Improved affinity for the neonatal Fc receptor (FcRn) is known to extend antibody half-life in vivo. However, this has never been linked with enhanced therapeutic efficacy. We tested whether antibodies with half-lives extended up to fivefold in human (h)FcRn transgenic mice and threefold in cynomolgus monkeys retain efficacy at longer dosing intervals. We observed that prolonged exposure due to FcRn-mediated enhancement of half-life improved antitumor activity of Fcengineered antibodies in an hFcRn/Rag1 −/− mouse model. This bridges the demand for dosing convenience with the clinical necessity of maintaining efficacy.The well-established role of FcRn in IgG serum turnover has been the foundation for Fc engineering efforts aimed at improving the pharmacokinetic (PK) properties of antibodies 1, 2. Despite contrary results about the relationship between FcRn affinity and half-life3 , 4, a number of such PK engineering studies in non-human primates, whose FcRn is similar to that of human, have demonstrated increased half-life by engineered antibody variants [5][6][7][8] . Yet while the successful extension of half-life in PK experiments bodes well for the prospect of improving clinical dosing, a critical gap remains. For half-life extension technologies to be of practical use, efficacy of a biotherapeutic with longer half-life must be preserved at longer dosing intervals. Although the relationship between drug exposure and efficacy is well-established, this correlation has not thus far been established for antibodies Fc-engineered for longer halflife.Rational design methods coupled with high-throughput protein screening were used to engineer a series of Fc variants with greater affinity for human FcRn. Variants were constructed in the context of the humanized anti-VEGF IgG1 antibody bevacizumab 9 (Avastin®, Genentech/ Roche), which is currently approved for the treatment of colorectal, lung, breast, and renal cancers. A description of the construction, production, and binding studies of the antibodies is provided in the Supplementary Methods. Antibodies were screened for binding to human FcRn at pH 6.0 using Biacore. Engineered variants provide between 3 and 20-fold greater binding to FcRn at pH 6.0, with improvements due almost exclusively to slower off-rate (k off ) ( Supplementary Fig. 1, Supplementary Table 1). A lead variant M428L/N434S, subsequently selected principally based on its PK performance (see below), provided an 11-fold improvement in FcRn affinity at pH 6.0. This double substitution in the context of bevacizumab is referred to as Xtend™-VEGF. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptA PK study was carried out in cynomolgus monkeys (macaca fascicularis) in order to evaluate the capacity of the variants to improve serum half-life in monkeys. A description of these experiments is provided in the Supplementary Methods. Binding improvements of the variants to monkey FcRn at pH 6.0 were comparable to improvements for human FcRn, and the rank order of the variants i...

Despite the general observation that single domain proteins denature in a completely cooperative manner, amide hydrogen exchange of ribonuclease H in low levels of denaturant demonstrates the existence of two partially folded species. The structures of these marginally stable species resemble kinetic folding intermediates and the molten globule state of the protein. These data suggest that the first region to fold is the thermodynamically most stable portion of the protein and that the molten globule is a high free energy conformation present at equilibrium in the native state.

No abstract

Robust generation of IgG bispecific antibodies has been a long-standing challenge. Existing methods require extensive engineering of each individual antibody, discovery of common light chains, or complex and laborious biochemical processing. Here we combine computational and rational design approaches with experimental structural validation to generate antibody heavy and light chains with orthogonal Fab interfaces. Parental monoclonal antibodies incorporating these interfaces, when simultaneously co-expressed, assemble into bispecific IgG with improved heavy chain-light chain pairing. Bispecific IgGs generated with this approach exhibit pharmacokinetic and other desirable properties of native IgG, but bind target antigens monovalently. As such, these bispecific reagents may be useful in many biotechnological applications.

Atomic force microscopy has been employed to investigate the structural organization of amyloid fibrils produced in vitro from three very different polypeptide sequences. The systems investigated are a 10-residue peptide derived from the sequence of transthyretin, the 90-residue SH3 domain of bovine phosphatidylinositol-3'-kinase, and human wild-type lysozyme, a 130-residue protein containing four disulfide bridges. The results demonstrate distinct similarities between the structures formed by the different classes of fibrils despite the contrasting nature of the polypeptide species involved. SH3 and lysozyme fibrils consist typically of four protofilaments, exhibiting a left-handed twist along the fibril axis. The substructure of TTR(10-19) fibrils is not resolved by atomic force microscopy and their uniform appearance is suggestive of a regular self-association of very thin filaments. We propose that the exact number and orientation of protofilaments within amyloid fibrils is dictated by packing of the regions of the polypeptide chains that are not directly involved in formation of the cross-beta core of the fibrils. The results obtained for these proteins, none of which is directly associated with any human disease, are closely similar to those of disease-related amyloid fibrils, supporting the concept that amyloid is a generic structure of polypeptide chains. The detailed architecture of an individual fibril, however, depends on the manner in which the protofilaments assemble into the fibrillar structure, which in turn is dependent on the sequence of the polypeptide and the conditions under which the fibril is formed.