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1 Supporting Information Klein et al /pnas SI Text Cloning, Expression, and Protein Purification. Sequences encoding variable light and heavy (V L and V H ) domains were amplified from genes encoding the b12 and 4E10 antibodies (gifts from D. R. Burton, Scripps Research Institute, La Jolla, CA). The V L and V H genes were fused using a linker encoding Gly 4 Ser or (Gly 4 Ser) 3 by bridge PCR to create 6 constructs (Fig. 1). Two were the monovalent b12 and 4E10 scfvs, which were constructed by using a (Gly 4 Ser) 3 linker, and 4 were bivalent architectures derived from scfv genes. One of the bivalent architectures was a diabody, in which a shorter linker of only one Gly 4 Ser repeat between the variable domains resulted in dimerization (diabodies b12 and 4E10) through 3D domain swapping (1 3). The other bivalent architecture was a single-chain bivalent Fv (scbvfv) consisting of a first scfv (V L joined to V H with a (Gly 4 Ser) 3 linker) followed by (Gly 4 Ser) 3 and a second scfv (V H joined to V L with a (Gly 4 Ser) 3 linker). For expression of monomeric scfvs, the b12 and 4E10 scfv genes were subcloned into the pet-22b( ) vector (EMD Biosciences) and transformed into E. coli strain BL21(DE3). Inclusion bodies containing unfolded scfv were solubilized and refolded as described previously (4). Briefly, bacterial cultures were grown to an OD 600 of 0.9 at 37 C, at which point IPTG was added to 1 mm, and the cultures were incubated for an additional 4 h. Inclusion bodies were isolated by 5 rounds of sonication and centrifugation, solubilized in 7 M guanidine with 10 mm reduced glutathione and 1 mm oxidized glutathione and refolded by rapid dilution at 4 C in 0.1 M Tris HCl (ph 8.0), 0.4 M arginine-hcl, 10 mm reduced glutathione, and 1 mm oxidized glutathione. After concentration, refolded scfv was further purified with Ni-NTA metal affinity Sepharose (Qiagen). The gene encoding diabody 4E10 was subcloned into the bicistronic pac- -Fc vector (PROGEN Biotechnik) for expression in baculovirus-infected insect cells. Genes encoding diabody b12 and the scbvfv constructs were subcloned into pacgp67-a (BD PharMingen). Recombinant baculoviruses were generated by cotransfection of a transfer vector with linearized Baculogold viral DNA (BD PharMingen) and used to infect High Five cells (Invitrogen). Supernatants were then concentrated, bufferexchanged with 50 mm Tris HCl (ph 7.4), and 150 mm NaCl (TBS), and purified over Ni-NTA Sepharose. IgGs were transiently expressed in mammalian cells. Fulllength 4E10 heavy-chain (IgG1 subclass) and light-chain ( ) genes were subcloned separately into pcdna3.1( ) (Invitrogen) and cotransfected into HEK 293T cells (American Type Culture Collection; ATCC) using Lipofectamine 2000 (Invitrogen). Constructs encoding the heavy chain and light chain of IgG b12 in the bicistronic vector pdr12 (a gift from D. R. Burton) were also expressed by transient transfection in HEK 293T cells. To produce Fab 4E10, a truncated 4E10 heavy-chain gene (terminated after residue Thr-252) was cotransfected into EBNA cells (ATCC) along with the 4E10 light-chain gene. Fab b12 was prepared by papain digestion of IgG b12 expressed in HEK 293F cells (Invitrogen) using 25-kDa linear PEI as a transfection reagent (Polysciences). Intact IgGs were purified by protein A chromatography (Pierce Biotechnology), and Fabs were purified using goat anti-human Fab polyclonal antibody (Sigma Aldrich) cross-linked to Protein A beads. All proteins were further purified by size-exclusion chromatography using Superdex 75 10/30, Superdex 75 16/60, or Superdex /60 columns (Amersham Biosciences) running in TBS. The final yields for bacterially expressed proteins were 2 and 0.5 mg/l for scfv b12 and scfv 4E10, respectively. The yields for scbvfv b12, diabody b12, scbvfv 4E10, and diabody 4E10 were 2.5, 1, 1.5, 0.7, mg/l of insect cell supernatant, respectively. The yields for IgG b12, IgG 4E10, and Fab 4E10, were 6.5, 0.8, and 2.6 mg/l, respectively. Protein concentrations were determined by absorbance at 280 nm using extinction coefficients valid for denatured protein calculated from the secreted protein sequences using the online tool PlotParam ( (5). Extinction coefficients were: scfv b12, M 1 cm 1 ; scbvfv b12, M 1 cm 1 ; diabody b12, M 1 cm 1 ; Fab b12, M 1 cm 1 ; IgG b12, M 1 cm 1 ; scfv 4E10, M 1 cm 1 ; scbvfv 4E10, M 1 cm 1 ; diabody 4E10, M 1 cm 1 ; Fab 4E10, M 1 cm 1 ; IgG 4E10, M 1 cm 1. No significant differences were observed when absorbance measurements were compared for scfv proteins diluted either in guanidine-hcl to 6.0 M or in 10 mm Hepes (ph 7.4) and 150 mm NaCl. All proteins were 95% pure and stable at concentrations of 1 2 mg/ml at 4 C for at least several weeks as assessed by unchanged gel filtration profiles. Purified recombinant full-length gp120 (clade B, strain HxBc2) expressed in Chinese hamster ovary cells and recombinant gp41 ectodomain (clade B, strain MN) expressed in bacteria were purchased from Immunodiagnostics. The recombinant gp41 is a 25-kDa fragment expressed as a fusion protein and purified by affinity chromatography and preparative electrophoresis (product specifications for Immunodiagnostics catalog no. 1091). To validate the antigenicity of the recombinant gp41, scfv and IgG versions of 2F5, a monoclonal antibody that binds an epitope near the 4E10-binding site on gp41 (6), were injected over the gp41 surface in the binding assay, resulting in a 10 nm affinity for the scfv, comparable with the 5.3 nm affinity observed when binding to the MPER peptide from gp41 and a low picomolar apparent affinity for IgG binding to gp41. No significant binding of scfv b12 to gp41 was observed at a concentration of 1 M. Strain Selection for in Vitro Neutralization Assays. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health: SVPB5, SVPB6, SVPB8, SVPB11, and SVPB12 (David Montefiori and Feng Gao); SVPB14, SVPB16, SVPB18 (B. H. Hahn and J. F. Salazar-Gonzalez); SVPB15 (B. H. Hahn and D. L. Kothe); SF162 (L. Stamatatos and C. Cheng-Mayer); SVPB17 (B. H. Hahn, X. Wei, and G. M. Shaw); psg3 env (John C. Kappes and Xiaoyun Wu); TZM-bl cells (John C. Kappes, Xiaoyun Wu, and Tranzyme Inc). HIV-1 pseudovirus particles from 10 pseudotyped primary virus strains [ (SVPB5), QH (SVPB6), REJO (SVPB16), RHPA (SVPB14), SC (SVPB8), SF162, THRO (SVPB15), TRJO (SVPB17), TRO.11 (SVPB12), and WITO (SVPB18)] were prepared, and in vitro neutralization assays were performed as described previously (7 9). To be able to generalize our results across an entire clade, we examined the sensitivities of various clades to both b12 and 4E10 using previously published results (10) to select appropriate strains. 4E10 neutralizes most/all strains regardless of clade, so choosing appropriate clade(s) for our comparison depended on b12, which is less broadly neutralizing. It was observed that clades A, B, C, D, AE, and BG contained at least 1 strain that was sensitive to IgG b12 (10). To be useful in our experiments 1of10

2 in which potentially weakly neutralizing reagents (e.g., monovalent constructs) would be tested, we estimated that a strain would need to have an IC g/ml for IgG b12 in order for it to be possible for us to accurately derive an IC 50 for a monovalent reagent. Clade B was the only clade in which a majority of strains met this criterion (Clade A: 2 of 12 strains; clade B: 19 of 29; clade C: 5 of 12; clade D: 3 of 11; clade AE: 1 of 10; clade BG: 1 of 1). We therefore chose to confine our comparative studies to clade B. In Vitro Neutralization Assay Methods. Briefly, TZM-bl cells (8, 11, 12), a HeLa cell line expressing CD4, the HIV-1 coreceptors CCR5 and CXCR4, and Tat-responsive firefly luciferase, were infected by pseudotyped viruses, and single rounds of infection were detected as luminescence from luciferase. Each antibody reagent was tested for inhibition of infection in triplicate by preincubating 5,000 infectious viral units per well with a 3-fold dilution series of the potential inhibitor for 1hat37 C.Ten thousand TZM-bl cells were then added to each well. After 48 h at 37 C, the cells were lysed in the presence of Bright-Glo (Promega), and relative luminescence was recorded by using a Victor3 luminometer (PerkinElmer). Percent neutralization was calculated as [1 [( )/( )]] 100, where is the relative luminescence observed for each sample well, is the background luminescence observed for cells without virus or antibody reagent, and is the maximum luminescence observed for cells with virus only. Neutralization curves were plotted as the percentage of neutralization (y axis) versus concentration of potential inhibitor (x axis). Each data point on a neutralization curve is the mean of a triplicate measurement SEM. IC 50 values were calculated as described (13). Briefly, neutralization curves were fit to the equation N 100/[1 (IC 50 /c) H ] where N is percent neutralization and c is the concentration of the reagent being tested, which constrains the maximum and minimum of each curve to 100% and 0% neutralization, respectively, and H represents the Hill coefficient (KaleidaGraph v3.6; Synergy Software). Fitting the data with a Hill coefficient constrained to a value of 1 does not change the results. Errors reported for the IC 50 values in Table 1 were the asymptotic standard error calculated from nonlinear regression analyses and therefore represent the goodness-of-fit. An IC 50 value could not be determined for diabody b12 neutralization of strain WITO because the protein was not stable at the concentration necessary to achieve neutralization above 50%. To evaluate the reproducibility of the IC 50 values, we used either scfv b12 or IgG b12 as internal controls. When data from these multiple replicates were available, we reported the IC 50 value in Table 1 as the average, and a representative curve is shown in Fig. S2. The error reported in Table 1 was then calculated as the product of the average IC 50 value and the square root of the average of the sum of squares of the fractional errors from each of the replicates. The results from these multiple independent replicates are summarized in Table S2 and a comparison of our IC 50 values with published IC 50 values for IgG b12 and IgG 4E10 are presented in Table S3. These comparisons demonstrate that our neutralization curves yielded reproducible IC 50 values that are in agreement with those published by others. 1. Bennett MJ, Eisenberg D (2004) The evolving role of 3D domain swapping in proteins. Structure (London) 12: Perisic O, Webb PA, Holliger P, Winter G, Williams RL (1994) Crystal structure of a diabody, a bivalent antibody fragment. Structure (London) 2: Holliger P, Prospero T, Winter G (1993) Diabodies : Small bivalent and bispecific antibody fragments. Proc Natl Acad Sci USA 90: Steinle A, et al. (2001) Interactions of human NKG2D with its ligands MICA, MICB, and homologs of the mouse RAE-1 protein family. Immunogenetics 53: Gasteiger E, et al. (2005) Protein Identification and Analysis Tools on the ExPASy Server. The Proteomics Protocols Handbook, ed Walker JM (Humana Press, Totowa, NJ), pp Muster T, et al. (1993) A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J Virol 67: Li M, et al. (2005) Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. J Virol 79: Wei X, et al. (2002) Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother 46: Wei X, et al. (2003) Antibody neutralization and escape by HIV-1. Nature 422: Binley JM, et al. (2004) Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies. J Virol 78: Derdeyn CA, et al. (2000) Sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by coreceptor specificity defined by the V3 loop of gp120. J Virol 74: Platt EJ, Wehrly K, Kuhmann SE, Chesebro B, Kabat D (1998) Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1. J Virol 72: Gustchina E, Bewley CA, Clore GM (2008) Sequestering of the prehairpin intermediate of gp41 by peptide N36Mut(e,g) potentiates the human immunodeficiency virus type 1 neutralizing activity of monoclonal antibodies directed against the N-terminal helical repeat of gp41. J Virol 82: of10

3 Fig. S1. Surface plasmon resonance sensorgrams for binding to immobilized antigens. Analytes were injected with the range of concentrations listed above the sensorgrams. (A) Sensorgrams for 2-fold dilution series of the indicated analyte binding to immobilized monomeric gp120. (B) Sensorgrams for 2-fold dilution series of the indicated analyte binding to immobilized gp41 ectodomain. All sensorgrams were fit with a 1-to-1 binding model. For IgG 4E10 binding to gp41, the off-rate (a concentration-independent parameter) was fit separately from the on-rate. Residual plots are shown beneath each set of fitted sensorgrams. 3 of 10

4 Fig. S2. In vitro pseudovirus neutralization curves. (A) b12 constructs. 4of10

5 Fig. S2B. 4E10 constructs. 5of10

6 Fig. S3. Modeling of the structural requirements for IgG b12 to achieve intraspike cross-linking. Using the coordinates of the HIV gp120 trimer in its b12-bound state (orange) derived from tomographic reconstructions of intact HIV trimers (PDB ID code 3DNL) (1), we aligned the coordinates for Fab b12 (heavy chain in blue and light chain in yellow) using the crystal structure of a Fab b12/gp120 complex (PDB ID code 2NY7) (2). The Fc domain from the crystal structure of IgG b12 (PDB ID code 1HZH) (3) was then placed between and approximately equidistant from the 2 Fabs. The distance between the N-terminal Cys residue of each Fc chain (red dot) and the C-terminal Cys residue of each Fab heavy chain (red dot) is 9 nm, which is 8 nm longer than a typical IgG hinge (e.g., see IgG in Fig. 1). 1. Liu J, Bartesaghi A, Borgnia MJ, Sapiro G, Subramaniam S (2008) Molecular architecture of native HIV-1 gp120 trimers. Nature 455: Zhou T, et al. (2007) Structural definition of a conserved neutralization epitope on HIV-1 gp120. Nature 445(7129): Saphire EO, et al. (2001) Crystal structure of a neutralizing human IGG against HIV-1: A template for vaccine design. Science 293(5532): of10

7 Fig. S4. Bar graph of ratios of average molar IC 50 values (geometric means) for b12 constructs (blue) and 4E10 constructs (orange). Reagent pairs with an average ratio of 1.0 (black line) are equal in average potencies. Ratios 1.0 indicate that reagent b is more potent than reagent a. Ratios 1.0 indicate that reagent a is more potent than reagent b. Error bars represent the standard errors calculated from the variability in strain-specific ratios for each pair of reagents. 7of10

8 Table S1. Strain-specific IC 50 neutralization ratios IC 50 ratio by virus strain b QH RHPA SC SF162 THRO REJO WITO Monovalent/bivalent scfv/igg Fab/IgG scfv/scbvfv Fab/scBvFv scfv/diabody n.d. Fab/diabody n.d. Bivalent/bivalent scbvfv/igg diabody/igg n.d. diabody/scbvfv n.d. Monovalent/monovalent Fab/scFv E QH RHPA SC SF162 THRO TRJO TRO.11 Monovalent/bivalent scfv/igg Fab/IgG scfv/scbvfv Fab/ scbvfv scfv/diabody Fab/diabody Bivalent/ bivalent scbvfv/igg diabody/igg diabody/scbvfv Monovalent/monovalent Fab/scFv The average IC 50 ratios presented in Fig. 3 of the main text were calculated as the ratio of their respective arithmetic mean IC 50 values as opposed to an arithmetic mean of the individual ratios presented here. n.d., not done. See discussion in SI Text. 8of10

9 Table S2. IgG b12 and scfv b12 were used as internal controls to examine the reproducibility of independently determined IC 50 values IC 50 replicates, nm Isolate Average IgG b QH RHPA SC SF THRO scfv b QH RHPA SC SF THRO of10

10 Table S3. Comparison of IC 50 values for IgG b12 and IgG 4E10 to previously published results Isolate Ours ( g/ml) Li, et al. ( g/ml)* Fold difference IgG b QH RHPA SC SF (0.03 ) 4.1 (1.4 ) THRO REJO WITO IgG 4E QH RHPA SC SF (4.0 ) 9.0 (0.7 ) THRO TRJO TRO *With the exception of SF162, the HIV isolates used in our study were initially characterized in Li, et al. (1). Our IC 50 values for this strain more closely match those reported by Binley, et al. (2), the laboratory in which the original characterization of this strain was conducted. 1. Li M,etal.(2005) Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. J Virol 79: Binley JM,etal.(2004) Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies. J Virol 78: of 10