IgA Nephropathy: Interventions with Generation of Nephritogenic Immune Complexes

描述(申请人提供)：IgA肾病(IgAN)是世界上最常见的肾小球肾炎。最近，我们已经为IgAN的自身免疫特性提供了证据：肾炎免疫复合体是由重(H)链的唯一铰链区(HR)上具有改变的O-连接糖链的IgA1分子作为抗原而形成的，这些抗原被自然产生的糖特异性抗体识别。我们还证明了形成的IC的致病潜能强烈依赖于分子质量：我们在体外和体内都证明了只有分子质量~700-1000 kDa的大IC才表现出肾性。在这一应用中，我们建议探索一种新的、高度非常规的新兴方法，利用仅包含H链可变区(VHH片段)的骆驼单域抗体作为肾炎性IC形成的抑制物。骆驼抗体的VHH片段含有仅由H的互补决定区贡献的抗原结合部位，而不是轻链，对抗原具有很高的亲和力，含有~100个氨基酸(~16 kDa)，不激活补体级联，非常稳定，高度水溶性，在包括人类在内的不同物种中注射时不具有抗原性。对于我们的建议来说，最重要的是，VHH是单价的，因此没有交联性，所以形成的IC将是低分子质量的，因此不会导致肾病。此外，我们还建立了可用于检测IC肾毒性的体外和体内检测方法。重要的是，我们的研究结果可能适用于并推广到最终干扰其他人类IC疾病的致病IC的形成，VHH片段也可能因其精致的特异性和稳定性而被用作优秀的诊断工具。

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.