Safety and efficacy of a humanized CD19 chimeric antigen receptor T cells for relapsed/refractory acute lymphoblastic leukemia.

CD19-targeted chimeric antigen receptor T (CAR-T) cells using murine single-chain variable fragment (scFv) has shown substantial clinical efficacy in treating relapsed/refractory acute lymphoblastic leukemia (R/R ALL). However, potential immunogenicity of the murine scFv domain may limit the persistence of CAR-T cells. In this study, we treated 52 consecutive subjects with R/R ALL with humanized CD19-specific CAR-T cells (hCART19s). Forty-six subjects achieved complete remission (CR) (N = 43) or CR with incomplete count recovery (CRi) (N = 3) within 1 month post infusion. During the follow-up with a median time of 20 months, the 1-year cumulative incidence of relapse was 25% (95% confidence interval [CI] 13-46), and 1-year event-free survival was 45% (95% CI 29-60). To the cutoff date, 20 patients presented CD19+ relapse and 2 had CD19- relapse. Among the 22 relapsed patients, 14 had treatment-mediated and treatment-boosted antidrug antibodies (ADA) as detected in a sensitive and specific cell-based assay. ADA positivity was correlated with the disease relapse risk. ADA-positive patients had a significantly lower CAR copy number than ADA-negative patients at the time of recurrence (p < .001). In conclusion, hCART19s therapy is safe and highly active in R/R ALL patients, and the hCART19s treatment could induce the emergence of ADA, which is related to the recurrence of the primary disease.

A novel nanobody-heavy chain antibody against Angiopoietin-like protein 3 reduces plasma lipids and relieves nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a metabolic disease mainly on account of hypercholesterolemia and may progress to cirrhosis and hepatocellular carcinoma. The discovery of effective therapy for NAFLD is an essential unmet need. Angiopoietin-like protein 3 (ANGPTL3), a critical lipid metabolism regulator, resulted in increased blood lipids and was elevated in NAFLD. Here, we developed a nanobody-heavy chain antibody (VHH-Fc) to inhibit ANGPTL3 for NAFLD treatment. RESULTS: In this study, we retrieved an anti-ANGPTL3 VHH and Fc fusion protein, C44-Fc, which exhibited high affinities to ANGPTL3 proteins and rescued ANGPLT3-mediated inhibition of lipoprotein lipase (LPL) activity. The C44-Fc bound a distinctive epitope within ANGPTL3 when compared with the approved evinacumab, and showed higher expression yield. Meanwhile, C44-Fc had significant reduction of the triglyceride (~ 44.2%), total cholesterol (~ 36.6%) and LDL-cholesterol (~ 54.4%) in hypercholesterolemic mice and ameliorated hepatic lipid accumulation and liver injury in NAFLD mice model. CONCLUSIONS: We discovered a VHH-Fc fusion protein with high affinity to ANGPTL3, strong stability and also alleviated the progression of NAFLD, which might offer a promising therapy for NAFLD.

Coagulation Disorders after Chimeric Antigen Receptor T Cell Therapy: Analysis of 100 Patients with Relapsed and Refractory Hematologic Malignancies.

Chimeric antigen receptor (CAR)-T cell therapy, a new immunotherapy for relapsed and refractory (R/R) hematologic malignancies, can be accompanied by adverse events, including coagulation disorders. Here, we performed a comprehensive analysis of coagulation parameters in 100 patients with R/R hematologic malignancies after receiving CAR-T cell therapy to illuminate the profiles of coagulation disorders and to facilitate the management of coagulation disorders. A high incidence of coagulation disorders was observed, including elevated D-dimer (50/100, 50%), increased fibrinogen degradation product (45/100, 45%), decreased fibrinogen (23/100, 23%), prolonged activated partial thromboplastin time (16/100, 16%), and prolonged prothrombin time (10/100, 10%). Coagulation disorders occurred mainly during day 6 to day 20 after CAR-T cell infusion. The changes in coagulation parameters were associated with high tumor burden in acute lymphoblastic leukemia, more lines of prior therapies, lower baseline platelet count, and especially cytokine release syndrome (CRS). Disseminated intravascular coagulation (DIC) was found in 7 patients with grade ≥3 CRS and indicated a poor prognosis. Our study suggests that coagulation disorders are manageable in most patients after CAR-T cell therapy. Coexistence of DIC and severe CRS is closely related to nonrelapsed deaths during the acute toxicity phase, and effective and timely treatment is the key to reduce nonrelapse mortality for patients with DIC and severe CRS.

Potent anti-leukemia activities of humanized CD19-targeted Chimeric antigen receptor T (CAR-T) cells in patients with relapsed/refractory acute lymphoblastic leukemia.

Chimeric antigen receptor T (CAR-T) cell therapy has shown promising results for relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL). The immune response induced by murine single-chain variable fragment (scFv) of the CAR may limit CAR-T cell persistence and thus increases the risk of leukemia relapse. In this study, we developed a novel humanized scFv from the murine FMC63 antibody. A total of 18 R/R ALL patients with or without prior murine CD19 CAR-T therapy were treated with humanized CD19-targeted CAR-T cells (hCART19s). After lymphodepletion chemotherapy with cyclophosphamide and fludarabine, the patients received a single dose (1 × 106 /kg) of autologous hCART19s infusion. Among the 14 patients without previous CAR-T therapy, 13 (92.9%) achieved complete remission (CR) or CR with incomplete count recovery (CRi) on day 30, whereas 1 of the 3 patients who failed a second murine CAR-T infusion achieved CR after hCART19s infusion. At day 180, the overall and leukemia-free survival rates were 65.8% and 71.4%, respectively. The cumulative incidence of relapse was 22.6%, and the nonrelapse mortality rate was 7.1%. During treatment, 13 patients developed grade 1-2 cytokine release syndrome (CRS), 4 patients developed grade 3-5 CRS, and 1 patient experienced reversible neurotoxicity. These results indicated that hCART19s could induce remission in patients with R/R B-ALL, especially in patients who received a reinfusion of murine CAR-T.

Simultaneous blockade of VEGF-B and IL-17A ameliorated diabetic kidney disease by reducing ectopic lipid deposition and alleviating inflammation response.

The pathogenesis of diabetic kidney disease (DKD) is complicated. Current clinical treatments fail to achieve satisfactory efficacy in the prevention of DKD progression, it urgently needs novel and effective treatment for DKD. In this study, we firstly demonstrated that renal lipid metabolism abnormality and inflammation significantly changed in DKD conditions by mining public transcriptomic data of DKD patient samples. KEGG analysis further exhibited the critical role of vascular endothelial growth factor B (VEGF-B) and interleukin 17A (IL-17A) signal pathways in DKD progression, indicating that VEGF-B and IL-17A might be the promising targets for DKD treatment. Then the potential of a novel combination therapy, anti-VEGF-B plus anti-IL-17A antibody, was evaluated for DKD treatment. Our results demonstrated that simultaneous blockade of VEGF-B and IL-17A signaling with their neutralizing antibodies alleviated renal damage and ameliorated renal function. The therapeutic effectiveness was not only related to the reduced lipid deposition especially the neutral lipids in kidney but also associated with the decreased inflammation response. Moreover, the therapy alleviated renal fibrosis by reducing collagen deposition and the expression of fibronectin and α-SMA in kidney tissues. RNA-seq analysis indicated that differential expression genes (DEGs) in db/db mice were significantly clustered into lipid metabolism, inflammation, fibrosis and DKD pathology-related pathways, and 181 of those DEGs were significantly reversed by the combinatory treatment, suggesting the underlying mechanism of administration of anti-VEGF-B and anti-IL-17A antibodies in DKD treatment. Taken together, this study identified that renal lipid metabolism abnormality and inflammation were critically involved in the progression of DKD, and simultaneous blockade of VEGF-B and IL-17A signaling represents a potential DKD therapeutic strategy.

Long-Term Follow-Up of Combination of B-Cell Maturation Antigen and CD19 Chimeric Antigen Receptor T Cells in Multiple Myeloma.

PURPOSE: A combination of anti-B-cell maturation antigen (BCMA) and anti-CD19 chimeric antigen receptor (CAR) T cells induced high response rates in patients with relapsed or refractory (R/R) multiple myeloma (MM), but long-term outcomes have not been assessed yet. PATIENTS AND METHODS: In this single-arm, phase II trial, patients with R/R MM received a combination of anti-BCMA CAR T cells and anti-CD19 CAR T cells at a dose of 1 × 106 cells/kg, after receiving a conditioning chemotherapy consisting of cyclophosphamide and fludarabine. The overall response, long-term outcomes, and safety were assessed, as were their associations with clinical and disease characteristics. RESULTS: Of 69 enrolled patients, 62 received the combined infusion of anti-BCMA and anti-CD19 CAR T cells with a median follow-up of 21.3 months. The overall response rate was 92% (57/62), and complete response or better was observed in 37 patients (60%). Minimal residual disease-negativity was confirmed in 77% (43/56) of the patients with available minimal residual disease detection. The estimated median duration of response was 20.3 months (95% CI, 9.1 to 31.5). The median progression-free survival was 18.3 months (95% CI, 9.9 to 26.7), and the median overall survival was not reached. Patients with extramedullary disease had significantly inferior survival. Fifty-nine patients (95%) had cytokine release syndrome, with 10% grade 3 or higher. Neurotoxic events occurred in seven patients (11%), including 3% grade 3 or higher. Late adverse effects were rare, except for B-cell aplasia, hypogammaglobulinemia, and infections. CONCLUSION: The combination of anti-BCMA and anti-CD19 CAR T cells induced durable response in patients with R/R MM, with a median progression-free survival of 18.3 months and a manageable long-term safety profile.

The localization of hnRNP A2/B1 in nuclear matrix and the aberrant expression during the RA-induced differentiation of human neuroblastoma SK-N-SH cells.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is involved in the synthesis of RNA. Its expression is up-regulated in many tumor cell lines. In this study, we investigated the distribution of hnRNP A2/B1 in the nuclear matrix, including its co-localization with expression products of related genes. Results from 2-DE PAGE and MS showed that hnRNP A2/B1 is involved with components of nuclear matrix proteins of SK-N-SH cells, and that its expression level is down-regulated after retinoic acid (RA) treatment. Protein immunoblotting results further confirm the existence of hnRNP A2/B1 in the nuclear matrix, as well as its down-regulation after RA treatment. Immunofluorescence microscopy observation showed that hnRNP A2/B1 localized in nuclear matrix of SK-N-SH cells and its distribution regions were altered after RA treatment. Laser scanning confocal microscopy observation showed that hnRNP A2/B1 co-localized with c-Myc, c-Fos, P53, and Rb in SK-N-SH cells. The co-localized region was altered as a result of RA treatment. Our data proved that hnRNP A2/B1 is a nuclear matrix protein and can be up-regulated in human neuroblastoma. The expression and distribution of hnRNP A2/B1 can affect the differentiation of SK-N-SH cells, as well as its co-localization with related oncogenes and tumor suppressor genes.

Localization of prohibitin in the nuclear matrix and alteration of its expression during differentiation of human neuroblastoma SK-N-SH cells induced by retinoic acid.

The nuclear matrix-intermediate filament system of human neuroblastoma SK-N-SH cells before and after retinoic acid (RA) treatment was selectively extracted and the distribution of prohibitin (PHB) in the nuclear matrix, as well as its colocalization with related genes, was observed. Results of two-dimensional gel electrophoresis (2-DE), mass spectrometry (MS) identification, and protein immunoblotting all confirm that PHB was present in the components of SK-N-SH nuclear matrix proteins and was down-regulated after RA treatment. Immunofluorescence microscopy observations show that PHB was localized in the nuclear matrix and its distribution was altered due to RA treatment. Laser confocal microscopy results reveal that PHB colocalized with the expression products of c-myc, c-fos, p53, and Rb, but the colocalization region was altered after RA treatment. Our results prove that PHB is a nuclear matrix protein and is localized in nuclear matrix fibers. The distribution of PHB in SK-N-SH cells and its colocalization with related proto-oncogenes and tumor suppressor genes suggest that PHB plays pivotal roles in the differentiation of SK-N-SH cells and deserves further study.

Aberrant expression and localization of hnRNP-A2/B1 is a common event in human gastric adenocarcinoma.

BACKGROUND AND AIM: Nuclear-matrix proteins can be proteomic markers for cancer lesions. The present study aimed to determine the roles of heterogeneous nuclear ribonucleoproteins--A2 and B1 (hnRNP-A2/B1) in human gastric carcinogenesis. METHODS: Human gastric cancer and non-cancerous tissues were collected for immunohistochemical analysis. Proteomics technique, Western blot, laser confocal microscope, and real-time quantitative reverse transcription-polymerase chain reaction were performed to determine the aberrant expression of nuclear-matrix proteins. RESULTS: hnRNP-A2/B1 existed in the nuclear matrix of gastric cancer cells, and its expression was enhanced in human gastric cancer and decreased by hexamethylene bisacetamide. The colocalization of hnRNP-A2/B1 with c-myc, c-fos, p53, and Rb was translocated from the nucleolus to the cytoplasm during the differentiation of tumor cells. CONCLUSIONS: hnRNP-A2/B1 affected tumor cell differentiation through interaction with oncogenes and tumor-suppressor genes, and it was overexpressed in human gastric cancer. We postulate that hnRNP-A2/B1 could serve as a biomarker for the diagnosis of human gastric cancer.

Kinetics of immune reconstitution after anti-CD19 chimeric antigen receptor T cell therapy in relapsed or refractory acute lymphoblastic leukemia patients.

INTRODUCTION: Anti-CD19 chimeric antigen receptor (CAR) -T cells, which recognize and kill both B lymphoblasts and normal B cells, result in B cell aplasia and humoral immunodeficiency. However, there were only a few detailed reports on the profile of immune reconstitution after anti-CD19 CAR-T cell therapy. METHODS: Thirty nine patients with relapsed or refractory (R/R) B cell acute lymphoblastic leukemia (ALL) receiving anti-CD19 CAR-T cell therapy were enrolled. Subjects died, relapsed, received other treatment, or lost to follow-up within 60 days post-infusion were excluded. 21 patients were finally selected. Laboratory and clinical data were collected for analysis of immune reconstitution. RESULTS: CD8+ cells were the first to recover with a median time on day 21(7-87), followed by CD16/CD56+ cells on day 28(14-87), and finally CD4+ cells with only 5(23.81%) patients recovered within 60 days post-infusion. CD4/CD8 ratio was inverted, sustaining for at least 1 year. B cell aplasia occurred in all patients and CD19+ cells returned to normal on a median time of day 79(41-118). All patients developed hypogammaglobulinemia with a median onset time of 2 weeks post-infusion. IgG recovered in 6 patients with a median time on day 184(89-346). IgM recovered on days 212, 242, and 346 in 3 patients. IgA recovered most slowly and remained low >1 year postinfusion. A total of 9 infections occurred in 6(28.57%) patients. CONCLUSIONS: Our data showed prolonged reconstitution of immune function, especially humoral immunity, in R/R B cell ALL patients receiving anti-CD19 CAR-T cell therapy.

Efficacy and Safety of Chimeric Antigen Receptor T-Cell Therapy for Relapsed/Refractory Immunoglobulin D Multiple Myeloma.

Immunoglobulin D (IgD) multiple myeloma (MM) is a rare subtype of MM that carries a worse prognosis than non-IgD subtypes. Compared with non-IgD subtypes, IgD MM is associated with a shorter survival time. The application of chimeric antigen receptor (CAR) T-cell therapy for patients with relapsed or refractory multiple myeloma (R/R MM) has increasing evidence as an efficacious treatment. This study was designed to investigate efficacy and safety of chimeric antigen receptor (CAR) T-cell therapy for relapsed/refractory IgD MM (R/R IgD MM). In this single-arm, phase 2 trial, patients diagnosed with R/R IgD MM were infused with either a combination of anti-B-cell maturation antigen and anti-CD19 CAR T-cells or anti-CD19 CAR T-cells alone, with subsequent evaluation of therapeutic response and treatment-related toxicities. At the data cutoff date, 7 patients were enrolled in our study, and all patients achieved response based on the International Myeloma Working Group Uniform Response Criteria. Six patients achieved stringent complete remission (sCR) within 60 days after CAR T-cell infusion (median time 58 days, range 18 to 90 days), and 1 patient with extramedullary disease achieved minimal response (MR) at 30 days after infusion. Bone marrow minimal residual disease (MRD) negativity was achieved in all patients, and the median time to achieve MRD negativity was 22 days (range 14 to 60 days). The most common grade 3 to 4 treatment-related toxicities were hematological toxicities. All patients experienced cytokine release syndrome (CRS), although CAR T-cell-related neurotoxicity was not observed. In our study, CAR T-cell therapy showed encouraging efficacy in the patients with R/R IgD MM, achieving high rates of sCR and MRD negativity. Aside from CRS and prolonged hematologic toxicities, other adverse reactions were mild, suggesting that this is a well-tolerated treatment with a high therapeutic potential.

Humoral immune reconstitution after anti-BCMA CAR T-cell therapy in relapsed/refractory multiple myeloma.

Systematic and dynamic humoral immune reconstitution is little-known for patients with relapsed/refractory (R/R) multiple myeloma (MM) who received anti-B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T-cell therapy. We investigated the kinetics of B-cell, normal plasma cell, and immunoglobulin recovery in 40 patients who achieved ongoing response after anti-BCMA CAR T-cell therapy. All patients developed B-cell aplasia and the median duration of B-cell aplasia was 70 days (range, 23-270). The B-cell count reached its nadir on median day 7 and returned to baseline level on median day 97. BCMA+ cells in bone marrow turned undetectable on median day 28 (13-159) in 94.87% (37 of 39) of patients. Normal plasma cells in bone marrow were first redetected on median day 212. All patients developed a significant decrease in serum IgG, IgA, and IgM on median day 60. At year 1, recovery of serum IgG, IgM, and IgA was observed in 53.33% (8 of 15; non-IgG MM), 73.08% (19 of 26; non-IgM MM), and 23.81% (5 of 21;non-IgA MM) of the patients, respectively. Median time to IgG, IgM, and IgA recovery were days 386, 254, and not reached during follow-up, respectively. Virus-specific IgG levels decreased with loss of protection. Twenty-three of 40 (57.5%) patients had a total of 44 infection events. There were no infection-related deaths. These results reveal a 7-month aplasia of bone marrow normal plasma cells and longer period of hypogammaglobulinemia, suggesting a profound and lasting humoral immune deficiency after anti-BCMA CAR T-cell therapy, especially for IgA.

Development and evaluation of a novel nano-scale vector for siRNA.

To synthesize a lipid-cationic polymer (LCP) containing brassidic acid side chain and to investigate its transfection efficiency and characteristics as a siRNA gene vector. The LCP was chemically synthesized and its nucleic acid binding capacity was determined by gel electrophoresis. HeLa-EGFP and TH1080-EGFP cell lines were transfected with siRNA against enhanced green fluorescent protein (EGFP) gene using a LCP to investigate the transfection efficiency. An MTT assay was performed to evaluate the cellular toxicity of the LCP vector. Its degradability and stability under acidic conditions were also investigated. The LCP vector possessed high DNA binding capacity. More than 73% of the cellular fluorescence was inhibited by the LCP-mediated transfection of siRNA against EGFP gene, indicating that vector had high transfection efficiency. Cellular viability was about 95% at the optimum transfection efficiency of LCP, suggesting that the cellular toxicity of LCP was very low. The LCP was also observed to be degradable; moreover, it could be easily stored at normal temperature. A gene vector used for the transfection of siRNA was successfully fabricated from synthesized LCP. Its numerous excellent properties entitle values for further scientific research.

Localization of nucleophosmin in nuclear matrix and changes in its expression during the differentiation of human neuroblastoma induced by retinoic acid.

In this article, we selectively extracted the nuclear matrix and intermediate filament system of human neuroblastoma SK-N-SH cells pre- and post-treated with retinoic acid (RA). The distribution of nucleophosmin (NPM) in the nuclear matrix and its colocalization with several products of related genes were investigated. Results from two-dimensional gel electrophoresis and MALDI-TOF showed that NPM was a component of the nuclear matrix and its expression in SK-N-SH cells post-treated with RA was down-regulated. Immunofluorescent microscopy observations further showed that NPM was localized in the nuclear matrix of SK-N-SH cells, and its expression level and distribution were altered after treatment with RA. The colocalization of NPM with c-myc, c-fos, p53, and Rb in SK-N-SH cells was observed under a laser scanning confocal microscope, but the colocalization region was changed by RA. Our results prove that NPM is a nuclear matrix protein, which is localized in nuclear matrix fibers. The colocalization of NPM with its related genes and oncogenes affect the differentiation of SK-N-SH cells. The expression of NPM and its distribution in the process of cell differentiation deserve more intensive investigation.

Safety and efficacy of chimeric antigen receptor (CAR)-T-cell therapy in persons with advanced B-cell cancers and hepatitis B virus-infection.

Chimeric antigen receptor (CAR)-T-cell is a safe and effective therapy of B-cell cancers but it is unknown if this is so in persons with prior hepatitis B virus (HBV) infection. We studied 70 subjects with advanced B-cell cancers receiving CAR-T-cell therapy, 12 of whom had chronic HBV-infection (HBsAg positive) and 29 with resolved HBV-infection (HBsAg negative and anti-HBc positive). Safety and efficacy were compared with 29 subjects without HBV-infection. HBV was reactivated in 2 subjects with chronic HBV-infection and 1 with resolved HBV-infection. There was no HBV-related hepatitis flare. Responses to CAR-T-cell therapy in the three cohorts were not significantly different. There was no significant difference in the incidence or severity of cytokine release syndrome (CRS) and neurologic toxicity between the cohorts. Our data suggest that chronic and resolved HBV-infection do not affect the safety and efficacy of CAR-T-cell therapy.

Differential expression of nuclear matrix proteins during the differentiation of human neuroblastoma SK-N-SH cells induced by retinoic acid.

To investigate the alteration of nuclear matrix proteins (NMPs) during the differentiation of neuroblastoma SK-N-SH cells induced by retinoic acid (RA), differentiation markers were detected by immunocytochemistry and NMPs were selectively extracted and subjected to two-dimensional gel electrophoresis analysis. Immunocytochemical observation demonstrated that the expression of neuronal markers was up-regulated in SK-N-SH cells following RA treatment. Meanwhile, 52 NMPs (41 of which were identified) changed significantly during SK-N-SH differentiation; four of these NMPs were further confirmed by immunoblotting. This study suggests that the differentiation of neuroblastoma cells was accompanied by the altered expression of neuronal markers and NMPs. The presence of some differentially expressed NMPs was related to the proliferation and differentiation of neuroblastomas. Our results may help to reveal the relationship between NMPs and neuroblastoma carcinogenesis and reversion, as well as elucidate the regulatory principals driving neural cell proliferation and differentiation.

An Antibody Fab Fragment-based Chimeric Antigen Receptor Could Efficiently Eliminate Human Thyroid Cancer Cells.

Thyroid cancer remains a significant health problem worldwide. Traditional chemotherapy does generate long-term benefit but are usually accompanied by severe side effects. Immunotherapy by adoptive infusion of T cells is now an attractive alternative to chemotherapy. Chimeric antigen receptor engineered lymphocytes have produced tremendous clinical outcomes in treating leukemia or lymphoma, but not in solid tumors, which is in part due to the low affinity of single chain Fv fragment or the rapid loss of transfused T cells. In present research, we designed a novel Fab based chimeric antigen receptor, which inherits the advantages of Fab fragment as well as the natural TCR receptor. The novel Fab CAR could recognize the tumor antigens independent of MHC/peptide complex, and mimic the natural activation process of endogenous TCR, therefore extend the life span of CAR-engineered T cells and generate durable clinical effects.

A combination of humanised anti-CD19 and anti-BCMA CAR T cells in patients with relapsed or refractory multiple myeloma: a single-arm, phase 2 trial.

BACKGROUND: Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T-cell therapy has been shown to have activity in patients with relapsed or refractory multiple myeloma. Reports have suggested that a small subgroup of less differentiated myeloma clones express CD19 and anti-CD19 CAR T-cell therapy has shown activity in some of these patients. We aimed to assess the activity and safety of a combination of humanised anti-CD19 and anti-BCMA CAR T cells in patients with relapsed or refractory multiple myeloma. METHODS: We did a single-centre, single-arm, phase 2 trial at the Affiliated Hospital of Xuzhou Medical University in China. Patients were eligible if they were aged 18-69 years, had histologically confirmed multiple myeloma, a Karnofsky Performance Score of 50 points or more, and met the International Myeloma Working Group diagnostic criteria for relapsed or refractory disease. Fludarabine (three daily doses of 30mg/m2) and cyclophosphamide (one daily dose of 750 mg/m2) were used to deplete lymphocytes before infusion of humanised anti-CD19 CAR T cells (1 × 106 cells per kg) and murine anti-BCMA CAR T cells (1 × 106 cells per kg). The primary outcome was the proportion of patients who achieved an overall response. Responses were assessed according to the International Myeloma Working Group criteria. This study is registered with the Chinese Clinical Trial Registration Center, number ChiCTR-OIC-17011272. FINDINGS: From May 1, 2017, to Jan 20, 2019, 22 patients were enrolled and 21 received an infusion of CAR T cells and were evaluable for safety and activity analyses. At a median follow-up of 179 days (IQR 72-295), 20 (95%) of 21 patients had an overall response, including nine (43%) stringent complete responses, three (14%) complete responses, five (24%) very good partial responses, and three (14%) partial responses. The most common adverse events included cytokine release syndrome (19 [90%] of 21), including 18 patients (86%) with grade 1-2 cytokine release syndrome. The most common serious adverse events were haematological toxicities, which occurred in 20 (95%) of 21 patients. Common grade 3 or higher adverse events included neutropenia (18 [86%]), anaemia (13 [62%]), and thrombocytopenia (13 [62%]). One patient died due to cerebral hemorrhage, which was considered related to sustained thrombocytopenia. No deaths were judged to be treatment-related. INTERPRETATION: Our results confirm that combined infusion of humanised anti-CD19 and anti-BCMA CAR T cells is feasible in patients with relapsed or refractory multiple myeloma, and the preliminary activity observed warrants further investigation in randomised trials. This dual CAR-T cell combinations might be a promising treatment option for relapsed or refractory multiple myeloma. FUNDING: National Natural Science Foundation of China, Natural Science Foundation, Key Research and Development Plan of Jiangsu.

ATF4 is a novel regulator of MCP-1 in microvascular endothelial cells.

BACKGROUND: Monocyte chemoattractant protein-1 (MCP-1) is a major chemokine that recruits monocyte/macrophage to the site of tissue injury and plays a critical role in microvascular complications of diabetes. However, the mechanisms underlying the regulation of MCP-1 are not fully understood. The present study aims to explore the role of activating transcription factor 4 (ATF4), an ER stress-inducible transcription factor, in regulation of MCP-1 expression and production in brain and retinal microvascular endothelial cells. METHODS: For in vitro study, primary brain microvascular endothelial cells isolated from ATF4 knockout mice or mouse retinal endothelial cells were treated with lipopolysaccharide (LPS) to induce MCP-1 expression. ATF4 expression/function was manipulated by adenoviruses expressing wild type ATF4 (Ad-ATF4) or a dominant negative mutant of the protein (Ad-ATF4DN). For in vivo study, MCP-1 expression was induced by intravitreal injection of LPS or Ad-ATF4 in heterozygous ATF4 knockout or wild type mice. RESULTS: LPS treatment induced a dose- and time-dependent increase in ATF4 expression, ER stress and MCP-1 production in brain and retinal microvascular endothelial cells. Overexpression of ATF4 in endothelial cells significantly increased the secretion of MCP-1 and promoted THP-1 monocyte-endothelial cell adhesion. Conditioned medium from ATF4-overexpressiing endothelial cells significantly enhanced THP-1 cell migration. Consistently, intravitreal injection of Ad-ATF4 remarkably enhanced retinal levels of MCP-1 and promoted inflammatory cell infiltration into the vitreous and retina. In contrast, LPS-induced MCP-1 upregulation was markedly attenuated in ATF4-deficient endothelial cells and in retinas of ATF4 knockout mice, suggesting that ATF4 is essential for LPS-induced MCP-1 production in endothelial cells and in the retina. Mechanistically, overexpression of ATF4 enhanced, while inhibition of ATF4, attenuated the basal and LPS-stimulated phosphorylation of NF-κB, P38, and JNK. Furthermore, pharmacological inhibition of NF-κB, P38, or JNK significantly reduced ATF4-stimulated MCP-1 secretion from endothelial cells. CONCLUSIONS: Taken together, our results suggest a critical role of ATF4 in the regulation of MCP-1 production in retinal and brain microvascular endothelial cells, which may contribute to inflammation-related endothelial injury in diseases such as diabetic retinopathy.