Immunosuppressive properties of anti-CD3 single-chain Fv and diabody

• Published in: Journal of immunological methods 2004-02, Vol.285 (1), p.111-127
• Main Authors: Le Gall, Fabrice, Reusch, Uwe, Moldenhauer, Gerhard, Little, Melvyn, Kipriyanov, Sergey M.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antibody engineering; Base Sequence; Biological and medical sciences; CD3 Complex - immunology; Cell Division; Cytokines - biosynthesis; Diabody; Dimerization; DNA, Complementary - genetics; Drug Design; Drug Stability; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Human CD3; Humans; Immunoglobulin Variable Region - chemistry; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - pharmacology; Immunosuppression; Immunosuppressive Agents - chemistry; Immunosuppressive Agents - pharmacology; Jurkat Cells; Mice; Models, Molecular; Molecular immunology; Molecular Sequence Data; Muromonab-CD3 - chemistry; Muromonab-CD3 - genetics; Muromonab-CD3 - pharmacology; Receptors, Antigen, T-Cell - metabolism; Receptors, Interleukin-2 - metabolism; Single-chain Fvs; T lymphocyte; T-Lymphocytes - cytology; T-Lymphocytes - immunology; Techniques
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2003.11.007

The mouse anti-human CD3 monoclonal antibody OKT3 is a potent immunosuppressive agent used in clinical transplantation. However, OKT3 therapy is associated with unpleasant and often serious side effects which appear to result from cytokine release, complement activation and a human anti-mouse antibody (HAMA) response. To decrease these adverse side effects, we constructed antibody fragments comprising OKT3 variable domains without any constant domains. Single-chain Fv (scFv) monomers, dimers and trimers were generated by changing the linker length between the V H and V L domains. The linkers used were the natural extensions of the V H into the C H1 domain. The dimeric molecules (diabodies) demonstrated the best CD3-binding activity. The diabody with the six amino acid linker was produced in bacteria with a tenfold higher yield than other scFvs and possessed CD3-binding affinity approaching that of the parental mAb. In contrast to OKT3 mAb, the anti-CD3 diabody and scFv monomer did not cause any T-cell activation and cytokine release in vitro, while demonstrating CD3 modulation. In mixed lymphocyte cultures, both diabody and scFv, but not the monoclonal antibody OKT3, were able to suppress T-cell activation and secretion of IL-2 and IFN-γ in a dose-dependent manner. The anti-CD3 diabody may provide a potent immunosuppressive drug with low toxicity and immunogenicity.