Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics

• Published in: Journal of molecular biology Vol. 293; no. 1; pp. 41 - 56
• Main Authors: Kipriyanov, Sergey M, Moldenhauer, Gerhard, Schuhmacher, Jochen, Cochlovius, Björn, Von der Lieth, Claus-Wilhelm, Matys, E.Ronald, Little, Melvyn
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 15.10.1999
• Subjects: Animals; Antibodies, Bispecific - genetics; Antibodies, Bispecific - pharmacokinetics; Antibodies, Neoplasm - genetics; Antibodies, Neoplasm - metabolism; Antigens, CD19 - immunology; Antigens, Neoplasm - immunology; bispecific diabody; Cancer Vaccines - pharmacokinetics; CD3 Complex - immunology; Cell Line; Dimerization; domain swapping; Escherichia coli; Escherichia coli - metabolism; Flow Cytometry; human CD19; human CD3; Humans; Immunoglobulin Fragments - immunology; Immunoglobulin Fragments - metabolism; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - immunology; Jurkat Cells; Lymphoma, B-Cell; Mice; Models, Molecular; Neoplasms - immunology; Neoplasms - therapy; Protein Binding; Recombinant Proteins - immunology; Recombinant Proteins - metabolism; tandem diabody; LL; TCR; IMAC; Tandab; sc-diabody; human CD19; BsAb; AUC; domain swapping; scFv; bispecific diabody; SL; tandem diabody; human CD3
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1999.3156

To increase the valency, stability and therapeutic potential of bispecific antibodies, we designed a novel recombinant molecule that is bispecific and tetravalent. It was constructed by linking four antibody variable domains (V H and V L) with specificities for human CD3 (T cell antigen) or CD19 (B cell marker) into a single chain construct. After expression in Escherichia coli, intramolecularly folded bivalent bispecific antibodies with a mass of 57 kDa (single chain diabodies) and tetravalent bispecific dimers with a molecular mass of 114 kDa (tandem diabodies) could be isolated from the soluble periplasmic extracts. The relative amount of tandem diabodies proved to be dependent on the length of the linker in the middle of the chain and bacterial growth conditions. Compared to a previously constructed heterodimeric CD3 × CD19 diabody, the tandem diabodies exhibited a higher apparent affinity and slower dissociation from both CD3 + and CD19 + cells. They were also more effective than diabodies in inducing T cell proliferation in the presence of tumor cells and in inducing the lysis of CD19 + cells in the presence of activated human PBL. Incubated in human serum at 37 °C, the tandem diabody retained 90 % of its antigen binding activity after 24 hours and 40 % after one week. In vivo experiments indicated a higher stability and longer blood retention of tandem diabodies compared to single chain Fv fragments and diabodies, properties that are particularly important for potential clinical applications.