CLONING AND MODIFICATION OF ANTI-TUMOR ANTIGEN IMMUNOGLOBULIN GENES

正在进行的研究包括设计和使用用于癌症治疗和诊断的新型免疫试剂。本实验室已研制出几种对胰腺癌抗原肿瘤相关糖蛋白(TAG)-72具有选择性反应性的小鼠单克隆抗体（mab）。编码这些抗体的基因已经被克隆。在与TAG-72抗原发生反应的单克隆抗体中，单克隆抗体CC49在临床前研究和临床试验中显示出最大的诊断和治疗潜力。为了优化治疗效果，目前的重点是新型重组Ig形式CC49的设计和转化研究。这种单抗的鼠人嵌合和人源化版本已经产生。为了便于体外转染和体内表达重组CC49用于治疗，目前已经设计、生成和表征了单基因编码的MAb CC49单链衍生物。其中一个单基因结构编码一个完整的cCC49单抗。从转染瘤分泌的同二聚体分子SCacCC49与TAG-72抗原的结合与nCC49相似，其细胞溶解活性与cCC49相似。我们还开发了一种编码单链免疫球蛋白- il -2融合蛋白的单基因构建体。利用经皮基因枪和肌肉注射成功地证实了融合蛋白在Balb/c小鼠体内的表达。为了最大限度地减少毒性和宿主免疫反应，人源化单克隆抗体CC49 （HuCC49）被用作开发转基因单克隆抗体的原型。我们构建了一个人源化的CH2结构域缺失CC49，它比完整的CC49具有更快的血浆清除率和更好的肿瘤靶向性。在另一项研究中，为了尽量减少患者对HuCC49的独特型反应，鉴定了对抗原结合不重要的小鼠cdr，并用人类cdr代替。此外，通过评估一组HuCC49变异体的相对亲和力，通过用人类cdr的相应残基取代必需cdr的一个或多个氨基酸残基，确定了必需小鼠cdr的可缺性区域。在先前的临床试验中，使用这些变体和来自给予小鼠CC49的患者的血清，已经确定了那些有助于患者免疫反应目标的独特型的氨基酸残基。在这些研究的基础上，我们开发出了HuCC49的最终变体，它保持适度的抗原反应性，并且不与小鼠CC49治疗患者血清中的抗独特型抗体发生反应。在另一项提高肿瘤靶向性的研究中，正在尝试增强HuCC49单抗的亲和力。已经构建了一个编码单链Ig的单基因，该单链Ig由一个通过铰链区连接到人γ - 1 Fc的HuCC49糖体组成。机体是一种二价抗原结合结构，由VH/VL和VL/VH结构域组成；这两个抗原位点通过一对的VL与另一对的VH的横向非共价结合连接。合作研究正在进行中，以产生特定的重定向抗肿瘤的人类t细胞群。为此，通过逆转录病毒基因转移，构建了含有抗肿瘤抗体单链Fv和T细胞CD3复合体zeta链的mhc非限制性抗原受体编码基因，并将其引入T淋巴细胞。由huc49衍生的通用受体（UR）基因逆转录转导的CD8+淋巴细胞对tag -72阳性靶细胞表现出特异性的细胞溶解作用。重定向T细胞也已产生，对cea阳性的人类肿瘤细胞表现出特异性的细胞溶解作用。编码该UR的构建体来源于HuCOL-1单链Ig -癌、免疫诊断、免疫治疗、重组抗体、

Ongoing studies involve the design and use of novel immunological reagents for cancer therapy and diagnosis. Several murine monoclonal antibodies (MAbs) with selective reactivity to the pancarcinoma antigen, tumor associated glycoprotein (TAG)-72, have been developed in this laboratory. The genes encoding these antibodies have been cloned. Among those MAbs that react with the TAG-72 antigen, MAb CC49 has shown greatest potential for diagnostic and therapeutic utility in preclinical studies and clinical trials. To optimize therapeutic efficacy, emphasis is now being placed on the design and translational research of novel recombinant Ig forms of CC49. The mouse-human chimeric and humanized versions of this MAb have been generated. To faciliate 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. To minimize toxicity and host immune responses, 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. 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 by evaluating the relative affinity of a panel of HuCC49 variants generated by replacing one or more amino acid residues of the essential CDRs by corresponding residues of the human CDRs. Using these variants and sera from patients administered murine CC49 in a previous clinical trial, those amino acid residues which contribute to the idiotopes that are the targets of patients immune responses have been identified. Based on these studies, a final variant of the HuCC49 has been developed that maintains moderate antigen reactivity and does not react with the anti-idiotypic antibodies of the sera from patients treated with murine CC49. In a separate study to improve tumor targeting, attempts are being made to enhance the avidity of the HuCC49 MAb. A single gene has been constructed that encodes a single-chain Ig consisting of a HuCC49 diabody attached to a human gamma 1 Fc via the hinge region. The diabody, a bivalent antigen binding structure, is made up of VH/VL and VL/VH domains; the two antigen sites are linked by lateral and noncovalent association of VL of one pair with the VH of the other. Collaborative studies are under way to generate specific redirected antitumor human T-cell populations. To that end, genes encoding MHC-unrestricted antigen receptors containing single-chain Fv of antitumor antibody and the zeta chain of the CD3 complex of the T cell have been constructed and introduced into T lymphocytes by retroviral gene transfer. CD8+ lymphocytes retrovirally transduced by the HuCC49-derived Universal receptor (UR) gene have shown specific cytolysis against TAG-72-positive target cells. Redirected T cells have also been generated that show specific cytolysis against CEA-positive human tumor cells. The construct encoding this UR is derived from HuCOL-1 single-chain Ig - carcinoma, immunodiagnosis, immunotherapy, recombinant antibodies,