Allotype Structure, Organization, & Ig Gene Expression

同种异型结构、组织、

• 批准号: 7189437
• 负责人: rose G. mage
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7189437
• 关键词: B lymphocyte; antibody formation; developmental immunology; gene conversion; gene expression; gene rearrangement; gut associated lymphoid tissue; human tissue; immunogenetics; immunoglobulin genes; immunoglobulin structure; immunoregulation; isoantibody; laboratory rabbit; laser capture microdissection; micromanipulator; molecular genetics; monoclonal antibody; nucleic acid sequence; peptide library; phage display; polymerase chain reaction; technology /technique development; vascular endothelial growth factors

Summary: Rabbit Preimmune Repertoire Development We study genes of the rabbit immune system using techniques of molecular biology and immunology (1). In species such as mouse and human, generation of combinatorial diversity through use of different VH and VL genes in immunoglobulin VHDJH and VLJL rearrangements can be a major contributor to the primary antibody repertoire. In rabbits, the contribution of the combinatorial mechanism to heavy chain diversity is minimal as only a few VH genes are rearranged and expressed. This resembles chicken antibody formation. Rabbit appendix and chicken bursa of Fabricius are primary lymphoid organs where the B cell antibody repertoire develops in germinal centers mainly by a gene conversion-like process. As in the chicken, the 3-prime most VH1 gene is rearranged in most rabbit B lymphocytes. Somatic hypermutation and gene conversion contribute to primary diversification in appendix of young rabbits or in bursa of Fabricius of embryonic and young chickens and also to secondary diversification during immune responses in germinal centers (GCs)(2). We previously showed that diversification patterns in the clones from appendix were strikingly different from those found in splenic GCs where an immunizing antigen was driving the expansion and selection process toward high affinity. Clonally related appendix B cells developed different amino acid sequences in each complementarity-determining region (CDR) including CDR3 whereas dominant clones from spleen underwent few changes in CDR3. The variety of combining sites generated by diversification within individual appendix clones suggests that at least some clonal expansion and selection, known to require normal gut flora, may be driven through indirect effects of microbial components rather than solely by their recognition as specific foreign antigens. In collaboration with Dr. Ramit Mehr, we used a novel method for analyzing lineage tree shapes, using terms from graph theory to study diversification in rabbits and chickens. When lineage tree shapes were analyzed to quantify the differences between primary and secondary diversification in rabbits, the analyses indicated that primary diversification appears to occur at a constant rate in the appendix and the type of antigen-specific selection seen in splenic GCs is absent. This supports the view that a primary repertoire is being generated within the expanding clonally related B cells in appendix of young rabbits and emphasizes the important role that gut associated lymphoid tissues may play in early development of mammalian immune repertoires. The data also indicate a higher rate of hypermutation in rabbit during immune responses in splenic GCs, such that the balance between hypermutation and selection tends more towards mutation and less towards selection in rabbit compared to murine GCs (3). Rabbit Immune Repertoires for Generation and Humanization of Therapeutic Monoclonal Antibodies The rabbit immune repertoire has long been a rich source of diagnostic polyclonal antibodies. Now it also holds great promise as a source of therapeutic monoclonal antibodies. Rabbits with the rare b9 and bas allotypes are excellent sources for therapeutic monoclonal antibodies. We generated rabbit polyclonal antibodies and from these rabbits, the laboratory of Dr. C. Rader isolated monoclonal antibodies selective for all three members of the Nogo receptor (NgR) family. NgR family members are cell surface proteins involved in the development, plasticity, and regeneration of the central nervous system. The use of our antibodies to characterize NgR2 was recently published (4). Vascular endothelial growth factor (VEGF) and its receptors have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. Murine tumor angiogenesis models and receptor-specific antibodies are required to facilitate evaluation of the roles of VEGF receptors in mouse models of human cancer. We developed rabbit antibodies that cross-react with mouse (Flk-1) and human (KDR) VEGFR2. High-affinity, species cross-reactive, VEGFR2-specific Fabs were selected from an antibody phage display library generated from an immunized b9 allotype rabbit. The selected chimeric rabbit/human Fabs were found to bind to KDR and Flk-1 with nanomolar affinity. Three selected Fabs detected KDR expression on human endothelial cells as well as Flk-1 on murine endothelial cells. The availability of anti-VEGFR2 Fab with species cross-reactivity will help to decipher functional role of KDR/Flk-1 in tumor biology as well as facilitate the preclinical evaluation of the suitability of KDR/Flk-1 for drug targeting. This report further underscores our earlier finding that b9 allotype rabbits are excellent sources for generating high affinity cross-reactive antibodies with therapeutic potential (5).

总结：兔免疫前库开发 我们使用分子生物学和免疫学技术研究兔子免疫系统的基因（1）。在诸如小鼠和人的物种中，通过在免疫球蛋白VHDJH和VLJL重排中使用不同的VH和VL基因产生组合多样性可以是一抗库的主要贡献者。在兔中，组合机制对重链多样性的贡献最小，因为只有少数VH基因重排和表达。这类似于鸡抗体的形成。兔阑尾和鸡法氏囊是主要的淋巴器官，其中B细胞抗体库主要通过基因转换样过程在生殖中心发育。与鸡一样，在大多数兔B淋巴细胞中，最大的3-引物VH 1基因重排。体细胞超突变和基因转换有助于幼兔阑尾或胚胎和幼鸡腔上囊的初级多样化，也有助于生殖中心（GC）免疫应答期间的次级多样化（2）。我们之前表明，来自阑尾的克隆的多样化模式与在脾脏GC中发现的多样化模式截然不同，在脾脏GC中，免疫抗原推动扩增和选择过程趋向高亲和力。克隆相关的阑尾B细胞在每个互补决定区（CDR）（包括CDR 3）中产生不同的氨基酸序列，而来自脾脏的显性克隆在CDR 3中发生很少的变化。单个阑尾克隆内多样化产生的结合位点的多样性表明，至少有一些克隆扩增和选择，已知需要正常的肠道植物群，可能是通过微生物成分的间接作用，而不仅仅是通过识别特异性外来抗原。在与Ramit梅尔博士的合作中，我们使用了一种新的方法来分析谱系树的形状，使用图论中的术语来研究兔子和鸡的多样化。当谱系树的形状进行了分析，以量化初级和二级多样化之间的差异，在兔子，分析表明，初级多样化似乎发生在一个恒定的速度在附录和脾GC中看到的抗原特异性选择的类型是不存在的。这支持了这样的观点，即一个主要的剧目正在扩大克隆相关的B细胞在幼兔阑尾内产生，并强调了重要的作用，肠道相关的淋巴组织可能在哺乳动物免疫剧目的早期发展。数据还表明，在脾GC中的免疫应答期间，家兔中的超突变率较高，因此与鼠GC相比，家兔中的超突变和选择之间的平衡更倾向于突变，而更不倾向于选择（3）。 用于制备和人源化治疗性单克隆抗体的兔免疫库 兔免疫库长期以来一直是诊断性多克隆抗体的丰富来源。现在，它作为治疗性单克隆抗体的来源也有很大的希望。具有罕见b 9和bas同种异型的兔子是治疗性单克隆抗体的极好来源。我们制备了兔多克隆抗体，从这些兔中，C. Rader分离出对Nogo受体（NgR）家族所有三个成员具有选择性的单克隆抗体。NgR家族成员是参与中枢神经系统发育、可塑性和再生的细胞表面蛋白。最近发表了使用我们的抗体来表征NgR 2（4）。血管内皮生长因子（VEGF）及其受体通过刺激肿瘤相关的血管生成而促进实体瘤的生长和转移。需要小鼠肿瘤血管生成模型和受体特异性抗体来促进评价VEGF受体在人类癌症小鼠模型中的作用。我们开发了与小鼠（Flk-1）和人（KDR）VEGFR 2交叉反应的兔抗体。从由免疫的b 9同种异型兔产生的抗体噬菌体展示文库中选择高亲和力、物种交叉反应性、VEGFR 2特异性Fab。发现所选择的嵌合兔/人Fab以纳摩尔亲和力结合KDR和Flk-1。三个选择的Fab检测人内皮细胞上的KDR表达以及鼠内皮细胞上的Flk-1。具有物种交叉反应性的抗VEGFR 2 Fab的可用性将有助于破译KDR/Flk-1在肿瘤生物学中的功能作用，并促进KDR/Flk-1用于药物靶向的适用性的临床前评估。这份报告进一步强调了我们早期的发现，即b 9同种异型兔是产生具有治疗潜力的高亲和力交叉反应抗体的极好来源（5）。