In vitro and in vivo properties of a dimeric bispecific single-chain antibody IgG-fusion protein for depletion of CCR2+ target cells in mice.
The chemokine receptor CCR2 is highly expressed on leukocytes in several inflammatory diseases of both mice and men. Apart from blockade of CCR2 to prevent chemokine-dependent cell migration, depletion of CCR2(+) cells might be a promising strategy for treatment of inflammatory diseases. We therefore designed a bispecific antibody construct with the ability to deplete CCR2(+) target cells in vitro and in vivo. The bispecific antibody construct consists of two single-chain antibody variable fragments (scFv) - one recognizing murine CD3epsilon and the other recognizing murine CCR2 - joined by a short linker and fused to a modified hinge region and the C(H)2 and C(H)3 domains of murine IgG1 for dimerization. The protein was expressed in mammalian cells and purified via its C-terminal histidine tail. In vitro this construct leads to efficient antigen-specific and costimulation-independent activation of T cells and strong lysis of CCR2(+) target cells. In vivo the construct induces an almost complete depletion of CCR2(+)CD11b(+) monocytes from the peripheral blood and spleens of BALB/c mice within 24 h. This recombinant protein construct is a dimeric, bispecific antibody with markedly improved serum levels compared to conventional bispecific single-chain antibodies and the ability to deplete CCR2(+)CD11b(+) monocytes in vivo.
Schneider
Martin A
MA
Medical Policlinic, University of Munich, Munich, Germany.
Brühl
Hilke
H
Wechselberger
Alexandra
A
Cihak
Josef
J
Stangassinger
Manfred
M
Schlöndorff
Detlef
D
Mack
Matthias
M
D000818:Animals; D018033:Antibodies, Bispecific / Q000276:immunology*; D016466:CHO Cells; D018920:Coculture Techniques; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D005434:Flow Cytometry; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000276:immunology*; D047589:Leukocyte Reduction Procedures / Q000379:methods*; D008213:Lymphocyte Activation / Q000276:immunology; D008212:Lymphocyte Depletion / Q000379:methods*; D051379:Mice; D054390:Receptors, CCR2; D019707:Receptors, Chemokine / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000276:immunology
D018033:Antibodies, Bispecific; C515335:Ccr2 protein, mouse; D017931:DNA Primers; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D054390:Receptors, CCR2; D019707:Receptors, Chemokine; D011994:Recombinant Proteins