Recombinant Antibodies from the Sea Lamprey

说明(申请人提供)：抗体在生物技术和生物医学应用中是非常有用的试剂，然而重组抗体有其局限性。这些问题包括它们的抗原结合部位的复杂结构，这使得合成抗体库的生成复杂化，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)支架的形式密集寻找抗体的替代品，如锚蛋白重复序列、纤维连接蛋白、脂蛋白和蛋白A。然而，这些人工支架中只有几个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的抗原受体。事实上，唯一已知的天然非Ig抗原受体是七鳃鳗和七鳃鳗的可变淋巴细胞受体(VLR)。VLRs由高度多样化的富含亮氨酸的重复模块组成，这些重复模块通过基因转换通过DNA重组组装在一起，产生了超过1014个独特受体的潜在谱系。用不同抗原免疫的七鳃鳗通过产生针对该抗原的高亲和力的血浆VLRs来应答。因此，VLRs是生物技术中作为非Ig单链抗体的理想候选者。我们利用酵母表面展示技术和定制的酵母展示载体，开发了一个从大型VLR文库(&gt；107个独立克隆)中筛选重组抗原结合VLR的高通量平台。我们已经分离出对鸡蛋溶菌酶(HEL)具有低微摩尔亲和力、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VLR与HEL结合的解离常数降低了13倍，VLR-HEL复合体已经结晶。我们建议建立作为一类新的分子识别分子的单克隆性VLRs的生物技术潜力。我们的目标是：1.克隆与代表抗原世界的不同配体特异结合的VLR，包括蛋白质、碳水化合物和整个细菌。将构建NAOVE和免疫库，并通过酵母表面展示筛选结合蛋白。2.用表面等离子体共振结合X射线结晶学表征VLR-相互作用的生化和结构性质。3.通过随机或定点诱变的方法设计高亲和力配体结合的VLRs。总之，这些研究将确定VLR对不同抗原的亲和力范围，确定NAOVVLR谱系是否足够多样化，以允许从非免疫库中分离特定的结合蛋白，确定VLRs识别抗原的结构基础，并为这些新的抗原受体的亲和力成熟建立有效的策略。公共卫生相关性：虽然哺乳动物抗体在生物技术中是非常有用的试剂，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，用于生物技术中的诊断、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.