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Updated: Jul 10, 2026
Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody
Published on: May 16, 2020
Construction of humanized carcinoembryonic antigen specific single chain variable fragment and mitomycin conjugate
De-Jie Chen1, Zui Tan, Feng Chen
1Department of General Surgery, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071, Hubei Province, China.
Aim:
To construct a new target-oriented conjugate of humanized carcinoembryonic antigen (CEA) specific single chain variable fragment (scFv) and mitomycin (MMC) against colorectal cancer, and to investigate its influence on the growth and apoptosis of colorectal cancer cells.
Methods:
The primer was designed according to the gene sequence described in reference 16, which respectively contains restriction enzyme cleavage sites BamHI and EcoRI in its upstream and downstream. PCR was performed with the plasmid as template containing genes of humanized anti-CEA scFv. The product was digested by BamHI and EcoRI, and connected to an expression vector which also has the restriction enzyme cleavage sites BamHI and EcoR. Expression of the reaction was induced by isopropy-beta-D-thiogalactoside (IPTG). Then the expression product was covalently coupled with MMC by dextran T-40. The immunoreactivity of the conjugate against colorectal cancer cells as well as CEA was measured by enzyme linked immunosorbent assay (ELISA). The inhibiting ratio of conjugate on the growth of colorectal cancer cells was also measured by ELISA. The effect of conjugate on the apoptosis of colorectal cancer cells was determined by flow cytometry (FCM).
Results:
Restriction endonuclease cleavage and gene sequencing confirmed that the expression vector was successfully constructed. Sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) confirmed that this vector correctly expressed the fusion protein. ELISA confirmed that the conjugate had quite a strong immunoreactivity against colorectal cancer cells and CEA. The conjugate had inhibitory effects on colorectal cancer cells in a concentration-dependent manner and could induce apoptosis of colorectal cancer cells in a concentration-dependent manner.
Conclusion:
The CEA-scFv-MMC conjugate can be successfully constructed and is able to inhibit the growth and induce apoptosis of colorectal cancer cells.
Insights
A novel conjugate linking a humanized carcinoembryonic antigen (CEA) specific single chain variable fragment (scFv) with mitomycin (MMC) was successfully developed. This CEA-scFv-MMC conjugate effectively inhibits colorectal cancer cell growth and induces apoptosis.
Area of Science:
- Oncology
- Molecular Biology
- Bioconjugation
Background:
- Colorectal cancer (CRC) remains a significant health challenge globally.
- Targeted therapies offer improved efficacy and reduced systemic toxicity.
- Carcinoembryonic antigen (CEA) is a validated biomarker for CRC.
Purpose of the Study:
- To construct a novel targeted conjugate of humanized anti-CEA single chain variable fragment (scFv) and mitomycin (MMC).
- To evaluate the efficacy of this conjugate in inhibiting colorectal cancer cell growth and inducing apoptosis.
Main Methods:
- Gene cloning and expression of humanized anti-CEA scFv.
- Covalent coupling of scFv with mitomycin (MMC) via dextran T-40.
- Assessment of conjugate immunoreactivity, growth inhibition, and apoptosis induction using ELISA and flow cytometry.
Main Results:
- Successful construction and expression of the CEA-scFv-MMC conjugate confirmed by sequencing and SDS-PAGE.
- The conjugate demonstrated significant immunoreactivity against colorectal cancer cells and CEA.
- Concentration-dependent inhibition of colorectal cancer cell growth and induction of apoptosis were observed.
Conclusions:
- The developed CEA-scFv-MMC conjugate is a promising targeted therapeutic agent for colorectal cancer.
- The conjugate effectively inhibits cancer cell proliferation and promotes cell death.
