Abstract:abYsis is a web-based antibody research system that includes an integrated database of antibody sequence and structure data. The system can be interrogated in numerous ways -from simple text and sequence searches to sophisticated queries that apply 3D structural constraints. The publicly available version includes pre-analysed sequence data from EMBL-ENA and Kabat as well as structure data from the PDB. A researcher's own sequences can also be analysed through the web interface.A defining characteristic of abY… Show more
“…Structural studies of V1V2 bNAbs showed that the protruding CDRH3 is used to penetrate through the glycan shield on Env surrounding the Asn160 gp120 and Asn156 gp120 N-glycans to contact basic residues in the V2 loop (Gorman et al, 2016; McLellan et al, 2011; Pancera et al, 2013; Pan et al, 2015; Pancera et al, 2010; Pejchal et al, 2010). The BG1 CDRH3 length, 22 residues, is shorter than CDRH3 loops in other V1V2 bNAbs (Figure 1A), but longer than typical human antibody CDRH3s for which the most common length is 14 residues (Swindells et al, 2017). A 2.0 Å crystal structure of unbound BG1 Fab (Figure 1B; Table 1) revealed that, in contrast to the protruding CDRH3s in other V1V2 bNAbs, the BG1 CDRH3 does not extend notably beyond the antigen-binding site surface (Figure 1C).…”
Section: Resultsmentioning
confidence: 83%
“…10.7554/eLife.27389.002Figure 1.Comparison of BG1 and other V1V2 bNAbs.( A ) Sequences of the HCs of BG1 and representative other V1V2 bNAbs. Residue numbering (Kabat) refers to BG1, and the CDRs were defined using AbM (Swindells et al, 2017). ( B ) Crystal structure of BG1 Fab with highlighted CDRs.…”
The HIV-1 envelope (Env) glycoprotein binds to host cell receptors to mediate membrane fusion. The prefusion Env trimer is stabilized by V1V2 loops that interact at the trimer apex. Broadly neutralizing antibodies (bNAbs) against V1V2 loops, exemplified by PG9, bind asymmetrically as a single Fab to the apex of the symmetric Env trimer using a protruding CDRH3 to penetrate the Env glycan shield. Here we characterized a distinct mode of V1V2 epitope recognition by the new bNAb BG1 in which two Fabs bind asymmetrically per Env trimer using a compact CDRH3. Comparisons between cryo-EM structures of Env trimer complexed with BG1 (6.2 Å resolution) and PG9 (11.5 Å resolution) revealed a new V1V2-targeting strategy by BG1. Analyses of the EM structures provided information relevant to vaccine design including molecular details for different modes of asymmetric recognition of Env trimer and a binding model for BG1 recognition of V1V2 involving glycan flexibility.DOI:
http://dx.doi.org/10.7554/eLife.27389.001
“…Structural studies of V1V2 bNAbs showed that the protruding CDRH3 is used to penetrate through the glycan shield on Env surrounding the Asn160 gp120 and Asn156 gp120 N-glycans to contact basic residues in the V2 loop (Gorman et al, 2016; McLellan et al, 2011; Pancera et al, 2013; Pan et al, 2015; Pancera et al, 2010; Pejchal et al, 2010). The BG1 CDRH3 length, 22 residues, is shorter than CDRH3 loops in other V1V2 bNAbs (Figure 1A), but longer than typical human antibody CDRH3s for which the most common length is 14 residues (Swindells et al, 2017). A 2.0 Å crystal structure of unbound BG1 Fab (Figure 1B; Table 1) revealed that, in contrast to the protruding CDRH3s in other V1V2 bNAbs, the BG1 CDRH3 does not extend notably beyond the antigen-binding site surface (Figure 1C).…”
Section: Resultsmentioning
confidence: 83%
“…10.7554/eLife.27389.002Figure 1.Comparison of BG1 and other V1V2 bNAbs.( A ) Sequences of the HCs of BG1 and representative other V1V2 bNAbs. Residue numbering (Kabat) refers to BG1, and the CDRs were defined using AbM (Swindells et al, 2017). ( B ) Crystal structure of BG1 Fab with highlighted CDRs.…”
The HIV-1 envelope (Env) glycoprotein binds to host cell receptors to mediate membrane fusion. The prefusion Env trimer is stabilized by V1V2 loops that interact at the trimer apex. Broadly neutralizing antibodies (bNAbs) against V1V2 loops, exemplified by PG9, bind asymmetrically as a single Fab to the apex of the symmetric Env trimer using a protruding CDRH3 to penetrate the Env glycan shield. Here we characterized a distinct mode of V1V2 epitope recognition by the new bNAb BG1 in which two Fabs bind asymmetrically per Env trimer using a compact CDRH3. Comparisons between cryo-EM structures of Env trimer complexed with BG1 (6.2 Å resolution) and PG9 (11.5 Å resolution) revealed a new V1V2-targeting strategy by BG1. Analyses of the EM structures provided information relevant to vaccine design including molecular details for different modes of asymmetric recognition of Env trimer and a binding model for BG1 recognition of V1V2 involving glycan flexibility.DOI:
http://dx.doi.org/10.7554/eLife.27389.001
“…For example, whereas the CDR L1 loop of eOD01 is 15 amino acids in length, characteristic of the mouse IGKV3 germline family, the corresponding CDR L1 region of GD01 is derived from a different mouse germline group IGKV6 and is 11 amino acids (SI Appendix, Fig. S7), a length more commonly observed both in mouse and human antibodies (29) (Table 1). Interestingly, the eOD01 light-chain protein sequence shows little deviation from the germline IGKV3-2*01 V-gene (SI Appendix, Fig.…”
Transmission of hemorrhagic fever New World arenaviruses from their rodent reservoirs to human populations poses substantial public health and economic dangers. These zoonotic events are enabled by the specific interaction between the New World arenaviral attachment glycoprotein, GP1, and cell surface human transferrin receptor (hTfR1). Here, we present the structural basis for how a mouse-derived neutralizing antibody (nAb), OD01, disrupts this interaction by targeting the receptor-binding surface of the GP1 glycoprotein from Junín virus (JUNV), a hemorrhagic fever arenavirus endemic in central Argentina. Comparison of our structure with that of a previously reported nAb complex (JUNV GP1-GD01) reveals largely overlapping epitopes but highly distinct antibody-binding modes. Despite differences in GP1 recognition, we find that both antibodies present a key tyrosine residue, albeit on different chains, that inserts into a central pocket on JUNV GP1 and effectively mimics the contacts made by the host TfR1. These data provide a molecular-level description of how antibodies derived from different germline origins arrive at equivalent immunological solutions to virus neutralization. . These viruses are endemic to rodent populations in rural areas of Argentina, Bolivia, and Venezuela, respectively, and viral spillover into human populations can result in severe hemorrhagic fever (HF) (3). Novel pathogenic NW arenaviruses continue to be identified (4-6), underscoring a wide-scale need for effective vaccines and therapeutics.JUNV, the etiological agent of Argentine hemorrhagic fever (AHF), constitutes one of the most dangerous NW arenaviruses, putting an estimated 5 million people at risk (7,8). JUNV infection typically exhibits a rapid onset of disease (7-14 d) and high mortality rates (15-30%) (7, 9, 10). There are no internationally approved drugs for preventing or treating NW arenavirus HF. However, the successful development of a live, attenuated JUNV vaccine, Candid#1, has proven that AHF can be controlled (11). Similarly, virus-neutralizing immune plasma from convalescent individuals has been successfully used for the treatment of AHF (10, 12).The JUNV envelope surface is decorated by trimeric multifunctional glycoprotein complex (GPC) spikes. Each protomer in the trimer consists of: a myristoylated (13) stable signal peptide, an attachment subunit (GP1), and a transmembrane fusion subunit (GP2) (14-19). Host-cell entry of JUNV and other clade B arenaviruses is initiated by the interaction between the arenaviral GP1 and the host transferrin receptor (TfR1) (20). The GP1 subunit interacts with the apical domain of TfR1, distal from natural transferrin and hereditary hemochromatosis protein recognition sites (21, 22). A primary determinant of zoonotic spread of clade B arenaviruses is the ability of the GP1 to recognize the human TfR1 ortholog (20, 23).The JUNV GPC spike comprises the primary target for neutralizing immune responses (24) and three GPC-specific mousederived nAbs-GD01, OD01, and GB03-have shown prom...
“…Given that the insertions and deletions at VDJ junctions would generate complementarity-determining region (CDR)-H3 sequences that could not be found in human antibody germline sequences, we searched for the thrombin recognition sequence in 2 additional databases: 1) a MedImmune proprietary next-generation sequencing database of over 1.4 million unique CDR-H3 sequences obtained from primary B cells from 5 healthy human donors, and 2) sequences retrieved from the abYsis database. 35 Both database searches revealed a very low frequency for the thrombin recognition sequence for the CDR-H3 (Fig. 1E).…”
Section: Imab Designmentioning
confidence: 98%
“…34 For the abYsis database, the thrombin recognition sequence was searched online using Homo sapiens as organism. 35 Sequence repertoires for the CDR-H3 were also obtained from human blood samples. One million B cells from 5 healthy donors were isolated and used to generate V H and V L amplicons that were sequenced using Illumina MiSeq 2£300 bp (SeqMagic).…”
Section: Frequency Of Thrombin Recognition Sequence In Human Antibodiesmentioning
We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies.
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