Lead Engineering

For the development of therapeutic antibodies, optimization of the identified Hits is recommended to generate early lead candidates. Two in-vitro evolution technologies are available for the optimization of the antibody’s affinity, stability, specificity or selectivity:

  • in-vitro evolution by YUlight shuffling
  • in-vitro evolution by mutagenesis

in-vitro evolution by YUlight shuffling:

Goal: Optimization of individual antibody Hits with promising binding characteristics with regards to affinity, specificity, selectivity or stability.

Principal: For most antibodies the VH domain is predominantly responsible for the antigen binding. However, the VL domain stabilizes the interaction and therefore, a perfect VH:VL pairing is most often needed to achieve the best antibody binding and overall biochemical properties.

Technology: The VH domain of antibody Hits (or small pools thereof) is isolated and shuffled with the full YUMAB library VL repertoire. The new VH:VL combinations are used to generate a new antibody sublibrary (>108 additional VL diversity) with predefined antigen specificity. Compared to the initial Hit discovery, this library can be used for in-vitro selection under more stringent conditions to identify antibodies with improved characteristics.

Advantage: The introduced diversity is based on fully human, nature-derived sequences. Major class-switches can occur.

Disadvantage: If the early antibody lead candidate contains a potent VH:VL combination, VL germline switches will be limited. In these cases, the VL germline will remain identical and differences are mainly restricted to the LCDR3.

Application: Improvement of tool antibodies and potential therapeutic candidates (internally generated or external antibodies).

The antibody Lead Engineering by YUlight shuffling includes the following modules that can be combined individually:

in-vitro-evolution-by-YUlight-shuffling

Generation of YUlight shuffling library

  • Isolation of VH of antibody Hits
  • Shuffling with the YUMAB library VL repertoire and library cloning (>108 additional VL diversity)
  • Antibody-phage packaging and QC

Discovery of optimized antibodies by in-vitro selection under highly stringent conditions to improve:

  • Affinity
  • Stability
  • Specificity
  • Selectivity

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

  • DNA sequencing of antigen specific antibodies and bioinformatic 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 reformatting and production

  • Reformatting into a mono- or bivalent antibody format (e.g. scFv, Fab, scFv-Fc, IgG; each format with different isoforms, Fc species or tags available).
  • Production by transient transfection of mammalian cells (typically HEK293, CHO on request)
  • Up to 100 mg for selected lead candidate(s)v
  • Low endotoxin production available (< 1 EU/mg)
  • Stable cell line generation with external partner

Validation and analytics

  • ELISA
  • Flow cytometry
  • Affinity measurement (Kinexa, BLI)
  • Gel filtration/Size exclusion chromatography (with/without stress conditions)
  • Immunofluorescence
  • Immunohistochemistry
  • Functional bioassays (assay dependent, on request)

In-vitro evolution by mutagenesis

Goal: Optimization of individual antibody Hits that show promising binding characteristics with regards to affinity, specificity, selectivity or stability.

Principal: In the last years it was more and more recognized that the antibody binding is not necessarily determined solely by the CDRs but also by the framework regions. Mutations in the frameworks may lead to a shifted CDR orientation which impacts the antibody binding. Furthermore, framework mutations may lead to a better binding by decreasing the entropy. Similarly, mutations in the framework regions also affect the antibody’s overall biochemical properties like stability, charge and hydrophobicity.

Technology: The VH:VL domain of antibody Hits (or small pools thereof) are isolated and randomly mutated by error-prone PCR. Typically, a low (0-2 aa substitutions), medium (3-5 aa substitutions) and high mutation rate (5-7 aa substitutions) are targeted. The mutated VH:VL combinations are used to generate a new antibody sublibrary (>108 additional diversity) with predefined antigen specificity. Compared to the initial Hit discovery, this library can be used for in-vitro selection under more stringent conditions to identify antibodies with improved characteristics.

Advantage: The introduced diversity is equally distributed and occurs in both, CDR and framework regions.

Disadvantage: The introduced mutations are not nature-derived and might lead to immunogenic neoepitopes.

Application: Improvement of tool antibodies and potential therapeutic candidates (internally generated or external antibodies).

The antibody Lead Engineering by mutagenesis includes the following modules that can be combined individually:

in-vitro-evolution-by-mutagenesis

Generation of mutation library

  • Isolation of VH:VL of antibody Hits
  • Mutation of VH:VL by error-prone PCR at different mutation rates (low, medium, high) and library cloning (>108 additional diversity)
  • Antibody-phage packaging and QC

Discovery of optimized antibodies by in-vitro selection under highly stringent conditions to improve:

  • Affinity
  • Stability
  • Specificity
  • Selectivity

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

  • DNA sequencing of antigen specific antibodies & bioinformatic 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 reformatting and production

  • Reformatting into the mono- or bivalent antibody format (e.g. scFv, Fab, scFv-Fc, IgG; each format with different isoforms, Fc species or tags available).
  • Production by transient transfection of mammalian cells (typically HEK293, CHO on request)
  • Up to 100 mg for selected lead candidate(s)
  • Low endotoxin production available (< 1 EU/mg)
  • Stable cell line generation with external partner

Validation and analytics

  • ELISA
  • Flow cytometry
  • Affinity measurement (Kinexa, BLI)
  • Gel filtration/Size exclusion chromatography (with/without stress conditions)
  • Immunofluorescence
  • Immunohistochemistry
  • Functional bioassays (assay dependent, on request)

YUMAB® Hit Optimization
– YUlight shuffling and/or mutagenesis increases the diversity of antibody Hits
– Identification of improved antibodies by highly stringent in-vitro selection
– Optimization of affintiy, stability, specificity or selectivity

Contact

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

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