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/farmacocinéticaRESUMEN
Introduction MCLA-128 is a bispecific monoclonal antibody targeting the HER2 and HER3 receptors. Pharmacokinetics (PK) and pharmacodynamics (PD) of MCLA-128 have been evaluated in preclinical studies in cynomolgus monkeys and mice. The aim of this study was to characterize the PK and PD of MCLA-128 and to predict a safe starting dose and efficacious clinical dose for the First-In-Human study. Methods A PK-PD model was developed based on PK data from cynomolgus monkeys and tumor growth data from a mouse JIMT-1 xenograft model. Allometric scaling was used to scale PK parameters between species. Simulations were performed to predict the safe and efficacious clinical dose, based on AUCs, receptor occupancies and PK-PD model simulations. Results MCLA-128 PK in cynomolgus monkeys was described by a two-compartment model with parallel linear and nonlinear clearance. The xenograft tumor growth model consisted of a tumor compartment with a zero-order growth rate and a first-order dying rate, both affected by MCLA-128. Human doses of 10 to 480 mg q3wk were predicted to show a safety margin of >10-fold compared to the cynomolgus monkey AUC at the no-observed-adverse-effect-level (NOAEL). Doses of ≥360 mg resulted in predicted receptor occupancies above 99% (Cmax and Cave). These doses showed anti-tumor efficacy in the PK-PD model. Conclusions This analysis predicts that a flat dose of 10 to 480 mg q3wk is suitable as starting dose for a First-in-Human study with MCLA-128. Flat doses ≥360 mg q3wk are expected to be efficacious in human, based on receptor occupancies and PK-PD model simulations.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/farmacocinética , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/farmacocinética , Inmunoglobulina G/farmacología , Modelos Biológicos , Investigación Biomédica Traslacional , Animales , Área Bajo la Curva , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Simulación por Computador , Relación Dosis-Respuesta Inmunológica , Femenino , Humanos , Macaca fascicularis , Ratones , Ratones SCID , Resultado del Tratamiento , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Monoclonal antibodies are successful biologics in treating a variety of diseases, including the prevention or treatment of viral infections. CL184 is a 1:1 combination of two human monoclonal IgG1 antibodies (CR57 and CR4098) against rabies virus, produced in the PER.C6 human cell line. The two antibodies are developed as replacements of human rabies immune globulin (HRIG) and equine rabies immune globulin (ERIG) in postexposure prophylaxis (PEP). The rapid fluorescent focus inhibition test (RFFIT) is a cell-based virus neutralization assay which is usually performed to determine the biological potency of a vaccine and to measure the levels of protection against rabies in humans and animals. In order to confirm the suitability of this assay as a pharmacodynamic assay, we conducted a validation using both HRIG- and CL184-spiked serum samples and sera from vaccinated donors. The validation results met all analytical acceptance criteria and showed that HRIG and CL184 serum concentrations can be compared. Stability experiments showed that serum samples were stable in various suboptimal conditions but that rabies virus should be handled swiftly once thawed. We concluded that the assay is suitable for the measurement of polyclonal and monoclonal rabies neutralizing antibodies in clinical serum samples.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Pruebas de Neutralización/métodos , Virus de la Rabia/inmunología , Anticuerpos Monoclonales/inmunología , Línea Celular , Humanos , Inmunoglobulinas/inmunología , Vacunas Antirrábicas/inmunologíaRESUMEN
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.
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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 & purificaciónRESUMEN
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.
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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 fármacosRESUMEN
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.
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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/metabolismoRESUMEN
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 MolecularRESUMEN
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.
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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 ViralRESUMEN
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.
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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 , TransfecciónRESUMEN
The currently recommended treatment for individuals exposed to rabies virus is the combined administration of rabies vaccine and rabies immune globulin (RIG). This review sets out the criteria used to guide development of a cocktail of human monoclonal antibodies as a replacement for RIG. Using this process as a model, the general requirements for development of safe and efficacious monoclonal antibody alternatives to currently used polyclonal serum products are discussed.
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Anticuerpos Monoclonales/uso terapéutico , Factores Inmunológicos/uso terapéutico , Rabia/prevención & control , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulinas/uso terapéutico , Virus de la Rabia/inmunologíaRESUMEN
The World Health Organization estimates human mortality from endemic canine rabies to be 55,000 deaths/year. Limited supply hampers the accessibility of appropriate lifesaving treatment, particularly in areas where rabies is endemic. Anti-rabies antibodies are key to protection against lethal rabies. Currently, only human and equine polyclonal anti-rabies immune globulin (HRIG and ERIG) is available. Replacement of HRIG and ERIG with a safer and more widely available product is recommended. We have recently identified a combination of 2 human monoclonal antibodies (MAbs), CR57 and CR4098, that has high potential. We here describe a head-to-head comparison between an CR57/CR4098 MAb cocktail and HRIG. The MAb cocktail neutralized all viruses from a panel of 26 representative street rabies virus isolates. In combination with vaccine, the MAb cocktail protected Syrian hamsters against lethal rabies when administered 24 h after exposure, comparable with the results obtained with HRIG. Furthermore, the MAb cocktail did not interfere with rabies vaccine differently from HRIG. These results demonstrate that the human MAb cocktail of CR57 and CR4098 is a safe and efficacious alternative to RIG in rabies postexposure prophylaxis.
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Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Antivirales/administración & dosificación , Inmunoglobulinas/administración & dosificación , Virus de la Rabia/inmunología , Rabia/terapia , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Línea Celular , Cricetinae , Humanos , Inmunoglobulinas/efectos adversos , Inmunoterapia , Mesocricetus , Rabia/inmunología , Rabia/prevención & control , Vacunas Antirrábicas/administración & dosificación , Virus de la Rabia/genéticaRESUMEN
The need to replace rabies immune globulin (RIG) as an essential component of rabies postexposure prophylaxis is widely acknowledged. We set out to discover a unique combination of human monoclonal antibodies (MAbs) able to replace RIG. Stringent criteria concerning neutralizing potency, affinity, breadth of neutralization, and coverage of natural rabies virus (RV) isolates and in vitro escape mutants were set for each individual antibody, and the complementarities of the two MAbs were defined at the onset. First, we identified and characterized one human MAb (CR57) with high in vitro and in vivo neutralizing potency and a broad neutralization spectrum. The linear antibody binding site was mapped on the RV glycoprotein as antigenic site I by characterizing CR57 escape mutants. Secondly, we selected using phage display a complementing antibody (CR4098) that recognized a distinct, nonoverlapping epitope (antigenic site III), showed similar neutralizing potency and breadth as CR57, and neutralized CR57 escape mutants. Reciprocally, CR57 neutralized RV variants escaping CR4098. Analysis of glycoprotein sequences of natural RV isolates revealed that the majority of strains contain both intact epitopes, and the few remaining strains contain at least one of the two. In vitro exposure of RV to the combination of CR57 and CR4098 yielded no escape mutants. In conclusion, a novel combination of human MAbs was discovered suitable to replace RIG.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Virus de la Rabia/inmunología , Animales , Antígenos Virales/inmunología , Cricetinae , Genotipo , Glicoproteínas/inmunología , Inmunoglobulina G/inmunología , Mesocricetus , Ratones , Mutación , Pruebas de Neutralización , Rabia/prevención & control , Virus de la Rabia/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Antibody phage display technology was used to identify human monoclonal antibodies that neutralize rabies virus (RV). A phage repertoire was constructed using antibody genes harvested from the blood of vaccinated donors. Selections using this repertoire and three different antigen formats of the RV glycoprotein (gp) resulted in the identification of 147 unique antibody fragments specific for the RV gp. Analysis of the DNA sequences of these antibodies demonstrated a large variation in the heavy- and light-chain germ-line gene usage, suggesting that a broad antibody repertoire was selected. The single-chain variable fragment (scFv) antibodies were tested in vitro for RV neutralization, resulting in 39 specificities that neutralize the virus. Of the scFv clones, 21 were converted into full-length human IgG(1) format. Analysis of viral escape variants and binding competition experiments indicated that the majority of the neutralizing antibodies are directed against antigenic site III of the RV gp. The obtained specificities expand the set of human anti-RV antibodies eligible for inclusion in an antibody cocktail aimed for use in rabies post-exposure prophylaxis.
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Anticuerpos Antivirales/análisis , Antígenos Virales/inmunología , Glicoproteínas/inmunología , Biblioteca de Péptidos , Virus de la Rabia/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/metabolismo , Humanos , Fragmentos de Inmunoglobulinas/análisis , Fragmentos de Inmunoglobulinas/biosíntesis , Inmunoglobulina G/análisis , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/metabolismo , Región Variable de Inmunoglobulina/análisis , Región Variable de Inmunoglobulina/biosíntesis , Datos de Secuencia Molecular , Mapeo PeptídicoRESUMEN
Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.
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Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Glicoproteínas de Membrana/inmunología , Proteínas de la Nucleocápside/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Antivirales/química , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Sitios de Unión , Chlorocebus aethiops , Proteínas de la Nucleocápside de Coronavirus , Mapeo Epitopo , Humanos , Datos de Secuencia Molecular , Pruebas de Neutralización , Glicoproteína de la Espiga del Coronavirus , Células VeroRESUMEN
Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We produced two previously described potent rabies virus-neutralizing human MAbs, CR57 and CRJB, in human PER.C6 cells. The two MAbs competed for binding to rabies virus glycoprotein. Using CR57 and a set of 15-mer overlapping peptides covering the glycoprotein ectodomain, a neutralization domain was identified between amino acids (aa) 218 and 240. The minimal binding region was identified as KLCGVL (aa 226 to 231), with key residues K-CGV- identified by alanine replacement scanning. The critical binding region of this novel nonconformational rabies virus epitope is highly conserved within rabies viruses of genotype 1. Subsequently, we generated six rabies virus variants escaping neutralization by CR57 and six variants escaping CRJB. The CR57 escape mutants were only partially covered by CRJB, and all CRJB-resistant variants completely escaped neutralization by CR57. Without exception, the CR57-resistant variants showed a mutation at key residues within the defined minimal binding region, while the CRJB escape viruses showed a single mutation distant from the CR57 epitope (N182D) combined with mutations in the CR57 epitope. The competition between CR57 and CRJB, the in vitro escape profile, and the apparent overlap between the recognized epitopes argues against including both CR57 and CRJB in a MAb cocktail aimed at replacing classical immunoglobulin preparations.