High-throughput, high-titer CHO production of miniproteins with high yields and low endotoxin levels, surpassing traditional E. coli production methods.
Pioneering TCR Optimization via Targeted Engineering & HTP Screening Wenya Shan, Huihui He, Donghui Wu, Gennady Gololobov, Yu Tian, Lei Guo, Dawei Zhang, Jiansheng Wu
WuXi Biologics, No.240 Hedan Road, Pudong New District, Shanghai, China (Contact: PS_Marketing@wuxibiologics.com)
T-cell receptor (TCR) therapy is a promising immunotherapy that engineers TCRs to precisely recognize tumor antigens, pathogens, or abnormal cell surface molecules. While this innovation advances the treatment of cancer and diverse diseases, the low a ff inity of natural TCRs o ft en limits their capability to activate T cells and drive cytotoxic e ff ects. To solve this problem, WuXi Biologics has developed a state-of-the-art phage display platform ( Fig.1 ) for TCR optimization. In this workflow, site-directed mutagenesis libraries are constructed, and comprehensive screening of complementarity-determining regions (CDRs) of TCR molecules are performed. The modified TCRs are then displayed on bacteriophages. A ft er 3-4 rounds of selection, high-a ff inity variants are rapidly isolated from extensive mutant libraries. Following this, the innovative T Cell Mate platform ( Fig.2 ) enables high-titer, HTP production of sTCRs with premium purity and low endotoxin levels, while HTP SPR k o ff ranking system streamlines the screening of TCR-engineered molecules with enhanced a ff inity and specificity. Introduction Fig.1 High-quality phage display library construction enabling the enrichment of specific and diverse binders via high-throughput panning Abstract T-cell receptor (TCR) therapy holds great promise for treating cancer, autoimmune, and infectious diseases. However, the inherently low a ff inity of natural TCRs for target antigens, typically at the micromolar level, remains a major barrier to achieving optimal therapeutic e ff icacy. To address this challenge, WuXi Biologics has established a robust, e ff icient TCR optimization platform that combines high-quality phage display, targeted engineering, and high-throughput (HTP) production and screening. Through site-directed mutagenesis, high-a ff inity TCRs via phage can be e ff iciently isolated. TCR molecules are then rapidly produced from CHO cells and screened using HTP SPR k o ff system. Together, these capabilities streamline TCR discovery and optimization, paving the way for developing next-gen TCR-based therapies.
Fig.5 Screening of multi-site mutant library
High-Throughput Screening Phage ELISA screening using antigen coating and TCR phage detection
Sequence Analysis
Laed Identification Delivery of TCR-engineered molecules with K D ranging from 10 - ⁷ to 10 - ⁹ M
Results
Fig.6 Identification of TCR-engineered molecules with enhanced binding activity and specificity
V α Chain output screening
V β Chain output screening Combinatorial library output screening
Concentration (nM)
Antigen
Target pMHC
12.5
Phage Display Library Design & Construction
Non-Target pMHC
50
Solid-Phase Panning
Library Design & Construction
Panning 3-4 Rounds
Liquid-Phase Panning
This case study demonstrates that high-throughput screening enabled identification of TCR-engineered molecules with improved nanomolar a ff inity to targets over non-target controls.
Cell-Based Panning
Fig.7 High-Throughput TCR production in CHO Cells
Phage Display
Sequencing & Characterization
Fig.2 High-titer protein production platform for T-cell-related proteins
This case study showcases high-throughput transient CHO expression of TCR molecules that achieved an average yield of 0.85 g/L and purity of 92.4% in 96-well DWP.
sTCR
TCR-Ab Fusion RF-pMHC, E.coli
SCT
Fig.8 A ff inity improvement of TCRs against target antigens over non-target antigens
Method Fig.3 WuXi Biologics’ proprietary platforms for TCR optimization
Target Antigen Ligand (ID) k a (1/Ms)
Analyte
k d (1/s)
K D (M)
1.84E-09 2.59E-09
138 158
2.10E+05 3.87E-04 2.06E+05 5.32E-04 4.91E+05 1.12E-03
WBP1200_pMHC-02 HLA-A*02:01-NY-ESO-1
161 2.28E-09 1G4-c113 6.88E+05 1.51E-03 2.19E-09 1G4-WT NA NA 4.33E-05 2.19E-09
Non-Target Antigen
Analyte
Ligand (ID) k a (1/Ms)
k d (1/s)
K D (M) K (M)
β CDR1
β CDR2 β CDR3
138
158 161 1G4-c113 1G4-WT
WBP1200_pMHC-04 HLA-A*02:01-gp100
No or weak binding
α CDR1
α CDR2
α CDR3
Control TCR (c113) doi: 10.1038/nbt1070
14,347-folds a ff inity improved
In this case study, engineered TCRs achieved up to 14,347-fold a ff inity improvement for target antigens, compared to no detectable or weak binding to non-target antigens.
Multi-site tailored mutant library design
Multi-site tailored mutant library design
Conclusions WuXi Biologics has established a robust, versatile platform for TCR optimization, enabling rapid identification of TCRs from phase display library with enhanced a ff inity and specificity. In addition, coupling HTP transient CHO production with SPR-based K o ff ranking system accelerates the selection of optimal TCR-engineered candidates for e ff icient downstream therapeutics development. Acknowledgment 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 TCR optimization platforms. Additionally, we extend our appreciation to WuXi Biologics' legal, public relations, and marketing departments for their assistance in patent filings and the preparation and production of written materials. About WuXi Biologics CRO Services WuXi Biologics is a leading global CRDMO, with the R represented by our premium-quality CRO Services. With expertise in diverse drug modalities, we deliver comprehensive research services, including antibody discovery, protein production and engineering, and in vitro / in vivo testing, to seamlessly advance our clients’ molecules from concept to CMC development.
Display rate detection of mutant library
Sequencing of mutant library
Fig.4 QC data of multi-site mutant library
Drug Development Expertise Empowering Research Services for Biologics For more information see us at https://www.wuxibiologics.com/cro-services
TCR Optimization_Poster_202510_V3
To discuss this poster: PS_Marketing@wuxibiologics.com
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