Rabbit Allotypes--Structure, Organization and Regulated Expression of Ig Genes

兔同种异型——Ig 基因的结构、组织和调控表达

• 批准号: 7732428
• 负责人: rose G. mage
• 金额: $ 18.24万
• 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/7732428
• 关键词: Affinity; Amino Acid Sequence; Antibodies; Antibody Formation; Antibody Repertoire; Appendix; Ascaridil; B-Lymphocytes; Basic Science; Binding; Biological Assay; Biology; Bursa of Fabricius; Cell Surface Proteins; Cell surface; Chickens; Cocrystallography; Collaborations; DNA Sequence Rearrangement; Development; Diagnostic; Embryo; Epitopes; Family; Family member; Gene Conversion; Generations; Genes; Gut associated lymphoid tissue; HIV; HIV-1; Hepatitis B e Antigens; Homologous Gene; Human; Immune; Immune response; Immune system; Immunization; Immunofluorescence Microscopy; Immunoglobulin G; Immunoglobulin Genes; Immunoglobulin Somatic Hypermutation; Immunoglobulin Variable Region; Immunoglobulins; Immunology; Immunoprecipitation; Individual; Infection; Investigation; Kinetics; Knowledge; Laboratories; Libraries; Ligand Binding; Lymphoid; Manuscripts; Molecular; Molecular Biology Techniques; Molecular Genetics; Molecular Structure; Monoclonal Antibodies; Mus; Myelin Associated Glycoprotein; National Institute of Diabetes and Digestive and Kidney Diseases; Natural regeneration; Nature; Neuraxis; Neurons; Organ; Orthologous Gene; Oryctolagus cuniculus; Phage Display; Preparation; Process; Production; Property; Proteins; Range; Rattus; Reagent; Recombinants; Research; Role; Secondary to; Sialic Acids; Source; Specificity; Structure; Structure of germinal center of lymph node; Technology; Therapeutic; Therapeutic Monoclonal Antibodies; Time; Vaccine Design; Variant; Viral; Virus; Work; axon growth; base; central nervous system injury; combinatorial; design; insight; interest; member; mimetics; neutralizing antibody; polyclonal antibody; receptor; vector

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 allotype are excellent sources for therapeutic monoclonal antibodies. We generated rabbit polyclonal antibodies from these rabbits. Broadly neutralizing antibodies against receptor-activated epitopes on the N-heptad repeat region (NHR) of HIV-1 gp41 are elicited during natural infection and by immunization. Our collaborator Dr. Michael Zwick rescued HIV-1-neutralizing anti-NHR antibodies from immune phage display libraries that were prepared from an HIV-1 infected individual, and from b9 rabbits that we immunized with a mimetic of the NHR inner trimeric coiled-coil previously described by Dr. C. Bewley, NIDDK. The human and rabbit monoclonal antibodies bind to overlapping epitopes on the NHR trimer, generate distinct binding profiles against a panel of gp41 mimetic proteins and neutralize primary HIV-1 from various clades with modest potency using a pseudotyped virus assay. An examination of the differing abilities of the antibodies to neutralize different viruses bearing identical NHR sequences has revealed a particular restriction to neutralization that appears to be steric in nature, rather than dependent on viral entry kinetics. Taken together, the results indicate that broadly neutralizing antibodies, albeit currently of limited potency, are elicited against the NHR trimer of HIV-1 gp41 during natural infection and by immunization. For vaccine design, the newly selected antibody panel can be used in optimizing the NHR trimeric coiled-coil to favorably display neutralizing epitopes (Nelson, J. D., Jensen, R., Bewley, C. A., Brunel, F. M., Louis, J. M., Clore, G. M., Mage, R. G., Dawson, P. E., Burton, D. R., and Zwick, M. B. 2008 (1) The laboratory of Dr. C. Rader (NCI) isolated monoclonal antibodies selective for all three members of the Nogo-66 receptor (NgR) family. NgR family members are cell surface proteins involved in the development, plasticity, and regeneration of the central nervous system. NgR1, NgR2, and NgR3 are primarily expressed by neurons in the central nervous system (CNS) and believed to limit axonal growth and sprouting following CNS injury. In an attempt to define the expression and decipher the function of individual members of the Nogo-66 receptor family, we generated selective rabbit polyclonal antibodies (Venkatesh K, Chivatakarn O, Lee H, Joshi PS, Kantor DB, Newman BA, Mage R, Rader C, Giger RJ, The Nogo-66 receptor homolog NgR2 is a sialic acid-dependent receptor selective for myelin-associated glycoprotein. J Neurosci. 25:808-22, 2005). We then used phage display technology to generate rabbit monoclonal antibodies (mAbs) from the same immune repertoires. We obtained mAbs with nanomolar affinity to epitopes that are specific for NgR1 and NgR2, respectively, but at the same time conserved between mouse, rat, and human orthologs. Employing phage display vector pC3C, a newly designed phagemid optimized for the generation and selection of Fab libraries with human constant domains, rabbit mAbs were selected from chimeric rabbit/human Fab libraries, characterized in terms of specificity, affinity, and amino acid sequence, and finally converted to chimeric rabbit/human IgG. Strong and specific recognition of cell surface bound Nogo-66 receptor family members by chimeric rabbit/human IgG was demonstrated using immunofluorescence microscopy and immunoprecipitation. The rabbit mAbs together with their amino acid sequences constitute a defined panel of species cross-reactive reagents in infinite supply for investigations toward a functional role of the Nogo-66 receptor family in and beyond the CNS (1) and Venkatesh K, Raiker S, Lee A, Lee-Osbourne J, Wychowski T, Hofer T, Mage R, Rader C, and Giger RJ, Development of Nogo Receptor Variants with enhanced Ligand Binding Properties: Insights into the structural basis of the Myelin Associated Glycoprotein Nogo Receptor 2 association-- manuscript in preparation. In a collaboration with Drs. Wingfield, Stahl and Watts, we immunized b9 rabbits with recombinant HIV rev and HBeAg (Core e-antigen of Hepatitis B) for phage display and selection of Fab against conformational epitopes to be used for cocrystallization. Libraries generated from these immunized rabbits have been screened and Fab of interest recovered and sequenced. The above studies derive from work that characterized rabbit pre-immune and post-immune repertoire development. We studied genes of the rabbit immune system using techniques of molecular biology and immunology. 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)(Mage RG, Lanning D, Knight KL,B cell and antibody repertoire development in rabbits: The requirement of gut-associated lymphoid tissues. Dev Comp Immunol 30:137-53, 2006).

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同种异型的兔子是治疗性单克隆抗体的极好来源。我们从这些兔子中产生了兔多克隆抗体。针对HIV-1 gp 41的N-七肽重复区（NHR）上的受体活化表位的广泛中和抗体在自然感染期间和通过免疫接种引起。我们的合作者Michael Zwick博士从免疫噬菌体展示文库中拯救了HIV-1中和性抗NHR抗体，所述免疫噬菌体展示文库是从HIV-1感染个体制备的，并且从我们用先前由C. Bewley，NIDDK.人和兔单克隆抗体结合NHR三聚体上的重叠表位，产生针对一组gp 41模拟蛋白的不同结合谱，并使用假型病毒测定以适度的效力中和来自各种进化枝的原代HIV-1。对抗体中和携带相同NHR序列的不同病毒的不同能力的检查已经揭示了对中和的特定限制，其在本质上似乎是空间的，而不是依赖于病毒进入动力学。两者合计，结果表明，广泛中和抗体，虽然目前的效力有限，引起对HIV-1 gp 41的NHR三聚体在自然感染和免疫。对于疫苗设计，新选择的抗体组可用于优化NHR三聚卷曲螺旋以有利地展示中和表位（纳尔逊，J.D.，詹森，R.，伯利角一、Brunel，F. M.，路易斯，J.M.，克洛尔M.，法师河G.，道森，体育，Burton，D. R.，和Zwick，M. B。2008年（1） C博士的实验室Rader（NCI）分离了对Nogo-66受体（NgR）家族的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。NgR 1、NgR 2和NgR 3主要由中枢神经系统（CNS）中的神经元表达，并被认为限制CNS损伤后的轴突生长和发芽。为了试图定义Nogo-66受体家族的单个成员的表达和破译其功能，我们产生了选择性兔多克隆抗体（Venkatesh K，Chivatakarn O，Lee H，Joshi PS，Kantor DB，纽曼BA，法师R，Rader C，Giger RJ，The Nogo-66 receptor homolog NgR 2 is a sialic acid-dependent receptor selective for myelin-associated glycoprotein.神经科学杂志25：808-22，2005）。然后，我们使用噬菌体展示技术从相同的免疫库产生兔单克隆抗体（mAb）。我们获得了对NgR 1和NgR 2分别具有特异性的表位具有纳摩尔亲和力的mAb，但同时在小鼠、大鼠和人直系同源物之间是保守的。利用噬菌体展示载体pC 3C（一种新设计的噬菌粒，针对具有人恒定结构域的Fab库的生成和选择进行了优化），从嵌合兔/人Fab库中选择兔mAb，对其特异性、亲和力和氨基酸序列进行表征，最终转化为嵌合兔/人IgG。使用免疫荧光显微镜和免疫沉淀证实了嵌合兔/人IgG对细胞表面结合的Nogo-66受体家族成员的强和特异性识别。兔mAb与它们的氨基酸序列一起构成了一组确定的物种交叉反应试剂，其无限供应用于研究Nogo-66受体家族在CNS中和CNS以外的功能作用（1）和Venkatesh K，Raiker S，Lee A，Lee-Osbourne J，Wychowski T，霍费尔T，法师R，Rader C和Giger RJ，开发具有增强配体结合特性的Nogo受体变体：深入了解髓磷脂相关糖蛋白Nogo受体2相关性的结构基础-手稿正在编写中。在与温菲尔德、斯塔尔和瓦茨博士的合作中，我们用重组HIV rev和HBeAg（B型肝炎核心e抗原）免疫b 9兔，用于噬菌体展示和选择用于共结晶的针对构象表位的FaB。筛选从这些免疫的兔产生的文库，回收感兴趣的Fab并测序。 上述研究源自表征兔免疫前和免疫后库发育的工作。 我们利用分子生物学和免疫学技术研究了兔免疫系统的基因。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的阑尾或胚胎和幼鸡的法氏囊中的初级多样化，并且还有助于在生发中心（GC）中的免疫应答期间的次级多样化（法师RG，Lanning D，Knight KL，B cell and antibody repertoire development in rabbits：The requirement of gut-associated lymphatic tissues. Dev Comp Immunol 30：137-53，2006）。