Safety, efficacy and determinants of response of allogeneic CD19-specific CAR-NK cells in CD19+ B cell tumors: a phase 1/2 trial.

There is a pressing need for allogeneic chimeric antigen receptor (CAR)-immune cell therapies that are safe, effective and affordable. We conducted a phase 1/2 trial of cord blood-derived natural killer (NK) cells expressing anti-CD19 chimeric antigen receptor and interleukin-15 (CAR19/IL-15) in 37 patients with CD19+ B cell malignancies. The primary objectives were safety and efficacy, defined as day 30 overall response (OR). Secondary objectives included day 100 response, progression-free survival, overall survival and CAR19/IL-15 NK cell persistence. No notable toxicities such as cytokine release syndrome, neurotoxicity or graft-versus-host disease were observed. The day 30 and day 100 OR rates were 48.6% for both. The 1-year overall survival and progression-free survival were 68% and 32%, respectively. Patients who achieved OR had higher levels and longer persistence of CAR-NK cells. Receiving CAR-NK cells from a cord blood unit (CBU) with nucleated red blood cells ≤ 8 × 107 and a collection-to-cryopreservation time ≤ 24 h was the most significant predictor for superior outcome. NK cells from these optimal CBUs were highly functional and enriched in effector-related genes. In contrast, NK cells from suboptimal CBUs had upregulation of inflammation, hypoxia and cellular stress programs. Finally, using multiple mouse models, we confirmed the superior antitumor activity of CAR/IL-15 NK cells from optimal CBUs in vivo. These findings uncover new features of CAR-NK cell biology and underscore the importance of donor selection for allogeneic cell therapies. ClinicalTrials.gov identifier: NCT03056339 .

First-in-human clinical trial of personalized neoantigen vaccines as early intervention in untreated patients with lymphoplasmacytic lymphoma.

Lymphoplasmacytic lymphoma (LPL) is an incurable low-grade B-cell lymphoma of the bone marrow. Despite a cumulative risk of progression, there is no approved therapy for patients in the asymptomatic phase. We conducted a first-in-human clinical trial of a novel therapeutic DNA idiotype neoantigen vaccine in nine patients with asymptomatic LPL. Treatment was well tolerated with no dose limiting toxicities. One patient achieved a minor response, and all remaining patients experienced stable disease, with median time to disease progression of 61+ months. Direct interrogation of the tumor microenvironment by single-cell transcriptome analysis revealed an unexpected dichotomous antitumor response, with significantly reduced numbers of clonal tumor mature B-cells, tracked by their unique BCR, and downregulation of genes involved in signaling pathways critical for B-cell survival post-vaccine, but no change in clonal plasma cell subpopulations. Downregulation of HLA class II molecule expression suggested intrinsic resistance by tumor plasma cell subpopulations and cell-cell interaction analyses predicted paradoxical upregulation of IGF signaling post vaccine by plasma cell, but not mature B-cell subpopulations, suggesting a potential mechanism of acquired resistance. Vaccine therapy induced dynamic changes in bone marrow T-cells, including upregulation of signaling pathways involved in T-cell activation, expansion of T-cell clonotypes, increased T-cell clonal diversity, and functional tumor antigen-specific cytokine production, with little change in co-inhibitory pathways or Treg. Vaccine therapy also globally altered cell-cell communication networks across various bone marrow cell types and was associated with reduction of protumoral signaling by myeloid cells, principally non-classical monocytes. These results suggest that this prototype neoantigen vaccine favorably perturbed the tumor immune microenvironment, resulting in reduction of clonal tumor mature B-cell, but not plasma cell subpopulations. Future strategies to improve clinical efficacy may require combinations of neoantigen vaccines with agents which specifically target LPL plasma cell subpopulations, or enable blockade of IGF-1 signaling or myeloid cell checkpoints.

A randomized phase 2 trial of idiotype vaccination and adoptive autologous T-cell transfer in patients with multiple myeloma.

We hypothesized that combining adoptively transferred autologous T cells with a cancer vaccine strategy would enhance therapeutic efficacy by adding antimyeloma idiotype (Id)-keyhole limpet hemocyanin (KLH) vaccine to vaccine-specific costimulated T cells. In this randomized phase 2 trial, patients received either control (KLH only) or Id-KLH vaccine, autologous transplantation, vaccine-specific costimulated T cells expanded ex vivo, and 2 booster doses of assigned vaccine. In 36 patients (KLH, n = 20; Id-KLH, n = 16), no dose-limiting toxicity was seen. At last evaluation, 6 (30%) and 8 patients (50%) had achieved complete remission in KLH-only and Id-KLH arms, respectively (P = .22), and no difference in 3-year progression-free survival was observed (59% and 56%, respectively; P = .32). In a 594 Nanostring nCounter gene panel analyzed for immune reconstitution (IR), compared with patients receiving KLH only, there was a greater change in IR genes in T cells in those receiving Id-KLH relative to baseline. Specifically, upregulation of genes associated with activation, effector function induction, and memory CD8+ T-cell generation after Id-KLH but not after KLH control vaccination was observed. Similarly, in responding patients across both arms, upregulation of genes associated with T-cell activation was seen. At baseline, all patients had greater expression of CD8+ T-cell exhaustion markers. These changes were associated with functional Id-specific immune responses in a subset of patients receiving Id-KLH. In conclusion, in this combination immunotherapy approach, we observed significantly more robust IR in CD4+ and CD8+ T cells in the Id-KLH arm, supporting further investigation of vaccine and adoptive immunotherapy strategies. This trial was registered at www.clinicaltrials.gov as #NCT01426828.

Generation of glucocorticoid-resistant SARS-CoV-2 T cells for adoptive cell therapy.

Adoptive cell therapy with virus-specific T cells has been used successfully to treat life-threatening viral infections, supporting application of this approach to coronavirus disease 2019 (COVID-19). We expand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) T cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observe that the choice of cytokines modulates the expansion, phenotype, and hierarchy of antigenic recognition by SARS-CoV-2 T cells. Culture with interleukin (IL)-2/4/7, but not under other cytokine-driven conditions, results in more than 1,000-fold expansion in SARS-CoV-2 T cells with a retained phenotype, function, and hierarchy of antigenic recognition compared with baseline (pre-expansion) samples. Expanded cytotoxic T lymphocytes (CTLs) are directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T cells cannot be expanded efficiently from the peripheral blood of non-exposed controls. Because corticosteroids are used for management of severe COVID-19, we propose an efficient strategy to inactivate the glucocorticoid receptor gene (NR3C1) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing.

Third-Party BK Virus-Specific Cytotoxic T Lymphocyte Therapy for Hemorrhagic Cystitis Following Allotransplantation.

PURPOSE: BK virus-associated hemorrhagic cystitis (BKV-HC) is a common complication of allogenic hematopoietic stem cell transplantation (AHSCT), particularly in recipients of alternative donor transplants, which are being performed in increasing numbers. BKV-HC typically results in painful hematuria, urinary obstruction, and renal dysfunction, without a definitive therapeutic option. METHODS: We performed a clinical trial (ClinicalTrials.gov identifier: NCT02479698) to assess the feasibility, safety, and efficacy of administering most closely HLA-matched third-party BKV-specific cytotoxic T lymphocytes (CTLs), generated from 26 healthy donors and banked for off-the-shelf use. The cells were infused into 59 patients who developed BKV-HC following AHSCT. Comprehensive clinical assessments and correlative studies were performed. RESULTS: Response to BKV-CTL infusion was rapid; the day 14 overall response rate was 67.7% (40 of 59 evaluable patients), which increased to 81.6% among evaluable patients at day 45 (40 of 49 evaluable patients). No patient lost a previously achieved response. There were no cases of de novo grade 3 or 4 graft-versus-host disease, graft failure, or infusion-related toxicities. BKV-CTLs were identified in patient blood samples up to 3 months postinfusion and their in vivo expansion predicted for clinical response. A matched-pair analysis revealed that, compared with standard of care, after accounting for prognostic covariate effects, treatment with BKV-CTLs resulted in higher probabilities of response at all follow-up timepoints as well as significantly lower transfusion requirement. CONCLUSION: Off-the-shelf BKV-CTLs are a safe and effective therapy for the management of patients with BKV-HC after AHSCT.

Generation of glucocorticoid resistant SARS-CoV-2 T-cells for adoptive cell therapy.

Adoptive cell therapy with viral-specific T cells has been successfully used to treat life-threatening viral infections, supporting the application of this approach against COVID-19. We expanded SARS-CoV-2 T-cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observed that the choice of cytokines modulates the expansion, phenotype and hierarchy of antigenic recognition by SARS-CoV-2 T-cells. Culture with IL-2/4/7 but not other cytokine-driven conditions resulted in >1000 fold expansion in SARS-CoV-2 T-cells with a retained phenotype, function and hierarchy of antigenic recognition when compared to baseline (pre-expansion) samples. Expanded CTLs were directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T-cells could not be efficiently expanded from the peripheral blood of non-exposed controls. Since corticosteroids are used for the management of severe COVID-19, we developed an efficient strategy to inactivate the glucocorticoid receptor gene ( NR3C1 ) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing.

Fracture resistance of teeth obturated with two different types of mineral trioxide aggregate cements / Resistência à fratura de dentes obturados com dois tipos diferentes de cimentos agregados de trióxido minera

Objective: Endodontically obturated teeth have lower fracture resistance depending on the obturating material and technique. The purpose of this study was therefore to evaluate the influence of ProRoot MTA (Dentsply Sirona, Tulsa Division) and OrthoMTA III (BioMTA, Daejeon, Korea) as an obturating material on the fracture resistance of endodontically treated teeth. Material and Methods: Thirty extracted human maxillary central incisors were decoronated and instrumented using Protaper instruments (size F5). Irrigation was performed with 2.5% sodium hypochlorite between each instrument change followed by 7% maleic acid for one minute. Finally, canals were flushed with 5 ml of PBS solution for one minute. Samples were then divided into three groups. Group I- positive control (no root canal filling); Group II- obturation with ProRoot MTA; Group III- obturation with OrthoMTA III. Ten teeth were randomly selected as a negative control in which no treatment was performed. All the specimens were then subjected to fracture strength testing using universal testing machine. For evaluation of biomineralization, six maxillary central incisors were divided into two groups. Group I obturated with ProRoot MTA and group II obturated with OrthoMTA III. These samples were subjected to SEM analysis. Results: Positive control group demonstrated the least fracture resistance, while OrthoMTA III group showed the highest fracture resistance. There was no significant difference between negative control group and ProRoot MTA groups (p=0.821). OrthoMTA III group showed better tubular biomineralization when compared to ProRoot MTA. Conclusions: Root canals obturated with OrthoMTA III had better fracture resistance and increased tubular biomineralization compared to ProRoot MTA. Since root canals obturated with OrthoMTA III had better fracture resistance, it can be used as a promising obturating material.(AU)

Objetivo: Dentes obturados endodonticamente apresentam menor resistência à fratura, dependendo do material e da técnica de obturação. Portanto, o objetivo deste estudo foi avaliar a influência do ProRoot MTA (Dentsply Sirona, Tulsa Division) e OrthoMTA III (BioMTA, Daejeon, Coréia) como material obturador na resistência à fratura de dentes tratados endodonticamente. Material e Métodos: Trinta incisivos centrais superiores humanos extraídos foram decoronados e instrumentados com instrumentos Protaper (tamanho F5). A irrigação foi realizada com hipoclorito de sódio a 2,5% entre cada troca de instrumento, seguida por ácido maleico a 7% por um minuto. Finalmente, os canais foram lavados com 5 ml de solução de PBS por um minuto. As amostras foram então divididas em três grupos. Grupo I - controle positivo(sem preenchimento do canal radicular); Grupo II - obturação com ProRoot MTA; Grupo III -obturação com OrthoMTA III. Dez dentes foram selecionados aleatoriamente como controle negativo, no qual nenhum tratamento foi realizado. Todas as amostras foram então submetidas atestes de resistência à fratura usando uma máquina de teste universal. Para avaliação da biomineralização, seis incisivos centrais superiores foram divididos em dois grupos: grupo Iobturado com ProRoot MTA e grupo II obturado com OrthoMTA III. Essas amostras foram submetidas à análise SEM. Resultados: O grupo controle positivo demonstrou a menor resistência à fratura, enquanto o grupo OrthoMTA III apresentou a maior resistência à fratura. Não houve diferença significativa entre o grupo controle negativo e os grupos ProRoot MTA (p= 0,821). O grupo OrthoMTA III apresentou melhor biomineralização tubular quando comparado ao ProRoot MTA. Conclusões: Os canais radiculares obturados com OrthoMTA III apresentaram melhor resistência à fratura e maior biomineralização tubular em comparação como ProRoot MTA. Como os canais radiculares obturados com OrthoMTA III apresentaram melhor resistência à fratura, podendo ser utilizado como um material obturador promissor.(AU)

The efficacy of different irrigation protocols in removing tricalcium silicate-based sealers from simulated root canal irregularities.

OBJECTIVE: The aim of this study was to evaluate the efficacy of different irrigation protocols in removing two tricalcium silicate-based sealers from simulated root canal irregularities and root canal walls. MATERIAL AND METHODS: Root canals of 140 single-rooted teeth were instrumented. In one-half of each root, an apical groove was created. The samples were divided into two main groups (n = 70) based on the sealer used. In group 1, the grooves were filled with MTA Fillapex; in group 2, BioRoot RCS. The reassembled root halves were divided into six experimental and one control groups: 2.5% NaOCl-17% EDTA (Passive ultrasonic irrigation [PUI]), 5% NaOCl/9% DualRinse HEDP (PUI), 2.5% NaOCl-7% Maleic acid (PUI), 2.5% NaOCl-17% EDTA (Er:YAG laser activated irrigation [LAI]), 2.5% NaOCl/9% DualRinse HEDP (LAI), 2.5% NaOCl-7% Maleic acid (LAI), Distilled water (Control). Specimens were scored using SEM. The data were analysed using Kruskal-Wallis and Mann Whitney U tests. RESULTS: Maleic acid and DualRinse HEDP removed higher amounts of MTA Fillapex from the grooves compared to EDTA, when used with both activation methods (p < .001). CONCLUSIONS: Ultrasonically activated maleic acid or DualRinse HEDP can be an effective irrigation regimen in removing tricalcium silicate-based sealers.

Evaluation of SmearOFF, maleic acid and two EDTA preparations in smear layer removal from root canal dentin.

Objectives: To evaluate SmearOFF, 7% maleic acid (MA) and two different preparations of ethylenediaminetetraacetic acid (EDTA) in smear layer removal.Materials and methods: Fifty single-rooted teeth were separated into five groups, instrumented and irrigated as follows: (1) SmearOFF, (2) 7% MA, (3) 18% EDTA (pH 11.4), (4) 17% EDTA (pH 8.5) and (5) 0.9% saline. Teeth samples were blinded and examined by scanning electron microscopy with Image J software.Results: Eighteen percent EDTA was less efficient when compared to SmearOFF and MA at all thirds of the root canal system. There was no difference between SmearOFF and MA in the coronal and middle thirds. In the apical third, MA removed more smear layer. Seventeen percent EDTA was as efficient as SmearOFF and MA in coronal and middle third but not in the apical third. Eighteen percent EDTA removed smear layer less efficiently in the coronal and middle thirds than 17% EDTA; in the apical third, there was no difference observed. In the saline group, all specimens were heavily smeared. There was no significant difference between 18% EDTA and saline at all canal thirds.Conclusions: SmearOFF and 17% EDTA (pH 8.5) had better smear layer removal capability in the coronal and middle thirds of the root canal system. In the apical third, 7% MA was superior. 18% EDTA (pH 11.4) and saline had poor smear layer removal ability.

Phase I study of an active immunotherapy for asymptomatic phase Lymphoplasmacytic lymphoma with DNA vaccines encoding antigen-chemokine fusion: study protocol.

BACKGROUND: There is now a renewed interest in cancer vaccines. Patients responding to immune checkpoint blockade usually bear tumors that are heavily infiltrated by T cells and express a high load of neoantigens, indicating that the immune system is involved in the therapeutic effect of these agents; this finding strongly supports the use of cancer vaccine strategies. Lymphoplasmacytic lymphoma (LPL) is a low grade, incurable disease featuring an abnormal proliferation of Immunoglobulin (Ig)-producing malignant cells. Asymptomatic patients are currently managed by a "watchful waiting" approach, as available therapies provide no survival advantage if started before symptoms develop. Idiotypic determinants of a lymphoma surface Ig, formed by the interaction of the variable regions of heavy and light chains, can be used as a tumor-specific marker and effective vaccination using idiotypes was demonstrated in a positive controlled phase III trial. METHODS: These variable region genes can be cloned and used as a DNA vaccine, a delivery system holding tremendous potential for streamlining vaccine production. To increase vaccination potency, we are targeting antigen-presenting cells (APCs) by fusing the antigen with a sequence encoding a chemokine (MIP-3α), which binds an endocytic surface receptor on APCs. Asymptomatic phase LPL is an excellent model to test our vaccine since patients have not received chemotherapeutics that interfere with innate immune function and have low tumor burden. We are evaluating the safety of this next-generation DNA vaccine in a first-in-human clinical trial currently enrolling asymptomatic LPL patients. To elucidate the mode of action of this vaccine, we will assess its ability to generate tumor-specific immune responses and examine changes in the immune profile of both the peripheral blood and bone marrow. DISCUSSION: This vaccine could shift the current paradigm of clinical management for patients with asymptomatic LPL and inform development of other personalized approaches. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT01209871; registered on September 24, 2010.

MMP-9 in Dentinal Fluid Correlates with Caries Lesion Depth.

The analysis of molecular cues in dentinal fluid from an excavated cavity could improve diagnostics in the context of minimally invasive caries treatment. In the current clinical trial we assessed whether the dentinal fluid levels of MMP-9 (matrix metalloproteinase-9; neutrophil gelatinase) would increase with the progression of carious lesions. MMP-9 is associated with neutrophil-related tissue breakdown in the pulp. Absolute MMP-9 levels were contrasted against the levels of MMP-2, an enzyme related to normal tissue turnover. Dentinal fluid was collected below deep and shallow caries from molars and premolars within the same patients aged 18 years and older (n = 30, 1 tooth per group/patient). Experimental teeth were isolated under a rubber dam prior to excavation. Dentinal fluid was collected from the bottom of the cavity using a size 25 paper point. MMP levels were assessed using an enzyme-linked immunosorbent assay. Nonparametric methods were applied to test for differences between groups. Significantly more (p < 0.05, Wilcoxon test) MMP-9 was collected from the deep carious lesions than from the shallow counterparts. Pairwise comparison of MMP-9 values within patients revealed that there was more MMP-9 collected from deep lesions than from shallow counterparts in 27 of the 30 individuals under investigation (pairwise Wilcoxon test, p < 0.001). In contrast, no such difference existed for MMP-2. There was a high correlation between MMP-9 from deep and shallow lesions (Spearman's ρ = 0.72, p < 0.001), indicating that patients with more MMP-9 in the deep carious lesion also tended to have more MMP-9 in the shallow lesion.

Generation of a new therapeutic peptide that depletes myeloid-derived suppressor cells in tumor-bearing mice.

Immune evasion is an emerging hallmark of cancer progression. However, functional studies to understand the role of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are limited by the lack of available specific cell surface markers. We adapted a competitive peptide phage display platform to identify candidate peptides binding MDSCs specifically and generated peptide-Fc fusion proteins (peptibodies). In multiple tumor models, intravenous peptibody injection completely depleted blood, splenic and intratumoral MDSCs in tumor-bearing mice without affecting proinflammatory immune cell types, such as dendritic cells. Whereas control Gr-1-specific antibody primarily depleted granulocytic MDSCs, peptibodies depleted both granulocytic and monocytic MDSC subsets. Peptibody treatment was associated with inhibition of tumor growth in vivo, which was superior to that achieved with Gr-1-specific antibody. Immunoprecipitation of MDSC membrane proteins identified S100 family proteins as candidate targets. Our strategy may be useful to identify new diagnostic and therapeutic surface targets on rare cell subtypes, including human MDSCs.

Methods for genome-wide analysis of gene expression changes in polyploids.

Polyploidy is an evolutionary innovation, providing extra sets of genetic material for phenotypic variation and adaptation. It is predicted that changes of gene expression by genetic and epigenetic mechanisms are responsible for novel variation in nascent and established polyploids (Liu and Wendel, 2002; Osborn et al., 2003; Pikaard, 2001). Studying gene expression changes in allopolyploids is more complicated than in autopolyploids, because allopolyploids contain more than two sets of genomes originating from divergent, but related, species. Here we describe two methods that are applicable to the genome-wide analysis of gene expression differences resulting from genome duplication in autopolyploids or interactions between homoeologous genomes in allopolyploids. First, we describe an amplified fragment length polymorphism (AFLP)--complementary DNA (cDNA) display method that allows the discrimination of homoeologous loci based on restriction polymorphisms between the progenitors. Second, we describe microarray analyses that can be used to compare gene expression differences between the allopolyploids and respective progenitors using appropriate experimental design and statistical analysis. We demonstrate the utility of these two complementary methods and discuss the pros and cons of using the methods to analyze gene expression changes in autopolyploids and allopolyploids. Furthermore, we describe these methods in general terms to be of wider applicability for comparative gene expression in a variety of evolutionary, genetic, biological, and physiological contexts.

Defensive chemicals of two species of Trachypachus Motschulski.

Analyses of pygidial gland contents of two species of a previously uninvestigated family of beetles (Trachypachidae) by Gas Chromatography-Mass Spectrometry (GC-MS) revealed that their chemistry is similar to that reported from many members of the family Carabidae. Nevertheless, the composition of defensive gland fluids of the two species Trachypachus slevini and T. gibbsii differs sufficiently to distinguish between the two species solely on the basis of their defensive chemistry. The major components of T. slevini glandular fluid are methacrylic, tiglic, and octanoic (= caprylic) acids, together with the hydrocarbon (Z)-9-pentacosene. In contrast, the glandular contents of T. gibbsii contain a rather unique mixture of polar and nonpolar compounds, the principal constituents of which are methacrylic and ethacrylic acids (= 2-ethylacrylic acid), together with 2-phenylethanol, 2-phenylethyl methacrylate, 2-phenylethyl ethacrylate, and (Z)-9-pentacosene.