Schanzer Juergen M, Fichtner Iduna, Baeuerle Patrick A, Kufer Peter
Micromet AG, Staffelseestrasse 2, 81477 Munich, Germany.
J Immunother. 2006 Sep-Oct;29(5):477-88. doi: 10.1097/01.cji.0000210080.60178.fd.
Cytokine targeting to tumor-associated antigens via antibody cytokine fusion proteins has demonstrated potent antitumor activity in numerous animal models and has led to the clinical development of 2 antibody-interleukin-2 (IL-2) fusion proteins. We previously reported on the construction and in vitro properties of a "dual" cytokine fusion protein for simultaneous targeted delivery of human granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-2 to human tumors. The fusion protein is based on a heterodimerized core structure formed by human CH1 and Ckappa domains (heterominibody) with C-terminally fused human cytokines and N-terminally fused single-chain antibody fragments specific for the tumor-associated surface antigen epithelial cell adhesion molecule (Ep-CAM). For testing the antitumor activity in syngeneic mouse xenograft models, we developed "dual cytokine heterominibodies" with murine cytokines (mDCH). mDCH fusion proteins and, as controls, "single cytokine heterominibodies" (SCH) carrying either murine GM-CSF (mGM-CSF) or murine IL-2 (mIL-2) were constructed, of which all retained the specific activities of cytokines and binding to the Ep-CAM antigen on human Ep-CAM transfected mouse colon carcinoma CT26-KSA cells. Over a 5-day treatment course, DCH fusion proteins induced significant inhibition of established pulmonary CT26-KSA metastases in immune-competent Balb/c mice at low daily doses of 1 mug of fusion protein per mouse. However, with the tested dosing schemes, antitumor activity of mDCH was largely independent of cytokine targeting to tumors as demonstrated by a control protein with mutated Ep-CAM binding sites. Single cytokine fusion proteins mSCH-GM-CSF and mSCH-IL-2 showed similar antitumor activity as the dual cytokine fusion protein mDCH, indicating that GM-CSF and IL-2 in one molecule did not significantly synergize in tumor rejection under our experimental conditions. Our results seem to contradict the notion that IL-2 and GM-CSF can synergize in antitumor activity and that with conventional dose regimens, their specific targeting to tumors, as tested here with 2 antibodies of different affinities, enhances their antitumor activity.
通过抗体细胞因子融合蛋白将细胞因子靶向肿瘤相关抗原，已在众多动物模型中展现出强大的抗肿瘤活性，并推动了两种抗体 - 白细胞介素 - 2（IL - 2）融合蛋白的临床开发。我们之前报道了一种“双功能”细胞因子融合蛋白的构建及其体外特性，该融合蛋白可将人粒细胞巨噬细胞集落刺激因子（GM - CSF）和IL - 2同时靶向递送至人类肿瘤。该融合蛋白基于由人CH1和Cκ结构域形成的异源二聚化核心结构（异源微抗体），其C末端融合人细胞因子，N末端融合对肿瘤相关表面抗原上皮细胞粘附分子（Ep - CAM）具有特异性的单链抗体片段。为了在同基因小鼠异种移植模型中测试抗肿瘤活性，我们开发了携带鼠细胞因子的“双细胞因子异源微抗体”（mDCH）。构建了mDCH融合蛋白，以及作为对照的携带鼠GM - CSF（mGM - CSF）或鼠IL - 2（mIL - 2）的“单细胞因子异源微抗体”（SCH），所有这些都保留了细胞因子的特异性活性以及与转染人Ep - CAM的小鼠结肠癌CT26 - KSA细胞上Ep - CAM抗原的结合能力。在为期5天的治疗过程中，DCH融合蛋白在每只小鼠每日低剂量1μg融合蛋白的情况下，可显著抑制免疫健全的Balb / c小鼠中已形成的肺CT26 - KSA转移瘤。然而，在所测试的给药方案中，如具有突变Ep - CAM结合位点的对照蛋白所示，mDCH的抗肿瘤活性在很大程度上与细胞因子对肿瘤的靶向作用无关。单细胞因子融合蛋白mSCH - GM - CSF和mSCH - IL - 2表现出与双细胞因子融合蛋白mDCH相似的抗肿瘤活性，这表明在我们的实验条件下，一个分子中的GM - CSF和IL - 2在肿瘤排斥中并未显著协同作用。我们的结果似乎与IL - 2和GM - CSF在抗肿瘤活性中可协同作用以及在传统剂量方案下它们对肿瘤的特异性靶向（如在此用两种不同亲和力的抗体测试）可增强其抗肿瘤活性的观点相矛盾。
