Molecular basis for the dynamic strength of the integrin alpha4beta1/VCAM-1 interaction

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

Molecular for the dynamic strength of the integrin α 4β1/VCAM-1 interaction

Intercellular adhesion mediated by integrin α(4)β(1) 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 α(4)β(1)/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 α(4)β(1)/VCAM-1 complex. Our force measurements revealed that the dissociation of the α(4)β(1)/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 Mg(2+) ion at the N-terminal A-domain of the β(1) subunit of α(4)β(1) 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 α(4)β(1)/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 α(4)β(1)/VCAM-1 complex was stabilized by integrin activation. Together, these findings provide a molecular explanation for the functionally relevant kinetic properties of the α(4)β(1)/VCAM-1 interaction

A fit-for-purpose strategy for the risk-based immunogenicity testing of biotherapeutics - a European industry perspective

There is much debate in the pharmaceutical industry on how to translate the current guidelines on immunogenicity testing for biotherapeutics into a testing strategy that suits the specific requirements of individual drug candidates. In this paper, member companies from the European immunogenicity platform (EIP) present a consensus view on the essential requirements for immunogenicity testing of a biotherapeutic throughout the various phases of drug development, to ensure patient safety and to enable successful market entry. Our aim is to open the debate and provoke discussion on this important topic which is unique to biotherapeutic drug development. The scope of this paper is limited to aspects relevant to biotherapeutic drug development and does not include fundamental academic studies of immunogenicity. Here, we propose two pre-defined testing strategies for the detection and characterization of anti-drug antibody (ADA) responses where the different strategies are based on the phase of development for a biotherapeutic, a. without (category 1) and b. with (category 2) the expected potential to elicit ADA mediated severe clinical consequences. The harm of a potential ADA response determines which of the two testing strategies is adopted. The scientific rationale on which the "case-by-case" approach advocated in white papers and guidance documents may be translated for each individual drug development program is provided and, underpins the recommendations made here

ABIRISK Consortium Recommendations for Terms and Definitions for Describing and Interpreting Unwanted Immunogenicity of Biopharmaceuticals

Biopharmaceuticals (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 responses that affect clinical safety or efficacy, remain among the most common negative effects associated with this important class of drug therapies. Therefore, there is a growing need to elucidate the underlying causes and to evaluate methods for predicting and mitigating immunogenicity. Supported by the Innovative Medicines Initiative (IMI; www.imi-europe.org), the ABIRISK consortium (Anti-Biopharmaceutical [BP] Immunization Prediction and Clinical Relevance to Reduce the Risk; www.abirisk.eu), was formed in 2012 with participants representing a network of leaders from various communities who have an interest in furthering the understanding and reducing the risk of BP immunogenicity: clinicians involved in the care of patients treated with various type of BPs, academic scientists researching mechanisms of immunogenicity, and EFPIA (European Federation of Pharmaceutical Industries and Associations) scientists involved in development of BPs. ABIRISK’s goal of improving the understanding of BP immunization should also lead to the generation of guidelines for drug development and the clinical care of patients. A prerequisite to understanding the impact of immunogenicity is to provide clear definitions around terms and concepts related to immunogenicity, its prediction, and associated clinical events. An overview of the concepts behind key terms and definitions adopted to date by ABIRISK is provided in this document along with a link to the documented ABIRISK terms and definitions that will be maintained on the ABIRISK website throughout the duration of the consortium. It is anticipated that refinement of definitions as well as introduction of new terms will be needed over the 5 year duration of the consortium’s work. In addition, ABIRISK will invite comment on these terms, definitions and concepts from other immunogenicity experts within the broader community of medical, academic, and pharmaceutical scientists outside of the ABIRISK consortium. Therefore, it is the consortium’s intention to maintain the most current version on the ABIRISK site for public use, review and comment

ABIRISK Consortium Recommendations for Terms and Definitions for Describing and Interpreting Unwanted Immunogenicity of Biopharmaceuticals

Biopharmaceuticals (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 responses that affect clinical safety or efficacy, remain among the most common negative effects associated with this important class of drug therapies. Therefore, there is a growing need to elucidate the underlying causes and to evaluate methods for predicting and mitigating immunogenicity. Supported by the Innovative Medicines Initiative (IMI; www.imi-europe.org), the ABIRISK consortium (Anti-Biopharmaceutical [BP] Immunization Prediction and Clinical Relevance to Reduce the Risk; www.abirisk.eu), was formed in 2012 with participants representing a network of leaders from various communities who have an interest in furthering the understanding and reducing the risk of BP immunogenicity: clinicians involved in the care of patients treated with various type of BPs, academic scientists researching mechanisms of immunogenicity, and EFPIA (European Federation of Pharmaceutical Industries and Associations) scientists involved in development of BPs. ABIRISK’s goal of improving the understanding of BP immunization should also lead to the generation of guidelines for drug development and the clinical care of patients. A prerequisite to understanding the impact of immunogenicity is to provide clear definitions around terms and concepts related to immunogenicity, its prediction, and associated clinical events. An overview of the concepts behind key terms and definitions adopted to date by ABIRISK is provided in this document along with a link to the documented ABIRISK terms and definitions that will be maintained on the ABIRISK website throughout the duration of the consortium. It is anticipated that refinement of definitions as well as introduction of new terms will be needed over the 5 year duration of the consortium’s work. In addition, ABIRISK will invite comment on these terms, definitions and concepts from other immunogenicity experts within the broader community of medical, academic, and pharmaceutical scientists outside of the ABIRISK consortium. Therefore, it is the consortium’s intention to maintain the most current version on the ABIRISK site for public use, review and comment