RESUMO
OBJECTIVE: To assess the ability of an anti-lysozyme antibody to stabilize the plasma pharmacokinetics of lysozyme through a 'buffering' effect. METHODS: Hen egg lysozyme was radiolabeled with (14)C and infused with or without the murine anti-lysozyme antibody D1.3 into the jugular vein of rats. The dosages of antibody and lysozyme were varied, and the antibody and lysozyme were administered either together or in separate infusions. RESULTS: Buffering of lysozyme with an equimolar amount of D1.3 more than doubled the plasma half-life of lysozyme and addition of more antibody buffer prolonged the half-life to an even greater extent. D1.3 bound lysozyme that was infused several minutes after the D1.3 administration, indicating that D1.3 can be charged with drug in vivo. CONCLUSION: Antibody buffering of monovalent therapeutic enzymes is possible and may have potential for clinical use.
Assuntos
Anticorpos Monoclonais/farmacologia , Muramidase/farmacocinética , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/sangue , Reações Antígeno-Anticorpo , Soluções Tampão , Galinhas , Relação Dose-Resposta Imunológica , Meia-Vida , Infusões Intravenosas , Veias Jugulares , Masculino , Camundongos , Muramidase/administração & dosagem , Muramidase/sangue , Muramidase/imunologia , Ratos , Ratos Sprague-DawleyRESUMO
The efficacy of chemotherapy on brain tumors is often hindered by the presence of the blood brain barrier. This barrier keeps many systemically administered substances from entering the cerebrospinal fluid (CSF), while allowing intrathecally administered drugs free passage out of that compartment. Therefore, achieving a therapeutic concentration of a cell cycle inhibitor in the CSF for a time long enough to have a cytotoxic effect on slow-growing tumor cells has proven difficult. The ability of an antibody to prolong ligand half-life and bioactivity has been previously described occurring in the plasma. This phenomenon has not yet been described or exploited for use in the CSF compartment. Antibodies often have a longer residence time in the CSF than small-molecule drugs, so antibody buffering, administration of a drug with its specific antibody, can prolong the bioactive lifetime of a drug in the CSF. Here we describe antibody buffering of the small molecule hapten 2-phenyl-oxazol-5-one-methylene-gamma-amino butyrate in the CSF of a rats. Not only does the presence of an antibody buffer increase the half-life of both total and free hapten in the CSF, but the antibody can be re-charged in situ with fresh hapten, even days after the initial antibody infusion. Antibody buffering may provide a viable option for delivering a stable, bio-available concentration of a drug that is normally rapidly eliminated from the CSF.
Assuntos
Anticorpos Monoclonais/farmacocinética , Encéfalo/metabolismo , Haptenos/líquido cefalorraquidiano , Aminobutiratos/química , Animais , Anticorpos Monoclonais/imunologia , Barreira Hematoencefálica , Encéfalo/imunologia , Soluções Tampão , Meia-Vida , Haptenos/imunologia , Ligantes , Lipoproteínas LDL/imunologia , Lipoproteínas LDL/metabolismo , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Apo chicken liver bile acid-binding protein has been structurally characterized by NMR. The dynamic behavior of the protein in its apo- and holo-forms, complexed with chenodeoxycholate, has been determined via (15)N relaxation and steady state heteronuclear (15)N((1)H) nuclear Overhauser effect measurements. The dynamic parameters were obtained at two pH values (5.6 and 7.0) for the apoprotein and at pH 7.0 for the holoprotein, using the model free approach. Relaxation studies, performed at three different magnetic fields, revealed a substantial conformational flexibility on the microsecond to millisecond time scales, mainly localized in the C-terminal face of the beta-barrel. The observed dynamics are primarily caused by the protonation/deprotonation of a buried histidine residue, His(98), located on this flexible face. A network of polar buried side chains, defining a spine going from the E to J strand, is likely to provide the long range connectivity needed to communicate motion from His(98) to the EF loop region. NMR data are accompanied by molecular dynamics simulations, suggesting that His(98) protonation equilibrium is the triggering event for the modulation of a functionally important motion, i.e. the opening/closing at the protein open end, whereas ligand binding stabilizes one of the preexisting conformations (the open form). The results presented here, complemented with an analysis of proteins belonging to the intracellular lipid-binding protein family, are consistent with a model of allosteric activation governing the binding mechanism. The functional role of this mechanism is thoroughly discussed within the framework of the mechanism for the enterohepatic circulation of bile acids.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Ligação Proteica , Regulação Alostérica , Sequência de Aminoácidos , Animais , Apolipoproteínas/química , Apolipoproteínas/metabolismo , Ácidos e Sais Biliares/metabolismo , Sítios de Ligação , Galinhas , Ligantes , Fígado/enzimologia , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação ProteicaRESUMO
Autoantibodies to the 65-kDa isoform of glutamate decarboxylase GAD65 (GAD65Ab) are strong candidates for a pathological role in Stiff-Person syndrome (SPS). We have analyzed the binding specificity of the GAD65Ab in serum and cerebrospinal fluid (CSF) of 12 patients with SPS by competitive displacement studies with GAD65-specific rFab-derived from a number of human and mouse mAbs specific for different determinants on the Ag. We demonstrate considerable differences in the epitope specificity when comparing paired serum and CSF samples, suggesting local stimulation of B cells in the CSF compartment of these patients. Moreover, these autoantibodies strongly inhibit the enzymatic activity of GAD65, thus blocking the formation of the neurotransmitter gamma-aminobutyric acid. The capacity of the sera to inhibit the enzymatic activity of GAD65 correlated with their binding to a conformational C-terminal Ab epitope. Investigation of the inhibitory mechanism revealed that the inhibition could not be overcome by high concentrations of glutamate or the cofactor pyridoxal phosphate, suggesting a noncompetitive inhibitory mechanism. Finally, we identified a linear epitope on amino acids residues 4-22 of GAD65 that was recognized solely by autoantibodies from patients with SPS but not by serum from type 1 diabetes patients. A mAb (N-GAD65 mAb) recognizing this N-terminal epitope was successfully humanized to enhance its potential therapeutic value by reducing its overall immunogenicity.
Assuntos
Especificidade de Anticorpos , Autoanticorpos/imunologia , Glutamato Descarboxilase/imunologia , Isoenzimas/imunologia , Rigidez Muscular Espasmódica/imunologia , Adulto , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Autoanticorpos/sangue , Autoanticorpos/líquido cefalorraquidiano , Ligação Competitiva , Mapeamento de Epitopos , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Administration of a therapeutic antibody can lead to an anti-antibody response (AAR). Much effort has been applied to engineering antibodies with as little as possible non-human structure to minimize such responses. Here, we review reported AAR to murine, mouse-human chimeric, and humanized antibodies. Replacement of mouse immunoglobulin constant regions with human ones effects the largest immunogenicity reduction. Humanization of variable domains effects a further decrease.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Especificidade de Anticorpos , Humanos , Regiões Constantes de Imunoglobulina/genética , Regiões Constantes de Imunoglobulina/imunologia , Camundongos , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
We report a new method of humanizing antibodies by complementarity determining region (CDR) grafting. Our method differs from others in that we choose human framework sequences from the set of human germline genes based on the structural similarity of the human CDRs to those of the mouse antibody to be humanized. The structural similarity is evaluated by scoring residue-to-residue homology of the mouse CDRs to human candidates with the same Chothia canonical structures. The method is illustrated with the humanization of the anti-lysozyme antibody D1.3.
Assuntos
Anticorpos Monoclonais/genética , Regiões Determinantes de Complementaridade/genética , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/imunologia , Escherichia coli/genética , Genes de Imunoglobulinas , Células Germinativas/fisiologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/metabolismo , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Homologia de Sequência de AminoácidosRESUMO
Clearance is the practical limit on drug action. Here we propose a means of slowing clearance, thereby extending drug lifetime in vivo by "antibody buffering." In this process, a drug and an anti-drug antibody are coadministered. Most of the drug is bound to the antibody, preventing the drug from acting, but also preventing its elimination. A dynamic free drug pool is established by reversible dissociation from the antibody. The free drug is active and can be eliminated, but the free pool is constantly replenished by reequilibration from the antibody-drug complex, giving a long effective lifetime. Here we explore antibody buffering experimentally by using a model compound, 2-phenyloxazol-5-one-gamma-aminobutyrate (Ox), as a drug proxy. We show that antibody buffering can extend by an order of magnitude the plasma lifetime of Ox in rats, and that the steady-state Ox level depends on the molecular properties of the antibody used to buffer the Ox. In addition, the anti-Ox antibody can be recharged with drug in vivo to extend Ox lifetime without additional antibody administration, making this technique even more suitable for possible clinical application.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Oxazolona/análogos & derivados , Preparações Farmacêuticas , Farmacocinética , Anticorpos Monoclonais/imunologia , Ligantes , Oxazolona/imunologia , Oxazolona/farmacocinética , Oxazolona/uso terapêutico , Fatores de Tempo , Trítio , Ácido gama-Aminobutírico/imunologia , Ácido gama-Aminobutírico/farmacocinética , Ácido gama-Aminobutírico/uso terapêuticoRESUMO
Autoantibodies to the 65-kDa isoform of GAD (GAD65Abs) are associated with type 1 diabetes development, but the conformational nature of the GAD65Ab epitopes complicates the evaluation of disease risk. Six GAD65-specific recombinant Fabs (rFabs) were cloned from monoclonal antibodies b96.11, DP-C, DP-A, DPD, 144, and 221-442. The binding of GAD65Abs in 61 type 1 diabetic patients to GAD65 was analyzed by competitive radioimmunoassays with the six rFabs to ascertain disease-specific GAD65Ab binding specificities. The median binding was reduced significantly by rFab b96.11 (72%) (P < 0.0001), DP-A (84%) (P < 0.0001), DP-C (84%) (P < 0.0001), 221-442 (79%) (P < 0.0001), and DP-D (80%) (P < 0.0001). The competition pattern in type 1 diabetic patients differed from that in GAD65Ab-positive late autoimmune diabetes in adults (LADA) patients (n = 44), first-degree relatives (n = 38), and healthy individuals (n = 14). Whereas 87 and 72% of the type 1 diabetic sera were competed by rFab b96.11 and DP-C, respectively, only 34 and 26% of LADA patients, 18 and 25% of first-degree relatives, and 7 and 28% of healthy individuals showed competition (P < 0.0001). These findings support the view that type 1 diabetes is associated with disease- and epitope-specific GAD65Abs and supports the notion that the middle epitope is disease associated. These GAD65-specific rFabs should prove useful in predicting type 1 diabetes and in the study of conformational GAD65Ab epitopes.
Assuntos
Anticorpos Monoclonais/imunologia , Autoanticorpos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Glutamato Descarboxilase/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Isoenzimas/imunologia , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Especificidade de Anticorpos , Criança , Glutamato Descarboxilase/antagonistas & inibidores , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Região Variável de Imunoglobulina/química , Isoenzimas/antagonistas & inibidores , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Recombinantes/imunologia , Valores de Referência , Alinhamento de Sequência , Homologia de Sequência de AminoácidosAssuntos
Especificidade de Anticorpos , Antígenos/imunologia , Imunoglobulina E/química , Imunoglobulina E/imunologia , 2,4-Dinitrofenol/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Diversidade de Anticorpos , Complexo Antígeno-Anticorpo , Reações Antígeno-Anticorpo , Sítios de Ligação de Anticorpos , Reações Cruzadas , Cristalografia por Raios X , Haptenos/imunologia , Isomerismo , Testes de Neutralização , Biblioteca de Peptídeos , Conformação Proteica , Proteínas Recombinantes/imunologiaRESUMO
Humanized Abs are created by combining, at the genetic level, the complementarity-determining regions of a murine mAb with the framework sequences of a human Ab variable domain. This leads to a functional Ab with reduced immunogenic side effects in human therapy. In this study, we report a new approach to humanizing murine mAbs that may reduce immunogenicity even further. This method is applied to humanize the murine anti-human CD28 Ab, 9.3. The canonical structures of the hypervariable loops of murine 9.3 were matched to human genomic V gene sequences whose hypervariable loops had identical or similar canonical structures. Framework sequences for those human V genes were then used, unmodified, with the 9.3 complementarity-determining regions to construct a humanized version of 9.3. The humanized 9.3 and a chimeric 9.3 control were expressed in Escherichia coli as Fab. The humanized Fab showed a moderate loss in avidity in a direct binding ELISA with immobilized CD28-Ig fusion protein (CD28-Ig). Humanized 9.3 blocked ligation of CD28-Ig to cells expressing the CD28 receptor CD80. Lastly, the humanized 9.3 showed biological activity as an immunosuppressant by inhibiting a MLR.