The tertiary structure of the integrin heterodimer is currently unknown, although several predictive models have been generated. Detailed structural studies of integrins have been consistently hampered for several reasons, including the small amounts of purified protein available, the large size and conformational flexibility of integrins, and the presence of transmembrane domains and N-linked glycosylation sites in both receptor subunits. As a first step toward obtaining crystals of an integrin receptor, we have expressed a minimized dimer. By using the Fc dimerization and mammalian cell expression system designed and optimized by Stephens et al. Integrins are ␣,␤ heterodimeric transmembrane receptors that play central roles in cell adhesion, migration, differentiation, and survival (1). Several lines of evidence indicate that integrins also contribute to the progression of a wide variety of diseases, including inflammatory, thrombotic, and neoplastic conditions (2-4), and that the integrin families are valid therapeutic targets. The rational design of integrin antagonists based on ligand peptide motifs such as RGD and LDV is currently well advanced. Although the tertiary structure of the integrin heterodimer is unknown, this information would aid the process of drug development, and it represents one of the most important outstanding questions in the field.The overall shape and dimensions of the ␣IIb␤ 3 and ␣ 5 ␤ 1 integrin heterodimers have been revealed by rotary shadowing electron microscopy (5-7). Both receptors consisted of an Nterminal globular head of 8 -12 nm with two extended tails of 18 -20 nm that corresponded to the C termini (7). Similarly, a soluble ␣IIb␤ 3 integrin, generated by removal of the ␣IIb and ␤ 3 transmembrane and cytoplasmic domains, and the ␤ 3 cysteine-rich repeats also contained a globular head, but its tails were 4 -6 nm shorter (8).In the absence of a tertiary structure for the integrin heterodimer, several predictive models have been generated (9 -12), and these have subsequently been supported by biochemical analyses (13)(14)(15)(16). Whereas the ␣-and ␤-subunits are unrelated in primary sequence, they share common structural features including an N-terminal globular ligand-binding domain, C-terminal stalk regions, transmembrane domains, and short cytoplasmic domains (17,18). The N-terminal portion of ␣-subunits contains seven homologous repeats, each 60 -70 amino acid residues in length. These repeats are quite similar in sequence, and repeat four in some integrins and repeats five to seven in all receptors contain EF-hand-like divalent cationbinding motifs. The seven repeats have been predicted to fold cooperatively, forming an all-␤ structure known as a ␤-propeller fold (9) (Fig.

Intercellular adhesion mediated by integrin alpha4beta1 and vascular cell adhesion molecule-1 (VCAM-1) plays a crucial role in both the rolling and firm attachment of leukocytes onto the vascular endothelium. Essential to the alpha4beta1/VCAM-1 interaction is its mechanical strength that allows the complex to resist the large shear forces imposed by the bloodstream. Herein we employed single-molecule dynamic force spectroscopy to investigate the dynamic strength of the alpha4beta1/VCAM-1 complex. Our force measurements revealed that the dissociation of the alpha4beta1/VCAM-1 complex involves overcoming at least two activation potential barriers: a steep inner barrier and a more elevated outer barrier. The inner barrier grants the complex the tensile strength to withstand large pulling forces (>50 pN) and was attributed to the ionic interaction between the chelated Mg2+ ion at the N-terminal A-domain of the beta1 subunit of alpha4beta1 and the carboxyl group of Asp-40 of VCAM-1 through the use of site-directed mutations. In general, additional mutations within the C-D loop of domain 1 of VCAM-1 suppressed both inner and outer barriers of the alpha4beta1/VCAM-1 complex, while a mutation at Asp-143 of domain 2 of VCAM-1 resulted in the suppression of the outer barrier, but not the inner barrier. In contrast, the outer barrier of alpha4beta1/VCAM-1 complex was stabilized by integrin activation. Together, these findings provide a molecular explanation for the functionally relevant kinetic properties of the alpha4beta1/VCAM-1 interaction.

SummaryBiopharmaceuticals (BPs) represent a rapidly growing class of approved and investigational drug therapies that is contributing significantly to advancing treatment in multiple disease areas, including inflammatory and autoimmune diseases, genetic deficiencies and cancer. Unfortunately, unwanted immunogenic responses to BPs, in particular those affecting clinical safety or efficacy, remain among the most common negative effects associated with this important class of drugs. To manage and reduce risk of unwanted immunogenicity, diverse communities of clinicians, pharmaceutical industry and academic scientists are involved in: interpretation and management of clinical and biological outcomes of BP immunogenicity, improvement of methods for describing, predicting and mitigating immunogenicity risk and elucidation of underlying causes. Collaboration and alignment of efforts across these communities is made difficult due to lack of agreement on concepts, practices and standardized terms and definitions related to immunogenicity. The Innovative Medicines Initiative (IMI; www.imi-europe.org), ABIRISK consortium [Anti‐Biopharmaceutical (BP) Immunization Prediction and Clinical Relevance to Reduce the Risk; www.abirisk.eu] was formed by leading clinicians, academic scientists and EFPIA (European Federation of Pharmaceutical Industries and Associations) members to elucidate underlying causes, improve methods for immunogenicity prediction and mitigation and establish common definitions around terms and concepts related to immunogenicity. These efforts are expected to facilitate broader collaborations and lead to new guidelines for managing immunogenicity. To support alignment, an overview of concepts behind the set of key terms and definitions adopted to date by ABIRISK is provided herein along with a link to access and download the ABIRISK terms and definitions and provide comments (http://www.abirisk.eu/index_t_and_d.asp).

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Background: IgG4 antibodies are able to undergo a process termed Fab arm exchange (FAE). Results: A novel method for quantifying FAE in physiologically relevant matrices was developed. Conclusion: A hinge mutation in IgG4 antibodies inhibits FAE to undetectable levels in the immunoassays described herein. Significance: These methods are valuable for investigating and assessing the factors affecting and controlling IgG4 FAE.