Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation

Abstract

Background: Humanization of murine monoclonal antibodies by CDR grafting is a widely used technique. Results: In vitro somatic hypermutation was coupled with minimal CDR grafting to produce potent, pM affinity antibodies. Conclusion: This methodology can rapidly generate potent, humanized antibodies containing a minimum of donor sequence. Significance: Antibodies produced using this approach contain reduced rodent antibody donor content and possess potential advantages in manufacturability and immunogenicity.

Generation of a high-fidelity antibody against nerve growth factor using library scanning mutagenesis and validation with structures of the initial and optimized Fab-antigen complexes

“…SHM mutations are directed based on in vivo sequence and phenotypic observations using flow cytometry and mammalian cell culture. 28 By comparison, LSM is likely to be of general applicability as a low-cost, minimal technology affinity maturation of antibodies that recognize a subset of highly homologous protein families, such as the fibroblast growth factors (FGFs), because it maintains fidelity of the binding epitope. Broader libraries, for example, risk the possibility of PCR errors, or dramatic epitope drifts in the individual CDR sequences or results in loss of a synergistic relationship between variable domain framework pairings.…”

“…34 Other methods have used lower homology antibody frameworks with CDR grafting in combination with a softer mutagenesis technique called In Vitro Somatic Hypermutation. 28 Bowers and coworkers used this sophisticated and challenging method requiring mammalian HEK293 cells expressing activation-induced cytidine deaminase (AID) resulting in what is called somatic hypermutation mutagenesis (SHM). SHM mutations are directed based on in vivo sequence and phenotypic observations using flow cytometry and mammalian cell culture.…”

“…Following this, a query of the antibody IMGT database allowed creation of an ad hoc alignment to guide the selection of minor mutations in CDRs 1 and 2 for further humanization, which is commonly performed. 28 This non-exhaustive preparatory step before LSM was completed in order to decrease the chance of immunogenicity and to improve the overall antibody structural fitness. For example, 911 L2 I51 was converted to a small amino acid in the human sequence alignment, where threonine was experimentally better than other small conserved residues, and this was retained in the optimized tanezumab.…”

“…Amino acid positions that were conserved in human CDRs were identified and replaced with the most commonly occurring amino acids at those few positions, and amino acids likely to be used during in vivo affinity maturation were left alone based on alignment of the selected germline with Igblast. 25,28 These criteria were determined by taking the human germline frameworks selected and using them to query the IMGT database and protein data bank with Igblast to create a non-exhaustive framework alignment from the database. Focusing in CDRs, positions that were not conserved in the H1, H2, L1 and L2 CDRs across the alignment were identified; these are likely optimized for epitope recognition during affinity maturation.…”

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody

“…21 This method can also be combined with affinity optimization using somatic hypermutation. 22 Finally, immunologically relevant antibody humanness, or human string content, is also a method that has been used to select humanized antibodies. 23 Here, framework segments are selected from multiple germlines to make the overall variable region more humanlike.…”