Singer I I, Kawka D W, DeMartino J A, Daugherty B L, Elliston K O, Alves K, Bush B L, Cameron P M, Cuca G C, Davies P
Department of Biochemical and Molecular Pathology, Merck Research Laboratories, Rahway, NJ 07065.
J Immunol. 1993 Apr 1;150(7):2844-57.
The murine anti-CD18 mAb 1B4 has been humanized using CDR grafting. Three VH (Gal, Jon, and New) and two VL (Rei and Len) human frameworks, whose selection was based exclusively on their sequence identity with m1B4, were used to construct five human gamma 4/kappa recombinant antibodies: Gal/Rei, Gal/Len, Jon/Rei, and New/Rei, and a "hemichimeric" antibody pairing the VH of m1B4 with grafted Rei. Each of these h1B4 constructs completely inhibited the binding of m1B4 to activated human leukocytes with avidities (IC50) ranging from 1.5 to 8.0 nM, compared to 0.5 nM for m1B4. Replacement of three VH residues in the best VH framework, Gal, with the corresponding m1B4 "packing" (nonsolvent exposed) residues gave an h1B4 (mutant Gal/Rei) with the same avidity as m1B4. Avidity correlated with overall percent identity between the human and murine VH frameworks and, in particular, with conservation of "packing" residues. Rei and Len VL frameworks proved to be interchangeable. Further characterization showed that the Gal/Rei prototype was equipotent to m1B4 in blocking adhesion of polymorphonuclear leukocytes and monocytes to human vascular endothelium in vitro, and polymorphonuclear leukocyte extravasation into C5a-injected rabbit or monkey skin sites. Dual-label immunofluorescence microscopy of bone marrow cells with Gal/Rei h1B4 and m1B4 demonstrated that the fine specificity of the combining sites had not been altered by humanization. Reduced immunogenicity was demonstrated in rhesus monkeys that tolerated weekly treatment with h1B4 for 6 wk, whereas m1B4 induced profound anaphylaxis at 3 wk. Anti-1B4 titers in h1B4-treated rhesus were 50 to 66% lower and developed 1 wk later than in m1B4-treated monkeys. Crucially, the anti-h1B4 antibodies were anti-idiotypic while the anti-m1B4 antibodies were directed against constant and framework regions. We conclude that sequence identity searches are sufficient to identify suitable human frameworks for CDR-grafting of m1B4, yielding functionally equivalent humanized antibodies that are tolerated better in primates.
鼠抗 CD18 单克隆抗体 1B4 已通过 CDR 移植进行人源化。基于与 m1B4 的序列同一性专门选择了三个 VH（Gal、Jon 和 New）和两个 VL（Rei 和 Len）人源框架，用于构建五种人γ4/κ重组抗体：Gal/Rei、Gal/Len、Jon/Rei 和 New/Rei，以及一种将 m1B4 的 VH 与移植的 Rei 配对的“半嵌合”抗体。与 m1B4 的 0.5 nM 相比，这些 h1B4 构建体中的每一个都能以 1.5 至 8.0 nM 的亲和力（IC50）完全抑制 m1B4 与活化的人白细胞的结合。用相应的 m1B4“堆积”（非溶剂暴露）残基替换最佳 VH 框架 Gal 中的三个 VH 残基，得到了与 m1B4 具有相同亲和力的 h1B4（突变体 Gal/Rei）。亲和力与人源和鼠源 VH 框架之间的总体序列同一性相关，特别是与“堆积”残基的保守性相关。事实证明 Rei 和 Len VL 框架是可互换的。进一步的表征表明，Gal/Rei 原型在体外阻断多形核白细胞和单核细胞与人血管内皮的粘附以及多形核白细胞向注射 C5a 的兔或猴皮肤部位的渗出方面与 m1B4 等效。用 Gal/Rei h1B4 和 m1B4 对骨髓细胞进行双标记免疫荧光显微镜检查表明，结合位点的精细特异性并未因人源化而改变。在恒河猴中证明了免疫原性降低，这些恒河猴耐受每周一次的 h1B4 治疗 6 周，而 m1B4 在 3 周时诱导严重过敏反应。h1B4 治疗的恒河猴中的抗 1B4 滴度比 m1B4 治疗的猴子低 50%至 66%，且在 1 周后出现。至关重要的是，抗 h1B4 抗体是抗独特型的，而抗 m1B4 抗体是针对恒定区和框架区的。我们得出结论，序列同一性搜索足以识别适合 m1B4 的 CDR 移植的人源框架，产生在灵长类动物中耐受性更好的功能等效的人源化抗体。
