Electron microscopic and immunochemical analysis of the broadly neutralizing HIV-1-specific, anti-carbohydrate antibody, 2G12

Roux, Kenneth H.; Zhu, Ping; Seavy, Margaret; Katinger, Hermann; Kunert, Renate; Seamon, Vanessa

doi:10.1016/j.molimm.2004.05.008

Cited by 19 publications

(17 citation statements)

References 38 publications

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“…This is an important result because Fc and lower hinge plays a key role in recruitment of C1q to initiate Fc-mediated neutralization events. Among the mAbs studied here, the shape of 2G12 supported the presence of strong Fab-Fab interaction along with Fab-Fc, an observation that has been reported before (7,9). In summary, all SAXS data-based modeling approaches provided similar results indicating that the global shapes of mAbs differ significantly; non-nmAbs adopt an open shape, and known nmAbs adopt a shape where at least one or both Fabs are unextended from the central Fc, which was found in a twisted orientation.…”

supporting

confidence: 83%

“…In contrast, gp140-bound 2G12 appeared to adopt a shape where the two Fab arms were locked above the Fc. The closed shape of 2G12 was supported in another EM image analysis-based study, where the mAb was studied in unliganded form (9). Further support for the Fab-Fab interactions leading to conjoined status of the Fab arms was seen from papain digestion of this mAb, where the Fab arms "cleaved off" as dimer.…”

mentioning

confidence: 71%

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Global Shape and Ligand Binding Efficiency of the HIV-1-neutralizing Antibodies Differ from Those of Antibodies That Cannot Neutralize HIV-1

Solanki

Rathore

Badmalia

et al. 2014

Journal of Biological Chemistry

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Background: It remains unaddressed whether varying neutralizing potency of mAbs is somehow correlated with differences in their global shapes. Results: Non-neutralizing mAbs have an open shape, whereas Fab-Fab and Fab-Fc interactions induce a closed shape in HIV-1-neutralizing mAbs. Conclusion:An unopen shape appears to be a hallmark of neutralizing potency, at least for HIV-1. Significance: This work provides new insight into the shape-function relationship of mAbs.

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supporting

confidence: 83%

mentioning

confidence: 71%

Global Shape and Ligand Binding Efficiency of the HIV-1-neutralizing Antibodies Differ from Those of Antibodies That Cannot Neutralize HIV-1

Solanki

Rathore

Badmalia

et al. 2014

Journal of Biological Chemistry

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“…2G12 protein has been reported to aggregate under strongly acidic conditions (16). To address whether the low pH used during elution from the protein A column affected the ability of 2G12 to dimerize, we purified 2G12 from transfected cell supernatants by using a neonatal Fc receptor affinity column in which samples were loaded at pH 6.0 and eluted at pH 8.0 (8).…”

Section: Resultsmentioning

confidence: 99%

Design and Expression of a Dimeric Form of Human Immunodeficiency Virus Type 1 Antibody 2G12 with Increased Neutralization Potency

West¹,

Galimidi²,

Foglesong³

et al. 2009

J Virol

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The antigen-binding fragment of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) antibody 2G12 has an unusual three-dimensional (3D) domain-swapped structure with two aligned combining sites that facilitates recognition of its carbohydrate epitope on gp120. When expressed as an intact immunoglobulin G (IgG), 2G12 formed typical IgG monomers containing two combining sites and a small fraction of a higher-molecular-weight species, which showed a significant increase in neutralization potency (50-to 80-fold compared to 2G12 monomer) across a range of clade A and B strains of HIV-1. Here we show that the higher-molecular-weight species corresponds to a 2G12 dimer containing four combining sites and present a model for how intermolecular 3D domain swapping could create a 2G12 dimer. Based on the structural model for a 3D domain-swapped 2G12 dimer, we designed and tested a series of 2G12 mutants predicted to increase the ratio of 2G12 dimer to monomer. We report a mutation that effectively increases the 2G12 dimer/monomer ratio without decreasing the expression yield. Increasing the proportion of 2G12 dimer compared to monomer could lead to a more potent reagent for gene therapy or passive immunization.Broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) have attracted attention not only for the lessons they provide for designing vaccine antigens to induce a more robust immunological response (2) but also as potential therapeutic reagents. Although HIV infection leads to a vigorous antibody response, most antibodies fail to control the virus due to targeting of non-neutralizing epitopes or the ability of escape mutants to quickly develop against neutralizing antibodies (23). Correlating with the ability of the virus to elude antibodies, the majority of neutralizing antibodies are highly strain specific. Nevertheless, a small set of broadly neutralizing antibodies has been isolated from the blood of HIVinfected individuals, and these reagents have been extensively studied (2). Clinical trials using a cocktail of three such antibodies-2G12, 4E10, and 2F5-have demonstrated a partial ability to suppress viral replication (13,20,21).The 2G12 antibody has an unusual structure that facilitates recognition of its carbohydrate epitope on gp120 (4). Whereas typical immunoglobulin G (IgG) antibodies contain two flexibly attached antigen-binding fragments (Fabs), resulting in two antigen-binding sites separated by distances ranging from 120 to 150 Å in structures of intact IgGs (6,7,17), the Fab arms of 2G12 are entwined in such a way as to create a single antigenbinding region with two rigidly arranged antigen-binding sites separated by ϳ35 Å (4) (Fig. 1A and B). The entwined structure of the 2G12 Fabs results from three-dimensional (3D) domain swapping (1) in which each 2G12 light chain associates with both heavy chains: the light-chain variable domain (V L ) is paired with the variable domain of one heavy chain (V H ), while the light constant domain (C L ) is paired with cons...

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“…In order to assess domain exchange by other means than SEC, we tested the anti-2G12 idiotypic antibody M1G1 (34) for binding to different versions of 2G12. It was speculated that M1G1 binds to the elbow region of the light chain and that the epitope contains a pronounced conformational component (34). ELISA data showed that M1G1 bound well to the domain-exchanged wt2G12 but not to a conventional antibody, as represented by wt2G12K/gl2G12H, and with intermediate affinity to different 2G12 variants depending on the percentage of domain exchange (Fig.…”

Section: Construction Of a Germ Line Version Of 2g12mentioning

confidence: 95%

Very Few Substitutions in a Germ Line Antibody Are Required To Initiate Significant Domain Exchange

Huber

Le²,

Doores

et al. 2010

J Virol

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2G12 is a broadly neutralizing anti-HIV-1 monoclonal human IgG1 antibody reactive with a high-mannose glycan cluster on the surface of glycoprotein gp120. A key feature of this very highly mutated antibody is domain exchange of the heavy-chain variable region (V H ) with the V H of the adjacent Fab of the same immunoglobulin, which assembles a multivalent binding interface composed of two primary binding sites in close proximity. A non-germ line-encoded proline in the elbow between V H and C H 1 and an extensive network of hydrophobic interactions in the V H /V H interface have been proposed to be crucial for domain exchange. To investigate the origins of domain exchange, a germ line version of 2G12 that behaves as a conventional antibody was engineered. Substitution of 5 to 7 residues for those of the wild type produced a significant fraction of domain-exchanged molecules, with no evidence of equilibrium between domain-exchanged and conventional forms. Two substitutions not previously implicated, A H14 and E H75 , are the most crucial for domain exchange, together with I H19 at the V H /V H interface and P H113 in the elbow region. Structural modeling gave clues as to why these residues are essential for domain exchange. The demonstration that domain exchange can be initiated by a small number of substitutions in a germ line antibody suggests that the evolution of a domainexchanged antibody response in vivo may be more readily achieved than considered to date.

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Electron microscopic and immunochemical analysis of the broadly neutralizing HIV-1-specific, anti-carbohydrate antibody, 2G12

Cited by 19 publications

References 38 publications

Global Shape and Ligand Binding Efficiency of the HIV-1-neutralizing Antibodies Differ from Those of Antibodies That Cannot Neutralize HIV-1

Global Shape and Ligand Binding Efficiency of the HIV-1-neutralizing Antibodies Differ from Those of Antibodies That Cannot Neutralize HIV-1

Design and Expression of a Dimeric Form of Human Immunodeficiency Virus Type 1 Antibody 2G12 with Increased Neutralization Potency

Very Few Substitutions in a Germ Line Antibody Are Required To Initiate Significant Domain Exchange

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