RESUMEN
Bacterial capsular polysaccharides are important vaccine immunogens. However, the study of polysaccharide-specific immune responses has been hindered by technical restrictions. Here, we developed and validated a high-throughput method to analyse antigen-specific B cells using combinatorial staining with fluorescently-labelled capsular polysaccharide multimers. Concurrent staining of 25 cellular markers further enables the in-depth characterization of polysaccharide-specific cells. We used this assay to simultaneously analyse 14 Streptococcus pneumoniae or 5 Streptococcus agalactiae serotype-specific B cell populations. The phenotype of polysaccharide-specific B cells was associated with serotype specificity, vaccination history and donor population. For example, we observed a link between non-class switched (IgM+) memory B cells and vaccine-inefficient S. pneumoniae serotypes 1 and 3. Moreover, B cells had increased activation in donors from South Africa, which has high-incidence of S. agalactiae invasive disease, compared to Dutch donors. This assay allows for the characterization of heterogeneity in B cell immunity that may underlie immunization efficacy.
Asunto(s)
Inmunización , Vacunas , Citometría de Flujo , Polisacáridos Bacterianos , InmunidadRESUMEN
Acute myeloid leukemia (AML) is a high-risk disease with a poor prognosis, particularly in elderly patients. Because current AML treatment relies primarily on untargeted therapies with severe side effects that limit patient eligibility, identification of novel therapeutic AML targets is highly desired. We recently described AT1413, an antibody produced by donor B cells of a patient with AML cured after allogeneic hematopoietic stem cell transplantation. AT1413 binds CD43s, a unique sialylated epitope on CD43, which is weakly expressed on normal myeloid cells and overexpressed on AML cells. Because of its selectivity for AML cells, we considered CD43s as a target for a bispecific T-cell-engaging antibody (bTCE) and generated a bTCE by coupling AT1413 to two T-cell-targeting fragments using chemo-enzymatic linkage. In vitro, AT1413 bTCE efficiently induced T-cell-mediated cytotoxicity toward different AML cell lines and patient-derived AML blasts, whereas endothelial cells with low binding capacity for AT1413 remained unaffected. In the presence of AML cells, AT1413 bTCE induced upregulation of T-cell activation markers, cytokine release, and T-cell proliferation. AT1413 bTCE was also effective in vivo. Mice either coinjected with human peripheral blood mononuclear cells or engrafted with human hematopoietic stem cells [human immune system (HIS) mice] were inoculated with an AML cell line or patient-derived primary AML blasts. AT1413 bTCE treatment strongly inhibited tumor growth and, in HIS mice, had minimal effects on normal human hematopoietic cells. Taken together, our results indicate that CD43s is a promising target for T-cell-engaging antibodies and that AT1413 holds therapeutic potential in a bTCE-format. SIGNIFICANCE: These findings offer preclinical evidence for the therapeutic potential of a bTCE antibody that targets a sialylated epitope on CD43 in AML.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Epítopos/inmunología , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucosialina/inmunología , Activación de Linfocitos/inmunología , Ácido N-Acetilneuramínico/metabolismo , Linfocitos T/inmunología , Animales , Apoptosis , Proliferación Celular , Citotoxicidad Inmunológica , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Epítopos/efectos de los fármacos , Epítopos/metabolismo , Femenino , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Pronóstico , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Most patients with acute myeloid leukemia (AML) can only be cured when allogeneic hematopoietic stem-cell transplantation induces a graft-versus-leukemia immune response (GVL). Although the role of T cells and natural killer cells in tumor immunology has been established, less is known about the contribution of B cells. From B cells of high-risk patients with AML with potent and lasting GVL responses, we isolated monoclonal antibodies directed against antigens expressed on the cell surface of AML cells but not on normal hematopoietic and nonhematopoietic cells. A number of these donor-derived antibodies recognized the U5 snRNP200 complex, a component of the spliceosome that in normal cells is found in the cell. In AML however, the U5 snRNP200 complex is exposed on the cell membrane of leukemic blasts. U5 snRNP200 complex-specific antibodies induced death of AML cells in an Fc receptor-dependent way in the absence of cytotoxic leukocytes or complement. In an AML mouse model, treatment with U5 snRNP200 complex-specific antibodies led to significant tumor growth inhibition. Thus, donor-derived U5 snRNP200 complex-recognizing AML-specific antibodies may contribute to antitumor responses.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Apoptosis/inmunología , Efecto Injerto vs Leucemia/inmunología , Células Asesinas Naturales/inmunología , Leucemia Mieloide Aguda/inmunología , Ribonucleoproteína Nuclear Pequeña U5/inmunología , Linfocitos T Citotóxicos/inmunología , Adulto , Animales , Terapia Combinada , Femenino , Trasplante de Células Madre Hematopoyéticas , Humanos , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/terapia , Masculino , Ratones SCID , Persona de Mediana Edad , PronósticoRESUMEN
Immunotherapy has proven beneficial in many hematologic and nonhematologic malignancies, but immunotherapy for acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) is hampered by the lack of tumor-specific targets. We took advantage of the tumor-immunotherapeutic effect of allogeneic hematopoietic stem cell transplantation and searched the B-cell repertoire of a patient with a lasting and potent graft-versus-AML response for the presence of AML-specific antibodies. We identified an antibody, AT1413, that was of donor origin and that specifically recognizes a novel sialylated epitope on CD43 (CD43s). Strikingly, CD43s is expressed on all World Health Organization 2008 types of AML and MDS. AT1413 induced antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity of AML cells in vitro. Of note, AT1413 was highly efficacious against AML cells in a humanized mouse model without affecting nonmalignant human myeloid cells, suggesting AT1413 has potential as a therapeutic antibody.
RESUMEN
Monoclonal antibodies are essential therapeutics and diagnostics in a large number of diseases. Moreover, they are essential tools in all sectors of life sciences. Although the great majority of monoclonal antibodies currently in use are of mouse origin, the use of human B cells to generate monoclonal antibodies is increasing as new techniques to tap the human B cell repertoire are rapidly emerging. Cloned lines of immortalized human B cells are ideal sources of monoclonal antibodies. In this review, we summarize our studies to the regulation of the replicative life span, differentiation, and maturation of B cells that led to the development of a platform that uses immortalization of human B cells by in vitro genetic modification for antibody development. We describe a number of human antibodies that were isolated using this platform and the application of the technique in other species. We also discuss the use of immortalized B cells as antigen-presenting cells for the discovery of tumor neoantigens.
Asunto(s)
Células Productoras de Anticuerpos/inmunología , Células Productoras de Anticuerpos/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Autorrenovación de las Células , Animales , Anticuerpos Monoclonales/biosíntesis , Formación de Anticuerpos/inmunología , Células Productoras de Anticuerpos/citología , Presentación de Antígeno , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Linfocitos B/citología , Técnicas de Cultivo de Célula , Diferenciación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Descubrimiento de Drogas , Regulación de la Expresión Génica , Centro Germinal/citología , Centro Germinal/fisiología , Humanos , Memoria Inmunológica , Interleucinas/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Vacunas/inmunología , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Antibody based therapies are increasingly applied to prevent and treat human disease. While the majority of antibodies currently on the market are chimeric or humanized antibodies from rodents, the focus has now shifted to the isolation and development of fully human antibodies. By retroviral transduction of B cell lymphoma-6 (BCL-6), which prevents terminal differentiation of B cells and, the anti-apoptotic gene B-cell lymphoma-extra large (Bcl-xL) into primary human B cells we efficiently immortalize antibody-producing B cells allowing the isolation of therapeutic antibodies. Selection of antigen-specific B cell clones was greatly facilitated because the transduced B cells retain surface immunoglobulin expression and secrete immunoglobulin into the culture supernatant. Surface immunoglobulin expression can be utilized to stain and isolate antigen specific B cell clones with labeled antigen. Immunoglobulins secreted in culture supernatant can directly be tested in functional assays to identify unique B cell clones. Here we describe the key features of our Bcl-6/Bcl-xL culture platform (AIMSelect).
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Linfocitos B/fisiología , Animales , Anticuerpos Monoclonales/biosíntesis , Técnicas de Cultivo de Célula , Separación Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Descubrimiento de Drogas , Ingeniería Genética , Humanos , Proteínas Proto-Oncogénicas c-bcl-6 , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Proteína bcl-X/genéticaRESUMEN
Today it is generally accepted that B cells require cognate interactions with CD4(+) T cells to develop high-affinity antibodies against proteins. CD4(+) T cells recognize peptides (epitopes) presented by MHC class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4(+) T cells that can bind to the MHC class II-peptide complex. We used a new humanized hemophilic mouse model to identify FVIII peptides presented by HLA-DRB1*1501. This model carries a knockout of all murine MHC class II molecules and expresses a chimeric murine-human MHC class II complex that contains the peptide-binding sites of the human HLA-DRB1*1501. When mice were treated with human FVIII, the proportion of mice that developed antibodies depended on the application route of FVIII and the activation state of the innate immune system. We identified 8 FVIII peptide regions that contained CD4(+) T-cell epitopes presented by HLA-DRB1*1501 to CD4(+) T cells during immune responses against FVIII. CD4(+) T-cell responses after intravenous and subcutaneous application of FVIII involved the same immunodominant FVIII epitopes. Interestingly, most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays.
Asunto(s)
Formación de Anticuerpos/inmunología , Linfocitos T CD4-Positivos/inmunología , Modelos Animales de Enfermedad , Epítopos de Linfocito T/inmunología , Factor VIII/administración & dosificación , Factor VIII/inmunología , Cadenas HLA-DRB1/inmunología , Hemofilia A/inmunología , Animales , Presentación de Antígeno , Linfocitos T CD4-Positivos/metabolismo , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Cadenas HLA-DRB1/metabolismo , Haplotipos/genética , Hemofilia A/metabolismo , Hemofilia A/patología , Humanos , Inyecciones Intravenosas , Inyecciones Subcutáneas , Masculino , Ratones , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Replacement of the missing factor VIII (FVIII) is the current standard of care for patients with hemophilia A. However, the short half-life of FVIII makes frequent treatment necessary. Current efforts focus on the development of longer-acting FVIII concentrates by introducing chemical and genetic modifications to the protein. Any modification of the FVIII protein, however, risks increasing its immunogenic potential to induce neutralizing antibodies (FVIII inhibitors), and this is one of the major complications in current therapy. It would be highly desirable to identify candidates with a high risk for increased immunogenicity before entering clinical development to minimize the risk of exposing patients to such altered FVIII proteins. In the present study, we describe a transgenic mouse line that expresses a human F8 cDNA. This mouse is immunologically tolerant to therapeutic doses of native human FVIII but is able to mount an antibody response when challenged with a modified FVIII protein that possesses altered immunogenic properties. In this situation, immunologic tolerance breaks down and antibodies develop that recognize both the modified and the native human FVIII. The applicability of this new model for preclinical immunogenicity assessment of new FVIII molecules and its potential use for basic research are discussed.
Asunto(s)
Factor VIII/genética , Factor VIII/inmunología , Hemofilia A/genética , Tolerancia Inmunológica/genética , Memoria Inmunológica/genética , Animales , Formación de Anticuerpos/genética , Formación de Anticuerpos/fisiología , Modelos Animales de Enfermedad , Factor VIII/antagonistas & inhibidores , Femenino , Hemofilia A/inmunología , Hemofilia A/patología , Humanos , Tolerancia Inmunológica/fisiología , Memoria Inmunológica/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Especificidad de la EspecieRESUMEN
The eradication of inhibitory antibodies in patients with haemophilia A can be accomplished by frequent administration of high or intermediate doses of factor VIII (FVIII), so-called immune tolerance induction (ITI). This study monitored the distribution of IgG subclasses of anti-FVIII antibodies during ITI. FVIII-specific antibodies of subclass IgG1 were detected in all inhibitor patients tested, anti-FVIII IgG4 in 16, IgG2 in 10 and IgG3 in one of 20 patients analysed. Levels of anti-FVIII IgG1 and IgG4 correlated well with inhibitor titres as measured by Bethesda assay. In low-titre inhibitor patients, anti-FVIII antibodies consisted primarily of subclass IgG1 whereas, anti-FVIII antibodies of subclass IgG4 were more prominent in patients with high titre inhibitors who needed prolonged treatment or who failed ITI. Longitudinal analysis of 14 patients undergoing ITI revealed that the relative contribution of IgG subclasses was constant for most of the patients analysed. In two patients, the relative contribution of IgG4 increased during ITI. Overall, our findings document the distribution and dynamics of anti-FVIII IgG subclasses during ITI. Future studies will need to address whether monitoring the relative contribution of anti-FVIII subclasses IgG1 and IgG4 may be useful for the identification of patients who are at risk of failing ITI.
Asunto(s)
Factor VIII/inmunología , Hemofilia A/inmunología , Tolerancia Inmunológica , Inmunoglobulina G/sangre , Adolescente , Adulto , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Factor VIII/administración & dosificación , Hemofilia A/complicaciones , Hemofilia A/tratamiento farmacológico , Humanos , LactanteRESUMEN
In its most severe form, haemophilia A is a life-threatening haemorrhagic bleeding disorder that is caused by mutations in the factor VIII (FVIII) gene. About 25% of patients who receive replacement therapy with intravenous FVIII products develop neutralising antibodies (FVIII inhibitors) that inhibit the function of substituted FVIII. Long-term application of high or low doses of FVIII has evolved as an effective strategy for eradicating antibodies and inducing long-lasting immune tolerance. Despite clinical experience with the therapy, little is known about the immunological mechanisms that cause the down modulation of FVIII-specific immune responses or the induction of long-lasting immune tolerance against FVIII. This review summarises current knowledge of the immunological mechanisms that might be involved in the induction of immune tolerance against FVIII in patients with haemophilia A who have FVIII inhibitors. In addition to data from patients with haemophilia A, data from patients who have had organ transplants or have immune-related disorders, such as autoimmune diseases, are considered as well as data from animal models.
Asunto(s)
Inhibidores de Factor de Coagulación Sanguínea/inmunología , Factor VIII/inmunología , Hemofilia A/inmunología , Animales , Formación de Anticuerpos , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Coagulantes/uso terapéutico , Factor VIII/uso terapéutico , Hemofilia A/terapia , Humanos , Tolerancia Inmunológica , Memoria Inmunológica , Modelos AnimalesRESUMEN
Inhibitory antibodies develop in approximately 25% of patients with severe hemophilia. A following treatment with factorVIII. In E-16KO or E-17KO mice, in which the factor VIII gene has been inactivated by insertion of a neo cassette, inhibitors develop following administration of factor VIII. Here, we describe the generation of transgenic mice expressing human factor VIII-R593C (huFVIII-R593C). Human factor VIII-R593C cDNA under control of a mouse albumin enhancer/promoter was injected into fertilized oocytes. Analysis of transgenic mice revealed that human factor VIII-R593C was expressed in the liver. Transgenic mice were crossed with factor VIII-deficient mice (E-16KO mice). In plasma of E-16KO mice antibodies were detected after five serial intravenous injections of factor VIII, while plasma of huFVIII-R593C/E-16KO mice did not contain detectable levels of antibodies. No antibody secreting cells were observed in either spleen or bone marrow of huFVIII-R593C/E-16KO mice. Also, factor VIII-specific memory B cells were not observed in the spleen of huFVIII-R593C/E-16KO mice. Analysis of T cell responses revealed that splenocytes derived of E-16KO mice secreted IL-10 and IFN-gamma following restimulation with factor VIII in vitro. In contrast, no factor VIII-specific T cell responses were observed in huFVIII-R593C/E-16KO mice. These results indicate that huFVIII-R593C/E-16KO mice are tolerant to intravenously administered factor VIII. It is anticipated that this model may prove useful for studying immune responses in the context of factor VIII gene therapy.