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1.
J Biol Chem ; 292(35): 14706-14717, 2017 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-28655766

RESUMEN

Bispecific antibodies combine two different antigen-binding sites in a single molecule, enabling more specific targeting, novel mechanisms of action, and higher clinical efficacies. Although they have the potential to outperform conventional monoclonal antibodies, many bispecific antibodies have issues regarding production, stability, and pharmacokinetic properties. Here, we describe a new approach for generating bispecific antibodies using a common light chain format and exploiting the stable architecture of human immunoglobulin G1 We used iterative experimental validation and computational modeling to identify multiple Fc variant pairs that drive efficient heterodimerization of the antibody heavy chains. Accelerated stability studies enabled selection of one Fc variant pair dubbed "DEKK" consisting of substitutions L351D and L368E in one heavy chain combined with L351K and T366K in the other. Solving the crystal structure of the DEKK Fc region at a resolution of 2.3 Å enabled detailed analysis of the interactions inducing CH3 interface heterodimerization. Local shifts in the IgG backbone accommodate the introduction of lysine side chains that form stabilizing salt-bridge interactions with substituted and native residues in the opposite chain. Overall, the CH3 domain adapted to these shifts at the interface, yielding a stable Fc conformation very similar to that in wild-type IgG. Using the DEKK format, we generated the bispecific antibody MCLA-128, targeting human EGF receptors 2 and 3. MCLA-128 could be readily produced and purified at industrial scale with a standard mammalian cell culture platform and a routine purification protocol. Long-term accelerated stability assays confirmed that MCLA-128 is highly stable and has excellent biophysical characteristics.


Asunto(s)
Anticuerpos Biespecíficos/metabolismo , Anticuerpos Monoclonales Humanizados/metabolismo , Antineoplásicos/metabolismo , Inmunoglobulina G/metabolismo , Modelos Moleculares , Ingeniería de Proteínas , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-3/antagonistas & inhibidores , Animales , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/farmacocinética , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/farmacocinética , Afinidad de Anticuerpos , Antineoplásicos/sangre , Antineoplásicos/química , Antineoplásicos/farmacocinética , Reactores Biológicos , Células CHO , Biología Computacional , Cricetulus , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Femenino , Semivida , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/genética , Ratones Endogámicos BALB C , Simulación del Acoplamiento Molecular , Conformación Proteica , Estabilidad Proteica , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Proteínas Recombinantes/sangre , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética
2.
Anal Chem ; 84(16): 7227-32, 2012 Aug 21.
Artículo en Inglés | MEDLINE | ID: mdl-22882109

RESUMEN

Native mass spectrometry was evaluated for the qualitative and semiquantitative analysis of composite mixtures of antibodies representing biopharmaceutical products coexpressed from single cells. We show that by using automated peak fitting of the ion signals in the native mass spectra, we can quantify the relative abundance of each of the antibodies present in mixtures, with an average accuracy of 3%, comparable to a cation exchange chromatography based approach performed in parallel. Moreover, using native mass spectrometry we were able to identify, separate, and quantify 9 antibodies present in a complex mixture of 10 antibodies, whereas this complexity could not be unraveled by cation exchange chromatography. Native mass spectrometry presents a valuable alternative to existing analytical methods for qualitative and semiquantitative profiling of biopharmaceutical products. It provides both the identity of each species in a mixture by mass determination and the relative abundance through comparison of relative ion signal intensities. Native mass spectrometry is a particularly effective tool for characterization of heterogeneous biopharmaceutical products such as bispecific antibodies and antibody mixtures.


Asunto(s)
Anticuerpos/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Anticuerpos/aislamiento & purificación , Células CHO , Cromatografía por Intercambio Iónico , Cricetinae , Cricetulus , Inmunoglobulina G/análisis , Inmunoglobulina G/aislamiento & purificación
3.
Nat Cancer ; 3(4): 418-436, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35469014

RESUMEN

Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.


Asunto(s)
Anticuerpos Biespecíficos , Neoplasias Glandulares y Epiteliales , Anticuerpos Biespecíficos/farmacología , Receptores ErbB/metabolismo , Humanos , Imidazoles , Neoplasias Glandulares y Epiteliales/metabolismo , Células Madre Neoplásicas/metabolismo , Organoides , Pirazinas , Receptores Acoplados a Proteínas G/metabolismo
4.
Nat Commun ; 12(1): 4445, 2021 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-34290245

RESUMEN

Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages.


Asunto(s)
Ligando 4-1BB/agonistas , Anticuerpos Biespecíficos/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Linfocitos T CD8-positivos/efectos de los fármacos , Inhibidores de Puntos de Control Inmunológico/farmacología , Ligando 4-1BB/inmunología , Animales , Anticuerpos Biespecíficos/inmunología , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/inmunología , Epítopos , Humanos , Inhibidores de Puntos de Control Inmunológico/inmunología , Tolerancia Inmunológica/efectos de los fármacos , Memoria Inmunológica/efectos de los fármacos , Inmunoterapia , Activación de Linfocitos/efectos de los fármacos
5.
Biotechnol Bioeng ; 106(5): 741-50, 2010 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-20564612

RESUMEN

Therapeutic monoclonal antibodies, a highly successful class of biological drugs, are conventionally manufactured in mammalian cell lines. A recent approach to increase the therapeutic effectiveness of monoclonal antibodies has been to combine two or more of them; however this increases the complexity of development and manufacture. To address this issue a method to efficiently express multiple monoclonal antibodies from a single cell has been developed and we describe here the generation of stable cell clones that express high levels of a human monoclonal antibody mixture. PER.C6 cells were transfected with a combination of plasmids containing genes encoding three different antibodies. Clones that express the three corresponding antibody specificities were identified, subcloned, and passaged in the absence of antibiotic selection pressure. At several time points, batch production runs were analyzed for stable growth and IgG production characteristics. The majority (11/12) of subclones analyzed expressed all three antibody specificities in constant ratios with total IgG productivity ranging between 15 and 20 pg/cell/day under suboptimal culture conditions after up to 67 population doublings. The growth and IgG production characteristics of the stable clones reported here resemble those of single monoclonal antibody cell lines from conventional clone generation programs. We conclude that the methodology described here is applicable to the generation of stable PER.C6(R) clones for industrial scale production of mixtures of antibodies.


Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Biotecnología/métodos , Expresión Génica , Anticuerpos Monoclonales/genética , Técnicas de Cultivo de Célula , Línea Celular , Vectores Genéticos , Humanos , Plásmidos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética
6.
Methods Mol Biol ; 562: 45-60, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19554286

RESUMEN

A method for the construction of West Nile virus immune donor antibody repertoires is described. B cells are harvested from a suitable donor and the antibody variable genes are amplified using polymerase chain reaction (PCR). The PCR fragments are cloned in a phage display vector to construct a repertoire that can be used in panning procedures to identify many unique monoclonal antibodies.


Asunto(s)
Anticuerpos Antivirales/inmunología , Bacteriófagos/inmunología , Donantes de Sangre , Biblioteca de Péptidos , Fiebre del Nilo Occidental/sangre , Virus del Nilo Occidental/inmunología , Antígenos Virales/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Ingeniería de Proteínas , ARN Viral/genética , ARN Viral/metabolismo
7.
Methods Mol Biol ; 528: 141-58, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19153691

RESUMEN

The discovery of novel target antigens for antibody-based immunotherapy is still a major challenge. Antibody phage display is one of the technologies that is widely applied for the identification of novel cell surface molecules on intact eukaryotic cells and many reports describe the isolation of phage-antibodies binding to restricted cell populations such as cells in a certain pathological condition. However, the transition from cell-specific phage antibodies to the identification of the target antigens is still a major hurdle. Herein a method is described for the identification of these cell surface molecules using two complementary technologies. A genomic approach based on expression cloning can be used when cDNA libraries and antigen-negative cells are available. Otherwise, a proteomic approach based on small scale immunoprecipitation followed by large scale purification and mass-spectrometry-based identification can be applied. Correct identification of the antigens is confirmed using technologies such as recombinant expression of the target antigen followed by immunoprecipitation or cDNA transfection and FACS analysis.


Asunto(s)
Anticuerpos Antivirales/inmunología , Genómica/métodos , Proteínas de la Membrana/análisis , Proteómica/métodos , Biotinilación , Línea Celular , Clonación Molecular , Citometría de Flujo , Biblioteca de Genes , Inmunoprecipitación , Espectrometría de Masas , Proteínas de la Membrana/inmunología , Biblioteca de Péptidos , Plásmidos , Transfección
8.
Expert Opin Biol Ther ; 19(7): 721-733, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31286786

RESUMEN

Objective: We report the characterization of MCLA-117, a novel T cell-redirecting antibody for acute myeloid leukaemia (AML) treatment targeting CD3 on T cells and CLEC12A on leukaemic cells. In AML, CLEC12A is expressed on blasts and leukaemic stem cells. Methods: The functional capacity of MCLA-117 to redirect resting T cells to eradicate CLEC12APOS tumor cells was studied using human samples, including primary AML samples. Results: Within the normal hematopoietic compartment, MCLA-117 binds to cells expressing CD3 and CLEC12A but not to early myeloid progenitors or hematopoietic stem cells. MCLA-117 induces T cell activation (EC50 = 44 ng/mL), T cell proliferation, mild pro-inflammatory cytokine release, and redirects T cells to lyse CLEC12APOS target cells (EC50 = 68 ng/mL). MCLA-117-induced targeting of normal CD34POS cells co-cultured with T cells spares erythrocyte and megakaryocyte differentiation as well as preserves mono-myelocytic lineage development. In primary AML patient samples with autologous T cells, MCLA-117 robustly induced AML blast killing (23-98%) at low effector-to-target ratios (1:3-1:97). Conclusion: These findings demonstrate that MCLA-117 efficiently redirects T cells to kill tumour cells while sparing the potential of the bone marrow to develop the full hematological compartment and support further clinical evaluation as a potentially potent treatment option for AML.


Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Linfocitos T/inmunología , Animales , Anticuerpos Biespecíficos/metabolismo , Anticuerpos Biespecíficos/farmacocinética , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular , Citocinas/análisis , Citocinas/metabolismo , Células HL-60 , Semivida , Humanos , Lectinas Tipo C/inmunología , Leucemia Mieloide Aguda/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Receptores Mitogénicos/inmunología , Linfocitos T/metabolismo
9.
Cancer Cell ; 33(5): 922-936.e10, 2018 05 14.
Artículo en Inglés | MEDLINE | ID: mdl-29763625

RESUMEN

HER2-driven cancers require phosphatidylinositide-3 kinase (PI3K)/Akt signaling through HER3 to promote tumor growth and survival. The therapeutic benefit of HER2-targeting agents, which depend on PI3K/Akt inhibition, can be overcome by hyperactivation of the heregulin (HRG)/HER3 pathway. Here we describe an unbiased phenotypic combinatorial screening approach to identify a bispecific immunoglobulin G1 (IgG1) antibody against HER2 and HER3. In tumor models resistant to HER2-targeting agents, the bispecific IgG1 potently inhibits the HRG/HER3 pathway and downstream PI3K/Akt signaling via a "dock & block" mechanism. This bispecific IgG1 is a potentially effective therapy for breast cancer and other tumors with hyperactivated HRG/HER3 signaling.


Asunto(s)
Anticuerpos Biespecíficos/administración & dosificación , Inmunoglobulina G/administración & dosificación , Neoplasias/tratamiento farmacológico , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-3/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Anticuerpos Biespecíficos/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Inmunoglobulina G/farmacología , Células MCF-7 , Ratones , Modelos Moleculares , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/química , Receptor ErbB-3/química , Ensayos Antitumor por Modelo de Xenoinjerto
10.
PLoS Med ; 3(7): e237, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16796401

RESUMEN

BACKGROUND: Experimental animal data show that protection against severe acute respiratory syndrome coronavirus (SARS-CoV) infection with human monoclonal antibodies (mAbs) is feasible. For an effective immune prophylaxis in humans, broad coverage of different strains of SARS-CoV and control of potential neutralization escape variants will be required. Combinations of virus-neutralizing, noncompeting mAbs may have these properties. METHODS AND FINDINGS: Human mAb CR3014 has been shown to completely prevent lung pathology and abolish pharyngeal shedding of SARS-CoV in infected ferrets. We generated in vitro SARS-CoV variants escaping neutralization by CR3014, which all had a single P462L mutation in the glycoprotein spike (S) of the escape virus. In vitro experiments confirmed that binding of CR3014 to a recombinant S fragment (amino acid residues 318-510) harboring this mutation was abolished. We therefore screened an antibody-phage library derived from blood of a convalescent SARS patient for antibodies complementary to CR3014. A novel mAb, CR3022, was identified that neutralized CR3014 escape viruses, did not compete with CR3014 for binding to recombinant S1 fragments, and bound to S1 fragments derived from the civet cat SARS-CoV-like strain SZ3. No escape variants could be generated with CR3022. The mixture of both mAbs showed neutralization of SARS-CoV in a synergistic fashion by recognizing different epitopes on the receptor-binding domain. Dose reduction indices of 4.5 and 20.5 were observed for CR3014 and CR3022, respectively, at 100% neutralization. Because enhancement of SARS-CoV infection by subneutralizing antibody concentrations is of concern, we show here that anti-SARS-CoV antibodies do not convert the abortive infection of primary human macrophages by SARS-CoV into a productive one. CONCLUSIONS: The combination of two noncompeting human mAbs CR3014 and CR3022 potentially controls immune escape and extends the breadth of protection. At the same time, synergy between CR3014 and CR3022 may allow for a lower total antibody dose to be administered for passive immune prophylaxis of SARS-CoV infection.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos Virales/inmunología , Inmunización Pasiva , Glicoproteínas de Membrana/inmunología , Síndrome Respiratorio Agudo Grave/prevención & control , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Proteínas del Envoltorio Viral/inmunología , Sustitución de Aminoácidos , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Variación Antigénica , Secuencia de Bases , Sitios de Unión , Células Cultivadas/virología , Chlorocebus aethiops , Brotes de Enfermedades , Relación Dosis-Respuesta Inmunológica , Sinergismo Farmacológico , Epítopos/inmunología , Humanos , Sueros Inmunes , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/inmunología , Macrófagos/virología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Datos de Secuencia Molecular , Mutación Missense , Nandiniidae/virología , Pruebas de Neutralización , Mutación Puntual , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Síndrome Respiratorio Agudo Grave/tratamiento farmacológico , Síndrome Respiratorio Agudo Grave/epidemiología , Síndrome Respiratorio Agudo Grave/terapia , Síndrome Respiratorio Agudo Grave/virología , Glicoproteína de la Espiga del Coronavirus , Resonancia por Plasmón de Superficie , Células Vero , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/fisiología , Replicación Viral
11.
Nucleic Acids Res ; 31(11): e59, 2003 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-12771223

RESUMEN

Phage display is a widely used technology for the isolation of peptides and proteins with specific binding properties from large libraries of these molecules. A drawback of the common phagemid/helper phage systems is the high infective background of phages that do not display the protein of interest, but are propagated due to non-specific binding to selection targets. This and the enhanced growth rates of bacteria harboring aberrant phagemids not expressing recombinant proteins leads to a serious decrease in selection efficiency. Here we describe a VCSM13-derived helper phage that circumvents this problem, because it lacks the genetic information for the infectivity domains of phage coat protein pIII. Rescue of a library with this novel CT helper phage yields phages that are only infectious when they contain a phagemid-encoded pIII-fusion protein, since phages without a displayed protein carry truncated pIII only and are lost upon re-infection. Importantly, the CT helper phage can be produced in quantities similar to the VCSM13 helper phage. The superiority of CT over VCSM13 during selection was demonstrated by a higher percentage of positive clones isolated from an antibody library after two selection rounds on a complex cellular target. We conclude that the CT helper phage considerably improves the efficiency of selections using phagemid-based protein libraries.


Asunto(s)
Bacteriófagos/genética , Biblioteca de Péptidos , Proteínas Recombinantes de Fusión/genética , Proteínas de la Cápside , Proteínas de Unión al ADN/genética , Humanos , Región Variable de Inmunoglobulina/genética , Mutación , Técnicas de Amplificación de Ácido Nucleico , Células U937 , Proteínas Virales de Fusión/genética
12.
Cancer Res ; 64(22): 8443-50, 2004 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-15548716

RESUMEN

Acute myeloid leukemia (AML) has a poor prognosis due to treatment-resistant relapses. A humanized anti-CD33 antibody (Mylotarg) showed a limited response rate in relapsed AML. To discover novel AML antibody targets, we selected a panel of single chain Fv fragments using phage display technology combined with flow cytometry on AML tumor samples. One selected single chain Fv fragment broadly reacted with AML samples and with myeloid cell lineages within peripheral blood. Expression cloning identified the antigen recognized as C-type lectin-like molecule-1 (CLL-1), a previously undescribed transmembrane glycoprotein. CLL-1 expression was analyzed with a human anti-CLL-1 antibody that was generated from the single chain Fv fragment. CLL-1 is restricted to the hematopoietic lineage, in particular to myeloid cells present in peripheral blood and bone marrow. CLL-1 is absent on uncommitted CD34(+)/CD38(-) or CD34(+)/CD33(-) stem cells and present on subsets of CD34(+)/CD38(+) or CD34(+)/CD33(+) progenitor cells. CLL-1 is not expressed in any other tissue. In contrast, analysis of primary AMLs demonstrated CLL-1 expression in 92% (68 of 74) of the samples. As an AML marker, CLL-1 was able to complement CD33, because 67% (8 of 12) of the CD33(-) AMLs expressed CLL-1. CLL-1 showed variable expression (10-60%) in CD34(+) cells in chronic myelogenous leukemia and myelodysplastic syndrome but was absent in 12 of 13 cases of acute lymphoblastic leukemia. The AML reactivity combined with the restricted expression on normal cells identifies CLL-1 as a novel potential target for AML treatment.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Lectinas Tipo C/metabolismo , Leucemia Mieloide/metabolismo , Enfermedad Aguda , Secuencia de Aminoácidos , Secuencia de Bases , Membrana Celular/metabolismo , Citometría de Flujo , Humanos , Lectinas Tipo C/química , Lectinas Tipo C/genética , Datos de Secuencia Molecular
14.
Lancet ; 363(9427): 2139-41, 2004 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-15220038

RESUMEN

SARS coronavirus continues to cause sporadic cases of severe acute respiratory syndrome (SARS) in China. No active or passive immunoprophylaxis for disease induced by SARS coronavirus is available. We investigated prophylaxis of SARS coronavirus infection with a neutralising human monoclonal antibody in ferrets, which can be readily infected with the virus. Prophylactic administration of the monoclonal antibody at 10 mg/kg reduced replication of SARS coronavirus in the lungs of infected ferrets by 3.3 logs (95% CI 2.6-4.0 logs; p<0.001), completely prevented the development of SARS coronavirus-induced macroscopic lung pathology (p=0.013), and abolished shedding of virus in pharyngeal secretions. The data generated in this animal model show that administration of a human monoclonal antibody might offer a feasible and effective prophylaxis for the control of human SARS coronavirus infection.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Síndrome Respiratorio Agudo Grave/prevención & control , Animales , Femenino , Hurones , Humanos , Inmunización Pasiva , Inmunoglobulina G/inmunología , Pulmón/patología , Pulmón/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/aislamiento & purificación , Síndrome Respiratorio Agudo Grave/patología , Síndrome Respiratorio Agudo Grave/virología , Replicación Viral
15.
Eur J Cancer ; 41(1): 178-87, 2005 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-15618003

RESUMEN

Tumour-associated cell surface markers are potential targets for antibody-based therapies. We have obtained a panel of myeloid cell binding single chain variable fragments (scFv) by applying phage display selection on myeloid cell lines followed by a selection round on freshly isolated acute myeloid leukaemia (AML) blasts using flow cytometry. To identify the target antigens, the scFv were recloned and expressed in an IgG(1) format and tested for their ability to immunoprecipitate cell surface proteins. The IgGs that reacted with distinct cell membrane extractable proteins were used in large-scale affinity purification of the target antigen followed by mass-spectrometry-based identification. Well-characterised cell surface antigens, such as leukocyte antigen-related receptor protein tyrosine phosphatase (LAR PTP) and activated leukocyte adhesion molecule (ALCAM) in addition to several unknown proteins, like ATAD3A, were identified. These experiments demonstrate that phage antibody selection in combination with affinity chromatography and mass spectrometry can be exploited successfully to identify novel antibody target molecules on malignant cells.


Asunto(s)
Antígenos de Neoplasias/análisis , Leucemia Mieloide/genética , Proteómica , Molécula de Adhesión Celular del Leucocito Activado , Enfermedad Aguda , Antígenos de Superficie/metabolismo , Bacteriófagos/metabolismo , Línea Celular Tumoral , Células Clonales , Citometría de Flujo/métodos , Humanos , Inmunoglobulina G/metabolismo , Leucocitos Mononucleares/metabolismo , Espectrometría de Masas , Células Mieloides/metabolismo , Transfección
16.
J Immunol Methods ; 302(1-2): 68-77, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15992810

RESUMEN

Application of antibody phage display to the identification of cell surface antigens with restricted expression patterns is often complicated by the inability to demonstrate specific binding to a certain cell type. The specificity of an antibody can only be properly assessed when the antibody is of sufficient high affinity to detect low-density antigens on cell surfaces. Therefore, a robust and simple assay for the prediction of relative antibody affinities was developed and compared to data obtained using surface plasmon resonance (SPR) technology. A panel of eight anti-CD46 antibody fragments with different affinities was selected from phage display libraries and reformatted into complete human IgG1 molecules. SPR was used to determine K(D) values for these antibodies. The association and dissociation of the antibodies for binding to CD46 expressed on cell surfaces were analysed using FACS-based assays. We show that ranking of the antibodies based on FACS data correlates well with ranking based on K(D) values as measured by SPR and can therefore be used to discriminate between high- and low-affinity antibodies. Finally, we show that a low-affinity antibody may only detect high expression levels of a surface marker while failing to detect lower expression levels of this molecule, which may lead to a false interpretation of antibody specificity.


Asunto(s)
Anticuerpos/metabolismo , Afinidad de Anticuerpos , Bacteriófagos/inmunología , Citometría de Flujo/métodos , Biblioteca de Péptidos , Animales , Antígenos CD/inmunología , Sitios de Unión de Anticuerpos , Humanos , Proteína Cofactora de Membrana , Glicoproteínas de Membrana/inmunología , Ratones , Resonancia por Plasmón de Superficie
17.
MAbs ; 6(1): 197-203, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24351421

RESUMEN

Composite antibody mixtures designed to combat diseases present a new, rapidly emerging technology in the field of biopharmaceuticals. The combination of multiple antibodies can lead to increased effector response and limit the effect of escape variants that can propagate the disease. However, parallel development of analytical technologies is required to provide fast, thorough, accurate, and robust characterization of these mixtures. Here, we evaluate the utility of native mass spectrometry on an Orbitrap platform with high mass resolving power to characterize composite mixtures of up to 15 separate antibodies. With this technique, unambiguous identification of each antibody in the mixtures was achieved. Mass measurements of the intact antibodies varied 7 ppm on average, allowing highly reproducible identification and quantitation of each compound in these complex mixtures. We show that with the high mass-resolving power and robustness of this technology, high-resolution native mass spectrometry can be used efficiently even for batch-to batch characterization.


Asunto(s)
Espectrometría de Masas/métodos , Anticuerpos de Cadena Única/química , Células HEK293 , Humanos
18.
J Mol Biol ; 387(3): 548-58, 2009 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-19361421

RESUMEN

To study the contribution of antibody light (L) chains to the diversity and binding properties of immune repertoires, a phage display repertoire was constructed from a single human antibody L chain and a large collection of antibody heavy (H) chains harvested from the blood of two human donors immunized with tetanus toxoid (TT) vaccine. After selection for binding to TT, 129 unique antibodies representing 53 variable immunoglobulin H chain (V(H)) gene rearrangements were isolated. This panel of anti-TT antibodies restricted to a single variable immunoglobulin L chain (V(L)) could be organized into 17 groups binding non-competing epitopes on the TT molecule. Comparison of the V(H) regions in this V(L)-restricted panel with a previously published repertoire of anti-TT V(H) regions with cognate V(H)-V(L) pairing showed a very similar distribution of V(H), D(H) and J(H) gene segment utilization and length of the complementarity-determining region 3 of the H chain. Surface plasmon resonance analysis of the single-V(L) anti-TT repertoire unveiled a range of affinities, with a median monovalent affinity of 2 nM. When the single-V(L) anti-TT V(H) repertoire was combined with a collection of naïve V(L) regions and again selected for binding to TT, many of the V(H) genes were recovered in combination with a diversity of V(L) regions. The affinities of a panel of antibodies consisting of a single promiscuous anti-TT V(H) combined with 15 diverse V(L) chains were determined and found to be identical to each other and to the original isolate restricted to a single-V(L) chain. Based on previous estimates of the clonal size of the human anti-TT repertoire, we conclude that up to 25% of human anti-TT-encoding V(H) regions from an immunized repertoire have promiscuous features. These V(H) regions readily combine with a single antibody L chain to result in a large panel of anti-TT antibodies that conserve the expected epitope diversity, V(H) region diversity and affinity of a natural repertoire.


Asunto(s)
Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/inmunología , Toxoide Tetánico/inmunología , Secuencia de Aminoácidos , Afinidad de Anticuerpos , Epítopos/química , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Datos de Secuencia Molecular , Biblioteca de Péptidos , Toxoide Tetánico/química
19.
PLoS One ; 3(12): e3942, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-19079604

RESUMEN

BACKGROUND: The hemagglutinin (HA) glycoprotein is the principal target of protective humoral immune responses to influenza virus infections but such antibody responses only provide efficient protection against a narrow spectrum of HA antigenic variants within a given virus subtype. Avian influenza viruses such as H5N1 are currently panzootic and pose a pandemic threat. These viruses are antigenically diverse and protective strategies need to cross protect against diverse viral clades. Furthermore, there are 16 different HA subtypes and no certainty the next pandemic will be caused by an H5 subtype, thus it is important to develop prophylactic and therapeutic interventions that provide heterosubtypic protection. METHODS AND FINDINGS: Here we describe a panel of 13 monoclonal antibodies (mAbs) recovered from combinatorial display libraries that were constructed from human IgM(+) memory B cells of recent (seasonal) influenza vaccinees. The mAbs have broad heterosubtypic neutralizing activity against antigenically diverse H1, H2, H5, H6, H8 and H9 influenza subtypes. Restriction to variable heavy chain gene IGHV1-69 in the high affinity mAb panel was associated with binding to a conserved hydrophobic pocket in the stem domain of HA. The most potent antibody (CR6261) was protective in mice when given before and after lethal H5N1 or H1N1 challenge. CONCLUSIONS: The human monoclonal CR6261 described in this study could be developed for use as a broad spectrum agent for prophylaxis or treatment of human or avian influenza infections without prior strain characterization. Moreover, the CR6261 epitope could be applied in targeted vaccine strategies or in the design of novel antivirals. Finally our approach of screening the IgM(+) memory repertoire could be applied to identify conserved and functionally relevant targets on other rapidly evolving pathogens.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Linfocitos B/virología , Inmunoglobulina M/inmunología , Memoria Inmunológica/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Gripe Humana/prevención & control , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Especificidad de Anticuerpos/inmunología , Linfocitos B/inmunología , Sitios de Unión de Anticuerpos , Reacciones Cruzadas , Perros , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Gripe Humana/inmunología , Gripe Humana/virología , Ratones , Datos de Secuencia Molecular , Pruebas de Neutralización , Biblioteca de Péptidos , Estructura Terciaria de Proteína , Donantes de Tejidos
20.
Expert Rev Vaccines ; 6(2): 183-91, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17408368

RESUMEN

Seasonal epidemics of West Nile virus (WNV) infection now occur throughout North America, causing clinical symptoms ranging from fever to encephalitis. There are no specific treatment options or licensed vaccines. Several classically developed vaccine candidates are being evaluated in clinical trials. However, questions of safety and/or immunogenicity may limit their usefulness. Mapping of human and murine antibody repertoires against the WNV envelope protein after WNV infection have revealed important insights into the protective immune response against the virus. This review will give an overview of vaccines under development and summarize current data on E-protein antigenicity that could aid in the design of next generation WNV vaccines.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Mapeo Epitopo , Proteínas del Envoltorio Viral/inmunología , Fiebre del Nilo Occidental/prevención & control , Vacunas contra el Virus del Nilo Occidental/uso terapéutico , Virus del Nilo Occidental/inmunología , Animales , Diseño de Fármacos , Humanos , Epítopos Inmunodominantes , Ratones , Vacunación/tendencias , Fiebre del Nilo Occidental/inmunología , Fiebre del Nilo Occidental/terapia , Vacunas contra el Virus del Nilo Occidental/inmunología
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