Riso Mariapia, Shah Rohan N, Koide Akiko, Ruthenburg Alexander J, Koide Shohei, Hattori Takamitsu
Laura and Isaac Perlmutter Cancer Center, New York University Langone Health, New York, NY 10016.
Pritzker School of Medicine, The University of Chicago, Chicago, IL 60637.
Proc Natl Acad Sci U S A. 2025 Jan 7;122(1):e2411720121. doi: 10.1073/pnas.2411720121. Epub 2024 Dec 30.
Posttranslational modifications (PTMs) of proteins play critical roles in regulating many cellular events. Antibodies targeting site-specific PTMs are essential tools for detecting and enriching PTMs at sites of interest. However, fundamental difficulties in molecular recognition of both PTM and surrounding peptide sequence have hindered the efficient generation of highly sequence-specific anti-PTM antibodies. Furthermore, the widespread use of potentially inconsistent, nonrenewable, and molecularly undefined antibodies presents experimental challenges thought to contribute to the reproducibility problem in biomedical research. In this study, we describe the binding mode-guided development of a platform that efficiently generates potent and selective recombinant antibodies to PTMs that are molecularly defined and renewable. Our platform is built on our previous discovery of an unconventional binding mode of anti-PTM antibodies, antigen clasping, where two antigen binding sites cooperatively sandwich a single antigen, creating extensive interactions with the antigen and leading to high selectivity and potency. We designed the platform that generates clasping antibodies with two distinct binding units, resulting in efficient generation of antibodies to a set of trimethylated histone H3 with high levels of specificity and affinity. Performance comparison in chromatin immunoprecipitation, a common application in epigenomics, revealed that a clasping antibody to trimethylated histone H3 at lysine 27 exhibited superior specificity to a widely used conventional antibody and captured symmetric and asymmetric nucleosomes in a less biased manner. We further generated clasping antibodies to phosphotyrosine antigens by using the same principle. These results suggest the broad applicability of our platform to generating high-performance clasping antibodies to diverse PTMs.
蛋白质的翻译后修饰（PTM）在调节许多细胞事件中起着关键作用。靶向位点特异性PTM的抗体是检测和富集感兴趣位点PTM的重要工具。然而，对PTM及其周围肽序列进行分子识别存在的根本困难阻碍了高效产生高度序列特异性的抗PTM抗体。此外，广泛使用的可能不一致、不可再生且分子定义不明确的抗体带来了实验挑战，这些挑战被认为是生物医学研究中可重复性问题的原因之一。在本研究中，我们描述了一种平台的开发，该平台基于结合模式引导，能够高效产生针对分子定义且可再生的PTM的强效和选择性重组抗体。我们的平台基于我们之前发现的抗PTM抗体的一种非常规结合模式——抗原夹合，即两个抗原结合位点协同夹着单个抗原，与抗原产生广泛相互作用，从而导致高选择性和强效性。我们设计的平台能产生具有两个不同结合单元的夹合抗体，从而高效产生针对一组三甲基化组蛋白H3的抗体，且具有高水平的特异性和亲和力。在染色质免疫沉淀（表观基因组学中的一种常见应用）中的性能比较表明，针对赖氨酸27处三甲基化组蛋白H3的夹合抗体对广泛使用的传统抗体表现出更高的特异性，并且以较少偏差的方式捕获对称和不对称核小体。我们还利用相同原理产生了针对磷酸酪氨酸抗原的夹合抗体。这些结果表明我们的平台在产生针对多种PTM的高性能夹合抗体方面具有广泛的适用性。
