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Antibody Humanization Strategy

Left panel illustrates the strategy to graft CDRs from mouse antibody into human antibody. The framework of human antibody carries degenerate amino acid sequences allowing multiple variations at the hypermutated site (bold font). Right panel shows the de novo alignment of the heavy chain Fv domain in human antibody from germline expression.

Yeast Display System for Affinity Maturation

Left panel illustrates the yeast display system that features a fusion protein presenting the scFv domain of humanized antibody on the surface of yeast cell. HA tag and Myc tag allow validation of scFv expression. Right panel shows the antigen binding data of yeast cells. Antigen is labeled with FITC and bound with yeast cells. P4 region in top image is shown at the bottom.

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Antibody Humanization & Maturation

$18,950.00

Item Cat No.: BCHUMA

Antibody: Monoclonal Antibody

Concentration: 0.25 mg/ml purified IgG

Application: In Vitro and In Vivo

Reactivity: Human, Mouse, Rat

BiCell Scientific’s antibody humanization & maturation service utilizes a unique and proprietary yeast display platform to experimentally select from a database of mature human frameworks to generate safe and effective humanized antibodies while maintaining their binding affinities.

Humanized antibodies are antibodies from non-human species whose amino acid sequences have been modified to increase their similarity to antibodies naturally produced in human.

The first generation of humanization refers to the creation of a mouse-human antibody chimera, in which the entire Fv domains of both heavy chain and light chain of a mouse antibody are grafted into a human antibody. Such a strategy of humanization is incomplete – because the majority of amino acid sequences in the Fv domain does not participate in the antibody-antigen interaction, these sequences, if carried over from mouse or other species, may still trigger a strong immune response in human.

The second generation of humanization refers to the grafting of the complementarity-determining regions (CDRs) within the Fv domain of a mouse antibody to a human antibody, which are responsible for the ability of the antibody to bind to its antigen. Such a strategy of humanization offers greatly improved safety. Nevertheless, the xenogenic grafting of mouse CDRs to human framework alters the overall 3D structure of the Fv domain, causing the humanized antibodies to either structurally collapse or significantly lose their binding affinities.

BiCell Scientific’s antibody humanization & maturation service utilizes a greater diversity of human antibody framework sequences that are from both human genome (germline) and single B cell RNA-seq database. BiCell Scientific Inc has developed a unique and proprietary yeast display platform to incorporate the framework sequence variations and experimentally select the best human framework that is able to seamlessly fuse with the grafted CDRs from mouse or other species.

  • Molecular cloning of CDRs (CDR1, CDR2, and CDR3) from IgG heavy chain and light chain of mouse or other species into yeast display library vector pYSD series, expressing the scFv form of fused Fv domains from heavy chain and light chain.
  • Transform pYSD libraries into EBY100 YSD yeast strain.
  • Harvest yeast cells with induced expression of scFv. Perform antigen based enrichment with magnetic bead sorting.
  • Recover sorted yeast cells and plate them to form single colonies.
  • Clone the scFv gene sequences from recovered yeast cells into lentiviral expression vector.
  • Transfection of HEK293T cells with lentiviral expression vectors as well as vectors expressing gag–pol, rev and vsv–g
  • Harvesting and purification of lentivirus from cell culture medium
  • Transducing naive Sp2/0-Ag14 hybridoma cells with lentivirus encoding humanized IgG
  • Clonal selection and stable cell line establishment in 96-well plate
  • Validation of humanized IgG antibody expression in 96-well plate against antigen and selection of the highest expressing clone
  • Affinity chromatography purification with protein A column

Customers will receive the following deliverables at the end of each project:

  • Fully sequenced Fv domains of heavy chain and light chain of the selected humanized antibody
  • One stable Sp2/0-Ag14 hybridoma cell line expressing humanized monoclonal antibody with the highest yield
  • 1 mg of affinity purified humanized monoclonal antibody
  • Molecular cloning of CDRs (CDR1, CDR2, and CDR3) from IgG heavy chain and light chain of mouse or other species into yeast display library vector – 2-3 weeks
  • Yeast cell transformation and expression validation  – 2-3 weeks
  • Yeast display and affinity maturation – 3-4 weeks
  • Cloning and sequencing of affinity selected scFv genes from yeast – 2-3 weeks
  • Assembling humanized IgG in lentiviral expression system – 2-3 weeks
  • Naive Sp2/0-Ag14 hybridoma cell transduction – 1-2 weeks
  • Clonal selection and stable cell line establishment – 3-4 weeks
  • Humanized antibody expression validation – 2-3 weeks
  • Cell culture expansion and affinity purification – 2-3 weeks

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"The assay is a homophilic interaction mediated cell adhesion on purified protein (in this case, immobilized purified Pcdhga9 to Pcdhga9 expressed on cell surface). Compared to control, cell adhesion is reduced in the presence of Pcdhga9 monoclonal antibody supernatants!"

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I have tested the rat polyclonal IgGs to ABCD1 by immunoflourescence on cells overexpressing ABCD1. The antibodies successfully detected the protein (either untagged or tagged with GFP) at a 1:500 dilution and there was little background. The antibodies did not detect ABCD2. So this is very good news, and you may now go ahead and clone out a monoclonal!

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