A single-chain IL-12 IgG3 antibody fusion protein retains antibody specificity and IL-12 bioactivity and demonstrates antitumor activity.
A single-chain IL-12 IgG3 antibody fusion protein retains antibody specificity and IL-12 bioactivity and demonstrates antitumor activity.
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DOI：
10.4049/jimmunol.163.1.250
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发表时间：
1999-07
影响因子：
4.4
通讯作者：
Lisan S. Peng;M. Penichet;S. L. Morrison
中科院分区：
文献类型：
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作者：
Lisan S. Peng;M. Penichet;S. L. Morrison
IL-12是一种异源二聚体细胞因子，对先天免疫和细胞免疫具有多种作用，可能具有抗肿瘤和抗转移作用。然而，全身施用IL-12可能是有毒的。肿瘤特异性抗体提供了一种选择性靶向转移性/残留结节并递送治疗量的免疫刺激分子如IL-12的方法，其具有较低的全身水平和理想的毒性。我们报告了抗体融合蛋白的构建和表征，其中单链鼠IL-12在氨基末端与抗Her 2/neu Ab融合（mscIL-12.her2.IgG3）。在融合蛋白中使用单链IL-12简化了载体构建，确保了两个IL-12亚基的等摩尔浓度，并且可以赋予融合蛋白更大的稳定性。SDS-PAGE分析显示该320-kDa蛋白被分泌并正确组装。FACS分析表明，该融合蛋白结合用Her 2/neu Ag转染的细胞，从而保留Ab特异性;该融合蛋白还结合细胞系和表达IL-12 R的PHA活化的PBMC，从而证明细胞因子受体特异性。T细胞增殖试验和NK细胞毒性试验表明，该融合蛋白具有与重组鼠IL-12相当的IL-12生物活性。体内研究表明该融合蛋白具有抗肿瘤活性。这些结果是显著的，并且表明该IL-12 Ab融合蛋白可以有效地将IL-12的治疗潜力与Ab的肿瘤靶向能力联合收割机结合，并且可以提供IL-12的全身施用的可行替代方案。
IL-12 is a heterodimeric cytokine with many actions on innate and cellular immunity that may have antitumor and antimetastatic effects. However, systemic administration of IL-12 can be toxic. Tumor-specific Abs provide a means to selectively target a metastatic/residual nodule and deliver therapeutic quantities of an immunostimulatory molecule like IL-12 with lower systemic levels and ideally, toxicity. We report the construction and characterization of an Ab fusion protein in which single-chain murine IL-12 is fused to an anti-Her2/neu Ab at the amino terminus (mscIL-12.her2.IgG3). The use of single-chain IL-12 in the fusion protein simplifies vector construction, ensures equimolar concentrations of the two IL-12 subunits, and may confer greater stability to the fusion protein. SDS-PAGE analysis shows this 320-kDa protein is secreted and correctly assembled. FACS analysis demonstrates that this fusion protein binds to cells transfected with the Her2/neu Ag, thus retaining Ab specificity; this fusion protein also binds to a cell line and to PHA-activated PBMC that express the IL-12R, thus demonstrating cytokine receptor specificity. T cell proliferation assays and NK cytotoxicity assays demonstrate that this fusion protein exhibits IL-12 bioactivity comparable to recombinant murine IL-12. In vivo studies demonstrate that this fusion protein has antitumor activity. These results are significant and suggest that this IL-12 Ab fusion protein can effectively combine the therapeutic potential of IL-12 with the tumor-targeting ability of the Ab and may provide a viable alternative to systemic administration of IL-12.