Optimal Design Features of Camelized Human Single-domain Antibody Libraries*

Jamshid TanhaDagger , Ping Xu§, Zhigang Chen§, Feng Ni§, Howard Kaplan, Saran A. NarangDagger , and C. Roger MacKenzieDagger ||

From the Dagger  Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada, the § Biotechnology Research Institute, National Research Council of Canada, Montréal Québec H4P 2R2, Canada, and  Viventia Biotech Inc., Winnipeg, Manitoba R3T 3Z1, Canada

Received for publication, January 26, 2001, and in revised form, April 25, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have constructed a human VH library 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 the 1H-15N HSQC spectra, indicated that the VH sequence for library construction was a good choice. These results should be useful in the design of VH libraries with optimal features.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heavy chain antibodies, found in camelids (1, 2), 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 (3, 4). This explanation is supported by experiments in which an insoluble human VH was made soluble by introducing these substitutions (5). Second, in the solved structures of two camel dAbs, the CDR3s fold back on the VH surface, masking a significant surface area of the VL interface (3-6).

Two other features of VHHs are noteworthy. One is the frequent occurrence of cysteine residues in CDR1 and CDR3 (7-10). 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 (3, 11). 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 (3). A second feature is the longer average length of the VHH CDR3, relative to human or murine VHs (8). 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 (3).

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 (4, 9, 12-15). 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 (5, 16, 17).

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 (6, 9, 14). 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 (13, 15), 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 (18). In prior experimental studies with this VH,2 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 (1). 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 (3). The library was evaluated by panning against two monoclonal antibody targets.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Library Construction and Panning-- Wild type dAb (Fig. 1) was constructed from BT32/A6 (18). Two camelized versions, BT32/A6.ERG and BT32/A6.ERI (Table I), were constructed by standard protocols (19). 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 and BstEII, and ligated to NheI/BstEII-treated pSJF6-BT32/A6VH phagemid.3 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. (20). 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 (21). Following this, 1.5 µg of the desalted ligated product was mixed with 40 µl of competent E. coli strain 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 (20). 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.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 1.   Sequence of BT32/A6 dAb. The locations of the CDRs and the NheI and BstEII sites are bold and underlined. The Kabat numbering system (37) is used.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Variable residues and solution properties of BT32/A6 VHs

The library was panned against 3B1, a scFv specific for a bacterial polysaccharide (22), and M2 anti-FLAG IgG (Sigma). Immuno MaxiSorpTM (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 (1012 transducing 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 (19), mixed in 300-µl aliquots with 3 ml of 0.7% agarose in LB (19) 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 (23). The dAbs were purified from periplasmic fractions (24) by immobilized metal affinity chromatography (25) 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) (26).

Alkylation Reactions-- 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 M guanidine 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).

Binding Studies-- 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) (27). 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).

NMR Sample Preparation-- Isotopically labeled proteins were prepared from cells grown in 15N- or 15N/13C-enriched Bioexpress medium (Cambridge Isotopes Laboratory). Six-ml amounts of LB (19) containing 100 µg/ml ampicillin were inoculated with single colonies and incubated at 37 °C and 260 rpm until an A600 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 an A600 = 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 mM NaCl, 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 Spectroscopy-- NMR experiments were performed at 298 and/or 308 K on a Bruker Avance-800 spectrometer equipped with pulse field gradient accessories. Two-dimensional 1H-15N HSQC spectra (28) were acquired using solvent suppression via the WATERGATE method implemented through the 3-9-19 pulse train (29, 30). Triple-resonance experiments (Ref. 31 and references therein) included HNCalpha , HNCalpha Cbeta , and Cbeta Calpha (CO)NH for both R3A10.G47I and M2R2-1.Cys-. The NMR data were processed using NMRPipe/NMRDraw (32) and analyzed by the use of the NMRView software program (33). Sequence-specific assignments of the backbone NH, 15N, and 13Calpha /13Cbeta resonances were achieved only for the residues having strong 1H-15N HSQC cross-peaks through a combined analysis of the HNCalpha , HNCalpha Cbeta , and Cbeta Calpha (CO)NH spectra. Chemical-shift values were referenced internally to the proton resonance of sodium 2,2-dimethyl-2-silapentane-5-sulfonate and indirectly for 15N and 13C assuming gamma 15N/gamma 1H = 0.101329118 and gamma 13C/gamma 1H = 0.251449530 (34).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Panning-- 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 (Table II).

                              
View this table:
[in this window]
[in a new window]
 
Table II
The CDR3 sequences of dAbs isolated by panning against 3B1 scFv
The consensus sequence is shown in bold.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Specificity of the dAbs listed in Table II for 3B1 scFv. Phage displayed dAbs were tested in an enzyme-linked immunosorbent assay format using bovine serum albumin-coated wells and the anti-M2 dAb M2R2 as controls.

In the phage vector format, the library was also panned against M2 IgG, an antibody raised against the FLAG peptide DYKDDDDK (35) and shown to recognize the consensus sequence XYKXXD (36). 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).

                              
View this table:
[in this window]
[in a new window]
 
Table III
The CDR3 sequences of dAbs isolated by panning against M2 IgG in the absence of DTT
The FLAG consensus sequence is shown in bold.

Size Exclusion Chromatography-- 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 (5, 12). Camelized BT32/A6 dAbs gave products that were exclusively monomer and which eluted at the expected volume.

Verification of CDR1-CDR3 Disulfide Linkage Formation-- 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. 3A) 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. 3B). 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. 3A a minor peak corresponding to the Mr 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.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 3.   Deconvoluted molecular mass profiles obtained by mass spectrometry of M2R2-5 after treatment with iodoacetic acid alone (A) and DTT (B) followed by iodoacetic acid. The minor peak referred to in the text is indicated by its molecular mass.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   1H-15N NMR HSQC spectra of R3A10.G47I/Cys- (A) and M2R2-1.Cys- (B), both with CDR1-CDR3 disulfide bridge deletion (Table I). The spectrum for R3A10.G47I/Cys- was acquired at pH 6.8 and a temperature of 308 K and that for M2R2-1.Cys- at pH 6.8 and a temperature of 298 K. Cross-peaks from the CDR1 and CDR2 loops are boxed and labeled by italicized letters.

Binding Kinetics-- 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 presence of DTT fit much better to a 1:1 interaction model. Similarly, the data for the binding of a M2R2-1 mutant lacking the CDR1-CDR3 disulfide bridge to immobilized M2 IgG fit reasonably well to the simple interaction model. Global analysis of the data gave an association rate constant of 340 M-1 s-1 and a dissociation rate constant of 3.4 × 10-4 s-1. From these rate constants the dissociation constant of the interaction was determined to be 1 × 10-6 M.

NMR Studies-- Yields of at least 10 mg/liter of bacterial culture of soluble product were obtained for native and camelized forms of BT32/A6 dAb, facilitating labeling studies. The BT32/A6 VH fragment (BT32/A6, Table I) was reasonably soluble in aqueous solution but exhibited broad NMR signals in both one-dimensional proton and two-dimensional NOESY spectra (data not shown), indicating partial aggregation. Various schemes of camelization resulted in variant VH molecules with significantly enhanced solubility and improved NMR spectral quality (Table I). However, with the VH fragments studied here, including BT32/A6.ERI with the Y47I substitution, the NMR data quality was not improved by the presence of CHAPS detergent, which was found to be essential for the improved solution properties of the human VH fragment studied previously (10, 38, 39). On the other hand, both the proton and HSQC spectra of R3A10.G47I responded to the addition of DTT with a sharpening of some selected resonances (spectra not shown). The localized spectral changes in the presence of DTT are very likely induced by the selective reduction of the disulfide bond between CDR1 and CDR3 (see next section).

In dramatic contrast, the R3A10.G47I/Cys- VH fragment had significantly enhanced solubility and dramatically improved spectral properties (Table I). Specifically, all the HSQC peaks of R3A10.G47I influenced by the presence of DTT were well resolved in the R3A10.G47I/Cys- spectrum; also HSQC peaks were detected and assigned to residues such as Ser31, Tyr32, and Ala33 from the CDR1 loop (Figs. 1 and 4A). In other words, removal of the CDR1-CDR3 disulfide bridge in this fragment appears to eliminate the conformational heterogeneity leading to resonance doubling or broadening experienced by R3A10.G47I. Also, the absence of this disulfide bond does not interfere with the formation of an intact tertiary structure as a majority of the HSQC peaks remained at similar positions in both the R3A10.G47I and R3A10.G47I/Cys- molecules. Fig. 4B shows the HSQC spectrum of M2R2-1.Cys-, a VH fragment derived from the panel of binding hits after panning the phage library using M2 antibody as the "antigen" (Table III). Excluding those from the side chain amides, ~120 HSQC cross-peaks were observed for both R3A10.G47I/Cys- and M2R2-1.Cys- and most (>90%) of these HSQC peaks were assigned, as shown in part in Fig. 4. Very importantly, both CDR1 and CDR2 residues were at least partially assigned for R3A10.G47I/Cys- and M2R2-1.Cys- while the CDR3 residues in both molecules were still missing in the HSQC spectra. Therefore, the engineered CDR1-CDR3 disulfide bond appears to affect the conformational behavior of the CDR1 residues, but still leaves the large CDR3 loop unstructured in solution and mostly undetectable in NMR HSQC experiments.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Minimizing the size of antigen-binding proteins to a single immunoglobulin domain has been one of the primary goals of antibody engineering over the past decade. However, low levels of soluble expression in Escherichia coli and solubility problems have hampered development of such molecules. The discovery of camelid heavy chain antibodies (1) opened up new opportunities for development of single domain antibodies, including the incorporation of features of these antibodies into human VH frameworks. Camelization of human VHs is a promising technology for the generation of small antigen-binding fragments that should be useful for therapeutic purposes in humans. However, while the camelized antibodies described in the literature (5, 13, 38, 39) have tremendously improved physical properties relative to their non-camelized counterparts, these properties are still less than ideal.

Davies and Reichmann (5) camelized a human VH by introducing G44E, L45R, and W47G mutations. However, the yields in E. coli of soluble camelized product were low (typically less than 1 mg/l) and in order to obtain the yields and stability required for NMR studies they opted for a W47I mutation instead of the W47G mutation (5, 13). This resulted in yields of up to 5 mg/liter, which is an order of magnitude lower than the yields reported here for camelized BT32/A6. A NMR structure of a human VH camelized in this manner has been described (39) but, in order to reduce aggregation and achieve sufficient solubility, the addition of CHAPS detergent to the sample during NMR data collection was a requirement. In contrast, we were able to collect high quality NMR data in the absence of detergent, establishing that BT32/A6 VH is an ideal template for the generation of camelized dAbs. The NMR spectra and size exclusion chromatograms indicated that the camelized BT32/A6 VH molecules had properties that were indistinguishable from those of dAbs derived from natural heavy chain antibodies.

In addition to the mutations introduced at positions 44, 45, and 47, it is obvious that some features inherent in the wild type VH contribute to the solubility of camelized BT32/A6 VH. Several observations support this. First, it has been observed that a murine VH existed as a monomer at millimolar concentrations without the need for camelization (15) while camelization of a different VH failed to eliminate aggregation (40). Second, x-ray crystallographic experiments have shown that, in addition to residues 44, 45, and 47, several others at positions scattered throughout the VHH surface also contribute to solubility (4). Third, long CDR3s like that present in BT32/A6 VH are known to contribute to VHH solubility (3, 6).

As a template for camelized VH library construction, the unusually long CDR3 of BT32/A6 offers the option of introducing a CDR1-CDR3 disulfide bridge and provides a larger antigen-binding surface, features which increase library diversity. Formation of the disulfide bond was confirmed for several sequences and introduction of the two cysteines did not have a negative impact on the yield of soluble product. For anti-M2 dAbs the presence of the disulfide imposed a constraint that prevented optimal interaction with M2. The SPR data indicated that the binding of dAbs containing the introduced disulfide bond to M2 was heterogeneous with reduction of the bond resulting in binding data that fit well to a 1:1 interaction model. This was consistent with NMR data showing that elimination of the R3A10 disulfide bridge changed CDR1 and CDR2 conformation and eliminated structural heterogeneity without any change in the overall fold of the protein. The structural heterogeneity of the disulfide form was apparently not attributable to incomplete disulfide bond formation. Construction and pooling of two libraries, one with and one without the bridge, would appear to be desirable. The presence of the bridge would probably be desirable in some instances since this is a common feature of heavy chain antibodies and it introduces structural diversity into the library.

Synthetic VH libraries described to date have been based on randomization of CDR3s comprised of 12 amino acids or less (13, 15) and, therefore, covered only the lower range of camelid CDR3 lengths. Employing VHs with longer CDR3s, such as BT32/A6, may provide the level of complexity required for isolation of lead antibodies specific for a variety of antigens. Libraries containing shorter and partially randomized CDR3s, such as the C-terminal residues, could be constructed and pooled with the library described here to further increase diversity. While exclusion of CDR1 and CDR2 randomization compromises library complexity, construction of primary libraries based only on CDR3 randomization remains an attractive choice because of design simplicity. In addition, limited randomization minimizes the problem of generating theoretical library sizes that exceed practical library sizes. Libraries constructed on a single framework offer the advantage of relatively simple affinity maturation of lead antibodies in that secondary CDR1 and CDR2 libraries can be generated using single primer pairs.

Our data demonstrated that, for the antigens described here at least, only a small region of the long CDR3 was available for antigen binding. The structural basis for this observation could not be determined since the CDR3 was unstructured, regardless of whether or not the CDR1-CDR3 disulfide bridge was present. It was somewhat surprising to find that only the C-terminal portion of the CDR3 was involved in antigen binding since the crystal structure of a camel anti-lysozyme dAb with a similar CDR3 length showed that the N-terminal half of the CDR3 was involved in binding (3). Based on this finding it had been suggested that more efficient camelized dAb libraries can be constructed in which the N-terminal region of CDR3 is randomized leaving the C-terminal side unchanged. Our finding clearly shows that using camelid dAbs as a guide to construct camelized libraries can be misleading. It is therefore wise, before committing to larger libraries, to construct smaller test libraries in order to map the antigen binding contact area of CDR3. Larger libraries then can be constructed by only randomizing those amino acids in CDR3 which are involved in antigen recognition.

As more and more information accumulates on the properties of native and camelized VHs, it is becoming clear that there are still unresolved issues surrounding factors that contribute to the solubility and monomeric nature of isolated VHs. The work described here indicates that BT32/A6 VH is a useful molecule for addressing these issues. Unmodified BT32/A6 VH is unusually soluble and camelization converts the molecule to one with excellent solution properties. More detailed structural analysis of the camelized VHs described here is underway and it is expected that the resulting data will provide useful information for the design of human single domain antibodies that contain a minimum of non-human residues, thereby making them particularly valuable as therapeutic reagents.

    ACKNOWLEDGEMENTS

We thank Doris Bilous for oligonucleotide synthesis, Joe Michniewicz for DNA sequencing, and Simon Foote for providing the expression and phagemid vectors. We thank Dr. Pierre Thibault and Don Krajcarski for assistance with the mass spectrometry and Drs. Darren Fast, Joycelyn Entwistle, Keith Lewis, and Martin Young for helpful discussions. We acknowledge the technical assistance of Ginette Dubuc.

    FOOTNOTES

* This is National Research Council of Canada publication 42431.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed. Tel.: 613-990-0833; Fax: 613-941-1327; E-mail: roger.mackenzie@nrc.ca.

Published, JBC Papers in Press, May 2, 2001, DOI 10.1074/jbc.M100770200

2 J. Tanha, S. Narang, H. Kaplan, M. Dan, and C. R. MacKenzie, unpublished results.

3 S. J. Foote, personal communication.

    ABBREVIATIONS

The abbreviations used are: dAb, single domain antibody; CDR, complementarity determining region; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DTT, dithiothreitol; Fab, antigen-binding fragment; HSQC, heteronuclear single-quantum coherence spectroscopy; IgG, immunoglobulin G; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy; PBS, phosphate-buffered saline; scFv, single chain variable fragment of an antibody; SPR, surface plasmon resonance; VH, antibody heavy chain variable domain; VHH, variable domain of a heavy chain antibody; VL, antibody light chain variable domain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hamers, C., Songa, Baiyana, B., Bendahman, N., and Hamers, R. (1993) Nature 363, 446-448[CrossRef][Medline] [Order article via Infotrieve]
2. Sheriff, S., and Constantine, K. L. (1996) Nat. Struct. Biol. 3, 733-736[Medline] [Order article via Infotrieve]
3. Desmyter, A., Transue, T. R., Ghahroudi, M. A., Thi, M. H., Poortmans, F., Hamers, R., Muyldermans, S., and Wyns, L. (1996) Nat. Struct. Biol. 3, 803-811[Medline] [Order article via Infotrieve]
4. Spinelli, S., Frenken, L., Bourgeois, D., de Ron, L., Bos, W., Verrips, T., Anguille, C., Cambillau, C., and Tegoni, M. (1996) Nat. Struct. Biol. 3, 752-757[Medline] [Order article via Infotrieve]
5. Davies, J., and Riechmann, L. (1994) FEBS Lett. 339, 285-290[CrossRef][Medline] [Order article via Infotrieve]
6. Decanniere, K., Desmyter, A., Lauwereys, M., Ghahroudi, M. A., Muyldermans, S., and Wyns, L. (1999) Structure 7, 361-370[CrossRef][Medline] [Order article via Infotrieve]
7. Nguyen, V. K., Hamers, R., Wyns, L., and Muyldermans, S. (2000) EMBO J. 19, 921-930[Abstract/Free Full Text]
8. Muyldermans, S., Atarhouch, T., Saldanha, J., Barbosa, J. A., and Hamers, R. (1994) Protein Eng. 7, 1129-1135[Abstract]
9. Lauwereys, M., Arbabi, G. M., Desmyter, A., Kinne, J., Holzer, W., De Genst, E., Wyns, L., and Muyldermans, S. (1998) EMBO J. 17, 3512-3520[Abstract/Free Full Text]
10. Vu, K. B., Ghahroudi, M. A., Wyns, L., and Muyldermans, S. (1997) Mol. Immunol. 34, 1121-1131[CrossRef][Medline] [Order article via Infotrieve]
11. Davies, J., and Riechmann, L. (1996) Protein Eng 9, 531-537[Abstract]
12. Ward, E. S., Gussow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989) Nature 341, 544-546[CrossRef][Medline] [Order article via Infotrieve]
13. Davies, J., and Riechmann, L. (1995) Bio/Technology 13, 475-479[Medline] [Order article via Infotrieve]
14. Arbabi, G. M., Desmyter, A., Wyns, L., Hamers, R., and Muyldermans, S. (1997) FEBS Lett. 414, 521-526[CrossRef][Medline] [Order article via Infotrieve]
15. Reiter, Y., Schuck, P., Boyd, L. F., and Plaksin, D. (1999) J. Mol. Biol. 290, 685-698[CrossRef][Medline] [Order article via Infotrieve]
16. Constantine, K. L., Goldfarb, V., Wittekind, M., Anthony, J., Ng, S. C., and Mueller, L. (1992) Biochemistry 31, 5033-5043[Medline] [Order article via Infotrieve]
17. Constantine, K. L., Goldfarb, V., Wittekind, M., Friedrichs, M. S., Anthony, J., Ng, S. C., and Mueller, L. (1993) J. Biomol. NMR 3, 41-54[Medline] [Order article via Infotrieve]
18. Dan, M. D., Earley, E. M., Griffin, M. C., Maiti, P. K., Prashar, A. K., Yuan, X. Y., Friesen, A. D., and Kaplan, H. A. (1995) J. Neurosurg. 82, 475-480[Medline] [Order article via Infotrieve]
19. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
20. Harrison, J. L., Williams, S. C., Winter, G., and Nissim, A. (1996) Methods Enzymol. 267, 83-109[Medline] [Order article via Infotrieve]
21. MacKenzie, R., and To, R. (1998) J. Immunol. Methods 220, 39-49[CrossRef][Medline] [Order article via Infotrieve]
22. Deng, S. J., MacKenzie, C. R., Sadowska, J., Michniewicz, J., Young, N. M., Bundle, D. R., and Narang, S. A. (1994) J. Biol. Chem. 269, 9533-9538[Abstract/Free Full Text]
23. Anand, N. N., Mandal, S., MacKenzie, C. R., Sadowska, J., Sigurskjold, B., Young, N. M., Bundle, D. R., and Narang, S. A. (1991) J. Biol. Chem. 266, 21874-21879[Abstract/Free Full Text]
24. Anand, N. N., Dubuc, G., Phipps, J., MacKenzie, C. R., Sadowska, J., Young, N. M., Bundle, D. R., and Narang, S. A. (1991) Gene (Amst.) 100, 39-44[Medline] [Order article via Infotrieve]
25. MacKenzie, C. R., Sharma, V., Brummell, D., Bilous, D., Dubuc, G., Sadowska, J., Young, N. M., Bundle, D. R., and Narang, S. A. (1994) Bio/Technology 12, 390-395[Medline] [Order article via Infotrieve]
26. Deng, S. J., MacKenzie, C. R., Hirama, T., Brousseau, R., Lowary, T. L., Young, N. M., Bundle, D. R., and Narang, S. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4992-4996[Abstract]
27. Jönsson, 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., and Malmqvist, M. (1991) BioTechniques 11, 620-627[Medline] [Order article via Infotrieve]
28. Bodenhausen, G., and Ruben, D. J. (1980) Chem. Phys. Lett. 69, 185-188[CrossRef]
29. Piotto, M., Saudek, V., and Sklenar, V. (1992) J. Biomol. NMR 2, 661-665[Medline] [Order article via Infotrieve]
30. Sklenar, V., Piotto, M., Leppik, R., and Saudek, V. (1993) J. Magn. Reson. Sect. A 102, 241-245[CrossRef]
31. Slatter, M., Schleucher, J., and Griesinger, C. (1999) Progr. NMR Spectrosc. 34, 93-158
32. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) J. Biomol. NMR 6, 277-293[Medline] [Order article via Infotrieve]
33. Johnson, B. A., and Blevins, R. A. (1994) J. Chem. Phys. 29, 1012-1014
34. Wishart, D. S., Bigam, C. G., Yao, J., Abildgaard, F., Dyson, H. J., Oldfield, E., Markley, J. L., and Sykes, B. D. (1995) J. Biomol. NMR 6, 135-140[Medline] [Order article via Infotrieve]
35. Knappik, A., and Plückthun, A. (1994) BioTechniques 17, 754-761[Medline] [Order article via Infotrieve]
36. Miceli, R. M., Degraaf, M. E., and Fischer, H. D. (1994) J. Immunol. Methods 167, 279-287[CrossRef][Medline] [Order article via Infotrieve]
37. Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller, C. (1991) Sequences of Proteins of Immunological Interest , United States Department of Health and Human Services, United States Public Health Service, Bethesda, MD
38. Riechmann, L., and Davies, J. (1995) J. Biomol. NMR 6, 141-152[Medline] [Order article via Infotrieve]
39. Riechmann, L. (1996) J. Mol. Biol. 259, 957-969[CrossRef][Medline] [Order article via Infotrieve]
40. Voordijk, S., Hansson, T., Hilvert, D., and van Gunsteren, W. F. (2000) J. Mol. Biol. 300, 963-973[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.