Recombinant Antibodies from the Sea Lamprey

描述（由申请人提供）：抗体是生物技术和生物医学应用中非常有用的试剂，但重组抗体有其局限性。其中包括抗原结合位点的复杂结构，这使得合成抗体库的生成变得复杂，以及重组抗体开发的主要专利限制。因此，人们一直在以非免疫球蛋白（Ig）支架的形式寻找抗体的替代品，如锚蛋白重复序列、纤维连接蛋白、脂钙蛋白和蛋白a。然而，只有少数这些人工支架实际上对不同类型的靶标产生了特异性，可能是因为它们都不是天然抗原受体。事实上，唯一已知的天然非ig抗原受体是七鳃鳗和盲鳗的可变淋巴细胞受体（VLRs）。VLRs由高度多样化的富含亮氨酸的重复序列模块组成，这些模块通过基因转换通过DNA重组组装而成，从而产生超过1014个独特受体的潜在库。用各种抗原免疫的七鳃鳗通过产生对抗原特异性的高亲和力血浆VLRs来应答。因此，VLRs是生物技术中用作非ig单链抗体的理想候选物。我们利用酵母表面展示技术和定制酵母展示载体，开发了一个高通量平台，用于从大型VLR文库（bbbb107个独立克隆）中选择重组抗原结合VLR。我们分离到了对鸡蛋溶菌酶（HEL）具有低微摩尔亲和力和对r -藻红蛋白具有低纳摩尔亲和力的VLRs。一轮体外亲和成熟使与hel结合的VLR解离常数降低了13倍，并形成了VLR- hel复合物。我们建议建立单克隆VLRs作为一类新的分子识别分子的生物技术潜力。我们的目标是：1；克隆特异结合不同抗原配体的VLRs，包括蛋白质、碳水化合物和整个细菌。通过酵母的表面展示，构建天然文库和免疫文库，筛选结合物。2. 利用表面等离子体共振结合x射线晶体学表征vlr相互作用的生化和结构特性。3. 通过随机或靶向诱变设计高亲和力配体结合VLRs。总的来说，这些研究将确定VLR对不同抗原的亲和力范围，确定原始VLR库是否足够多样化以允许从非免疫文库中分离特异性结合物，确定VLR识别抗原的结构基础，并建立这些新型抗原受体的亲和力成熟的有效策略。公共卫生相关性：尽管哺乳动物抗体是生物技术中非常有用的试剂，但由于其大小和复杂性，它们有一定的局限性。我们建议从海七鳃鳗中开发高亲和力的重组抗体，作为一类全新的识别分子，用于生物技术的诊断、分析和生物传感器应用。

DESCRIPTION (provided by applicant): Antibodies are highly useful reagents in biotechnological and biomedical applications, yet recombinant antibodies have their limitations. These include the complex architecture of their antigen- binding sites, which complicates the generation of synthetic antibody libraries, and major patent restrictions on recombinant antibody development. As a result, there has been an intensive search for alternatives to antibodies in the form of non-immunoglobulin (Ig) scaffolds such as ankyrin repeats, fibronectins, lipocalins and protein A. However, only a few of these artificial scaffolds have actually yielded specificities towards different types of targets, possibly because none of them are natural antigen receptors. Indeed, the only known natural non-Ig antigen receptors are the variable lymphocyte receptors (VLRs) of lamprey and hagfish. VLRs consist of highly diverse leucine-rich repeat modules, which are assembled by DNA recombination via gene conversion, resulting in a potential repertoire of over 1014 unique receptors. Lamprey immunized with various antigens respond by production of high avidity plasma VLRs specific for the antigen. VLRs are thus ideal candidates to serve as non-Ig single chain antibodies in biotechnology. We have developed a high-throughput platform for the selection of recombinant antigen-binding VLRs from large VLR libraries (>107 independent clones) using yeast surface display technology and a customized yeast display vector. We have isolated VLRs with low micromolar affinity to hen egg lysozyme (HEL) and low nanomolar affinity to R-phycoerythrin. One round of in vitro affinity maturation decreased by 13-fold the dissociation constant of an HEL-binding VLR, and a VLR-HEL complex has been crystallized. We propose to establish the biotechnological potential of monoclonal VLRs as a new class of molecular recognition molecules. Our goals are: 1. Clone VLRs that specifically bind diverse ligands representing the antigenic world, including proteins, carbohydrates and whole bacteria. Both naove and immune libraries will be constructed and screened for binders by yeast surface display. 2. Characterize the biochemical and structural properties of VLR-interactions using surface plasmon resonance combined with X-ray crystallography. 3. Engineer high affinity ligand-binding VLRs by random or targeted mutagenesis. Collectively, these studies will define the affinity range of VLRs for diverse antigens, determine whether the naove VLR repertoire is sufficiently diverse to allow isolation of specific binders from nonimmune libraries, define the structural basis for antigen recognition by VLRs, and establish efficient strategies for affinity maturation of these novel antigen receptors. PUBLIC HEALTH RELEVANCE: Although mammalian antibodies are highly useful reagents in biotechnology, they have certain limitations related to their size and complexity. We propose to develop high affinity recombinant antibodies from the sea lamprey as a completely new class of recognition molecules for diagnostic, analytic, and biosensor applications in biotechnology.