FUSION PROTEINS AGAINST CARCINOEMBRYONIC ANTIGEN

本研究的总体目标是开发抗体融合蛋白，用于将细胞因子和/或共刺激分子靶向表达癌胚抗原（CEA）的肿瘤，用于免疫治疗。这些研究将使用高亲和力的抗CEA单克隆抗体T84.66，用于在CEA转基因小鼠模型中研究的基因工程融合蛋白。在第一个目的中，将构建含有IL-2、IL-12、IL-18、GM-CSF、CD 40 L或Flt 3L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、分子大小和功能特性进行表征。将通过酶免疫测定、与细胞表面抗原的反应和表面等离子体共振来检查抗原结合特性。融合蛋白的生物活性将在增殖、IFN-γ产生和效应细胞重靶向测定中测定。对于第二个目标，将研究放射性碘标记的融合蛋白的药代动力学、正常组织分布和肿瘤靶向特性。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测量肿瘤靶向的特异性。在最终目标中，将在携带具有低免疫原性的肿瘤的CEA转基因小鼠中研究融合蛋白单独或以所选组合的治疗性质。将检查源自协同作用或具有不同免疫细胞靶标的融合蛋白的共同施用的治疗益处。将在淋巴细胞群消融后的小鼠或免疫剥夺动物中研究肿瘤排斥的机制。靶向表达CEA的肿瘤并具有诱导和增强抗肿瘤应答的能力的重组抗体融合蛋白将为人类癌症的更有效的免疫治疗提供机会。

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.