Dual Site-Specific Chemoenzymatic Antibody Fragment Conjugation Using CRISPR-Based Hybridoma Engineering,Bioconjugate Chemistry

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Dual Site-Specific Chemoenzymatic Antibody Fragment Conjugation Using CRISPR-Based Hybridoma Engineering
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2021-01-21 , DOI: 10.1021/acs.bioconjchem.0c00673
Camille M Le Gall _{1,

2} , Johan M S van der Schoot _{1,

2} , Iván Ramos-Tomillero _{1,

3} , Melek Parlak Khalily ₄ , Floris J van Dalen ₁ , Zacharias Wijfjes _{1,

3} , Liyan Smeding ₁ , Duco van Dalen ₁ , Anna Cammarata ₁ , Kimberly M Bonger _{3,

4} , Carl G Figdor _{1,

2,

3} , Ferenc A Scheeren ₅ , Martijn Verdoes _{1,

3}

Affiliation

Functionalized antibodies and antibody fragments have found applications in the fields of biomedical imaging, theranostics, and antibody–drug conjugates (ADC). In addition, therapeutic and theranostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labeling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogeneous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab′ fragments bearing two distinct site-specific labeling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab′ molecule (DTFab′), which can be easily isolated. To demonstrate feasibility, we functionalized the DTFab′ with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theranostics, and next-generation ADCs.

中文翻译：

使用基于 CRISPR 的杂交瘤工程进行双位点特异性化学酶抗体片段缀合

功能化抗体和抗体片段已在生物医学成像、治疗诊断学和抗体药物偶联物 (ADC) 领域得到应用。此外，治疗和治疗诊断方法受益于将不止一种类型的货物输送到靶细胞的可能性，这进一步挑战了随机标记策略。因此，需要生物缀合方法以可重复地获得具有多种不同货物分子的确定的均质缀合物，而不会影响目标亲和力。在这里，我们描述了一种简单的基于 CRISPR/Cas9 的策略，以快速设计杂交瘤细胞以分泌带有两个不同位点特异性标记基序的 Fab' 片段，这些基序可以被两个不同的分选酶 A 突变体分别修饰。我们表明，重链 (HC) 和轻链 (LC) 基因座的顺序遗传编辑能够生成稳定的细胞系，该细胞系分泌双标记的 Fab' 分子 (DTFab')，该分子可以轻松分离。为了证明可行性，我们以特定于站点的方式用两种不同的货物对 DTFab' 进行了功能化。该技术平台将在多模态显像剂、治疗诊断学和下一代 ADC 的开发中发挥重要作用。

更新日期：2021-02-17

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