Explore the
Ultimate Humanization® Platform

By combining the best of existing in vivo and in vitro technologies, the Ultimate Humanization® technology allows the generation of lead-drug antibodies with high affinity, low predicted cross-reactivity in humans (“off-target recognition”) and outstanding low immunogenicity. As a result, toxicity risks and therapeutic effects are optimized early during therapeutic antibody development which positively contributes to preclinical and clinical sucess.

BIOTEM Ultimate Humanization® Platform takes advantage of:

  • Non Human Primate (NHP) Immunization: Generation of high affinity antibodies with strong homology to their human counterparts.
  • Hyper-Immune Library: Construction of highly representative libraries of NHP immune response (or derived from human donors, when available) with large epitope coverage.
  • Phage Display Screening: High throughput screening based on predefined criteria (affinity, recognition of a particular epitope, stability, etc.).
  • Antibody Germinalization: Facilitated and extensive germinalization of candidate antibodies in FR and CDR regions thanks to innate high Germinality Index (see box).
  • Sequence Optimization: Improvement of physicochemical properties, codon optimization and maturation of antibody affinity & activity to potentiate drugability and anticipate antibody manufacturing.


The Germinality Index (GI) quantifies the identity of an antibody variable regions with its most homologous human germinal sequences. Human antibodies encoded by germline genes (IgM, GI = 100%) form the true immunological self and are usually perfectly tolerated. Mature human antibodies (IgG) underwent affinity maturation, leading to antibodies with higher affinity and specificity exhibiting a GI comprised between 88 and 96 %.

During antibody engineering non-germline mutations may be introduced to optimize antibody sequence, thereby lowering antibody GI. This is a potential risk since it has been observed that engineered antibodies with low GI (< 88 %) are are often associated with severe side effects.

In order to circumvent such tolerance issues, BIOTEM guarantees a minimum GI of 92% (typically>95 %) while retaining parental affinity.

Species GI* Germinalization
Human IgG 88 - 96 % Facilitated & extensive(FR & CDR)
PNHs 80 - 90 %
Lama 74 - 82 % Medium & Case dependent
Mouse 70 - 80 % Difficult & Partial(FR only)


Immunization &
Library Construction

Immunization Strategy

With more than 30 years of expertise, BIOTEM offers a large panel of immunization strategies enabling to generate immune responses against particular epitopes or challenging antigens (conserved proteins, haptens, small modifications,…). After an in depth analysis of client’s specifications, BIOTEM will define the best immunization strategies.

NHP Immunization

Active immunization of outbred NHPs elicits strong and natural immune responses compared to inbred or transgenic animals in which the immune system is often impaired. BIOTEM’s team benefits from a long experience in NHP immunization and isolation of its antibody-secreting cells (B-cells) which requires strong technical know-how.

  • BIOTEM’s added value
  • Long-lasting experience in NHP immunization
  • Optimized strategies for immunization with phylogenitically conserved targets
  • Animal immunization requiring BSL-2 or BSL-3 (Biosafety level) conditions

Immune Library Generation

For each project, BIOTEM builds a tailored and highly representative hyper-immune library which allows obtaining:

  • High affinity antibodies: 10 to 1000-fold superior affinities compared to antibodies usually obtained from naive or synthetic libraries.
  • Large epitope coverage: with possible targeting of specific antigen epitopes.
  • Antibodies with low predicted toxicity: Specific NHP immunization strategies allows generating immune responses strongly focused on the therapeutic targets and not against NHP self-antigens (thanks to well-known tolerance mechanisms). Since NHP and human proteins are highly homologous (>95% identity on average), the risk of cross-reactivity between NHP antibodies and human antigens is very low.


Phage Display &
ScFv Characterization

Phage Display Screening

Screening by phage display aims at isolating antibodies of interest by an enrichment and high throughput process, called biopanning (see figure on the left)

Depending on project specifications and the nature of immune response, BIOTEM will implement the most adapted screening strategy: direct, competitive or subtractive.

The isolated scFv candidates from the phage display screening are selected and sequenced before being produced and characterized.

Hence, the best candidates are identified thanks to:

  • The Germinality Index
  • Antibody affinity
  • Biological activity
  • Biophysical characterization
  • Expression yield


Germinalization &
Optimization

Antibody Germinalization

Unlike germinalization of non-NHP antibodies, BIOTEM Ultimate Humanization® technology allows FR and CDR germinalization. Therapeutic antibodies generated through this technology exhibit therefore exceptionally high GI (>95%) while retaining their parental affinity and specificity. Most notably, extensive humanization is accomplished through germinalisation of CDR regions which are often predicted to be highly immunogenic but essential for antigen recognition.

Sequence Optimization

To potentiate drugability and anticipate antibody manufacturing, BIOTEM services include state of the art sequence optimization.

  • Improvement of physiochemical properties: Specific amino acid substitutions will be proposed in order to avoid post-translational modifications susceptible to affect antibody production, stability, solubility, aggregation, heterogeneity etc.
  • Antibody maturation: Depending on specifications, in vitro affinity maturation and/or optimization of biological functions will be proposed.
  • Codon optimization: Antibody coding sequence will be optimized to increase expression level in client’s expression system.

Bioinformatic analysis

Identification of potential residues on VL and VH to be mutated for:

  • Extensive germinalization (FR and CDR)
  • Sequence optimization

In vitro evaluation

In vitro evaluation

  • Variant production by site directed mutagenesis
  • Affinity determination

Integration of validated mutations

  • Preserved parental affinity
  • Guaranteed GI > 92 % (typically ≈ 95 %)
  • Optimized manufacturability


Reformatting &
Production

Depending on project specifications, BIOTEM offers different options for lead-candidate production in mammalian cells (before and/or after antibody germinalization).

  • ScFv- or full immunoglobulin-format (various isotypes available)
  • Small and large scale production batch
  • Custom purification protocols
  • Endotoxin-free preparation
  • BIOTEM’s Quality Control
  • A comprehensive set of quality controls are available on lead antibodies:
  • Purity analysis
  • Affinity constant determination
  • Endotoxin analysis
  • Aggregation and oligomerization (AUC, DLS & SEC-HPLC)
  • Stability (DSC & Circular Dichroism)
  • Other quality controls upon request