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Optimal His-Tag Design for Efficient [^99mTc(CO)₃]⁺ and [¹⁸⁸Re(CO)₃]⁺ Labeling of Proteins for Molecular Imaging and Radionuclide Therapy by Analysis of Peptide Arrays.

ABSTRACT: Hexahistidine tags (His-tags), incorporated into recombinant proteins to facilitate purification using metal-affinity chromatography, are useful binding sites for radiolabeling with [^99mTc(CO)₃]⁺ and [¹⁸⁸Re(CO)₃]⁺ for molecular imaging and radionuclide therapy. Labeling efficiencies vary unpredictably, and the method is therefore not universally useful. To overcome this, we have made quantitative comparisons of radiolabeling of a bespoke Celluspots array library of 382 His-tag-containing peptide sequences with [^99mTc(CO)₃]⁺ and [¹⁸⁸Re(CO)₃]⁺ to identify key features that enhance labeling. A selected sequence with 10-fold enhanced labeling efficiency compared to the most effective literature-reported sequences was incorporated into an exemplar protein and compared biologically with non-optimized analogues, in vitro and in vivo. Optimal labeling with either [^99mTc(CO)₃]⁺ or [¹⁸⁸Re(CO)₃]⁺ required six consecutive His residues in the protein sequence, surrounded by several positively charged residues (Arg or Lys), and the presence of phosphate in the buffer. Cys or Met residues in the sequence were beneficial, to a lesser extent. Negatively charged residues were deleterious to labeling. His-tags with adjacent positively charged residues could be labeled as much as 40 times more efficiently than those with adjacent negatively charged residues. ³¹P NMR of [Re(CO)₃(H₂O)₃]⁺ and electrophoresis of solutions of [^99mTc(CO)₃(H₂O)₃]⁺ suggest that phosphate bridges form between cationic residues and the cationic metal synthon during labeling. The trial optimized protein, a scFv targeted to the PSMA antigen expressed in prostate cancer, was readily labeled in >95% radiochemical yield, without the need for subsequent purification. Labeling occurred more quickly and to higher specific activity than comparable non-optimized proteins, while retaining specific binding to PSMA and prostate cancer in vivo. Thus, optimized His-tags greatly simplify radiolabeling of recombinant proteins making them potentially more widely and economically available for imaging and treating patients.

SUBMITTER: Williams JD

PROVIDER: S-EPMC7611356 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposeSentinel lymph node (SLN) biopsy has proven to reliably stage the clinically negative neck in early-stage oral squamous cell carcinoma (OSCC). [99mTc]Tc-tilmanocept may be of benefit in OSCC with complex lymphatic drainage patterns and close spatial relation to SLNs.MethodsA prospective within-patient evaluation study was designed to compare [99mTc]Tc-tilmanocept with [99mTc]Tc-nanocolloid for SLN detection. A total of 20 patients with early-stage OSCC were included, who underwent lymphoscintigraphy with both tracers. Both lymphoscintigraphic images of each patient were evaluated for SLN detection and radiotracer distribution at 2-4 h post-injection.ResultsThe injection site's remaining radioactivity was significantly lower for [99mTc]Tc-tilmanocept (29.9%), compared with [99mTc]Tc-nanocolloid (60.9%; p < 0.001). Radioactive uptake in SLNs was significantly lower for [99mTc]Tc-tilmanocept (1.95%) compared with [99mTc]Tc-nanocolloid (3.16%; p = 0.010). No significant difference was seen in SLN to injection site ratio in radioactivity between [99mTc]Tc-tilmanocept (0.066) and [99mTc]Tc-nanocolloid (0.054; p = 0.232). A median of 3.0 and 2.5 SLNs were identified with [99mTc]Tc-tilmanocept and [99mTc]Tc-nanocolloid, respectively (p = 0.297). Radioactive uptake in higher echelon nodes was not significantly different between [99mTc]Tc-tilmanocept (0.57%) and [99mTc]Tc-nanocolloid (0.86%) (p = 0.052). A median of 2.0 and 2.5 higher echelon nodes was identified with [99mTc]Tc-tilmanocept and [99mTc]Tc-nanocolloid, respectively (p = 0.083).Conclusion[99mTc]Tc-tilmanocept had a higher injection site clearance, but at the same time a lower uptake in the SLN, resulting in an SLN to injection site ratio, which was not significantly different from [99mTc]Tc-nanocolloid. The relatively low-radioactive uptake in SLNs of [99mTc]Tc-tilmanocept may limit intraoperative detection of SLNs, but can be overcome by a higher injection dose.

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Optimal His-Tag Design for Efficient [^99mTc(CO)₃]⁺ and [¹⁸⁸Re(CO)₃]⁺ Labeling of Proteins for Molecular Imaging and Radionuclide Therapy by Analysis of Peptide Arrays.

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Optimal His-Tag Design for Efficient [99mTc(CO)3]+ and [188Re(CO)3]+ Labeling of Proteins for Molecular Imaging and Radionuclide Therapy by Analysis of Peptide Arrays.

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OmicsDI is part of the ELIXIR infrastructure

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Optimal His-Tag Design for Efficient [^99mTc(CO)₃]⁺ and [¹⁸⁸Re(CO)₃]⁺ Labeling of Proteins for Molecular Imaging and Radionuclide Therapy by Analysis of Peptide Arrays.