Therapeutic efficacy of anti-CD19 CAR-T cells in a mouse model of systemic lupus erythematosus.

Dysregulated B-cell activation plays pivotal roles in systemic lupus erythematosus (SLE), which makes B-cell depletion a potential strategy for SLE treatment. The clinical success of anti-CD19 CAR-T cells in treating B-cell malignancies has attracted the attention of researchers. In this study, we aimed to investigate the feasibility of applying anti-CD19 CAR-T cell therapy to SLE treatment in a mouse disease model. We constructed murine anti-CD19 CARs with either CD28 or 4-1BB as the intracellular costimulatory motif and evaluated the therapeutic function of the corresponding CAR-T cells by infusing them into MRL-lpr mice. Furthermore, anti-CD19 CAR-T cells were transferred to MRL-lpr mice before the onset of disease to determine their role in SLE prevention. According to our observations, compared with antibody treatment, the adoptive transfer of our anti-CD19 CAR-T cells showed a more sustained B-cell-depletion effect in MRL-lpr mice. The transfer of syngeneic anti-CD19 CAR-T cells not only prevented disease pathogenesis before the onset of disease symptoms but also displayed therapeutic benefits at a later stage after disease progression. We also tried to optimize the treatment strategy and found that compared with CAR-T cells with the CD28 costimulatory motif, CAR-T cells with the 4-1BB costimulatory motif showed better therapeutic efficiency without cell enrichment. Taken together, these results show that anti-CD19 CAR-T cell therapy was effective in the prevention and treatment of a murine model of SLE, indicating its potential for clinical use in patients.

Preclinical safety evaluation of chimeric antigen receptor-modified T cells against CD19 in NSG mice.

BACKGROUND: With the increase of chimeric antigen receptor-modified T (CAR-T) cell therapy, serious complications initiated by CAR-T cells have garnered wide attention. We have previously developed a 4-1BB/CD3-ζ-costimulated CAR-T cells against CD19 (CART19) for adult acute lymphoblastic leukemia (ALL). In this study, a preclinical safety assessment of CART19 was performed on NSG mice, to evaluate the preclinical toxicity along with its efficacy and tissue distribution. METHODS: A total of 120 NSG mice were used for a combined pharmacodynamics and toxicity study for 56 days. Ninety-six mice of which were single dosed with Raji-Luc (5×105 per animal, i.p.) and different concentrations of CART19 (0.2×107, 0.6×107 and 1.8×107 per animal, i.v.), while the rest were assigned to the Untreated group. Optical intensity of Raji-Luc in mice, clinical symptoms, body mass, hematological analysis, humanized cytokine, lymphocyte subset counting, necropsy and histopathological examinations were performed. In addition, a single dose of 0.6×107 CART19 was intravenously administered to 48 NSG mice, and the distribution of CART19 in different tissues was analyzed using quantitative PCR. RESULTS: CART19 is widely distributed in organs well-perfused with blood, including the lungs, blood, bone marrow, liver and spleen. Significant proliferation of CART19 was also found in the blood by through recognition using humanized CD3+ for T lymphocytes. The survival rate and leukemia related clinical symptoms in mice administered CART19 were markedly ameliorated, and the proliferation of Raji cells in mice was effectively inhibited. However, CART19 had no obvious effects on either the mean body mass or the blood cell counts, and no cytokine release syndrome and graft versus host disease were observed. CONCLUSIONS: NSG mice given CART19 treatment demonstrated a longer survival period without significant immunotoxicity, suggesting encouraging clinical prospects for CART19 in patients with R/R ALL. Our study shed light on evaluation and supervision strategies for CAR-T products for the treatment of hematological diseases or leukemia.

Delayed Terminal Ileal Perforation in a Relapsed/Refractory B-Cell Lymphoma Patient with Rapid Remission Following Chimeric Antigen Receptor T-Cell Therapy.

Chimeric antigen receptor T-cell strategy targeting CD19 (CART19) has prominent anti-tumor effect for relapsed/refractory B-cell lymphomas. CART19-associated complications have been gradually recognized, however, late-onset complications have not been extensively studied. Herein, for the first time we report a diffuse large B-cell lymphoma patient with terminal ileum involvement obtained rapid remission and developed spontaneous terminal ileal perforation 38 days following CART19 infusion. The late-onset perforation reminds us that, for the safety of CART treatment, more cautions are warranted for the management of delayed GI complications.

CD19 targeted CAR-T therapy versus chemotherapy in re-induction treatment of refractory/relapsed acute lymphoblastic leukemia: results of a case-controlled study.

Chimeric antigen receptor modified T cells against CD19 (CART19s) have potent anti-leukemia activities in patients with refractory/relapsed acute lymphoblastic leukemia (R/R ALL). This study was designed to investigate the correlation between safety/efficacy and therapeutic modalities including chemotherapy and CART19 therapy. Total 23 and 69 patients were enrolled in the CART19 group and in the chemotherapy group, respectively. The safety and efficacy profiles of 66 and 22 patients in the 2 groups were evaluated. The complete remission (CR) rate was higher in the CART19 group than that in the chemotherapy group (90.9 vs 37.9%, P = 0.000). For patients relapsed after allo-HSCT and chemotherapy, CR rates were 100% (8/8) vs 48.0% (12/25) (P = 0.009) and 85.7% (12/14) vs 31.7% (13/41) (P = 0.000), respectively. Moreover, a higher percentage in the CART19 group had results below the threshold for minimal residual disease (100 vs 7.58%, P = 0.000). In survival analysis, the overall survival rate at 12 months was higher in the CART19 group than that in the chemotherapy group (60.9 vs 10.1%, P = 0.000). For post-transplant patients achieving CR, 25.0% (2/8) and 75.0% (9/12) complicated with GVHD (P = 0.04) in the CART19 group and chemotherapy group, respectively. For all CR patients, the median duration of absolute neutrophil count less than 500/µL and platelet count less than 20,000/µL were longer in the CART19 group than in the chemotherapy group (p = 0.0047 and 0.0003, respectively). Our data demonstrated that patients with CART19s therapy acquired higher rates of remission and longer survival, confirming the encouraging application of CART19 therapy in R/R ALL.

Potent Anti-leukemia Activities of Chimeric Antigen Receptor-Modified T Cells against CD19 in Chinese Patients with Relapsed/Refractory Acute Lymphocytic Leukemia.

Purpose: Patients with relapsed/refractory acute lymphocytic leukemia (R/R ALL) have a poor prognosis. Chimeric antigen receptor-modified T cells against CD19 (CART19) have displayed anti-leukemia activities. However, data from systemic trials in Chinese patients are limited.Experimental Design: T cells transduced with CD19-directed CAR lentiviral vectors were infused in patients with R/R ALL under fludarabine- and cyclophosphamide-based lymphodepletion. The postinfusion responses, toxicities, expansion, and persistence of CART19s in enrolled patients were observed and monitored.Results: We enrolled 15 patients with R/R ALL. The median transduction efficiency of CART19s was 33%. In vitro cytotoxicity assays were conducted and showed prominent antileukemia activities with CART19s. The patients received CART19s infusion at doses of 1.1 × 106/kg to 9.8 × 106/kg. Twelve patients achieved complete remission 1 month after CART19s infusion. CART19s expanded and persisted in peripheral blood and bone marrow. At 150 days, the overall survival rate and leukemia-free survival rate were 65.5% and 37.8%, respectively. The cumulative incidence of relapse and the nonrelapse mortality rate were 54.5% and 7.7%, respectively. Four patients underwent subsequent haploidentical hematopoietic stem cell transplantation. In this trial, 10 patients experienced cytokine release syndrome (CRS). Grade 3 CRS developed in 40% of patients and was associated with a higher disease burden on day -1 and a higher number of previous relapses.Conclusions: This trial demonstrated potent antileukemia activities of CART19s in Chinese patients with R/R ALL. Disease relapse remained the main obstacle. However, patients with a high risk of relapse after CART19s might benefit from subsequent haploidentical hematopoietic stem cell transplantation. Clin Cancer Res; 23(13); 3297-306. ©2016 AACR.

Anti-CD19 chimeric antigen receptor T-cell therapy for adult Philadelphia chromosome-positive acute lymphoblastic leukemia: Two case reports.

RATIONALE: The presence of the Philadelphia chromosome (Ph) in acute lymphoblastic leukemia (ALL) has been associated with a high risk of disease relapse and a poor prognosis. Allogeneic hematopoietic stem cell transplantation (HSCT) is an established treatment for adults with Ph-positive ALL, but relapse remains the primary cause of treatment failure, and is associated with an extremely poor prognosis. The emergence of resistance to tyrosine kinase inhibitors (TKIs) poses a challenge for patients with disease relapses after initial treatment with TKI-containing regimens. PATIENT CONCERNS: Two patients with TKI-resistant recurrent Ph-positive ALL. DIAGNOSES: Ph-positive ALL. INTERVENTIONS: Anti-CD19 CAR T-cell infusion. OUTCOMES: One patient's bone marrow blasts decreased significantly, and the other reached negative minimal residual disease (MRD). However, we first recorded the development of new-onset acute graft-versus-host disease (aGVHD) after anti-CD19 CAR T-cell infusion in a patient who received allogeneic HSCT. Our 2 case reports also demonstrate the efficacy of anti-CD19 CAR T-cell therapy in the treatment of TKI-resistant Ph-positive ALL. LESSONS: Our report suggests that anti-CD19 CAR T-cell therapy may be a promising option for the treatment of relapsed Ph-positive ALL after conventional chemotherapy or allogeneic HSCT. However, caution is due given the possibility of the adverse effects of cytokine release syndrome (CRS)-induced aGVHD for patients receiving allogeneic HSCT.

Predominant cerebral cytokine release syndrome in CD19-directed chimeric antigen receptor-modified T cell therapy.

UNLABELLED: Chimeric antigen receptor-modified (CAR) T cells targeting CD19 (CART19) have shown therapeutical activities in CD19+ malignancies. However, the etiological nature of neurologic complications remains a conundrum. In our study, the evidence of blood-brain barrier (BBB)-penetrating CAR T cells as a culprit was revealed. A patient with acute lymphocytic leukemia developed sustained pyrexia with tremors about 6 h after CART19 infusion, followed by a grade 2 cytokine release syndrome (CRS) and neurological symptoms in the next 3 days. Contrast-enhanced magnetic resonance showed signs of intracranial edema. Lumbar puncture on day 5 showed an over 400-mmH2O cerebrospinal pressure. The cerebrospinal fluid (CSF) contained 20 WBCs/µL with predominant CD3+ T cells. qPCR analysis for CAR constructs showed 3,032,265 copies/µg DNA in CSF and 988,747 copies/µg DNA in blood. Cytokine levels including IFN-γ and IL-6 in CSF were extremely higher than those in the serum. Methyprednisone was administrated and the symptoms relieved gradually. The predominance of CART19 in CSF and the huge discrepancies in cytokine distributions indicated the development of a cerebral CRS, presumably featured as CSF cytokines largely in situ produced by BBB-penetrating CAR T cells. For the first time, we reported the development of cerebral CRS triggered by BBB-penetrating CAR T cells. TRIAL REGISTRATION: ChiCTR-OCC-15007008 .

RHOBTB3 promotes proteasomal degradation of HIFα through facilitating hydroxylation and suppresses the Warburg effect.

Hypoxia-inducible factors (HIFs) are master regulators of adaptive responses to low oxygen, and their α-subunits are rapidly degraded through the ubiquitination-dependent proteasomal pathway after hydroxylation. Aberrant accumulation or activation of HIFs is closely linked to many types of cancer. However, how hydroxylation of HIFα and its delivery to the ubiquitination machinery are regulated remains unclear. Here we show that Rho-related BTB domain-containing protein 3 (RHOBTB3) directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation. RHOBTB3 also directly interacts with the von Hippel-Lindau (VHL) protein, a component of the E3 ubiquitin ligase complex, facilitating ubiquitination of HIFα. Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα. Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα. Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth. Our work thus reveals that RHOBTB3 serves as a scaffold to organize a multi-subunit complex that promotes the hydroxylation, ubiquitination and degradation of HIFα.