Abstract
Carcinoembryonic antigen (CEA) is the only blood based protein biomarker at present, used for preoperative screening of advanced colorectal cancer (CRC) patients to determine the appropriate curative treatments and post-surveillance screening for tumour recurrence. Current diagnostics for CRC detection have several limitations and development of a highly sensitive, specific and rapid diagnostic device is required. The majority of such devices developed to date are antibody-based and suffer from shortcomings including multimeric binding, cost and difficulties in mass production. To circumvent antibody-derived limitations, the present study focused on the development of Affimer proteins as a novel alternative binding reagent for CEA detection. Here, we describe the selection, from a phage display library, of Affimers specific to CEA protein. Characterization of three anti-CEA Affimers reveal that these bind specifically and selectively to protein epitopes of CEA from cell culture lysate and on fixed cells. Kinetic binding analysis by SPR show that the Affimers bind to CEA with high affinity and within the nM range. Therefore, they have substantial potential for used as novel affinity reagents in diagnostic imaging, targeted CRC therapy, affinity purification and biosensor applications.
Highlights
Colorectal cancer (CRC) is the fourth leading cause of cancer-related death and the third most commonly diagnosed malignancy w orldwide[1]
48 randomly picked positive Affimer clones were evaluated for their binding ability to Carcinoembryonic antigen (CEA) by phage enzyme-linked immunosorbent assay (ELISA) (Fig. 1A) and subsequently their DNA was sent for DNA sequencing to allow analysis of the binding loop sequences
The purity and molecular mass of the Affimer proteins was determined using liquid chromatography mass spectrometry (LC–MS) analysis and results showed that all proteins were pure and the molecular masses of CEA-Affimer-I, II and III were 12,676, 12, 499 and 12,612 Da, respectively (Fig. 1B–D)
Summary
Colorectal cancer (CRC) is the fourth leading cause of cancer-related death and the third most commonly diagnosed malignancy w orldwide[1]. Antibodies have been used as excellent natural binding reagents in therapeutic and diagnostic applications They are known for their high specificity and sensitivity, strong affinity towards their target and long serum half-life[7,8]. The latter is a crucial feature of targeted drug carriers and biotherapeutic agents Their complex protein structure, batch-to-batch variability, expensive and slow production, complex chemical modification for oriented immobilisation in immunoassay applications and poor tissue penetration due to their size (~ 150 KDa)[9] have rendered them of limited use in some biomedical and bioanalytical applications. There have been several reports on the development of anti-CEA reagents from recombinant antibody technology[26,27], based on the promising potential of Affimers as alternative affinity reagents, we postulated that generation of CEA binding Affimers could facilitate the acceleration of diagnostics and treatment of CRC and other cancers
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