BIOENGINEERED ANTIBODIES

抗CEA抗体的分子工程 将努力提高其效用， 腺癌的放射免疫治疗和放射免疫成像。 三 蛋白质工程可以改善整体的关键领域 将研究放射性标记抗体的质量和性能。 首先，尽管通过产生抗病毒抗体实现了免疫原性的降低， 嵌合T84.66在上一个项目期间，在临床治疗中 试验中，相当一部分患者已经证明了人类抗- 嵌合抗体（HACA）。 因此，抗体的进一步人源化 可变区域具有高度优先性。 T84.66将使用 重铺路面的方法：Fy的内部将被保留， 选择的表面残基将被诱变为匹配的保守表面残基， 人抗体中的残基。 因此，该方法极有可能 保留亲本抗体的高亲和力和特异性， 尽管降低的免疫原性将允许给予多剂量 在临床试验阶段 第二，开发位点特异性缀合物， 抗体将允许精确控制病毒的位置和程度 放射性标记，导致可重复生产 具有高免疫反应性和低免疫原性的放射免疫缀合物。 独特的半胱氨酸残基（提供反应性巯基）或N-连接的 糖基化位点（以提供反应性醛基， 高碘酸盐氧化）将被引入VL-VH-VH的CH 3结构域中。 CH 3“微抗体”先前显示出优异的异种移植 面向. 这些微抗体将使用新的双功能 项目3开发的螯合剂。 第三，融合生物活性 肽或抗原结合结构域将被研究作为一种手段， 增加放射性标记抗体的肿瘤积聚。 这些措施包括： 抗CEA-γ-IFN融合物，以局部增加 肿瘤;抗CEA-IL-2融合，以局部增加血管 双特异性抗CEA-抗TAG 72 抗体片段以增加具有异质性的肿瘤中的总体摄取 两种抗原的表达。 抗体-细胞因子预处理 融合物应该增强肿瘤对随后施用的 放射性标记抗体。 双特异性片段的肿瘤靶向将 与相应的单特异性试剂进行比较。 该组合 多学科的方法应该产生放射免疫缀合物， 提高功效。

Molecular engineering of anti-CEA (carcinoembryonic antigen) antibodies will be undertaken in a effort to increase their utility in radioimmunotherapy and radioimmunoimaging of adenocarcinomas. Three critical areas in which protein engineering can improve the overall quality and performance of radiolabeled antibodies will be investigated. First, despite the reduction in immunogenicity achieved by production of chimeric T84.66 during the previous project period, in clinical therapy trials a substantial fraction of patients have demonstrated human anti- chimeric antibodies (HACA). Thus, further humanization of the antibody variable regions is of high priority. T84.66 will be humanized using a resurfacing approach: the interior of the Fy will be retained and selected surface residues will be mutagenized to matched conserved surface residues in human antibodies. Thus approach has a high likelihood of retaining the high affinity and specificity of the parental antibody, while reduced immunogenicity will allow administration of multiple doses in clinical trials. Secondly, development of site-specific conjugation of antibodies will allow precise control over the location and extent of radiolabeling, resulting in reproducible production of radioimmunoconjugates with high immunoreactivity and low immunogenicity. Unique cysteine residues (providing reactive thiol groups) or N-linked glycosylation sites (to provide reactive aldehyde groups following periodate oxidation) will be introduced into the CH3 domain of a VL-V H - CH3 "minibody" previously shown to demonstrate excellent xenograft targeting. these minibodies will be conjugated using novel bifunctional chelating agents developed in project 3. Thirdly, fusion to bioactive peptides or antigen-binding domains will be investigated as a means to increase tumor accretion of radiolabeled antibodies. These will include: an anti-CEA-gamma-IFN fusion to locally increase CEA antigen expression in the tumor; an anti-CEA-IL-2 fusion to locally increase vascular permeability at the tumor site; and a bispecific anti-CEA-anti-TAG72 antibody fragment to increase overall uptake in tumors with heterogeneous expression of both antigens. Preadministration of the antibody-cytokine fusions should enhance tumor uptake of subsequently administered radiolabeled antibodies. Tumor targeting by the bispecific fragment will be compared with the corresponding monospecific agent. This combined multidisciplinary approach should yield radioimmunoconjugates with greatly improved efficacy.