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Background <p>Mesothelin is a 40&#xa0;kDa glycoprotein overexpressed in several cancers, including triple-negative breast cancer (TNBC). The anti-mesothelin single-domain antibody (sdAb, or nanobody) A1 can serve as a radio-theranostic agent, but random DOTA conjugation on lysines yields heterogeneous products.</p> Results <p>We reengineered A1-His by directed mutagenesis to produce four single-lysine variants (A1K1-His, A1K2-His, A1K3-His, and A1K4-His). Each was site-specifically conjugated with p-SCN-Bn-DOTA, radiolabeled with <sup>68</sup>Ga, and evaluated by PET imaging in mice bearing HCC70 TNBC xenografts, followed by ex vivo biodistribution at 1&#xa0;h post-injection. All mutants were successfully produced and site-specifically conjugated. A1K1-His showed lower conjugation efficiency and increased liver/spleen retention, whereas A1K3-His exhibited reduced stability. A1K2-His and A1K4-His performed best overall. Removing the His-tag and administering gelofusin further lowered renal uptake. Notably, A1K2 displayed tumor-to-kidney and tumor-to-liver ratios 2.4 and 1.9 times higher, respectively, than A1K4 (<i>p</i> &lt; 0.01).</p> Conclusions <p>For the first time, site-specific DOTA conjugation using sdAb derivatives containing a single lysine was achieved, avoiding the production of mixed final compounds. These findings identify <sup>68</sup>Ga-DOTA-A1K2 as the leading candidate for mesothelin-expressing tumor imaging with minimal off-target uptake. Ongoing studies will assess its therapeutic utility with <sup>177</sup>Lu-DOTA-A1K2. Since these four lysines are conserved in many sdAbs, this strategy may be broadly applicable for site-specific sdAb labeling.</p>

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Enhancing theranostic potential of anti-mesothelin sdAb through site-specific labeling at a unique conserved lysine by molecular engineering

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年4月28日

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  • Émilien N’Guessan,
  • Florian Raes,
  • Mitra Ahmadi,
  • Sandrine Bacot,
  • Laurent Dumas,
  • Julien Leenhardt,
  • Marlène Debiossat,
  • Clémence André,
  • Jean-Luc Lenormand,
  • Catherine Ghezzi,
  • Daniel Fagret,
  • Charlotte Lombardi,
  • Alexis Broisat

摘要

Background

Mesothelin is a 40 kDa glycoprotein overexpressed in several cancers, including triple-negative breast cancer (TNBC). The anti-mesothelin single-domain antibody (sdAb, or nanobody) A1 can serve as a radio-theranostic agent, but random DOTA conjugation on lysines yields heterogeneous products.

Results

We reengineered A1-His by directed mutagenesis to produce four single-lysine variants (A1K1-His, A1K2-His, A1K3-His, and A1K4-His). Each was site-specifically conjugated with p-SCN-Bn-DOTA, radiolabeled with 68Ga, and evaluated by PET imaging in mice bearing HCC70 TNBC xenografts, followed by ex vivo biodistribution at 1 h post-injection. All mutants were successfully produced and site-specifically conjugated. A1K1-His showed lower conjugation efficiency and increased liver/spleen retention, whereas A1K3-His exhibited reduced stability. A1K2-His and A1K4-His performed best overall. Removing the His-tag and administering gelofusin further lowered renal uptake. Notably, A1K2 displayed tumor-to-kidney and tumor-to-liver ratios 2.4 and 1.9 times higher, respectively, than A1K4 (p < 0.01).

Conclusions

For the first time, site-specific DOTA conjugation using sdAb derivatives containing a single lysine was achieved, avoiding the production of mixed final compounds. These findings identify 68Ga-DOTA-A1K2 as the leading candidate for mesothelin-expressing tumor imaging with minimal off-target uptake. Ongoing studies will assess its therapeutic utility with 177Lu-DOTA-A1K2. Since these four lysines are conserved in many sdAbs, this strategy may be broadly applicable for site-specific sdAb labeling.