Inhibiting HER3-mediated tumor cell growth with affibody molecules engineered to low picomolar affinity by position-directed error-prone PCR-like diversification.
Inhibiting HER3-mediated tumor cell growth with affibody molecules engineered to low picomolar affinity by position-directed error-prone PCR-like diversification.
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DOI：
10.1371/journal.pone.0062791
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Löfblom J
中科院分区：
文献类型：
--
作者：
Malm M;Kronqvist N;Lindberg H;Gudmundsdotter L;Bass T;Frejd FY;Höidén-Guthenberg I;Varasteh Z;Orlova A;Tolmachev V;Ståhl S;Löfblom J
HER3受体与各种癌症的进展以及对几种目前使用的药物的耐药性有关，因此是开发新疗法的潜在靶点。我们先前已经产生了抑制heregulin诱导的HER3途径信号传导的亲和体分子。本研究的目的是提高结合剂的亲和力，以有望提高受体抑制功效，并实现肿瘤中受体介导的高摄取。我们探索了一种亲和体分子亲和力成熟的新策略，该策略基于丙氨酸扫描，然后设计文库多样化以模拟易错PCR反应的结果，但完全控制突变位置，因此偏差较小。使用成熟文库的细菌表面展示和流式细胞术分选，对HER3的亲和力提高了30倍以上，降至21 pM。亲和力是已经报道的亲和体分子的较高亲和力之一，并且我们相信成熟策略应该普遍适用于亲和力蛋白的改进。新的粘合剂还表现出改进的热稳定性以及变性后的完全再折叠。此外，与先前表现最好的克隆相比，HER3阳性乳腺癌细胞的配体诱导增殖的抑制提高了两个数量级以上。放射性标记的亲和体分子在体外显示出对许多HER3阳性细胞系的特异性靶向以及在体内小鼠模型中对HER3的靶向，并且代表了用于靶向治疗和诊断的未来开发的有希望的候选物。
The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 pM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.
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影响因子：
3.7
作者：
Kong A;Calleja V;Leboucher P;Harris A;Parker PJ;Larijani B
通讯作者：
Larijani B
影响因子：
3.7
作者：
Göstring L;Malm M;Höidén-Guthenberg I;Frejd FY;Ståhl S;Löfblom J;Gedda L
通讯作者：
Gedda L
影响因子：
4.7
作者：
Ekerljung, Lina;Wallberg, Helena;Gedda, Lars
通讯作者：
Gedda, Lars
影响因子：
2.4
作者：
Kronqvist, Nina;Lofblom, John;Stahl, Stefan
通讯作者：
Stahl, Stefan
DOI：
10.1073/pnas.91.17.8132
发表时间：
1994-08-16
影响因子：
11.1
作者：
GUY, PM;PLATKO, JV;CARRAWAY, KL
通讯作者：
CARRAWAY, KL