CLONING AND MODIFICATION OF ANTI-TUMOR ANTIGEN IMMUNOGLOBULIN GENES

本研究项目的主要目的是研究结构- 重组免疫球蛋白分子的功能关系 开发新的免疫试剂用于诊断和治疗 人类癌 几种鼠单克隆抗体（MAb）， 对癌相关抗原的选择性反应性，包括肿瘤 相关糖蛋白（TAG）-72，已在这方面的发展， 实验室 在与TAG-72抗原反应的那些MAb中，MAb CC 49在最近的临床试验中显示出极好的肿瘤定位 并因此用作原型重组免疫球蛋白。的 我们努力的重点是解决免疫原性问题， 的小鼠CC 49在患者中，并优化IG phamacoketine和 新陈代谢.为此，我们cDNA克隆了编码重链的基因， 和单克隆抗体CC 49的轻链，并随后开发了小鼠- 人嵌合体（c）CC 49。我们现在已经开发，纯化， 特征在于“人源化”CC 49（HuCC 49），其预期具有 甚至在人体中免疫原性的降低更大。 既往临床 用鼠CC 49进行的试验也显示了抗可变区域应答。 因此，目前的努力之一是确定那些 MAb CC 49 CDR，其部分或全部可以是单克隆抗体或单克隆抗体。 用于结合TAG-72抗原。最大限度地提高 在HuCC 49中，我们希望替换CDRs或其部分， 具有衍生自人抗体CDR的序列。优化 单克隆抗体CC 49的药代动力学，我们已经开发了一种CH 2结构域缺失的 cCC 49，并评估其血浆清除率和肿瘤靶向性 特性.最近，我们还开发了一种CH 2结构域缺失的 HuCC 49。也正在尝试使用杆状病毒系统来 表达和纯化这些潜在的临床试剂， 有效的方式。为了促进离体转染和体内转染， 表达的重组CC 49，我们已经开发了单基因 编码单链免疫球蛋白（SCIg）分子的构建体。 这些SCIg已经从两种细胞中表达、纯化和表征， 转染的SP2/0小鼠骨髓瘤和昆虫细胞。一种这样的分子， 单链免疫球蛋白-IL-2融合蛋白，SCIg-IL-2， 在小鼠中表达，使用经皮基因枪和 肌肉注射 融合蛋白在200 nm处容易地检测到。 基因传递的位点以及动物血清中。 在 此外，细胞和体液免疫反应的体内 表达的融合蛋白。因此，这项技术可以 还提供了一种独特的方法， 用于靶向携带抗原的癌细胞的淋巴细胞。

The main objective of this research project is to study structure- function relationships of recombinant immunoglobulin molecules and to develop novel immunological reagents for the diagnosis and therapy of human carcinomas. Several murine monoclonal antibodies (MAbs) with selective reactivity to carcinoma associated antigens, including tumor associated glycoprotein (TAG)-72, have been developed in this laboratory. Among those MAbs which react with the TAG-72 antigen, MAb CC49 has shown excellent tumor localization in recent clinical trials and is thus serving as a prototype recombinant immunoglobulin. The thrust of our effort is to obviate the problem of the immunogenicity of the murine CC49 in patients, and to optimize Ig phamacokinetics and metabolism. To this end, we cDNA cloned the genes encoding the heavy and light chains of the MAb CC49, and subsequently developed a mouse- human chimeric (c) CC49. We have now developed, purified and characterized a "humanized" CC49 (HuCC49), which is expected to have even a greater reduced immunogenicity in humans. Previous clinical trials with murine CC49 have also shown antivariable region responses. Therefore, one of the current efforts is aimed at identifying those MAb CC49 CDRs, which, in part or in their entirety, may be dispensable for binding to the TAG-72 antigen. To maximize the human content of the HuCC49, we wish to replace the dispensable CDRs, or parts of them, with sequences derived from human antibody CDRs. To optimize the pharmacokinetics of MAb CC49, we have developed a CH2 domain-deleted cCC49 and evaluated its plasma clearance and tumor targeting properties. More recently, we have also developed a CH2 domain-deleted HuCC49. Attempts are also underway to use the baculovirus system to express and purify these potential clinical reagents, in a cost effective way. To facilitate ex vivo transfection and in vivo expression of the recombinant CC49, we have developed single-gene constructs encoding single-chain immunoglobulin (SCIg) molecules. These SCIgs have been expressed, purified and characterized from both transfected SP2/0 mouse myeloma and insect cells. One such molecule, a single-chain immunoglobulin-IL-2 fusion protein, SCIg-IL-2, has been expressed in mice, using both a transcutaneous gene gun and intramuscular injection. The fusion protein was readily detected at the site of the gene delivery as well as in the animal sera. In addition, cellular and humoral immune responses to the in vivo expressed fusion protein have been detected. This technique could thus also provide a unique approach for the activation of cytotoxic lymphocytes for targeting antigen bearing cancer cells.