Related Experiment Videos
Construction and characterization of a bispecific diabody for retargeting T cells to human carcinomas
W Helfrich1, B J Kroesen, R C Roovers
1GUIDE, University Hospital, Department of Clinical Immunology, Groningen, The Netherlands.
Abstract:
We describe the construction of a recombinant bispecific antibody fragment in the diabody format with specificity for both the well-established human pancarcinoma associated target antigen EGP2 (epithelial glycoprotein 2, also known as the CO17-1A antigen or KSA) and the CD3epsilon chain of human TCR/CD3 complex. The murine anti-EGP2 (MOC31) single chain variable fragment (scFv) and the humanized anti-CD3 (Ucht1v9) scFv were cast into a diabody format (designated Dia5v9) using a short 5 amino acid Gly-Ser linker between immunoglobulin heavy-chain and light-chain variable domains. Purification of the poly-histidine tagged Dia5v9 was achieved from extracts of protease deficient Escherichia coli by IMAC chromatography. The Dia5v9 diabody showed strong binding to both EGP2 and CD3 in transfected cells. The in vitro efficacy of Dia5v9 in mediating tumor cell lysis by interleukin-2 activated human T cells appeared to be similar to that of the hybrid-hybridoma-derived BsF(ab')2 Bis1 (anti-EGP2/anti-CD3) in a standard 4-hr 51Cr-release assay. This small and partially humanized recombinant bispecific antibody fragment may be valuable for T-cell-based immunotherapeutical treatment protocols, retargeting activated peripheral blood T lymphocytes to lyse various human carcinomas in vivo.
Insights
Researchers developed a novel bispecific antibody fragment (Dia5v9) targeting epithelial glycoprotein 2 (EGP2) on cancer cells and CD3 on T cells. This engineered antibody shows potential for cancer immunotherapy by redirecting T cells to attack tumors.
Area of Science:
- Immunology
- Biotechnology
- Oncology
Background:
- Epithelial glycoprotein 2 (EGP2) is a target antigen on human carcinomas.
- T-cell receptor/CD3 complex (TCR/CD3) is crucial for T-cell activation.
- Bispecific antibodies can retarget T cells for cancer therapy.
Purpose of the Study:
- To construct and characterize a recombinant bispecific antibody fragment (diabody) targeting EGP2 and CD3.
- To evaluate the in vitro efficacy of this diabody in mediating tumor cell lysis.
Main Methods:
- Construction of a diabody (Dia5v9) format using anti-EGP2 and anti-CD3 single-chain variable fragments (scFv).
- Purification of poly-histidine tagged Dia5v9 from Escherichia coli using IMAC chromatography.
- In vitro efficacy assessment using a 51Cr-release assay with interleukin-2 activated T cells.
Main Results:
- Dia5v9 demonstrated strong binding to both EGP2 and CD3 on transfected cells.
- The in vitro efficacy of Dia5v9 in mediating tumor cell lysis was comparable to a hybrid-hybridoma-derived bispecific antibody fragment.
- Purification from protease-deficient E. coli was successful.
Conclusions:
- The Dia5v9 diabody is a small, partially humanized bispecific antibody fragment with potential for T-cell-based cancer immunotherapy.
- This agent can retarget peripheral blood T lymphocytes to lyse various human carcinomas in vivo.
- Further investigation for therapeutic protocols is warranted.