Integrins are transmembrane multi-conformation receptors that mediate interactions with the extracellular matrix. In cancer, integrins influence metastasis, proliferation, and survival. Collagen-binding integrin-α11/β1, a marker of aggressive tumors that is involved in stroma-tumor crosstalk, may be an attractive target for anti-cancer therapeutic antibodies. We performed selections with phage-displayed synthetic antibody libraries for binding to either purified integrin-α11/β1 or in situ on live cells. The in-situ strategy yielded many diverse antibodies, and strikingly, most of these antibodies did not recognize purified integrin-α11/β1. Conversely, none of the antibodies selected for binding to purified integrin-α11/β1 were able to efficiently recognize native cell-surface antigen. Most importantly, only the in-situ selection yielded functional antibodies that were able to compete with collagen-I for binding to cell-surface integrin-α11/β1, and thus inhibited cell adhesion. In-depth characterization of a subset of in situ-derived clones as full-length immunoglobulins revealed high affinity cellular binding and inhibitory activities in the single-digit nanomolar range. Moreover, the antibodies showed high selectivity for integrin-α11/β1 with minimal cross-reactivity for close homologs. Taken together, our findings highlight the advantages of in-situ selections for generation of anti-integrin antibodies optimized for recognition and inhibition of native cell-surface proteins, and our work establishes general methods that could be extended to many other membrane proteins.

中文翻译：

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原位抗体噬菌体展示可产生整合素 α11/β1 的最佳抑制剂。

整合素是跨膜多构象受体，介导与细胞外基质的相互作用。在癌症中，整合素影响转移、增殖和存活。胶原结合整合素-α11/β1 是参与基质-肿瘤串扰的侵袭性肿瘤的标志物，可能是抗癌治疗抗体的一个有吸引力的靶标。我们使用噬菌体展示的合成抗体文库进行选择，以结合纯化的整合素-α11/β1 或在活细胞上进行原位结合。原位策略产生了许多不同的抗体，令人惊讶的是，这些抗体中的大多数不识别纯化的整合素-α11/β1。相反，选择与纯化的整联蛋白-α11/β1 结合的抗体均不能有效识别天然细胞表面抗原。最重要的是，只有原位选择才能产生功能性抗体，这些抗体能够与 I 型胶原竞争与细胞表面整合素-α11/β1 的结合，从而抑制细胞粘附。作为全长免疫球蛋白的原位衍生克隆子集的深入表征揭示了单位数纳摩尔范围内的高亲和力细胞结合和抑制活性。此外，这些抗体对整合素-α11/β1 显示出高选择性，并且与密切同源物的交叉反应性极小。总而言之，我们的研究结果强调了原位选择用于生成针对天然细胞表面蛋白的识别和抑制而优化的抗整合素抗体的优势，并且我们的工作建立了可以扩展到许多其他膜蛋白的通用方法。