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We recently performed specific mutagenesis of all six CDRs in a single scFv antibody sequence with Ranomics. We were very pleased with the mutation diversity that was obtained and were impressed with how specific (and fast) the mutations could be introduced to make a new library.

Adam Adler

CSO, Gigagen

Revolutionizing Antibody Affinity Maturation Of Therapeutic Monoclonal Antibodies 

In this study, researchers employed yeast surface display to screen eight scFv libraries against key immuno-oncology markers such as PD-1, PD-L1, CTLA-4, and OX40. The goal of the screen is to identify affinity enhancing mutations on each scFv. The study compared scFv libraries synthesized using error-prone PCR and combinatorial codon mutagenesis by Ranomics. While the error-prone PCR library resulted in randomly dispersed mutations along the scFv, the Ranomics combinatorial mutagenesis library resulted in targeted mutations in the scFv CDRs. Screening of the yeast libraries for improved affinity was conducted, and long-read high-throughput sequencing before and after sorting provided precise quantification of amino acid mutations across the full heavy and light chain V(D)J sequences. The optimization process, guided by sequencing data, aimed at refining the libraries and identifying promising antibody variants through an iterative approach.

The collaboration's success is documented in the publication titled:

"Affinity Maturation of Antibodies by Combinatorial Codon Mutagenesis versus Error-Prone PCR"

Key findings:

 Ranomics VariantFind Combinatorial Mutagenesis Libraries:

  • Multiple mutations distributed along the entire open reading frame.

  • No restrictions on spatial separation between mutated codons.

  • Absence of strong positional biases.

  • Substantial representation of amino acids observed across all templates.

  Comparative Analysis with epPCR

  • Similar patterns across all four targets.

  • Slightly higher tendency for epPCR to incorporate negatively charged and polar side chains.

  • More substantial differences for individual amino acids.

  Amino Acid Class Distribution:

  • VariantFind reflects the expected NNK codon distribution.

  • Not detectably influenced by the original amino acid.

  epPCR amino acid base

  • Biases observed for particular amino acids.

  • Likelihood of a derived amino acid codon influenced by the parental sequence's codon.

  Key Distinction:

  • VariantFind lacks parental sequence codon influence on derived amino acid codons.

  • Flexibility and unbiased representation distinguish VariantFind from epPCR mutagenesis.

This case study highlights the pivotal role played by Ranomics VariantFind in creating variant libraries for accelerating antibody engineering. The key findings from the Ranomics VariantFind Site-directed Mutagenesis, compared with epPCR, highlight its unique capabilities. Specifically, VariantFind demonstrates the ability to distribute multiple mutations along an entire open reading frame without any restrictions on spatial separation between mutated codons.

Ranomics excels in synthesizing diverse mutagenesis libraries, ranging from degenerate NNK to codon-optimized versions, effectively addressing low diversity challenges. For a comprehensive understanding of VariantFind's capabilities, we invite you to download our informative brochure.

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