Search

Chimeric antigen receptor T cells (CAR-T) therapy has achieved remarkable therapeutic success in treating a variety of hematopoietic malignancies. However, the high relapse rate and poor in vivo persistence, partially caused by CAR-T cell exhaustion, are still important barriers against CAR-T therapy. It remains largely elusive on the mechanisms of CAR-T exhaustion and how to attenuate exhaustion to achieve better therapeutic efficacy. In this study, we initially observed that CAR-T cells showed rapid differentiation and increased exhaustion after co-culture with tumor cells in vitro, and then performed single-cell ATAC-seq to depict the comprehensive and dynamic landscape of chromatin accessibility of CAR-T cells during tumor cell stimulation. Analyses of differential chromatin accessible regions and motif accessibility revealed that TFs were distinct in each cell type and reconstituted a coordinated regulatory network to drive CAR-T exhaustion. Furthermore, we performed scATAC-seq in patient-derived CAR-T cells and identified BATF and IRF4 as pivotal regulators in CAR-T cell exhaustion. Finally, knockdown of BATF or IRF4 enhanced the killing ability, inhibited exhaustion, and prolonged the persistence of CAR-T cells in vivo. Together, our study unraveled the epigenetic regulatory mechanisms of CAR-T exhaustion and provided new insights into CAR-T engineering to achieve better clinical treatment benefits.

Supplementary materials

A, A representative diagram of the transduction efficiency evaluation of CART19s 5 days after lentivirus infection. B, Specific target cell lysis by CART19s. T cells or CART19s were co-cultured with K562-RFP cells or CD19-RFP-K562 cells at the indicated E:T ratio for overnight co-culture (24 hours). C, IFN-γ secretion measurements in supernatants in T cells or CART19 cells co-cultured with K562-RFP cells or CD19-RFP-K562 cells at the indicated E:T ratios for 24 hours.

Comparison of CART19 levels in different groups. A, Levels of CART19 cells in PB were shown as assessed by means of qPCR assay at serial time points before and after infusion of CART19 respectively. B, CAR DNA copies in BM and PB on day 7 to 9 after CART19 infusion were compared. CAR DNA copies in BM (Patient 2, 6, 11, 13) were higher than in PB. C, Paired measurements of CAR DNA copies in BM and PB on day 7 to 9 after CART19 infusion, percentage of leukemia cells after FC chemotherapy shows strong correlations (CAR DNA copies in BM VS in PB, Spearman r=0.956, p

Relapsed leukemia cells analysis by FACS.A, leukemia cells with bright CD19 expression obtained CR with MRD negative after CART19 therapy and relapsed with CD19 + leukemia cells (Patient 1). B, leukemia cells with dim CD19 expression obtained CR with MRD negative after CART19 therapy but relapsed with 2 subsets of CD19 + and - leukemia cells (Patient 2 and 3).

Supplementary Tables including Table 1, Table 2 and Table 3

CRS complication after CART19 therapy. A, the peak serum concentration of IL-6, IFN-γ, IL-10, CRP, D-dimer and ferritin of a representative patient (Patient 15) during CRS at the indicated time-points after CART19 infusion are showed. B, D, Peak serum levels of CRP, ferritin and D-dimer in patients who developed grade 3 CRS (n=6) compared with those without CRS or with 1 or 2 CRS (n=10). Data represented the mean{plus minus}SEM. The Mann-Whitney U test was used for statistical analysis. C, Paired measurements of peak serum IL-6, CRP, ferritin and D-dimer showed strong correlations (IL-6 VS CRP, Spearman r=0.617, p=0.014; IL-6 VS ferritin, Spearman r=0.574, p=0.028; IL-6 VS D-dimer, Spearman r=0.789, p

CART19 identification and quantification

The cumulative incidence of non-relapse mortality are shown.

Patient 13 with extramedullary leukemia relapse compromising testis obtained CR after CART19 infusion. A, Ultrasound showed complete elimination of extramedullary relapsed masses involved in testis (red arrows). Images are from before treatment and 90 days after CART19 infusion. B, Pathology showed complete elimination of extramedullary relapsed masses involved in testis. Images are from before treatment and 30 days after CART19 infusion. Left: 10×10, right: 10×40.

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.

A rapid and efficient base mediated synthesis of 1,3,5-trisubstituted 1,2,4-triazoles has been developed using the annulation of nitriles with hydrazines, which can be expanded to a wide range of triazoles in good to excellent yields. Ammonia gas is liberated during the reaction, and halo and hetero functional groups as well as free hydroxyl and amino groups are tolerated in this transformation. A variety of alkyl and aryl-substituted nitriles can be functionalized with aromatic and aliphatic hydrazines employing this procedure. This finding provides a practical and useful strategy for the synthesis of various 15N-labeled 1,2,4-triazole derivatives, and two types of mGlu5 receptor pharmaceuticals can be easily assembled in a one-pot manner.

A base-mediated cascade amidination/N-alkylation reaction of amines by alcohols has been developed. For the first time, nitriles have been identified as an efficient and benign water acceptor reagent in N-alkylation. Notably, the procedure tolerates a series of functional groups, such as methoxyl, halo, vinyl and hetero groups, providing a convenient method to construct different substituted diamino compounds, 15N labeled amine and could be scaled up to 1 mol scale offering 138.7 g of the desired product in good yield in one-pot. Mechanistic studies provided strong evidence for the amidination of amines with nitriles facilitated by t-BuOK.

A practical and efficient base mediated synthesis of free 3-aminoindazoles has been developed from the reaction of nitriles with hydrazines, which successfully overcomes the difficulty of using aromatic hydrazines as substrates and allows for the synthesis of a wide range of N-aryl substituted free 3-aminoindazoles in moderate to excellent yields under mild conditions in one-pot. This finding provides a rapid and useful strategy for the synthesis of various functionalized 3-aminoindazole derivatives.

Novel and efficient base-mediated N-alkylation and amidation of amidines with alcohols have been developed, which can be carried out in one-pot reaction conditions, which allows for the synthesis of a wide range of N-alkyl amines and free amides in good to excellent yields with high atom economy. In contrast to borrowing hydrogen/hydrogen autotransfer or oxidative-type N-alkylation reactions, in which alcohols are activated by transition-metal-catalyzed or oxidative aerobic dehydrogenation, the use of amidines provides an effective surrogate of amines. This circumvents the inherent necessity in N-alkylation of an oxidant or a catalyst to be stabilized by ligands.

Lung cancer in nonsmokers tends to be driven by a single somatic mutation or a gene fusion. KIF5B-RET fusion is an oncogene identified in non-small cell lung cancers. In this study, we verified the oncogenic activity of KIF5B-RET fusion and investigated how KIF5B-RET activates the specific signaling pathways for cellular transformation. We aimed to provide a basis for the further development of the therapy for KIF5B-RET positive lung cancer patients.RT-PCR was used to screen for KIF5B-RET fusions in Chinese lung cancer patients. To verify the oncogenic activity of KIF5B-RET kinase in lung cancer cells, we manipulated its expression genetically followed by colony formation and tumor formation assays. The mechanism by which KIF5B-RET kinase induces proliferation was investigated by western blot, coimmunoprecipitation, and administration of RET, MAPK and STAT3 inhibitors.Our study identified a KIF5B-RET fusion in Chinese NSCLC patients and demonstrated that KIF5B-RET transfected cells showed a significantly increased proliferation rate and colony-forming ability. Furthermore, we found that KIF5B-RET fusion kinase induced multilevel activation of STAT3 at both Tyr705 and Ser727, and KIF5B-RET-STAT3 signaling related inhibitors repressed the proliferation and tumorigenicity of lung cancer cells significantly.Our data suggest that KIF5B-RET promotes the cell growth and tumorigenicity of non-small cell lung cancers through multilevel activation of STAT3 signaling, providing possible strategies for the treatment of KIF5B-RET positive lung cancers.

A new porous organic polymer supported rhodium catalyst (Rh/POL-BINAPa&PPh3) has been developed for the hydroformylation of various alkynes to afford the corresponding α,β-unsaturated aldehydes with high chem- and stereoselectivity, excellent catalytic activity and good reusability (10 cycles). The heterogeneous catalyst exhibited more catalytic activity than the comparable homogeneous Rh/BINAPa/PPh3 system.

A new porous organic polymer has been prepared via copolymerization of divinyl-functionalized phosphoramidite ligand and tris(4-vinylphenyl)phosphine. The porous polymer was loaded with Rh(acac)CO2...

Introduction: Chimeric antigen receptor T cells (CAR-T) targeting CD19 have shown great potential for treatment of B-cell malignancies. For those patients who can not achieve complete remission (CR) or suffer from relapse after CAR-T therapy, further therapeutic strategies still remain elusive. Whether existing CAR-T cells can revitalize in vivo and eradicate tumor cells is still unknown. Patient concerns: We report a case of diffused large B-cell lymphoma patient who had achieved CR after CD19 targeted CAR-T therapy but relapsed after 5 months. Diagnosis: Five months after CAR-T cell infusion, the patient was confirmed a relapse by follow-up PET/CT scan and a mass biopsy. Flow cytometry showed a dramatically decreased percentage of CAR-T cells in peripheral blood (PB). Interventions: A second anti-CD19 CAR-T therapy was planned with deliberation. Firstly, the patient received lymphodepletion chemotherapy with fludarabine (25 mg/m2, d1–d3) and cyclophosphamide (500 mg/m2 d2–d3). Outcomes: After fludarabine and cyclophosphamide (FC) lymphodepletion chemotherapy, pre-existing CAR-T cells were revitalized and the patient developed grade 2 cytokine release syndrome (CRS) contributing to the regression of relapsed B-cell lymphoma. Conclusions: This case suggested that FC chemotherapy could revitalize CAR-T cells contributing to the regression of relapsed B-cell lymphoma. Nevertheless, further researches are required in the future as this report described only a single case.

Successful chimeric Ag receptor modified T cell therapy for isolated testicular relapse after hematopoietic cell transplantation in an acute lymphoblastic leukemia patient

Chronic myeloid leukemia (CML) is a clonal disorder of multipotent hematopoietic progenitor cells and is characterized by t (9; 22) (q34; q11) that produce the BCR-ABL fusion protein with constitut...

Objective: Chimeric antigen receptor T cells (CART) emerged as a robust therapeutic approach for refractory or relapsed B acute lymphoblastic leukaemia (B-ALL) in recent years. However, 30-50% of patients experienced leukaemia relapse within 1 year after CART cells therapy. Bone marrow derived mesenchymal stromal cells (MSCs) have been demonstrated to have immunosuppressive properties on T cell-mediated immune responses, but the impacts of MSCs on CART cells are not clear. Here we address the role of galectin-9 secreted from MSCs in immunosuppression of CART cells. Methods: MSCs and T cells were isolated from bone marrow and peripheral blood of healthy donors, respectively, and CD19 targeted CART cells containing 4-1BB costimulatory construction were prepared as previously reported. The proliferation of CART cells was evaluated by cell counting and Ki-67 expression. The expression of PD1, TIM3, LAG3 and FasL was detected by flow cytometry. The cytotoxicity of CART cells was determined by luciferase-based assays. Transwell was used to assess the contribution of soluble factors. The mRNA expression of COX-2, IL-6, IL-10, TGFβ and galectin-9 was detected by real time PCR. We used the lentivirus-based shRNA interference to assess the role of Galetin-9 on immunosuppression of CART cells. Results: In this work, we discovered that CAR-T cells co-cultured with MSCs for 72 hours in vitro showed a decreased proliferation rate and Ki-67 expression. And the inhibition effect became more significant with the increase in the proportion of MSCs (Fig.a,b,c,d). Notably, the expression of inhibitory receptor TIM3 upregulated remarkably while no change of PD1, LAG3 and FasL were observed(Fig.e,f). We routinely use luciferase-based cytotoxic assay for functional testing. By this measure, we found that with the increase in the proportion of MSC, CAR-T cells behave even weaker cytotoxic effect, achieving lower cytolysis rate of Nalm6 cells(Fig.g).To further determine the possible factors that contribute to the changes mentioned above, CART cells were separated from MSCs by transwell. As expected, the phenomenon of inhibited CART cells proliferation and cytotoxicity, increased expression of TIM3 was observed as well, although without direct contact of CART cells with MSCs. The contribution of soluble factors was in consideration and further experiments of real time PCR revealed that COX-2 and galectin-9 were strongly induced by CART cells and pro-inflammatory cytokines(IFNγ, TNFα). In light of that galectin-9 is the ligand of inhibitory receptor TIM3, which increased obviously in the presence of MSCs, we speculated that galectin-9 might be responsible for immunosuppression of CART cells. A knockdown approach with shRNA demonstrated the immunosuppressive activity of galectin-9 deficient MSCs on CART cells decreased significantly. We provided experimental evidence that galectin-9 may contribute to MSC mediated immunosuppression on CART cells by binding to its receptor TIM-3. Conclusion: Our findings demonstrated for the first time that galectin-9 is involved in MSC mediated immunosuppression on CART cells, and represented a potential therapeutic target for enhancing the efficacy of CART cells and reducing the incidence of leukaemia relapse after CART cells therapy. Key words: Chimeric antigen receptor T cells; mesenchymal stromal cells; immunosuppression; galectin-9. Fig. a. Co-culture of CART cells and MSCs. b,c,d. Cell proliferation and Ki-67 expression of CART cells co-cultured with MSCs at different ratios. e,f. Expression of TIM3, PD1, LAG3, FasL of CART cells co-cultured with MSCs at different ratios. g. Cytotoxicity of CART cells co-cultured with MSCs at different ratios (CART without MSC, CART:MSC=1:5, CART:MSC=1:10, CART:MSC=1:20). Figure Disclosures No relevant conflicts of interest to declare.

GRB2-associated-binding protein 1 (Gab1) belongs to Gab adaptor family, which integrates multiple signals in response to the epithelial growth factors. Recent genetic studies identified genetic variants of human Gab1 gene as potential risk factors of asthmatic inflammation. However, the functions of Gab1 in lungs remain largely unknown. Alveolar type-II cells (AT-IIs) are responsible for surfactant homeostasis and essentially regulate lung inflammation following various injuries (3). In this study, in vitro knockdown of Gab1 was shown to decrease the surfactant proteins (SPs) levels in AT-IIs. We further examined in vivo Gab1 functions through alveolar epithelium-specific Gab1 knockout mice (Gab1Δ/Δ). In vivo Gab1 deficiency leads to a decrease in SP synthesis and the appearance of disorganized lamellar bodies. Histological analysis of the lung sections in Gab1Δ/Δmice shows no apparent pathological alterations or inflammation. However, Gab1Δ/Δmice demonstrate inflammatory responses during the LPS-induced acute lung injury. Similarly, in mice challenged with bleomycin, fibrotic lesions were found to be aggravated in Gab1Δ/Δ. These observations suggest that the abolishment of Gab1 in AT-IIs impairs SP homeostasis, predisposing mice to lung injuries. In addition, we observed that the production of surfactants in AT-IIs overexpressing Gab1 mutants, in which Shp2 phosphatase and PI3K kinase binding sites have been mutated (Gab1ΔShp2, Gab1ΔPI3K), has been considerably attenuated. Together, these findings provide the direct evidence about the roles of docking protein Gab1 in lungs, adding to our understanding of acute and interstitial lung diseases caused by the disruption of alveolar SP homeostasis.

Introduction: Chimeric antigen receptor (CAR) T-cell therapy has displayed potent anti-leukemia activity in refractory/relapsed acute lymphocytic leukemia (ALL). However, the influence of CAR-T therapy on host systemic and local immunity has not been well examined. We therefore applied high-throughput T cell receptor β chain sequencing to track dynamic change of T-cell repertoire in vivo induced by CAR-T therapy in B-cell acute lymphocytic leukemia patients. Patients and Methods: Six patients with 45 samples were under observation. The samples obtained to be tested were the peripheral blood mononuclear cell (PBMC) samples and bone marrow mononuclear cell (BMMC) samples before and after CAR-T administration, as well as the CAR-transduced autologous T cell samples on the day when they were to be infused to patients. The information of samples and patients was summarized in Table 1. The TCR full length mRNAs of these samples were deeply sequenced using the ImmunHubTM TCR profiling system (ImmunQuad Biotech). Briefly, a 5'RACE unbiased amplification protocol was used. An algorithm was applied to raw sequencing data for PCR and sequencing errors correction and V, D, J, C gene segments mapping with IMGT®. The inverse Simpson index and the clonality index was calculated to estimate TCRβ clonotype diversity and the state of clonal proliferation of T cells. The donut chart and clone tracking heat map were generated by R. Result: Compared to preinfusion TCR diversity, we observed inverse Simpson's index of 4 of 6 patients' PBMC samples (Fig. 1A) and of 3 of 4 patients' BMMC samples (Fig. 1B) had been increasing to day 7 after CAR-T treatment. As time went by, a decline of TCR diversity from day 7 to day 28 was detected in both PBMC and BMMC samples (Fig. 1). Of note, a relative rising tide of TCR diversity was observed after the decline in PBMC samples (Fig. 1A). The decreased TCR diversity leaded us to test the change of TCR clonality. As shown by Fig. 2A and 2B, in comparison with the pre-treatment and the CAR-T samples, we detected highly clonal proliferation of T cells in both blood and bone marrow. Next, we applied the clonality index to quantitatively define T cell clonal expansion (Fig. 2C and 2D). The clonality index was raised from 0.16±0.07 and 0.14±0.05 before treatment to 0.27±0.09 and 0.3±0.15 at last time point post infusion in PBMCs and BMMCs, respectively (mean ± SD, student's t test, P=0.03 and P=0.17, respectively).CAR-T-induced T-cell clonal expansion triggered us to trace back the original clonal source. For tracking clonal evolution, we selected the 100 most prevalent T cell clonotypes found at the last time point in both PBMCs and BMMCs and tracked their frequencies at earlier time points. As was displayed by the heat map, the top 100 T cell clones dominating in PBMCs and BMMCs at the last time point were barely found in CAR-T cell pool and were the low-frequency clonotypes in preinfusion samples (Fig. 3A and 3B). In order to quantitatively describe this phenomenon, we compared the total frequency of top 10 T cell clones in PBMC and BMMC samples at the last time point to their corresponding total frequency in preinfusion samples and the infused CAR-T pool (Fig. 3C, 3D, 3E and 3F). The total frequency of the top 10 T cell clones was 23%±11% and 27%±14% at last time point, 4%±6% and 8%±7% before treatment and 0.05%±0.09% and 0.01%±0.01% in the infused CAR-T samples in PBMCs and BMMCs, respectively (mean ± SD, Fig. 3C, 3D, 3E and 3F; student's t test, P=0.01, P=0.01, P=0.08 and P=0.03, respectively). Only one patient's (patient 6's) top 10 T cell clones in PBMCs at last time point were not found in both CAR-T pool and preinfusion sample, which may be caused by the limitation of sequencing detection (Fig. 3C). Conclusion: For the first time, we demonstrated CAR-T therapy could stimulate the clonal proliferation of endogenous non-CAR T cells in patients. Along with other groups' animal results (Barber A et al. J Immunol. 2009) indicating that CAR-T therapy could facilitate the infiltrating of tumor antigen-specific T cells, these expanded T cell clones of patients in our trial are most likely tumor antigen specific, which could provide synergistic anti-tumor effect following CAR-T adoptive transfer. The trial was registered in Chinese Clinical Trial Registry and the registration number is ChiCTR-OCC-15007008. Disclosures No relevant conflicts of interest to declare.

Background Lung cancer in nonsmokers tends to be driven by a single somatic mutation or a gene fusion. KIF5B-RET fusion is an oncogene identified in non-small cell lung cancers. In this study, we verified the oncogenic activity of KIF5B-RET fusion and investigated how KIF5B-RET activates the specific signaling pathways for cellular transformation. We aimed to provide a basis for the further development of the therapy for KIF5B-RET positive lung cancer patients. Methods RT-PCR was used to screen for KIF5B-RET fusions in Chinese lung cancer patients. To verify the oncogenic activity of KIF5B-RET kinase in lung cancer cells, we manipulated its expression genetically followed by colony formation and tumor formation assays. The mechanism by which KIF5B-RET kinase induces proliferation was investigated by western blot, coimmunoprecipitation, and administration of RET, MAPK and STAT3 inhibitors. Results Our study identified a KIF5B-RET fusion in Chinese NSCLC patients and demonstrated that KIF5B-RET transfected cells showed a significantly increased proliferation rate and colony-forming ability. Furthermore, we found that KIF5B-RET fusion kinase induced multilevel activation of STAT3 at both Tyr705 and Ser727, and KIF5B-RET-STAT3 signaling related inhibitors repressed the proliferation and tumorigenicity of lung cancer cells significantly. Conclusions Our data suggest that KIF5B-RET promotes the cell growth and tumorigenicity of non-small cell lung cancers through multilevel activation of STAT3 signaling, providing possible strategies for the treatment of KIF5B-RET positive lung cancers.