Construction and Expression of Mouse-human Chimeric Antibody SZ-51 Specific for Activated Platelet P-selectin

Abstract

SummaryA murine monoclonal (mAb) SZ-51 specific for human P-selectin may be used for in vivo thrombus imaging and for the targeting of fibrinolytic agents to thrombi. In order to reduce the immunogenicity of the murine mAb SZ-51 in humans, we cloned and sequenced the cDNAs encoding the variable region of mAb SZ-51 in order to develop mouse/human chimeric reagents. The E. coli expression vector. pHENl-SZ51 Fab/Hu was constructed by fusing the variable regions of mAb SZ-51 with human IgG γICHI and Cκ genes. The constructs were introduced into E. coli HB2151 for expression of soluble chimeric Fab fragment. We also constructed two fusion products by joining the variable regions of mouse antibody to the appropriate constant regions of human Igγl and κ. These chimeras were cloned into two eukaryotic selectable expression vectors separately, which were then cotransfected into a non-Ig secreting murine myeloma line SP2/0 with lipofectin reagent. Six cell lines remained positive for Ig secretion. The highest producing cell line, which showed stable integration and expression at 5 mg/1 of culture, was selected for the large scale production of chimeric antibody. Immunoblotting analysis demonstrated that both of the chimeric antibodies (SZ51Fab/Hu, SZ51/Hu) in the culture supernatants, like the native mAb SZ-51, bind P-selectin. In addition, the whole chimeric antibody can compete for binding to activated platelets with murine SZ-51. Therefore, the SZ-51 chimeric antibody may be a potential agent for diagnosis and treatment of thrombotic diseases in the future.

An unmet clinical need: The history of thrombus imaging

“…Another monoclonal antibody (SZ-51) was developed against a platelet epitope, alpha granule membrane protein (GMP-140), which was later characterized to be P-selectin. 65–131 I-SZ-51 was initially shown to be effective against acute dog thrombus 66 and was later modified into a mouse-human chimeric ligand that was characterized in vitro. 65,67 However, both monoclonal approaches failed to progress significantly.…”

“…65–131 I-SZ-51 was initially shown to be effective against acute dog thrombus 66 and was later modified into a mouse-human chimeric ligand that was characterized in vitro. 65,67 However, both monoclonal approaches failed to progress significantly. In parallel with the events in fibrin targeting, platelet homing ligands targeting the GP IIbIIIa receptor took many forms over the years including: radiolabeled small molecule receptor antagonists, 68–70 disintegrins, 71–73 and peptides.…”

Radiation-Guided P-Selectin Antibody Targeted to Lung Cancer

“…However, very few recombinant antibodies are available to study the clinical function of P-selectin except for therapeutic attempts in the form of monoclonal antibodies and chimeric antibodies. 10,15,19,32,34 Presently, there have not been any studies using P-selectin as an imaging target in cancer, partly due to the lack of specific desired antibodies to the protein. For these reasons, the aim of this study is to develop antihuman P-selectin scFvs that can be used for radiation-guided tumor imaging.…”

New radiotracers for imaging of vascular targets in angiogenesis-related diseases

“…In one study, the light chain (LC) of SZ-51 (a mouse mAb against human P-selectin [210]) was expressed in P. pastoris [206]. SZ-LC was shown to bind activated platelet specifically.…”