Engineering of monobody conjugates for human EphA2-specific optical imaging

Abstract

In a previous study, we developed an E1 monobody specific for the tumor biomarker hEphA2 [PLoS ONE (2015) 10(7): e0132976]. E1 showed potential as a molecular probe for in vitro and in vivo targeting of cancers overexpressing hEphA2. In the present study, we constructed expression vectors for E1 conjugated to optical reporters such as Renilla luciferase variant 8 (Rluc8) or enhanced green fluorescent protein (EGFP) and purified such recombinant proteins by affinity chromatography in E. coli. E1-Rluc8 and E1-EGFP specifically bound to hEphA2 in human prostate cancer PC3 cells but not in human cervical cancer HeLa cells, which express hEphA2 at high and low levels, respectively. These recombinant proteins maintained >40% activity in mouse serum at 24 h. In vivo optical imaging for 24 h did not detect E1-EGFP signals, whereas E1-Rluc8 showed tumor-specific luminescence signals in PC3 but not in HeLa xenograft mice. E1-Rluc8 signals were detected at 4 h, peaked at 12 h, and were undetectable at 24 h. These results suggest the potential of E1-Rluc8 as an EphA2-specific optical imaging agent.

Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy

“…CRT3-Rluc8, CRT4-Rluc8, and #DGR-Rluc8 showed similar photon counts at each time-point, with a maximum value of 2.3 × 10 7 photons/sec/cm 2 /sr. Their luciferase activities decreased to approximately 40% of the control after 12 h and to 24% after 24 h. This stability data is similar to that of other Rluc8-fused monobodies in mouse serum [ 52 ].…”

“…These two monobodies showed similar stability in serum (an approximately 12 h half-life). The stability of CRT3 and CRT4 in serum is similar to the reported stability of monobodies against other targets [ 52 ]. Therefore, our peptide-grafted monobodies showed the expected high affinity and stability.…”

“…After agarose gel separation and purification of PCR fragments, monobody genes were amplified with the 94 old-F: 94 old-R primer set against a mixture of BC, DE, and FG fragments. The amplified fragments were digested with Nhe 1 and Bam H1 enzymes and cloned into the same sites of the pETh vector [ 52 ]. The resulting plasmids were named according to insertion peptide sequences in FG and BC loops as pETh-CRT1, pETh-CRT2, pETh-CRT3, pETh-CRT4, pETh-CRT5, and pETh-CRT6, respectively (abbreviated as CRT1–CRT6).…”

“…First, the monobody megaprimers were amplified against the pETh-CRT plasmid with the T7-F: FG-R primer set for 30 cycles. After purification in agarose gels, EMP PCR was performed with the T7-F: T7-R primer set using a mixture of the megaprimers and pETh-E1-Rluc8 [ 52 ]. The amplified PCR fragments were phosphorylated with polynucleotide kinase, digested with Dpn I, and ligated with T4 DNA ligase.…”

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

“…As a sensory component of hybrids, various biospecific polypeptides were applied—antibodies or their fragments [ 111 , 112 , 113 , 114 ], antigens [ 115 , 116 , 117 , 118 ], polypeptides with special binding ability [ 119 , 120 , 121 , 122 ], GFP and its color variants [ 123 , 124 , 125 , 126 , 127 , 128 , 129 ], etc. Based on the phenomenon of assembling split luciferase derivatives, a whole spectrum of homogeneous methods has been developed [ 125 , 126 , 127 , 128 , 129 ] and applied for studying protein–protein/ligand interaction [ 130 , 131 , 132 , 133 ], protein activity [ 134 , 135 ], and protein folding [ 136 , 137 ].…”