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Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization
Published on: December 23, 2015
[Construction, expression and purification of a mammalian secretory recombinant fusion protein rPC]
Chunchun Li1, Yuqiong Xie1, Jiang Cao1
1Clinical Research Center, the 2nd Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, Zhejiang, China.
Abstract:
Drugs targeting immune checkpoint are used for cancer treatment, but resistance to single drug may occur. Combination therapy blocking multiple checkpoints simultaneously can improve clinical outcome. Therefore, we designed a recombinant protein rPC to block multiple targets, which consists of extracellular domains of programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). The coding sequence was inserted into expression vector and stably transfected into HEK293 cells. The culture supernatant was collected and rPC was affinity-purified. Real-time quantitative PCR was used to evaluate the expression levels of ligands for PD-1 and CTLA-4 in several human cancer cell lines. The binding of rPC with cancer cells was examined by immunofluorescence cell staining, the influence of rPC on cancer cell growth was assayed by CCK-8. The results showed that rPC could be expressed and secreted by stably transfected HEK293 cells, the purified rPC could bind to lung cancer NCI-H226 cells which have high levels of ligands for PD-1 and CTLA-4, no direct impact on cancer cell growth could be observed by rPC treatment. The recombinant protein rPC can be functionally assayed further for developing novel immunotherapeutic drugs for cancer.
Insights
Researchers developed a novel recombinant protein, rPC, to block multiple immune checkpoints simultaneously. This dual-target approach shows potential for enhancing cancer immunotherapy by overcoming resistance to single-agent treatments.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Cancer immunotherapy often utilizes drugs targeting immune checkpoints like PD-1 and CTLA-4.
- Resistance to single-target immune checkpoint inhibitors can limit clinical efficacy.
- Combination therapies blocking multiple checkpoints may improve patient outcomes.
Purpose of the Study:
- To design and express a novel recombinant protein, rPC, targeting both PD-1 and CTLA-4.
- To evaluate the binding capability and functional effects of rPC on cancer cells.
- To explore the potential of rPC as a dual-target immunotherapeutic agent.
Main Methods:
- Constructed a recombinant protein (rPC) with extracellular domains of PD-1 and CTLA-4.
- Expressed and purified rPC using stably transfected HEK293 cells.
- Assessed PD-1 and CTLA-4 ligand expression, rPC binding to cancer cells, and impact on cell growth.
Main Results:
- Successfully expressed and secreted rPC from transfected cells.
- Purified rPC demonstrated binding to lung cancer cells with high PD-1 and CTLA-4 ligand expression.
- rPC treatment did not directly inhibit cancer cell proliferation.
Conclusions:
- The recombinant protein rPC is a viable candidate for further investigation in cancer immunotherapy.
- rPC's ability to bind to cancer cells warrants further functional studies for therapeutic development.
- Dual-targeting of PD-1 and CTLA-4 via rPC may offer a strategy to overcome treatment resistance.
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