An engineered lipocalin specific for CTLA-4 reveals a combining site with structural and conformational features similar to antibodies
An engineered lipocalin specific for CTLA-4 reveals a combining site with structural and conformational features similar to antibodies
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DOI：
10.1073/pnas.0813399106
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发表时间：
2009-05-19
影响因子：
11.1
通讯作者：
Skerra, A.
中科院分区：
文献类型：
--
作者：
Schoenfeld, D.;Matschiner, G.;Skerra, A.
能够对蛋白质进行特异性分子识别的生物分子试剂在基础研究以及医学领域都起着至关重要的作用。到目前为止，抗体（免疫球蛋白）已被广泛用于此目的。它们的主要特征是由6个高变环组成的大量抗原结合位点。然而，由于其复杂的结构、较大的尺寸和多种功能，抗体存在一些实际的缺点。另一方面，脂质运载蛋白已经进化成为一个主要用于结合小分子的蛋白质家族。在此，我们表明一种由人脂质运载蛋白2（Lcn2）改造而来的脂质运载蛋白能够以亚纳摩尔级亲和力特异性结合T细胞共受体细胞毒性T淋巴细胞相关抗原4（CTLA - 4）这一特定蛋白质靶点。晶体学分析显示，其由4个可变环形成的重新塑造的杯状结合位点与该“抗原”具有完美的结构互补性。此外，与未结合复合物的改造脂质运载蛋白的晶体结构相比，表明存在一种显著的诱导契合机制，这是一种迄今为止被认为是抗体所特有的现象。通过识别CTLA - 4上与抗原呈递细胞上的反受体B7.1/B7.2相互作用的相同表位，改造后的Lcn2表现出强大的跨物种拮抗活性，其生物学效应与一种CTLA - 4特异性抗体相当。凭借其在体内对T细胞已被证实的刺激活性，阻断CTLA - 4的脂质运载蛋白为癌症和传染病的免疫治疗提供了潜力。除此之外，具有改造的抗原结合位点的脂质运载蛋白，即所谓的“抗运载蛋白”（Anticalins），提供了一类小（约180个残基）、结构简单且稳定的结合蛋白，在生命科学领域具有普遍的应用。
Biomolecular reagents that enable the specific molecular recognition of proteins play a crucial role in basic research as well as medicine. Up to now, antibodies (immunoglobulins) have been widely used for this purpose. Their predominant feature is the vast repertoire of antigen-binding sites that arise from a set of 6 hypervariable loops. However, antibodies suffer from practical disadvantages because of their complicated architecture, large size, and multiple functions. The lipocalins, on the other hand, have evolved as a protein family that primarily serves for the binding of small molecules. Here, we show that an engineered lipocalin, derived from human Lcn2, can specifically bind the T cell coreceptor CTLA-4 as a prescribed protein target with subnanomolar affinity. Crystallographic analysis reveals that its reshaped cup-like binding site, which is formed by 4 variable loops, provides perfect structural complementarity with this "antigen.'' Furthermore, comparison with the crystal structure of the uncomplexed engineered lipocalin indicates a pronounced induced-fit mechanism, a phenomenon so far considered typical for antibodies. By recognizing the same epitope on CTLA-4 that interacts with the counterreceptors B7.1/B7.2 on antigen-presenting cells the engineered Lcn2 exhibits strong, cross-species antagonistic activity, as evidenced by biological effects comparable with a CTLA-4-specific antibody. With its proven stimulatory activity on T cells in vivo, the CTLA-4 blocking lipocalin offers potential for immunotherapy of cancer and infectious disease. Beyond that, lipocalins with engineered antigen-binding sites, so-called Anticalins, provide a class of small (approximate to 180 residues), structurally simple, and robust binding proteins with applications in the life sciences in general.