Abstract
We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1). This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs) isolated from immunized OmniFlic animals. Using next-generation sequencing antibody repertoire analysis, we measured heavy chain variable gene usage and the diversity of clonotypes present in the lymph node germinal centers of 75 OmniFlic rats immunized with 9 different protein antigens. Furthermore, we expressed 2,560 unique heavy chain sequences sampled from a diverse set of clonotypes as fixed light chain antibody proteins and measured their binding to antigen by ELISA. Finally, we measured patterns and overall levels of somatic hypermutation in the full B-cell repertoire and in the 2,560 mAbs tested for binding. The results demonstrate that OmniFlic animals produce an abundance of antigen-specific antibodies with heavy chain clonotype diversity that is similar to what has been described with unrestricted light chain use in mammals. In addition, we show that sequence-based discovery is a highly effective and efficient way to identify a large number of diverse monoclonal antibodies to a protein target of interest.
Highlights
Human monoclonal antibodies are a promising and rapidly growing class of therapeutic molecules for various disease indications [1, 2]
In this study we show that the OmniFlic rat model produces a large sequence diversity of antibodies through the use of various VH genes and V–D–J recombination leading to a large number of distinct clonotypes despite each antibody using a common light chain
Based on the frequency of VH-gene usage and clonotype diversity measured by NGS, we show in this study that the OmniFlic animal generates heavy chain diversity during a response to immunogen comparable to animals with unrestricted light chain use [9, 11, 23]
Summary
Human monoclonal antibodies (mAbs) are a promising and rapidly growing class of therapeutic molecules for various disease indications [1, 2]. A significant technical achievement in creating therapeutic antibodies was the generation of transgenic mice [3, 4] and rats [5, 6] that express fully human Ig loci. Such systems enable the development of fully human mAbs with reduced risk of antidrug immune reactions in human patients. Sequence-Based Antibody Discovery been created that express a full heavy chain repertoire with a fixed rearranged light chain [7, 8] Toward this end, we created a novel transgenic Sprague-Dawley rat strain called OmniFlic that expresses a fixed human kappa light chain in combination with a chimeric IgH locus including human VHs, Ds, JHs, and rat CHs in a triple Ig KO background [9]. The human IgH V genes were covered by two bacterial artificial chromosomes (BACs): BAC6-VH3-11 containing the region spanning from VH4-39 to VH3-23 followed by VH3-11 and BAC3 containing the region spanning from VH3-11 to VH6-1 (811L16 RPCI-11) as described by Osborn et al [9]
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