title: The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-Cβ antibody.
abstract: The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-Cβ antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 × 10(-5)M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-Cβ antibody, its binding affinity for p/MHC increased by 5-fold (2.2 × 10(-6)M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-Cβ antibody, which is probably due to the stabilization of the Vα/Vβ region of the TCR. These findings provide new insights into the binding of sTCRs to p/MHCs and will hopefully be instrumental in establishing functional sTCR as a diagnostic and therapeutic tool for cancer.
authors.affiliation: Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Ozawa
authors.forename: Masae
authors.initials: M
authors.lastname: Horii
authors.forename: Eiji
authors.initials: E
authors.lastname: Kobayashi
authors.forename: Aishun
authors.initials: A
authors.lastname: Jin
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kishi
authors.forename: Atsushi
authors.initials: A
authors.lastname: Muraguchi
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000276:immunology*; D007518:Isoantibodies / Q000737:chemistry / Q000276:immunology*; D017434:Protein Structure, Tertiary; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology*
chemical_list: D000951:Antigens, Neoplasm; D007141:Immunoglobulin Fc Fragments; D007518:Isoantibodies; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C051138:anti-c antibody