To enhance CAR T therapies, scientists are actively studying TCR signaling and T-cell exhaustion mechanisms to improve CAR T persistence and function. Additionally, there is a growing trend towards developing TCR-CAR hybrids to enhance the effectiveness of T-cell therapies.1 These advancements require the expression of T cell-related proteins, such as T-cell receptors (TCRs) and peptide-MHC (pMHC) complexes. However, producing functional TCRs and pMHC complexes in sufficient yield, quality, and efficacy remains challenging.
Traditional E. coli expression systems often suffer from low protein yields, limited throughput, and endotoxin contamination, limiting their applicability in therapeutic research. To overcome these limitations, WuXi Biologics’ Protein Sciences team has developed the T Cell Mate platform, enabling high-titer CHO expression of T cell-related proteins, including soluble TCRs (sTCRs), TCR fusion proteins, and single chain trimers (SCTs), while leveraging E. coli-based refolding for pMHC complex production. Furthermore, high-throughput SPR assays are employed to assess TCR-pMHC binding interactions, ensuring specificity and function for downstream applications.
Soluble TCRs (sTCRs) are essential for studying antigen recognition and immune response mechanisms.2 However, conventional E. coli expression systems often exhibit low yields, low throughput, and high endotoxin levels, making them less suitable for research and therapeutic applications. The T Cell Mate platform addresses these challenges, enabling a high-titer, high-throughput CHO production of sTCRs while maintaining high purity and low endotoxin levels.
The production process begins with codon optimization of the DNA sequence, followed by cloning into WuXi Biologics’ proprietary vector. Transfection into CHO cells and a seven-day culture period allow for protein expression. The culture medium is then clarified, and the sTCRs are purified using Affinity Chromatograph and SEC. Final characterization includes SDS-PAGE, SEC-HPLC, LC-MS, and endotoxin detection assays.
Using this approach, various TCR formats have been successfully produced. Shown here are three sTCR molecules produced in CHO cells (Figure 1). One-arm TCR-Fc fusion, His-tagged TCR, and scFv-fused TCR yielded 1,100 mg/L, 130 mg/L, and 100 mg/L, respectively. These results demonstrate the superior productivity of our high-titer CHO transient expression system for sTCR.
Figure 1. SDS-PAGE (NR/R) analysis for one-step affinity purification of 3 sTCRs (one-arm TCR-Fc fusion, His-tagged TCR, and scFv-fused TCR).
Properly folded peptide-MHC (pMHC) complexes are essential for TCR specificity screening. However, the quality of the refolded (RF) pMHC complexes may not be consistent across different labs, which frequently leads to poor performance in subsequent assays. Optimizing refolding methods in E. coli allows for the generation of high-quality peptide-MHC (RF-pMHC) complexes. More importantly, stringent QC such as LC-MS analysis can verify structural integrity before downstream assays.
The process begins with codon optimization and cloning into expression vectors, followed by HLA and B2M protein expression in inclusion body form. Inclusion bodies are then solubilized, and an optimized process is applied to facilitate the refolding of the HLA heavy chain, B2M light chain, and target peptide. Purification involves AEX, in vitro biotinylation, and SEC, followed by characterization via SDS-PAGE and LC-MS.
By applying this optimized refolding workflow, over 300 RF-pMHC complexes were successfully generated in E. coli across batch sizes ranging from 0.1 L to 50 L, demonstrating the scalability and reproducibility of this approach. LC-MS also confirmed their structural integrity for quality control (Figure 2).
Figure 2. RF-pMHCI production workflow, outlining the purification and QC methods (LC-MS and SDS-PAGE) for
purity assessment.
Peptide-MHCII (pMHCII) complexes are essential for CD4+ T-cell activation,3 but they are often challenging to express at high yields and quality in traditional expression systems. The T Cell Mate platform leverages a high-titer CHO transient production system with optimized zipper-fusion constructs, significantly enhancing both expression levels and protein quality of pMHCII complexes.
The production workflow begins with the cloning of optimized DNA sequences into WuXi Biologics’ proprietary vector, followed by transient transfection into CHO cells and a seven-day culture. After clarification of the culture medium, purification is carried out through a multi-step purification workflow, including Ni affinity chromatography, zipper peptide cleavage (optional), AEX chromatography, and SEC. The final product is characterized by SDS-PAGE, SEC-HPLC, LC-MS, and endotoxin detection assays.
With this approach, CHO-expressed pMHCII complexes were successfully generated with yields exceeding 500 mg/L (Figure 3). The zipper-fusion constructs enhanced both expression levels and structural integrity. Following affinity purification, the pMHCII complexes exhibited high purity, suitable for downstream applications.
Figure 3. SDS-PAGE (NR/R) analysis and yield comparison for one-step purification of 4 pMHCII constructs.
Evaluating TCR-pMHC binding interactions is essential for selecting high-affinity TCR candidates for the development of TCR-related therapeutics. High-throughput SPR assays provide real-time kinetics measurements of binding affinity, on/off rates, and interaction specificity, allowing for the screening and selection of optimal TCR candidates.
Figure 4. SPR sensorgrams for TCR-pMHC interactions with pMHC generated from CHO and E. coli expression systems.
In a high-throughput SPR binding study, multiple TCRs were tested against pMHC complexes derived from both CHO and E. coli expression systems (Figure 4). The kinetics data of ka, kd, KD (Table 1) allowed for the screening and selection of high-affinity TCR candidates.
Table 1. Summary of SPR Kinetics Data
The T Cell Mate platform offers a solution to the challenges of producing T cell-related proteins, such as sTCRs and pMHCII complexes, which are essential for advancing
T cell-based therapies. The platform utilizes high-titer CHO expression to produce these proteins with high yields, purity, and low endotoxin levels. Additionally, E. coli refolding workflows enable the efficient generation of RF-pMHC complexes, while high-throughput SPR binding assays provide kinetics data for evaluating TCR-pMHC interactions, aiding in TCR screening, and candidate selection. By leveraging the T Cell Mate platform, researchers can enhance protein production efficiency, streamline TCR screening, and accelerate the development of next-generation T-cell therapeutics.
At WuXi Biologics, Dawei Zhang is a senior director of CRO services, Lei Guo, PhD, serves as a director of CRO services, and Jiansheng Wu, PhD, is head of CRO services and senior vice president.
References
1. Posey, Avery D. et al. Future perspectives on engineered T Cells for cancer. Trends in Cancer. 2024 Volume 10, Issue 8, 687 – 695.
2. Robinson RA, McMurran C, McCully ML, Cole DK. Engineering soluble T-cell receptors for therapy. FEBS J. 2021 Nov;288(21):6159-6173. doi: 10.1111/febs.15780.
3. Vyasamneni R, Kohler V, Karki B, Mahimkar G, Esaulova E, McGee J, Kallin D, Sheen JH, Harjanto D, Kirsch M, Poran A, Dong J, Srinivasan L, Gaynor RB, Bushway ME, Srouji JR. A universal MHCII technology platform to characterize antigen-specific CD4+ T cells. Cell Rep Methods. 2023 Jan 13;3(1):100388. doi: 10.1016/j.crmeth.2022.100388.
The post High-Titer CHO Expression of T Cell-Related Proteins appeared first on GEN - Genetic Engineering and Biotechnology News.
Traditional E. coli expression systems often suffer from low protein yields, limited throughput, and endotoxin contamination, limiting their applicability in therapeutic research. To overcome these limitations, WuXi Biologics’ Protein Sciences team has developed the T Cell Mate platform, enabling high-titer CHO expression of T cell-related proteins, including soluble TCRs (sTCRs), TCR fusion proteins, and single chain trimers (SCTs), while leveraging E. coli-based refolding for pMHC complex production. Furthermore, high-throughput SPR assays are employed to assess TCR-pMHC binding interactions, ensuring specificity and function for downstream applications.
Soluble TCR Generation in CHO
Soluble TCRs (sTCRs) are essential for studying antigen recognition and immune response mechanisms.2 However, conventional E. coli expression systems often exhibit low yields, low throughput, and high endotoxin levels, making them less suitable for research and therapeutic applications. The T Cell Mate platform addresses these challenges, enabling a high-titer, high-throughput CHO production of sTCRs while maintaining high purity and low endotoxin levels.
The production process begins with codon optimization of the DNA sequence, followed by cloning into WuXi Biologics’ proprietary vector. Transfection into CHO cells and a seven-day culture period allow for protein expression. The culture medium is then clarified, and the sTCRs are purified using Affinity Chromatograph and SEC. Final characterization includes SDS-PAGE, SEC-HPLC, LC-MS, and endotoxin detection assays.
Using this approach, various TCR formats have been successfully produced. Shown here are three sTCR molecules produced in CHO cells (Figure 1). One-arm TCR-Fc fusion, His-tagged TCR, and scFv-fused TCR yielded 1,100 mg/L, 130 mg/L, and 100 mg/L, respectively. These results demonstrate the superior productivity of our high-titer CHO transient expression system for sTCR.
Figure 1. SDS-PAGE (NR/R) analysis for one-step affinity purification of 3 sTCRs (one-arm TCR-Fc fusion, His-tagged TCR, and scFv-fused TCR).
Peptide-MHC Complex Refolding in E. coli
Properly folded peptide-MHC (pMHC) complexes are essential for TCR specificity screening. However, the quality of the refolded (RF) pMHC complexes may not be consistent across different labs, which frequently leads to poor performance in subsequent assays. Optimizing refolding methods in E. coli allows for the generation of high-quality peptide-MHC (RF-pMHC) complexes. More importantly, stringent QC such as LC-MS analysis can verify structural integrity before downstream assays.
The process begins with codon optimization and cloning into expression vectors, followed by HLA and B2M protein expression in inclusion body form. Inclusion bodies are then solubilized, and an optimized process is applied to facilitate the refolding of the HLA heavy chain, B2M light chain, and target peptide. Purification involves AEX, in vitro biotinylation, and SEC, followed by characterization via SDS-PAGE and LC-MS.
By applying this optimized refolding workflow, over 300 RF-pMHC complexes were successfully generated in E. coli across batch sizes ranging from 0.1 L to 50 L, demonstrating the scalability and reproducibility of this approach. LC-MS also confirmed their structural integrity for quality control (Figure 2).
Figure 2. RF-pMHCI production workflow, outlining the purification and QC methods (LC-MS and SDS-PAGE) for
purity assessment.
Peptide-MHCII Complex Generation in CHO
Peptide-MHCII (pMHCII) complexes are essential for CD4+ T-cell activation,3 but they are often challenging to express at high yields and quality in traditional expression systems. The T Cell Mate platform leverages a high-titer CHO transient production system with optimized zipper-fusion constructs, significantly enhancing both expression levels and protein quality of pMHCII complexes.
The production workflow begins with the cloning of optimized DNA sequences into WuXi Biologics’ proprietary vector, followed by transient transfection into CHO cells and a seven-day culture. After clarification of the culture medium, purification is carried out through a multi-step purification workflow, including Ni affinity chromatography, zipper peptide cleavage (optional), AEX chromatography, and SEC. The final product is characterized by SDS-PAGE, SEC-HPLC, LC-MS, and endotoxin detection assays.
With this approach, CHO-expressed pMHCII complexes were successfully generated with yields exceeding 500 mg/L (Figure 3). The zipper-fusion constructs enhanced both expression levels and structural integrity. Following affinity purification, the pMHCII complexes exhibited high purity, suitable for downstream applications.
Figure 3. SDS-PAGE (NR/R) analysis and yield comparison for one-step purification of 4 pMHCII constructs.
High-Throughput SPR Assays of TCR-pMHC Binding
Evaluating TCR-pMHC binding interactions is essential for selecting high-affinity TCR candidates for the development of TCR-related therapeutics. High-throughput SPR assays provide real-time kinetics measurements of binding affinity, on/off rates, and interaction specificity, allowing for the screening and selection of optimal TCR candidates.
Figure 4. SPR sensorgrams for TCR-pMHC interactions with pMHC generated from CHO and E. coli expression systems.
In a high-throughput SPR binding study, multiple TCRs were tested against pMHC complexes derived from both CHO and E. coli expression systems (Figure 4). The kinetics data of ka, kd, KD (Table 1) allowed for the screening and selection of high-affinity TCR candidates.
Table 1. Summary of SPR Kinetics Data
Conclusion
The T Cell Mate platform offers a solution to the challenges of producing T cell-related proteins, such as sTCRs and pMHCII complexes, which are essential for advancing
T cell-based therapies. The platform utilizes high-titer CHO expression to produce these proteins with high yields, purity, and low endotoxin levels. Additionally, E. coli refolding workflows enable the efficient generation of RF-pMHC complexes, while high-throughput SPR binding assays provide kinetics data for evaluating TCR-pMHC interactions, aiding in TCR screening, and candidate selection. By leveraging the T Cell Mate platform, researchers can enhance protein production efficiency, streamline TCR screening, and accelerate the development of next-generation T-cell therapeutics.
At WuXi Biologics, Dawei Zhang is a senior director of CRO services, Lei Guo, PhD, serves as a director of CRO services, and Jiansheng Wu, PhD, is head of CRO services and senior vice president.
References
1. Posey, Avery D. et al. Future perspectives on engineered T Cells for cancer. Trends in Cancer. 2024 Volume 10, Issue 8, 687 – 695.
2. Robinson RA, McMurran C, McCully ML, Cole DK. Engineering soluble T-cell receptors for therapy. FEBS J. 2021 Nov;288(21):6159-6173. doi: 10.1111/febs.15780.
3. Vyasamneni R, Kohler V, Karki B, Mahimkar G, Esaulova E, McGee J, Kallin D, Sheen JH, Harjanto D, Kirsch M, Poran A, Dong J, Srinivasan L, Gaynor RB, Bushway ME, Srouji JR. A universal MHCII technology platform to characterize antigen-specific CD4+ T cells. Cell Rep Methods. 2023 Jan 13;3(1):100388. doi: 10.1016/j.crmeth.2022.100388.
The post High-Titer CHO Expression of T Cell-Related Proteins appeared first on GEN - Genetic Engineering and Biotechnology News.