FUSION PROTEINS AGAINST CARCINOEMBRYONIC ANTIGEN

本研究的总体目标是开发抗体融合蛋白，用于靶向细胞因子和/或共刺激分子到表达癌胚抗原(CEA)的肿瘤，用于免疫治疗。这些研究将使用高亲和力的抗CEA单抗T84.66来对融合蛋白进行基因工程，这些融合蛋白将在CEA转基因小鼠模型中进行研究。第一个目的是构建含有IL-2、IL-12、IL-18、GM-CSF、CD40L或FIt3L的抗CEA融合蛋白。将生产单基因编码或多链融合蛋白，并对其稳定性、分子大小和功能性质进行表征。抗原结合特性将通过酶免疫分析、与细胞表面抗原的反应和表面等离子体合理性来检测。融合蛋白的生物活性将在增殖、产生干扰素-γ和效应细胞重定向试验中确定。对于第二个目标，将研究放射性碘化融合蛋白的药代动力学、正常组织分布和肿瘤靶向性。将在携带CEA表达海洋结肠癌的CEA转基因小鼠中确定肿瘤定位。在同一动物中生长的CEA阴性亲本肿瘤中的抗体积累将衡量肿瘤靶向的特异性。在最终目标中，将在CEA转基因小鼠身上研究融合蛋白单独或选择组合的治疗特性，这些转基因小鼠携带低免疫原性肿瘤。我们将考察联合应用协同作用或具有不同免疫细胞靶点的融合蛋白所产生的治疗益处。肿瘤排斥的机制将在淋巴组织消融后的小鼠身上或在免疫剥夺的动物身上进行研究。针对CEA表达的肿瘤并具有诱导和增强抗肿瘤反应能力的重组抗体融合蛋白将为更有效地治疗人类癌症提供机会。

The overall objective of this research is to develop antibody fusion proteins for the targeting of cytokines and/or co-stimulatory molecules to tumors expressing carcinoembryonic antigen (CEA) for immunotherapy. These studies will use a high affinity anti-CEA Mab, T84.66, for genetically engineering fusion proteins that will be studied in a CEA transgenic mouse model. In the first aim, anti-CEA fusion proteins will be constructed that contain IL-2, IL-12, IL-18, GM-CSF, CD40L, or FIt3L. Single gene-encoded or multi-chain fusion proteins will be produced and characterized for their stability, molecular size, and functional properties. Antigen binding properties will be examined by enzyme immunoassay, reaction with cell surface antigen, and surface plasmon reasonance. The biological activity of fusion proteins will be determined in proliferation, IFN-gamma production, and effector cell retargeting assays. For the second aim, the pharmacokinetics, normal tissue distribution, and tumor targeting properties of radioiodinated fusion proteins will be studied. Tumor localization will be determined in CEA transgenic mice bearing CEA-expressing marine colon carcinomas. Antibody accumulation in CEA-negative parental tumors growing in the same animal will measure the specificity of tumor targeting. In the final aim, the therapeutic properties of fusion proteins alone or in selected combinations will be studied in CEA transgenic mice bearing tumors with low immunogenicity. The therapeutic benefit derived from co-administration of fusion proteins that act synergistically or have different immune cell targets will be examined. Mechanisms of tumor rejection will be investigated in mice following ablation of lymphoid populations or in immunodeprived animals. Recombinant antibody fusion proteins that are targeted to CEA- expressing tumors and have the capacity to induce and augment antitumor responses will provide the opportunity for more effective immunotherapy of human carcinomas.