Martin F, Toniatti C, Salvati A L, Ciliberto G, Cortese R, Sollazzo M
Department of Biotechnology IRBM P. Angeletti, Pomezia (Rome), Italy.
J Mol Biol. 1996 Jan 12;255(1):86-97. doi: 10.1006/jmbi.1996.0008.
The minibody is a designed small beta-protein conceived to enable the construction of large libraries of minimal discontinuous epitopes displayed on the surface of filamentous phage. The 61 residue molecule consists of three strands from each of the two beta-sheets of the variable domain of immunoglobulins packed face to face, along with the exposed H1 and H2 hypervariable regions. We have previously shown that from a minibody repertoire of more than 50 million molecules displayed on phage, we were able to select a minibody with micromolar affinity for human interleukin-6 that behaves as a selective cytokine antagonist. The minibody exposes a surface composed of two constrained loops, which provides the possibility of improving IL-6 binding and specificity by swapping the hypervariable regions, followed by further selection. We established experimental conditions for "stringent" selection such as monovalent phage display, competitive selection and epitope masking. Here, we show that by virtue of the optimization/selection process, we have isolated a minibody with improved antagonistic potency and greater specificity. Furthermore, using hIL-6 mutants carrying amino acid substitutions in distinct surface sites it was possible to carefully define the cytokine region that binds the minibody.
微型抗体是一种经过设计的小型β蛋白，旨在构建丝状噬菌体表面展示的最小不连续表位的大型文库。这个由61个残基组成的分子由免疫球蛋白可变区两个β折叠片中的各三条链面对面堆积而成，还包括暴露的H1和H2高变区。我们之前已经表明，从噬菌体展示的超过5000万个分子的微型抗体库中，我们能够筛选出对人白细胞介素-6具有微摩尔亲和力的微型抗体，其表现为一种选择性细胞因子拮抗剂。该微型抗体暴露了一个由两个受限环组成的表面，这使得通过交换高变区并进一步筛选来提高白细胞介素-6结合力和特异性成为可能。我们建立了“严格”筛选的实验条件，如单价噬菌体展示、竞争性筛选和表位掩蔽。在这里，我们表明，通过优化/筛选过程，我们分离出了一种具有更高拮抗效力和更高特异性的微型抗体。此外，使用在不同表面位点携带氨基酸取代的人白细胞介素-6突变体，能够精确界定与微型抗体结合的细胞因子区域。
