No abstract

For complex diseases in which multiple mediators contribute to overall disease pathogenesis by distinct or redundant mechanisms, simultaneous blockade of multiple targets may yield better therapeutic efficacy than inhibition of a single target. However, developing two separate monoclonal antibodies for clinical use as combination therapy is impractical, owing to regulatory hurdles and cost. Multi-specific, antibody-based molecules have been investigated; however, their therapeutic use has been hampered by poor pharmacokinetics, stability and manufacturing feasibility. Here, we describe a generally applicable model of a dual-specific, tetravalent immunoglobulin G (IgG)-like molecule--termed dual-variable-domain immunoglobulin (DVD-Ig)--that can be engineered from any two monoclonal antibodies while preserving activities of the parental antibodies. This molecule can be efficiently produced from mammalian cells and exhibits good physicochemical and pharmacokinetic properties. Preclinical studies of a DVD-Ig protein in an animal disease model demonstrate its potential for therapeutic application in human diseases.

Loricrin is the major protein of the cornified cell envelope of terminally differentiated epidermal keratinocytes which functions as a physical barrier. In order to understand its properties and role in cornified cell envelope, we have expressed human loricrin from a fulllength cDNA clone in bacteria and purified it to homogeneity. We have also isolated loricrin from newborn mouse epidermis. By circular dichroism and fluorescence spectroscopy, the in vivo mouse and bacterially expressed human loricrins possess no ␣ or ␤ structure but have some organized structure in solution associated with their multiple tyrosines and can be reversibly denatured by either guanidine hydrochloride or temperature. The transglutaminase (TGase) 1, 2, and 3 enzymes expressed during epidermal differentiation utilized loricrin in vitro as a complete substrate, but the types of cross-linking were different. The TGase 3 reaction favored certain lysines and glutamines by forming mostly intrachain cross-links, whereas TGase 1 formed mostly large oligomeric complexes by interchain crosslinks involving different lysines and glutamines. Together, the glutamines and lysines used in vitro are almost identical to those seen in vivo. The data support a hypothesis for the essential and complementary roles of both TGase 1 and TGase 3 in cross-linking of loricrin in vivo. Failure to cross-link loricrin by TGase 1 may explain the phenotype of lamellar ichthyosis, a disease caused by mutations in the TGase 1 gene.Terminal differentiation in the epidermis involves the expression of a number of specific proteins that ultimately fulfill different structural roles in the cornified, dead stratum corneum cell. One set of proteins is the keratin intermediate filaments and the interfilamentous matrix protein filaggrin (1-3). A second set of proteins is used to construct the cornified cell envelope (CE), 1 a 15-nm-thick layer of protein deposited on the inner surface of the cell periphery, which serves as a physical barrier for the epidermis (4, 5). The CE proteins are rendered insoluble by cross-linking by both disulfide bonds and the N ⑀ -(␥-glutamyl)lysine isopeptide bond formed by the action of one or more of the three known epidermal transglutaminases (TGases) (4 -6). Several proteins have now been documented as CE constituents by direct sequencing analyses of cross-linked peptides (7), including loricrin, small proline-rich proteins 1 and 2 (SPR1 and SPR2), elafin, keratins, filaggrin, and desmoplakin. The proteins involucrin and cystatin ␣ are also likely constituents, but direct sequencing of cross-linked peptides involving these proteins has not yet been reported (reviewed in Ref. 8).In particular, a variety of data have suggested that loricrin comprises about 75% of the total CE protein mass (reviewed in Ref. 9), or 85-95% of the cytoplasmic two-thirds of the CE. In fact, amino acid sequencing of many peptides recovered by the proteolysis has now provided rigorous support for this idea (7). About 90% of the molar mass of peptides from the cytoplasmic...

Peptidylarginine deiminases, which are commonly found in mammalian cells, catalyze the deimination of protein-bound arginine residues to citrullines. However, very little is known about their substrate requirements and the significance or consequences of this postsynthetic modification. We have explored this reaction in vitro with two known substrates filaggrin and trichohyalin. First, the degree and rate of modification of arginines to citrullines directly correlates with the structural order of the substrate. In filaggrin, which has little structural order, the reaction proceeded rapidly to >95% completion. However, in the highly ␣-helical protein trichohyalin, the reaction proceeded slowly to about 25% and could be forced to a maximum of about 65%. Second, the rate and degree of modification depends on the sequence location of the target arginines. Third, we show by gel electrophoresis, circular dichroism, and fluorescence spectroscopy that the reaction interferes with organized protein structure: the net formation of 10% citrulline results in protein denaturation. Cyanate modification of the lysines in model ␣-helix-rich proteins to homocitrullines also results in loss of organized structure. These data suggest that the ureido group on the citrulline formed by the peptidylarginine deiminase enzyme modification functions to unfold proteins due to decrease in net charge, loss of potential ionic bonds, and interference with H bonds.