Optimal Design Features of Camelized Human Single-domain Antibody Libraries

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 The second results in the randomization of the 19 residues in CDR3, with the of a Cys is introduced to the formation of disulfide linkage between and in CDR1 and CDR3, respectively. The were used as in a of PCR primers A6VH.33C and to a site The were with NheI and J. The was using and used to of the library was performed as by S.C. Winter G. A. 1996; PubMed Google Scholar). To the library into a phage library template and two primers which were to the and of the dAb and and were used to PCR amplify the dAb The products were with and and to the phage R. To R. J. Immunol. 1998; PubMed Scopus Google Scholar). of the was with of E. and the were by standard the of the and phage libraries were determined to be and respectively. Phage were and as S.C. Winter G. A. 1996; PubMed Google Scholar). One was from the phage library because of its higher of soluble Two mutated of and were constructed by standard residues and properties of BT32/A6 VH properties of the VH molecules are as soluble and with or NMR was soluble to a of and to The properties of the VH molecules were from HSQC in the NMR of to the addition of to a of which changes were complete sequence in The properties of the VH molecules are as soluble and with or NMR was soluble to a of and to The properties of the VH molecules were from HSQC in the NMR of to the addition of to a of which changes were The complete sequence in in a The library was panned against a for a J. J. J. Biol. 1994; Full Text PDF PubMed Google Scholar), and were by of antigen in were with with of at for and as Phage in were and at for which phage were The were with and with phage were by of and with of were with of phage at for of were used to the of phage as The of the were in of (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), in with of in (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google at and the phage on at Phage were the were and a of of phage were used for of for were to an that C-terminal and and were as S. J. B. J. Biol. Full Text PDF PubMed Google Scholar). The dAbs were from G. J. J. PubMed Scopus (25) Google by V. D. D. G. J. Bio/Technology. 1994; PubMed Scopus Google that the was To the of dAb in protein was performed using T. R. S. A. 1995; PubMed Scopus Google Scholar). Cold was to of dAb and the were and in a at at for The was in of and of were a of relative to Cys was and the was at for A relative to of made was and the was as The was in of using an experiments were that was replaced with The of and dAbs were determined by using phage were by standard using a monoclonal antibody as the plasmon resonance was performed using a L. B. B. R. K. S. B. H. S. E. R. C. H. M. Google Scholar). resonance of or were on by antibodies were the in at and at a of To the effect of the CDR1-CDR3 disulfide bridge on dAb to dAbs were with prior to and the was with an of The of the disulfide bridge on was also by construction and of a that the were with data were using the were from in of (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google containing were with single and at and an A of was The were and in of were to of in to and at at for The dAbs were as and in at either or NMR were by the protein to using a NMR experiments were performed at on a with HSQC G. Lett. Scopus Google were using the the M. V. V. J. Biomol. NMR. 1992; PubMed Scopus Google Scholar, V. M. R. V. J. A. 1993; Scopus Google Scholar). experiments M. J. C. NMR 1999; 34: Full Text Full Text PDF Scopus Google and and for and The NMR data were using F. S. G. J. A. J. Biomol. NMR. 1995; PubMed Scopus Google and by the use of the J. 1994; Scopus Google Scholar). of the were for the residues HSQC a of the and were to the resonance of and and and J. F. E. J. Biomol. NMR. 1995; PubMed Scopus Google Scholar). The library was in against the panning to for and PCR of at of the panning of the dAb This is the result of with the phage dAbs that to As in these dAbs to the target in experiments with to In the sequence was at the C-terminal of CDR3 CDR3 sequences of dAbs by panning against sequence is in in a The sequence is in In the phage the library was also panned against an antibody against the A. A. 1994; 17: Google and to the sequence J. Immunol. 1994; PubMed Scopus Google Scholar). dAbs with the sequence were by of of panning sequence other be the the sequences in the C-terminal of CDR3 with two occupied positions. To this was to the of CDR1-CDR3 disulfide the of the library was also panned against the was performed in the and In and for dAbs by panning in the of and the sequence was of CDR3 not CDR3 sequences of dAbs by panning against in the absence of sequence is in in a The sequence is in of the to and higher on the basis of their The that the dAb with the This is a of human and dAbs that is not and which has been (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). BT32/A6 dAbs products that were and which at the of the CDR1-CDR3 disulfide linkage was by spectrometric of dAbs from the The for with a with a of which to the of the In the and a of The increase of of Cys residues. of should a increase of The cysteine residues are the conserved at positions and In A a to the of a dAb at position have from a of as experiments were performed for dAbs as well as and in formation of the CDR1-CDR3 disulfide bridge was This that disulfide linkage formation is of the CDR3 sequence and is a of the fold of the The to of of the dAbs in and was by surface plasmon The data to a in the of and However, studies were in the of was that the of particularly for Furthermore, data in the of much to a the data for the of a the CDR1-CDR3 disulfide bridge to well to the of the data an of and a of these the of the was determined to be of at of of soluble were for and camelized of BT32/A6 The BT32/A6 VH fragment was soluble in NMR in and not of in VH molecules with solubility and NMR quality However, with the VH BT32/A6.ERI with the the NMR data quality was not by the of which was found to be for the properties of the human VH fragment K.B. Ghahroudi M.A. Wyns L. Muyldermans S. Mol. Immunol. 1997; 34: 1121-1131Crossref PubMed Scopus (250) Google Scholar, L. J. J. Biomol. NMR. 1995; PubMed Scopus (28) Google Scholar, L. J. Mol. Biol. 1996; PubMed Scopus Google Scholar). the other the and HSQC of to the addition of with a of not The changes in the of are by the of the disulfide between CDR1 and CDR3 In the solubility and properties the HSQC of by the of were well in the also HSQC were and to residues as and from the CDR1 loop In other of the CDR1-CDR3 disulfide bridge in this fragment to the conformational to resonance or by the absence of this disulfide not with the formation of an structure as a of the HSQC at positions in the and the HSQC of a VH fragment derived from the of panning the phage library using antibody as the those from the chain HSQC were for and and of these HSQC were as in in Very CDR1 and residues were at partially for and the CDR3 residues in molecules were in the HSQC the CDR1-CDR3 disulfide to the conformational of the CDR1 the CDR3 loop in and in NMR HSQC the size of antigen-binding to a single immunoglobulin domain has been of the of antibody the However, of soluble in and solubility problems have of The 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 for of single domain antibodies, the of features of these antibodies into human VH of human VHs is a for the of antigen-binding that should be useful for in However, the camelized antibodies in the (5Davies J. Riechmann L. FEBS Lett. 1994; 339: 285-290Crossref PubMed Scopus (144) Google Scholar, 13Davies J. Riechmann L. Bio/Technology. 1995; 13: 475-479Crossref PubMed Scopus (136) Google Scholar, L. J. J. Biomol. NMR. 1995; PubMed Scopus (28) Google Scholar, L. J. Mol. Biol. 1996; PubMed Scopus Google have properties relative to their these properties are and (5Davies J. Riechmann L. FEBS Lett. 1994; 339: 285-290Crossref PubMed Scopus (144) Google camelized a human VH by introducing and However, the yields of soluble camelized were and in to the yields and for NMR studies they for a of the (5Davies J. Riechmann L. FEBS Lett. 1994; 339: 285-290Crossref PubMed Scopus (144) Google Scholar, 13Davies J. Riechmann L. Bio/Technology. 1995; 13: 475-479Crossref PubMed Scopus (136) Google Scholar). This in yields of to which is an of the yields for camelized A NMR structure of a human VH camelized in this has been L. J. Mol. Biol. 1996; PubMed Scopus Google in to and the addition of to the NMR data was a In we were to high quality NMR data in the absence of that BT32/A6 VH is an template for the of camelized The NMR and size indicated that the camelized BT32/A6 VH molecules properties that were from those of dAbs derived from heavy chain In addition to the introduced at positions and is obvious that features in the type to the solubility of camelized BT32/A6 First, has been that a murine VH as a at without the for Y. Schuck P. Boyd L.F. Plaksin D. J. Mol. Biol. 1999; 290: 685-698Crossref PubMed Scopus (75) Google of a VH to S. T. D. J. Mol. Biol. 2000; PubMed Scopus Google Scholar). Second, experiments have in addition to residues and at positions the VHH surface also to solubility (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). CDR3s that in BT32/A6 VH are to to VHH solubility (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, 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). As a template for camelized VH library the CDR3 of BT32/A6 the of introducing a CDR1-CDR3 disulfide bridge and a antigen-binding features which increase library of the disulfide was for sequences and of the two not have a on the of soluble dAbs the of the disulfide imposed a constraint that optimal with The data indicated that the of dAbs containing the introduced disulfide to was with of the in data that well to a This was with NMR data that of the disulfide bridge CDR1 and and structural without in the fold of the The structural of the disulfide form was not to disulfide and of two with and without the would to be desirable. The of the bridge would be in this is a feature of heavy chain antibodies and introduces structural into the Synthetic VH libraries to have been based on randomization of CDR3s of or (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 the of camelid CDR3 VHs with longer as the level of for of antibodies for a of containing shorter and partially randomized as the C-terminal be constructed and with the library to increase While of CDR1 and randomization library construction of libraries based on CDR3 randomization an attractive because of design In randomization the problem of library that practical library constructed on a single offer the advantage of of antibodies in that CDR1 and libraries can be using single data demonstrated for the antigens at a region of the CDR3 was for antigen The structural basis for this not be determined the CDR3 was of or not the CDR1-CDR3 disulfide bridge was was to that the C-terminal of the CDR3 was in antigen the crystal structure of a camel dAb with a CDR3 length showed that the of the CDR3 was in (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). on this been that more efficient camelized dAb libraries can be constructed in which the region of CDR3 is randomized the C-terminal that using camelid dAbs as a to camelized libraries can be is to to smaller libraries in to the antigen contact area of libraries can be constructed by those in CDR3 which are in antigen As more and more on the properties of and camelized is that are that to the solubility and nature of The that BT32/A6 VH is a useful for these BT32/A6 soluble and the to with structural of the camelized VHs is and is that the data useful for the design of human single domain antibodies that a of non-human particularly as We for for and for the and We and for with the and and for We the of

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.