Supporting InformationDudgeon et al. 10.1073/pnas.1202866109SI TextSI Methods. Generation of mutant VH and VL domains and repertoires.Genes encoding human variable domains (V3–23/DP47, JH4b)(VH3) (1, 2) (O12/O2/DPK9, Jκ1) (Vκ1) (2, 3) and Trastuzumab(Herceptin) variant huMAb4D5-5 (4) were cloned into the phagedisplay vector FdMyc. Mutations were introduced using methodsdescribed by Kunkel and others (5). Phage display repertoiresof human V3-23/DP47 were constructed using synthetic oligo-nucleotide-meditated diversification at H2 and H3 positions.Specifically, H2 was diversified using the degenerate codonKMT (Y/A/D/S) at positions 50, 52, and 54; RRT (S/N/D/G) atposition 52a; and SMT (P/A/E/H) at position 53 [numbering ac-cording to IUPAC-IUB and Kabat (6)]. H3 was diversified usingtrinucleotide phosphoramidite oligonucleotides (7) encoding(20% Y; 17% G; 15% S; 7% D/A; 4% T/P/V/R/I/L; 2% W/F/M/Q/N/H/K/E) at positions 95–100A/C. H1 was not diversifiedbut restricted to either the germline V3-23/DP47 sequence(WT) or aspartate at positions 32D/33D. Phage display reper-toires of human Vκ1/DPK9 were generated as described above:Diversity was introduced into L1 using the degenerate codonKMT (Y/A/D/S) at positions 28, 30, 31, and 32. L3 diversity wasintroduced at positions 91, 92, 93, 94, and 96 using trinucleotidephosphoramidite oligonucleotides as described above. L2 was re-stricted to either the germline O12/O2/DPK9 sequence (WT) oraspartate at positions 52D/53D. From each repertoire 48 cloneswere randomly selected, confirmed by sequencing, and tested foraggregation resistance on phage.

Analysis of aggregation resistance on phage. The aggregation resis-tance of human antibody variable domains were analyzed as de-scribed by Jespers et al. (8). The method is based on measuringsuperantigen binding after heat-induced aggregation of antibodyvariable domains displayed on phage. In brief, a Maxisorp Immu-no-plate (Nunc) was coated with 5 μg∕mL superantigen in100 mM sodium carbonate buffer pH 9.6 [S. aureus protein A(Sigma-Aldrich) or P. magnus protein L (Biovision)]. After wash-ing with phosphate buffered saline (PBS) and blocking with 4%(w∕v) milk powder in PBS, signal was detected as follows: Phagewere biotinylated in culture supernatant by addition of 50 μM ofbiotin-PEO4-hydroxysuccinimide (Pierce). To induce aggregationsupernatant was heated at 80 °C for 10 min, followed by 10 min at4 °C, added to the blocked ELISA plate and superantigen bindingdetected by sequentially adding Extravidin-HRP conjugate(Sigma-Aldrich) and 3,3′,5,5′-tetramethylbenzidine substrate.The level of retained superantigen binding after treatment wasexpressed as a percentage of the untreated sample. Statisticalanalyses of means were based on Student’s t tests (two-tailed).

Protein expression and purification. Antibody fragments were ex-pressed using the periplasmic expression vector pET12a (Nova-gen) and Escherichia coli BL21-Gold (Stratagene) at 30 °C.Filtered supernatant was added to protein A resin (GE Health-care) or protein L resin (Genscript). After washes with PBS, frag-ments were eluted with 0.1 M glycine-HCl pH 2.7, neutralizedwith 100 mM TrisHCl pH 8.0 and dialyzed against PBS. For IgGvariants transient expression in HEK 293 cells was utilized (hu-man IgG1 isotype), followed by protein A purification.

Expression levels, elution volumes, refolding yields, and turbiditymeasurements. Representative mutant variable domains wereexpressed and purified as described above (DP47: WT, A31D,Y32E, S33D, 31/32DE, 31/33DD, 32/33DD, 31-31DED;

DPK9: WT, A50D, A51D, S52D, S53D, 50/52DD, 50/53DD,51/53DD, 52/53DD, 50,52–53DDD). Expression levels were de-termined by ELISA using superantigen capture. Proteins werebiotinylated in culture supernatant by adding 50 μM of biotin-PEO4-hydroxysuccinimide (Pierce) and added to a superantigen-coated ELISA plate (expression and coating conditions asabove). Signal was detected by sequentially adding Extravidin-HRP conjugate (Sigma-Aldrich) and 3,3′,5,5′-tetramethylbenzi-dine substrate. The assay was calibrated using purified proteinfor the generation of standard curves for each variant. Solubleexpression levels were calculated using linear regression analyses.Elution volumes were determined by size-exclusion chromatogra-phy on Superdex-G75 using an ÄKTA Purifier (GE Healthcare).Samples were analyzed using a flow rate of 0.5 mL∕min in PBS.Heat refolding yields were calculated by heating purified proteinpreparations. Sample conditions were as follows: VH at 20 μM inPBS; VL at 100 μM in 20 mM phosphate buffer pH 7.4; scFv at10 μM in PBS. Yields for each variant were determined by chro-matography on Superdex-G75 as above and calculated as ratios ofareas under the curve (heated/unheated). Turbidity was measuredby absorbance at 360 nm, using sample conditions as above. Mea-surements were made on a Varian Cary 50 Bio UV–Vis spectro-photometer (Agilent Technologies) using a 1-cm path length.Photographs of mutants were generated using sample conditionsas above, heating for 5/60/10 min for VH∕VL∕scFv, respectively.

Crystal growth, structure solution, refinement, and analysis.A humanVL triple mutant (DPK9 50,52–53DDD)was crystallized by sitting-drop vapor diffusion. Equal volumes of protein at 3.1 mg∕mL inPBS and precipitant solution (100 mM Tris (pH 7.5), 26% (w∕v)PEG 10,000) were combined and incubated at room temperaturefor several weeks. Conditions were identified using the sparsematrix JBScreen Classic HTS screen (Jena Bioscience GmbH). In-itial crystals of a human VH triple mutant (DP47 31–33DED) wereobtained under many conditions of JCSG-plus and PACT premierscreens (Molecular Dimensions). However, crystals were small(approximately 5–10 μm) and resisted attempts at further refine-ment by vapor diffusion. Larger crystals (approximately 50 × 50 ×10 μm) were obtained by counter-diffusion using a Crystal Harp(SWISSCI) incubating at room temperature for several weeks.The structure was solved from a crystal grown using protein at6.3 mg∕mL in PBS and precipitant solution comprising 100 mMcitrate (pH 5.5) and 20% (w∕v) PEG 3,350. Diffraction data wascollected at 100 K on beamline MX2 at the Australian Synchro-tron. Crystals were snap frozen in a cold nitrogen stream prior todata collection. Structures were solved by molecular replacement(see Table S1). Structures of representative human variable do-mains used for comparative purposes in this study were 3QOS,2VXS, 3KDM, 3BN9 (VH, 10 chains total), and 2BX5 (VL, 15chains total). Rmsd values were calculated based on Cα coordi-nates using residues 3–97 of VH and residues 1–109 of VL. Coor-dinates for the mutant VH and VL structures have been depositedin the Protein Data Bank as entries 3UPC and 3UPA, respectively.

Determination of thermodynamic stabilities. Fluorescence spectrawere recorded at 20 °C using a Cary Eclipse spectrofluorimeter(Varian). Proteins were mixed with guanidinium chloride dena-turant and, after overnight incubation, fluorescence emissionspectra were recorded in the 300-nm to 400-nm range, using anexcitation wavelength of 280 nm. From these spectra, free energyof unfolding (ΔGN-U) was calculated as described (9).

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Affinity measurements, cellular binding, inhibition of proliferation,and serum clearance. The binding affinities of scFv variants weremeasured using surface plasmon resonance (BIAcore, GEHealthcare). Biotinylated HER2 extracellular domain was immo-bilized on a streptavidin sensor chip. Dilution series of each scFvvariant were injected at a flow rate of 20 μL∕min and curvesfitted to a 1∶1 langmuir binding model. Cellular binding assayswere performed using the SK-BR-3 human breast cancer cell line(ATCC) and IgG variants. Varying concentrations of variantswere added to cells (2.5 × 104 cells∕sample) for 1 h on ice. Fol-lowing two washes with PBS supplemented with 1% bovine serumalbumin, bound IgG was detected using anti-human IgG Fab’2-FITC conjugate. Fluorescence intensity of cells was analyzedusing a FACSCalibur analyzer (BD Biosciences) and FlowJo7.6.5 software (Tree Star). For measurements of proliferation,

SK-BR-3 cells were maintained in RPMI-1640 medium (Invitro-gen) supplemented with 10% fetal bovine serum (FBS). Cellswere detached using 0.05% trypsin/EDTA, resuspended at2 × 104 cells∕mL, and added to a 48-well cell culture plate. Cellswere allowed to adhere for 30 min and IgG variants added at10 μg∕mL. After seven days, wells were washed with RPMI-1640 medium (without FBS), detached (as above) and live cellscounted. Proliferation levels were calculated as a percentage ofcells grown in the absence of antibody. Serum clearance was de-termined by intra-peritoneal injection of IgG into C57/BL6 miceat 1 mg∕kg. Concentrations were determined by ELISA usinganti-human IgG for capture. Signal was detected by sequentiallyadding anti-human-IgG conjugate and 3,3′,5,5′-tetramethylbenzi-dine substrate.

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5. Kunkel TA, Roberts JD, Zakour RA (1987) Rapid and efficient site-specific mutagenesiswithout phenotypic selection. Methods Enzymol 154:367–382.

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Immunological Interest (Diane Publishing, Darby, PA), 5th Ed, p 2719.

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8. Jespers L, Schon O, FammK,Winter G (2004) Aggregation-resistant domain antibodies

selected on phage by heat denaturation. Nat Biotechnol 22:1161–1165.

9. Pace CN, Scholz JM (1997) Protein Structure: A Practical Approach, (Oxford Univ Press,

New York), 2nd Ed, pp 299–321.

Fig. S1. Effect of mutations in human antibody variable domains on superantigen binding. Surface residues in variable heavy and light domains (human VH3,human Vκ1) are targeted for substitution with aspartic acid (aspartate). Superantigen binding of the domains is determined by phage ELISA (8) (wild-typeresidue: WT; means, standard deviation (SD) shown, n ¼ 2). Numbering according to Kabat (6). (A) Single mutations in human VH. (B) Single mutations inhuman VL. Graphs show binding to superantigen before (Upper) and after (Lower) heating to 80 °C on phage. Mutations in human antibody variable domainsdisplay only minor effects on superantigen binding before heating, but considerably improve superantigen binding after heating.

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Fig. S2. Effects of charged substitutions on aggregation-resistance of human antibody variable domains. Surface residues in human VH3 are targeted.Aggregation-resistance of the domains is determined bymeasuring retained binding to superantigen after heating to 80 °C on phage (1, –3) (wild-type residue:WT; means, standard deviation shown, n ≥ 2). Residue numbering according to Kabat (4). (A) Single substitutions in CDR1 (H1) with negatively charged aminoacids increase resistance against heat-induced aggregation. A preference for aspartate over glutamate is observed at all analyzed positions. (B) Similarly, astrong preference for aspartate over the positively charged amino acids lysine and arginine is observed for single and multiple substitutions.

1 Jespers L, Schon O, Famm K, Winter G (2004) Aggregation-resistant domain antibodies selected on phage by heat denaturation. Nat Biotechnol 22:1161–1165.2 Jansson B, Uhlen M, Nygren PA (1998) All individual domains of staphylococcal protein A show Fab binding. FEMS Immunol Med Microbiol 20:69–78.3 Bjorck L (1988) Protein L. A novel bacterial cell wall protein with affinity for Ig L chains. J Immunol 140:1194–1197.4 Kabat E, Wu TT, Perry HM, Kay S, Gottesman CF (1992) Sequences of Proteins of Immunological Interest (Diane Publishing, Darby, PA), 5th Ed, p 2719.

Fig. S3. Aggregation upon freeze/drying or filtration: effects of mutations in human antibody variable domains. Representative human variable domains aretargeted for substitution with aspartate/glutamate in CDR H1 (VH) or L2 (VL), expressed and purified (see SI Text for details). Numbering according to Kabat (6).Sample conditions are as follows: VH at 20 μM in PBS; VL at 100 μM in 20 mM phosphate buffer pH 7.4 (seeMethods for details). For freeze/drying, samples arefrozen in liquid nitrogen, dried in a vacuum centrifugal evaporator for 2 h and reconstituted in water. For filtration, samples are spun in an Amicon Ultracelldevice at room temperature for 20 min at 13;200 × g (Millipore, 10 kDa cut-off). Aggregation is determined by measuring sample turbidity at 320 nm. (A)Human VH. (B) Human VL.

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Fig. S4. Structures of human antibody variable domain mutants. Shown are cartoon (Upper) and surface-charge representations (Lower). Mutant residues inCDR H1 and L2 are highlighted (sticks, Upper). Electrostatic surface potential is indicated [colored from −3 kT∕e (red) to þ3 kT∕e (blue)]. Representations aregenerated using APBS (1), PDB2PQR (2) and PyMol (3). (A) Structure of human VH triple mutant. The molecule crystallized as a domain-swapped dimer [anarrangement previously reported for other antibody domains (4)]. Tenmolecules were present in the asymmetric unit (chains A–J), arranged as five pairs (singlepair shown here). (B) Structure of human VL triple mutant. The protein crystallized as a dimer (chains A–B) in a canonical Bence–Jones arrangement (5). Areas ofnegative electrostatic surface potential are centered on the mutant residues in CDR H1 and L2 (in red).

1 Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA (2001) Electrostatics of nanosystems: Application to microtubules and the ribosome. Proc Natl Acad Sci USA 98:10037–10041.2 Dolinsky TJ, Nielsen JE, McCammon JA, Baker NA (2004) PDB2PQR: An automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Res 32:

W665–667.3 Delano WL (2002) The PyMOL Molecular Graphics System (Delano Scientific, San Carlos, CA).4 Spinelli S, et al. (2004) Domain swapping of a llama VHH domain builds a crystal-wide beta-sheet structure. FEBS Lett 564:35–40.5 Epp O, Lattman EE, SchifferM, Huber R, PalmW (1975) Themolecular structure of a dimer composed of the variable portions of the Bence-Jones protein REI refined at 2.0-A resolution.

Biochemistry 14:4943–4952.

Fig. S5. Antigen binding of Herceptin derived scFv fragment and VH and VL variable domains on phage. Binding is detected by ELISA using biotinylated HER2extracellular domain immobilized on a streptavidin plate (as measured by absorbance at 450 nm; means, SD shown, n ¼ 4).

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Fig. S6. Effects of mutations in CDR H1 and L2 in the absence of net charge. Shown are synthetic antibody repertoires human VH3, human Vk1, with diversityclosely mimicking the amino acid distribution in the natural antibody repertoire (1). The domains shown here carry no net charge (Qnet ¼ 0) (2). Aggregationresistance of repertoires is determined by measuring retained binding to superantigen after heating on phage (3, 4, 5) (wild-type residue: WT). Doubleaspartate substitutions in H1 (32D/33D) and L2 (52D/53D) significantly increase mean aggregation resistance of the repertoires (***, p < 0.001).

1 Knappik A, et al. (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol296:57–86.

2 Lawrence MS, Phillips KJ, Liu DR (2007) Supercharging proteins can impart unusual resilience. J Am Chem Soc 129:10110–10112.3 Jespers L, Schon O, Famm K, Winter G (2004) Aggregation-resistant domain antibodies selected on phage by heat denaturation. Nat Biotechnol 22:1161–1165.4 Jansson B, Uhlen M, Nygren PA (1998) All individual domains of staphylococcal protein A show Fab binding. FEMS Immunol Med Microbiol 20:69–78.5 Bjorck L (1988) Protein L. A novel bacterial cell wall protein with affinity for Ig L chains. J Immunol 140:1194–1197.

Fig. S7. Structural determinants of aggregation resistance in humanVH domains.Mutations in CDRH1 (VH triple mutant) do not induce conformational changesof framework residues. (A) Structures of previously reported soluble human VH single domains. Shown are the crystal structures of HEL4 model domain (in red;PDB ID code 10HQ; chains A/B) and of a mutant 4D5 single domain (in orange; PDB ID code 3B9V; chain D) (1, 2). The structures are characterized by conforma-tional changes centered on a framework residue at position 47 (tryptophanW47, sticks). The residue is highly conserved in human VH and forms part of the VH–VL

interface. (B) Structure of VH triple mutant determined in this study (in tan; chains A–J; PDB ID code 3UPC). Highlighted are CDR H1/H2/H3 in yellow/blue/red. (C)Structures of representative humanVH domains (in gray). Shown are domains from previously reported antibody Fab structures (PDB ID codes 3QOS, 2VXS, 3KDM,3BN9; 10 chains total) (3–6). (D) Overlay of structures shown in A–C. The structure of the VH triple mutant reported here (in tan) does not display the conforma-tional changes previously reported for soluble human model VH single domains (in red/orange). Rather, the VH triple mutant structure closely follows the W47“consensus” conformation observed in the structures of representative human domains (in gray). This consensus conformation is compatible with VL and antigenbinding, as highlighted by the interaction of the representative VH domains with light chain partners and hapten/protein targets (3–6).

1 Jespers L, Schon O, James LC, Veprintsev D, Winter G (2004) Crystal structure of HEL4, a soluble, refoldable human V(H) single domain with a germ-line scaffold. J Mol Biol 337:893–903.2 Barthelemy PA, et al. (2008) Comprehensive analysis of the factors contributing to the stability and solubility of autonomous human VH domains. J Biol Chem 283:3639–3654.3 Gerhardt S, et al. (2009) Structure of IL-17A in complex with a potent, fully human neutralizing antibody. J Mol Biol 394:905–921.4 Malia TJ, Obmolova G, Almagro JC, Gilliland GL, Teplyakov A (2011) Crystal structure of human germline antibody 3–23/B3. Mol Immunol 48:1586–1588.5 Niemi MH, et al. (2011) The testosterone binding mechanism of an antibody derived from a naive human scFv library. J Mol Recognit 24:209–219.

6 Farady CJ, Egea PF, Schneider EL, Darragh MR, Craik CS (2008) Structure of an Fab-protease complex reveals a highly specific non-canonical mechanism of inhibition. J Mol Biol

380:351–360.

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Table S1. Diffraction data and structure refinement statistics

Diffraction Data

Dataset Human VH Human VL

Spacegroup P21212 P43212Unit cell dimensions (a,b,c)(Å) 82.6, 143.1, 145.6 45.0, 45.0, 173.2Wavelength (Å) 0.95369 0.95369Resolution range (Å) 43.4–2.8 45.4–1.8Observed reflections* 311,782 206,540Unique reflections* 43,312 17,458Completeness*†(%) 100.0 (100.0) 99.8 (99.6)Multiplicity*† 7.2 (7.4) 11.8 (12.5)Rmeas*

† 0.16 (0.84) 0.15 (0.82)Mean (I∕sd)*† 10.5 (2.7) 11.1 (3.0)Wilson B (Å2) 58.0 19.2RefinementProtein molecules/asu 10 2Amino acids/asu 1,131 214Waters modeled 0 94PEGs modeled 7 0Ramachandran‡—favored (%) 98.29 95.7Ramachandran‡—outliers(%) 0.54 0.48R 0.21 0.23Rfree (5% data) 0.25 0.28rmsd bond lengths (Å) 0.012 0.011rmsd bond angles (°) 1.45 1.39PDB ID code 3UPC 3UPA

Data was processed using iMOSFLM (1), POINTLESS (2), SCALA (2) and CCP4i(3). Structures were solved by molecular replacement using PHASER (4).Search models utilized were 3QOS (VH excluding CDR3) and 1F6L (VL).Structures were refined by REFMAC5 (5) and torsion-libration-screw (TLS)parameterization (6) and analyzed using COOT (7) and MOLPROBITY (8).*As output by SCALA.†Values in parentheses are of the highest resolution shell.‡As calculated by the Molprobity validation server.

1 Battye TG, Kontogiannis L, Johnson O, Powell HR, Leslie AG (2011) iMOSFLM: A new graphical interface for diffraction-imageprocessing with MOSFLM. Acta Crystallogr D Biol Crystallogr 67:271–281.

2 Evans P (2006) Scaling and assessment of data quality. Acta Crystallogr D Biol Crystallogr 62:72–82.3 Potterton E, Briggs P, Turkenburg M, Dodson E (2003) A graphical user interface to the CCP4 program suite. Acta Crystallogr D Biol

Crystallogr 59:1131–1137.4 McCoy AJ, et al. (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674.5 Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta

Crystallogr D Biol Crystallogr 53:240–255.6 WinnMD, IsupovMN, Murshudov GN (2001) Use of TLS parameters to model anisotropic displacements in macromolecular refinement.

Acta Crystallogr D Biol Crystallogr 57:122–133.7 Emsley P, Cowtan K (2004) COOT: Model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132.8 Chen VB, et al. (2010) MolProbity: All-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr

66:12–21.

Table S2. Thermodynamic stability of mutant humanantibody variable domains

WT S31D Y32E A33D 31-33DED

VH 40.4 40.2 24.7 34.3 26.8*WT A50D S52D S53D 50,52-53DDD

VL 28.9 29.9 24.7 26.8 24.0

Shown are free energies of unfolding (ΔGN−U) (in kJ mol−1).Representative human variable domains are targeted forsubstitution with aspartate/glutamate in CDR H1 (VH) or L2 (VL),expressed and purified (see SI Text for details). Values are obtainedby guanidinium chloride unfolding and fitted according to a two-state model (1).*For VH triple mutant, data could not be fitted to a two-state modeland apparent free energy of unfolding was instead determined bythermal denaturation using circular dichroism and a ΔCp of 12 calper residue (1, 2).

1 Pace CN, Scholz JM (1997) Protein Structure: A Practical Approach, (Oxford Univ Press, New York), 2nd Ed, pp 299–321.2Myers JK, Pace CN, Scholtz JM (1995) Denaturantm values and heat capacity changes: relation to changes in accessible surface areasof protein unfolding. Protein Sci 4:2138–2148.

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