Abstract
BackgroundCyclin-dependent kinase 4 (CDK4) when hyperactivated drives development and maintenance of most tumour types, thus prompting its use as an essential cancer treatment target and a diagnostic tool. Target-binding molecules, such as single-chain variable fragment (scFv) antibodies, hold tremendous potential for use in a wide range of cancer diagnostic and therapeutic applications.ResultsA human anti-CDK4 scFv antibody (AK2) derived from a human phage display library was expressed in soluble form in Escherichia coli and shown to be secreted into the culture supernatant. Next, soluble AK2 within culture supernatant was successfully purified using affinity chromatography then was shown, using enzyme-linked immunosorbent assays, to bind to recombinant human CDK4 with high affinity and specificity. Further analyses of AK2 interactions with intracellular components demonstrated that AK2 recognised and interacted specifically with endogenous CDK4 and thus could be useful for detection of CDK4 within tumour cells.ConclusionsA novel anti-CDK4 scFv antibody that can recognise and interact specifically with recombinant human CDK4 and endogenous CDK4 in tumour cells was expressed and purified successfully. These results suggest that the anti-CDK4 scFv antibody may serve as a new and promising tool for achieving CDK4-targeted diagnosis, prognosis and treatment of numerous types of cancers.
Highlights
The timing of eukaryotic cell cycle progression is precisely controlled by a regulatory network that primarily involves activities and specificities of cyclin-dependent kinases, including Cyclin-dependent kinase 4 (CDK4), which plays a key role in cell cycle initiation [1]
AK2 and determine its binding activity, the supernatant of an E. coli AK2/HB2151 culture was collected after overnight Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction of AK2 expression and was added to microtiter plates that had been precoated with recombinant human CDK4 (rhCDK4) or an irrelevant protein to conduct a non-competitive Enzyme linked immunosorbent assay (ELISA)
Binding of the abovementioned culture supernatant without AK2 to rhCDK4-coated wells was similar to binding to wells coated with irrelevant proteins (P > 0.05)
Summary
The timing of eukaryotic cell cycle progression is precisely controlled by a regulatory network that primarily involves activities and specificities of cyclin-dependent kinases, including CDK4 (cyclin-dependent kinase 4), which plays a key role in cell cycle initiation [1]. An observed association of cyclin D-CDK4/6 activity with tumour maintenance has been noted in several previous studies. Based on these results, research efforts are currently being directed toward the development of anti-cancer treatment strategies based on utilisation of CDK4 as a target [10]. Application and development of anti-tumour treatments targeting CDK4 still require more extensive and in-depth studies. New strategies are needed to solve the problem of emerging drug resistance and side effects associated with use of small molecule-based anti-cancer drugs [14,15,16,17]. Cyclin-dependent kinase 4 (CDK4) when hyperactivated drives development and maintenance of most tumour types, prompting its use as an essential cancer treatment target and a diagnostic tool. Target-binding molecules, such as single-chain variable fragment (scFv) antibodies, hold tremendous potential for use in a wide range of cancer diagnostic and therapeutic applications
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