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。本实验室研制了几种对肿瘤相关糖蛋白(TAG)-72具有选择性反应的鼠源性单抗(MAb)。编码这些抗体的基因已经被克隆。在与TAG-72抗原反应的单抗中，CC49单抗在临床前研究和临床试验中显示出最大的诊断和治疗作用潜力。为了优化治疗效果，目前的重点是新型重组免疫球蛋白CC49的设计和翻译研究。这种单抗的鼠-人嵌合体和人源化版本已经产生。为了便于重组CC49的体外转染和体内表达用于治疗，现已设计、制备和鉴定了三种单基因编码的CC49单链单链衍生物。其中一个单基因构建体编码完整的cCC49单抗。转染瘤分泌的同源二聚体分子SCacCC49与TAG-72抗原的结合与nCC49相似，对细胞的杀伤活性与cCC49相似。我们还开发了编码单链免疫球蛋白-IL-2融合蛋白的单基因结构。通过经皮基因枪和肌肉注射，成功地证明了融合蛋白在Balb/c小鼠体内的表达。为了将毒性和宿主免疫反应降到最低，以人源化单抗CC49(HuCC49)为原型研制了转基因单抗。人源化的CH2结构域缺失的CC49已经被构建，并显示出比完整的CC49更快的血浆清除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，确定了对抗原结合不关键的小鼠CDR，并用人CDR取代。此外，还通过评估一组HuCC49变异体的相对亲和力来识别基本CDR的可有可区，这些变异是通过将基本CDR的一个或多个氨基酸残基替换为相应的人CDR残基而产生的。使用这些变异体和先前临床试验中注射小鼠CC49的患者的血清，已经确定了那些对作为患者免疫反应目标的独特位的贡献的氨基酸残基。在这些研究的基础上，已经开发出HuCC49的最终变体，该变体保持中等的抗原反应性，并且不与接受小鼠CC49治疗的患者的血清中的抗独特型抗体发生反应。在另一项旨在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HuCC49 Diabody通过铰链区连接到人的Gamma 1 Fc上。Diabody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VL与另一对VH的侧向非共价结合而连接。合作研究正在进行中，以产生特定的重定向抗肿瘤人类T细胞群。为此，构建了编码MHC-非限制性抗原受体的基因，该受体含有抗肿瘤抗体的单链抗体和T细胞CD3复合体的Zeta链，并通过逆转录病毒基因转移将其导入T淋巴细胞。由HuCC49衍生的通用受体(UR)基因逆转录病毒转导的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。还产生了重定向的T细胞，显示出对CEA阳性的人类肿瘤细胞的特异性细胞溶解作用。编码该UR的构建物来源于HuCOL-1单链免疫球蛋白癌、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,