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• W2170269585 abstract "We have constructed a human VHlibrary based on a camelized VH sequence. The library was constructed with complete randomization of 19 of the 23 CDR3 residues and was panned against two monoclonal antibody targets to generate VH sequences for determination of the antigen contact residue positions. Furthermore, the feasibility and desirability of introducing a disulfide bridge between CDR1 and CDR3 was investigated. Sequences derived from the library showed a bias toward the use of C-terminal CDR3 residues as antigen contact residues. Mass spectrometric analyses indicated that CDR1-CDR3 disulfide formation was universal. However, surface plasmon resonance and NMR data showed that the CDR3 constraint imposed by the disulfide bridge was not always desirable. Very high yields of soluble protein products and lack of protein aggregation, as demonstrated by the quality of the1H-15N HSQC spectra, indicated that the VH sequence for library construction was a good choice. These results should be useful in the design of VHlibraries with optimal features. We have constructed a human VHlibrary based on a camelized VH sequence. The library was constructed with complete randomization of 19 of the 23 CDR3 residues and was panned against two monoclonal antibody targets to generate VH sequences for determination of the antigen contact residue positions. Furthermore, the feasibility and desirability of introducing a disulfide bridge between CDR1 and CDR3 was investigated. Sequences derived from the library showed a bias toward the use of C-terminal CDR3 residues as antigen contact residues. Mass spectrometric analyses indicated that CDR1-CDR3 disulfide formation was universal. However, surface plasmon resonance and NMR data showed that the CDR3 constraint imposed by the disulfide bridge was not always desirable. Very high yields of soluble protein products and lack of protein aggregation, as demonstrated by the quality of the1H-15N HSQC spectra, indicated that the VH sequence for library construction was a good choice. These results should be useful in the design of VHlibraries with optimal features. single domain antibody complementarity determining region 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate dithiothreitol antigen-binding fragment heteronuclear single-quantum coherence spectroscopy immunoglobulin G nuclear magnetic resonance nuclear Overhauser effect spectroscopy phosphate-buffered saline single chain variable fragment of an antibody surface plasmon resonance antibody heavy chain variable domain variable domain of a heavy chain antibody antibody light chain variable domain Heavy chain antibodies, found in camelids (1Hamers-Casterman C. Atarhouch T. Muyldermans S. Robinson G. Hamers C. Songa Baiyana B. Bendahman N. Hamers R. Nature. 1993; 363: 446-448Crossref PubMed Scopus (2205) Google Scholar, 2Sheriff S. Constantine K.L. Nat. Struct. Biol. 1996; 3: 733-736Crossref PubMed Scopus (59) Google Scholar), lack light chains and as a result have variable domains that reflect the absence of a VL partner. Single domain antibodies (dAbs)1 derived from the variable domains (VHHs) of these antibodies are highly soluble and the structural basis of solubility has been partially elucidated. First, conserved human/murine interface residues are generally replaced in heavy chain antibodies by residues that increase the hydrophilicity of the VL interface either by non-polar to polar substitutions or, in a more subtle way, by inducing local conformational changes (3Desmyter A. Transue T.R. Ghahroudi M.A. Thi M.H. Poortmans F. Hamers R. Muyldermans S. Wyns L. Nat. Struct. Biol. 1996; 3: 803-811Crossref PubMed Scopus (417) Google Scholar, 4Spinelli S. Frenken L. Bourgeois D. de Ron L. Bos W. Verrips T. Anguille C. Cambillau C. Tegoni M. Nat. Struct. Biol. 1996; 3: 752-757Crossref PubMed Scopus (131) Google Scholar). This explanation is supported by experiments in which an insoluble human VH was made soluble by introducing these substitutions (5Davies J. Riechmann L. FEBS Lett. 1994; 339: 285-290Crossref PubMed Scopus (144) Google Scholar). Second, in the solved structures of two camel dAbs, the CDR3s fold back on the VHsurface, masking a significant surface area of the VLinterface (3Desmyter A. Transue T.R. Ghahroudi M.A. Thi M.H. Poortmans F. Hamers R. Muyldermans S. Wyns L. Nat. Struct. Biol. 1996; 3: 803-811Crossref PubMed Scopus (417) Google Scholar, 4Spinelli S. Frenken L. Bourgeois D. de Ron L. Bos W. Verrips T. Anguille C. Cambillau C. Tegoni M. Nat. Struct. Biol. 1996; 3: 752-757Crossref PubMed Scopus (131) Google Scholar, 5Davies J. Riechmann L. FEBS Lett. 1994; 339: 285-290Crossref PubMed Scopus (144) Google Scholar, 6Decanniere K. Desmyter A. Lauwereys M. Ghahroudi M.A. Muyldermans S. Wyns L. Structure. 1999; 7: 361-370Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Two other features of VHHs are noteworthy. One is the frequent occurrence of cysteine residues in CDR1 and CDR3 (7Nguyen V.K. Hamers R. Wyns L. Muyldermans S. EMBO J. 2000; 19: 921-930Crossref PubMed Scopus (216) Google Scholar, 8Muyldermans S. Atarhouch T. Saldanha J. Barbosa J.A. Hamers R. Protein Eng. 1994; 7: 1129-1135Crossref PubMed Scopus (399) Google Scholar, 9Lauwereys M. Arbabi G.M. Desmyter A. Kinne J. Holzer W. De Genst E. Wyns L. Muyldermans S. EMBO J. 1998; 17: 3512-3520Crossref PubMed Scopus (398) Google Scholar, 10Vu K.B. Ghahroudi M.A. Wyns L. Muyldermans S. Mol. Immunol. 1997; 34: 1121-1131Crossref PubMed Scopus (250) Google Scholar). While the location of the CDR1 cysteine is typically fixed, that of the CDR3 cysteine varies. These two residues form a disulfide linkage between CDR1 and CDR3 (3Desmyter A. Transue T.R. Ghahroudi M.A. Thi M.H. Poortmans F. Hamers R. Muyldermans S. Wyns L. Nat. Struct. Biol. 1996; 3: 803-811Crossref PubMed Scopus (417) Google Scholar, 11Davies J. Riechmann L. Protein Eng. 1996; 9: 531-537Crossref PubMed Scopus (135) Google Scholar). In the crystal structure of a dAb-lysozyme complex, the disulfide linkage imparts rigidity on the CDR3 loop that extends out of the combining site and penetrates deep into the active site of lysozyme (3Desmyter A. Transue T.R. Ghahroudi M.A. Thi M.H. Poortmans F. Hamers R. Muyldermans S. Wyns L. Nat. Struct. Biol. 1996; 3: 803-811Crossref PubMed Scopus (417) Google Scholar). A second feature is the longer average length of the VHH CDR3, relative to human or murine VHs (8Muyldermans S. Atarhouch T. Saldanha J. Barbosa J.A. Hamers R. Protein Eng. 1994; 7: 1129-1135Crossref PubMed Scopus (399) Google Scholar). A longer CDR3 increases the antigen-binding surface and partially compensates for the absence of the antigen-binding surface provided by the VL in conventional antibodies (3Desmyter A. Transue T.R. Ghahroudi M.A. Thi M.H. Poortmans F. Hamers R. Muyldermans S. Wyns L. Nat. Struct. Biol. 1996; 3: 803-811Crossref PubMed Scopus (417) Google Scholar). As antigen-binding fragments, dAbs are an attractive alternative to scFvs because of their much smaller size and the fact that they have affinities comparable to those of scFvs (4Spinelli S. Frenken L. Bourgeois D. de Ron L. Bos W. Verrips T. Anguille C. Cambillau C. Tegoni M. Nat. Struct. Biol. 1996; 3: 752-757Crossref PubMed Scopus (131) Google Scholar, 9Lauwereys M. Arbabi G.M. Desmyter A. Kinne J. Holzer W. De Genst E. Wyns L. Muyldermans S. EMBO J. 1998; 17: 3512-3520Crossref PubMed Scopus (398) Google Scholar, 12Ward E.S. Gussow D. Griffiths A.D. Jones P.T. Winter G. Nature. 1989; 341: 544-546Crossref PubMed Scopus (891) Google Scholar, 13Davies J. Riechmann L. Bio/Technology. 1995; 13: 475-479Crossref PubMed Scopus (136) Google Scholar, 14Arbabi G.M. Desmyter A. Wyns L. Hamers R. Muyldermans S. FEBS Lett. 1997; 414: 521-526Crossref PubMed Scopus (590) Google Scholar, 15Reiter Y. Schuck P. Boyd L.F. Plaksin D. J. Mol. Biol. 1999; 290: 685-698Crossref PubMed Scopus (75) Google Scholar). Smaller size is an advantage in applications requiring tissue penetration and rapid blood clearance. Smaller molecules also offer a tremendous advantage in terms of structural studies (5Davies J. Riechmann L. FEBS Lett. 1994; 339: 285-290Crossref PubMed Scopus (144) Google Scholar, 16Constantine K.L. Goldfarb V. Wittekind M. Anthony J. Ng S.C. Mueller L. Biochemistry. 1992; 31: 5033-5043Crossref PubMed Scopus (25) Google Scholar, 17Constantine K.L. Goldfarb V. Wittekind M. Friedrichs M.S. Anthony J. Ng S.C. Mueller L. J. Biomol. NMR. 1993; 3: 41-54Crossref PubMed Scopus (28) Google Scholar). Phage antibody library construction is much simpler and more efficient with dAbs as compared with Fabs or scFvs. Randomization can be introduced at a much higher percentage of CDR positions without exceeding practical library size. The problem of shuffling original VL-VH pairings is also avoided. Camelid phage dAb libraries constructed from the VHH repertoire of camels immunized with target antigens have performed well (6Decanniere K. Desmyter A. Lauwereys M. Ghahroudi M.A. Muyldermans S. Wyns L. Structure. 1999; 7: 361-370Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 9Lauwereys M. Arbabi G.M. Desmyter A. Kinne J. Holzer W. De Genst E. Wyns L. Muyldermans S. EMBO J. 1998; 17: 3512-3520Crossref PubMed Scopus (398) Google Scholar, 14Arbabi G.M. Desmyter A. Wyns L. Hamers R. Muyldermans S. FEBS Lett. 1997; 414: 521-526Crossref PubMed Scopus (590) Google Scholar). However, in addition to the obvious problems of this approach, the non-human nature of products from these libraries limits their usefulness. Synthetic dAb libraries (13Davies J. Riechmann L. Bio/Technology. 1995; 13: 475-479Crossref PubMed Scopus (136) Google Scholar, 15Reiter Y. Schuck P. Boyd L.F. Plaksin D. J. Mol. Biol. 1999; 290: 685-698Crossref PubMed Scopus (75) Google Scholar), particularly those based on a human VH framework, alleviate these problems. Here we describe the construction of camelized human dAb library that is based on the VH of the human monoclonal antibody BT32/A6 (18Dan M.D. Earley E.M. Griffin M.C. Maiti P.K. Prashar A.K. Yuan X.Y. Friesen A.D. Kaplan H.A. J. Neurosurg. 1995; 82: 475-480Crossref PubMed Scopus (5) Google Scholar). In prior experimental studies with this VH, 2J. Tanha, S. Narang, H. Kaplan, M. Dan, and C. R. MacKenzie, unpublished results. the length of its CDR3 and its solubility properties were found to be remarkable, features which were later determined to be characteristic of camelid heavy chain antibodies (1Hamers-Casterman C. Atarhouch T. Muyldermans S. Robinson G. Hamers C. Songa Baiyana B. Bendahman N. Hamers R. Nature. 1993; 363: 446-448Crossref PubMed Scopus (2205) Google Scholar). To generate the library, the CDR3 was randomized and cysteine residues were introduced at key positions with the expectation that the residues would form the CDR1-CDR3 disulfide bridge found in the camel antibody cAb-Lys3 (3Desmyter A. Transue T.R. Ghahroudi M.A. Thi M.H. Poortmans F. Hamers R. Muyldermans S. Wyns L. Nat. Struct. Biol. 1996; 3: 803-811Crossref PubMed Scopus (417) Google Scholar). The library was evaluated by panning against two monoclonal antibody targets. Wild type dAb (Fig.1) was constructed from BT32/A6 (18Dan M.D. Earley E.M. Griffin M.C. Maiti P.K. Prashar A.K. Yuan X.Y. Friesen A.D. Kaplan H.A. J. Neurosurg. 1995; 82: 475-480Crossref PubMed Scopus (5) Google Scholar). Two camelized versions, BT32/A6.ERG and BT32/A6.ERI (TableI), were constructed by standard protocols (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). BT32/A6.ERG was used as the template in PCR to amplify a shorter fragment using primers 5′-TGTTCAGCTAGCGGATTCACCTTCAGTAGCTATTGTATGCACTGGGTCCGC-3′ (A6VH.33C) containing the NheI site (underlined) and 5′-TGCTGCACAGTAATACACAGCCGT-3′. At the protein level this introduces Cys and two Ala residues at positions 33, 93, and 94, respectively. In camelid VHHs, positions 93 and 94 are predominantly occupied by Ala residues and Cys is frequently found at position 33 (8, 10). The mutated fragment was used as the template in a second PCR using the primers A6VH.33C and 5′-GCCCCAGATATCAAA(A/CNN)9GCA(A/CNN)10TGCTGCACAGTAATA-3′. The second primer results in the randomization of the first 19 residues in CDR3, with the exception of 100e where a Cys is introduced to facilitate the formation of intramolecular disulfide linkage between 33Cys and 100eCys in CDR1 and CDR3, respectively. The amplified fragments were used as templates in a third round of PCR employing primers A6VH.33C and 5′-TGAGGAGACGGTGACCGTTGTCCCTTGG-CCCCAGATATCAAA-3′ to incorporate a 3′ end BstEII site (underlined). The amplified fragments were purified, digested with NheI andBstEII, and ligated toNheI/BstEII-treated pSJF6-BT32/A6VH phagemid. 3S. J. Foote, personal communication. The product was desalted using spin columns and used to transform Escherichia coli strain XL1-Blue. Growth of the library was performed as described by Harrison et al. (20Harrison J.L. Williams S.C. Winter G. Nissim A. Methods Enzymol. 1996; 267: 83-109Crossref PubMed Google Scholar). To sub-clone the library into a phage vector, library phagemid DNA template (180 pmol) and two primers which were complimentary to the 5′ and 3′ ends of the dAb genes, and incorporated flanking ApaLI and NotI restriction sites, were used to PCR amplify the dAb genes. The products were purified, cut with ApaLI and NotI, purified, and ligated to the ApaLI/NotI-digested phage vector fd-tetGIIID (21MacKenzie R. To R. J. Immunol. Methods. 1998; 220: 39-49Crossref PubMed Scopus (31) Google Scholar). Following this, 1.5 μg of the desalted ligated product was mixed with 40 μl of competent E. colistrain TG1 and the cells were transformed by electroporation. Using standard methods, the sizes of the phagemid and phage libraries were determined to be 2.1 × 107 and 6.6 × 107, respectively. Phage were produced and purified as described previously (20Harrison J.L. Williams S.C. Winter G. Nissim A. Methods Enzymol. 1996; 267: 83-109Crossref PubMed Google Scholar). One clone, R3A10 (Table I), was selected from the phage library because of its higher yield of soluble product. Two mutated versions of R3A10, R3A10.G47I and R3A10.G47I/Cys− (Table I), were constructed by standard protocols.Table IVariable residues and solution properties of BT32/A6 VH sDesignationVariable residuesCDR3Solution properties1-aThe solution properties of the various VH molecules are characterized as soluble (s) and structurally folded (f) with broad (b), sharp (s), or partially-sharp (ps) NMR spectra. R3A10.G47I was soluble up to a concentration of 1 mm and R3A10.G47I/Cys− up to 2 mm. The folding properties of the VH molecules were established from two-dimensional NOESY and/or 1H-15N HSQC spectra. Selected resonances in the NMR spectra of R3A10.G47I responded to the addition of DTT up to a concentration of 4 mm DTT beyond which no further spectral changes were detected.334445479293BT32/A61-bThe complete sequence given in Fig.1.AGLYVKDRLKVEYYDSSGYYVSRFGAFDIs/f/bBT32/A6.ERGAERGVKDRLKVEYYDSSGYYVSRFGAFDIs/f/sBT32/A6.ERIAERIVKDRLKVEYYDSSGYYVSRFGAFDIs/f/sR3A10.G47ICERIAALNLMEWTGDGCRLSLDARQRFDIs/f/psR3A10.G47I/Cys−AERIAALNLMEWTGDGSRLSLDARQRFDIs/f/sM2R2–1.Cys−AERGAAVQYGKHRRGSSIEVHPEYKDFDIs/f/s1-a The solution properties of the various VH molecules are characterized as soluble (s) and structurally folded (f) with broad (b), sharp (s), or partially-sharp (ps) NMR spectra. R3A10.G47I was soluble up to a concentration of 1 mm and R3A10.G47I/Cys− up to 2 mm. The folding properties of the VH molecules were established from two-dimensional NOESY and/or 1H-15N HSQC spectra. Selected resonances in the NMR spectra of R3A10.G47I responded to the addition of DTT up to a concentration of 4 mm DTT beyond which no further spectral changes were detected.1-b The complete sequence given in Fig.1. Open table in a new tab The library was panned against 3B1, a scFv specific for a bacterial polysaccharide (22Deng S.J. MacKenzie C.R. Sadowska J. Michniewicz J. Young N.M. Bundle D.R. Narang S.A. J. Biol. Chem. 1994; 269: 9533-9538Abstract Full Text PDF PubMed Google Scholar), and M2 anti-FLAG IgG (Sigma). Immuno MaxiSorp™ (Nunc) microtiter plate wells were coated overnight by adding 150 μl of 100 μg/ml antigen in PBS. Plates were rinsed three times with PBS, blocked with 400 μl of PBS, 2% (w/v) skim milk (2% MPBS) at 37 °C for 2 h and rinsed as above. Phage (1012transducing units in 2% MPBS) were added and incubated at room temperature for 1.5 h after which unbound phage were removed. The wells were rinsed 10 times with PBS, 0.1% (v/v) Tween 20 and then 10 times with PBS. Bound phage were eluted by adding 200 μl of freshly prepared 100 mm triethylamine and neutralized with 100 μl of 1 m Tris-HCl, pH 7.4. Exponentially growing TG1 cultures (10 ml) were infected with 150 μl of eluted phage at 37 °C for 30 min. Serial dilutions of infected cells were used to determine the titers of eluted phage as described above. The remainders of the infected cells were pelleted, re-suspended in 900 μl of 2 × YT (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), mixed in 300-μl aliquots with 3 ml of 0.7% agarose in LB (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) at 50 °C and the phage propagated on plates overnight at 37 °C. Phage were purified, the titers were determined, and a total of 1011 transducing units of phage were used for further rounds of selection. Clones selected for expression were transferred to an expression vector that added C-terminal c-Myc and His5 tags and were grown as described previously (23Anand N.N. Mandal S. MacKenzie C.R. Sadowska J. Sigurskjold B. Young N.M. Bundle D.R. Narang S.A. J. Biol. Chem. 1991; 266: 21874-21879Abstract Full Text PDF PubMed Google Scholar). The dAbs were purified from periplasmic fractions (24Anand N.N. Dubuc G. Phipps J. MacKenzie C.R. Sadowska J. Young N.M. Bundle D.R. Narang S.A. Gene ( Amst .). 1991; 100: 39-44Crossref PubMed Scopus (25) Google Scholar) by immobilized metal affinity chromatography (25MacKenzie C.R. Sharma V. Brummell D. Bilous D. Dubuc G. Sadowska J. Young N.M. Bundle D.R. Narang S.A. Bio/Technology. 1994; 12: 390-395Crossref PubMed Scopus (18) Google Scholar) except that the starting buffer was 10 mm HEPES, 10 mm imidazole, 500 mm NaCl, pH 7.0. To detect the presence of dimer/multimer dAb in protein preparations, gel filtration chromatography was performed using Superdex 75 (Amersham Phamacia Biotech) (26Deng S.J. MacKenzie C.R. Hirama T. Brousseau R. Lowary T.L. Young N.M. Bundle D.R. Narang S.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4992-4996Crossref PubMed Scopus (53) Google Scholar). Cold acetone (5 × volume) was added to 200 μg of dAb solution and the contents were mixed and then centrifuged in a microcentrifuge at maximum speed at 4 °C for 10 min. The pellet was dissolved in 500 μl of 6 mguanidine hydrochloride and 55 μl of 1 m Tris buffer, pH 8.0, were added. Subsequently, a 25 m excess of DTT, relative to Cys residues, was added and the mixture was incubated at room temperature for 30 min. A 2.2 m excess, relative to DTT, of freshly made iodoacetic acid was added and the reaction was incubated as described above. The alkylated product was then concentrated in 50 μl of distilled water using an Ultrafree-MC 10,000 NMWL filter unit (Millipore). Control experiments were identical except that DTT was replaced with water. The molecular sizes of native and iodoacetate-treated dAbs were determined by infusion-electrospray ionization mass spectrometry (positive ion mode) using Quatro triple quadrupole mass spectrometer (Micromass). Following panning, phage clones were screened by standard enzyme-linked immunosorbent assay procedures using a horseradish peroxidase/anti-M13 monoclonal antibody conjugate (Amersham Pharmacia Biotech) as the detection reagent. Surface plasmon resonance was performed using a BIACORE Upgrade (Biacore AB) (27Jönsson U. Fågerstam L. Ivarsson B. Johnsson B. Karlsson R. Lundh K. Löfås S. Persson B. Roos H. Rönnberg I. Sjölander S. Sternberg E. Ståhlberg R. Urbaniczky C. Östlin H. Malmqvist M. BioTechniques. 1991; 11: 620-627PubMed Google Scholar). Approximately 14,000 resonance units of anti-FLAG M2 IgG or control IgG were immobilized on research grade CM5 sensor chips by amine coupling. Single-domain antibodies were passed over the sensor chips surfaces in 10 mm HEPES buffer, pH 7.4, 150 mm NaCl, 3.4 mm EDTA, 0.005% P-20 (Biacore AB) at 25 °C and at a flow rate of 5 μl/min. To assess the effect of the CDR1-CDR3 disulfide bridge on dAb binding to M2, dAbs were incubated with DTT prior to injection and the above buffer was supplemented with an appropriate amount of DTT. The influence of the disulfide bridge on binding was also investigated by construction and characterization of a mutant, M2R2–1.Cys− (Table I), that lacks the bridge. Surfaces were regenerated with 10 mm HCl. Sensorgram data were analyzed using the BIAevaluation 3.0 software package (Biacore AB). Isotopically labeled proteins were prepared from cells grown in 15N- or15N/13C-enriched Bioexpress medium (Cambridge Isotopes Laboratory). Six-ml amounts of LB (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) containing 100 μg/ml ampicillin were inoculated with single colonies and incubated at 37 °C and 260 rpm until an A 600 of ∼5 was reached. The cells were centrifuged and re-suspended in 3 ml of sterile PBS. Aliquots were transferred to 25 ml of Bioexpress, 100 μg/ml ampicillin in 125-ml Erlenmeyer flasks, to give anA 600 = 0.06, and incubated at 37 °C at 200 rpm for 9–10 h. The dAbs were purified as described above and dialyzed extensively in 10 mm sodium phosphate, 150 mmNaCl, 0.5 mm EDTA, at either pH 5.5 or 6.8. NMR samples were prepared by concentrating the protein to ∼1 mm using a YM10 membrane (Amicon). NMR experiments were performed at 298 and/or 308 K on a Bruker Avance-800 spectrometer equipped with pulse field gradient accessories. Two-dimensional1H-15N HSQC spectra (28Bodenhausen G. Ruben D.J. Chem. Phys. Lett. 1980; 69: 185-188Crossref Scopus (2435) Google Scholar) were acquired using solvent suppression via the WATERGATE method implemented through the 3-9-19 pulse train (29Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3539) Google Scholar, 30Sklenar V. Piotto M. Leppik R. Saudek V. J. Magn. Reson. Sect. A. 1993; 102: 241-245Crossref Scopus (1116) Google Scholar). Triple-resonance experiments (Ref. 31Slatter M. Schleucher J. Griesinger C. Progr. NMR Spectrosc. 1999; 34: 93-158Abstract Full Text Full Text PDF Scopus (1399) Google Scholar and references therein) included HNCα, HNCαCβ, and CβCα(CO)NH for both R3A10.G47I and M2R2–1.Cys−. The NMR data were processed using NMRPipe/NMRDraw (32Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11638) Google Scholar) and analyzed by the use of the NMRView software program (33Johnson B.A. Blevins R.A. J. Chem. Phys. 1994; 29: 1012-1014Crossref Scopus (1096) Google Scholar). Sequence-specific assignments of the backbone NH,15N, and13Cα/13Cβresonances were achieved only for the residues having strong1H-15N HSQC cross-peaks through a combined analysis of the HNCα, HNCαCβ, and CβCα(CO)NH spectra. Chemical-shift values were referenced internally to the proton resonance of sodium 2,2-dimethyl-2-silapentane-5-sulfonate and indirectly for15N and 13C assuming γ15N/γ1H = 0.101329118 and γ13C/γ1H = 0.251449530 (34Wishart D.S. Bigam C.G. Yao J. Abildgaard F. Dyson H.J. Oldfield E. Markley J.L. Sykes B.D. J. Biomol. NMR. 1995; 6: 135-140Crossref PubMed Scopus (2083) Google Scholar). The library was panned, in both formats, against 3B1 scFv. With the phagemid format, panning failed to enrich for binders and PCR analysis of clones selected at different stages of the panning process revealed almost universal deletion of the dAb inserts. This is probably the result of monovalent display since multivalent display with the phage vector format gave seven different dAbs that bound to 3B1 (Table II). As shown in Fig.2, these dAbs bound to the target antigen, 3B1, in enzyme-linked immunosorbent assay experiments with no detectable binding to control BSA. In each instance, the consensus sequence was present at the extreme C-terminal end of CDR3 (TableII).Table IIThe CDR3 sequences of dAbs isolated by panning against 3B1 scFvdAbCDR3 sequence3B1R3–1PITGGAPRAVCKHAKAWFLPFDI3B1R2–3SSQPRVTSSPCVASKSWFLPFDI3B1R2–2PTTGIRGEKDCTPKKMWRLPFDI3B1R2–4RDPSVTDTGCCTPRWQAWLPFDI3B1R3–3PGEPPEASAPCLRHRVGWLPFDI3B1R3–15KTVKMRDDEVCTKRTNWLLPFDI3B1R3–19PGNVASQQNLCGLRATRWLPFDIThe consensus sequence is shown in bold. Open table in a new tab The consensus sequence is shown in bold. In the phage vector format, the library was also panned against M2 IgG, an antibody raised against the FLAG peptide DYKDDDDK (35Knappik A. Plückthun A. BioTechniques. 1994; 17: 754-761PubMed Google Scholar) and shown to recognize the consensus sequence XYKXXD (36Miceli R.M. Degraaf M.E. Fischer H.D. J. Immunol. Methods. 1994; 167: 279-287Crossref PubMed Scopus (50) Google Scholar). Twenty-four different dAbs with the FLAG consensus sequence were identified by sequencing of clones randomly selected after 3 rounds of panning (Table III). No consensus sequence other than XYKXXD could be identified. Interestingly, like the 3B1 binders, all the FLAG consensus sequences occurred in the C-terminal half of CDR3 and, with two exceptions, occupied identical positions. To ascertain if this observation was related to the presence of CDR1-CDR3 disulfide linkage, the reduced version of the same library was also panned against M2 IgG. Panning, including the washing steps, was performed in the presence 1 μm, 1 mm, 10 mm, and 100 mm DTT. In 13/13, 6/8, 8/8, and 10/10 instances for dAbs obtained by panning in the presence of 1 μm, 1 mm, 10 mm, and 100 mm DTT, respectively, the FLAG consensus sequence was located C terminus of CDR3 (data not shown).Table IIIThe CDR3 sequences of dAbs isolated by panning against M2 IgG in the absence of DTTVQYGKHRRGSCIEVHPEYKDFDI3-aM2R2–1.PRPARTGHKTCFVRPKNYKDFDINPPKPGAQARCVTTVKDYKEFDI3-bM2R2–2.AEAHSQLPPRCRRKTDEYKIFDIAAIQTETARWCDRHPVSYKMFDI3-cM2R2–4.SHKTSQPVRNCSATDNSYKLFDIQTETQPLYNDCILRQAGYKWFDI3-dM2R2–5.TMGTLHSPHECMKSLVTYKNFDIMHTLQHYRNLCSYQLADYKHFDIGRYFQSKITSCENNDRDYKLFDIGLSGSRPNEQCDYKTGDHVQFDI3-eM2R2–10.ELGWRPRVQACHYSRNDYKYFDILSGQNYTKTRCLVMQNDYKMFDI3-fM2R2–13.KDVTRTNTVSCSKDRQDYKMFDITAEPALSPQACMTKERQYKDFDIYSATAKWRDKCYEKSRDYKMFDIETYMYTRGKYCRALSADYKLFDIYEIVPFIASRCVIERADYKLFDIESKASRTADQCSGPTPGYKNFDIADAPNRQKERCVVAVHGYKRFDIGSQAIKNLSECLVRSDDYKKFDI3-gM2R3–4.NEEKFSVYSECELYLPTYKMFDIGRYFQSKITSCENNDRDYKLFDIIWEGEKHYAECVTGTYKQPDFDIThe FLAG consensus sequence is shown in bold.3-a M2R2–1.3-b M2R2–2.3-c M2R2–4.3-d M2R2–5.3-e M2R2–10.3-f M2R2–13.3-g M2R3–4. Open table in a new tab The FLAG consensus sequence is shown in bold. Size exclusion chromatography of the non-camelized product revealed three components corresponding to monomer, dimer, and higher oligomer on the basis of their elution volumes. The monomer peak eluted unusually late suggesting that the dAb interacted nonspecifically with the gel matrix. This is a property of human and murine-derived dAbs that is not unusual and which has been documented previously (5Davies J. Riechmann L. FEBS Lett. 1994; 339: 285-290Crossref PubMed Scopus (144) Google Scholar, 12Ward E.S. Gussow D. Griffiths A.D. Jones P.T. Winter G. Nature. 1989; 341: 544-546Crossref PubMed Scopus (891) Google Scholar). Camelized BT32/A6 dAbs gave products that were exclusively monomer and which eluted at the expected volume. Formation of the CDR1-CDR3 disulfide linkage was verified by mass spectrometric analysis of alkylated dAbs isolated from the library. The mass spectrum obtained for M2R2-5 after treatment with iodoacetic acid had a major peak with a mass of 16,545.19 ± 2.4 Da (Fig. 3 A) which corresponds to the mass of the untreated protein. In contrast, the major peak observed following reduction and alkylation had a mass of 16,783.29 ± 2.94 Da (Fig.3 B). The mass increase of 237.70 ± 2.94 Da indicates alkylation of 4 Cys residues. Theoretically, alkylation of four cysteines should give a mass increase of 236.16 Da. The 2 additional cysteine residues are the conserved ones at positions 22 and 92. In Fig. 3 A a minor peak corresponding to the M r of a dAb alkylated at one position may have resulted from a side reaction involving alkylation of another amino acid such as histidine. Identical experiments were performed for eight additional anti-M2 dAbs as well as R3A10.G47I and in all instances formation of the CDR1-CDR3 disulfide bridge was confirmed. This demonstrates that disulfide linkage formation is independent of the CDR3 sequence and is likely a function of the overall fold of the protein. The binding to M2 IgG of six of the dAbs listed in Table III (M2R2-1, M2R2-2, M2R2-4, M2R2-10, M2R2-13, and M2R3-4) was investigated by surface plasmon resonance. The binding data fit poorly to a 1:1 interaction model in all instances, making the derivation of kinetic and affinity constants impossible. However, when binding studies were conducted in the presence of DTT it was observed that the amount of binding increased significantly, particularly for M2R2-2. Furthermore, data collected in the pr" @default.
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• W2170269585 title "Optimal Design Features of Camelized Human Single-domain Antibody Libraries" @default.
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