Clinical analysis of long-term survival and influencing factors of chimeric antigen receptor T-cell therapy in relapsed/refractory acute B-cell lymphoblastic leukemia / 中华血液学杂志

Objective: To analyze the survival and influencing factors of chimeric antigen receptor (CAR) T-cell therapy in relapsed/refractory acute B-cell lymphoblastic leukemia (R/R B-ALL) . Methods: Clinical information of patients who received CAR-T-cell therapy and achieved complete remission of R/R B-ALL between May 2015 and June 2018 at the Shaanxi Provincial People's Hospital was obtained. Kaplan-Meier analysis was used to evaluate the overall survival (OS) and leukemia-free survival (LFS) times of patients, and Cox regression analysis was performed to analyze the prognostic factors that affect patient survival after CAR-T therapy. Results: Among the 38 patients with R/R B-ALL, 21 were men, with a median age of 25 (6-59) years and a median OS time of 18 (95% CI 3-33) months. Multivariate Cox regression analysis showed that positive MLL-AF4 fusion gene expression was an independent risk factor for OS and LFS (OS: HR=4.888, 95% CI 1.375-17.374, P=0.014; LFS: HR=6.683, 95% CI 1.815-24.608, P=0.004). Maintenance therapy was a protective factor for OS and LFS (OS: HR=0.153, 95% CI 0.054-0.432, P<0.001; LFS: HR=0.138, 95% CI 0.050-0.382, P<0.001). In patients with MRD negative conversion, LFS benefit (HR=0.209, 95% CI 0.055-0.797, P=0.022) and OS difference was statistically insignificant (P=0.111). Moreover, patients with high tumor burden were risk factors for OS and LFS at the level of 0.1 (OS: HR=2.662, 95% CI 0.987-7.184, P=0.053; LFS: HR=2.452, 95% CI 0.949-6.339, P=0.064) . Conclusion: High tumor burden and high-risk genetics may affect the long-term survival rate of patients with R/R B-ALL receiving CAR-T, and lenalidomide-based maintenance therapy may improve their prognosis.

Research progress of CAR-T in the treatment of hepatocellular carcinoma / 中华肝胆外科杂志

In recent years, chimeric antigen receptor-modified T-cell (CAR-T) immunotherapy, as a new idea of tumor immunotherapy, has been proved to be effective in hematological diseases. More and more studies have been focusing on this field. At present, some progress have been made in the treatment of hepatocellular carcinoma with CAR-T, but some problems such as solid tumor inherent barrier, tumor microenvironment, immune escape and specific tumor associated antigens still need to be further figure out. Nevertheless, CAR-T immunotherapy will provide a more cutting-edge treatment for hepatocellular carcinoma immunotherapy. In addition, the combination of CAR-T and other methods may also be the direction to be explored in the next step.

Preclinical study of suicide gene as a safety switch to control CAR-T cell cytotoxicity / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate whether AP1903, a small-molecule chemical inducer, can terminate the cytotoxicity of CD19CAR-T cells over-expressing iCasp9 suicide gene in vivo and in vitro. Methods: CD19CAR-T cells over-expressing iCasp9 (iCasp9-CD19CAR-T) were constructed and co-incubated with AP1903. Then, the cell phenotype and apoptosis were detected by Flow cytometry, and the iCasp9/CID suicide gene system was verified on K562 and T cells, respectively. The cytotoxicity of iCasp9-CD19CAR-T cells was detected in vivo (survival rate of NCG mice bearing Raji cell transplanted xenograft) and in vitro (cell killing function was detected by Flow cytometry) under the administration of AP1903. Results: Compared with CD19CAR-T cells, iCasp9-CD19CAR-T cells showed in significant difference in proliferation, phenotype and cytotoxicity both in vitro and in vivo (all P>0.05). At 2 h after AP1903 administration, the apoptosis rates of K562 and T cells co-expressing iCasp9 and CD19CAR were (33.8±0.9)% and (27.95±0.35)%, respectively; and at 24 h after AP1903 administration, the apoptosis rates reached 100% in both cell lines. The in vitro cytotoxicity of iCasp9-CD19CAR-T cells induced by AP1903 was significantly lower than that without AP1903 treatment (P<0.01); the 60-day survival rate of mice bearing Raji cell transplanted tumor treated with AP1903-induced iCasp9-CD19CAR-T cells was also significantly lower than those treated with iCasp9-CD19CAR-T cells alone (P<0.01). Conclusion: AP1903 can effectively terminate the cytotoxicity of CD19CAR-T cells over-expressing iCasp9 suicide gene in vitro and in vivo.

An experimental study of CD4 targeted chimeric antigen receptor modified T cell with anti-lymphoma activity / 中华血液学杂志

Objective@#To study the specific killing effect of CD4 membrane protein targeted chimeric antigen receptor modified T (CAR-T) cell.@*Methods@#The second generation CD4 targeted chimeric antigen receptor containing 4-1BB costimulation domain was insert into lentiviral vector through recombinant DNA technology. Lentivirus was prepared and packaged by 293T cells with four plasmids. Beads activated T cells were transduced with lentivirus and the transduction efficiency was checked with Protein L and flow cytometry. T cell subsets and IFN-γ concentrations were detected with probe-tagged antibody and cytometric bead assay.@*Results@#①The transduction efficiency of activated T cells with prepared lentivirus were 50.0%-70.0%. A subset of CD8+ T cell acquired dim expression of CD4 membrane protein after activation. CD4+T cell and CD8+CD4dim T cell were gradually killed by CD4 targeted CAR-T post lentivirus transduction. ②The kill efficacy of CD4 targeted CAR-T cell and control T cell toward KARPAS 299 T cell at an E∶T ratio of 8∶1 for 24 h was (96.9±2.1)% and (11.2±3.1)%, CAR-T cell has a higher killing efficacy than control T cell (t=7.137, P=0.028). The IFN-γ concentrations in culture supernatant of CAR-T cell with K562-CD4 cell, CAR-T cell with K562 cell and CAR-T cell alone were (15 648±2 168), (1 978±354) and (1 785±268) pg/ml, CAR-T cell cocultured with K562-CD4 cell produced more IFN-γ than the other two controls (P<0.01).@*Conclusions@#CD4 targeted CAR-T has an immunophenotype of CD8+CD4-T cell. CD4 targeted CAR-T cell has killing efficacy toward normal CD4+T cell and CD4+T lymphoma cell. CD4 targeted CAR-T cell also has a killing efficacy toward CD4dim target cell.

Challenges of minimal residual disease monitoring in patients with leukemia during chimeric antigen receptor modified T cell immunotherapy / 中华检验医学杂志

Chimeric antigen receptor modified T cell(CAR-T)immunotherapy has achieved remarkable success in the treatment of hematological malignancies,especially for relapsed/refractory acute B lymphocytic leukemia(B-ALL)leukemia patients,most of whom could obtain complete remissions or partial remissions.However,clinical studies also found a as high as 50%total recurrence rate of these patients,in which more than 20% patients relapsed with tumor cells not expressing the primary CAR-T-targeted CD molecules.Increased relapses with negative targeted-molecules or even lost primary abnormal tumor gene, suggesting that immune escape and even clonal evolution events increased rapidly under the high -precision CAR-T immunotherapy pressure.Monitoring of minimal residual disease(MRD)is important to assess the efficacy and find recurrence trends early in a timely manner to guide further clinical intervention.The new features of disease recurrence after CAR-T treatment have put forward higher requirements for traditional and emerging MRD detection methods so as to ensure their effectiveness in the CAR-T treatment era.

Regulatory perspective for chemistry manufacturing and controls considerations in chimeric antigen receptor-modified T cell therapy / 药学学报

As a living cell product, chimeric antigen receptor (CAR)-T cell therapy displays multiple characteristics including the diversity of raw materials, the complexity of manufacturing process and the complementarity of quality control set. Pharmaceutical research and evaluation of CAR-T cell therapy are fundamentally different from small molecule and macromolecular recombinant proteins. Chemistry manufacturing and controls (CMC) review of investigational new drug (IND) submission for CAR-T therapy should especially pay attention to above unique characteristics and focus on potential risks to ensure clinical safety. Based on questions and concerns from recent CMC review practice and workshop on CAR-T cell therapy IND application, the critical points to consider for CMC study is proposed, and questions related to supplementation are also discussed in this review to accelerate the clinic translation of CAR-T therapy.

An experimental study of CD4 targeted chimeric antigen receptor modified T cell with anti-lymphoma activity / 中华血液学杂志

Objective: To study the specific killing effect of CD4 membrane protein targeted chimeric antigen receptor modified T (CAR-T) cell. Methods: The second generation CD4 targeted chimeric antigen receptor containing 4-1BB costimulation domain was insert into lentiviral vector through recombinant DNA technology. Lentivirus was prepared and packaged by 293T cells with four plasmids. Beads activated T cells were transduced with lentivirus and the transduction efficiency was checked with Protein L and flow cytometry. T cell subsets and IFN-γ concentrations were detected with probe-tagged antibody and cytometric bead assay. Results: ①The transduction efficiency of activated T cells with prepared lentivirus were 50.0%-70.0%. A subset of CD8+ T cell acquired dim expression of CD4 membrane protein after activation. CD4+T cell and CD8+CD4dim T cell were gradually killed by CD4 targeted CAR-T post lentivirus transduction. ②The kill efficacy of CD4 targeted CAR-T cell and control T cell toward KARPAS 299 T cell at an E∶T ratio of 8∶1 for 24 h was (96.9±2.1)% and (11.2±3.1)%, CAR-T cell has a higher killing efficacy than control T cell (t=7.137, P=0.028). The IFN-γ concentrations in culture supernatant of CAR-T cell with K562-CD4 cell, CAR-T cell with K562 cell and CAR-T cell alone were (15 648±2 168), (1 978±354) and (1 785±268) pg/ml, CAR-T cell cocultured with K562-CD4 cell produced more IFN-γ than the other two controls (P<0.01). Conclusions: CD4 targeted CAR-T has an immunophenotype of CD8+CD4-T cell. CD4 targeted CAR-T cell has killing efficacy toward normal CD4+T cell and CD4+T lymphoma cell. CD4 targeted CAR-T cell also has a killing efficacy toward CD4dim target cell.