ABSTRACT
BACKGROUND: Nanobodies(®) are therapeutic proteins derived from the smallest functional fragments of heavy chain-only antibodies. The development and validation of an LC-MS/MS-based method for the quantification of an IgE binding Nanobody in cynomolgus monkey plasma is presented. RESULTS: Nanobody quantification was performed making use of a proteotypic tryptic peptide chromatographically enriched prior to LC-MS/MS analysis. The validated LLOQ at 36 ng/ml was measured with an intra- and inter-assay precision and accuracy <20%. The required sensitivity could be obtained based on the selectivity of 2D LC combined with MS/MS. No analyte specific tools for affinity purification were used. Plasma samples originating from a PK/PD study were analyzed and compared with the results obtained with a traditional ligand-binding assay. Excellent correlations between the two techniques were obtained, and similar PK parameters were estimated. CONCLUSION: A 2D LC-MS/MS method was successfully developed and validated for the quantification of a next generation biotherapeutic.
Subject(s)
Immunoglobulin E/blood , Immunoglobulin E/immunology , Single-Domain Antibodies/blood , Single-Domain Antibodies/immunology , Animals , Binding Sites , Chromatography, High Pressure Liquid , Immunoglobulin E/chemistry , Macaca fascicularis , Single-Domain Antibodies/chemistry , Tandem Mass SpectrometryABSTRACT
ALX-0681 is a therapeutic Nanobody targeting the A1-domain of VWF. It inhibits the interaction between ultra-large VWF and platelet GpIb-IX-V, which plays a crucial role in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). In the present study, we report the efficacy and safety profile of ALX-0681 in a baboon model of acquired TTP. In this model, acute episodes of TTP are induced by administration of an ADAMTS13-inhibiting mAb. ALX-0681 completely prevented the rapid onset of severe thrombocytopenia and schistocytic hemolytic anemia. After induction of TTP, platelet counts also rapidly recovered on administration of ALX-0681. This effect was corroborated by the full neutralization of VWF activity. The schistocytic hemolytic anemia was also halted and partially reversed by ALX-0681 treatment. Brain CT scans and post mortem analysis did not reveal any sign of bleeding, suggesting that complete neutralization of VWF by ALX-0681 under conditions of thrombocytopenia was not linked with an excessive bleeding risk. The results obtained in this study demonstrate that ALX-0681 can successfully treat and prevent the most important hallmarks of acquired TTP without evidence of a severe bleeding risk. Therefore, ALX-0681 offers an attractive new therapeutic option for acquired TTP in the clinical setting.
Subject(s)
Anemia, Hemolytic/drug therapy , Antibodies, Monoclonal/therapeutic use , Fibrinolytic Agents/therapeutic use , Purpura, Thrombotic Thrombocytopenic/drug therapy , von Willebrand Factor/antagonists & inhibitors , ADAM Proteins/antagonists & inhibitors , ADAM Proteins/metabolism , Anemia, Hemolytic/complications , Anemia, Hemolytic/metabolism , Anemia, Hemolytic/pathology , Animals , Antibodies, Monoclonal/pharmacology , Blood Platelets/drug effects , Blood Platelets/metabolism , Blood Platelets/pathology , Disease Models, Animal , Drug Evaluation, Preclinical , Fibrinolytic Agents/pharmacology , Male , Multimodal Imaging , Papio , Platelet Count , Positron-Emission Tomography , Purpura, Thrombotic Thrombocytopenic/complications , Purpura, Thrombotic Thrombocytopenic/metabolism , Purpura, Thrombotic Thrombocytopenic/pathology , Tomography, X-Ray Computed , Treatment Outcome , von Willebrand Factor/metabolismABSTRACT
Accumulating evidence indicates that neutralizing antibodies play an important role in protection from chronic hepatitis C virus (HCV) infection. Efforts to elicit such responses by immunization with intact heterodimeric E1E2 envelope proteins have met with limited success. To determine whether antigenic sites, which are not exposed by the combined E1E2 heterodimer structure, are capable of eliciting neutralizing antibody responses, we expressed and purified each as separate recombinant proteins E1 and E2, from which the immunodominant hypervariable region (HVR-1) was deleted. Immunization of chimpanzees with either E1 or E2 alone induced antigen-specific T-helper cytokines of similar magnitude. Unexpectedly, the capacity to neutralize HCV was observed in E1 but not in animals immunized with E2 devoid of HVR-1. Furthermore, in vivo only E1-vaccinated animals exposed to the heterologous HCV-1b inoculum cleared HCV infection.
Subject(s)
Antibodies, Neutralizing/blood , Hepacivirus/immunology , Hepatitis C/therapy , Viral Envelope Proteins/immunology , Viral Hepatitis Vaccines/immunology , Animals , Disease Models, Animal , Genotype , Hepacivirus/genetics , Pan troglodytes , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Viral Envelope Proteins/genetics , Viral Hepatitis Vaccines/administration & dosage , Viral Hepatitis Vaccines/geneticsABSTRACT
Neutralizing the interaction of the platelet receptor gpIb with VWF is an attractive strategy to treat and prevent thrombotic complications. ALX-0081 is a bivalent Nanobody which specifically targets the gpIb-binding site of VWF and interacts avidly with VWF. Nanobodies are therapeutic proteins derived from naturally occurring heavy-chain-only Abs and combine a small molecular size with a high inherent stability. ALX-0081 exerts potent activity in vitro and in vivo. Perfusion experiments with blood from patients with acute coronary syndrome on standard antithrombotics demonstrated complete inhibition of platelet adhesion after addition of ALX-0081, while in the absence of ALX-0081 residual adhesion was observed. In a baboon efficacy and safety model measuring acute thrombosis and surgical bleeding, ALX-0081 showed a superior therapeutic window compared with marketed antithrombotics. Pharmacokinetic and biodistribution experiments demonstrated target-mediated clearance of ALX-0081, which leads to a self-regulating disposition behavior. In conclusion, these preclinical data demonstrate that ALX-0081 combines a high efficacy with an improved safety profile compared with currently marketed antithrombotics. ALX-0081 has entered clinical development.
Subject(s)
Antibodies, Bispecific/pharmacokinetics , Fibrinolytic Agents/pharmacology , Immunoglobulin Heavy Chains/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Single-Chain Antibodies/pharmacokinetics , Thrombosis/drug therapy , Animals , Antibody Specificity , Binding Sites/immunology , Fibrinolytic Agents/immunology , Humans , In Vitro Techniques , Macaca fascicularis , Papio , Platelet Adhesiveness/drug effects , Platelet Adhesiveness/immunology , Platelet Glycoprotein GPIb-IX Complex/immunology , Platelet Glycoprotein GPIb-IX Complex/metabolism , Pulsatile Flow/physiology , Thrombosis/immunology , von Willebrand Factor/immunology , von Willebrand Factor/metabolismABSTRACT
The relative importance of humoral and cellular immunity in the prevention or clearance of hepatitis C virus (HCV) infection is poorly understood. However, there is considerable evidence that neutralizing antibodies are involved in disease control. Here we describe the detailed analysis of human monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly neutralize HCVpp bearing the envelope glycoproteins of genotype 2a. Genotype 3a was not neutralized. The epitopes for both MAbs were mapped to the region encompassing amino acids 313 to 327. In addition, robust neutralization was also observed against cell culture-adapted viruses of genotypes 1a and 2a. Results from this study suggest that these MAbs may have the potential to prevent HCV infection.