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Tumor endothelial marker 1 (TEM1) has been identified as a novel surface marker upregulated on the blood vessels and stroma in many solid tumors. We previously isolated a novel single-chain variable fragment (scFv) 78 against TEM1 from a yeast display scFv library. Here we evaluated the potential applications of scFv78 as a tool for tumor molecular imaging, immunotoxin-based therapy and nanotherapy. Epitope mapping, three-dimensional (3D) structure docking and affinity measurements indicated that scFv78 could bind to both human and murine TEM1, with equivalent affinity, at a well-conserved conformational epitope. The rapid internalization of scFv78 and scFv78-labeled nanoparticles was triggered after specific TEM1 binding. The scFv78-saporin immunoconjugate also exerted dose-dependent cytotoxicity with high specificity to TEM1-positive cells in vitro. Finally, specific and sensitive tumor localization of scFv78 was confirmed with optical imaging in a mouse tumor model that has highly endogenous mTEM1 expression in the vasculature. Our data indicate that scFv78, the first fully human anti-TEM1 recombinant antibody, recognizes both human and mouse TEM1 and has unique and favorable features that are advantageous for the development of imaging probes or antibody-toxin conjugates for a large spectrum of human TEM1-positive solid tumors.
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Mesothelin is an epithelial marker highly expressed at the cell surface of cancer cells from diverse origins, including ovarian and pancreatic adenocarcinomas and mesotheliomas. Previously, we identified and characterized an antimesothelin nanobody (NbG3a) for in vitro diagnostic applications. The main goal of this research was to establish the potential of NbG3a as a molecular imaging agent. Site-specific biotinylated NbG3a (bNbG3a) was bound to streptavidin-conjugated reagents for in vitro and in vivo assays. Initially, we performed microscale thermophoresis to determine the binding affinity between bNbG3a and human ( Kd = 46 ± 8 nM) or mouse ( Kd = 4.8 ± 0.4 nM) mesothelin protein. The human and mouse cross-reactivity was confirmed by in vivo optical imaging using bNbG3a bound to fluorescent streptavidin. We also localized the binding site of nNbG3a on human mesothelin using overlapping peptide scan. NbG3a recognized an epitope within residues 21-65 of the mature membrane bound form of human mesothelin, which is part of the N-terminal region of mesothelin that is important for interactions between mesothelin on peritoneal cells and CA125 on tumor cells. Next, the bNbG3a in vivo half-life after intravenous injection in healthy mice was estimated by ELISA assay to be 5.3 ± 1.3 min. In tumor-bearing animals, fluorescent bNbG3a accumulated in a subcutaneous ovarian xenograft (A1847) and in two syngeneic, orthotopic ovarian tumors (intraovary and intraperitoneal ID8) within an hour of intravenous injection that peaked by 4 h and persisted up to 48 h. MRI analysis of bNbG3a-targeted streptavidin-labeled iron oxides showed that the MRI signal intensity decreased 1 h after injection for a subcutaneous xenograft model of ovarian cancer for bNbG3a-labeled iron oxides compared to unlabeled iron oxides. The signal intensity differences continued up to the final time point at 24 h post injection. Finally, in vivo immunofluorescence 24 or 48 h after bNbG3a intravenous injection showed bNbG3a diffuse distribution of both xenograft and syngeneic ovarian tumors, with local areas of high concentration throughout A1847 human tumor. The data support the use of NbG3a for continued preclinical development and translation to human applications for cancers that overexpress mesothelin.
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Reacciones Cruzadas/inmunología , Proteínas Ligadas a GPI/metabolismo , Neoplasias Ováricas/patología , Anticuerpos de Dominio Único/inmunología , Animales , Antígeno Ca-125/metabolismo , Línea Celular Tumoral , Femenino , Compuestos Férricos/metabolismo , Colorantes Fluorescentes/metabolismo , Proteínas Ligadas a GPI/inmunología , Xenoinjertos , Humanos , Imagen por Resonancia Magnética/métodos , Mesotelina , Ratones , Ratones Endogámicos C57BL , Imagen Molecular/métodos , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/metabolismo , Estreptavidina/metabolismoRESUMEN
B7-H4 protein is frequently overexpressed in ovarian cancer. Here, we engineered T cells with novel B7-H4-specific chimeric antigen receptors (CARs) that recognized both human and murine B7-H4 to test the hypothesis that B7-H4 CAR T cell therapy can be applied safely in preclinical models. B7-H4 CAR T cells specifically secreted IFN-γ and lysed B7-H4(+) targets. In vivo, B7-H4 CAR T cells displayed antitumor reactivity against B7-H4(+) human ovarian tumor xenografts. Unexpectedly, B7-H4 CAR T cell treatment reproducibly showed delayed, lethal toxicity 6-8 weeks after therapy. Comprehensive assessment of murine B7-H4 protein distribution uncovered expression in ductal and mucosal epithelial cells in normal tissues. Postmortem analysis revealed the presence of widespread histologic lesions that correlated with B7-H4(+) expression, and were inconsistent with graft versus host disease. Lastly, expression patterns of B7-H4 protein in normal human tissue were comparable to distribution in mice, advancing our understanding of B7-H4. We conclude that B7-H4 CAR therapy mediates control of cancer outgrowth. However, long-term engraftment of B7-H4 CAR T cells mediates lethal, off-tumor toxicity that is likely due to wide expression of B7-H4 in healthy mouse organs. This model system provides a unique opportunity for preclinical evaluation of safety approaches that limit CAR-mediated toxicity after tumor destruction in vivo.
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Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Neoplasias Ováricas/terapia , Receptores de Antígenos/metabolismo , Linfocitos T/inmunología , Inhibidor 1 de la Activación de Células T con Dominio V-Set/inmunología , Animales , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Neoplasias Ováricas/inmunología , Linfocitos T/trasplante , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
With the notable exception of B-cell malignancies, the efficacy of chimeric antigen receptor (CAR) T cells has been limited, and CAR T cells have not been shown to expand and persist in patients with nonlymphoid tumors. Here we demonstrate that redirection of primary human T cells with a CAR containing the inducible costimulator (ICOS) intracellular domain generates tumor-specific IL-17-producing effector cells that show enhanced persistence. Compared with CARs containing the CD3ζ chain alone, or in tandem with the CD28 or the 4-1BB intracellular domains, ICOS signaling increased IL-17A, IL-17F, and IL-22 following antigen recognition. In addition, T cells redirected with an ICOS-based CAR maintained a core molecular signature characteristic of TH17 cells and expressed higher levels of RORC, CD161, IL1R-1, and NCS1. Of note, ICOS signaling also induced the expression of IFN-γ and T-bet, consistent with a TH17/TH1 bipolarization. When transferred into mice with established tumors, TH17 cells that were redirected with ICOS-based CARs mediated efficient antitumor responses and showed enhanced persistence compared with CD28- or 4-1BB-based CAR T cells. Thus, redirection of TH17 cells with a CAR encoding the ICOS intracellular domain is a promising approach to augment the function and persistence of CAR T cells in hematologic malignancies.
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Proteína Coestimuladora de Linfocitos T Inducibles/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Células TH1/inmunología , Células Th17/inmunología , Animales , Antígenos CD28/genética , Antígenos CD28/inmunología , Antígenos CD28/metabolismo , Complejo CD3/genética , Complejo CD3/inmunología , Complejo CD3/metabolismo , Línea Celular Tumoral , Células Cultivadas , Citometría de Flujo , Humanos , Inmunoterapia Adoptiva/métodos , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Subunidad gamma Común de Receptores de Interleucina/genética , Interleucina-17/inmunología , Interleucina-17/metabolismo , Interleucinas/inmunología , Interleucinas/metabolismo , Células K562 , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Células TH1/metabolismo , Células Th17/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Interleucina-22RESUMEN
Nanoparticles are diligently crafted with exacting control over size, shape, and composition. The pristine nanoparticles are rigorously characterized in vitro by numerous physical and materials science techniques. Immediately after being exposed to body fluids, nanoparticles interact with a heterogeneous mixture of proteins and numerous different cell types that modify the nanoparticle surface and affect their bioavailability. Understanding the mechanisms behind the recognition and elimination of these modified nanoparticles is the key to the successful translation of nanomaterials from preclinical to clinical applications. This paper reviews the anatomy of the primary organs (kidney, liver, and spleen) responsible for nanoparticle bioelimination and the components of the innate immune system (complement system and scavenger receptors) that indirectly and directly recognize nanoparticles as foreign. Recent results using PEG as a steric barrier, generating biomimetic nanoparticles, and the effect of nanoparticle material properties to increase the bioavailability of nanoparticles are presented.
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Materiales Biomiméticos/química , Materiales Biomiméticos/farmacocinética , Diseño de Fármacos , Riñón/metabolismo , Hígado/metabolismo , Nanopartículas/química , Bazo/metabolismo , Animales , Disponibilidad Biológica , Humanos , Riñón/química , Hígado/química , Bazo/químicaRESUMEN
Durable response to chimeric antigen receptor T (CART) cell therapy remains limited in part due to CART cell exhaustion. Here, we investigate the regulation of CART cell exhaustion with three independent approaches including: a genome-wide CRISPR knockout screen using an in vitro model for exhaustion, RNA and ATAC sequencing on baseline and exhausted CART cells, and RNA and ATAC sequencing on pre-infusion CART cell products from responders and non-responders in the ZUMA-1 clinical trial. Each of these approaches identify interleukin (IL)-4 as a regulator of CART cell dysfunction. Further, IL-4-treated CD8+ CART cells develop signs of exhaustion independently of the presence of CD4+ CART cells. Conversely, IL-4 pathway editing or the combination of CART cells with an IL-4 monoclonal antibody improves antitumor efficacy and reduces signs of CART cell exhaustion in mantle cell lymphoma xenograft mouse models. Therefore, we identify both a role for IL-4 in inducing CART exhaustion and translatable approaches to improve CART cell therapy.
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Linfocitos T CD8-positivos , Interleucina-4 , Humanos , Animales , Interleucina-4/metabolismo , Interleucina-4/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Ratones Endogámicos NOD , FemeninoRESUMEN
Cancer regression by gene-modified T cells bearing a chimeric antigen receptor (CAR) exodomain of mouse origin can be limited by the induction of transgene immunogenicity resulting in poor persistence and function in vivo. The development of functionally-active CAR of human origin can address this issue. Here, we constructed and evaluated fully human anti-mesothelin CARs comprised of a human mesothelin-specific single-chain antibody variable fragment (P4 scFv) coupled to T cell signaling domains. Primary human T cells expressing P4 CAR specifically produced proinflammatory cytokines, degranulated and exerted potent cytolytic functions when cultured with mesothelin-expressing tumors in vitro. P4 CAR T cells also mediated bystander killing of mesothelin-negative cancer cells during coculture. CAR reactivity was not abrogated by soluble tumor-secreted or recombinant mesothelin protein even at supraphysiological levels. Importantly, adoptive transfer of P4 CAR-expressing T cells mediated the regression of large, established tumor in the presence of soluble mesothelin in a xenogenic model of human ovarian cancer. Thus, primary human T cells expressing fully human anti-mesothelin CAR efficiently kill mesothelin-expressing tumors in vitro and in vivo and have the potential to overcome the issue of transgene immunogenicity that may limit CAR T cell trials that utilize scFvs of mouse origin.
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Proteínas Ligadas a GPI/inmunología , Neoplasias Ováricas/inmunología , Receptores de Antígenos de Linfocitos T/genética , Anticuerpos de Cadena Única/genética , Linfocitos T/inmunología , Animales , Efecto Espectador/inmunología , Línea Celular , Citotoxicidad Inmunológica , Epítopos/inmunología , Femenino , Proteínas Ligadas a GPI/metabolismo , Orden Génico , Vectores Genéticos/genética , Humanos , Lentivirus/genética , Mesotelina , Ratones , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Anticuerpos de Cadena Única/inmunología , Linfocitos T/metabolismo , Transducción Genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Glypican-3 (GPC3) is a heparan-sulfate proteoglycan frequently expressed on the cell membrane of malignant hepatocytes in hepatocellular carcinoma. The capacity for screening potential antibodies in vitro using human hepatocellular lines is critical to ensure binding to this highly post-translationally modified glycophosphatidylinositiol-linked protein. We hypothesized that we could utilize a recently described paired display/secretory yeast library to isolate human-derived scFv against glypican-3 for potential diagnostic and/or therapeutic application. RESULTS: Using two different biotinylated antigen targets, a synthesized 29mer fragment GPC3(550-558) and a truncated GPC3(368-548) fused with glutathione S-transferase (GST) we enriched the yeast display library to greater than 30% target-specific yeast with both positive selection and depletion of streptavidin- and GST-specific clones. After cloning of scFv cDNA from the enriched sub-library, scFv specificity was validated by ELISA for binding to recombinant protein from prokaryotic and eukaryotic sources and ultimately naturally presented human protein on the cell membrane of human hepatocellular cell lines. Specificity was confirmed using non-expressing cell lines and shRNA knockdown. Ultimately, five unique scFv with affinity EC(50) ranging from 5.0-110.9 nM were identified. CONCLUSIONS: Using a paired display/secretory yeast library, five novel and unique scFvs for potential humoral or chimeric therapeutic development in human hepatocellular carcinoma were isolated and characterized.
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Glipicanos/metabolismo , Anticuerpos de Cadena Única/inmunología , Reacciones Antígeno-Anticuerpo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Glipicanos/antagonistas & inhibidores , Glipicanos/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismoRESUMEN
OBJECTIVE: To evaluate the utility of serum (HE4) as a marker for high risk disease in patients with endometrial cancer (EC). METHODS: Preoperative serum HE4 levels were measured from a cohort of 75 patients surgically treated for EC. Cases were compared to matched controls without a history of cancer. HE4 levels were analyzed as a function of primary tumor diameter, grade, stage and histological subtype. Wilcoxon rank-sum test, ROC curve, Spearman rank correlation coefficient and contingency tables were used for statistical analyses. RESULTS: Stage distribution was as follows: 49 stage I, 2 stage II, 20 stage III, 4 stage IV. Type I EC was present in 54 patients, type II in 21. Median HE4 was significantly elevated in both types I and II EC compared to controls (P<0.001 and P=0.019, respectively). There was significant correlation between type I EC, median HE4, deep myometrial invasion (MI) (>50%, P<0.001) and primary tumor diameter (PTD) (>2 cm, P=0.002). Low risk patients (type I, MI ≤ 50% and PTD ≤ 2 cm) had significantly lower median HE4 compared to all other type I EC patients (P<0.01). In comparison to prior investigations, HE4 (cutoff of 8 mfi) was more sensitive than CA125 in detecting advanced stage disease. CONCLUSION: Our data suggest that HE4 is elevated in a high proportion of EC patients, is correlated with PTD and MI, and is more sensitive than CA125 in EC. These observations suggest potential utility of HE4 in the preoperative prediction of high risk disease and the necessity for definitive surgical staging.
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Biomarcadores de Tumor/sangre , Neoplasias Endometriales/sangre , Neoplasias Endometriales/patología , Miometrio/patología , Proteínas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Endometriales/cirugía , Femenino , Humanos , Persona de Mediana Edad , Clasificación del Tumor , Invasividad Neoplásica , Estadificación de Neoplasias , Proyectos Piloto , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAPRESUMEN
Axicabtagene ciloleucel (axi-cel) is an anti-CD19 chimeric antigen receptor (CAR) T cell therapy approved for relapsed/refractory large B cell lymphoma (LBCL) and has treatment with similar efficacy across conventional LBCL subtypes. Toward patient stratification, we assessed whether tumor immune contexture influenced clinical outcomes after axi-cel. We evaluated the tumor microenvironment (TME) of 135 pre-treatment and post-treatment tumor biopsies taken from 51 patients in the ZUMA-1 phase 2 trial. We uncovered dynamic patterns that occurred within 2 weeks after axi-cel. The biological associations among Immunoscore (quantification of tumor-infiltrating T cell density), Immunosign 21 (expression of pre-defined immune gene panel) and cell subsets were validated in three independent LBCL datasets. In the ZUMA-1 trial samples, clinical response and overall survival were associated with pre-treatment immune contexture as characterized by Immunoscore and Immunosign 21. Circulating CAR T cell levels were associated with post-treatment TME T cell exhaustion. TME enriched for chemokines (CCL5 and CCL22), γ-chain receptor cytokines (IL-15, IL-7 and IL-21) and interferon-regulated molecules were associated with T cell infiltration and markers of activity. Finally, high density of regulatory T cells in pre-treatment TME associated with reduced axi-cel-related neurologic toxicity. These findings advance the understanding of LBCL TME characteristics associated with clinical responses to anti-CD19 CAR T cell therapy and could foster biomarker development and treatment optimization for patients with LBCL.
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Productos Biológicos , Linfoma de Células B Grandes Difuso , Receptores Quiméricos de Antígenos , Antígenos CD19 , Recuento de Células , Humanos , Inmunoterapia Adoptiva/efectos adversos , Interferones/uso terapéutico , Interleucina-15 , Interleucina-7/uso terapéutico , Linfoma de Células B Grandes Difuso/terapia , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/uso terapéutico , Microambiente TumoralRESUMEN
T cell-based therapies have induced cancer remissions, though most tumors ultimately progress, reflecting inherent or acquired resistance including antigen escape. Better understanding of how T cells eliminate tumors will help decipher resistance mechanisms. We used a CRISPR/Cas9 screen and identified a necessary role for Fas-FasL in antigen-specific T-cell killing. We also found that Fas-FasL mediated off-target "bystander" killing of antigen-negative tumor cells. This localized bystander cytotoxicity enhanced clearance of antigen-heterogeneous tumors in vivo, a finding that has not been shown previously. Fas-mediated on-target and bystander killing was reproduced in chimeric antigen receptor (CAR-T) and bispecific antibody T-cell models and was augmented by inhibiting regulators of Fas signaling. Tumoral FAS expression alone predicted survival of CAR-T-treated patients in a large clinical trial (NCT02348216). These data suggest strategies to prevent immune escape by targeting both the antigen expression of most tumor cells and the geography of antigen-loss variants. SIGNIFICANCE: This study demonstrates the first report of in vivo Fas-dependent bystander killing of antigen-negative tumors by T cells, a phenomenon that may be contributing to the high response rates of antigen-directed immunotherapies despite tumoral heterogeneity. Small molecules that target the Fas pathway may potentiate this mechanism to prevent cancer relapse.This article is highlighted in the In This Issue feature, p. 521.
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Citotoxicidad Inmunológica , Inmunoterapia , Linfocitos T/inmunología , Linfocitos T/metabolismo , Receptor fas/metabolismo , Animales , Antígenos de Neoplasias/inmunología , Efecto Espectador/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Edición Génica , Ingeniería Genética , Humanos , Inmunoterapia/efectos adversos , Inmunoterapia/métodos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Ratones , Ratones Noqueados , Neoplasias/etiología , Neoplasias/terapia , Receptores Quiméricos de Antígenos , Especificidad del Receptor de Antígeno de Linfocitos T , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Prior studies suggest that combining the Symptom Index (SI) with a serum HE4 test or a CA125 test may improve prediction of ovarian cancer. However, these three tests have not been evaluated in combination. METHODS: A prospective case-control study design including 74 women with ovarian cancer and 137 healthy women was used with logistic regression analysis to evaluate the independent contributions of HE4 and CA125, and the SI to predict ovarian cancer status in a multivariate model. The diagnostic performance of various decision rules for combinations of these tests was assessed to evaluate potential use in predicting ovarian cancer. RESULTS: The SI, HE4, and CA125 all made significant independent contributions to ovarian cancer prediction. A decision rule based on any one of the three tests being positive had a sensitivity of 95% with specificity of 80%. A rule based on any two of the three tests being positive had a sensitivity of 84% with a specificity of 98.5%. The SI alone had sensitivity of 64% with specificity of 88%. If the SI index is used to select women for CA125 and HE4 testing, specificity is 98.5% and sensitivity is 58% using the 2-of-3-positive decision rule. CONCLUSIONS: A 2-of-3-positive decision rule yields acceptable specificity, and higher sensitivity when all 3 tests are performed than when the SI is used to select women for screening by CA125 and HE4. If positive predictive value is a high priority, testing by CA125 and HE4 prior to imaging may be warranted for women with ovarian cancer symptoms.
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Antígeno Ca-125/sangre , Proteínas Secretorias del Epidídimo/metabolismo , Neoplasias Ováricas/diagnóstico , Estudios de Casos y Controles , Femenino , Humanos , Modelos Logísticos , Persona de Mediana Edad , Neoplasias Ováricas/sangre , Valor Predictivo de las Pruebas , Estudios Prospectivos , Índice de Severidad de la Enfermedad , beta-DefensinasRESUMEN
Anti-CD19 chimeric antigen receptor (CAR)-expressing T cells are an effective treatment for B-cell lymphoma, but often cause neurologic toxicity. We treated 20 patients with B-cell lymphoma on a phase I, first-in-human clinical trial of T cells expressing the new anti-CD19 CAR Hu19-CD828Z (NCT02659943). The primary objective was to assess safety and feasibility of Hu19-CD828Z T-cell therapy. Secondary objectives included assessments of blood levels of CAR T cells, anti-lymphoma activity, second infusions and immunogenicity. All objectives were met. Fifty-five percent of patients who received Hu19-CD828Z T cells obtained complete remission. Hu19-CD828Z T cells had clinical anti-lymphoma activity similar to that of T cells expressing FMC63-28Z, an anti-CD19 CAR tested previously by our group, which contains murine binding domains and is used in axicabtagene ciloleucel. However, severe neurologic toxicity occurred in only 5% of patients who received Hu19-CD828Z T cells, whereas 50% of patients who received FMC63-28Z T cells experienced this degree of toxicity (P = 0.0017). T cells expressing Hu19-CD828Z released lower levels of cytokines than T cells expressing FMC63-28Z. Lower levels of cytokines were detected in blood from patients who received Hu19-CD828Z T cells than in blood from those who received FMC63-28Z T cells, which could explain the lower level of neurologic toxicity associated with Hu19-CD828Z. Levels of cytokines released by CAR-expressing T cells particularly depended on the hinge and transmembrane domains included in the CAR design.
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Antígenos CD19/inmunología , Inmunoterapia Adoptiva , Linfoma de Células B/inmunología , Linfoma de Células B/terapia , Receptores Quiméricos de Antígenos/inmunología , Adolescente , Adulto , Anciano , Citocinas/metabolismo , Estudios de Factibilidad , Femenino , Humanos , Células K562 , Masculino , Persona de Mediana Edad , Fenotipo , Dominios Proteicos , Inducción de Remisión , Adulto JovenRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
OBJECTIVE: To evaluate the effect of ovarian cancer risk on the performance of the serum biomarkers mesothelin, human epididymis protein 4 (HE4), and CA125. METHODS: We measured mesothelin, HE4, and CA125 levels from women with invasive ovarian cancer (n = 143), benign gynecologic conditions (n = 124), and controls (n = 344). Demographic, epidemiologic, reproductive, medical, and family history data were collected using a standardized questionnaire. Pedigree and BRCA 1/2 test results were used to stratify women into average and high-risk groups. The diagnostic accuracy of each biomarker was characterized using receiver operating characteristic curve methods. RESULTS: Baseline characteristics did not vary by risk or case status. The distribution of stage and histology was similar in average and high-risk women. All three markers discriminated ovarian cancer cases from risk-matched healthy and benign controls. Marker performance did not vary by risk status. The sensitivity at 95% specificity for discriminating cases from risk-matched healthy control women in the average and high-risk groups, respectively, was 53.9% and 39.0% for mesothelin, 80.4% and 87.8% for HE4, and 79.4% and 82.9% for CA125. The performance of the markers was not as robust when cases were compared with benign controls. Area under the curve values for cases versus healthy and benign controls did not vary by risk status. CONCLUSIONS: The ability of serum mesothelin, HE4, and CA 125 levels to discriminate ovarian cancer cases from healthy and benign controls is not influenced by risk status. Our findings support the pursuit of additional studies evaluating the early detection potential of these markers in high-risk populations.
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Biomarcadores de Tumor/sangre , Antígeno Ca-125/sangre , Proteínas Secretorias del Epidídimo/metabolismo , Glicoproteínas de Membrana/sangre , Neoplasias Ováricas/diagnóstico , Adulto , Anciano , Área Bajo la Curva , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , Femenino , Proteínas Ligadas a GPI , Humanos , Mesotelina , Persona de Mediana Edad , Invasividad Neoplásica , Neoplasias Ováricas/sangre , Neoplasias Ováricas/patología , Medición de Riesgo , Sensibilidad y Especificidad , beta-DefensinasRESUMEN
A 10-fold improvement in the signal-to-noise (S/N) ratio of an optically encoded silica particle-based immunoassay was achieved through incorporating a protein resistant poly(ethylene glycol) (PEG) surface layer and optimizing antibody immobilization conditions. PEG was activated using 2,2,2-trifluoroethanesulfonyl chloride (tresyl) and required a minimum reaction time of 1.5 h. The activated PEG had a reactive half-life of approximately 5 h when stored in acidified dimethyl sulfoxide (DMSO). By increasing the protein incubation time and concentration, a maximum antibody loading on the particle surface of 1.6 x 10(-2) molecules per nm(2) was achieved. The assay S/N ratio was assessed using a multiplexed multicomponent optically encoded species-specific immunoassay. Encoded particles were covalently grafted or nonspecifically coated with either bovine or mouse IgG for the simultaneous detection of complementary anti-IgG "target" or uncomplementary anti-IgG "noise". The versatility and potential as a serum-based assay platform was demonstrated by immobilizing either a polyclonal antibody or an engineered single-chain variable fragment (scFv) capture probe on particles for the detection of the ovarian cancer biomarker, mesothelin (MSLN). The MLSN antigen was spiked into PBS buffer or 50% human serum. Both capture probe orientations, and media conditions showed similar low level detection limits of 5 ng/mL; however, a 40% decrease in maximum signal intensity was observed for assays run in 50% serum.
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Inmunoensayo/métodos , Animales , Bovinos , Proteínas Ligadas a GPI , Humanos , Glicoproteínas de Membrana/análisis , Mesotelina , Ratones , Nanopartículas/química , Polietilenglicoles/química , Sulfonas/químicaRESUMEN
OBJECTIVE: Maternal morbidity and/or mortality (MM) is increased in pyelonephritis and influenza. Alterations in the immune response could account for the increase MM. We sought to determine whether the immune response is functionally different during pregnant and nonpregnant (NP) states. STUDY DESIGN: Mouse model of systemic and localized inflammation was used. Maternal serum was assessed for expression of T-helper cell type 1 and 2 cytokines. Maternal spleens were harvested for immunohistochemistry. RESULTS: Systemic administration of lipopolysaccharides resulted in no mortality to NP mice compared with 88% in preterm and 100% in term mice. A potent cytokine response was present in both NP and pregnancy. Systemic inflammation in pregnancy results in increased CD8 and CD11c expression in spleens. CONCLUSION: Differences in cytokine response to systemic inflammation is unlikley to modulate the increased MM during pregnancy. Altered T-cell and dendritic cell responses in pregnancy may be responsible for the increase in MM.
Asunto(s)
Formación de Anticuerpos , Inflamación/inmunología , Inflamación/mortalidad , Animales , Femenino , Inflamación/sangre , Mortalidad Materna , Ratones , EmbarazoRESUMEN
PURPOSE: To measure circulating antigens, sandwich ELISA assays require two complementary affinity reagents. Mouse monoclonal antibodies (mAb) and polyclonal antibodies (pAb) are commonly used, but because their production is lengthy and costly, recombinant antibodies are emerging as an attractive alternative. EXPERIMENTAL DESIGN: We developed a new class of recombinant antibodies called biobodies (Bb) and compared them to mAb for use in serodiagnosis. Bbs were secreted biotinylated in vivo by diploid yeast and used as affinity reagents after Ni purification. Bead-based assays for HE4 and mesothelin were developed using Bbs in combination with pAbs (Bb/pAb assays). To assess precision, reproducibility studies were done using four runs of 16 replicates at six analyte levels for each marker. Pearson correlations and receiver-operator characteristic analyses were done in 214 patient serum samples to directly compare the Bb/pAb assays to mAb assays. Diagnostic performance of the Bb/pAb assay was further assessed in an expanded set of 336 ovarian cancer cases and controls. RESULTS: On average across analyte levels, Bb/pAb assays yielded within-run and between-run coefficients of variations of 11.7 and 23.8, respectively, for HE4 and 14.0 and 14.5, respectively, for mesothelin. In the subset (n = 214), Pearson correlations of 0.95 for HE4 and 0.92 for mesothelin were observed between mAb and Bb/pAb assays. The area under the curves for the mAb and Bb/pAb assays were not significantly different for HE4 (0.88 and 0.84, respectively; P = 0.20) or mesothelin (0.74 and 0.72, respectively; P = 0.38). CONCLUSION: Yeast-secreted Bbs can be used reliably in cost-effective yet highly sensitive bead-based assays for use in large validation studies.
Asunto(s)
Anticuerpos/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas Secretorias del Epidídimo/metabolismo , Glicoproteínas de Membrana/sangre , Neoplasias Ováricas/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/inmunología , Biotinilación , Proteínas Secretorias del Epidídimo/inmunología , Femenino , Proteínas Ligadas a GPI , Humanos , Glicoproteínas de Membrana/inmunología , Mesotelina , Persona de Mediana Edad , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/metabolismo , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Levaduras/genética , beta-DefensinasRESUMEN
Tumor-specific glycosylation changes are an attractive target for the development of diagnostic and therapeutic applications. Periostin is a glycoprotein with high expression in many tumors of epithelial origin including ovarian cancer. Strategies to target the peptide portion of periostin as a diagnostic or therapeutic biomarker for cancer are limited due to increased expression of periostin in non-cancerous inflammatory conditions. Here, we have screened for antibody fragments that recognize the tumor-specific glycosylation present on glycoforms of periostin containing bisecting N-glycans in ovarian cancer using a yeast-display library of antibody fragments, while subtracting those that bind to the periostin protein with glycoforms found in non-malignant cell types. We generated a biotinylated form of a fully human scFv antibody (scFvC9) that targets the bisecting N-glycans expressed by cancer cells. Validation studies in vitro and in vivo using scFvC9 indicate this antibody can be useful for the development of diagnostic, imaging, and therapeutic applications for cancers that express the antigen.
Asunto(s)
Anticuerpos de Cadena Única/metabolismo , Animales , Western Blotting , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Femenino , Glicosilación , Humanos , Inmunoquímica , Fragmentos de Inmunoglobulinas/metabolismo , Imagen por Resonancia Magnética , Ratones , Neoplasias Ováricas/metabolismo , Biblioteca de Péptidos , Polisacáridos/metabolismoRESUMEN
BACKGROUND: The complexity and heterogeneity of the human plasma proteome have presented significant challenges in the identification of protein changes associated with tumor development. Refined genetically engineered mouse (GEM) models of human cancer have been shown to faithfully recapitulate the molecular, biological, and clinical features of human disease. Here, we sought to exploit the merits of a well-characterized GEM model of pancreatic cancer to determine whether proteomics technologies allow identification of protein changes associated with tumor development and whether such changes are relevant to human pancreatic cancer. METHODS AND FINDINGS: Plasma was sampled from mice at early and advanced stages of tumor development and from matched controls. Using a proteomic approach based on extensive protein fractionation, we confidently identified 1,442 proteins that were distributed across seven orders of magnitude of abundance in plasma. Analysis of proteins chosen on the basis of increased levels in plasma from tumor-bearing mice and corroborating protein or RNA expression in tissue documented concordance in the blood from 30 newly diagnosed patients with pancreatic cancer relative to 30 control specimens. A panel of five proteins selected on the basis of their increased level at an early stage of tumor development in the mouse was tested in a blinded study in 26 humans from the CARET (Carotene and Retinol Efficacy Trial) cohort. The panel discriminated pancreatic cancer cases from matched controls in blood specimens obtained between 7 and 13 mo prior to the development of symptoms and clinical diagnosis of pancreatic cancer. CONCLUSIONS: Our findings indicate that GEM models of cancer, in combination with in-depth proteomic analysis, provide a useful strategy to identify candidate markers applicable to human cancer with potential utility for early detection.