Antibody Discovery & Optimization Platforms

Robust antibody discovery (Hybridoma, Single B and Display) and optimization platforms to accelerate the identification of diverse binders

One-Stop Antibody Discovery & Optimization Platforms for Accelerated Biologic Drug Discovery

Donghui Wu, Jieying Liu, Jiansheng Wu WuXi Biologics, NO.240 Hedan Road, Pudong New District, Shanghai, China (Contact: PS_BD@wuxibiologics.com)

Abstract Hundreds of antibodies have been approved for clinics use in treating cancer, inflammation, autoimmune and infectious diseases. As essential building blocks, identifying monoclonal antibody (mAb) binders with desired properties is critical for downstream applications, such as constructing bispecific antibodies (bsAbs), multispecific antibodies and antibody-drug conjugates (ADCs). However, identifying a diverse set of mAb binders remains challenging when selecting for desired properties. Moreover, some discovered lead antibodies may exhibit issues, such as insufficient affinity or containing potential post-translational modification (PTM) sites, necessitating further optimization steps like affinity maturation, PTM removal, and pH-dependent engineering. To address these challenges, WuXi Biologics has developed three robust antibody discovery platforms (Hybridoma, Single B and Display) to accelerate the identification of diverse binders. Phage/yeast display platforms also support antibody engineering such as affinity optimization and PTM removal.

Results Fig 5. Single B Technology Coupled with High-Throughput SPR Ainity Screening to Accelerate Discovery of Ultra-High Ainity Rabbit mAbs B Cell L oading IgG Detection 2 Cell-Based Binding Assays B Cell Export & BCR Sequencing

Engineered Cell

Tumor Cell

HTP Ainity Screening

Full Kinetics

Introduction

WuXi Biologics has developed state-of-the-art platforms for mAb discovery and optimization, including rodent hybridoma, phage/yeast display ( Fig 1 ) and rapid, all-species single B cell technology ( Fig 2 ). Leveraging these advanced platforms, over 450 antibody discovery projects ( Fig 3 ) and 50 antibody affinity maturation projects ( Fig 4 ) have been successfully delivered. Fig 1. High-Quality Phage/Yeast Display Library Construction for Enrichment of Specific and Diverse Binders via High-Throughput Panning and FACS Sorting

Phage/Yeast Display Library Design and Construction

Antibody-Induced Function Assay

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Top/Max Inhibition %

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Antibody

K D (M)

IC 50 (nM)

Lead.1D3

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112

-12

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Lead.

1C6

9E 13 - 3E-11 7E-12

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BMK1 BMK2

111 116

Amplification

Binding

6A/,1B:2C/+,D/E

Liquid-phase Cell-based panning Solid-phase Epitope-guided Function-guided

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Isotype control

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Panning (2-4 Rounds)

Sequencing/ Characterization

Fig 6. Identification of a Humanized Synthetic VHH Lead Molecule for ADC Application from WuXi Biologics’ Proprietary High-Quality VHH Phage Display Library

Elution/ FACS Sorting

Washing

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Fig 2. Rapid, All-Species Single B Cell Technology for Accelerated mAb Discovery

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Internalization EC 50 (nM)

Human target X binding FACS EC 50 (nM)

Cyno target X binding ELISA EC 50 (nM)

Yield (mg/L)

DSFT m 1

1.3

1.5

1.6

Benchmark Ab WuXi Bio lead

63.4 ℃

0.6

1.4

1.7

490

Fig 7. Ainity Enhancement of mAb Against Mouse Antigen to Support In Vivo Eicacy Studies in Mouse Models

Proprietary methods for plasma cell enrichment and memory B cell activation MACS or FACS Enrichment (1 day)

Unique PCR primers to enable high natural VH/VL pairwise rate across multiple species Single B Cell Sequencing (1 week)

Improvement validated by SPR

Lead

Candidate

MouseAg Candidate

k on (1/Ms)

k o ff (1/s)

K D (M)

Mouse antigen

A ff inity 4622 fold

Immunization (1-3 months) Multiple immunization strategies (protein/DNA/cell/ peptide/VLP/mRNA) to ensure strong immune response

Beacon Single B Cell Selection (1 day) Miniaturized, high-throughput, functional screenings to select optimal leads

5.87E-07 1.27E-10

Lead

9.62E+06

1.23E-03

Fig 3. Cutting-Edge Platforms to Expedite mAb Discovery

Human Ag Candidate

k on (1/Ms) 2.51E+07 3.31E+07

k o ff (1/s) 3.14E-03 6.82E-04

K D (M)

Human antigen

A ff inity 6 fold

1.25E-10 2.06E-11

Lead

Antibody Discovery Hybridoma Single B Native/Synthetic/ Immunization Library

After affinity maturation, the affinity for mouse antigen target improves 4622-fold, from micromolar to sub-nano - molar levels, reducing the affinity gap between human and mouse targets from 4696-fold to just 6-fold.

Camelid 100+

Human 50+

Conclusions

Acknowledgments We want to express our gratitude to the scientists in the CRO Services Department at WuXi Biologics for their invaluable support in the development of our proprietary lead identification and lead optimization plat- forms. Additionally, we extend our appreciation to WuXi Biologics' legal, public relations, and marketing de- partments for their assistance in patent filings and the preparation and production of written materials. WuXi Biologics has established powerful platforms for mAb lead identification and optimization. Three state-of-the-art antibody discovery platforms (Hybridoma, Single B and Display) significantly expand the diversity of identified binder sequences and epitopes. Phage display and yeast display platforms are integrated with structure-aided, rational mutagenesis to enhance lead affinity, improve developability and remove PTM-risk sites. • •

Rodent 300+

Rabbit ~10

Fig 4. WuXi Biologics’ Proprietary Platforms for mAb Optimization

Sequencing of Mutant Library

Multi-Site Tailored Mutant Library

CDR1

CDR2 CDR3

Average Ainity Improvement Across 54 projects

Parsimonious or multi-site mutagenesis screening to identify hot-spot Design and construct hot-spot combinatorial library with tailored diversity in each position Extensive-diversity space (library size up to 109) with high-throughput panning/screening Simultaneous removal of PTM-risk sites

Ainity Improvement

10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5

WuXi Biologics is a global leading Contract Research, Development discover, develop and manufacture biologics from concept About WuXi Biologics For more information see us at wuxibiologics.com partners to

8.48E-9

366-fold

and Manufacturing Organization (CRDMO) o ffe ring end-to-end solutions to enable

to commercialization.

3.11E-6

To discuss this poster: PS_BD@wuxibiologics.com

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