Related Experiment Video
Updated: May 29, 2025
10:58
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Published on: July 25, 2013
17.0K
Plasticity of BioPhi-driven humanness optimization in ScFv-CD99 binding affinity validated through AlphaFold,
Kanokporn Sornsuwan1,2, Thanathat Pamonsupornwichit2, On-Anong Juntit1,2
1Office of Research Administration, Chiang Mai University, Chiang Mai 50200, Thailand.
Computational and Structural Biotechnology Journal
|February 3, 2025
Summary
BioPhi-guided humanization of anti-CD99 single-chain variable fragments (ScFvs) improved structural stability but showed varied binding affinities. Despite enhanced humanness, all variants posed a high immunogenicity risk, underscoring the need for a balance in therapeutic antibody development.
Area of Science:
- Biotechnology
- Immunology
- Structural Biology
Background:
- Humanization of antibody fragments is crucial for reducing immunogenicity in therapeutic applications.
- Single-chain variable fragments (ScFvs) targeting CD99 are being explored for therapeutic potential.
- Optimizing ScFv humanness while maintaining binding affinity and stability is a key challenge.
Purpose of the Study:
- To enhance the humanness of an anti-CD99 ScFv using BioPhi-guided humanization.
- To evaluate the impact of humanization on the structural stability, binding affinity, and immunogenicity of anti-CD99 ScFvs.
- To investigate the interplay between humanness, structural integrity, and functional performance of engineered ScFvs.
Main Methods:
- BioPhi-guided humanization to create HuScFvMT99/3BP and HuScFvMT99/3HY variants.
- Molecular dynamics simulations using AlphaFold2, AlphaFold3, and HADDOCK to assess binding free energy (ΔGbinding) and structural stability (RMSD).
- Biolayer interferometry, ELISA, and flow cytometry to experimentally validate binding affinity and interactions. PITHA analysis for immunogenicity risk assessment.
Main Results:
- HuScFvMT99/3BP and HuScFvMT99/3HY showed improved structural stability compared to the wild-type (WT).
- Binding affinity results were inconsistent across simulation methods; biolayer interferometry indicated WT had the highest affinity (KD = 1.35 × 10⁻⁷ M), followed by BP and HY variants.
- All variants exhibited a high immunogenicity risk, although HuScFvMT99/3HY demonstrated improved humanness and specific structural features in its complementarity-determining regions (CDRs).
Conclusions:
- BioPhi-guided humanization can enhance ScFv humanness and structural stability but may compromise binding affinity.
- Achieving optimal therapeutic antibody candidates requires balancing humanness with functional efficacy and minimizing immunogenicity.
- Further optimization strategies are needed to develop safe and effective humanized anti-CD99 ScFv therapeutics.
More Related Videos
08:31Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions
Published on: December 1, 2020
4.9K
09:15Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions
Published on: November 21, 2017
8.2K
Related Concept Videos
02:18The Equilibrium Binding Constant and Binding Strength
12.8K
The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:
12.8K
02:35Noncovalent Attractions in Biomolecules
47.7K
Noncovalent attractions are associations within and between molecules that influence the shape and structural stability of complexes. These interactions differ from covalent bonding in that they do not involve sharing of electrons.
Four types of noncovalent interactions are hydrogen bonds, van der Waals forces, ionic bonds, and hydrophobic interactions.
Hydrogen bonding results from the electrostatic attraction of a hydrogen atom covalently bonded to a strong-electronegative atom like oxygen,...
Four types of noncovalent interactions are hydrogen bonds, van der Waals forces, ionic bonds, and hydrophobic interactions.
Hydrogen bonding results from the electrostatic attraction of a hydrogen atom covalently bonded to a strong-electronegative atom like oxygen,...
47.7K
01:49Conserved Binding Sites
4.2K
Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally...
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally...
4.2K
00:49Ligand Binding and Linkage
4.7K
Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked. In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence...
4.7K
01:57Gene Families
8.7K
Gene families consist of groups of genes proposed to have originated from a common ancestor. Typically these arise through events in which a gene or genes are mistakenly duplicated during cell division. Unlike their parent genes (which are subject to selection pressure to maintain function), these gene copies do not need to preserve their sequences and may evolve at a relatively faster rate.
Occasionally these regions can be adapted to take on new roles within the organism, becoming novel genes...
Occasionally these regions can be adapted to take on new roles within the organism, becoming novel genes...
8.7K
01:58Cooperative Allosteric Transitions
7.8K
Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...
7.8K