Growth hormone (GH) has been implicated in cancer progression andis a potential target for anticancer therapy. Currently, pegvisomant is the only GH receptor (GHR) antagonist approved for clinical use. Pegvisomant is a mutated GH molecule (B2036) which is PEGylated on amine groups to extend serum half-life. However, PEGylation significantly reduces the bioactivity of the antagonist in mice. To improve bioactivity, we generated a series of B2036 conjugates with the site-specific attachment of 20, 30, or 40 kDa methoxyPEG maleimide (mPEG maleimide) by introduction of a cysteine residue at amino acid 144 (S144C). Recombinant B2036–S144C was expressed in Escherichia coli, purified, and then PEGylated using cysteine-specific conjugation chemistry. To avoid issues with dimerization due to the introduced cysteine, B2036–S144C was PEGylated while immobilized on an Ni-nitrilotriacetic (Ni-NTA) acid column, which effectively reduced disulfide-mediated dimer formation and allowed efficient conjugation to mPEG maleimide. Following PEGylation, the IC₅₀ values for the 20, 30, and 40 kDa mPEG maleimide B2036–S144C conjugates were 66.2 ± 3.8, 106.1 ± 7.1, and 127.4 ± 3.6 nM, respectively. The circulating half-life of the 40 kDa mPEG conjugate was 58.3 h in mice. Subcutaneous administration of the 40 kDa mPEG conjugate (10 mg/kg/day) reduced serum insulin-like growth factor I (IGF-I) concentrations by 50.6%. This in vivo reduction in serum IGF-I was at a considerably lower dose compared to the higher doses required to observe comparable activity in studies with pegvisomant. In conclusion, we have generated a novel PEGylated GHR antagonist by the solid-phase site-specific attachment of mPEG maleimide at an introduced cysteine residue, which effectively reduces serum IGF-I in vivo.

中文翻译：

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固相位点特异性聚乙二醇化产生的人类GHR拮抗剂的增强的生物活性

生长激素（GH）与癌症发展有关，是抗癌治疗的潜在靶标。目前，pegvisomant是唯一获准用于临床的GH受体（GHR）拮抗剂。培维索孟特是一种突变的GH分子（B2036），在胺基团上聚乙二醇化以延长血清半衰期。然而，聚乙二醇化显着降低了拮抗剂在小鼠中的生物活性。为了提高生物活性，我们通过在氨基酸144处引入一个半胱氨酸残基，生成了一系列具有20、30或40 kDa甲氧基PEG马来酰亚胺（mPEG马来酰亚胺）位点特异性连接的B2036缀合物（S144C）。重组B2036–S144C在大肠杆菌中表达，纯化，然后使用半胱氨酸特异性缀合化学进行PEG化。为避免由于引入的半胱氨酸而引起的二聚化问题，将B2036–S144C聚乙二醇化，同时将其固定在镍三乙酸（Ni-NTA）酸柱上，这有效地减少了二硫键介导的二聚体的形成并允许与mPEG马来酰亚胺的有效结合。聚乙二醇化后，20、30和40 kDa mPEG马来酰亚胺B2036–S144C共轭物的IC ₅₀值分别为66.2±3.8、106.1±7.1和127.4±3.6 nM。40 kDa mPEG缀合物在小鼠中的循环半衰期为58.3小时。皮下给药40 kDa mPEG缀合物（10 mg / kg /天）可使血清胰岛素样生长因子I（IGF-1）浓度降低50.6％。这种体内与观察到pegvisomant研究中具有可比活性的较高剂量相比，降低血清IGF-I的剂量要低得多。总之，我们通过在引入的半胱氨酸残基上固相mPEG马来酰亚胺固相位点特异性附着产生了一种新型的聚乙二醇化GHR拮抗剂，可有效降低体内的血清IGF-1 。