RESUMEN
Aging results in increased myelopoiesis, which is linked to the increased incidence of myeloid leukemias and production of myeloid-derived suppressor cells. Here, we examined the contribution of plasma cells (PCs) to age-related increases in myelopoiesis, as PCs exhibit immune regulatory function and sequester in bone marrow (BM). PC number was increased in old BM, and they exhibited high expression of genes encoding inflammatory cytokines and pathogen sensors. Antibody-mediated depletion of PCs from old mice reduced the number of myeloid-biased hematopoietic stem cells and mature myeloid cells to levels in young animals, but lymphopoiesis was not rejuvenated, indicating that redundant mechanisms inhibit that process. PCs also regulated the production of inflammatory factors from BM stromal cells, and disruption of the PC-stromal cell circuitry with inhibitors of the cytokines IL-1 and TNF-α attenuated myelopoiesis in old mice. Thus, the age-related increase in myelopoiesis is driven by an inflammatory network orchestrated by PCs.
Asunto(s)
Envejecimiento/fisiología , Médula Ósea/fisiología , Células Madre Hematopoyéticas/patología , Inflamación/metabolismo , Mielopoyesis/fisiología , Células Plasmáticas/fisiología , Animales , Células Cultivadas , Humanos , Interleucina-1/metabolismo , Depleción Linfocítica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Understanding how viruses subvert host immunity and persist is essential for developing strategies to eliminate infection. T cell exhaustion during chronic viral infection is well described, but effects on antibody-mediated effector activity are unclear. Herein, we show that increased amounts of immune complexes generated in mice persistently infected with lymphocytic choriomeningitis virus (LCMV) suppressed multiple Fcγ-receptor (FcγR) functions. The high amounts of immune complexes suppressed antibody-mediated cell depletion, therapeutic antibody-killing of LCMV infected cells and human CD20-expressing tumors, as well as reduced immune complex-mediated cross-presentation to T cells. Suppression of FcγR activity was not due to inhibitory FcγRs or high concentrations of free antibody, and proper FcγR functions were restored when persistently infected mice specifically lacked immune complexes. Thus, we identify a mechanism of immunosuppression during viral persistence with implications for understanding effective antibody activity aimed at pathogen control.
Asunto(s)
Anticuerpos Antivirales/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Evasión Inmune/inmunología , Coriomeningitis Linfocítica/inmunología , Receptores de IgG/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales de Origen Murino/farmacología , Antígenos CD20/biosíntesis , Antígenos CD20/inmunología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Tolerancia Inmunológica/inmunología , Factores Inmunológicos/farmacología , Activación de Linfocitos/inmunología , Depleción Linfocítica , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis/inmunología , Receptores de IgG/inmunología , RituximabRESUMEN
A critical step in the induction of adaptive mucosal immunity is antigen transcytosis, in which luminal antigens are transported to organized lymphoid tissues across the follicle-associated epithelium (FAE) of Peyer's patches. However, virtually nothing is known about intracellular signaling proteins and transcription factors that regulate apical-to-basolateral transcytosis. The FAE can transcytose a variety of luminal contents, including inert particles, in the absence of specific opsonins. Furthermore, it expresses receptors for secretory immunoglobulin A (SIgA), the main antibody in mucosal secretions, and uses them to efficiently transcytose SIgA-opsonized particles present in the lumen. Using a human FAE model, we show that the tyrosine kinase HCK regulates apical-to-basolateral transcytosis of non-opsonized and SIgA-opsonized particles. We also show that, in cultured intestinal epithelial cells, ectopic expression of the transcription factor SPIB or EHF is sufficient to activate HCK-dependent apical-to-basolateral transcytosis of these particles. Our results provide the first molecular insights into the intracellular regulation of antigen sampling at mucosal surfaces.
Asunto(s)
Proteínas de Unión al ADN/inmunología , Mucosa Intestinal/inmunología , Modelos Inmunológicos , Ganglios Linfáticos Agregados/inmunología , Proteínas Proto-Oncogénicas c-hck/inmunología , Factores de Transcripción/inmunología , Transcitosis/inmunología , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina A/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Ratones , Ganglios Linfáticos Agregados/citología , Ganglios Linfáticos Agregados/metabolismo , Proteínas Proto-Oncogénicas c-hck/genética , Proteínas Proto-Oncogénicas c-hck/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Chimeric Antigen Receptors (CARs) consist of the antigen-recognition portion of a monoclonal antibody fused to an intracellular signaling domain capable of activating T-cells. CARs displayed on the surface of transduced cells perform non-MHC-restricted antigen recognition and activating intracellular signaling pathways for induction of target cytolysis, cytokine secretion and proliferation. Clinical trials are in progress assessing the use of mature T-lymphocytes transduced with CARs targeting CD19 antigen to treat B-lineage malignancies. CD19 is an attractive target for immunotherapy because of its consistent and specific expression in most of the stages of maturation and malignancies of B-lymphocyte origin, but not on hematopoietic stem cells. Antibodies against the extracellular domain of the CAR molecule (anti-Fab, Fc or idiotype) have been used for detection of CAR expression in research and clinical samples by flow cytometry, but may need development for each construct and present significant background in samples from xenograft models. METHODS: A specific reagent for the detection of anti-CD19 CAR expression was developed, a fusion protein consisting of human CD19 extracellular domains and the Fc region of human IgG1 (CD19sIg). Genes encoding CD19sIg fusion proteins were constructed by fusing either exons 1 to 3 (CD19sIg1-3) or exons 1 to 4 (CD19sIg1-4) of the human CD19 cDNA to a human IgG1Fc fragment. These fusion proteins are intended to work in similar fashion as the MHC Tetramers used for identification of antigen-specific T-cells, and may also have other applications in studies of activation of anti-CD19 CAR bearing cells. The CD19sIg proteins were produced from 293 T cells by stable lentiviral vector transduction and purification from culture medium. RESULTS: ELISA assays using several different monoclonal antibodies to CD19 demonstrated dose-related specific binding by the fusion molecule CD19sIg1-4, but no binding by CD19sIg1-3. Conjugation of the CD19sIg1-4 fusion protein to Alexa Fluor 488 allowed specific and sensitive staining of anti-CD19 CAR-bearing cells for flow cytometry assays, detecting as low as 0.5% of CAR-modified primary cells with minimal background staining. CONCLUSIONS: This fusion molecule is a sensitive reagent for detection of anti-CD19 CAR derived from any monoclonal antibody present in CAR-modified T-cells.
Asunto(s)
Antígenos CD19/química , Fragmentos de Inmunoglobulinas/química , Receptores de Antígenos/química , Proteínas Recombinantes de Fusión/metabolismo , Animales , Anticuerpos Monoclonales/química , Proliferación Celular , Trasplante de Células , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Células HEK293 , Humanos , Inmunoglobulina G/química , Leucocitos Mononucleares/citología , Activación de Linfocitos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Linfocitos T/citologíaRESUMEN
The anti-CD20 antibody rituximab has substantially improved outcomes in patients with B-cell non-Hodgkin lymphomas. However, many patients are not cured by rituximab-based therapies, and overcoming de novo or acquired rituximab resistance remains an important challenge to successful treatment of B-cell malignancies. Interferon-alpha (IFNalpha) has potent immunostimulatory properties and antiproliferative effects against some B-cell cancers, but its clinical utility is limited by systemic toxicity. To improve the efficacy of CD20-targeted therapy, we constructed fusion proteins consisting of anti-CD20 and murine or human IFNalpha. Fusion proteins had reduced IFNalpha activity in vitro compared with native IFNalpha, but CD20 targeting permitted efficient antiproliferative and proapoptotic effects against an aggressive rituximab-insensitive human CD20(+) murine lymphoma (38C13-huCD20) and a human B-cell lymphoma (Daudi). In vivo efficacy was demonstrated against established 38C13-huCD20 grown in syngeneic immunocompetent mice and large, established Daudi xenografts grown in nude mice. Optimal tumor eradication required CD20 targeting, with 87% of mice cured of rituximab-insensitive tumors. Gene knockdown studies revealed that tumor eradication required expression of type I IFN receptors on the tumor cell surface. Targeting type I IFNs to sites of B-cell lymphoma by fusion to anti-CD20 antibodies represents a potentially useful strategy for treatment of B-cell malignancies.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Interferón-alfa/farmacología , Linfoma de Células B/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales de Origen Murino , Antivirales/farmacología , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Técnicas de Silenciamiento del Gen , Humanos , Interferón-alfa/genética , Ratones , Ratones Desnudos , Receptores de Interferón , Proteínas Recombinantes de Fusión/genética , Rituximab , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antibody-mediated tumor delivery of cytokines can overcome limitations of systemic administration (toxicity, short half-lives). Previous work showed improved antitumor potency of anti-CD20-IFNα fusion proteins in preclinical mouse models of B-cell lymphoma. Although tumor targeting is mediated by the antibody part of the fusion protein, the cytokine component might strongly influence biodistribution and pharmacokinetics, as a result of its affinity, size, valency, and receptor distribution. Here, we used immunoPET to study the in vivo biodistribution and tumor targeting of the anti-CD20 rituximab-murine IFNα1 fusion protein (Rit-mIFNα) and compared it with the parental mAb (rituximab, Rit). Rit-mIFNα and Rit were radiolabeled with zirconium-89 (89Zr, t1/2 78.4 hours) and injected into C3H mice bearing syngeneic B-cell lymphomas (38C13-hCD20). Dynamic [(2 hours post injection (p.i.)] and static (4, 24, and 72 hours) PET scans were acquired. Ex vivo biodistribution was performed after the final scan. Both 89Zr-Rit-mIFNα and 89Zr-Rit specifically target hCD20-expressing B-cell lymphoma in vivo. 89Zr-Rit-mIFNα showed specific uptake in tumors (7.6 ± 1.0 %ID/g at 75 hours p.i.), which was significantly lower than 89Zr-Rit (38.4 ± 9.9 %ID/g, P < 0.0001). ImmunoPET studies also revealed differences in the biodistribution, 89Zr-Rit-mIFNα showed rapid blood clearance and high accumulation in the liver compared with 89Zr-Rit. Importantly, immunoPET clearly revealed a therapeutic effect of the single 89Zr-Rit-mIFNα dose, resulting in smaller tumors and fewer lymph node metastases compared with mice receiving 89Zr-Rit. Mice receiving 89Zr-Rit-mIFNα had enlarged spleens, suggesting that systemic immune activation contributes to therapeutic efficacy in addition to the direct antitumoral activity of IFNα. In conclusion, immunoPET allows the noninvasive tracking and quantification of the antibody-cytokine fusion protein and helps understand the in vivo behavior and therapeutic efficacy.
Asunto(s)
Linfoma de Células B , Radioisótopos , Animales , Línea Celular Tumoral , Humanos , Linfoma de Células B/tratamiento farmacológico , Ratones , Ratones Endogámicos C3H , Tomografía de Emisión de Positrones/métodos , Radioisótopos/uso terapéutico , Distribución Tisular , Circonio/uso terapéuticoRESUMEN
Emergence of drug-resistant mutant viruses during the course of antiretroviral therapy is a major hurdle that limits the success of chemotherapeutic treatment to suppress human immunodeficiency virus type 1 (HIV-1) replication and AIDS progression. Development of new drugs and careful patient management based on resistance genotyping data are important for enhancing therapeutic efficacy. However, identifying changes leading to drug resistance can take years of clinical studies, and conventional in vitro assays are limited in generating reliable drug resistance data. Here we present an efficient in vitro screening assay for selecting drug-resistant variants from a library of randomly mutated HIV-1 strains generated by transposon-directed base-exchange mutagenesis. As a test of principle, we screened a library of mutant HIV-1 strains containing random mutations in the protease gene by using a reporter T-cell line in the presence of the protease inhibitor (PI) nelfinavir (NFV). Analysis of replicating viruses from a single round of infection identified 50 amino acid substitutions at 35 HIV-1 protease residue positions. The selected mutant viruses showed specific resistance to NFV and included most of the known NFV resistance mutations. Therefore, the new assay is efficient for identifying changes leading to drug resistance. The data also provide insights into the molecular mechanisms underlying the development of drug resistance.
Asunto(s)
Inhibidores de la Proteasa del VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/genética , Línea Celular , Farmacorresistencia Viral/genética , Humanos , Mutación , Nelfinavir/farmacologíaRESUMEN
Immunogenicity caused by the use of nonhuman enzymes in antibody-directed enzyme prodrug therapy has limited its clinical application. To overcome this problem, we have developed a mutant human purine nucleoside phosphorylase, which, unlike the wild-type enzyme, accepts (deoxy)adenosine-based prodrugs as substrates. Among the different mutants of human purine nucleoside phosphorylase tested, a double mutant with amino acid substitutions E201Q:N243D (hDM) is the most efficient in cleaving (deoxy)adenosine-based prodrugs. Although hDM is capable of using multiple prodrugs as substrates, it is most effective at cleaving 2-fluoro-2'-deoxyadenosine to a cytotoxic drug. To target hDM to the tumor site, the enzyme was fused to an anti-HER-2/neu peptide mimetic (AHNP). Treatment of HER-2/neu-expressing tumor cells with hDM-AHNP results in cellular localization of enzyme activity. As a consequence, harmless prodrug is converted to a cytotoxic drug in the vicinity of the tumor cells, resulting in tumor cell apoptosis. Unlike the nonhuman enzymes, the hDM should have minimal immunogenicity when used in antibody-directed enzyme prodrug therapy, thus providing a novel promising therapeutic agent for the treatment of tumors.
Asunto(s)
Anticuerpos/inmunología , Neoplasias/patología , Neoplasias/terapia , Péptidos/metabolismo , Profármacos/administración & dosificación , Profármacos/uso terapéutico , Purina-Nucleósido Fosforilasa/metabolismo , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/toxicidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Guanosina/genética , Humanos , Cinética , Imitación Molecular , Mutación/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Péptidos/genética , Profármacos/metabolismo , Ingeniería de Proteínas , Purina-Nucleósido Fosforilasa/genética , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Previous studies have shown that the alternative pathway of complement activation plays an important role in protection against infection with Cryptococcus neoformans. Cryptococcus gattii does not activate the alternative pathway as well as C. neoformans in vitro. The role of complement in C. gattii infection in vivo has not been reported. In this study, we used mice deficient in complement components to investigate the role of complement in protection against a C. gattii isolate from an ongoing outbreak in northwestern North America. While factor B-deficient mice showed an enhanced rate of death, complement component C3-deficient mice died even more rapidly, indicating that the alternative pathway was not the only complement pathway contributing to protection against disease. Both C3- and factor B-deficient mice had increased fungal burdens in comparison to wild-type mice. Histopathology revealed an overwhelming fungal burden in the lungs of these complement-deficient mice, which undoubtedly prevented efficient gas exchange, causing death. Following the fate of radiolabeled organisms showed that both factor B- and C3-deficient mice were less effective than wild-type mice in clearing organisms. However, opsonization of C. gattii with complement components was not sufficient to prolong life in mice deficient in complement. Killing of C. gattii by macrophages in vitro was decreased in the presence of serum from factor B- and C3-deficient versus wild-type mice. In conclusion, we have demonstrated that complement activation is crucial for survival in C. gattii infection. Additionally, we have shown that the alternative pathway of complement activation is not the only complement pathway contributing to protection.
Asunto(s)
Complemento C3/inmunología , Factor B del Complemento/inmunología , Criptococosis/inmunología , Animales , Activación de Complemento/inmunología , Complemento C3/deficiencia , Factor B del Complemento/deficiencia , Criptococosis/patología , Cryptococcus/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The systemic administration of an agonist antibody against glucocorticoid-induced tumor necrosis factor receptor related (GITR) protein has been shown to be effective in overcoming immune tolerance and promoting tumor rejection in a variety of murine tumor models. However, little is known regarding the functional consequence of ligation of GITR with its natural ligand (GITR-L) in the context of regulatory T cell (Treg) suppression in vivo. To determine the mechanism of GITR-L action in vivo, we generated a panel of tumor cell clones that express varying levels of GITR-L. The ectopic expression of GITR-L on the tumor cell surface was sufficient to enhance anti-tumor immunity and delay tumor growth in syngeneic BALB/c mice. Within the range examined, the extent of anti-tumor activity in vivo did not correlate with the level of GITR-L expression, as all clones tested exhibited a similar delay in tumor growth. The localized expression of GITR-L on tumor cells led to a significant increase in CD8+ T cell infiltration compared to the levels seen in control tumors. The increased proportion of CD8+ T cells was only observed locally at the tumor site and was not seen in the tumor draining lymph node. Depletion studies showed that CD8+ T cells, but not CD4+ T cells, were required for GITR-L mediated protection against tumor growth. These studies demonstrate that signaling between GITR-L and GITR in the tumor microenvironment promotes the infiltration of CD8+ T cells, which are essential for controlling tumor growth.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Neoplasias del Colon/inmunología , Receptores de Factor de Crecimiento Nervioso/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Linfocitos T Reguladores/inmunología , Factores de Necrosis Tumoral/metabolismo , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Femenino , Vectores Genéticos , Proteína Relacionada con TNFR Inducida por Glucocorticoide , Tolerancia Inmunológica , Ratones , Ratones Endogámicos BALB C , Receptores de Factor de Crecimiento Nervioso/inmunología , Receptores del Factor de Necrosis Tumoral/inmunología , Linfocitos T Reguladores/metabolismo , Transfección , Factores de Necrosis Tumoral/inmunologíaRESUMEN
Anti-Rh alloantibodies are used in research and clinic laboratories to define the Rh antigenic profile of human blood samples. IgM anti-Rh antibodies directly agglutinate Rh-positive RBCs. Anti-Rh antibodies of the IgG isotype bind to Rh antigens with a higher intrinsic affinity than IgM and sensitize RBCs, but do not induce direct hemagglutination. The aim of this work was to produce IgG anti-Rh possessing direct hemagglutinating properties of IgM. To achieve this goal, recombinant antibody technology was used to construct genes encoding Ig light and heavy chains that will form polymers with anti-Rh specificity. Expression vectors and liposome-mediated DNA transfer were used to generate transfectomas secreting human recombinant IgG3 anti-Rh. ELISA, SDS-PAGE, and hemagglutination were used to identify and characterize the recombinant antibody produced. Thus, a recombinant polymeric IgM-like IgG3 anti-Rh antibody was produced that directly agglutinates RBCs with specificity identical to that of the parent non-agglutinating IgG. The results obtained suggest that the technology used here to generate polymeric IgM-like IgG3 anti-Rh antibodies can be applied to produce Rh blood typing reagents. This approach might also be used to develop reagents for which cell surface antigen binding and agglutination or aggregation is required.
Asunto(s)
Anticuerpos Monoclonales/química , Pruebas de Hemaglutinación/métodos , Inmunoglobulina G/química , Isoanticuerpos/química , Sistema del Grupo Sanguíneo Rh-Hr/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Células CHO , Cricetinae , Cricetulus , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Isoanticuerpos/biosíntesis , Isoanticuerpos/inmunología , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Parasitic infections, including schistosomiasis, are associated with high titres of specific and non-specific IgE antibody, and many reports show an in vitro role for IgE in parasite killing. Despite an active immune response, schistosomes survive for long periods in the human bloodstream, implying that the parasite is able to overcome or evade the IgE response mounted against it. One such mechanism is through cleavage of IgE into non-functional fragments by potent parasite derived enzymes. Using domain swap antibodies, recombinant Fcepsilon, and C-terminally tagged Cepsilon4 domains, we have narrowed down the principal cleavage sites to the Cepsilon2/Cepsilon3 and Cepsilon3/Cepsilon4 interdomain region of the IgE-Fc. Two serine proteases, one chymotrypsin-like and the second trypsin-like, have been proposed to be involved. Inhibition assays using selective inhibitors confirmed that both proteases contribute to Fc cleavage, although the chymotrypsin-like enzyme makes the greater contribution. Protein sequencing of IgE fragments cleaved by highly pure preparations of the chymotrypsin-like enzyme revealed that cleavage also occurred post Lys residues within kappa light chain dimers (LELK/GA). Related sequences are found in myosin, thrombospondin, collagen and actin-related proteins; macromolecules present in the skin and through which cercariae must penetrate to initiate an infection. Chemical knockout experiments using specific inhibitors and chromogenic substrates allowed us to show that the trypsin-like enzyme was responsible for light chain cleavage. The finding that pathogenic proteases can cleave the Fc of IgE may provide a useful biochemical tool for the further analysis of IgE structure. Indeed, the finding may raise new possibilities for treatment of IgE-mediated allergic reactions mediated through Fcepsilon-receptors.
Asunto(s)
Inmunoglobulina E/química , Inmunoglobulina E/metabolismo , Elastasa Pancreática/metabolismo , Schistosoma mansoni/enzimología , Solventes/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos , Proteína de Bence Jones/metabolismo , Biología Computacional , Dimerización , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Elastasa Pancreática/aislamiento & purificación , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Schistosoma mansoni/efectos de los fármacos , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Although the majority of adult tissues express only hexokinase 1 (HK1) for glycolysis, most cancers express hexokinase 2 (HK2) and many coexpress HK1 and HK2. In contrast to HK1+HK2+ cancers, HK1-HK2+ cancer subsets are sensitive to cytostasis induced by HK2shRNA knockdown and are also sensitive to synthetic lethality in response to the combination of HK2shRNA knockdown, an oxidative phosphorylation (OXPHOS) inhibitor diphenyleneiodonium (DPI), and a fatty acid oxidation (FAO) inhibitor perhexiline (PER). The majority of human multiple myeloma cell lines are HK1-HK2+. Here we describe an antisense oligonucleotide (ASO) directed against human HK2 (HK2-ASO1), which suppressed HK2 expression in human multiple myeloma cell cultures and human multiple myeloma mouse xenograft models. The HK2-ASO1/DPI/PER triple-combination achieved synthetic lethality in multiple myeloma cells in culture and prevented HK1-HK2+ multiple myeloma tumor xenograft progression. DPI was replaceable by the FDA-approved OXPHOS inhibitor metformin (MET), both for synthetic lethality in culture and for inhibition of tumor xenograft progression. In addition, we used an ASO targeting murine HK2 (mHK2-ASO1) to validate the safety of mHK2-ASO1/MET/PER combination therapy in mice bearing murine multiple myeloma tumors. HK2-ASO1 is the first agent that shows selective HK2 inhibition and therapeutic efficacy in cell culture and in animal models, supporting clinical development of this synthetically lethal combination as a therapy for HK1-HK2+ multiple myeloma. SIGNIFICANCE: A first-in-class HK2 antisense oligonucleotide suppresses HK2 expression in cell culture and in in vivo, presenting an effective, tolerated combination therapy for preventing progression of HK1-HK2+ multiple myeloma tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/10/2748/F1.large.jpg.
Asunto(s)
Hexoquinasa/genética , Mieloma Múltiple/patología , Oligonucleótidos Antisentido/farmacología , Mutaciones Letales Sintéticas , Animales , Línea Celular Tumoral , Proliferación Celular , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The tubular gland of the chicken oviduct is an attractive system for protein expression as large quantities of proteins are deposited in the egg, the production of eggs is easily scalable and good manufacturing practices for therapeutics from eggs have been established. Here we examined the ability of upstream and downstream DNA sequences of ovalbumin, a protein produced exclusively in very high quantities in chicken egg white, to drive tissue-specific expression of human mAb in chicken eggs. To accommodate these large regulatory regions, we established and transfected lines of chicken embryonic stem (cES) cells and formed chimeras that express mAb from cES cell-derived tubular gland cells. Eggs from high-grade chimeras contained up to 3 mg of mAb that possesses enhanced antibody-dependent cellular cytotoxicity (ADCC), nonantigenic glycosylation, acceptable half-life, excellent antigen recognition and good rates of internalization.
Asunto(s)
Anticuerpos Monoclonales/química , Animales , Southern Blotting , Western Blotting , Células CHO , Rastreo Diferencial de Calorimetría , Carbohidratos/química , Pollos , Cricetinae , ADN/metabolismo , Clara de Huevo , Embrión de Mamíferos/citología , Embrión no Mamífero , Ensayo de Inmunoadsorción Enzimática , Femenino , Vectores Genéticos , Genoma , Glicosilación , Humanos , Inmunoglobulina G , Inmunohistoquímica , Focalización Isoeléctrica , Ratones , Ratones Endogámicos BALB C , Modelos Genéticos , Monosacáridos/química , Oligosacáridos/química , Ovalbúmina/genética , Ovalbúmina/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Ionización de Electrospray , Células Madre/citologíaRESUMEN
Myeloma and Chinese hamster ovary (CHO) cells are frequently used for the production of recombinant antibodies. With increasing interest in producing recombinant IgA for protection against infectious agents, it is essential to characterize the IgA produced in these cells. Here we show that while myeloma cells secrete IgA2m(2) predominantly as H(2)L(2), CHO cells secrete H(2)L and H(2) in addition to fully assembled H(2)L(2). When the CHO cells also synthesize J chain and secretory component (SC), polymeric IgA and secretory IgA in which SC is disulfide bonded to the polymeric IgA are produced. Blocking cysteines on purified IgA2m(2) protein by alkylating with iodoacetamide stabilizes the disulfide bonds between the H and L chains suggesting that the disulfide bonds between H and L chains are unstable. Taken together our results suggest that the covalent assembly of IgA2m(2) is different in myeloma and CHO cells.
Asunto(s)
Inmunoglobulina A/clasificación , Inmunoglobulina A/metabolismo , Alotipos de Inmunoglobulinas/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Inmunoglobulina A/genética , Alotipos de Inmunoglobulinas/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/metabolismo , Mieloma Múltiple/genética , Mieloma Múltiple/inmunología , Mieloma Múltiple/metabolismo , Proteínas Recombinantes/clasificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
An important emerging form of immunotherapy targeting B cell malignancies is chimeric antigen receptor (CAR) T cell therapy. Despite encouraging response rates of anti-CD19 CAR T cell therapy in B cell lymphomas, limited durability of response necessitates further study to potentiate CAR T cell efficacy. Antibody-targeted interferon (IFN) therapy is a novel approach in immunotherapy. Given the ability of IFNs to promote T cell activation and survival, target cell recognition, and cytotoxicity, we asked whether antibody-targeted IFN could enhance the antitumor effects of anti-CD19 CAR T cells. We produced an anti-CD20-IFN fusion protein containing the potent type 1 IFN isoform alpha14 (α14), and demonstrated its ability to suppress proliferation and induce apoptosis of human B cell lymphomas. Indeed, with the combination of anti-CD20-hIFNα14 and CAR T cells, we found enhanced cell killing among B cell lymphoma lines. Importantly, for all cell lines pretreated with anti-CD20-hIFNα14, the subsequent cytokine production by CAR T cells was markedly increased regardless of the degree of cell killing. Thus, several activities of CD19 CAR T cells were enhanced in the presence of anti-CD20-hIFNα14. These data suggest that antibody-targeted IFN may be an important novel approach to improving the efficacy of CAR T cell therapy.
Asunto(s)
Antígenos CD19/inmunología , Inmunoterapia , Interferón-alfa/inmunología , Linfoma de Células B/inmunología , Linfoma de Células B/terapia , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Reacciones Antígeno-Anticuerpo , Línea Celular Tumoral , HumanosRESUMEN
In the version of this Article originally published, in Fig. 1a, all cells in the top schematic were missing, and in the bottom-left schematic showing multiple pattern shapes, two cells were missing in the bottom-right corner. This figure has now been updated in all versions of the Article.
RESUMEN
As cells with aberrant force-generating phenotypes can directly lead to disease, cellular force-generation mechanisms are high-value targets for new therapies. Here, we show that single-cell force sensors embedded in elastomers enable single-cell force measurements with ~100-fold improvement in throughput than was previously possible. The microtechnology is scalable and seamlessly integrates with the multi-well plate format, enabling highly parallelized time-course studies. In this regard, we show that airway smooth muscle cells isolated from fatally asthmatic patients have innately greater and faster force-generation capacity in response to stimulation than healthy control cells. By simultaneously tracing agonist-induced calcium flux and contractility in the same cell, we show that the calcium level is ultimately a poor quantitative predictor of cellular force generation. Finally, by quantifying phagocytic forces in thousands of individual human macrophages, we show that force initiation is a digital response (rather than a proportional one) to the proper immunogen. By combining mechanobiology at the single-cell level with high-throughput capabilities, this microtechnology can support drug-discovery efforts for clinical conditions associated with aberrant cellular force generation.
Asunto(s)
Elastómeros/química , Análisis de la Célula Individual/métodos , Asma/patología , Diferenciación Celular , Células Cultivadas , Colorantes Fluorescentes/química , Fumarato de Formoterol/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Microscopía Fluorescente , Contracción Miocárdica/efectos de los fármacos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Fagocitosis/efectos de los fármacosRESUMEN
An important question remains as to which FcRn binding parameters, if any, correlate with the serum half-life of antibodies. In the present study, we used a BIACore surface plasmon resonance (SPR) device to study kinetic properties of antibody binding to FcRn at different pHs and under different binding reaction conditions. The ability of many different models to fit the data was tested. The previous models could not adequately explain all of the data collected. We now present models that have intuitive appeal and fit a broader range of data than previous models. Specifically, the model assumes that there are two forms of FcRn on the BIAcore chip and that, in addition to monomeric IgG, there is some aggregated IgG that can function as ligand. Although this model represents an improvement over previous models, it is still not globally valid for the entire range of data that was collected. Even with these limitations, the model provides a powerful new tool to analyze not only FcRn-IgG interactions but also other complex protein-protein interactions.