Related Experiment Video
Updated: Aug 2, 2025
Tracking Bispecific Antibody-Induced T Cell Trafficking Using Luciferase-Transduced Human T Cells
Published on: May 12, 2023
Construction and Periplasmic Expression of a Bispecific Tandem scFv for Dual Targeting of Immune Checkpoints
Amirreza Rashti1, Vajihe Akbari1
1Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran.
Background:
Immune checkpoints are molecules that act as regulators of immune system pathways. However, some tumor cells can express the ligands of immune checkpoints to escape from antitumor immune responses. Some agents, such as antibodies, can inhibit these checkpoints that prevent the immune system from targeting and killing cancer cells. The aim of this study was to express a novel bispecific tandem scFv in periplasmic space of Escherichia coli for simultaneous targeting of two immune checkpoints, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1).
Materials And Methods:
The bispecific tandem scFv was constructed based on the variable regions gene of anti-PD1 and anti-CTLA-4 antibodies. The optimum codon for expression in E. coli was chemically synthesized and subcloned in periplasmic expression plasmid. After transformation, the effect of cultivation conditions on periplasmic expression of the protein in E. coli BL21(DE3) was evaluated. Then, the bispecific tandem scFv was purified and its binding ability to cells expressing PD-1 and CTLA-4 was evaluated.
Results:
Expression of tandem scFv with a molecular weight of 55 kDa was verified by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting analysis. The best condition for soluble periplasmic expression was obtained to be incubation with 0.5 mM isopropyl β-D-1-thiogalactopyranoside at 23°C. The protein was successfully purified using affinity chromatography with a final yield of 4.5 mg/L. Binding analysis confirmed the bioactivity of purified the tandem scFv.
Conclusion:
This bispecific tandem scFv could be a potential candidate to cancer immunotherapy, although more biological activity assessments are still required to be carried out.
Insights
Researchers developed a novel bispecific antibody fragment targeting both PD-1 and CTLA-4 immune checkpoints for cancer immunotherapy. This engineered protein, expressed in E. coli, shows potential for enhancing anti-tumor immune responses.
Area of Science:
- Immunology and Cancer Research
- Protein Engineering and Recombinant Expression
- Molecular Biology and Biotechnology
Background:
- Tumor cells exploit immune checkpoints, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1), to evade immune surveillance.
- Inhibiting these immune checkpoints with agents like antibodies can restore the immune system's ability to target and eliminate cancer cells.
- Developing novel therapeutic agents that simultaneously target multiple immune checkpoints offers a promising strategy for more effective cancer immunotherapy.
Purpose of the Study:
- To engineer and express a novel bispecific tandem single-chain variable fragment (scFv) capable of simultaneously targeting CTLA-4 and PD-1.
- To optimize the periplasmic expression of this bispecific tandem scFv in Escherichia coli (E. coli) for efficient production.
- To purify the expressed bispecific tandem scFv and confirm its binding capability to cells expressing the target immune checkpoints.
Main Methods:
- Construction of a bispecific tandem scFv gene using variable regions from anti-PD-1 and anti-CTLA-4 antibodies.
- Codon optimization for E. coli expression, chemical synthesis, and subcloning into a periplasmic expression plasmid.
- Evaluation of cultivation conditions for optimal periplasmic expression in E. coli BL21(DE3), followed by protein purification and binding assays.
Main Results:
- Successful expression of the bispecific tandem scFv (55 kDa) was confirmed via SDS-PAGE and Western blotting.
- Optimal soluble periplasmic expression was achieved with 0.5 mM isopropyl β-D-1-thiogalactopyranoside at 23°C.
- Purification via affinity chromatography yielded 4.5 mg/L of bioactive protein, confirmed by binding analysis.
Conclusions:
- The study successfully produced a novel bispecific tandem scFv targeting both PD-1 and CTLA-4.
- The optimized periplasmic expression in E. coli provides a viable method for producing this bispecific antibody fragment.
- This engineered bispecific scFv represents a potential candidate for advancing cancer immunotherapy, pending further biological activity assessments.
