BCMA-CD19 compound CAR T cells for systemic lupus erythematosus: a phase 1 open-label clinical trial.

OBJECTIVES: This study aims to evaluate the safety and efficacy of BCMA-CD19 compound chimeric antigen receptor T cells (cCAR) to dual reset the humoral and B cell immune system in patients with systemic lupus erythematosus (SLE) with lupus nephritis (LN). METHODS: This is a single-arm open-label multicentre phase 1 study of BCMA and CD19-directed cCAR in patients suffering from SLE/LN with autoantibodies produced by B cells and plasma/long-lived plasma cells. In this clinical trial, we sequentially assigned biopsy-confirmed (classes III-V) LN patients to receive 3×106 cCAR cells/kg postcessation of all SLE medications and conditioning. The primary endpoint of safety and toxicity was assessed. Complete immune reset was indicated by B cell receptor (BCR) deep sequencing and flow cytometry analysis. Patient 11 (P11) had insufficient lymphocyte counts and was underdosed as compassionate use. RESULTS: P1 and P2 achieved symptom and medication-free remission (MFR) from SLE and complete remission from lymphoma. P3-P13 (excluding P11) received an initial dose of 3×106 cCAR cells /kg and were negative for all autoantibodies, including those derived from long-lived plasma cells, 3 months post-cCAR and the complement returned to normal levels. These patients achieved symptom and MFR with post-cCAR follow-up to 46 months. Complete recovery of B cells was seen in 2-6 months post-cCAR. Mean SLE Disease Activity Index 2000 reduced from 10.6 (baseline) to 2.7 (3 months), and renal function significantly improved in 10 LN patients ≤90 days post-cCAR. cCAR T therapy was well tolerant with mild cytokine-release syndrome. CONCLUSIONS: Data suggest that cCAR therapy was safe and effective in inducing MFR and depleting disease-causing autoantibodies in patients with SLE.

Guidelines for the development of a critical software under emergency.

Context: During the first wave of the COVID-19 pandemic, an international and heterogeneous team of scientists collaborated on a social project to produce a mechanical ventilator for intensive care units (MVM). MVM has been conceived to be produced and used also in poor countries: it is open-source, no patents, cheap, and can be produced with materials that are easy to retrieve. Objective: The objective of this work is to extract from the experience of the MVM development and software certification a set of lessons learned and then guidelines that can help developers to produce safety-critical devices in similar emergency situations. Method: We conducted a case study. We had full access to source code, comments on code, change requests, test reports, every deliverable (60 in total) produced for the software certification (safety concepts, requirements specifications, architecture and design, testing activities, etc.), notes, whiteboard sketches, emails, etc. We validated both lessons learned and guidelines with experts. Findings: We contribute a set of validated lessons learned and a set of validated guidelines, together with a discussion of benefits and risks of each guideline. Conclusion: In this work we share our experience in certifying software for healthcare devices produced under emergency, i.e. with strict and pressing time constraints and with the difficulty of establishing a heterogeneous development team made of volunteers. We believe that the guidelines will help engineers during the development of critical software under emergency.

Role of neutral ceramidase in colon cancer.

Alterations in sphingolipid metabolism, especially ceramide and sphingosine 1-phosphate, have been linked to colon cancer, suggesting that enzymes of sphingolipid metabolism may emerge as novel regulators and targets in colon cancer. Neutral ceramidase (nCDase), a key enzyme in sphingolipid metabolism that hydrolyzes ceramide into sphingosine, is highly expressed in the intestine; however, its role in colon cancer has not been defined. Here we show that molecular and pharmacological inhibition of nCDase in colon cancer cells increases ceramide, and this is accompanied by decreased cell survival and increased apoptosis and autophagy, with minimal effects on noncancerous cells. Inhibition of nCDase resulted in loss of ß-catenin and inhibition of ERK, components of pathways relevant for colon cancer development. Furthermore, inhibition of nCDase in a xenograft model delayed tumor growth and increased ceramide while decreasing proliferation. It is noteworthy that mice lacking nCDase treated with azoxymethane were protected from tumor formation. Taken together, these studies show that nCDase is pivotal for regulating initiation and development of colon cancer, and these data suggest that this enzyme is a suitable and novel target for colon cancer therapy.-García-Barros, M., Coant, N., Kawamori, T., Wada, M., Snider, A. J., Truman, J.-P., Wu, B. X., Furuya, H., Clarke, C. J., Bialkowska, A. B., Ghaleb, A., Yang, V. W., Obeid, L. M., Hannun, Y. A. Role of neutral ceramidase in colon cancer.

Effect of sphingosine kinase 1 inhibition on blood pressure.

Accumulating evidence suggests that sphingosine kinase 1 (SphK1) plays a key role in carcinogenesis by regulating cyclooxygenase-2 (COX-2) expression. Recent clinical studies have revealed that COX-2 inhibitors cause adverse cardiovascular side effects, likely due to inhibition of prostacyclin (PGI(2)). In this work, we investigated the roles of SphK1 inhibition on blood pressure (BP). The results show that lack of SphK1 expression did not exacerbate angiotensin II (Ang II)-induced acute hypertension, whereas celecoxib, a COX-2 inhibitor, augmented and sustained higher BP in mice. Interestingly, SphK1-knockout mice inhibited prostaglandin E(2) (PGE(2)) but not PGI(2) production in response to Ang II, whereas celecoxib blocked both PGE(2) and PGI(2) production. Mechanistically, SphK1 down-regulation by siRNA in human umbilical vein endothelial cells decreased cytokine-induced PGE(2) production primarily through inhibition of microsomal PGE synthase-1 (mPGES-1), not COX-2. SphK1 down-regulation also decreased MKK6 expression, which phosphorylates and activates P38 MAPK, which, in turn, regulates early growth response-1 (Egr-1), a transcription factor of mPGES-1. Together, these data indicate that SphK1 regulates PGE(2) production by mPGES-1 expression via the p38 MAPK pathway, independent of COX-2 signaling, in endothelial cells, suggesting that SphK1 inhibition may be a promising strategy for cancer chemoprevention with lack of the adverse cardiovascular side effects associated with coxibs.

Sphingosine 1-phosphate induces filopodia formation through S1PR2 activation of ERM proteins.

Previously we demonstrated that the sphingolipids ceramide and S1P (sphingosine 1-phosphate) regulate phosphorylation of the ERM (ezrin/radixin/moesin) family of cytoskeletal proteins [Canals, Jenkins, Roddy, Hernande-Corbacho, Obeid and Hannun (2010) J. Biol. Chem. 285, 32476-3285]. In the present article, we show that exogenously applied or endogenously generated S1P (in a sphingosine kinase-dependent manner) results in significant increases in phosphorylation of ERM proteins as well as filopodia formation. Using phosphomimetic and non-phosphorylatable ezrin mutants, we show that the S1P-induced cytoskeletal protrusions are dependent on ERM phosphorylation. Employing various pharmacological S1PR (S1P receptor) agonists and antagonists, along with siRNA (small interfering RNA) techniques and genetic knockout approaches, we identify the S1PR2 as the specific and necessary receptor to induce phosphorylation of ERM proteins and subsequent filopodia formation. Taken together, the results demonstrate a novel mechanism by which S1P regulates cellular architecture that requires S1PR2 and subsequent phosphorylation of ERM proteins.

Protein tyrosine nitration and thiol oxidation by peroxynitrite-strategies to prevent these oxidative modifications.

The reaction product of nitric oxide and superoxide, peroxynitrite, is a potent biological oxidant. The most important oxidative protein modifications described for peroxynitrite are cysteine-thiol oxidation and tyrosine nitration. We have previously demonstrated that intrinsic heme-thiolate (P450)-dependent enzymatic catalysis increases the nitration of tyrosine 430 in prostacyclin synthase and results in loss of activity which contributes to endothelial dysfunction. We here report the sensitive peroxynitrite-dependent nitration of an over-expressed and partially purified human prostacyclin synthase (3.3 µM) with an EC50 value of 5 µM. Microsomal thiols in these preparations effectively compete for peroxynitrite and block the nitration of other proteins up to 50 µM peroxynitrite. Purified, recombinant PGIS showed a half-maximal nitration by 10 µM 3-morpholino sydnonimine (Sin-1) which increased in the presence of bicarbonate, and was only marginally induced by freely diffusing NO2-radicals generated by a peroxidase/nitrite/hydrogen peroxide system. Based on these observations, we would like to emphasize that prostacyclin synthase is among the most efficiently and sensitively nitrated proteins investigated by us so far. In the second part of the study, we identified two classes of peroxynitrite scavengers, blocking either peroxynitrite anion-mediated thiol oxidations or phenol/tyrosine nitrations by free radical mechanisms. Dithiopurines and dithiopyrimidines were highly effective in inhibiting both reaction types which could make this class of compounds interesting therapeutic tools. In the present work, we highlighted the impact of experimental conditions on the outcome of peroxynitrite-mediated nitrations. The limitations identified in this work need to be considered in the assessment of experimental data involving peroxynitrite.

Direct measurement of adhesion force between a yeast cell and a lactic acid bacterium cell with atomic force microscopy.

Lactic acid bacteria (LAB) and yeast coexist by providing nutrients as substrates for each other. LAB and yeast cells aggregate via specific interactions between mannose on the yeast surface and the mannose-binding protein (MBP) on the LAB surface. In addition to specific interactions via cell surface proteins, there are also nonspecific interactions related to microbial coaggregation; the extent of their contributions is not clear. Here, microbial coaggregation of yeast and LAB cells was investigated from the view point of particle technology. DLVO theory and thermodynamic approaches predicted that thermodynamically stable coaggregates are not formed because no hydrophobic interaction acts between yeast and LAB. In contrast, optical microscopy revealed that yeast with mannose and LAB with MBP were formed submillimeter-sized coaggregates, whereas deficient strains of yeast and/or LAB were dispersed. Single-cell force spectroscopy revealed that the median adhesion forces were less than 100 pN for all combinations of yeast and LAB. However, some of the adhesion forces between yeast with mannose and LAB with MBP were greater than 400 pN. Furthermore, in the presence of a microbial coaggregation inhibitor, coaggregation of yeast with mannose and LAB with MBP was suppressed, and the adhesion forces were less than 300 pN. These results indicate that the specific interaction rather than the nonspecific interactions acted between mannose on the yeast and the MBP on the LAB formed submillimeter-sized small aggregates. Understanding the contribution of predominant cell-cell interactions may help control microbial behavior in biotechnology.

Treatment of aggressive T-cell lymphoma/leukemia with anti-CD4 CAR T cells.

T-cell lymphomas are aggressive lymphomas that often resist current therapy options or present with relapsed disease, making the development of more effective treatment regimens clinically important. Previously, we have shown that CD4 CAR can effectively target T-cell malignancies in preclinical studies. As IL-15 has been shown to strengthen the anti-tumor response, we have modified CD4 CAR to secrete an IL-15/IL-15sushi complex. These CD4-IL15/IL15sushi CAR T cells and NK92 cells efficiently eliminated CD4+ leukemic cell lines in co-culture assays. Additionally, CD4-IL15/IL15sushi CAR out-performed CD4 CAR in in vivo models, demonstrating a benefit to IL-15/IL-15sushi inclusion. In a Phase I clinical trial, CD4-IL15/IL15sushi CAR T cells were tested for safety in three patients with different T-cell lymphomas. Infusion of CD4-IL15/IL15sushi CAR T cells was well-tolerated by the patients without significant adverse effects and led to the remission of their lymphomas. Additionally, infusion led to the depletion of CD4+ Treg cells and expansion of CD3+CD8+ T cells and NK cells. These results suggest that CD4-IL15/IL15sushi CAR T cells may be a safe and effective treatment for patients with relapsed or refractory T-cell lymphomas, where new treatment options are needed.

Treatment of Aggressive T Cell Lymphoblastic Lymphoma/leukemia Using Anti-CD5 CAR T Cells.

While treatment for B-cell malignancies has been revolutionized through the advent of CAR immunotherapy, similar strategies for T-cell malignancies have been limited. Additionally, T-cell leukemias and lymphomas can commonly metastasize to the CNS, where outcomes are poor and treatment options are associated with severe side effects. Consequently, the development of safer and more effective alternatives for targeting malignant T cells that have invaded the CNS remains clinically important. CD5 CAR has previously been shown to effectively target various T-cell cancers in preclinical studies. As IL-15 strengthens the anti-tumor response, we have modified CD5 CAR to secrete an IL-15/IL-15sushi complex. In a Phase I clinical trial, these CD5-IL15/IL15sushi CAR T cells were tested for safety and efficacy in a patient with refractory T-LBL with CNS infiltration. CD5-IL15/IL15sushi CAR T cells were able to rapidly ablate the CNS lymphoblasts within a few weeks, resulting in the remission of the patient's lymphoma. Despite the presence of CD5 on normal T cells, the patient only experienced a brief, transient T-cell aplasia. These results suggest that CD5-IL15/IL15sushi CAR T cells may be a safe and useful treatment of T-cell malignancies and may be particularly beneficial for patients with CNS involvement.Graphical Abstract.

Two pathways for cyclooxygenase-2 protein degradation in vivo.

COX-2, formally known as prostaglandin endoperoxide H synthase-2 (PGHS-2), catalyzes the committed step in prostaglandin biosynthesis. COX-2 is induced during inflammation and is overexpressed in colon cancer. In vitro, an 18-amino acid segment, residues 595-612, immediately upstream of the C-terminal endoplasmic reticulum targeting sequence is required for N-glycosylation of Asn(594), which permits COX-2 protein to enter the endoplasmic reticulum-associated protein degradation system. To determine the importance of this COX-2 degradation pathway in vivo, we engineered a del595-612 PGHS-2 (Delta 18 COX-2) knock-in mouse lacking this 18-amino acid segment. Delta 18 COX-2 knock-in mice do not exhibit the renal or reproductive abnormalities of COX-2 null mice. Delta 18 COX-2 mice do have elevated urinary prostaglandin E(2) metabolite levels and display a more pronounced and prolonged bacterial endotoxin-induced febrile response than wild type (WT) mice. Normal brain tissue, cultured resident peritoneal macrophages, and cultured skin fibroblasts from Delta 18 COX-2 mice overexpress Delta 18 COX-2 relative to WT COX-2 expression in control mice. These results indicate that COX-2 can be degraded via the endoplasmic reticulum-associated protein degradation pathway in vivo. Treatment of cultured cells from WT or Delta 18 COX-2 mice with flurbiprofen, which blocks substrate-dependent degradation, attenuates COX-2 degradation, and treatment of normal mice with ibuprofen increases the levels of COX-2 in brain tissue. Thus, substrate turnover-dependent COX-2 degradation appears to contribute to COX-2 degradation in vivo. Curiously, WT and Delta 18 COX-2 protein levels are similar in kidneys and spleens from WT and Delta 18 COX-2 mice. There must be compensatory mechanisms to maintain constant COX-2 levels in these tissues.

Targeting Two Antigens Associated with B-ALL with CD19-CD123 Compound Car T Cell Therapy.

The recent FDA approval of the first CAR immunotherapy marks a watershed moment in the advancement toward a cure for cancer. CD19 CAR treatment for B cell acute lymphocytic leukemia has achieved unprecedented remission rates. However, despite success in treating previously relapsed and refractory patients, CD19 CAR faces similar challenges as traditional chemotherapy, in that malignancy can adapt and overcome treatment. The emergence of both antigen positive and negative blasts after CAR treatment represents a need to bolster current CAR approaches. Here, we report on the anti-tumor activity of a CAR T cell possessing 2 discrete scFv domains against the leukemic antigens CD19 and CD123. We determined that the resulting compound CAR (cCAR) T cell possesses consistent, potent, and directed cytotoxicity against each target antigen population both in vitro and in vivo. Our findings indicate that targeting CD19 and CD123 on B-ALL cells may be an effective strategy for augmenting the response against leukemic blasts and reducing rates of relapse.

Characterization of an Anti-CD5 Directed CAR T-Cell against T-Cell Malignancies.

T-cell malignancies often result in poor prognosis and outcome for patients. Immunotherapy has recently emerged as a revolutionary treatment against cancer, and the success seen in CD19 CAR clinical trials may extend to T cell diseases. However, a shared antigen pool coupled with the impact of T-cell depletion incurred by targeting T cell disease remain concepts to be clinically explored with caution. Here we report on the ability of T cells transduced with a CD5CAR to specifically and potently lyse malignant T-cell lines and primary tumors in vitro in addition to significantly improving in vivo control and survival of xenograft models of T-ALL. To extensively explore and investigate the biological properties of a CD5 CAR, we evaluated multiple CD5 CAR constructs and constructed 3 murine models to characterize the properties of CD5 down-regulation, the efficacy and specificity produced by different CD5 CAR construct designs, and the impact of incorporating a CD52 safety switch using CAMPATH to modulate the persistency and function of CAR cells. These data support the potential use of CD5CAR T cells in the treatment of T cell malignancies or refractory disease in clinical settings.

Regulation of intracellular cyclooxygenase levels by gene transcription and protein degradation.

Cyclooxygenases-1 and -2 (COX-1 and -2) catalyze the committed step in prostaglandin formation. Each isozyme subserves different biological functions. This is, at least in part, a consequence of differences in patterns of COX-1 and COX-2 expression. COX-1 is induced during development, and COX-1 mRNA and COX-1 protein are very stable. These latter properties can explain why COX-1 protein levels usually remain constant in those cells that express this isozyme. COX-2 is usually expressed inducibly in association with cell replication or differentiation. Both COX-2 mRNA and COX-2 protein have short half-lives relative to those of COX-1. Therefore, COX-2 protein is typically present for only a few hours after its synthesis. Here we review and develop the concepts that (a) COX-2 gene transcription can involve at least six different cis-acting promoter elements interacting with trans-acting factors generated by multiple, different signaling pathways, (b) the relative contribution of each cis-acting COX-2 promoter element depends on the cell type, the stimulus and the time following the stimulus and (c) a unique 27 amino acid instability element located just upstream of the C-terminus of COX-2 targets this isoform to the ER-associated degradation system and proteolysis by the cytosolic 26S proteasome.

Preclinical Targeting of Human Acute Myeloid Leukemia Using CD4-specific Chimeric Antigen Receptor (CAR) T Cells and NK Cells.

Acute myeloid leukemia (AML) is an aggressive malignancy lacking targeted therapy due to shared molecular and transcriptional circuits as well as phenotypic markers with normal hematopoietic stem cells (HSCs). Identifying leukemia specific markers expressed on AML or AML subtypes for therapeutic targeting is of exquisite clinical value. Here we show that CD4, a T lymphocytes membrane glycoprotein that interacts with major histocompatibility complex class II antigens and is also expressed in certain AML subsets but not on HSCs is a proper target for genetically engineered chimeric antigen receptor T cells (CAR-T cells). Treatment with CD4 redirected CAR-T cell (CD4CAR) specifically eliminated CD4-expressing AML cell lines in vitro and exhibited a potent anti-leukemic effect in a systemic AML murine model in vivo. We also utilized natural killers as another vehicle for CAR engineered cells and this strategy similarly and robustly eliminated CD4- expressing AML cells in vitro and had a potent in vivo anti-leukemic effect and was noted to have shorter in vivo persistence. Our data offer a proof of concept for immunotherapeutic targeting of CD4 as a strategy to treat CD4 expressing refractory AML as a bridge to stem cell transplant (SCT) in a first in human clinical trial.

Targeting T Cell Malignancies Using CD4CAR T-Cells and Implementing a Natural Safety Switch.

T cell malignancies are aggressive diseases with no standard treatment available, often resulting in poor patient outcomes. Lately, the recent FDA approval of a CD19 CAR T cell therapy for B cell acute lymphoblastic leukemia has earned nationwide attention, leading to the possibility that success of CD19 CAR therapy can be extended to T cell malignancies. However, the impact of T cell depletion due to a shared antigen pool remains an issue to be resolved. Here, we describe a CD4CAR capable of eliminating CD4-positive T cell acute lymphoblastic leukemia in a systemic mouse model, with CAMPATH (alemtuzumab) as a natural safety switch to deplete the infused CD4CAR T cells to prevent toxicities associated with CD4 cell aplasia. Our data support the potential use of CD4CAR T cells for the treatment of CD4-postive T-cell acute lymphoblastic leukemia malignancies or refractory disease in clinical settings.

Anti-CD19 chimeric antigen receptor targeting of CD19 + acute myeloid leukemia.

Aberrant expression of CD19 in acute myeloid leukemia (AML) is commonly associated with t(8;21)(q22;q22), although AML cases lacking this translocation occasionally express CD19. Mixed-phenotype acute leukemia also frequently expresses CD19. Chimeric antigen receptor (CAR) technology is a major breakthrough for cancer treatment, with the recent approval of CD19-directed CAR (CD19CAR) for treating B-cell malignancies. However, little information exists on using CD19CAR for other CD19 positive neoplasms such as AML. Our findings indicate that CD19CAR therapy can potentially be used for those with mixed phenotype leukemia and a subset of AML cases.

Compound CAR T-cells as a double-pronged approach for treating acute myeloid leukemia.

Acute myeloid leukemia (AML) bears heterogeneous cells that can consequently offset killing by single-CAR-based therapy, which results in disease relapse. Leukemic stem cells (LSCs) associated with CD123 expression comprise a rare population that also plays an important role in disease progression and relapse. Here, we report on the robust anti-tumor activity of a compound CAR (cCAR) T-cell possessing discrete scFv domains targeting two different AML antigens, CD123, and CD33, simultaneously. We determined that the resulting cCAR T-cells possessed consistent, potent, and directed cytotoxicity against each target antigen population. Using four leukemia mouse models, we found superior in vivo survival after cCAR treatment. We also designed an alemtuzumab safety-switch that allowed for rapid cCAR therapy termination in vivo. These findings indicate that targeting both CD123 and CD33 on AML cells may be an effective strategy for eliminating both AML bulk disease and LSCs, and potentially prevent relapse due to antigen escape or LSC persistence.

Targeting T-cell malignancies using anti-CD4 CAR NK-92 cells.

Peripheral T-cell lymphomas (PTCLs) are a group of very aggressive non-Hodgkin's lymphomas (NHLs) with poor prognoses and account for a majority of T-cell malignancies. Overall, the standard of care for patients with T-cell malignancies is poorly established, and there is an urgent clinical need for a new approach. As demonstrated in B-cell malignancies, chimeric antigen receptor (CAR) immunotherapy provides great hope as a curative treatment regimen. Because PTCLs develop from mature T-cells, these NHLs are commonly CD4+, and CD4 is highly and uniformly expressed. Therefore, CD4 is an ideal target for PTCL CAR immunotherapy. To that effect, we created a robust third-generation anti-CD4 CAR construct (CD4CAR) and introduced it into clonal NK cells (NK-92). CD4CAR NK-92 cells specifically and robustly eliminated diverse CD4+ human T-cell leukemia and lymphoma cell lines (KARPAS-299, CCRF-CEM, and HL60) and patient samples ex vivo. Furthermore, CD4CAR NK-92 cells effectively targeted KARPAS-299 cells in vivo that modeled difficult-to-access lymphoma nodules, significantly prolonging survival. In our study, we present novel targeting of CD4 using CAR-modified NK cells, and demonstrate efficacy. Combined, our data support CD4CAR NK cell immunotherapy as a potential new avenue for the treatment of PTCLs and CD4+ T-cell malignancies.

Effect of Serum Cholesterol on Insulin Secretory Capacity: Shimane CoHRE Study.

OBJECTIVES: Previous studies indicate that, in addition to the blood glucose level, the lipid level in the blood may affect functions of pancreatic beta cells. In this study, we aimed to examine whether there was a relationship between the serum level of total cholesterol (TC) and the insulin secretory capacity in healthy subjects. SUBJECTS AND METHODS: In participants of health examinations conducted from 2006 to 2010, we analyzed data from a total of 2,499 subjects (1,057 men and 1,442 women) after exclusion of individuals with dyslipidemia, thyroid dysfunction, diabetes, HbA1c≥6.5%, or fasting blood glucose≥126 mg/dL. Homeostasis model assessment for beta cell function (HOMA-beta) was utilized as a model representing the pancreatic beta cell function. RESULTS: Although the serum TC level had a positive correlation with HOMA-beta in a univariate correlation analysis, after adjustment by confounding factors in a multiple regression analysis, HOMA-beta had a negative correlation with TC. This was further confirmed in a multiple logistic regression analysis, showing that higher TC was an independent risk factor for decreased insulin secretory capacity (defined as HOMA-beta≤30%) together with higher age, lower BMI, lower TG, male sex and regular alcohol intake. After the participants were stratified by BMI into three groups, the effect of TC on HOMA-beta increased along with the increase in BMI, and it was highly significant in the highest tertile. CONCLUSION: This cross-sectional study indicated that increased serum TC level might be related to the decrease of insulin secretory capacity in aged healthy population and that reduction of TC is more necessary in obese subjects to prevent diabetes.