Monobodies lack binding sites for metal ions and the central disulfide bond. This domain has a structure similar to antibody variable domains, with seven beta sheets forming a beta-sandwich and three exposed loops on each side corresponding to the three complementarity-determining regions. They are based on the structure of human fibronectin, more specifically on its tenth extracellular type III domain. The native FN3 scaffold consists of 94 amino acids and has a molecular mass of about 10 kDa, fifteen times smaller than an IgG type antibody and comparable to the size of a single variable domain of an antibody. An example is pegdinetanib (Angiocept), an antagonist of vascular endothelial growth factor receptor 2 (VEGFR-2), which has entered Phase II clinical trials investigating the treatment of glioblastoma in October 2007. The monobody technology has been adopted in the biotechnology industry, most notably by Adnexus, a biotechnology company which has been part of Bristol-Myers Squibb since 2007 under the name of Adnectins (originally as Trinectins by its predecessor, Phylos ). In contrast, most antibodies and antibody fragments depend on disulfide bonds formation and they must be produced under an oxidizing environment. This is because of the characteristics of the underlying FN3 scaffold: small (~90 residues), stable, easy to produce, and its lack of disulfide bonds that makes it possible to produce functional monobodies regardless of the redox potential of the cellular environment, including the reducing environment of the cytoplasm and nucleus. A major advantage of monobodies over conventional antibodies is that monobodies can readily be used as genetically encoded intracellular inhibitors, that is you can express a monobody inhibitor in a cell of choice by simply transfecting the cell with a monobody expression vector. Monobodies belong to the class of molecules collectively called antibody mimics (or antibody mimetics) and alternative scaffolds that aim to overcome shortcomings of natural antibody molecules. A large number of monobodies that have high affinity and high specificity to their respective targets have been reported. Monobodies are generated from combinatorial libraries in which portions of the FN3 scaffold are diversified using molecular display and directed evolution technologies such as phage display, mRNA display and yeast surface display. The hybrid term monobody was coined in 1998 by the Koide group who published the first paper demonstrating the monobody concept using the tenth FN3 domain of human fibronectin. Monobodies are a simple and robust alternative to antibodies for creating target-binding proteins. Specifically, this class of binding proteins are built upon a diversified library of the 10th FN3 domain of human fibronectin. Monobodies are synthetic binding proteins constructed using a fibronectin type III domain (FN3) as a molecular scaffold. Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, CZ-25250 Vestec, Czech Republic.Variable domain of an antibody's lambda light chain (human, PDB: 2RHE​) The procedure yielded scaffold-related variants with nanomolar affinity. We demonstrated its functionality by training it as a binder against human interleukin-10, a medically important cytokine. The newly developed scaffold, called ProBi (Protein Binder), contains two independently mutable surface patches. ![]() We proved the applicability of this systematic procedure by selecting a monomeric single-domain human protein with a fold different from previously known scaffolds. In the next step, we examined several variants of the candidate scaffolds including their wild types and alanine mutants. The candidate protein scaffolds were subjected to a thorough screening including computational evaluation of the mutability, and experimental determination of their expression yield in E. We hereby describe a process based on an analysis of protein structures from the Protein Data Bank and their experimental examination. This calls for a more systematic approach in designing new scaffolds suitable for use in one or more methods of directed evolution. The advantages include smaller size and a more robust, single-domain structural framework with a defined binding surface amenable to mutation. Diversity, Equity, Inclusion, and AccessĮngineered small non-antibody protein scaffolds are a promising alternative to antibodies and are especially attractive for use in protein therapeutics and diagnostics.Citation, Usage, Privacy Policies, Logo.Biologically Interesting Molecule Reference Dictionary (BIRD).
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