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X-linked Alport syndrome (XLAS) is a progressive kidney disease caused by genetic abnormalities of COL4A5. Lack of collagen IV α5 chain staining and “basket-weave” by electron microscopy (EM) in glomerular basement membrane (GBM) are its typical pathology. However, the causal relationship between GBM defects and progressive nephropathy is unknown. We analyzed sequential pathology in a mouse model of XLAS harboring a human nonsense mutation of COL4A5. In mutant mice, nephropathy commenced from focal GBM irregularity by EM at 6 weeks of age, prior to exclusive crescents at 13 weeks of age. Low-vacuum scanning EM demonstrated substantial ragged features in GBM, and crescents were closely associated with fibrinoid exudate, despite lack of GBM break and podocyte depletion at 13 weeks of age. Crescents were derived from two sites by different cellular components. One was CD44 + cells, often with fibrinoid exudate in the urinary space, and the other was accumulation of α-SMA + cells in the thickened Bowman’s capsule. These changes finally coalesced, leading to global obliteration. In conclusion, vulnerability of glomerular and capsular barriers to the structural defect in collagen IV may cause non-necrotizing crescents via activation of PECs and migration of interstitial fibroblasts, promoting kidney disease in this model.

Antibodies are composed of structurally and functionally independent domains that can be used as building blocks to construct different types of chimeric protein-format molecules. However, the generally used genetic fusion and chemical approaches restrict the types of structures that can be formed and do not give an ideal degree of homogeneity. In this study, we combined mutation techniques with chemical conjugation to construct a variety of homogeneous bivalent and bispecific antibodies. First, building modules without lysine residues&mdash, which can be chemical conjugation sites&mdash, were generated by means of genetic mutation. Specific mutated residues in the lysine-free modules were then re-mutated to lysine residues. Chemical conjugation at the recovered lysine sites enabled the construction of homogeneous bivalent and bispecific antibodies from block modules that could not have been so arranged by genetic fusion approaches. Molecular evolution and bioinformatics techniques assisted in finding viable alternatives to the lysine residues that did not deactivate the block modules. Multiple candidates for re-mutation positions offer a wide variety of possible steric arrangements of block modules, and appropriate linkages between block modules can generate highly bioactive bispecific antibodies. Here, we propose the effectiveness of the lysine-free block module design for site-specific chemical conjugation to form a variety of types of homogeneous chimeric protein-format molecule with a finely tuned structure and function.

The conjugation of polyethylene glycol (PEGylation) with downsized compact antibodies is an effective method for overcoming the problem of rapid elimination of the compact antibodies from the body. We integrated site-specific PEGylation with the refolding of a single-chain Fv (scFv) of humanized monoclonal antibody 528 with affinity for the epidermal growth factor receptor, to prepare active PEGylated scFv from insoluble aggregates produced in an Escherichia coli expression system. The insertion of a cysteine residue at the C-terminus of scFv to serve as the conjugation site for PEG led to the formation of highly multimeric scFv during the refolding process; however, PEGylation after refolding drastically dispersed the multimer into monomeric active scFv fragments. Further, the PEGylation of partially refolded scFv during the refolding process improved the PEGylation efficiency and suppressed the formation of highly multimeric scFv; consequently, monomeric active scFv fragments were obtained directly from the insoluble aggregates in E. coli. We show that in vitro refolding of PEGylated scFv should be useful for improving downstream processing performance in the production of clinically useful small antibodies from insoluble fractions.