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

兔治疗性单抗的制备及人源化免疫谱系 长期以来，兔的免疫系统一直是诊断多克隆抗体的丰富来源。现在，它也很有希望成为治疗性单抗的来源。具有罕见b9同种异型的兔是治疗性单抗的极好来源。我们从这些兔子身上产生了兔多克隆抗体。针对HIV-1gp41受体激活表位(NHR)的广谱中和抗体在自然感染和免疫过程中被激发。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中和抗NHR抗体，这些抗体来自HIV-1感染者的免疫噬菌体展示文库，以及我们用NHR内三聚体线圈的模拟物免疫的b9兔。人和兔的单抗与NHR三聚体上的重叠表位结合，与一组gp41模拟蛋白产生不同的结合特征，并使用伪型病毒分析以适度的效力中和来自不同分支的初级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发现，对中和的特殊限制似乎是立体的，而不是依赖于病毒进入动力学。综上所述，结果表明，在自然感染和免疫期间，针对HIV-1 gp41的NHR三聚体产生了广泛的中和抗体，尽管目前效力有限。对于疫苗设计，新选择的抗体小组可用于优化NHR三聚体螺旋线圈以有利地展示中和表位(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.，和Zwick，M.B.2008(1) C.Rader博士(NCI)的实验室分离出了对Nogo-66受体(NGR)家族的所有三个成员具有选择性的单抗。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。NgR1、NgR2和NgR3主要由中枢神经系统(CNS)中的神经元表达，被认为限制了中枢神经系统损伤后轴突的生长和萌发。为了确定Nogo-66受体家族单个成员的表达和功能，我们制备了选择性兔多克隆抗体(Venkatesh K，Chivatakarn O，Lee H，Joshi PS，Kantor DB，Newman BA，MAGE R，Rader C，Giger RJ，Nogo-66受体同系物NgR2是一种选择性髓鞘相关糖蛋白的唾液酸依赖受体)。J神经科。2005年25：808-22)。然后，我们利用噬菌体展示技术，从相同的免疫谱系中制备了兔单抗。我们获得了与NgR1和NgR2分别特异的表位具有纳摩尔亲和力的mAbs，但同时在小鼠、大鼠和人类同源物之间保守。利用新设计的噬菌体展示载体pC3C，从兔/人嵌合Fab库中筛选出兔/人嵌合抗体，对其特异性、亲和力和氨基酸序列进行鉴定，并转化为兔/人嵌合抗体。免疫荧光显微镜和免疫沉淀显示，兔/人嵌合抗体对细胞表面结合的Nogo-66受体家族成员有很强的特异性识别作用。兔单抗及其氨基酸序列构成了一组限定的物种交叉反应试剂，在无限供应中用于研究NOGO-66受体家族在中枢神经系统内外的功能作用(1)以及Venkatesh K、Raiker S、Lee A、Lee-Osbourne J、Wychowski T、Hofer T、MAGE R、Rader C和Giger RJ，Development of Nogo Receptor Variants with Enhanced Ligand Binding Properties：深入了解髓鞘相关糖蛋白Nogo受体2关联-手稿制备中的结构基础。在Wingfield、Stahl和Watts博士的合作下，我们用重组HIV rev和HBeAg(乙肝核心e抗原)免疫b9兔，用于噬菌体展示和针对用于共结晶的构象表位的Fab的选择。对这些免疫兔产生的文库进行了筛选，并对感兴趣的Fab进行了回收和测序。 上述研究源于对兔免疫前和免疫后曲谱系发展的研究。 我们利用分子生物学和免疫学技术研究了兔子免疫系统的基因。在小鼠和人类等物种中，通过使用免疫球蛋白VHDJH和VLJL重排中不同的VH和VL基因来产生组合多样性可能是初级抗体库的主要贡献。在兔中，组合机制对重链多样性的贡献很小，因为只有几个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鸡的初级淋巴器官，B细胞抗体库主要通过类似基因转换的过程在生发中心发育。与鸡一样，VH1基因在大多数兔B淋巴细胞中都有重排。体细胞的超突变和基因转换有助于幼兔阑尾或胚胎和幼鸡法氏囊的初级多样化，也有助于生发中心(GCs)免疫反应的次级多样化(MAGE RG、LANING D、Knight KL、B细胞和兔抗体库的发展：对肠道相关淋巴组织的要求。开发人员补充免疫30：137-53,2006)。

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).