Efficient Screening and Design of Variable Domain of Heavy Chain Antibody Ligands Through High Throughput Sequencing for Affinity Chromatography to Purify Fab Fragments.

To design an affinity ligand for purification of antigen-binding fragment (Fab) antibody, variable domain of heavy chain antibody (VHH) phage libraries were constructed from Fab-immunized Alpaca and subjected to biopanning against Fabs. To find the specific binders, we directly applied high-throughput sequencing (HTS) analysis of the VHH sequences in the panned phages on next-generation sequencer. The efficiently enriched sequences were aligned for construction of the phylogenetic tree to be categorized into five groups. VHHs from three major groups were first selected to analyze their properties as an affinity ligand. However, those VHHs were not suitable as an affinity ligand because of lack of resistance against alkaline pH and/or difficulty in acidic elution from the affinity column. So, we further searched the candidates from minor group sequences. Among five, one VHH showed the binding ability but with low affinity against Fabs. Therefore, we improved its affinity-by-affinity maturation through error-prone PCR library techniques. The final designed VHH showed highly alkaline pH resistance and easy acidic elution together with high affinity to Fabs. These results indicate that HTS techniques combined with biopanning and followed by error-prone PCR library techniques is powerful in designing specific binders with desired properties.

AppA: a web server for analysis, comparison, and visualization of contact residues and interfacial waters of antibody-antigen structures and models.

The study of contact residues and interfacial waters of antibody-antigen (Ab-Ag) structures could help in understanding the principles of antibody-antigen interactions as well as provide guidance for designing antibodies with improved affinities. Given the rapid pace with which new antibody-antigen structures are deposited in the protein databank (PDB), it is crucial to have computational tools to analyze contact residues and interfacial waters, and investigate them at different levels. In this study, we have developed AppA, a web server that can be used to analyze and compare 3D structures of contact residues and interfacial waters of antibody-antigen complexes. To the best of our knowledge, this is the first web server for antibody-antigen structures equipped with the capability for dissecting the contributions of interfacial water molecules, hydrogen bonds, hydrophobic interactions, van der Waals interactions and ionic interactions at the antibody-antigen interface, and for comparing the structures and conformations of contact residues. Various examples showcase the utility of AppA for such analyses and comparisons that could help in the understanding of antibody-antigen interactions and suggest mutations of contact residues to improve affinities of antibodies. The AppA web server is freely accessible at http://mspc.bii.a-star.edu.sg/minhn/appa.html.

Identification of multiple serine to asparagine sequence variation sites in an intended copy product of LUCENTIS® by mass spectrometry.

Patent expiration of first-generation biologics and the high cost of innovative biologics are 2 drivers for the development of biosimilar products. There are, however, technical challenges to the production of exact copies of such large molecules. In this study, we performed a head-to-head comparison between the originator anti-VEGF-A Fab product LUCENTIS® (ranibizumab) and an intended copy product using an integrated analytical approach. While no differences could be observed using size-exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate and potency assays, different acidic peaks were identified with cation ion exchange chromatography and capillary zone electrophoresis. Further investigation of the intact Fab, subunits and primary sequence with mass spectrometry demonstrated the presence of a modified light chain variant in the intended copy product batches. This variant was characterized with a mass increase of 27.01 Da compared to the originator sequence and its abundance was estimated in the range of 6-9% of the intended copy product light chain. MS/MS spectra interrogation confirmed that this modification relates to a serine to asparagine sequence variant found in the intended copy product light chain. We demonstrated that the integration of high-resolution and sensitive orthogonal technologies was beneficial to assess the similarity of an originator and an intended copy product.

Functional Characterization of AAV-Expressed Recombinant Anti-VEGF Single-Chain Variable Fragments In Vitro.

PURPOSE: Most retinal neovascular disorders are caused by upregulation of vascular endothelial growth factor (VEGF) expression. These disorders are treated with repeated injections of anti-VEGF molecules, which may have severe side effects. The expression of anti-VEGF molecules by the retina itself in a controlled manner following adeno-associated viral (AAV) gene transfer could be a replacement of this therapy. METHODS: The open reading frames (orf) of the light and the heavy chain of ranibizumab were cloned into an expression plasmid separated by an internal ribosomal entry site (IRES). The construct was mutated to generate ranibizumab single-chain variable fragments (scFv). Expression was verified by western blotting and the concentrations were measured with a custom-made ranibizumab ELISA. Biological activity, VEGF-binding properties, and the doxycycline-dependent induction of anti-VEGF expression were tested. An AAV2/5 vector was generated containing the optimal variant Ra02. RESULTS: Ra01-Ra05 molecules were detected in the cell culture medium. While the VEGF-binding affinity was significantly lower for Ra01 and Ra02 compared to Lucentis(®), the inhibition of cell migration was comparable and the maximum inhibition of Ra01 and Ra02 was reached at lower doses. The expression of Ra01 and Ra02 was shown to be regulable with the TetOn-system(®) as plasmid (Ra01, Ra02) and AAV vector construct (Ra02). CONCLUSION: Ra01 and Ra02 can be produced in eukaryotic cells after AAV-mediated gene transfer in a regulable manner in vitro and display comparable biological activity as Lucentis. These results are the basis for in vivo studies in human VEGF-overexpressing mice, a model for human neovascular disorders.