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Related Experiment Videos

Antibody engineering by parsimonious mutagenesis

R F Balint1, J W Larrick

  • 1Palo Alto Institute for Molecular Medicine, Mountain View, CA 94043.

Gene
|December 27, 1993
PubMed
Summary

Researchers developed parsimonious mutagenesis (PM) to enhance human monoclonal antibody (humAb) affinity for therapeutic use. This computer-assisted method efficiently identifies improved antibody variants by scanning antibody gene regions.

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Area of Science:

  • Biotechnology
  • Immunology
  • Molecular Biology

Background:

  • Human monoclonal antibody (humAb) development has advanced with techniques like polymerase chain reaction (PCR) and phage display.
  • Phage display enables recovery of humAbs with moderate affinities from non-immunized human B-cell repertoires.
  • Achieving therapeutic-grade affinities (> or = 10(9) M-1) requires effective in vitro affinity maturation.

Purpose of the Study:

  • To develop a method for efficient in vitro affinity maturation of human monoclonal antibodies.
  • To identify strategies for enhancing antibody-antigen interactions to achieve higher binding affinities.
  • To create a tool for improving antibody therapeutic potential by increasing affinity and reducing cross-reactivity.

Main Methods:

  • Developed parsimonious mutagenesis (PM), a computer-assisted oligodeoxyribonucleotide-directed scanning mutagenesis technique.
  • PM simultaneously searches all three complementarity-determining regions (CDRs) of a variable region (V-region) gene.
  • Utilized low-redundancy 'doping' codons and biased nucleotide mixtures to create manageable libraries for identifying high-affinity variants.

Main Results:

  • PM allows thorough searching of V-region genes for improved antibody variants in manageable library sizes.
  • The method enables probing antigen surfaces with diverse amino acid side chains to discover new high-affinity contacts.
  • Demonstrated potential for affinity maturation, removal of unwanted cross-reactivities, and reshaping of rodent mAbs.

Conclusions:

  • Parsimonious mutagenesis is an effective computer-assisted method for in vitro antibody affinity maturation.
  • PM facilitates the identification of novel high-affinity antibody-antigen interactions.
  • This technique holds promise for developing improved human monoclonal antibodies for therapeutic applications.

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