<br>Sortase 介导的位点特异性偶联制备氟 18 标记的纳米抗体,Bioconjugate Chemistry

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Sortase 介导的位点特异性偶联制备氟 18 标记的纳米抗体
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2024-08-22 , DOI: 10.1021/acs.bioconjchem.4c00264
Falguni Basuli ₁ , Jianfeng Shi ₁ , Eric Lindberg ₁ , Stanley Fayn _{2,

3,

4} , Woonghee Lee _{2,

3} , Mitchell Ho ₅ , Dima A Hammoud ₆ , Ross W Cheloha ₇ , Rolf E Swenson ₁ , Freddy E Escorcia _{2,

3}

Affiliation

单域抗体或纳米抗体（Nbs）因其出色的亲和力、特异性和从血液中快速清除而成为有前途用于分子成像的生物分子。鉴于它们的血液半衰期短，将 Nbs 与短寿命成像放射性同位素配对是可取的。由于氟 18 （¹⁸F）通常用于临床成像，因此它是 Nbs 的一种有吸引力的放射性同位素。我们报道了一种应用于三种纳米抗体的新型基于分选酶的位点特异性 ¹⁸F 标记方法。标记的纳米抗体通过一个罐中的两步间接放射性标记方法合成，或者使用放射性标记的螯合剂（H-GGGK（（±）-Al[¹⁸F]FH₃RESCA）-NH₂）或未标记的螯合剂（H-GGGK（（±）-H₃RESCA）-NH₂）的分选酶介导的偶联合成标记的纳米抗体，然后用 Al[¹⁸F]F 标记，分别。总放射化学产率在 70 分钟内为 15-43%（n = 22，衰变校正）（间接标记），在 50 分钟内为 23-58%（n = 12，衰变校正）（直接标记）。通过两种方法制备的标记纳米抗体的放射化学纯度为 >98%，比活性为 400–600 Ci/mmol （n = 22），并且在生理条件下表现出优异的稳定性。这种简单、位点特异性、可重现且可推广的 ¹⁸F 标记方法来制备纳米抗体（Nb-Al[¹⁸F]F-RESCA）或其他低分子量生物分子，可以很容易地在各种环境中用于临床前和临床研究。

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Sortase-Mediated Site-Specific Conjugation to Prepare Fluorine-18-Labeled Nanobodies

Single-domain antibodies, or nanobodies (Nbs), are promising biomolecules for use in molecular imaging due to their excellent affinity, specificity, and fast clearance from the blood. Given their short blood half-life, pairing Nbs with short-lived imaging radioisotopes is desirable. Because fluorine-18 (¹⁸F) is routinely used for clinical imaging, it is an attractive radioisotope for Nbs. We report a novel sortase-based, site-specific ¹⁸F-labeling method applied to three nanobodies. Labeled nanobodies were synthesized either by a two-step indirect radiolabeling method in one pot or by a one-step direct labeling method using a sortase-mediated conjugation of either the radiolabeled chelator (H-GGGK((±)-Al[¹⁸F]FH₃RESCA)-NH₂) or the unlabeled chelator (H-GGGK((±)-H₃RESCA)-NH₂) followed by labeling with Al[¹⁸F]F, respectively. The overall radiochemical yields were 15–43% (n = 22, decay-corrected) in 70 min (indirect labeling) and 23–58% (n = 12, decay-corrected) in 50 min (direct labeling). The radiochemical purities of the labeled nanobodies prepared by both methods were >98% with a specific activity of 400–600 Ci/mmol (n = 22) for each of the three Nbs tested and exhibited excellent stability profiles under physiological conditions. This simple, site-specific, reproducible, and generalizable ¹⁸F-labeling method to prepare nanobodies (Nb-Al[¹⁸F]F-RESCA) or other low molecular weight biomolecules can easily be adopted in various settings for preclinical and clinical studies.

更新日期：2024-08-22

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