCAR T cells receive activation signals from both T-cell receptor and CAR. The difference could affect CAR T-cell biology, resulting in different clinical outcomes. Methods: We retrospectively reviewed 31 consecutive patients who received CAR T-cell therapy in our center. Patients were divided into three groups, as autoCAR group, DD-alloCAR group and RD-alloCAR group. We compared the efficacy and safety profiles among the three groups. We also performed a subgroup analysis of patients received alloCAR. Results: Altogether there were 17 patients in the autoCAR, 11 in the RD-alloCAR and 3 in the DD-alloCAR groups. After a median follow-up of 9 months, the CR rate was 88.2% (95% CI 63.6-98.5%) in the autoCAR group and 100% (95% CI 71.5-100%) in the RD-alloCAR group, which had no significant difference (p=0.73). CR rate in DD-alloCAR was 66.7% (95% CI 9.43-99.1%). The median peak expansion of CAR T cells in the autoCAR was significantly higher than the RD-alloCAR group (p=0.007, Figure A). However, the median OS and EFS did not differ significantly among the groups. As far as adverse effects, RD-alloCAR group had significantly less patients with severe CRS (defined as Grade ≥ 3) than the autoCAR group (p=0.049), with significantly lower peak IL-6 level (p=0.021, Figure B). Neurotoxicity and the degree of cytopenia did not have significant difference among the groups. Acute GVHD occurred in 2 (18.2%) RD-alloCAR patients and 1 (33.3%) DD-alloCAR patients following CAR T-cell infusion. Univariate subgroup analysis of the patients who received alloCAR T-cell therapy showed the presence of cGVHD at the time of PBMC collection was significantly associated with less 6-month relapses (p=0.022); the median RFS in the no-cGVHD group was 2.00 (95% CI 0.72 to 3.28) months, compared to 12.0 (95% CI 2.40 to 21.6) months in the cGVHD group (p=0.084, Figure C); the median OS had no significant difference (Figure D). RD-alloCAR patients with or without cGVHD at PBMC collection did not differ in terms of the peak CAR T-cell expansion, CRS grades or OS. Conclusions: Compared with autoCAR, RD-alloCAR had similar efficacy but less severe CRS. For RD-alloCAR T-cell therapy, the presence of cGVHD at the time of PBMC collection was associated with fewer 6-month relapses. DD-alloCAR and RD-alloCAR T-cell therapies were effective and safe without causing significant GVHD. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Background: CD19-targeting chimeric antigen receptor (CAR) T-cell therapy has shown great efficacy in patients with refractory/relapsed non-Hodgkin lymphoma (NHL), but was associated with serious adverse effects such as cytokine release syndrome (CRS). It has been speculated that NHL baseline disease burden might affect clinical outcome and CRS, but such assumption has not been explored in detail in previous studies. Metabolic tumor volume (MTV) and total lesion glycolysis (TLG), calculated using FDG PET-CT, are quantitative indicators of baseline tumor burden. Methods: Utilizing FDG PET-CT, we calculated MTV and TLG at baseline and post CAR T-cell therapy in 15 patients with NHL. Results: Among all the patients, the median MTV was 72 (range 0.02-3024.9) cm3 and the median TLG was 610.1 (range 0.011-13156.3). After a median follow-up of 6 months, the overall response rate (ORR) was 66.7% (95% CI 38.4-88.2%). The baseline MTV and TLG did not have significant difference in patients with or without response (p=0.271 and 0.95, respectively). Cox-regression analysis did not find lower baseline MTV and TLG significantly associated with better overall survival (p=0.67 and 0.45, respectively). Patients with mild and moderate CRS (defined as Grade 0-2) had significantly lower MTV and TLG than those with severe CRS (Grade 3, 4) (median MTV: 49.3 v.s. 1137.7 cm3, p=0.012; median TLG: 379.1 v.s. 9384, p=0.012, Figure A, B). The median MTV in patients received tocilizumab, an IL-6 antagonist for the treatment of severe CRS, was 963.4 cm3, which was significantly higher than the patients not receiving tocilizumab (58.1 cm3, p=0.037). The median TLG in patients received tocilizumab was 9187.1, compared with 610.1 in patients not receiving tocilizumab (p=0.053). Using FDG PET-CT, we also demonstrated that CAR T-cell therapy in NHL patients could associate with severe local complications such as local compression and local inflammation. Conclusions: Low NHL baseline disease burden is not associated with better response rate or long-term outcome. Patients with higher baseline disease burden have more severe CRS Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Abstract Introduction: Patients (pts) with relapsed refractory B-ALL are mostly incurable by chemotherapy. The disease free survival (DFS) is low even treatment with allogeneic hematopoietic stem cell transplantation (allo-HSCT). We explored treatment of 47 cases of relapsed refractory B-ALL with low dose CD19 CAR-T cells and assessed the clinical safety and efficacy. Patients and Methods: (6-8)X107 pts' peripheral blood mononuclear cells(PBMCs) were activated with CD3 and CD28 antibodies for 24h, then transduced with the lentivirus encoding anti-CD19-CD3zeta-4-1BB CAR (Image1/Picture1) and cultured for 5-6 days in serum-free media containing IL2,IL7,IL15,IL21. All pts except one who had persistent cytopenia received cyclophosphamide 250 mg/m2/d X 3d, and fludarabine 30 mg/m2/d X 3d, then CAR-T cell infusion. Between Jul.31 2015 and Jul. 15 2016, a total of 47 cases were treated with CAR-T cells (Chart1). 37/47 cases had frank hematologically relapsed refractory B-ALL, who could not achieve complete remission (CR) after more than 1 cycles of chemotherapy. The median prior chemotherapy duration was 18 months. The median pre-treatment bone marrow blast percentage was 67% (6.5-98.5%). The most recently treated 15 cases had their PB blasts Chart 1: Characteristics of Patients Pre-CAR-T Image 1/Picture 1: Results: The pts received a median of 10 (0.5-140) X104cells/kg CAR-T cells, and the most recently treated 15 cases all received 10 X 104cells/kg. The median observation period was 201 days (20-368 days). On day 16-20 after CAR-T infusion, 31/35 (88.6%) relapsed hematological refractory cases achieved CR or incomplete CR(Cri), and 29/35 cases (82.9%) achieved negative MRD by FCM(CMR: complete molecular remission). No extramedullary leukemia was detected in any cases with residual disease. The most recently treated 15 consecutive cases all achieved CR with only mild ( Conclusion: Our anti-CD19 CAR-T cell therapy can result in a high CR/CRi /CMR rate in pts with refractory B-ALL and could overcome pre-existing risk factors for poor outcomes, including complex chromosome abnormalities, poor gene mutations, inherited predisposing gene mutation, extramedullary leukemia etc. Pts could be safely bridged into allo-HSCT for potential cure. The dose of infused CAR-T cells in our patients was far lower than previously reported in the literature and the culture period was only 6-7 days, which could dramatically reduce the cost of the CAR-T therapy. 10X104cells/kg of CAR-T cells was a safe and effective number for treating B-ALL. The major complication was CRS and the severity of CRS was directly correlated with the number of malignant B cells in the PB. The efficacy of CAR-T therapy was correlated to the infused number of CAR-T cells. The most recently treated 15 consecutive cases all achieved CR without severe CRS suggesting that the optimal number of CAR-T cells and patient selection are important for the efficacy and safety. Table. Table. Figure. Figure. Disclosures No relevant conflicts of interest to declare.