Sato K, Ohtomo T, Hirata Y, Saito H, Matsuura T, Akimoto T, Akamatsu K, Koishihara Y, Ohsugi Y, Tsuchiya M
Chugai Pharmaceutical Co. Ltd., Fuji-Gotemba Research Labs., Shizuoka, Japan.
Hum Antibodies Hybridomas. 1996;7(4):175-83.
Interleukin-6 (IL-6) inhibitors are good potential therapeutic agents in human patients, and anti-IL-6 antibodies are among the best candidates. Here, we have successfully humanized mouse monoclonal antibody SK2, which specifically binds to IL-6 and strongly inhibits IL-6 functions. Since this antibody possesses N-linked carbohydrates on Asn-30 of VH region, which seems to be very close to an antigen-binding site, influence of these carbohydrates on antigen-binding was investigated. A biosensor study showed that the mouse SK2 Fab and its deglycosylated fragments had almost equal Kd (Kon/Koff), 26.8 nM (1.05 x 10(6)/2.81 x 10(-2)) and 24.7 nM (1.28 x 10(6)/3.15 x 10(-2)), respectively. Furthermore, a mutant chimeric SK2 antibody, in which the N-glycosylation site was removed from the VH region, showed a Kd of 11 nM, almost similar to that of the original chimeric SK2 antibody, determined by Scatchard analysis with 125I-IL-6. These data indicate the carbohydrates of mouse SK2 VH region do not significantly influence antigen-binding activity. In the next step, two versions of each humanized SK2 VL and VH regions were carefully designed based on the amino acid sequences of human REI and DAW, respectively. Only one alteration, Tyr to Phe, was made at position 71 in the two light chains, according to the canonical residue for LI. A N-glycosylation site was introduced on the two heavy chains, by changing Ser to Asn at position 30. All four combinations of humanized light and heavy chains could bind to IL-6 as well as the chimeric SK2 antibody. The light chain first version, however, could not efficiently inhibit IL-6 binding to its receptor, indicating the importance of the LI loop conformation for the inhibitory activity of SK2 antibody. In contrast, both versions of the heavy chains were comparable, in yielding good humanized SK2 antibodies, suggesting that the glycosylation of the SK2 VH region has no influence in recreating a functional antigen-binding site in this humanization.
白细胞介素-6（IL-6）抑制剂在人类患者中是很有潜力的治疗药物，抗IL-6抗体是最佳候选药物之一。在此，我们成功地将小鼠单克隆抗体SK2人源化，该抗体特异性结合IL-6并强烈抑制IL-6的功能。由于该抗体在VH区域的Asn-30上具有N-连接碳水化合物，而该位置似乎非常靠近抗原结合位点，因此研究了这些碳水化合物对抗原结合的影响。生物传感器研究表明，小鼠SK2 Fab及其去糖基化片段的解离常数（Kd，即结合速率常数与解离速率常数之比）几乎相等，分别为26.8 nM（1.05×10⁶/2.81×10⁻²）和24.7 nM（1.28×10⁶/3.15×10⁻²）。此外，通过用¹²⁵I-IL-6进行Scatchard分析测定，一种从VH区域去除了N-糖基化位点的突变嵌合SK2抗体的Kd为11 nM，与原始嵌合SK2抗体的Kd几乎相似。这些数据表明小鼠SK2 VH区域的碳水化合物不会显著影响抗原结合活性。下一步，分别根据人REI和DAW的氨基酸序列，精心设计了两个人源化SK2 VL和VH区域的版本。根据LI的规范残基，在两条轻链的第71位仅进行了一处改变，即酪氨酸突变为苯丙氨酸。通过将第30位的丝氨酸变为天冬酰胺，在两条重链上引入了一个N-糖基化位点。人源化轻链和重链的所有四种组合都能与IL-6结合，与嵌合SK2抗体一样。然而，轻链的第一个版本不能有效地抑制IL-6与其受体的结合，这表明LI环构象对SK2抗体的抑制活性很重要。相比之下，两个版本的重链在产生良好的人源化SK2抗体方面相当，这表明在这种人源化过程中，SK2 VH区域的糖基化对重建功能性抗原结合位点没有影响。
