Crystal Structure of Human Antibody 2909 Reveals Conserved Features of Quaternary Structure-Speciﬁc Antibodies That Potently Neutralize HIV-1 (cid:1) †§

Monoclonal antibody 2909 belongs to a class of potently neutralizing antibodies that recognize quaternary epitopes on HIV-1. Some members of this class, such as 2909, are strain speciﬁc, while others, such as antibody PG16, are broadly neutralizing; all, however, recognize a region on the gp120 envelope glycoprotein that includes two loops (V2 and V3) and forms appropriately only in the oligomeric HIV-1 spike (gp120 3 /gp41 3 ). Here we present the crystal structure of 2909 and report structure-function analysis with antibody chimeras composed of 2909 and other members of this antibody class. The 2909 structure was dominated by a heavy-chain third-complementarity-determining region (CDR H3) of 21 residues, which comprised 36% of the combining surface and formed a (cid:1) -hairpin club extending (cid:1) 20 Å beyond the rest of the antibody. Sequence analysis and mass spectrometry identiﬁed sites of tyrosine sulfation at the middle and top of CDR H3; substitutions with phenylalanine either ablated (middle substitution) or substantially diminished (top substitution) neutralization. Chimeric antibodies composed of heavy and light chains, exchanged between 2909 and other members of the class, indicated a substantial lack of complementation. Comparison of 2909 to PG16 (which is tyrosine sulfated and the only other member of the class for which a structure has previously been reported) showed that both utilize protruding, anionic CDR H3s for recognition. Thus, despite some diversity, members of this class share structural and functional similarities, with conserved features of the CDR H3 subdomain likely reﬂecting prevalent solutions by the human immune system for recognition of a quaternary site of HIV-1 vulnerability. CDR H3 swap variants were designed in which the entire CDR H3 sequence was exchanged between the 2909 and PG16 heavy chains. To generate the CDR H3 swap variants, the 2909 variable heavy chain sequence with PG16 CDR H3 (2909H_PG16CDRH3) and the PG16 variable heavy chain sequence with 2909 CDR H3 (PG16H_2909CDRH3) were synthesized (Geneart) and subcloned into the pVRC8400 expression vector. To further stabilize the PG16H_2909CDRH3 variant, site-directed mutagenesis (GM Biosciences and ACGT) was used to generate two variants of the PG16 light chain (PG16L-R96V and PG16L-D50V/ L91W/R96V, which contained mutations to optimize interactions between the heavy and light chains. Site-directed mutagenesis (ACGT) was used to create 2909 heavy chain vari- ants in which potential sulfotyrosine sites in the CDR H3 were mutated to phenylalanine (2909H-Y 100a F and 2909H-Y 100c F). All 2909 CDR H3 mutants and swap variants were expressed and puriﬁed in the same manner as described above for wild-type 2909 immunoglobulin. Puri- ﬁed immunoglobulin was exchanged into PBS, ﬁltered through a 0.22- (cid:2) m ﬁlter, and concentrated to at least 1 mg/ml for neutralization assays. ESI-MS measurements. For electrospray ionization mass spectrometry (ESI-MS) measurements, 2909 Fab was generated by sequential reduction, al- kylation, and Lys-C digestion and puriﬁed by anion-exchange and size exclusion chromatography, as described above. ESI-MS data were collected using a Sciex 4000 Qtrap instrument at the Mass Spectrometry Lab, NIAID Research Technologies Branch. Cells and HIV-1 rev / env plasmids. TZM-bl cells were obtained from the NIH AIDS Research and Reference Reagent Program, as contributed of serially diluted test antibody in duplicate wells of a 96-well ﬂat-bottom culture plate. keep assay conditions constant, sham medium was used in place of antibody in speciﬁed control wells. The pseudovirus input was set at a multiplicity of infection of 0.01 to 0.1, which generally results in 100,000 to 400,000 relative light units (RLU) in a luciferase assay (Bright Glo; Promega, Madison, WI). The antibody concentrations were deﬁned at the point of incubation with pseudovirus supernatant. Neutralization curves were ﬁt by nonlinear regression using a 5-pa- rameter hill slope equation as previously described (37). 50% inhibitory concentrations were concentrations inhibit program we deﬁne Thus,