ABSTRACT
Phage display technology is frequently used to obtain antigen specific binders with predetermined characteristics. Phage display libraries are often constructed from animals immunized with the antigen of interest. An important point of consideration when making immune libraries is the availability of an appropriate antigen sources. When available, often either the amount is not sufficient for immunization or it is expensive to obtain. To overcome this problem, these antigens are typically obtained by over expression in prokaryotic or eukaryotic expression systems. While this could solve the problem of obtaining sufficient quantities of antigen for a reasonable price and effort, correct folding and differences in posttranslational modification could potentially lead to binders that recognize the recombinant, but not the endogenous protein. In addition, selection of binders against specific modifications or structural epitopes could be missed.In this chapter we describe a particular selection of VHH antibody fragments from phage display libraries that were constructed from llamas immunized with different complex protein samples containing the antigen of interest. We show that this can result in binders that preferentially recognize the target of interest when present in specific structures depending on the antigen source.
Subject(s)
Amyloid beta-Protein Precursor/immunology , Immunoglobulin Heavy Chains/immunology , Amyloid beta-Protein Precursor/metabolism , Animals , Antibodies/blood , Antibodies/immunology , Antibody Specificity/immunology , Camelids, New World/immunology , Humans , Immunoglobulin Heavy Chains/metabolism , Peptide Library , Protein Binding/immunologyABSTRACT
This study investigated the in vivo properties of two heavy chain antibody fragments (V(H)H), ni3A and pa2H, to differentially detect vascular or parenchymal amyloid-ß deposits characteristic for Alzheimer's disease and cerebral amyloid angiopathy. Blood clearance and biodistribution including brain uptake were assessed by bolus injection of radiolabeled V(H)H in APP/PS1 mice or wildtype littermates. In addition, in vivo specificity for Aß was examined in more detail with fluorescently labeled V(H)H by circumventing the blood-brain barrier via direct application or intracarotid co-injection with mannitol. All V(H)H showed rapid renal clearance (10-20 min). Twenty-four hours post-injection (99m)Tc-pa2H resulted in a small yet significant higher cerebral uptake in the APP/PS1 animals. No difference in brain uptake were observed for (99m)Tc-ni3A or DTPA((111)In)-pa2H, which lacked additional peptide tags to investigate further clinical applicability. In vivo specificity for Aß was confirmed for both fluorescently labeled V(H)H, where pa2H remained readily detectable for 24 hours or more after injection. Furthermore, both V(H)H showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aß. Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that V(H)H detect Aß deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aß deposits.
Subject(s)
Alzheimer Disease/diagnosis , Alzheimer Disease/immunology , Amyloid beta-Peptides/immunology , Immunoglobulin Fragments/immunology , Immunoglobulin Heavy Chains/immunology , Plaque, Amyloid/immunology , Amyloid beta-Peptides/blood , Animals , Autoradiography , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/pathology , Disease Models, Animal , Fluorescent Antibody Technique , Half-Life , Humans , Immunoglobulin Fragments/blood , Immunoglobulin Heavy Chains/blood , Mice , Mice, Transgenic , Protein Binding , Species Specificity , Tissue Distribution/immunologyABSTRACT
By phage display, llama-derived heavy chain antibody fragments were selected from non-immune and immune libraries and tested for their affinity and specificity for beta amyloid by phage-ELISA, immunohistochemistry and surface plasmon resonance. We identified eight distinct heavy chain antibody fragments specific for beta amyloid. While three of them recognized vascular and parenchymal beta amyloid deposits, the remaining five heavy chain antibody fragments recognized vascular beta amyloid specifically, failing to bind to parenchymal beta amyloid. These heavy chain antibody fragments, selected from different libraries, demonstrated differential affinity for different epitopes when used for immunohistochemistry. These observations indicate that the llama heavy chain antibody fragments are the first immunologic probes with the ability to differentiate between parenchymal and vascular beta amyloid aggregates. This indicates that vascular and parenchymal beta amyloid deposits are heterogeneous in epitope presence/availability. The properties of these heavy chain antibody fragments make them potential candidates for use in in vivo differential diagnosis of Alzheimer disease and cerebral amyloid angiopathy. Continued use and characterization of these reagents will be necessary to fully understand the performance of these immunoreagents.
