CLONING AND MODIFICATION OF ANTI-TUMOR ANTIGEN IMMUNOGLOBULIN GENES

这项研究工作的主要目标是设计，建造和 产生用于诊断的新型重组免疫试剂， 治疗人类癌症。已经开发了几种杂交瘤细胞系 在该实验室中，选择性地产生单克隆抗体（MAbs 与癌相关抗原反应。这些包括针对 癌胚抗原（CEA），肿瘤相关糖蛋白（TAG）-72， 高分子量粘蛋白存在于多种癌细胞上，单克隆抗体 D 612，其与表达在细胞表面上的48 kD抗原反应， 正常和恶性胃肠道上皮。正在进行的临床 试验中，抗TAG-72 MAb，B72.3和CC 49，D 612和抗CEA MAb， COL-1，已经显示出不同程度的潜力， 诊断和治疗试剂。然而，小鼠的有用性 用于体内诊断和治疗的单克隆抗体由于它们的局限性而受到限制。 免疫原性为了减少这个潜在的问题，我们开发了鼠标- 使用重组DNA的人嵌合（c）MAb，包括cB72.3（γ 1） 技术.为了优化血浆药物代谢动力学 清除和最大限度地提高本地化的效率， 我们已经开发出新型嵌合免疫球蛋白 变体;这些变体包括无糖基化cB72.3（γ 1）MAb和恒定 cB72.3（gamma 1）区域结构域缺失变体。相对于 cB72.3 MAb，CH 2结构域缺失的cMAb显示出更快的清除 速度和更快的肿瘤靶向。对于第二代抗TAG-72 单克隆抗体，单克隆抗体CC 49和CC 83的重链和轻链基因， 克隆、测序并插入逆转录病毒载体中。近日 cCC 49（gamma 1）已被表达和纯化。我们还开发了一个 具有保留效应子功能的单基因编码的IG分子。我们 还开发了一种cD 612单克隆抗体， 人T细胞系。分泌的免疫球蛋白保留了抗原- 结合特性及其介导抗人肿瘤ADCC的能力 细胞据我们所知，这是第一次展示生产 通过人类T细胞的IgG，并打开了治疗的可能性， T细胞分泌人源化抗肿瘤MAb的方法 在肿瘤部位介导ADCC。

The major goal of this research effort is to design, construct and generate novel recombinant immunological reagents for the diagnosis and therapy of human cancers. Several hybridoma cell lines have been developed in this laboratory that produce monoclonal antibodies (MAbs) selectively reactive with carcinoma associated antigens. These include MAbs against carcinoembryonic antigen (CEA), tumor associated glycoprotein (TAG)-72, a high-molecular weight mucin present on a variety of carcinomas, and MAb D612, which is reactive with a 48 kD antigen expressed on the surface of normal and malignant gastrointestinal epithelium. In ongoing clinical trials, the anti-TAG-72 MAbs, B72.3 and CC49, D612 and an anti-CEA MAb, COL-1, have shown various degrees of potential for being developed into diagnostic and therapeutic reagents. However, the usefulness of murine MAbs for in vivo diagnosis and therapy is limited because of their immunogenicity. To reduce this potential problem we have developed mouse- human chimeric (c) MAbs, including cB72.3 (gamma1), using recombinant DNA techniques. In an effort to optimize the pharmacokinetics of plasma clearance and to maximize the efficiency of localization of, and penetration into, tumors we have developed novel chimeric immunoglobulin variants; these include aglycosylated cB72.3 (gamma1) MAb and constant region domain-deleted variants of cB72.3 (gamma1). In comparison with the cB72.3 MAb, the CH2 domain-deleted cMAb demonstrated a faster clearance rate and a more rapid tumor targeting. For second generation anti-TAG-72 MAbs, the heavy and light chain genes of MAb CC49 and CC83 have been cloned, sequenced and inserted into retroviral vectors. Recently, the cCC49 (gamma1) has been expressed and purified. We have also developed a single gene enoded Ig molecule which has retained effector functions. We have also developed a cD612 MAb which has been expressed and secreted by a human T cell line. The secreted immunoglobulin retained its antigen- binding properties and its ability to mediate ADCC against human tumor cells. To our knowledge, this is the first demonstration of the production of an IgG by human T cells and opens the possibility of a therapeutic approach in which T-cells secrete humanized anti-tumor MAb capable of mediating ADCC at the tumor site.