CLONING AND MODIFICATION OF ANTI-TUMOR ANTIGEN IMMUNOGLOBULIN GENES

正在进行的研究涉及重组免疫球蛋白的设计和使用 (IG)癌症治疗和诊断表格。鼠源性单抗 (单抗)CC49选择性地与肿瘤抗原肿瘤反应 相关糖蛋白(TAG)-72。临床前研究和临床 试验已经证明了这种诊断和潜在的治疗方法 这种单抗的实用性。优化疗效，最大限度地减少 毒性和宿主免疫反应，现在的重点是 CC49新型重组免疫球蛋白的设计与翻译研究。 编码这些抗体的基因已经被克隆，并被老鼠-人 这些单抗的嵌合体和人源化版本已经产生。这个 以人源化单抗CC49(HuCC49)为原型研制 转基因单抗。人源化CH2结构域缺失的CC49已被 构建并展示了显著更快的等离子体清除和 比完整的CC49具有更好的肿瘤靶向性。为了促进体外实验 治疗性重组CC49的转染和体内表达 使用，单抗CC49的三个单基因编码的单链衍生物 现在已经被设计、生成和表征。其中一个单基因 构建了编码完整的cCC49单抗的载体。同源二聚体分子， 从转染瘤分泌的SCAcCC49与 TAG-72抗原为nCC49，其杀伤活性与cCC49相似。 我们还开发了一种编码单链的单基因结构 免疫球蛋白-IL-2融合蛋白。融合蛋白在体内的表达 在Balb/c小鼠中成功地证明了蛋白质的存在 经皮基因枪和肌肉注射。在一个单独的 研究，以减少患者对HuCC49的独特型反应 对抗原结合不是关键的CDR被识别并被替换为 人类CDR。此外，必要的小鼠CDR的可有可无的区域 鉴定并替换为人CDR的同源区。vbl.使用 这些变异体和来自接受小鼠CC49治疗的患者的血清 在之前的临床试验中，正在尝试识别这些氨基酸 对作为目标的独特位有贡献的酸性残留物 病人的免疫反应。 合作研究正在进行中，以产生特定的重定向 抗肿瘤人T细胞群。为此，编码MHC的基因- 含有单链抗肿瘤免疫球蛋白的非限制性抗原受体 T细胞CD3复合体的基因和Zeta链已经被 通过逆转录病毒基因转移将其导入T淋巴细胞。 通用受体逆转录病毒转导的CD8+淋巴细胞 针对TAG-72阳性tareget的基因表现出特异性的细胞溶解作用 细胞。抗CEA阳性人肿瘤重定向T细胞的产生 细胞正在进行中。人类免疫治疗的另一种途径 癌症的基础是结合肿瘤的抗原肽。 抗人FcgR1抗体的抗原。基因结构编码 两种不同多肽的融合蛋白及其人源化Fab 已经制备了针对人FcgR1位点的抗体，并 表达的融合蛋白目前正在鉴定中。这些 分子将被用来向专业人士靶向抗原肽 抗原提呈细胞，用于内化和提呈 在MHC类1的上下文中的肽。

Ongoing studies involve the design and use of recombinant immunoglobulin (Ig) forms for cancer therapy and diagnosis. Murine monoclonal antibody (MAb) CC49 selectively reacts with the pancarcinoma antigen, tumor associated glycoprotein (TAG)-72. Preclinical studies and clinical trials have demonstrated the diagnostic and potential therapeutic utility of this MAb. To optimize therapeutic efficacy and minimize toxicity and host immune responses, emphasis is now being placed on the design and translational research of novel recombinant Ig forms of CC49. The genes encoding these antibodies have been cloned and mouse-human chimeric and humanized versions of these MAbs have been generated. The humanized MAb CC49 (HuCC49) was used as a prototype to develop genetically altered MAbs. A humanized CH2 domain-deleted CC49 has been constructed and demonstrates significantly faster plasma clearance and better tumor targeting than the intact CC49. To facilitate ex vivo transfection and in vivo expression of recombinant CC49 for therapeutic use, three single-gene encoded single-chain derivatives of MAb CC49 have now been designed, generated and characterized. One of the single-gene constructs encoded an intact cCC49 MAb. The homodimeric molecule, SCAcCC49, secreted from the transfectoma showed similar binding to the TAG-72 antigen as nCC49 and similar cytolytic activity to that of cCC49. we have also developed a single-gene construct encoding a single-chain immunoglobulin-IL-2 fusion protein. In vivo expression of the fusion protein in Balb/c mice was successfully demonstrated using both a transcutaneous gene gun and intramuscular injection. In a separate study, to minimize idiotypic responses of patients to HuCC49, murine CDRs not critical for antigen binding were identified and replaced with human CDRs. Also, dispensable regions of the essential murine CDRs were identified and replaced with the homologous regions of human CDRs. Using these variants and sera from patients administered murine CC49 in a previous clinical trial, attempts are under way to identify those amino acid residues which contribute to the idiotopes that are the targets of patient's immune response. Collaborative studies are underway to generate specific redirected antitumor human T-cell populations. To that end, genes encoding MHC- unrestricted antigen receptors, containing single-chain antitumor Ig genes and the zeta chain of the CD3 complex of the T cell have been constructed introduced into T lymphocytes by retroviral gene transfer. CD8+ lymphocytes retrovirally transduced by the Universal receptor (UR) genes have shown specific cytolysis against TAG-72 positive tareget cells. Generation of redirected T cells against CEA positive human tumor cells is underway. Another approach for the immunotherapy of human carcinomas is based on conjugating an antigenic peptide of a tumor antigen to the anti-human FcgR1 antibody. Gene constructs encoding fusion proteins of two different peptides and the Fab of the humanized antibody specific for a site of the human FcgR1 have been made and expressed The fusion proteins are currently being characterized. These molecules will be used to target antigenic peptides to professional antigen presenting cells for internalization and presentation of the peptide in the context of MHC class 1.