IgA Nephropathy: Interventions with Generation of Nephritogenic Immune Complexes

描述（由申请人提供）：IgA肾病（IgAN）是世界上最常见的肾小球肾炎。最近，我们为IgAN的自身免疫特性提供了证据：形成肾源性免疫复合物，其中IgA1分子在重链(H)的独特铰链区（HR）具有改变的o -链聚糖作为抗原，被天然存在的聚糖特异性抗体识别。我们还证明了形成的IC的致病潜力强烈依赖于分子质量：我们在体外和体内证明，只有分子质量为700-1000 kDa的大IC才表现出肾原性。在这项应用中，我们提出探索一种新颖的、高度非常规的新兴方法，利用仅含有H链可变区域（VHH片段）的骆驼单域抗体的潜力，作为兔源性IC形成的抑制剂。骆驼抗体的VHH片段含有抗原结合位点，仅由H链的互补性决定区域贡献，而不是轻链，对抗原具有高亲和力。含有约100个氨基酸（约16 kDa），不激活补体级联，非常稳定，高度水溶性，注射到包括人类在内的多种物种时不具有抗原性。对于我们的建议最重要的是，VHH是单价的，因此不交联，因此形成的IC将具有低分子质量，因此不会致肾性。此外，我们还开发了体外和体内试验方法，可用于检测IC的肾毒性。重要的是，我们的研究结果可能适用并扩展到最终干扰其他人类IC疾病致病性IC的形成，VHH片段因其精湛的特异性和稳定性也可作为优秀的诊断工具。

DESCRIPTION (provided by applicant): IgA nephropathy (IgAN) is the most frequent glomerulonephritis in the world. Recently we have provided evidence for the autoimmune character of IgAN: nephritogenic immune complexes are formed in which IgA1 molecules with altered O-linked glycans in the unique hinge region (HR) of the heavy (H) chains serve as antigens that are recognized by naturally occurring, glycan-specific antibodies. We have also demonstrated that the pathogenic potential of IC formed is strongly dependent on the molecular mass: we demonstrated in vitro and in vivo that only large IC with molecular mass ~700-1000 kDa display nephritogenicity. In this application we propose to explore a novel, highly unconventional and emerging approach exploiting the potential of camelid single domain antibodies containing only the variable region of H chain (VHH fragments) as inhibitors of the formation of nephritogenic IC. VHH fragments of camelid antibodies contain the antigen- binding site contributed only by complementarity-determining regions of the H, but NOT light chains, display a high affinity for antigens, contain ~100 amino acids (~16 kDa), do not activate the complement cascade, are extremely stable, highly water-soluble, and not antigenic when injected in diverse species, including humans. Most importantly for our proposal, VHH are monovalent and consequently non-cross-linking, so that IC formed would be of low-molecular mass, and therefore, not nephritogenic. Furthermore, we have developed in vitro and in vivo assays which can be used for testing of the IC nephrotoxicity. Importantly, results emerging from our studies may be applicable and extended in the ultimate interference with the formation of pathogenic IC in other human IC diseases and VHH fragment may be also used as excellent diagnostic tools due their exquisite specificity and stability.