Antibody Humanization

The YUMAB® humanization platform provides two technologies for the humanization of animal-derived antibodies of any source (B-cells, Hybridoma cultures, antibody sequences) by:

  • Germline-optimized CDR grafting
  • Template-driven chain shuffling

The humanized antibodies can be directly used for further optimization by Lead Discovery and Lead Development.

Antibody-Humanization

Germline optimized CDR grafting:

Goal: Reduction of non-human sequences to a minimum and preservation of the binding properties of the parental antibody.

Principal: Yumab’s proprietary in-silico humanization tool identifies non-human amino acids in the VH and VL framework regions and replaces those with the human counterparts. Usually, for each domain different degrees of humanization are aimed (low, medium and high). Simultaneously, unwanted motifs (PTM, hydrophobic patches) can be removed. The optimized VH and VL sequence variants are combined individually with each other and produced in the final antibody format by mammalian cells. To identity functional antibodies with highest humanness, the antibody binding characteristics and biochemical properties are measured and correlated with the human germline identity.

Advantage: Preserves parental CDRs

Disadvantage: Potential CDR related IP issues with humanized version

The germline optimized CDR grafting includes the following modules that can be combined individually.

Germline-optimized-CDR-grafting

Antibody sequence identification

  • Different sources (B-cells, Hybridoma)
  • RNA isolation, cDNA synthesis and sequencing

Sequence analysis & optimization

  • Identification of framework and CDR sequences
  • Prediction of optimal human germlines
  • Replacement of non-human amino acids in the framework by human amino acids (with different degree of humanness)
  • in-silico sequence optimization to remove undesired patches and motifs
  • Codon-optimization

Antibody production

  • Combination of each optimized VH sequence variant with each optimized VL sequence variant (including non-optimized variants)
  • Production by transient transfection of mammalian cells (typically HEK293, CHO on request)

Antibody bioanalytics

  • Measurement of antibody binding properties and biochemical characteristics

ELISA

Flow cytometry

Affinity measurement (Kinexa, BLI)

Gelfiltration/Size exclusion chromatography (with/without stress conditions)

Immunofluorescence

Immunohistochemistry

Functional bioassays (assay dependent, on request)

Lead selection:

  • Correlation of binding/biochemical characteristics with human germline identity (and comparison to parental antibody)

Template driven chain shuffling

Goal: Based on the animal-derived antibody, an entirely novel, fully human antibody with similar or improved characteristics is generated.

Principle:

In a first step, the non-human VH domain is cloned into YUMAB® library vectors containing the full human VL repertoire. Vice versa, the non-human VL domain is cloned into YUMAB® library vectors that contain the full human VH repertoire. Since only one chain is replaced at a time, most antibodies from these hybrid libraries bind to the same or a similar epitope as the non-human antibody template. Subsequently, hybrid antibodies against the target structure are selected in vitro.

In a second step, the human VH and VL domains of the selected antibodies are isolated and shuffled with each other, generating a new library of fully human antibodies. Again, most antibodies bind to the same or similar epitope as the non-human template. After a final antibody discovery against the target structure, the fully human antibodies are sequence analyzed, functionally tested and compared to the non-human antibody.

Advantage:

  • novel, fully human antibody sequences (no CDR related IP issues)
  • same or similar epitope
  • different or improved functional properties.

The template driven chain shuffling includes the following modules that can be individually combined:

Template-driven-chain-shuffling

Antibody sequence identification

  • Different sources (B-cells, Hybridoma)
  • RNA isolation, cDNA synthesis and sequencing

Antibody library generation

  • Cloning of non-human VH with fully human VL library repertoire
  • Cloning of non-human VL with fully human VH library repertoire

Antibody discovery I

  • In vitro antibody selection:
    • on surface immobilized antigens
    • on antigens in solution
    • on whole cells
  • Control of binding conditions:
    • pH, salt, or buffer conditions
    • competitions with ligand or other antibodies for epitope specific antibodies
    • negative selection with isoform or homologous antigen to avoid cross-reactivity
    • overlapping panning with antigen of different species for interspecies cross-reactivity

Shuffling of antibody libraries

  • Isolation and shuffling of human VH and VL from output (Antibody discovery I)

Antibody discovery II

  • in-vitro antibody selection:
    • on surface immobilized antigens
    • on antigens in solution
    • on whole cells
  • Control of binding conditions:
    • pH, salt, or buffer conditions
    • competitions with ligand or other antibodies for epitope specific antibodies
    • negative selection with isoform or homologous antigen to avoid cross-reactivity
    • overlapping panning with antigen of different species for interspecies cross-reactivity
  • Antibody screening
    • Production of soluble antibodies instead of antibody-phage particles
    • ELISA on up to 4 different antigens in a single run
    • Flow cytometry on cells (up to 3 cell lines in a single run)
    • Off-rate ranking of antibodies (BLI)

Antibody sequence analysis

  • Framework and CDR analysis and annotation
  • Identification of potential PTMs in CDRs
  • Modelling of biochemical properties and developability ranking (e.g. surface and CDR hydrophobicity/charge, pI, germinality index, unusual amino acid residues)

Antibody production

  • Conversion of antibodies into final format
  • Production by transient transfection of mammalian cells (typically HEK293, CHO on request)

Antibody bioanalytics

  • Measurement of antibody binding properties and biochemical characteristics
    • ELISA
    • Flow cytometry
    • Affinity measurement (Kinexa, BLI)
    • Gelfiltration/Size exclusion chromatography (with/without stress conditions)
    • Immunofluorescence
    • Immunohistochemistry
    • Functional bioassays (assay dependent, on request)
  • Lead selection: Correlation of binding/biochemical characteristics with human germline identity (and comparison to parental antibody)

Contact

YUMAB GMBH
Science Campus Braunschweig-Süd
Inhoffenstr. 7
38124 Braunschweig – Germany

Email: info@yumab.com
Phone: +49 531 481170-0