RESUMO
Neutralizing anti-factor VIII (FVIII) antibodies that develop in patients with hemophilia A and in murine hemophilia A models, clinically termed "inhibitors," bind to several distinct surfaces on the FVIII-C2 domain. To map these epitopes at high resolution, 60 recombinant FVIII-C2 proteins were generated, each having a single surface-exposed residue mutated to alanine or a conservative substitution. The binding kinetics of these muteins to 11 monoclonal, inhibitory anti-FVIII-C2 antibodies were evaluated by surface plasmon resonance and the results compared with those obtained for wild-type FVIII-C2. Clusters of residues with significantly altered binding kinetics identified "functional" B-cell epitopes, defined as those residues contributing appreciable antigen-antibody avidity. These antibodies were previously shown to neutralize FVIII activity by interfering with proteolytic activation of FVIII by thrombin or factor Xa, or with its binding to phospholipid surfaces, von Willebrand factor, or other components of the intrinsic tenase complex. Fine mapping of epitopes by surface plasmon resonance also indicated surfaces through which FVIII interacts with proteins and phospholipids as it participates in coagulation. Mutations that significantly altered the dissociation times/half-lives identified functionally important interactions within antigen-antibody interfaces and suggested specific sequence modifications to generate novel, less antigenic FVIII proteins with possible therapeutic potential for treatment of inhibitor patients.
Assuntos
Mapeamento de Epitopos , Fator VIII/química , Mutação Puntual , Ressonância de Plasmônio de Superfície , Alanina/química , Aminoácidos/química , Animais , Anticorpos Monoclonais/química , Antígenos/química , Coagulação Sanguínea , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Epitopos de Linfócito B/química , Humanos , Camundongos , Mutagênese , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , SuínosRESUMO
Evidence of antibody isotype/subtype switching may provide prognostic value regarding the state of immune responses to therapeutic proteins, e.g. anti-factor VIII (FVIII) antibodies that develop in many hemophilia A patients, clinically termed "inhibitors". A sensitive, high- information-content surface plasmon resonance (SPR) assay has been developed to quantify IgG subtype distributions and the domain specificity of anti-drug antibodies. Plasma samples from 22 subjects with an allo- or auto-immune reaction to FVIII were analyzed. Pre-analytical treatment protocols were developed to minimize non-specific binding and specific matrix interference due to von Willebrand factor-FVIII interactions. The dynamic range for IgG quantification was 0.2-5 µg/ml (â¼1-33 nM), allowing characterization of inhibitor-positive samples. Subtype-specific monoclonal antibodies were used to quantify the IgG subtype distribution of FVIII-specific antibodies. Most samples obtained from multiply-infused inhibitor subjects contained IgG4 antibodies. Several distinct phenotypes were assigned based on the IgG subtype distribution: IgG1, IgG4, IgG1 & IgG4, and IgG1, IgG2 & IgG4. An IgG1-only response was found in mild/moderate HA subjects during early FVIII infusions, and analysis of serial samples followed antibody class switching as several subjects' immune responses developed. Competition studies utilizing a recombinant FVIII-C2 domain indicated 40-80% of FVIII-specific antibodies in most samples were directed against this domain.
Assuntos
Formação de Anticorpos/imunologia , Fator VIII/imunologia , Hemofilia A/imunologia , Imunoglobulina G/imunologia , Fenótipo , Ressonância de Plasmônio de Superfície/métodos , Animais , Anticorpos Monoclonais/imunologia , Hemofilia A/sangue , Humanos , Imunoglobulina G/classificação , CamundongosRESUMO
Interleukin-21 (IL-21) is a type I four-helical bundle cytokine that exerts a variety of significant effects on many hematopoietic cells, including T and B lymphocytes and natural killer cells. IL-21 is produced predominantly by CD4+ T cells and natural killer T cells and, when aberrantly overexpressed, appears to play important roles in a wide variety of autoimmune disorders. To generate potential therapeutic reagents capable of inhibiting IL-21 for clinical use, we immunized human immunoglobulin transgenic mice with IL-21 and then identified and cloned a panel of human anti-human IL-21 binding monoclonal antibodies. IL-21 neutralizing and IL-21-binding, non-neutralizing antibodies were assigned to distinct epitope "bins" based on surface plasmon resonance competition studies. The most potent neutralizing antibodies had extremely high (sub pM) affinity for IL-21 and were able to block IL-21 activity in various biological assays using either an IL-21R-transfected pre-B-cell line or primary human B cells, and their neutralizing activity was, in some cases, superior to that of a soluble form of the high affinity heterodimeric IL-21 receptor. Characterization of this panel of IL-21 antibodies provided the basis for the selection of a therapeutic candidate antibody capable of inhibiting IL-21 activity for the treatment of autoimmune and inflammatory diseases.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/imunologia , Interleucinas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , Autoimunidade , Linfócitos B/imunologia , Células CHO , Linhagem Celular , Cricetinae , Epitopos/imunologia , Humanos , Interleucinas/administração & dosagem , Interleucinas/química , Interleucinas/genética , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Transgênicos , Células Precursoras de Linfócitos B/imunologia , Coelhos , Ratos , Receptores de Interleucina-21/genética , Receptores de Interleucina-21/imunologia , Linfócitos T/imunologiaRESUMO
Metabolic profiles of hydrophilic and lipophilic cell extracts from three cancer cell lines, Miapaca-2, Panc-1 and AsPC-1, and a non-cancerous pancreatic ductal epithelial cell line, H6C7, were examined by proton nuclear magnetic resonance spectroscopy. Over twenty five hydrophilic metabolites were identified by principal component and statistical significance analyses as distinguishing the four cell types. Fifteen metabolites were identified with significantly altered concentrations in all cancer cells, e.g. absence of phosphatidylgrycerol and phosphatidylcholine, and increased phosphatidylethanolamine and cholesterols. Altered concentrations of metabolites involved in glycerophospholipid metabolism, lipopolysaccharide and fatty acid biosynthesis indicated differences in cellular membrane composition between non-cancerous and cancer cells. In addition to cancer specific metabolites, several metabolite changes were unique to each cancer cell line. Increased N-acetyl groups in AsPC-1, octanoic acids in Panc-1, and UDP species in Miapaca-2 indicated differences in cellular membrane composition between the cancer cell lines. Induced glutamine metabolism and protein synthesis in cancer cells were indicated by absence of glutamine other metabolites such as acetate, lactate, serine, branched amino acids, and succinate. Knowledge of the specifically altered metabolic pathways identified in these pancreatic cancer cell lines may be useful for identifying new therapeutic targets and studying the effects of potential new therapeutic drugs.
RESUMO
Elevated high mobility group A (HMGA) protein expression in pancreatic cancer cells is correlated with resistance to the chemotherapy agent gemcitabine. Here, we demonstrate use of HMGA-targeted AT-rich phosphorothioate DNA (AT-sDNA) aptamers to suppress HMGA carcinogenic activity. Cell growth of human pancreatic cancer cells (AsPC-1 and Miapaca-2) transfected with AT-sDNA were monitored after treatment with gemcitabine. Significant increases in cell death in AT-sDNA transfected cells compared to non-AT-rich sDNA treated cells were observed in both cell lines. The data indicate the potential use of HMGA targeted DNA aptamers to enhance chemotherapy efficacy in pancreatic cancer treatment.
Assuntos
Adenocarcinoma/terapia , Antimetabólitos Antineoplásicos/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Desoxicitidina/análogos & derivados , Proteínas HMGA/metabolismo , Neoplasias Pancreáticas/terapia , Oligonucleotídeos Fosforotioatos/farmacologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Oligonucleotídeos Fosforotioatos/genética , Oligonucleotídeos Fosforotioatos/metabolismo , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
INTRODUCTION: B-lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL) are members of the tumor necrosis factor (TNF) family that regulate B-cell maturation, survival, and function. They are overexpressed in a variety of autoimmune diseases and reportedly exist in vivo not only as homotrimers, but also as BLyS/APRIL heterotrimers. METHODS: A proprietary N-terminal trimerization domain was used to produce recombinant BLyS/APRIL heterotrimers. Heterotrimer biologic activity was compared with that of BLyS and APRIL in a 4-hour signaling assay by using transmembrane activator and CAML interactor (TACI)-transfected Jurkat cells and in a 4-day primary human B-cell proliferation assay. A bead-based immunoassay was developed to quantify native heterotrimers in human sera from healthy donors (n = 89) and patients with systemic lupus erythematosus (SLE; n = 89) or rheumatoid arthritis (RA; n = 30). Heterotrimer levels were compared with BLyS and APRIL homotrimer levels in a subset of these samples. RESULTS: The recombinant heterotrimers consisted mostly of one BLyS and two APRIL molecules. Heterotrimer signaling did not show any significant difference compared with APRIL in the TACI-Jurkat assay. Heterotrimers were less-potent inducers of B-cell proliferation than were homotrimeric BLyS or APRIL (EC(50), nMol/L: BLyS, 0.02; APRIL, 0.17; heterotrimers, 4.06). The soluble receptor fusion proteins atacicept and B-cell maturation antigen (BCMA)-immunoglobulin (Ig) neutralized the activity of BLyS, APRIL, and heterotrimers in both cellular assays, whereas B-cell activating factor belonging to the TNF family receptor (BAFF-R)-Ig neutralized only the activity of BLyS. In human sera, significantly more patients with SLE had detectable BLyS (67% versus 18%; P < 0.0001), APRIL (38% versus 3%; P < 0.0002), and heterotrimer (27% versus 8%; P = 0.0013) levels compared with healthy donors. Significantly more patients with RA had detectable APRIL, but not BLyS or heterotrimer, levels compared with healthy donors (83% versus 3%; P < 0.0001). Heterotrimer levels weakly correlated with BLyS, but not APRIL, levels. CONCLUSIONS: Recombinant BLyS/APRIL heterotrimers have biologic activity and are inhibited by atacicept and BCMA-Ig, but not by BAFF-R-Ig. A novel immunoassay demonstrated that native BLyS/APRIL heterotrimers, as well as BLyS and APRIL homotrimers, are elevated in patients with autoimmune diseases.
Assuntos
Doenças Autoimunes/sangue , Fator Ativador de Células B/sangue , Antígeno de Maturação de Linfócitos B/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/sangue , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Humanos , Interleucina-4/farmacologia , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Multimerização Proteica , Proteínas RecombinantesRESUMO
Bispecific antibodies (bsAbs) present an attractive opportunity to combine the additive and potentially synergistic effects exhibited by combinations of monoclonal antibodies (mAbs). Current challenges for engineering bsAbs include retention of the binding affinity of the parent mAb or antibody fragment, the ability to bind both targets simultaneously, and matching valency with biology. Other factors to consider include structural stability and expression of the recombinant molecule, both of which may have significant impact on its development as a therapeutic. Here, we incorporate selection of stable, potent single-chain variable fragments (scFvs) early in the engineering process to assemble bsAbs for therapeutic applications targeting the cytokines IL-17A/A and IL-23. Stable scFvs directed against human cytokines IL-23p19 and IL-17A/A were isolated from a human Fab phage display library via batch conversion of panning output from Fabs to scFvs. This strategy integrated a step for shuffling V regions during the conversion and permitted the rescue of scFv molecules in both the V(H)V(L) and the V(L)V(H) orientations. Stable scFvs were identified and assembled into several bispecific formats as fusions to the Fc domain of human IgG1. The engineered bsAbs are potent neutralizers of the biological activity of both cytokines (IC(50) < 1 nM), demonstrate the ability to bind both target ligands simultaneously and display stability and productivity advantageous for successful manufacture of a therapeutic molecule. Pharmacokinetic analysis of the bsAbs in mice revealed serum half-lives similar to human mAbs. Assembly of bispecific molecules using stable antibody fragments offers an alternative to reformatting mAbs and minimizes subsequent structure-related and manufacturing concerns.
Assuntos
Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Interleucina-17/imunologia , Interleucina-23/imunologia , Engenharia de Proteínas , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/farmacocinética , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Bases de Dados de Proteínas , Escherichia coli/genética , Feminino , Meia-Vida , Humanos , Cinética , Camundongos , Estabilidade Proteica , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismoRESUMO
Fab arm exchange by a stabilized anti-IL-31 IgG(4)S228P monoclonal antibody (mAb) was studied using physiologically relevant antibody concentrations and thiol exchange conditions, and directly compared to that of matched wild type IgG(4) (IgG(4)wt) and IgG(1) control antibodies. In vitro arm exchange between the test mAbs and a purified IgG(4)wt exchange partner was monitored using capillary isoelectric focusing and a size-exclusion peak shift assay. Arm exchange between the test mAbs and IgG exchange partners with unknown specificity was monitored using only the shift assay. Studies were performed using single isotype human and mouse mAbs, unfractionated human, mouse, and cynomolgus monkey IgG, and human serum as the sources of the exchange partners. In vitro studies using human serum demonstrated that anti-IL-31 IgG(4)S228P did not undergo significant Fab arm exchange with endogenous human IgG(4) whereas anti-IL-31 IgG(4)wt underwent rapid and extensive Fab arm exchange. The in vitro results were corroborated by in vivo studies in which mice were injected with a mixture of either form of the test mAb and an excess of non-specific human IgG(4) exchange partner.
Assuntos
Anticorpos Monoclonais/química , Fragmentos Fab das Imunoglobulinas/química , Imunoglobulina G/química , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Interleucinas/química , Interleucinas/imunologia , Macaca fascicularis , CamundongosRESUMO
Binding of Ag-Ab immune complexes to cellular FcgammaR promotes cell activation, release of inflammatory mediators, and tissue destruction characteristic of autoimmune disease. To evaluate whether a soluble FcgammaR could block the proinflammatory effects of immune complexes, recombinant human (rh) versions of FcgammaRIA, FcgammaRIIA, and FcgammaRIIIA were prepared. Binding of rh-FcgammaRIA to IgG was of high affinity (KD=1.7x10(-10) M), whereas rh-FcgammaRIIA and rh-FcgammaRIIIA bound with low affinity (KD=0.6-1.9x10(-6) M). All rh-FcgammaR reduced immune complex precipitation, blocked complement-mediated lysis of Ab-sensitized RBC, and inhibited immune complex-mediated production of IL-6, IL-13, MCP-1, and TNF-alpha by cultured mast cells. Local or systemic delivery only of rh-FcgammaRIA, however, reduced edema and neutrophil infiltration in the cutaneous Arthus reaction in mice. 125I-labeled rh-FcgammaRIA was cleared from mouse blood with a rapid distribution phase followed by a slow elimination phase with a t1/2gamma of approximately 130 h. The highest percentage of injected radioactivity accumulated in blood approximately liver approximately carcass>kidney. s.c. dosing of rh-FcgammaRIA resulted in lower serum levels of inflammatory cytokines and prevented paw swelling and joint damage in a murine model of collagen Ab-induced arthritis. These data demonstrate that rh-FcgammaRIA is an effective inhibitor of type III hypersensitivity.
Assuntos
Doenças do Complexo Imune/tratamento farmacológico , Receptores de IgG/uso terapêutico , Animais , Complexo Antígeno-Anticorpo/efeitos dos fármacos , Artrite Experimental/tratamento farmacológico , Artrite Experimental/patologia , Reação de Arthus/tratamento farmacológico , Reação de Arthus/patologia , Proteínas do Sistema Complemento/imunologia , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Humanos , Doenças do Complexo Imune/patologia , Imunoglobulina G/metabolismo , Mastócitos/imunologia , Camundongos , Receptores de IgG/biossínteseRESUMO
Bleeding can be a serious complication of surgery, and topical thrombin is widely used as an adjunct to hemostasis in diverse surgical settings. The potent hemostatic properties of thrombin derive from its ability to activate platelets directly to aggregate and adhere to damaged vessels and to catalyze the formation simultaneously of a fibrin matrix. Application of exogenous thrombin bypasses the physiological process of generating a thrombin burst by directly initiating the terminal reactions of blood clot formation. Currently, thrombin used to control surgical bleeding is primarily from bovine plasma, with a small percentage from human plasma. Human thrombin isolated from pooled plasma carries the risk of transmitting plasma-borne pathogens or prion diseases. The bovine preparations have been associated with protein and preparative contaminants that pose potential risks of developing cross-reacting antibodies. There is a need for a pure therapeutic preparation of human thrombin. Recombinant human thrombin (rhThrombin) has been efficiently produced from a prethrombin-1 precursor obtained from Chinese hamster ovary cell culture. This rhThrombin is substantially free of process-derived contaminants and has been characterized extensively in terms of composition, primary, secondary, and tertiary structure, enzymatic activity; and in vivo pharmacology. In vivo studies of topically applied rhThrombin have shown it is effective in achieving hemostasis in a rabbit liver excisional wound model. Clinical studies are ongoing to evaluate the safety and efficacy of rhThrombin as an adjunct to hemostasis in patients undergoing surgery.
Assuntos
Trombina/química , Trombina/uso terapêutico , Animais , Perda Sanguínea Cirúrgica/prevenção & controle , Humanos , Peso Molecular , Controle de Qualidade , Proteínas Recombinantes , Trombina/genética , Trombina/isolamento & purificaçãoRESUMO
Factor XIII (FXIII) is a thrombin-activated protransglutaminase responsible for fibrin clot stabilization and longevity. Deficiency in FXIII is associated with diffuse bleeding and wound-healing disorders in humans. This report summarizes results from several studies conducted in adult cynomolgus monkeys (M. fascicularis) to evaluate the safety and pharmacokinetics of recombinant human factor XIII A(2) dimer (rFXIII). Intravenous slow bolus injection of rFXIII resulted in the expected formation of the heterotetramer rA(2)cnB(2), prolonged circulating half-life (5-7 days), and increased plasma transglutaminase activity. Recombinant FXIII was well tolerated as a single dose up to 20 mg/kg rFXIII (2840 U/kg), as repeated daily doses up to 6 mg/kg (852 U/kg) for 14 days, and as 3 repeated doses of 8 mg/kg (1136 U/kg) separated by 14 days. Overt toxicity occurred after a single intravenous injection of = 22.5 mg/kg rFXIII (3150 U/kg), or with 2 doses of = 12.5 mg/kg (1775 U/kg) administered within 72 hours. The rFXIII-mediated toxicity was expressed as an acute systemic occlusive coagulopathy. Evaluation of plasma samples from dosed animals demonstrated formation of cross-linked fibrin/fibrinogen oligomers and higher-order protein aggregates, which are hypothesized to be responsible for the observed vessel occlusion and associated embolic sequelae. These results demonstrate that rFXIII-mediated toxicity results from exaggerated pharmacological activity of the molecule at supraphysiological concentrations. The absence of observed toxicological effect with repeated intravenous doses up to 8 mg/kg (1136 U/kg) was used to support an initial clinical dose range of 0.014 to 0.35 mg/kg (2-50 U/kg).