Abstract P-45: Structural Studies of Multispecific Antibody/Antigen Complexes by Cryo-EM

Background: Multispecific antibodies are artificially engineered molecules designed to bind simultaneously to several (different) antigens. Potential advantages of generating viable multispecific antibodies include the identification of malignant cells coupled with the concurrent recruitment of immu...

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Main Authors: David Fernandez-Martinez, Eaazhisai Kandiah, Magali Mathieu, Gordon Leonard
Format: Article
Language:English
Published: International Medical Research and Development Corporation 2019-06-01
Series:International Journal of Biomedicine
Subjects:
Online Access:http://ijbm.org/articles/IJBM_2019_9_S1_P45.pdf
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spelling doaj-b29f4c9729ab476e8b4e6363f1987b482020-11-25T02:22:46ZengInternational Medical Research and Development CorporationInternational Journal of Biomedicine2158-05102158-05292019-06-019Suppl_1S37S3710.21103/IJBM.9.Suppl_1.P45Abstract P-45: Structural Studies of Multispecific Antibody/Antigen Complexes by Cryo-EMDavid Fernandez-Martinez0Eaazhisai Kandiah1Magali Mathieu2Gordon Leonard3Structural Biology Group, European Synchrotron Radiation Facility, Grenoble, France; Sanofi R&D, IDD, Center de Recherche Vitry-sur-Seine, Vitry-sur-Seine Cedex, FranceStructural Biology Group, European Synchrotron Radiation Facility, Grenoble, FranceSanofi R&D, IDD, Center de Recherche Vitry-sur-Seine, Vitry-sur-Seine Cedex, FranceStructural Biology Group, European Synchrotron Radiation Facility, Grenoble, FranceBackground: Multispecific antibodies are artificially engineered molecules designed to bind simultaneously to several (different) antigens. Potential advantages of generating viable multispecific antibodies include the identification of malignant cells coupled with the concurrent recruitment of immune cells and the blocking of complex viral escape mechanisms. The cross-over dual-variable immunoglobulin (CODV-Ig) has been proposed as a universal bispecific therapeutic format. Its unique antigen-binding fragment (Fab) architecture provides pM affinities for ligands, no positional effect in target binding and a stable self-supporting structure. However, the three-dimensional arrangement of the constant and antigen-binding fragments in the CODV-Ig format may play a role in its in vivo effects. To further understand the structure and function of multispecific antibodies based on the CODV-Ig format high-resolution structural information is required. Towards this, we use cryo-electron microscopy (cryo-EM). Methods: We purified CODV-Ig both in an unbound state and in complex with a single antigen and validated sample quality using SDS-PAGE, Small Angle X-Ray Scattering (SAXS) and negative-stain electron microscopy (NSEM; Tecnai T12 and F20 microscopes at IBS, Grenoble). Data of sufficient quality for image analysis was obtained using a Titan Krios microscope (ESRF, Grenoble) equipped with a Quantum LS energy filter and K2 Summit direct electron detector. Results: NSEM of CODV-Ig resulted in low-resolution structural models and suggested a preferential orientation of the antibody under negative-stain conditions. Close-to-optimal vitrification conditions for CODV-Ig and antibody-antigen complexes have been identified. Efforts are in progress to reduce the antibody’s propensity to aggregation and aversion to conventional cryo-EM supports. Nevertheless, image processing of both CODV-Ig alone and in complex with antigens suggests very high flexibility and conformational heterogeneity. Conclusion: Particle heterogeneity may require additional data to be collected, in order to have sufficient signal that will lead to well-defined classes. An additional strategy may involve efforts to immobilize the molecules as to obtain fewer and better-defined classes.http://ijbm.org/articles/IJBM_2019_9_S1_P45.pdfantibodycryo-EMimage processing
collection DOAJ
language English
format Article
sources DOAJ
author David Fernandez-Martinez
Eaazhisai Kandiah
Magali Mathieu
Gordon Leonard
spellingShingle David Fernandez-Martinez
Eaazhisai Kandiah
Magali Mathieu
Gordon Leonard
Abstract P-45: Structural Studies of Multispecific Antibody/Antigen Complexes by Cryo-EM
International Journal of Biomedicine
antibody
cryo-EM
image processing
author_facet David Fernandez-Martinez
Eaazhisai Kandiah
Magali Mathieu
Gordon Leonard
author_sort David Fernandez-Martinez
title Abstract P-45: Structural Studies of Multispecific Antibody/Antigen Complexes by Cryo-EM
title_short Abstract P-45: Structural Studies of Multispecific Antibody/Antigen Complexes by Cryo-EM
title_full Abstract P-45: Structural Studies of Multispecific Antibody/Antigen Complexes by Cryo-EM
title_fullStr Abstract P-45: Structural Studies of Multispecific Antibody/Antigen Complexes by Cryo-EM
title_full_unstemmed Abstract P-45: Structural Studies of Multispecific Antibody/Antigen Complexes by Cryo-EM
title_sort abstract p-45: structural studies of multispecific antibody/antigen complexes by cryo-em
publisher International Medical Research and Development Corporation
series International Journal of Biomedicine
issn 2158-0510
2158-0529
publishDate 2019-06-01
description Background: Multispecific antibodies are artificially engineered molecules designed to bind simultaneously to several (different) antigens. Potential advantages of generating viable multispecific antibodies include the identification of malignant cells coupled with the concurrent recruitment of immune cells and the blocking of complex viral escape mechanisms. The cross-over dual-variable immunoglobulin (CODV-Ig) has been proposed as a universal bispecific therapeutic format. Its unique antigen-binding fragment (Fab) architecture provides pM affinities for ligands, no positional effect in target binding and a stable self-supporting structure. However, the three-dimensional arrangement of the constant and antigen-binding fragments in the CODV-Ig format may play a role in its in vivo effects. To further understand the structure and function of multispecific antibodies based on the CODV-Ig format high-resolution structural information is required. Towards this, we use cryo-electron microscopy (cryo-EM). Methods: We purified CODV-Ig both in an unbound state and in complex with a single antigen and validated sample quality using SDS-PAGE, Small Angle X-Ray Scattering (SAXS) and negative-stain electron microscopy (NSEM; Tecnai T12 and F20 microscopes at IBS, Grenoble). Data of sufficient quality for image analysis was obtained using a Titan Krios microscope (ESRF, Grenoble) equipped with a Quantum LS energy filter and K2 Summit direct electron detector. Results: NSEM of CODV-Ig resulted in low-resolution structural models and suggested a preferential orientation of the antibody under negative-stain conditions. Close-to-optimal vitrification conditions for CODV-Ig and antibody-antigen complexes have been identified. Efforts are in progress to reduce the antibody’s propensity to aggregation and aversion to conventional cryo-EM supports. Nevertheless, image processing of both CODV-Ig alone and in complex with antigens suggests very high flexibility and conformational heterogeneity. Conclusion: Particle heterogeneity may require additional data to be collected, in order to have sufficient signal that will lead to well-defined classes. An additional strategy may involve efforts to immobilize the molecules as to obtain fewer and better-defined classes.
topic antibody
cryo-EM
image processing
url http://ijbm.org/articles/IJBM_2019_9_S1_P45.pdf
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