Title:Generation and Characterization of a Bispecific Antibody Targeting Both PD-1 and c-MET
VOLUME: 24 ISSUE: 12
Author(s):Yi Wu, Min Yu*, Zujun Sun, Weihua Hou, Yuxiong Wang, Qingyun Yuan and Wei Mo
Affiliation:Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, and Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Fudan University, Shanghai, Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, and Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Fudan University, Shanghai, Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, and Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Fudan University, Shanghai, Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, and Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Fudan University, Shanghai, Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, and Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Fudan University, Shanghai, Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, and Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Fudan University, Shanghai, Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, and Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Fudan University, Shanghai
Keywords:Bispecific antibody, PD-1, c-MET, IFN-gamma, T cells, tumor.
Abstract:Background: Bispecific antibodies, BsAbs, are molecules with the ability to bind to two
different epitopes on the same or different antigens. c-MET, cellular-mesenchymal to epithelial
transition factor, is deregulated in many types of human malignancies. Abnormal c-MET activation
in cancer correlates with poor prognosis. PD-1, programmed death-1, is an additional inhibitory
receptor expressed by T cells. Blocking the interactions between PD-1 and PD-L1 has emerged as a
promising immunotherapy for treating cancer.
Objectives: The goal of this study was to identify a novel bispecific antibody targeting both c-MET
and PD-1 as an anti-cancer therapeutic candidate.
Methods: The BsAb was produced using 293E expression system and purified by Protein A affinity
chromatography. Then the binding specificity and affinity of the BsAb was examined by FACS and
biolayer light interferometry. The ability of the BsAb to inhibit the proliferation of tuman cells was
measured using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit; the potential
signaling pathway involved was identified by Western Blot. Cytokine secreted by PHA-L stimulated
PBMC was measured by ELISA. Effects of BsAb on PBMC-mediated lysis of MKN45 cells
was measured by LDH cytotoxicity assay.
Results: Based on the original sequences of PD-1 and c-MET mAb, a BsAb gene was designed,
cloned into pCEP4 vector for expression in 293E cells. The BsAb was obtained after purification of
the cell culture supernatant. It can bind to PD-1 and c-MET simultaneously, the calculated affinity
was 11.5 nM for PD-1 and 9.09 nM for c-MET. The BsAb enhanced IFN-γ production over control
IgG by 2-3 folds. It also inhibit the c-MET pathway activation and the proliferation of tumor cells
significantly, comparable to JnJ-38877605. The BsAb showed dose-dependent cytotoxic activity
against MKN45 cells.
Conclusion: Our results indicated that a novel BsAb recognizing PD-1 and c-MET was successfully
generated. It could redirect T cells to kill tumor cells, while retaining its inherent ability to
restore T cells and inhibit tumor cells. With this potential, this BsAb could be developed as a therapeutic
candidate for the treatment of various solid tumors.
