ABSTRACT
Local pulmonary delivery of biotherapeutics may offer advantages for the treatment of lung diseases. Delivery of the therapeutic entity directly to the lung has the potential for a rapid onset of action, reduced systemic exposure and the need for a lower dose, as well as needleless administration. However, formulation of a protein for inhaled delivery is challenging and requires proteins with favorable biophysical properties suitable to withstand the forces associated with formulation, delivery, and inhalation devices. Nanobodies are the smallest functional fragments derived from a naturally occurring heavy chain-only immunoglobulin. They are highly soluble, stable, and show biophysical characteristics that are particularly well suited for pulmonary delivery. This paper highlights a number of clinical and preclinical studies on antibodies delivered via the pulmonary route and describes the advantages of using Nanobodies for inhaled delivery to the lung. The latter is illustrated by the specific example of ALX-0171, a Nanobody in clinical development for the treatment of respiratory syncytial virus (RSV) infections.
Subject(s)
Drug Delivery Systems , Lung Diseases/drug therapy , Single-Domain Antibodies/administration & dosage , Administration, Inhalation , Animals , Drug Design , Humans , Lung Diseases/immunology , Lung Diseases/physiopathology , Respiratory Syncytial Virus Infections/drug therapy , Respiratory Syncytial Virus Infections/immunologyABSTRACT
Respiratory syncytial virus (RSV) is an important causative agent of lower respiratory tract infections in infants and elderly individuals. Its fusion (F) protein is critical for virus infection. It is targeted by several investigational antivirals and by palivizumab, a humanized monoclonal antibody used prophylactically in infants considered at high risk of severe RSV disease. ALX-0171 is a trimeric Nanobody that binds the antigenic site II of RSV F protein with subnanomolar affinity. ALX-0171 demonstrated in vitro neutralization superior to that of palivizumab against prototypic RSV subtype A and B strains. Moreover, ALX-0171 completely blocked replication to below the limit of detection for 87% of the viruses tested, whereas palivizumab did so for 18% of the viruses tested at a fixed concentration. Importantly, ALX-0171 was highly effective in reducing both nasal and lung RSV titers when delivered prophylactically or therapeutically directly to the lungs of cotton rats. ALX-0171 represents a potent novel antiviral compound with significant potential to treat RSV-mediated disease.
Subject(s)
Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , Respiratory Syncytial Virus Infections/drug therapy , Respiratory Syncytial Viruses/drug effects , Single-Domain Antibodies/pharmacology , Viral Fusion Proteins/antagonists & inhibitors , Administration, Inhalation , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Neutralizing/immunology , Antibodies, Viral/biosynthesis , Antibodies, Viral/immunology , Antiviral Agents/immunology , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Female , Gene Expression , Humans , Lung/drug effects , Lung/immunology , Lung/virology , Male , Models, Molecular , Nasal Cavity/drug effects , Nasal Cavity/immunology , Nasal Cavity/virology , Neutralization Tests , Palivizumab/biosynthesis , Palivizumab/immunology , Palivizumab/pharmacology , Pichia/genetics , Pichia/metabolism , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus Infections/pathology , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Viruses/immunology , Respiratory Syncytial Viruses/pathogenicity , Sigmodontinae , Single-Domain Antibodies/biosynthesis , Single-Domain Antibodies/immunology , Viral Fusion Proteins/chemistry , Viral Fusion Proteins/genetics , Viral Fusion Proteins/immunology , Viral Load/drug effects , Virus Replication/drug effectsABSTRACT
Protein sequences from multiple hepatitis B virus (HBV) isolates were analyzed for the presence of amino acid motifs characteristic of cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes with the goal of identifying conserved epitopes suitable for use in a therapeutic vaccine. Specifically, sequences bearing HLA-A1, -A2, -A3, -A24, -B7, and -DR supertype binding motifs were identified, synthesized as peptides, and tested for binding to soluble HLA. The immunogenicity of peptides that bound with moderate to high affinity subsequently was assessed using HLA transgenic mice (CTL) and HLA cross-reacting H-2(bxd) (BALB/c x C57BL/6J) mice (HTL). Through this process, 30 CTL and 16 HTL epitopes were selected as a set that would be the most useful for vaccine design, based on epitope conservation among HBV sequences and HLA-based predicted population coverage in diverse ethnic groups. A plasmid DNA-based vaccine encoding the epitopes as a single gene product, with each epitope separated by spacer residues to enhance appropriate epitope processing, was designed. Immunogenicity testing in mice demonstrated the induction of multiple CTL and HTL responses. Furthermore, as a complementary approach, mass spectrometry allowed the identification of correctly processed and major histocompatibility complex-presented epitopes from human cells transfected with the DNA plasmid. A heterologous prime-boost immunization with the plasmid DNA and a recombinant MVA gave further enhancement of the immune responses. Thus, a multiepitope therapeutic vaccine candidate capable of stimulating those cellular immune responses thought to be essential for controlling and clearing HBV infection was successfully designed and evaluated in vitro and in HLA transgenic mice.
