RESUMEN
Targeting bacterial virulence factors directly provides a new paradigm for the intervention and treatment of bacterial diseases. Pseudomonas aeruginosa produces a myriad of virulence factors to cause fatal diseases in humans. In this study, human single-chain antibodies (HuscFvs) that bound to P. aeruginosa exotoxin A (ETA) were generated by phage display technology using recombinant ETA, ETA-subdomains and the synthetic peptide of the ETA-catalytic site as baits for selecting ETA-bound-phages from the human-scFv phage display library. ETA-bound HuscFvs derived from three phage-transfected E. coli clones neutralized the ETA-induced mammalian cell apoptosis. Computerized simulation demonstrated that these HuscFvs used several residues in their complementarity-determining regions (CDRs) to form contact interfaces with the critical residues in ETA-catalytic domain essential for ADP-ribosylation of eukaryotic elongation factor 2, which should consequently rescue ETA-exposed-cells from apoptosis. The HuscFv-treated ETA-exposed cells also showed decremented apoptosis-related genes, i.e., cas3 and p53. The effective HuscFvs have high potential for future evaluation in animal models and clinical trials as a safe, novel remedy for the amelioration of exotoxin A-mediated pathogenesis. HuscFvs may be used either singly or in combination with the HuscFv cognates that target other P. aeruginosa virulence factors as an alternative therapeutic regime for difficult-to-treat infections.
Asunto(s)
ADP Ribosa Transferasas/antagonistas & inhibidores , Antibacterianos/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Exotoxinas/antagonistas & inhibidores , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Anticuerpos de Cadena Única/farmacología , Factores de Virulencia/antagonistas & inhibidores , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/inmunología , ADP Ribosa Transferasas/metabolismo , Antibacterianos/inmunología , Antibacterianos/uso terapéutico , Apoptosis/efectos de los fármacos , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/metabolismo , Dominio Catalítico/genética , Regiones Determinantes de Complementariedad/inmunología , Regiones Determinantes de Complementariedad/farmacología , Exotoxinas/genética , Exotoxinas/inmunología , Exotoxinas/metabolismo , Células HeLa , Humanos , Simulación del Acoplamiento Molecular , Biblioteca de Péptidos , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/patogenicidad , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/uso terapéutico , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismo , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Synthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Regiones Determinantes de Complementariedad/farmacología , Inmunoglobulina G/farmacología , Péptidos/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Antifúngicos/síntesis química , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Candida albicans/crecimiento & desarrollo , Regiones Determinantes de Complementariedad/química , Humanos , Inmunoglobulina G/química , Larva/efectos de los fármacos , Larva/microbiología , Pruebas de Sensibilidad Microbiana , Mariposas Nocturnas/efectos de los fármacos , Mariposas Nocturnas/microbiología , Péptidos/síntesis química , Fosfatidilserinas/análisis , Fosfatidilserinas/metabolismo , Relación Estructura-Actividad , Análisis de SupervivenciaRESUMEN
Lowering the isoelectric point (pI) through engineering the variable region or framework of an IgG can improve its exposure and half-life via a reduction in clearance mediated through non-specific interactions. As such, net charge is a potentially important property to consider in developing therapeutic IgG molecules having favorable pharmaceutical characteristics. Frequently, it may not be possible to shift the pI of monoclonal antibodies (mAbs) dramatically without the introduction of other liabilities such as increased off-target interactions or reduced on-target binding properties. In this report, we explored the influence of more subtle modifications of molecular charge on the in vivo properties of an IgG1 and IgG4 monoclonal antibody. Molecular surface modeling was used to direct residue substitutions in the complementarity-determining regions (CDRs) to disrupt positive charge patch regions, resulting in a reduction in net positive charge without affecting the overall pI of the mAbs. The effect of balancing the net positive charge on non-specific binding was more significant for the IgG4 versus the IgG1 molecule that we examined. This differential effect was connected to the degree of influence on cellular degradation in vitro and in vivo clearance, distribution and metabolism in mice. In the more extreme case of the IgG4, balancing the charge yielded an â¼7-fold improvement in peripheral exposure, as well as significantly reduced tissue catabolism and subsequent excretion of proteolyzed products in urine. Balancing charge on the IgG1 molecule had a more subtle influence on non-specific binding and yielded only a modest alteration in clearance, distribution and elimination. These results suggest that balancing CDR charge without affecting the pI can lead to improved mAb pharmacokinetics, the magnitude of which is likely dependent on the relative influence of charge imbalance and other factors affecting the molecule's disposition.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Especificidad de Anticuerpos/genética , Regiones Determinantes de Complementariedad , Inmunoglobulina G , Modelos Moleculares , Animales , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/farmacocinética , Anticuerpos Monoclonales Humanizados/farmacología , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Regiones Determinantes de Complementariedad/farmacología , Células HEK293 , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Punto Isoeléctrico , RatonesRESUMEN
Short synthetic peptides corresponding to sequences of complementarity-determining regions (CDRs) from different immunoglobulin families have been shown to induce antimicrobial, antiviral and antitumor activities regardless of the specificity of the original monoclonal antibody (mAb). Presently, we studied the in vitro and in vivo antitumor activity of synthetic peptides derived from conserved CDR sequences of different immunoglobulins against human tumor cell lines and murine B16F10-Nex2 melanoma aiming at the discovery of candidate molecules for cancer therapy. Four light- and heavy-chain CDR peptide sequences from different antibodies (C36-L1, HA9-H2, 1-H2 and Mg16-H2) showed cytotoxic activity against murine melanoma and a panel of human tumor cell lineages in vitro. Importantly, they also exerted anti-metastatic activity using a syngeneic melanoma model in mice. Other peptides (D07-H3, MN20v1, MS2-H3) were also protective against metastatic melanoma, without showing significant cytotoxicity against tumor cells in vitro. In this case, we suggest that these peptides may act as immune adjuvants in vivo. As observed, peptides induced nitric oxide production in bone-marrow macrophages showing that innate immune cells can also be modulated by these CDR peptides. The present screening supports the search in immunoglobulins of rather frequent CDR sequences that are endowed with specific antitumor properties and may be candidates to be developed as anti-cancer drugs.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/farmacología , Inmunoglobulinas/química , Melanoma/tratamiento farmacológico , Péptidos/farmacología , Animales , Antineoplásicos/síntesis química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Regiones Determinantes de Complementariedad/inmunología , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Inmunoglobulinas/inmunología , Inmunomodulación , Células MCF-7 , Melanoma/inmunología , Melanoma/patología , Ratones , Péptidos/síntesis química , Péptidos/química , Relación Estructura-ActividadRESUMEN
We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr(52) to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen-antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody.
Asunto(s)
Afinidad de Anticuerpos , Regiones Determinantes de Complementariedad/biosíntesis , Receptores ErbB/inmunología , Neoplasias/terapia , Anticuerpos de Cadena Única/biosíntesis , Sustitución de Aminoácidos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/farmacología , Humanos , Inmunoterapia , Concentración 50 Inhibidora , Cinética , Mutagénesis Sitio-Dirigida , Biblioteca de Péptidos , Unión Proteica , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/farmacología , TermodinámicaRESUMEN
Bovine herpesvirus 1 (BoHV-1) causes respiratory and genital diseases in cattle for which available vaccines do not confer adequate protection. Since passive immunization with antibodies permits disease prevention, single-chain fragment variable (scFv), originating from a monoclonal bovine IgG1 antibody against BoHV-1, were constructed and expressed in Pichia pastoris in V(lambda)-V(H) orientation via a flexible seven-amino acid linker. Similar to the intact IgG, the purified recombinant scFv neutralized BoHV-1 in vitro and recognized viral antigens in BoHV-1 infected MDBK cells by immunofluorescence. Homology modeling of the Fv predicts two distinct conformations for CDR3H. Firstly, a long protruding CDR3H conformation where no disulfide linkage occurred between two "non-canonical" Cys residues resulted in a large binding cavity between V(lambda) and V(H). Secondly, a smaller potential antigen-binding cavity is predicted with a disulfide linkage between the two Cys residues of CDR3H creating a six-membered loop in the ascending polypeptide, which fitted into the space between V(lambda) and V(H). Despite such potential configurational diversity of the antigen-binding site, the electrostatic surface potentials that would interact with the BoHV-1 epitope are largely similar for both the topographies where salt-bridge type electrostatic interactions likely occur at the edges of the binding site. Given that IgG1 antibody against BoHV-1 is clonally selected, it is likely that disulfide-stabilized broader and flatter surface topography is specifically generated to accommodate the predicted carbohydrate neutralizing B-epitope on BoHV-1. The specificity and neutralizing capacity for BoHV-1 of the scFv should make this bovine antibody fragment a useful diagnostic and potential therapeutic candidate for an important viral pathogen in cattle.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos/inmunología , Regiones Determinantes de Complementariedad/inmunología , Herpesvirus Bovino 1/inmunología , Inmunoglobulina G/inmunología , Rinotraqueítis Infecciosa Bovina/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/farmacología , Especificidad de Anticuerpos/genética , Sitios de Unión de Anticuerpos/genética , Sitios de Unión de Anticuerpos/inmunología , Bovinos , Línea Celular , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/farmacología , Inmunoglobulina G/genética , Inmunoglobulina G/farmacología , Rinotraqueítis Infecciosa Bovina/prevención & control , Ratones , Estructura Secundaria de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacologíaRESUMEN
BACKGROUND: It has been documented that, independently from the specificity of the native antibody (Ab) for a given antigen (Ag), complementarity determining regions (CDR)-related peptides may display differential antimicrobial, antiviral and antitumor activities. METHODOLOGY/PRINCIPAL FINDINGS: In this study we demonstrate that a synthetic peptide with sequence identical to V(H)CDR3 of a mouse monoclonal Ab (mAb) specific for difucosyl human blood group A is easily taken up by macrophages with subsequent stimulation of: i) proinflammatory cytokine production; ii) PI3K-Akt pathway and iii) TLR-4 expression. Significantly, V(H)CDR3 exerts therapeutic effect against systemic candidiasis without possessing direct candidacidal properties. CONCLUSIONS/SIGNIFICANCE: These results open a new scenario about the possibility that, beyond the half life of immunoglobulins, Ab fragments may effectively influence the antiinfective cellular immune response in a way reminiscent of regulatory peptides of innate immunity.
Asunto(s)
Inmunidad Adaptativa/inmunología , Regiones Determinantes de Complementariedad/inmunología , Inmunidad Innata/inmunología , Inmunidad Adaptativa/efectos de los fármacos , Animales , Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/inmunología , Candidiasis/inmunología , Candidiasis/patología , Regiones Determinantes de Complementariedad/farmacología , Activación Enzimática/efectos de los fármacos , Humanos , Inmunidad Innata/efectos de los fármacos , Cadenas Pesadas de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Inmunomodulación/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/inmunología , Ratones , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Péptidos/inmunología , Péptidos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Análisis de Supervivencia , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/biosíntesis , Regulación hacia Arriba/efectos de los fármacosRESUMEN
A tolerogenic peptide, hCDR1, ameliorated murine lupus via the upregulation of functional regulatory cells and by immunomodulating cytokine production. In the present study we analyzed the ability of hCDR1 to similarly affect gene expression and regulatory T cells when incubated with peripheral blood mononuclear cells (PBMC) of lupus patients. To this end, peripheral blood mononuclear cells (PBMC) of 11 lupus patients and five gender- and age-matched healthy controls were cultured with hCDR1 or a control peptide. Gene expression and regulatory T-cells were assessed. hCDR1 significantly downregulated interleukin (IL)-1beta, interferon (IFN)-gamma, and IL-10 gene expression. Furthermore, hCDR1 upregulated the expression of the anti-apoptotic Bcl-xL molecule and downregulated the pro-apoptotic caspase-3, resulting in reduced rates of apoptosis. hCDR1 increased the expression of transforming growth factor (TGF)-beta, FoxP3 and the negative regulators Foxj1 and Foxo3a. No significant effects were observed using a control peptide or when PBMC of healthy donors were incubated with hCDR1. The elevated gene expression of FoxP3 was due to hCDR1-induced upregulation of TGF-beta, resulting in an increase of CD4+CD25+FoxP3+ functional, regulatory cells. The ability of the regulatory cells to diminish IFN-gamma expression and to upregulate TGF-beta was abrogated after the addition of a neutralizing anti-CD25 antibody, confirming their role in the beneficial effects of hCDR1.
Asunto(s)
Regiones Determinantes de Complementariedad/inmunología , Citocinas/metabolismo , Leucocitos Mononucleares/inmunología , Lupus Eritematoso Sistémico/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Apoptosis/inmunología , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/farmacología , Citocinas/antagonistas & inhibidores , Regulación hacia Abajo/efectos de los fármacos , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Humanos , Tolerancia Inmunológica , Terapia de Inmunosupresión , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Lupus Eritematoso Sistémico/sangre , Activación de Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Péptidos/química , Péptidos/inmunología , Péptidos/farmacología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Veltuzumab is a humanized anti-CD20 monoclonal antibody with complementarity-determining regions (CDRs) identical to rituximab, except for one residue at the 101st position (Kabat numbering) in CDR3 of the variable heavy chain (V(H)), having aspartic acid (Asp) instead of asparagine (Asn), with framework regions of epratuzumab, a humanized anti-CD22 antibody. When compared with rituximab, veltuzumab has significantly reduced off-rates in 3 human lymphoma cell lines tested, as well as increased complement-dependent cytotoxicity in 1 of 3 cell lines, but no other in vitro differences. Mutation studies confirmed that the differentiation of the off-rate between veltuzumab and rituximab is related to the single amino acid change in CDR3-V(H). Studies of intraperitoneal and subcutaneous doses in mouse models of human lymphoma and in normal cynomolgus monkeys disclosed that low doses of veltuzumab control tumor growth or deplete circulating or sessile B cells. Low- and high-dose veltuzumab were significantly more effective in vivo than rituximab in 3 lymphoma models. These findings are consistent with activity in patients with non-Hodgkin lymphoma given low intravenous or subcutaneous doses of veltuzumab. Thus, changing Asn(101) to Asp(101) in CDR3-V(H) of rituximab is responsible for veltuzumab's lower off-rate and apparent improved potency in preclinical models that could translate into advantages in patients.
Asunto(s)
Sustitución de Aminoácidos , Anticuerpos Monoclonales/inmunología , Antígenos CD20/inmunología , Antineoplásicos/inmunología , Regiones Determinantes de Complementariedad/inmunología , Linfoma/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Anticuerpos Monoclonales de Origen Murino , Antígenos CD20/genética , Antineoplásicos/farmacología , Linfocitos B/inmunología , Línea Celular Tumoral , Chlorocebus aethiops , Proteínas del Sistema Complemento/genética , Proteínas del Sistema Complemento/inmunología , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/farmacología , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Linfoma/tratamiento farmacológico , Linfoma/genética , Ratones , Mutación Missense , Rituximab , Relación Estructura-ActividadRESUMEN
TLRs trigger immunity by detecting microbe-associated molecular patterns (MAMPs). Flagellin is a unique MAMP because it harbors 1) an antigenic hypervariable region and 2) a conserved domain involved in TLR5-dependent systemic and mucosal proinflammatory and adjuvant activities. In this study, the contribution of the flagellin domains in TLR5 activation was investigated. We showed that TLR5 signaling can be neutralized in vivo by flagellin-specific Abs, which target the conserved domain. However, deletions of flagellin's hypervariable region abrogated the protein's intrinsic ability to trigger the production of neutralizing Abs. The fact that MAMP-specific Abs block TLR-mediated responses shows that this type of neutralization is a novel mechanism for down-regulating innate immunity. The stimulation of mucosal innate immunity and adjuvancy to foreign Ag was not altered by the hypervariable domain deletions. In contrast, this domain is essential to trigger systemic innate immunity, suggesting that there are distinct mechanisms for TLR5 activation in systemic and mucosal compartments. In summary, specific MAMP determinants control the production of neutralizing Abs and the compartmentalization of innate responses.
Asunto(s)
Regiones Determinantes de Complementariedad/inmunología , Flagelina/inmunología , Flagelina/metabolismo , Receptor Toll-Like 5/inmunología , Receptor Toll-Like 5/metabolismo , Animales , Células CACO-2 , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/farmacología , Femenino , Flagelina/genética , Eliminación de Gen , Humanos , Inmunidad , Inmunidad Innata/inmunología , Ratones , Modelos Moleculares , Estructura Cuaternaria de Proteína , Salmonella enterica/genética , Salmonella enterica/inmunología , Salmonella enterica/metabolismoAsunto(s)
Regiones Determinantes de Complementariedad/farmacología , Lupus Eritematoso Sistémico/tratamiento farmacológico , Fragmentos de Péptidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Autoanticuerpos/inmunología , Regulación hacia Abajo/efectos de los fármacos , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/metabolismo , Lupus Eritematoso Sistémico/inmunología , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Reguladores/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Active and passive immunotherapy targeted at the amyloid-beta (Abeta) peptide has been proposed as therapeutic approach against Alzheimer's disease (AD), and efforts towards the generation and application of antibody-based reagents that are capable of preventing and clearing amyloid aggregates are currently under active investigation. Previously, we selected and characterized a new anti-Abeta1-42 phage-displayed scFv antibody, designated clone b4.4, using a non-immune human scFv antibody library and demonstrated that a peptide based on the sequence of the Ig heavy chain (VH) complementarity-determining region (HCDR3) of this antibody fragment bound to Abeta1-42)and had neuroprotective potential against Abeta1-42 mediated neurotoxicity in rat hippocampal cultured neurons. In the present study, using novel computational methods and in vitro experiments we demonstrated that b4.4 binds to the central region of Abeta1-42. We also demonstrated that this scFv antibody binds to Abeta-derived diffusible ligands (ADDLs) and neutralizes the toxicity of both fibrillar and oligomeric forms of Abeta1-42 tested in vitro in SH-SY5Y cell cultures.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Regiones Determinantes de Complementariedad/farmacología , Fragmentos de Inmunoglobulinas/farmacología , Cadenas Pesadas de Inmunoglobulina/farmacología , Fármacos Neuroprotectores/farmacología , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/inmunología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regiones Determinantes de Complementariedad/química , Mapeo Epitopo , Humanos , Fragmentos de Inmunoglobulinas/química , Cadenas Pesadas de Inmunoglobulina/química , Modelos Moleculares , Fármacos Neuroprotectores/química , Oligopéptidos/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Unión ProteicaRESUMEN
The humanized monoclonal antibody HzKR127 recognizes the preS1 domain of the human hepatitis B virus surface proteins with a broadly neutralizing activity in vivo. We present the crystal structures of HzKR127 Fab and its complex with a major epitope peptide. In the complex structure, the bound peptide forms a type IV beta-turn followed by 3(10) helical turn, the looped-out conformation of which provides a structural basis for broad neutralization. Upon peptide binding, the antibody undergoes a dramatic complementarity determining region H3 lid opening. To understand the structural implication of the virus neutralization, we carried out comprehensive alanine-scanning mutagenesis of all complementarity determining region residues in HzKR127 Fab. The functional mapping of the antigen-combining site demonstrates the specific roles of major binding determinants in antigen binding, contributing to the rational design for maximal humanization and affinity maturation of the antibody.
Asunto(s)
Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/inmunología , Anticuerpos contra la Hepatitis B/química , Anticuerpos contra la Hepatitis B/inmunología , Secuencia de Aminoácidos , Antígenos/química , Antígenos/inmunología , Sitios de Unión , Regiones Determinantes de Complementariedad/farmacología , Cristalografía por Rayos X , Anticuerpos contra la Hepatitis B/genética , Anticuerpos contra la Hepatitis B/farmacología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Péptidos/química , Estructura Cuaternaria de Proteína , Sensibilidad y Especificidad , Proteínas Virales/química , Proteínas Virales/inmunologíaRESUMEN
The interaction of fibrinogen with integrin alphaIIbbeta3 (GPIIb/IIIa), in part mediated by an RGD tripeptide motif, is an essential step in platelet aggregation. Based on their inhibition of platelet aggregation, three integrin alphaIIbbeta3 inhibitors are clinically approved. The clinically most widely used integrin alphaIIbbeta3 inhibitor abciximab is a chimeric mouse/human antibody that induces thrombocytopenia, often severe, in 1-2% of patients due to a human anti-mouse antibody (HAMA) response. In addition, unlike other ligands mimicking small molecular drugs, abciximab cross-reacts with integrin alphavbeta3 and alphaMbeta2. Here we used phage display to select monoclonal antibodies specific to integrin alphaIIbbeta3 from a synthetic human antibody library based on the randomized HCDR3 sequence VGXXXRADXXXYAMDV. The selected antibodies revealed a strong consensus in HCDR3 (V(V/W)CRAD(K/R)RC) and high specificity toward integrin alphaIIbbeta3 but not to other RGD binding integrins such as alphavbeta3, alphavbeta5, and alpha5beta1. The selected antibodies as well as three synthetic peptides (VWCRADRRC, VWCRADKRC, and VVCRADRRC) whose sequences were derived from the HCDR3 sequences of the selected antibodies strongly inhibited the interaction between integrin alphaIIbbeta3 and fibrinogen and platelet aggregation ex vivo. To our knowledge, these are the first fully human monoclonal antibodies that are specific to integrin alphaIIbbeta3 and can potently inhibit platelet aggregation.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Regiones Determinantes de Complementariedad/inmunología , Regiones Determinantes de Complementariedad/farmacología , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Regiones Determinantes de Complementariedad/química , Disulfuros/metabolismo , Fibrinógeno/antagonistas & inhibidores , Fibrinógeno/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/farmacología , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/síntesis química , Péptidos/química , Péptidos/inmunología , Péptidos/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Unión Proteica/efectos de los fármacosRESUMEN
Two peptides were derived from the structural analysis of a previously described monoclonal antibody [Mol. Immunol. 37 (2000) 423] against the tachykinin NK(1) receptor for the neuropeptide substance P. Here we show that these two peptides were able to inhibit the inositol phosphate transduction pathway triggered both by substance P and neurokinin A, another high-affinity endogenous ligand for the tachykinin NK(1) receptor. They also reduced the cAMP production induced by substance P. By contrast, only one antagonist peptide was able to prevent substance P and neurokinin A from binding the receptor, as revealed both by biochemical and autoradiographic studies. First, these results illustrate the generality of the antibody-based strategy for developing new bioactive peptides. Second, they indicate that antagonists, even exhibiting very close amino acid composition, can interact with the tachykinin NK(1) receptor at different contact sites, some of them clearly distinct from the contact domains for endogenous agonists.