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Drug Development Expertise Empowering Research Services for Biologics

Lead Optimization

Comprehensive Antibody Engineering Platforms for Humanization, Affinity Maturation, and Fc Engineering

Lead optimization represents a critical phase in biologics discovery. We provide comprehensive lead optimization services for antibody leads, designed to address key challenges early in drug discovery stage.

 

Our expertise encompasses a wide range of biologics, including monoclonal antibodies (mAbs), bispecific antibodies (bsAbs), and multispecific antibodies (msAbs), peptides, enzymes, soluble T-cell receptors (sTCRs), cytokines, and other complex biomolecules. Our scientists also work in close collaboration with clients to improve lead developability, binding affinity, stability, and manufacturability, ensuring every candidate is fully optimized for preclinical development and beyond.

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Antibody therapeutics optimization including humanization, affinity maturation, and Fc engineering.

Antibody Humanization: Optimal, Structure-Based CDR Grafting

WuXi Biologics offers antibody humanization services utilizing best-fit CDR grafting guided by advanced structural modeling techniques. With a success rate exceeding 98% across more than 200 completed projects, our antibody humanization services cover a wide range of non-human antibodies for improved developability, including those derived from mouse, rat, hamster, rabbit, and chicken, as well as VHH single-domain antibodies from llama and alpaca.

Antibody Humanization Service Details:

Service Item

Description

Turnaround Time

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Humanization

1. In silico CDR grafting

2. 3D model-guided design of back mutations

3. Humanized variants production

4. Affinity measurement (SPR/FACS) and lead selection

5. Optional: Developability assessment

4-6 weeks

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Humanization with PTM Risk Removal

1. In silico CDR grafting

2. 3D model-guided design of back mutations and PTM site mutations

3. Humanized & PTM-removal variants production

4. Affinity measurement (SPR/FACS) and lead selection

5. Combined lead production and affinity validation (SPR/FACS)

8-10 weeks

Case Study #1: Enhanced Binding Affinity and Developability Through Antibody Humanization

This case study highlights the effectiveness of our antibody humanization strategy in reducing immunogenicity risks while simultaneously maintaining or improving binding affinity and key developability attributes, such as stability, expression, and manufacturability.

Case study on antibody humanization process to minimize immunogenicity risks while improving binding affinity and key developability attributes.

Figure A: The results demonstrate a median 1.4-fold increase in antigen-binding affinity. Key developability metrics, including Protein A titer in HEK293 cells, thermal stability assessed by differential scanning fluorimetry (DSF), and T20 scores, further confirm the high quality and reliability of the humanization process.

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Affinity Maturation: Comprehensive Paratope Mapping to Identify Mutagenesis Hotspots

WuXi Biologics provides customized antibody affinity maturation services utilizing our validated parsimonious mutagenesis methods for each antigen-antibody pair with multiformat screening assays (ELISA, SPR, FACS). We identify critical mutagenesis hotspots and generate focused mutant libraries. This targeted approach enables efficient isolation of antibody variants with improved binding affinity and specificity.

Key Features of Our Affinity Optimization Services:

  • 1.5-4 months turnaround time
  • KD enhancement ranging from 10- to 10,000-fold, achieving pM levels
  • Minimized risks in epitope shifts, PTMs, poly-reactivity, and aggregation
  • pH dependency by parallel screening

Affinity Maturation Service Details:

Service Item

Description

Turnaround Time

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Antibody Paratope Mapping—Parsimonious Mutagenesis

1. Antibody reformatting and screening assay setup (ELISA)

2. Saturated mutagenesis screening of all CDR positions (Optional: Screening at two different conditions)

3. Hits ranking (ELISA/FACS) and combinatorial library design

4. Library screening and hits ranking (ELISA/FACS)

5. IgG reformatting and affinity ranking (SPR/FACS)

6. Optional: Developability screening of combinatorials hits

3-5 months

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Block Mutagenesis

1. Antibody reformatting and screening assay setup (FACS)

2. Block mutagenesis library design and construction

3. Block mutagenesis library panning and screening (FACS)

4. Hit ranking (FACS) and block combination design

5. IgG reformatting and affinity ranking (FACS).

6. Optional: Developability screening of combinatorials hits

3-5 months

pH-Dependency Engineering

1. Antibody reformatting and screening assay setup (ELISA at two pH conditions)

2. His-scanning library design and construction

3. pH-dependency screening (ELISA/FACS)

4. Hit ranking (ELISA/FACS) and mutant combination design

5. IgG reformatting and pH-dependency ranking (ELISA/SPR/FACS)

2-3 months

Parsimonious Mutagenesis Affinity Maturation Platform

WuXi Biologics parsimonious mutagenesis affinity maturation platform uses CDR mutagenesis, ELISA/FACS screening, and sequence validation.

Our parsimonious mutagenesis platform enables a 10- to 100-fold improvement in Koff  while minimizing the risk of epitope shift. This targeted approach also supports the elimination of liabilities (PTM, aggregation, and poly-reactivity) and facilitates the engineering of pH-sensitive binding profiles.

Case Study #1: Achieving a 456-Fold Increase in Antibody Affinity Through Parsimonious Mutagenesis

This case study illustrates the power of our targeted parsimonious mutagenesis approach, which enabled a 456-fold improvement in antibody binding affinity through precise identification and optimization of key paratope residues.

Case study on achieving a 456-fold increase in antibody binding affinity through a targeted parsimonious mutagenesis approach.

Figure A: The parental antibody exhibited a baseline binding affinity with a KD of 6.66 × 10−8 M. After optimization via parsimonious mutagenesis. The KD was improved to 1.46×10−10 M, representing a 456-fold increase in binding affinity.

Block Mutagenesis Affinity Maturation Platform for Cell-Based Panning and Screening

Block mutagenesis for high-affinity antibody maturation, enabling affinity-based panning/selection for cell surface targets.

Our block mutagenesis platform enables efficient affinity maturation through the construction of mutant libraries by randomizing overlapping five-amino-acid blocks across each CDR. With library sizes reaching up to 10⁸ variants, this approach supports affinity-based selection against cell surface targets via competitive, cell-based panning and screening, facilitating the identification of high-affinity antibody leads.

High-Throughput (HTP) pH Engineering Platform

High-throughput pH engineering platform improves pH-dependent binding, target selectivity, and in vivo pharmacokinetic profiles.

This platform enhances pH-dependent binding, thereby improving antibody-target selectivity and optimizing the in vivo pharmacokinetic (PK) profile.

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Fc Engineering: Strategic Design of Multiple Mutation Sets to Modulate Fc Effector Function

The Fc region is critical to antibody function, contributing not only to structural stability but also to immune effector activities, such as ADCC, CDC, ADCP, and antibody recycling. At WuXi Biologics, our Fc engineering platform employs rationally designed mutation sets to modulate Fc functions. This platform integrates comprehensive in vitro functional assays with in vivo PKPD studies, providing an end-to-end solution that reduces risk and enhances developability attributes across diverse therapeutic formats.

Key Features of Fc Engineering Services:

  • Customize Fc function to modulate Fc effector activity and half-life, support bispecific assembly, and enable ADC conjugation
  • Support a range of modalities, including monoclonal antibodies (mAbs), bispecific antibodies (bsAbs), ADCs, and fusion proteins
  • Comprehensive in vitro assays, such as Fc receptor binding (SPR), ADCC, CDC, and ADCP, combined with in vivo PKPD studies

Fc Engineering Service Details:

 

Service Items

Turnaround Time

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Fc Mutations Design and Production

3-4 weeks

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SPR Binding Characterization

1-2 weeks

In Vitro ADCC/CDC/ADCP Assays

2 weeks

Mouse PK

6-7 weeks

Rat PK

6-7 weeks

NHP PK

7-8 weeks

Case Study #1: Optimizing Fc Engineering for Enhanced Antibody Therapeutics

This case study highlights key engineering sites within the Fc region, categorized based on their functional impact: effector function modulation (red), half-life (blue), and Fc dimerization (green). These modifications could offer tailored solutions to required Fc functions in both therapeutic efficacy and pharmacokinetic properties.

Fc engineering for antibody therapeutics, highlighting key engineering sites for effector function modulation, half-life, and Fc dimerization to improve efficacy and pharmacokinetics.

 

Figure A: Fc engineering sites for Fc effector function (red), half-life (blue) and Fc dimerization (green).

Case Study #2: Modulating ADCC Effector Function via Fc Mutations

This study evaluated Fc-engineered IgG1 antibodies for enhanced or reduced ADCC function by measuring their binding affinity to human FcγRIIIa (F158 and V158) through SPR analysis. Mutations such as DE and DLE led to a significant increase in receptor binding, while LALA and N297A weakened interactions, highlighting their diverse roles in modulating ADCC effector function.

Table A: Binding affinities of Fc-engineered IgG1 variants to human FcγRIIIa (F158 and V158)

 

Ligand Analyte KD(M) ADCC effect
Human FcγRIIIa (V158) Wild type IgG1 antibody  7.55 × 10⁻⁷ +
IgG1 antibody with DE mutation 2.20 × 10⁻⁸ ++
IgG1 antibody with DLE mutation  1.56 × 10⁻⁸ ++
hIgG1 antibody with LALA mutation 2.89 × 10⁻⁵ –
hIgG1 antibody with N297A mutation No or weak binding –
Human FcγRIIIa (F158) Wild type IgG1 antibody  1.54 × 10⁻⁷ +
IgG1 antibody with DE mutation 9.54 × 10⁻⁹ ++
IgG1 antibody with DLE mutation  7.89 × 10⁻⁹ ++
hIgG1 antibody with LALA mutation 5.48 × 10⁻⁶ –
hIgG1 antibody with N297A mutation No or weak binding –

SPR binding curves of engineered IgG1 Fc variants showing differential FcγRIIIa binding affinities across wild-type, DE, DLE, LALA, and N297A mutations.

 

 

Figure A: SPR analysis of Fc-mutated IgG1 antibodies demonstrated that ADCC-enhancing mutations (DE, DLE) result in stronger binding to FcγRIIIa, while LALA and N297A mutations significantly reduce or eliminate receptor binding.

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Frequently Asked Questions for Lead Optimization

Q: Do you construct bispecific antibodies first before humanization?

A: Yes. Since humanization involves replacing the animal framework regions, it is a significant sequence modification. After humanization, we conduct binding analysis (e.g., SPR) and functional assays, along with Micro Developability assessments, as the changes can affect both function and biophysical properties.

Q: How does humanization improve the safety of therapeutic antibodies?

A: Humanization involves modifying non-human antibodies for improved developability and immunogenicity, including those derived from mouse, rat, hamster, rabbit, and chicken, as well as VHH single-domain antibodies from llama and alpaca. This process increases the likelihood of success in preclinical and clinical studies.

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