RESUMO
IgG molecules are widely used as therapeutic agents either in the form of intact Abs or as Fc fusion proteins. Although efficient binding of the IgG Fc fragment to cellular FcγRs may be essential to achieve a high cytolytic activity, it may be advantageous for other applications to limit or abolish this interaction. Genetic or biochemical approaches have been used to generate these non-FcγR-binding IgG variants. By using soluble versions of FcγRs and monomeric versions of these altered IgG molecules, it was demonstrated that these IgG variants no longer bind to FcγRs. Importantly, however, these assays do not reflect the physiologic interaction of IgG with low-affinity cellular FcγRs occurring in the form of multimeric immune complexes. In this study, we investigated how the size of an immune complex can affect the interaction of normal and various versions of potentially non-FcγR-binding IgG variants with cellular FcγRs. We show that neither the D265A mutation nor EndoS treatment resulting in IgG molecules with only one N-acetylglucosamine and a fucose residue was fully able to abolish the interaction of all IgG subclasses with cellular FcγRs, suggesting that IgG subclass-specific strategies are essential to fully interfere with human FcγR binding.
Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Sítios de Ligação de Anticorpos/imunologia , Imunoglobulina G/metabolismo , Receptores de IgG/metabolismo , Alelos , Animais , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/genética , Sítios de Ligação de Anticorpos/genética , Células CHO , Linhagem Celular , Cricetinae , Glicosídeo Hidrolases/farmacologia , Glicosilação , Células HEK293 , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Camundongos , Mutação/imunologia , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/farmacologia , Receptores de IgG/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
MAb 2G12 neutralizes HIV-1 by binding with high affinity to a cluster of high-mannose oligosaccharides on the envelope glycoprotein, gp120. Screening of phage-displayed peptide libraries with 2G12 identified peptides that bind specifically, with K(d)s ranging from 0.4 to 200 microM. The crystal structure of a 21-mer peptide ligand in complex with 2G12 Fab was determined at 2.8 A resolution. Comparison of this structure with previous structures of 2G12-carbohydrate complexes revealed striking differences in the mechanism of 2G12 binding to peptide vs. carbohydrate. The peptide occupies a site different from, but adjacent to, the primary carbohydrate-binding site on 2G12, and makes only slightly fewer contacts to the Fab than Man(9)GlcNAc(2) (51 vs. 56, respectively). However, only two antibody contacts with the peptide are hydrogen bonds in contrast to six with Man(9)GlcNAc(2), and only three of the antibody residues that interact with Man(9)GlcNAc(2) also contact the peptide. Thus, this mechanism of peptide binding to 2G12 does not support structural mimicry of the native carbohydrate epitope on gp120, since it neither replicates the oligosaccharide footprint on the antibody nor most of the contact residues. Moreover, 2G12.1 peptide is not an immunogenic mimic of the 2G12 epitope, since antisera produced against it did not bind gp120.
Assuntos
Anticorpos Monoclonais/metabolismo , Epitopos/química , Anticorpos Anti-HIV/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/metabolismo , Mimetismo Molecular , Peptídeos/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação de Anticorpos , Anticorpos Amplamente Neutralizantes , Cristalização , Cristalografia por Raios X , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Oligossacarídeos/química , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/imunologia , CoelhosRESUMO
Despite numerous attempts over many years to develop an HIV vaccine based on classical strategies, none has convincingly succeeded to date. A number of approaches are being pursued in the field, including building upon possible efficacy indicated by the recent RV144 clinical trial, which combined two HIV vaccines. Here, we argue for an approach based, in part, on understanding the HIV envelope spike and its interaction with broadly neutralizing antibodies (bnAbs) at the molecular level and using this understanding to design immunogens as possible vaccines. BnAbs can protect against virus challenge in animal models, and many such antibodies have been isolated recently. We further propose that studies focused on how best to provide T cell help to B cells that produce bnAbs are crucial for optimal immunization strategies. The synthesis of rational immunogen design and immunization strategies, together with iterative improvements, offers great promise for advancing toward an HIV vaccine.