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1.
Commun Biol ; 6(1): 828, 2023 08 09.
Article in English | MEDLINE | ID: mdl-37558752

ABSTRACT

Interleukin-2 (IL-2) engineered versions, with biased immunological functions, have emerged from yeast display and rational design. Here we reshaped the human IL-2 interface with the IL-2 receptor beta chain through the screening of phage-displayed libraries. Multiple beta super-binders were obtained, having increased receptor binding ability and improved developability profiles. Selected variants exhibit an accumulation of negatively charged residues at the interface, which provides a better electrostatic complementarity to the beta chain, and faster association kinetics. These findings point to mechanistic differences with the already reported superkines, characterized by a conformational switch due to the rearrangement of the hydrophobic core. The molecular bases of the favourable developability profile were tracked to a single residue: L92. Recombinant Fc-fusion proteins including our variants are superior to those based on H9 superkine in terms of expression levels in mammalian cells, aggregation resistance, stability, in vivo enhancement of immune effector responses, and anti-tumour effect.


Subject(s)
Directed Molecular Evolution , Interleukin-2 Receptor beta Subunit , Interleukin-2 , Peptide Library , Humans , Interleukin-2 Receptor beta Subunit/chemistry , Interleukin-2/chemistry , Interleukin-2/genetics , Interleukin-2/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Directed Molecular Evolution/methods , Protein Domains , Animals , Mice , Cell Line, Tumor
2.
Sci Rep ; 12(1): 12268, 2022 07 18.
Article in English | MEDLINE | ID: mdl-35851313

ABSTRACT

HER-1 and HER-2 are tumor-associated antigens overexpressed in several epithelial tumors, and successfully targeted by therapeutic approaches against cancer. Vaccination with their recombinant extracellular domains has had encouraging results in the pre-clinical setting. As complex humoral responses targeting multiple epitopes within each antigen are the ultimate goal of such active immunotherapy strategies, molecular dissection of the mixture of antibody specificities is required. The current work exploits phage display of antigenic versions of HER-1 and HER-2 domains to accomplish domain-level epitope mapping. Recognition of domains I, III and IV of both antigens by antibodies of immunized mice was shown, indicating diverse responses covering a broad range of antigenic regions. The combination of phage display and site-directed mutagenesis allowed mutational screening of antigen surface, showing polyclonal antibodies' recognition of mutated receptor escape variants known to arise in patients under the selective pressure of the anti-HER-1 antibody cetuximab. Phage-displayed HER domains have thus the potential to contribute to fine specificity characterization of humoral responses during future development of anti-cancer vaccines.


Subject(s)
Bacteriophages , Cancer Vaccines , Animals , Antibodies , Antigens, Neoplasm , Epitope Mapping/methods , Mice , Peptide Library , Technology
3.
MAbs ; 6(6): 1368-76, 2014.
Article in English | MEDLINE | ID: mdl-25484050

ABSTRACT

Antibody engineering must be accompanied by mapping strategies focused on identifying the epitope recognized by each antibody to define its unique functional identity. High throughput fine specificity determination remains technically challenging. We review recent experiences aimed at revisiting the oldest and most extended display technology to develop a robust epitope mapping platform, based on the ability to manipulate target-derived molecules (ranging from the whole native antigen to antigen domains and smaller fragments) on filamentous phages. Single, multiple and combinatorial mutagenesis allowed comprehensive scanning of phage-displayed antigen surface that resulted in the identification of clusters of residues contributing to epitope formation. Functional pictures of the epitope(s) were thus delineated in the natural context. Successful mapping of antibodies against interleukin-2, epidermal growth factor and its receptor, and vascular endothelial growth factor showed the versatility of these procedures, which combine the accuracy of site-directed mutagenesis with the high throughput potential of phage display.


Subject(s)
Antigens/immunology , Cell Surface Display Techniques/methods , Epitope Mapping/methods , Peptide Library , Antigens/chemistry , Antigens/genetics , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Humans , Models, Molecular , Mutagenesis, Site-Directed/methods , Protein Structure, Tertiary
4.
MAbs ; 6(3): 637-48, 2014.
Article in English | MEDLINE | ID: mdl-24589624

ABSTRACT

Although multiple different procedures to characterize the epitopes recognized by antibodies have been developed, site-directed mutagenesis remains the method of choice to define the energetic contribution of antigen residues to binding. These studies are useful to identify critical residues and to delineate functional maps of the epitopes. However, they tend to underestimate the roles of residues that are not critical for binding on their own, but contribute to the formation of the target epitope in an additive, or even cooperative, way. Mapping antigenic determinants with a diffuse energetic landscape, which establish multiple individually weak interactions with the antibody paratope, resulting in high affinity and specificity recognition of the epitope as a whole, is thus technically challenging. The current work was aimed at developing a combinatorial strategy to overcome the limitations of site-directed mutagenesis, relying on comprehensive randomization of discrete antigenic regions within phage-displayed antigen libraries. Two model antibodies recognizing epidermal growth factor were used to validate the mapping platform. Abrogation of antibody recognition due to the introduction of simultaneous replacements was able to show the involvement of particular amino acid clusters in epitope formation. The abundance of some of the original residues (or functionally equivalent amino acids sharing their physicochemical properties) among the set of mutated antigen variants selected on a given antibody highlighted their contributions and allowed delineation of a detailed functional map of the corresponding epitope. The use of the combinatorial approach could be expanded to map the interactions between other antigens/antibodies.


Subject(s)
Epidermal Growth Factor/genetics , Epidermal Growth Factor/immunology , Epitope Mapping/methods , Mutagenesis , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antigen-Antibody Reactions , Antigens/chemistry , Antigens/genetics , Antigens/immunology , Cell Line, Tumor , Epidermal Growth Factor/chemistry , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Humans , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Library , Protein Conformation
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