General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold

Nanobodies, single-domain antigen-binding fragments of camelid-specific heavy-chain only antibodies offer special advantages in therapy over classic antibody fragments because of their smaller size, robustness, and preference to target unique epitopes. A Nanobody differs from a human heavy chain variable domain in about ten amino acids spread all over its surface, four hallmark Nanobody-specific amino acids in the framework-2 region (positions 42, 49, 50, and 52), and a longer third antigen-binding loop (H3) folding over this area. For therapeutic applications the camelid-specific amino acid sequences in the framework have to be mutated to their human heavy chain variable domain equivalent, i.e. humanized. We performed this humanization exercise with Nanobodies of the subfamily that represents close to 80% of all dromedary-derived Nanobodies and investigated the effects on antigen affinity, solubility, expression yield, and stability. It is demonstrated that the humanization of Nanobody-specific residues outside framework-2 are neutral to the Nanobody properties. Surprisingly, the Glu-49 → Gly and Arg-50 → Leu humanization of hallmark amino acids generates a single domain that is more stable though probably less soluble. The other framework-2 substitutions, Phe-42 → Val and Gly/Ala-52 → Trp, are detrimental for antigen affinity, due to a repositioning of the H3 loop as shown by their crystal structures. These insights were used to identify a soluble, stable, well expressed universal humanized Nanobody scaffold that allows grafts of antigen-binding loops from other Nanobodies with transfer of the antigen specificity and affinity. Nanobodies, single-domain antigen-binding fragments of camelid-specific heavy-chain only antibodies offer special advantages in therapy over classic antibody fragments because of their smaller size, robustness, and preference to target unique epitopes. A Nanobody differs from a human heavy chain variable domain in about ten amino acids spread all over its surface, four hallmark Nanobody-specific amino acids in the framework-2 region (positions 42, 49, 50, and 52), and a longer third antigen-binding loop (H3) folding over this area. For therapeutic applications the camelid-specific amino acid sequences in the framework have to be mutated to their human heavy chain variable domain equivalent, i.e. humanized. We performed this humanization exercise with Nanobodies of the subfamily that represents close to 80% of all dromedary-derived Nanobodies and investigated the effects on antigen affinity, solubility, expression yield, and stability. It is demonstrated that the humanization of Nanobody-specific residues outside framework-2 are neutral to the Nanobody properties. Surprisingly, the Glu-49 → Gly and Arg-50 → Leu humanization of hallmark amino acids generates a single domain that is more stable though probably less soluble. The other framework-2 substitutions, Phe-42 → Val and Gly/Ala-52 → Trp, are detrimental for antigen affinity, due to a repositioning of the H3 loop as shown by their crystal structures. These insights were used to identify a soluble, stable, well expressed universal humanized Nanobody scaffold that allows grafts of antigen-binding loops from other Nanobodies with transfer of the antigen specificity and affinity. Minimizing the size of antigen-binding entities from a multidomain protein such as a monoclonal antibody to a single-chain variable fragment or even a single domain has been one of the primary goals of antibody engineering. For drug therapy, these smaller formats can be beneficial in various aspects such as immunogenicity, biodistribution, renal clearance, serum half-life, tissue penetration, and target retention. However, the minimal sized antibody fragments need to retain sufficiently high antigen specificity and affinity, be expressed in high yields, and should have a low tendency to aggregate so as to maintain maximal potency and reduce possible risks of immunogenicity. Moreover functionality in adverse environments such as high concentrations of denaturant or elevated temperatures, and a concomitant increased shelf-life are additional assets. A significant proportion of the functional antibodies within species of the Camelidae are devoid of light chains. These immunoglobulins are referred to as heavy-chain antibodies (1Hamers-Casterman C. Atarhouch T. Muyldermans S. Robinson G. Hamers C. Songa E.B. Bendahman N. Hamers R. Nature.. 1993; 363: 446-448Google Scholar), and their antigen-binding fragment is reduced to a single domain (referred to as VHH or Nanobody), with a molecular size of only ∼15 kDa, which is smaller in comparison to single-chain variable fragment fragments (30 kDa), Fab fragments (60 kDa), and whole antibodies (150 kDa). All Nanobodies belong to the same sequence family, closely related to that of the human VH 3The abbreviations used are: VH, heavy chain variable domain; LDA, ligation during amplification protocol; GdmCl, guanidinium chloride; r.m.s.d., root mean square deviation; CDR, complementarity determining region.3The abbreviations used are: VH, heavy chain variable domain; LDA, ligation during amplification protocol; GdmCl, guanidinium chloride; r.m.s.d., root mean square deviation; CDR, complementarity determining region. of family III, although different subfamilies can be distinguished in dromedary based on the CDR2 length and the position of an additional cysteine in the CDR1 or the framework-2 (2Nguyen V.K. Hamers R. Wyns L. Muyldermans S. EMBO J.. 2000; 19: 921-930Google Scholar). Because extra cysteines are rare in llama VHH sequences, they cannot be used as a subfamily hallmark and alternative subfamily divisions had to be proposed for llama VHHs (3Achour I. Cavelier P. Tichit M. Bouchier C. Lafaye P. Rougeon F. J. Immunol... 2008; 181: 2001-2009Google Scholar, 4Harmsen M.M. Ruuls R.C. Nijman I.J. Niewold T.A. Frenken L.G. de Geus B. Mol. Immunol... 2000; 37: 579-590Google Scholar). Following immunization of a llama or dromedary, VHH genes can be easily cloned in a phagemid vector and antigen-specific VHHs can then be selected via phage display against virtually any antigen (5Arbabi Ghahroudi M. Desmyter A. Wyns L. Hamers R. Muyldermans S. FEBS Lett... 1997; 414: 521-526Google Scholar). Their small size, natural soluble behavior, and unique ability to target alternative epitopes make Nanobodies very attractive tools for tumor targeting, diagnostics, or even for in vivo therapy (6Baral T.N. Magez S. Stijlemans B. Conrath K. Vanhollebeke B. Pays E. Muyldermans S. De Baetselier P. Nat. Med... 2006; 12: 580-584Google Scholar, 7Cortez-Retamozo V. Backmann N. Senter P.D. Wernery U. De Baetselier P. Muyldermans S. Revets H. Cancer Res... 2004; 64: 2853-2857Google Scholar, 8Huang L. Reekmans G. Saerens D. Friedt J.M. Frederix F. Francis L. Muyldermans S. Campitelli A. Hoof C.V. Biosens. Bioelectron... 2005; 21: 483-490Google Scholar, 9Rothbauer U. Zolghadr K. Muyldermans S. Schepers A. Cardoso M.C. Leonhardt H. Mol. Cell Proteomics.. 2008; 7: 282-289Google Scholar, 10Saerens D. Frederix F. Reekmans G. Conrath K. Jans K. Brys L. Huang L. Bosmans E. Maes G. Borghs G. Muyldermans S. Anal. Chem... 2005; 77: 7547-7555Google Scholar). Analysis of the amino acid sequence of the Nanobodies obtained from immunized camelids revealed frequent substitutions in regions that are conserved in the VH domain of conventional antibodies. These Nanobody hallmark amino acids, located mainly in framework-2, are essential adaptations to avoid the association with the variable light chain domain (11Muyldermans S. Atarhouch T. Saldanha J. Barbosa J.A. Hamers R. Protein Eng.. 1994; 7: 1129-1135Google Scholar). Most of these mutations are substitutions from a hydrophobic to a hydrophilic residue and are considered to increase the solubility of the isolated Nanobody (12Davies J. Riechmann L. FEBS Lett... 1994; 339: 285-290Google Scholar, 13Conrath K. Vincke C. Stijlemans B. Schymkowitz J. Decanniere K. Wyns L. Muyldermans S. Loris R. J. Mol. Biol... 2005; 350: 112-125Google Scholar). In some cases, several of these residues at the “former VL-side” may also affect the antigen specificity of the Nanobody (13Conrath K. Vincke C. Stijlemans B. Schymkowitz J. Decanniere K. Wyns L. Muyldermans S. Loris R. J. Mol. Biol... 2005; 350: 112-125Google Scholar). These differences between VHH and VH conserved residues are Phe/Tyr-42 → Val, Glu-49 → Gly, Arg-50 → Leu, and Gly-52 → Trp (numbers refer to the International ImMunoGeneTics information system amino acid numbering (imgt.cines.fr)). Additionally, in ∼10% of the Nanobodies, Trp-118 is substituted by an Arg (14Harmsen M.M. de Haard H.J. Appl. Microbiol. Biotechnol... 2007; 77: 13-22Google Scholar). The Trp-118 → Arg substitution was proposed as an alternative option to further increase the solubility of single-domain antibody fragments (15Desmyter A. Decanniere K. Muyldermans S. Wyns L. J. Biol. Chem... 2001; 276: 26285-26290Google Scholar). Because mouse antibody fragments require a humanization step to be accepted as human therapeutics, it is likely that Nanobodies from camelid origin should also pass a humanization process. It is our objective in this work to assess the biochemical properties of several Nanobodies after such humanization effort. We investigated therefore the humanization of a couple of representative Nanobodies into more human-like antibody fragments and tested their retention of antigen-binding specificity. Two Nanobodies, members of subfamily-2, were chosen for this analysis: NbHuL6 (16Dumoulin M. Conrath K. Van Meirhaeghe A. Meersman F. Heremans K. Frenken L.G. Muyldermans S. Wyns L. Matagne A. Protein Sci... 2002; 11: 500-515Google Scholar) and NbBcII10 (17Conrath K.E. Lauwereys M. Galleni M. Matagne A. Frere J.M. Kinne J. Wyns L. Muyldermans S. Antimicrob. Agents Chemother.. 2001; 45: 2807-2812Google Scholar). Subfamily-2 is the most frequently occurring of the seven VHH subfamilies accounting for 75% of all isolated dromedary antigen-specific Nanobodies (2Nguyen V.K. Hamers R. Wyns L. Muyldermans S. EMBO J.. 2000; 19: 921-930Google Scholar). We first focused on the framework-2 residues, because those mutations are expected to have the largest impact on expression, solubility, and antigen affinity of the isolated domain. We then analyzed the effect of the Trp-118 → Arg mutation on these antibody formats. Subsequently, we substituted the remaining “non-human” residues in the framework and resurfaced NbBcII10 to obtain a humanized Nanobody scaffold (h-NbBcII10FGLA) so as to exhibit the closest possible amino acid identity to a human VH (Fig. 1). It was previously demonstrated that the NbBcII10 accepts the CDR loops from a whole range of Nanobodies with transfer of the antigen specificity of the loop donor (18Saerens D. Pellis M. Loris R. Pardon E. Dumoulin M. Matagne A. Wyns L. Muyldermans S. Conrath K. J. Mol. Biol... 2005; 352: 597-607Google Scholar). Therefore, the CDR loops of two Nanobodies, NbHuL6 and NbHSA, were grafted on the resurfaced framework h-NbBcII10FGLA, to assess whether this framework can indeed be used as a universal humanized Nanobody scaffold. We determined and compared the antigen specificity, kinetic binding rate constants (kon/koff), the thermal (Tm), and conformational (Cm/ΔG0) stability parameters of multiple variants, humanized to different extents. To evaluate the impact of humanization on the scaffold architecture of Nanobodies, we solved the crystal structure of a partially humanized NbHul6 mutant (Phe-42, Gly-49, Leu-50, and Trp-52) in complex with its antigen and of the maximally humanized NbBcII10 (h-NbBcII10FGLA). Based on this knowledge, a general strategy is proposed to generate a humanized version of any Nanobody with maximal retention of stability and antigen-binding characteristics. Finally, a universal humanized Nanobody scaffold was identified that accommodates antigen-binding loops from Nanobodies, even those from other subfamilies or species. Site-directed Mutagenesis—Mutations were introduced by phosphorylated mutagenic oligonucleotides using the ligation during amplification protocol (LDA) (19Chen Z. Ruffner D.E. Nucleic Acids Res... 1998; 26: 1126-1127Google Scholar). Multiple mutations were introduced simultaneously using multiple mutagenic primers, designed so as to add or eliminate a particular restriction enzyme site. The introduction of the different mutations was analyzed by restriction enzyme analysis and confirmed by DNA sequencing (ABI prism 3100 genetic analyzer, Applied Biosystems). Expression and Purification of Nanobodies—The plasmid constructs were transformed into Escherichia coli WK6 cells. The expression in the periplasm and purification of recombinant His6-tagged Nanobodies was performed as described previously (17Conrath K.E. Lauwereys M. Galleni M. Matagne A. Frere J.M. Kinne J. Wyns L. Muyldermans S. Antimicrob. Agents Chemother.. 2001; 45: 2807-2812Google Scholar). The purity of the proteins was evaluated by Coomassie-stained SDS-polyacrylamide gels. The protein concentration was determined spectrophotometrically at 280 nm using the computed extinction coefficient of each Nanobody, as calculated from their amino acid sequence (20Gasteiger E. Gattiker A. Hoogland C. Ivanyi I. Appel R.D. Bairoch A. Nucleic Acids Res... 2003; 31: 3784-3788Google Scholar). Affinity Measurements—Different concentrations ranging from 500 nm to 7.8 nm of the NbHuL6, NbBcII10, and their respective variants were flown over a CM5 chip (Biacore) to which respectively human lysozyme (200 relative units) and BcII (800 relative units) had been coupled using the amine coupling chemistry (N-ethyl-N′-(dimethylaminopropyl)-carbodiimide/N-hydroxy succinimide) according to the manufacturer's descriptions. All measurements were performed at a flow rate of 30 μl/min in HBS buffer (10 mm Hepes, pH 7.5, 150 mm NaCl, 3.5 mm EDTA, and 0.005% Tween 20) and 10 mm glycine/HCl, pH 1.5, was used for regeneration. Data were fitted with the help of the BIAevaluation software version 4.1 (Biacore), on the basis of a 1:1 Langmuir binding model, with simultaneous fitting of the dissociation (koff) and association (kon) rate constants. The kinetic parameters kon and koff were subsequently used to calculate the KD values. Temperature-induced Unfolding—CD measurements were performed with a Jasco J715 spectropolarimeter in the far-UV (205-250 nm) region, using a protein concentration of 0.166 mg/ml and 0.1 cm cell path length. A total volume of 300 μl of each sample was heated in 50 mm phosphate (pH 7.0). Heat-induced unfolding was monitored by increasing the temperature from 35 °C to 95 °C at a rate of 1 °C/min, and recording the fluorescence intensity at 205 nm as a function of temperature. Data were acquired with a reading frequency of 1/20 s-1, a 1-s integration time, and a 2 nm bandwidth. Data analysis was performed assuming two-state unfolding mechanisms (21Pace C.N. Scholtz J.M. Creighton T.E. Protein Structure, A practical Approach. Oxford University Press, New York1997: 299-321Google Scholar). The reversibility of the unfolding was assessed by recording wave-length scans of the protein at 35 °C, 95 °C and again after cooling down the sample to 35 °C. At each temperature five spectra were measured and averaged. Enzyme-linked Immunosorbent Assay—To test the residual binding activity after thermal unfolding, the different mutants were incubated at concentrations between 250 nm and 2 μm for 4 h at 90 °C. Maxisorb 96-well plates (Nunc) were coated overnight at 4 °C with human lysozyme or BcII at a concentration of 2 μg/ml in phosphate-buffered saline. Residual protein-binding sites in the wells were blocked for 2 h at room temperature with 1% milk powder in phosphate-buffered saline. After incubation with either untreated or heat-treated and subsequently refolded His-tagged Nanobody, bound protein was detected using a mouse anti-histidine tag antibody (Serotec) followed by an alkaline phosphatase anti-mouse-IgG conjugate (Sigma) and p-nitrophenyl phosphate as substrate. The percentage of binding activity restored after temperature unfolding was calculated using triplicates at four different concentrations of Nanobody. Equilibrium Denaturation Experiments—GdmCl-induced unfolding followed by intrinsic fluorescence measurements was employed to determine the thermodynamic stability. Protein-GdmCl mixtures containing a final protein concentration of 25 μg/ml and denaturant concentrations ranging from 0 to 6.0 m GdmCl were prepared by adding a GdmCl stock solution (7.2 m, in 50 mm phosphate, pH 7.0) to the purified protein (stock 2 mg/ml in phosphate-buffered saline). After overnight incubation at room temperature, the intrinsic fluorescence was measured at 25 °C on an Aminco-Bowman spectrofluorometer from 300 to 400 nm after excitation at a wavelength of 280 nm. The center of spectral mass of each spectrum was calculated as the center of spectral mass =Σνi × Fi/ΣFi, where νi is the wave number (i.e. inverse wavelength) and Fi is the fluorescence intensity at νi (22Royer C.A. Methods Mol. Biol... 1995; 40: 65-89Google Scholar). Thermodynamic parameters for chemical unfolding were computed on the assumption of a two-state model for the unfolding reaction, as observed for Nanobodies studied so far (16Dumoulin M. Conrath K. Van Meirhaeghe A. Meersman F. Heremans K. Frenken L.G. Muyldermans S. Wyns L. Matagne A. Protein Sci... 2002; 11: 500-515Google Scholar, 23Ewert S. Cambillau C. Conrath K. Pluckthun A. Biochemistry.. 2002; 41: 3628-3636Google Scholar). On this basis, a first data analysis was performed to obtain the ΔG0 and m values using a six-parameter equation as previously described by Pace (24Pace C.N. Trends Biotechnol... 1990; 8: 93-98Google Scholar) and Santoro and Bolen (25Santoro M.M. Bolen D.W. Biochemistry.. 1988; 27: 8063-8068Google Scholar). The concentration of denaturant at which half of the protein is denatured (Cm) has been shown to be the most reproducible value when comparing the stability of wild-type and mutant proteins, because it can be determined quite accurately and is largely insensitive to the unfolding mechanism (26Pace C.N. Methods Enzymol.. 1986; 131: 266-280Google Scholar). Transition curves were also analyzed according to Clarke and Fersht (27Clarke J. Fersht A.R. Biochemistry.. 1993; 32: 4322-4329Google Scholar) and Kellis et al. (28Kellis J.T.J. Nyberg K. Fersht A.R. Biochemistry.. 1989; 28: 4914-4922Google Scholar) to obtain the Cm and m values. ΔG0 can then simply be calculated from ΔG0 = m·Cm. Both fitting procedures resulted in very similar ΔG0 values, we therefore report only the latter. Generation of Chimeric Nanobody Constructs—The CDR-H loops from loop donor Nanobodies NbHuL6 (dromedary) and NbHSA (llama) were transferred to the scaffold of h-NbBcII10FGLA (humanized recipient Nanobody) by PCR-based mutagenesis. The sequence of each CDR-H loop from the loop donor Nanobody was encompassed by two primers, one back and one forward primer, containing at the 5′ and the 3′ ends the sequences corresponding to the framework residues of the recipient Nanobody. The chimeras were constructed as described previously with some minor modifications (18Saerens D. Pellis M. Loris R. Pardon E. Dumoulin M. Matagne A. Wyns L. Muyldermans S. Conrath K. J. Mol. Biol... 2005; 352: 597-607Google Scholar). The chimeric Nanobody construct of NbHuL6 on h-NbBcII10FGLAwas digested with NcoI and NotI, whereas the chimeric construct with NbHSA as donor Nanobody was digested with Hind-III and NotI. Both fragments were cloned in the expression vector pHEN6 (17Conrath K.E. Lauwereys M. Galleni M. Matagne A. Frere J.M. Kinne J. Wyns L. Muyldermans S. Antimicrob. Agents Chemother.. 2001; 45: 2807-2812Google Scholar). The expression yield of both chimeras is comparable to the level of the humanized recipient Nanobody (2 mg/liter of culture). Crystal Structure Determination—Data for the free form of NbBcII10 (pdb entry 3DWT) were collected at ESRF beamline ID14-2 under cryogenic conditions. The crystals diffract to 2.9-Å resolution. Data were reduced using the HKL suite of programs (29Otwinowski Z. Minor W. Methods Enzymol.. 1997; 276: 307-326Google Scholar). Molecular replacement was done with the maximum-likelihood based program PHASER (30McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. D. Biol. Crystallogr... 2005; 61: 458-464Google Scholar, 31Storoni L.C. McCoy A.J. Read R.J. Acta Crystallogr. D. Biol. Crystallogr... 2004; 60: 432-438Google Scholar), which allowed the unambiguous positioning of all eight VHH domains in the asymmetric unit. The framework region of crystal structure of NbBcII10 grafted with the CDR loops from NbLys3 (pdb entry 1ZMY) (18Saerens D. Pellis M. Loris R. Pardon E. Dumoulin M. Matagne A. Wyns L. Muyldermans S. Conrath K. J. Mol. Biol... 2005; 352: 597-607Google Scholar, 32Desmyter A. Transue T.R. Ghahroudi M.A. Thi M.H. Poortmans F. Hamers R. Muyldermans S. Wyns L. Nat. Struct. Biol... 1996; 3: 803-811Google Scholar) was used as the starting model. Rounds of simulated annealing refinement and B-factor refinement with CNS 1.0 (33Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D. Biol. Crystallogr... 1998; 54: 905-921Google Scholar) were alternated with manual rebuilding using TURBO (34Roussel A. Cambillau C. Silicon Graphic Geometry Partners' Directory. Silicon Graphics, Mountain View, CA1989: 71-78Google Scholar). NCS restraints were used throughout the refinement, and the mli target function was used. Data collection and refinement statistics are given in Table 1. The final R and Rfree factors were 0.245 and 0.304, respectively, with the model fitting the electron density very well. Attempts to reduce the R factors by relaxing the NCS restraints or to build different CDR conformations in different monomers were unsuccessful.TABLE 1Crystallographic data and refinement statistics for NbHuL6FGLW, NbBcII10, and h-NbBcII10FGLANbHuL6FGLWNbBcII10h-NbBcII10FGLASpace groupP61P21212P21212Unit cella = b = 63.67 Åa = 76.72 Åa = 75.59 Åc = 119.51 Åb = 174.71 Åb = 70.75 Åc = 115.53 Åc = 50.37 ÅResolution (highest resolution shell)20.0-1.85 (1.92-1.85) Å20.0-2.9 (3.0-2.9)50.0-1.95 (2.02-1.95)Data collection temperature100 K100 K100 KNumber of measured reflections107,299 (10208)238,144 (23075)129,288 (13187)Number of unique reflections23,285 (2320)34,579 (3402)20,171 (1998)Completeness (highest resolution shell)99.9 (99.9)%99.9 (100.0)%99.9 (100.0)I/σ (I) (highest resolution shell)14.3 (3.6)11.4 (2.7)9.99 (2.3)Rmerge (highest resolution shell)0.069 (0.402)0.127 (0.485)0.196 (0.607)R-factor (highest resolution shell)0.211 (0.283)0.245 (0.331)0.215 (0.237)Rfree-factor (highest resolution shell)0.255 (0.335)0.305 (0.492)0.252 (0.265)Ramachandran plotCore region87.5%82.5%87.7%Additional allowed12.0%16.9%12.3%Generously allowed0.5%0.6%0.0%Disallowed0.0%0.0%0.0%r.m.s.d.Bond lengths0.0058 Å0.0075 Å0.0058 ÅBond angles1.325°1.523°1.37°pdb entry3EBA3DWT3EAK Open table in a new tab X-ray data for the maximally humanized mutant of NbBcII10 (h-NbBcII10FGLA, pdb entry 3EAK) were collected at beamline BW7A of the DESY synchrotron, Hamburg, to a resolution of 1.85 Å and processed as for the wild-type data. The same starting model was used for molecular replacement and refinement as for the wild-type NbBcII10 structure to minimize bias. Refinement was carried out using the same protocol as for the wild-type NbBcII10, except that no NCS restraints were applied. The same protocol was also applied to the partly humanized NbHul6 complex (NbHul6FGLW, pdb entry 3EBA), for which the data were also collected on beamline BW7A. The wild-type NbHuL6:HuL complex (pdb entry 1OP9) was used as the starting model for molecular replacement and refinement. All details of data collection and refinement are presented in Table 1. Humanizing the Framework-2 Region of Nanobodies—Two Nanobodies, NbHuL6 and NbBcII10, with specificity for human lysozyme (HuL) and the β-lactamase BcII of Bacillus cereus, respectively, have been selected to determine the involvement of the VHH hallmark amino acid residues in framework-2 on expression yield, affinity, and stability. The NbHuL6 was chosen for this study because of its potential therapeutic relevance, because it stabilizes an unstable human lysozyme mutant that forms fibrils and amyloids (35Dumoulin M. Last A.M. Desmyter A. Decanniere K. Canet D. Larsson G. Spencer A. Archer D.B. Sasse J. Muyldermans S. Wyns L. Redfield C. Matagne A. Robinson C.V. Dobson C.M. Nature.. 2003; 424: 783-788Google Scholar). The high stability (50.9 kJ mol-1) of NbBcII10 (17Conrath K.E. Lauwereys M. Galleni M. Matagne A. Frere J.M. Kinne J. Wyns L. Muyldermans S. Antimicrob. Agents Chemother.. 2001; 45: 2807-2812Google Scholar) and the successful use of its framework to graft the antigen specificity loops from donor Nanobodies of the VHH subfamily-2 (18Saerens D. Pellis M. Loris R. Pardon E. Dumoulin M. Matagne A. Wyns L. Muyldermans S. Conrath K. J. Mol. Biol... 2005; 352: 597-607Google Scholar), makes it a logical candidate as universal Nanobody scaffold. The two Nanobodies were mutated by LDA-PCR at the framework-2 hallmark amino acid positions 42, 49, 50, and 52 to the amino acids occurring at those positions in human VH domains, i.e. Val42, Gly49, Leu50 and Trp52, [ImMunoGeneTics numbering (imgt.cines.fr)]. These mutants, referred to as NbHuL6VGLW and NbBcII10VGLW, respectively, were expressed in the periplasm of E. coli and purified by immobilized metal ion chromatography and gel filtration. (Nanobody mutants humanized in framework-2 are designated with a four-letter single letter code for the amino acid at positions 42, 49, 50, and 52; these letters are italicized if they correspond to the human sequence at that position). The amount of wild-type protein recovered after size-exclusion chromatography corresponded to 3 mg/liter of culture for NbHuL6 and NbBcII10. The expression yields of the framework-2 humanized proteins were lower: 1 mg/liter of culture for NbHuL6VGLW and 1.8 mg/liter of culture for NbBcII10VGLW, probably due to the lower cell density attained after overnight growth or to cell lysis. Both mutant proteins are monomeric, as observed by size-exclusion chromatography (data not shown). However, a delay in elution time from the column is noticed for both framework-2-humanized formats, suggesting nonspecific interaction of the humanized proteins with the gel matrix. This feature was also noted for isolated VH domains of conventional antibodies (13Conrath K. Vincke C. Stijlemans B. Schymkowitz J. Decanniere K. Wyns L. Muyldermans S. Loris R. J. Mol. Biol... 2005; 350: 112-125Google Scholar, 23Ewert S. Cambillau C. Conrath K. Pluckthun A. Biochemistry.. 2002; 41: 3628-3636Google Scholar, 36Barthelemy P.A. Raab H. Appleton B.A. Bond C.J. Wu P. Wiesmann C. Sidhu S.S. J. Biol. Chem... 2008; 283: 3639-3654Google Scholar). In addition, the NbHuL6VGLW has a tendency to precipitate upon prolonged storage at 4 °C. The effect of humanizing the framework-2 region of NbHuL6 and NbBcII10 on the antigen-binding capacity was assessed by surface plasmon resonance. In both cases an increase in the equilibrium dissociation constants was observed (14.6 nm for NbHuL6VGLW compared with 0.32 nm for wild-type NbHuL6 and 16 μm for NbBcII10VGLW compared with 7.4 nm for wild-type NbBcII10 (Tables 2A and 3A)).TABLE 2Kinetic rate and equilibrium dissociation constants, changes in the free energies of unfolding by equilibrium denaturation (A) and overview of the heat-induced unfolding experiments (B) upon mutation of VHH hallmark residues of NbHuL6A) NbHuL6aPartially humanized mutants in framework-2 are designated with a four-letter label referring to the residues at, respectively, positions 42, 49, 50, and 52. The human hallmark residues at these positions are in italic font.konkoffKDCmm-valueΔG0ΔΔGH2ObΔΔG0 = ΔG0 (wild type) - ΔG0 (mutant) (27, 28). (relative to wild-type)m−1 s−1s−1nmmkJmol−1m−1kJmol−1kJmol−1FERG2.39E+067.68E-040.323.02 ± 0.0213.7 ± 1.041.4 ± 3.4FERW2.04E+066.85E-033.363.35 ± 0.0115.5 ± 0.551.9 ± 1.9−10.5VERG2.70E+061.61E-030.602.58 ± 0.0114.4 ± 0.637.1 ± 1.84.3VERW1.01E+061.59E-0215.743.05 ± 0.0116.7 ± 0.750.8 ± 2.4−9.4FGLG1.42E+065.78E-040.413.39 ± 0.0219.0 ± 1.964.7 ± 6.6−23.3FGLW1.63E+063.85E-032.363.83 ± 0.0214.3 ± 1.155.0 ± 4.4−13.6VGLG2.58E+061.24E-030.482.95 ± 0.0119.7 ± 1.358.1 ± 3.9−16.7VGLW9.31E+051.36E-0214.613.50 ± 0.0115.4 ± 0.854.0 ± 3.1−12.6FERG W118R8.74E+052.98E-033.411.75 ± 0.029.0 ± 0.415.8 ± 0.925.6VGLW W118R2.12E+051.95E-0291.983.11 ± 0.0114.9 ± 0.546.4 ± 1.7−5.0B) NbHuL6aPartially humanized mutants in framework-2 are designated with a four-letter label referring to the residues at, respectively, positions 42, 49, 50, and 52. The human hallmark residues at these positions are in italic font.TmReversibilityrestored activity°C%%FERG79.7 ± 0.293.386.8FERW84.2 ± 0.593.4NDVERG76.2 ± 0.293.058.8VERW79.4 ± 0.292.6100FGLG82.5 ± 0.396.364.9FGLWNDcND, not determined.103.1100VGLG78.4 ± 0.281.665.4VGLW79.5 ± 0.366.779.9FERG W118R70.0 ± 0.169.958.4VGLW W118R79.9 ± 0.286.355.5a Partially humanized mutants in framework-2 are designated with a four-letter label referring to the residues at, respectively, positions 42, 49, 50, and 52. The human hallmark residues at these positions are in italic font.b ΔΔG0 = ΔG0 (wild type) - ΔG0 (mutant) (27Clarke J. Fersht A.R. Biochemistry.. 1993; 32: 4322-4329Google Scholar, 28Kellis J.T.J. Nyberg K. Fersht A.R. Biochemistry.. 1989; 28: 4914-4922Google Scholar).c ND, not determined. Open table in a new tab TABLE 3Kinetic rate and equilibrium dissociation c