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Antibody Affinity Maturation

The affinity of antibody is a vital parameter that regulates antibody biological function and efficacy. Because of the in vivo affinity ceiling, the affinity of antibody from hybridomas often falls short of the level of effective clinical use and thus improving antibody affinity is often required. Affinity maturation and optimization of a pre-existing antibody can improve affinity, potency, developability, and cross-species binding. We have very strong expertise in affinity maturation and can provide a range of comprehensive and integrated solutions, typically based on creation and screening of a mutated full-length IgG library displayed on the yeast cell surface. In addition, we also provide affinity maturation services for single domain antibodies (ScFv).
Genekine apply “triple saturation mutagenesis library” and extensive recombination approaches to provides antibody affinity maturation service. Our approaches improves the screening efficiency and increases the probability of obtaining high affinity antibodies. It can routinely yield affinity improvement of 5-100 times or greater compared to the parental antibodies. In “triple saturation mutagenesis library”, three consecutive amino acids of the parent antibody are diversified by NNK saturation mutagenesis, resulting in a genotypic complexity of 32,768 variants per triple NNK mutagenesis. In triple NNK mutagenesis, three consecutive residues are fully diversified, which allow to identify: (1) the best single substitutions at each CDR residues that can contribute to affinity improvements, and (2) many adjacent residue combinations that might synergistically contribute to affinity improvements. Thus, the “triple saturation mutagenesis library” is a multidimensional mutagenesis method that simultaneously assesses and optimizes combinatorial mutations of selected amino acids. A set of triple saturation mutagenesis libraries are constructed that together “walk-through” and cover all CDR residues in the light and heavy chains.These triple saturation mutagenesis libraries are displayed on yeast surface in full-length IgG form, followed by FACS selection against the target antigen to identify affinity improving mutations. Subsequently, the affinity improving mutations are subjected to extensive recombination approaches to select the best combinations of additive or synergistic mutations. The improving mutations in CDRs are combined to generate a combinatorial antibody mutation library. The library is displayed on yeast cell surface in full-length IgG form, followed by FACS selection to find the best combinations for desired affinity. Given the large flexibility in yeast surface display and FACS screening, our approaches have clear applications to optimize other antibody properties, such as stability, selectivity, species cross-reactivity, expression yield, and to engineer proteins beyond antibodies.
Schematic illustration of “triple saturation mutagenesis library” of CDR3-L Three consecutive amino acid residues in CDR3-L are diversified by saturation mutagenesis where randomizing codons such as NNK (encoding all 20 amino acids within 32 codons) are included, resulting in a genotypic complexity of 32,768 variants. In each triple NNK mutagenesis, three consecutive residues are fully diversified, which allow to identify: (1) the best single substitutions at each CDR position that can contribute to affinity improvements, and (2) many adjacent residue combinations that might synergistically contribute to affinity improvements. A set of “triple saturation mutagenesis libraries” can be constructed that together “walk-through”: and cover all CDR residues.
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