期刊
BIOCONJUGATE CHEMISTRY
卷 15, 期 1, 页码 16-26出版社
AMER CHEMICAL SOC
DOI: 10.1021/bc030018+
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- NCI NIH HHS [P01 CA47829] Funding Source: Medline
- NATIONAL CANCER INSTITUTE [P01CA047829] Funding Source: NIH RePORTER
ScFv recombinant antibody fragments can provide specific tumor binding modules for targeting drugs. In the process of building multimeric tumor targeting pharmaceuticals, a prerequisite is the conservation of functional scFv antigen binding domains, thereby excluding scFv random conjugation to a carrier molecule or to another scFv. The pCANTAB 5E phage display/expression vector was genetically engineered to express any scFv gene as scFv with an additional C-terminal cysteine (scFv-Cys) such that the specific conjugation site is removed from the binding domain. Selected scFvs derived from an anti-MUC-1 scFv phage library were expressed in pCANTAB 5E and its modified version pCANTAB 5E Cys vectors, and compared for key characteristics. Production yields of scFv and scFv-Cys in shaker flask and biofermentor were compared. In the absence of a reducing agent, stable dimers (covalent scFv homodimers (scFv-Cys)(2)) were the major form of scFv-Cys. These diabodies provided substantial signal enhancement for immunohistochemical staining of tissues. In the presence of a reducing agent, scFv-Cys molecules remained monomeric, with the free SH available for conjugation to a PEG(maleimide)(2) scaffold to form immunoreactive PEG(scFv)(2) bioconjugates. ScFv expression from pCANTAB 5E Cys allowed for the production of soluble scFv-Cys protein from E.coli, either as stable scFv-Cys or (scFv-Cys)(2). ScFv-Cys can be used for conjugation to PEG to form bivalent PEG (scFv-Cys)(2) molecules or used as (scFv-Cys)(2) for increased sensitivity in IHC.
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