Surface display of IgG antibody, Saturation mutagenesis library, Affinity maturation service, FACS

Antibody Affinity Maturation

The affinity of antibody is a crucial factor that determines antibody biological function and efficacy. However, the affinity of antibody from hybridomas is often limited by the in vivo affinity ceiling, which makes it insufficient for effective clinical use and requires affinity enhancement. Affinity maturation and optimization of an existing antibody can improve affinity, potency, developability, and cross-species binding.
Genekine has very strong expertise in affinity maturation and can offer a variety of comprehensive and integrated solutions, mainly based on creating and screening a mutated full-length IgG library displayed on yeast cell surface. 
Genekine uses “triple saturation mutagenesis library” and extensive recombination approaches to provide antibody affinity maturation service. Our approaches improve the screening efficiency and increases the chance of obtaining high affinity antibodies. They can routinely achieve affinity improvement of 5-100 times or more 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 optimal single substitutions at each CDR residue that can contribute to affinity improvement, and (2) many adjacent residue combinations that might have synergistic effects on affinity improvement. 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 cell surface in full-length IgG form, followed by FACS selection against the target antigen to identify affinity enhancing mutations. The “triple saturation mutagenesis library” is a multidimensional mutagenesis method that simultaneously evaluates and optimizes combinatorial mutations of selected amino acids.
Next, the affinity enhancing mutations are subjected to extensive recombination approaches to select the best combinations of additive or synergistic mutations. The enhancing 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.
Due to the large flexibility in yeast cell surface display and FACS screening, our approaches can also be applied 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. 

Antibody Affinity Maturation

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