High-Titer CHO Production of T-Cell-Related Proteins

High-throughput, high-titer CHO production of miniproteins with high yields and low endotoxin levels, surpassing traditional E. coli production methods.

T Cell Mate Platform: High-Titer CHO Production of T-Cell-Related Proteins Dawei Zhang, Mengjie Lu, Zhenyang Zhao, Feng Li, Jiansheng Wu

Protein Sciences Department, WuXi Biologics, CRO Services, No.240 Hedan Road, Pudong New District, Shanghai, China (Contact: PS_Marketing@wuxibiologics.com)

Abstract T-cell receptors (TCRs) are important targets in the development of immunotherapies and cancer treat- ment. However, traditional E. coli production methods for TCRs faces challenges, o ft en leading to limited throughput, low yields, and high endotoxin levels. To address these challenges, WuXi Biologics’ Protein Sciences Department developed the T Cell Mate platform. Leveraging a high-titer CHO expression system, this platform enables high-throughput production of a broad range of T-cell-related proteins, including soluble TCRs (sTCRs), TCR-Ab fusions, and single-chain trimers (SCTs), all with high yield, superior quality, and exceptionally low endotoxin levels, meeting the stringent requirements of therapeutic development.

Fig.4 MHCI production workflow, detailing purification and QC methods for purity evaluation Case Study 3: Extensive Production of MHCI in E. coli

Over 300 of MHC production from 0.1 to 50 L E. coli α 2 α 1 α 2

α 1

MHC Biotiny lation

Purification

α 3

HLA B2M Peptide

MHC Complex

Introduction

Purification Profile

Mass Spectrum

Final Product .ĞHĞHĞHĞH

…

3 x10 0 1 2 3 4 5 2 x10 0 2 4 6 8

+ESI Scan (4.497-5.723 min, 74 Scans) Frag=200.0V H2-Db-1.d Subtr

37421.48

T-cell receptors (TCRs) recognize antigenic peptides presented by MHCs on target cells, triggering signaling pathways that activate T-cells and initiate a targeted immune response. This mechanism has made TCRs important therapeutic targets in immunotherapies and cancer treatment. However, expressing TCRs in traditional E. coli systems has posed challenges in throughput, yield and endotoxin levels. To tackle these challenges, WuXi Biologics’ PS Department developed the T Cell Mate platform, which utilizes a high-titer CHO expression system to e ff iciently produce a variety of T-cell-related molecules, including sTCRs, TCR fusion proteins, and SCTs, all achieving high yields, quality and throughput. Notably, the SCT format integrates HLA and β 2-macroglobulin into a single polypeptide, facilitating the secretion of well-folded pMHCs from CHO cells. The platform also supports the production of RF-pMHCs in E. coli ., providing a versatile and scalable solution for therapeutic T-cell-related proteins.

mAU

H2-Db Biotinylated

B2M

Q HP Gradient

3000 2500 2000 1500 1000 500 0

11861.29

10000 20000 30000 40000 50000 60000 70000 80000 90000 Counts vs. Deconvoluted Mass (amu)

…

+ESI Scan (4.497-5.723 min, 74 Scans) Frag=200.0V H2-Db-1.d Subtr

Peptide

1027.4328

1028.4333

1029.4316

100

200

300

400

500

600

700

Counts vs. Mass-to-Charge (m/z) 1026 1027 1028 1029 1030 1031 1032

Fig.5 SDS-PAGE (NR/R) analysis and average titers for two-step purification of 10 SCTs Case Study 4: High-Throughput (HTP) Production of 10 SCTs in CHO Cells

AC Final

0 100 200 300 400 500 600 700 800 900 1000

Fig.1 T-Cell-Related Proteins

250 150 75 100 kDa

The innovative T Cell Mate platform delivers all the proteins necessary for T-cell related projects, including sTCRs, TCR fusion proteins, RF-pMHCs (in E. coli ), and SCTs.

50 37 25 15 10 20

HCCF

554

· SCT in CHO has much higher expression than RF-pMHC from E. coli · SEC is required to remove aggregated SCT

250 150 75 100 kDa

306

AC-SEC

50 37 25 15 10 20

sTCR TCR-Ab Fusion RF-pMHCs, E. coli C C b i C li

SCT SC

Results

In this case study, the expression of SCTs in transient CHO cells significantly surpassed that of RF-pMHC in E. coli. Among 10 SCTs, the average yield was 554 mg/L using AC purification. Following SEC for aggregate removal, the average yield of final products reached 306 mg/L. Fig.6 SDS-PAGE and SEC-HPLC analysis for two-step purification of a mouse SCT Case Study 5: High-Purity, High-Yield Production of a Mouse SCT in CHO Cells

Many Formats of TCR Molecules Have Been Produced Successfully

V α

Human V α

Mouse V α

Mouse C α

Human C α

C α

Human V β

Mouse V β

V β

C β

Mouse C β

Human C β

M L FT E

250 kDa 75 100 150

Human-Mouse

Mouse-Human

SCT

37 50 25 15 10 20

RT: 8.340 minutes Purity: 82.42%

RT: 8.373 minutes Purity: 99.57%

37 50 25 15 10 20

One arm TCR-Fc

Bivalent TCR-Fc

Chimera TCR

SEC

V α

His

Ni:18.3mg

Final: 10 mg LAL: < 0.1 EU/mg

ScFv

C α

V α

V β

TCR with Fc Titer: 300-1500 mg/L

C β

From 20 mL CHO HCCF, a mouse SCT was yielded at 18.3 mg a ft er Ni purification. Subsequent SEC purification increased the target purity from 82.42% to 99.57%, with a yield of 10 mg and an endotoxin level of less than 0.1 EU/mg.

C α

V β

Titer: 50-150 mg/L

C β

His-TCR

ScFv-TCR (TCE)

Fig.7 SDS-PAGE (NR/R) analysis and titers for one-step purification of 4 MHCII molecules Case Study 6: High-Yield Production of MHCII with Di ff erent Constructs in CHO Cells

TCR-Fab BsAb

8EUQ: HLA-DRA*0101 Peptide-HLA-DRB1*0401 5V4M: HLA-DRA*0101 Peptide-HLA-DRB1*1501

Case Study 1: High-Titer Expression of sTCR in CHO Cells

HCCF

α 2 α 1

β 2 β 1

Fig.2 SDS-PAGE (NR/R) analysis for one-step a ff inity purification of 3 sTCRs

E FT

M L FT E

L E FT

M L FT E

Degly

L E FT M L FT E Degly

kDa 250 150 100

250 kDa 75 100 150

Zipper 1

Zipper 2

V α

His

ScFv His

C α

V β

V α

75 50 37 25 15 10 20

Fc- α

ScFv- β PNGase F

C β

C α

8EUQ

5V4M

37 50 25 15 10 20

V β

α β

37 50 25 15 10 20

V β

500

β /Fc

C β

Zipper1

Zipper2

Zipper1

Zipper2

AC yields: 1,100 mg/L

130 mg/L

100 mg/L

Method All experiments were conducted within the Protein Sciences (PS) Department at WuXi Biologics. The DNA sequence codons were optimized using WuXi Biologics’ laboratory data management system (LDMS), followed by plasmid construction into WuXi Biologics’ proprietary vector. Using the WuXian™ Transient expression system as per SOPs, the culture medium was collected a ft er 7 days. Following clarification of the culture medium, all supernatant was loaded onto appropriate purification columns based on titer. Purification involved either Nickel column or protein A purification, followed by size exclusion chromatography (SEC). The purified products were then characterized through SDS-PAGE, SEC-HPLC, intact mass analysis, and endotoxin detection. Conclusions WuXi Biologics has revolutionized the high-titer production of T-cell-related proteins with the T Cell Mate Platform, which encompasses sTCRs, TCR fusions, SCTs, and RF-pMHCs (in E. coli ). The ultra-high titer CHO expression system ensures outstanding yields and enhanced throughput. Flexible purification and advanced QC techniques guarantee exceptional protein quality, achieving high purity, low endotoxin levels, and robust complex integrity. E ff icient workflow allows for rapid project completion, typically within just 3-4 weeks. Acknowledgment We extend our gratitude to all scientists from the Department of Protein Sciences at WuXi Biologics for their invaluable support during the development of the T Cell Mate platform. Additionally, we express our appreciation to the legal department, public relations department, and marketing department for their assistance with patent filing, poster preparation, and revision. 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. · · ·

In this case study, three sTCRs were produced using the T Cell Mate platform. Following one-step a ff inity purification, the yields for one-arm Fc fusion, His-tagged TCR, and scFv-infused TCRs achieved 1100.0 mg/L, 130 mg/L, and 100 mg/L, respectively.

Fig.3 TCR-Fab BsAb purification and QC results for purity evaluation Case Study 2: High-Purity, High-Yield Production of a TCR-Fab BsAb in CHO Cells

Post AC Quality

M L E FT

Target:

V α

kDa 250 150 100

C α

HC1

HC2

TCR

Fab

V α

SEC-HPLC Purity: 57.05%

75 50 37 25 15 10 20

V β

C α

C β

V α

LC1

LC2

C α

CEX Gradient

SDS-PAGE

Intact Mass (Degly-NR)

Zoom in

In this case study, the TCR-Fab bsAb was produced in transient CHO cells with a titer of 1.72 g/L. Following MSS-SEC-CEX purifica- tion, 29 mg of purified protein was obtained from 100 mL HCCF, with >99% heterodimer purity confirmed by Intact Mass analysis and an endotoxin level below 0.1 EU/mg.

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T Cell Mate_Poster_202510_V2

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