FUSION PROTEINS AGAINST CARCINOEMBRYONIC ANTIGEN

本研究的总体目标是开发抗体融合蛋白，将细胞因子和/或共刺激分子靶向表达癌胚抗原（CEA）的肿瘤进行免疫治疗。这些研究将使用高亲和力的抗CEA单抗，T84.66，用于基因工程融合蛋白，将在CEA转基因小鼠模型中进行研究。在第一个目标中，将构建含有IL-2、IL-12、IL-18、GM-CSF、CD40L或FIt3L的抗cea融合蛋白。单基因编码或多链融合蛋白将产生并表征其稳定性，分子大小和功能特性。抗原结合特性将通过酶免疫测定、与细胞表面抗原的反应和表面等离子体推理来检查。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确定。第二个目的是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特性。肿瘤定位将在CEA转基因小鼠携带表达CEA的海洋结肠癌中确定。在同一动物体内生长的cea阴性亲代肿瘤的抗体积累可以衡量肿瘤靶向的特异性。在最后的目的，融合蛋白单独或选择的组合将研究CEA转基因小鼠携带低免疫原性肿瘤的治疗特性。将对协同作用或具有不同免疫细胞靶点的融合蛋白的共同施用所获得的治疗效益进行研究。肿瘤排斥反应的机制将在淋巴细胞群消融后的小鼠或免疫剥夺动物中进行研究。靶向表达CEA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的人类肿瘤免疫治疗提供机会。

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.