Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library
© Liu et al; licensee BioMed Central Ltd. 2007
Received: 06 September 2007
Accepted: 19 November 2007
Published: 19 November 2007
Shark heavy chain antibody, also called new antigen receptor (NAR), consists of one single Variable domain (VH), containing only two complementarity-determining regions (CDRs). The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs) make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences.
A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB), ricin, and botulinum toxin A (BoNT/A) complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications.
We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins.
Sharks, similar to camelids, possess unconventional heavy (H) chain antibodies, consisting of heavy chain homodimers in which each chain contains one single variable and five constant domains [1, 2]. The conserved amino acid residues between the shark heavy chain antibody and those involved in forming the core of immunoglobulin and T-cell receptor variable regions, gave impetus for naming shark heavy chain antibody Immunoglobulin New Antigen Receptor (IgNAR or NAR) [2, 3]. Structural analysis by electron microscopy, crystal structure, and 3D modeling revealed that there are three NAR isotypes. These isotypes are defined according to their pattern of inter-loop disulfide linkages within the variable region and the timing of their appearance during the animal's development [3–6].
Using genetic engineering a single NAR variable (V) antigen-binding domain can be expressed as a separate soluble protein often referred to as a shark single domain antibody (sdAb). Shark sdAbs contain four conserved frame regions (FRs) and two complementarity-determining regions (CDRs), making them the smallest (~12 kD) Ig-based recognition units with full capacity for antigen binding affinity and specificity. Due to their small size, they may be able to access antigen epitopes not generally recognized by recombinant conventional antibodies . Although both shark and camelid sdAbs share similar structural features and functional requirements , shark sdAb lack a conventional CDR2, and contain two hyper-variable regions (HVs), HV2 and HV4, which may contribute to antigen binding [3, 8, 9]. According to the crystallographic analysis of NAR V structure, the loop of HV2, located within the FR2-CDR2 region, is located across the middle of the molecules and may influence the conformation of the CDR3; moreover, the loop of HV4, located between HV2 and CDR3, is formed proximal to CDR1 and may influence the antigen binding interactions .
Similar to camelid sdAbs, shark sdAbs exhibit excellent solubility for protein production, superior to many recombinant conventional antibodies, and retain conformational stability when heated or refold correctly upon cooling, [10–13]. These intrinsic properties make sdAb exceptional alternatives for diagnostic applications. Using phage display technology and PCR amplification, the repertoire of the naturally occurring NAR V from either immunized or naïve (non-immunized) animals were established and used for panning against target antigens [10, 11]. High affinity binders to a specific target were obtained from immunized libraries however required a waiting period for suitable immunization to be achieved and an animal care facility . On the other hand, weak binders against a wide variety of target antigens were obtained from naïve libraries; they were selected rapidly and no immunization period was required [10, 14]. If higher affinity binders are desired than those obtained from the naïve library, the sdAb can be enhanced using in vitro affinity maturation [12, 15].
It is believed that the diversity of naturally occurring NAR V results from multiple rearrangements of the CDR genes and somatic hypermutations in vivo . Consistent with this finding, the complexity of shark NAR V usually resides in CDR1 with sequence variation within residues 28–33 and an extended CDR3, which varies in length (5–23 residues) and in amino acid composition. Routes to introduce diversity and increase the complexity of naïve libraries include: variation of CDRs via DNA shuffling , PCR using randomized primers, and random mutagenesis by error prone PCR  or dNTP analogs . Although DNA shuffling, which involves the recombination of several small DNA fragments within a whole variable region, has been successfully used to create a semi-synthetic llama library with giga diversity , it has not been used for constructing a more diverse shark display library due to the short NAR V DNA fragments, less than 400 bp in size. Instead, PCR using randomized CDR3 sequences has been used to construct more diverse synthetic libraries, resulting in binders with higher affinity and specificity to target antigens [10, 12].
Our previously described naïve NAR V library, SP, from spiny dogfish shark (Squalus acanthias) was successfully panned for the isolation of binders to cholera toxin; however our ability to obtain specific binders towards other targets was disappointing . In this study, we expanded the utility of the naive display library by introducing variations in CDR3 amino acid composition and length (13, 16, or 18 residues). In this manner, a semi-synthetic NAR V display library, SPSL1, with a complexity of ~1e9 was successfully constructed and used for panning against ricin, staphylococcal enterotoxin B (SEB) and BoNT/A complex toxoid. We successfully selected binders against these three toxins and characterized the purified sdAbs using ELISA and Luminex 100 assays. Our results suggest that this new diversified NAR V display library can serve as a fruitful source of sdAbs against a variety of toxins.
Results and Discussion
Library construction and selections
Randomized oligonucleotide primers used for amplification of randomized spNAR V CDR3 sequences.
#8406, #8407/8, #7554/6, #9686/7, forextra
#6974/7, 7553/5, #9688/9, revextra
The two SEB binders along with the BoNT/A complex toxoid binder, P4BF7-3, had no formation of inter-loop disulfide bonds and shared a conserved W31 within CDR1, which are major features of type 3 NAR V originally defined as appearing only in juvenile nurse sharks less than one year old [5, 18]. Previously, we discovered that our spiny dogfish NAR V display library derived from adult animals over one year old contained approximately 7% of type 3 NAR V fragments. This may be a unique feature of spiny dogfish shark . These type 3 spiny dogfish shark NAR V differ from ones found in nurse shark, since they exhibited more diverse length and composition with or without a conserved F96 in CDR3 [5, 20, 22]. Amino acid composition has a great influence on conformation and stability. As the spiny dogfish shark type3 NAR V binders lack the invariant aromatic Y86 and F96 found to participate in forming more rigid CDR3 loops, they likely exhibit different CDR conformational plasticity than type3 NAR V from nurse sharks . Ricin binder, P4RA7-1 is an atypical type 2 NAR V with no formation of inter-loop disulfide bonds due to the lack of Cys in the CDRs (Fig. 2C). One of the BoNT/A complex toxoid binders, P4BH8, is an atypical type 1 NAR V that lacks Cys residues in FR2 and FR4 but contains two CDR3 Cys residues, which allows the formation of an intra-loop disulfide bridge to stabilize CDR conformation. The mechanism of CDR conformation stability for these atypical NAR V should be different from their corresponding typical NAR V and is a topic for future investigation. It is likely that HV2 and HV4 may play extremely dominant roles to stabilize CDR conformation [3, 8, 23].
Expression of soluble monomeric SdAbs
Five selected binders were sub-cloned to the pEcan 22 expression plasmids, a gift from Dr. Andrew Hayhurst, for overproduction . Monomeric soluble protein was purified using a nickel affinity column followed by gel filtration . Expression levels for these five binders varied. Yields for P4BH8, P4BF7-3, and P4RA7-1 were about 1.0 mg per liter of bacterial culture; this is similar to the yield of hen egg white lysozyme binders isolated from a repertoire of nurse shark NAR V . The production amounts for the two SEB binders, P1SD3-3 and P2SC8, were about 6-fold less. The difference in expression levels may be due to overall conformation stability and domain surface charges resulting from sequence and structural diversity. However, we did not observe a good correlation pattern between the clone sequences and the expression levels based on the comparison of amino acid residues. Structural and 3D modeling studies for these clones are required to reveal more information about correlations between protein expression level and sequence variation.
Direct binding to determine affinity and specificity
Equilibrium dissociation constants
299 ± 49
50.0 ± 7.5
10.2 ± 7.2
107 ± 38
3.6 ± 3.6
BoNT/A complex toxoid
154 ± 26
390 ± 90
0.02 ± 0.003
BoNT/B complex toxoid
106 ± 22
332 ± 68
0.05 ± 0.007
BoNT/E complex toxoid
93 ± 18
309 ± 75
The SEB binders showed very different binding affinity and specificity despite their identical CDR1 and homologous CDR3 sequences (Figs. 2B, 3D and 3E). P1SD3-3 bound to SEB specifically relative to irrelevant targets, while P2SC8 exhibited more non-specific sticky interactions with irrelevant targets, CT and ricin (Figs 3D and 3E). Moreover, P1SD3-3, has ten-fold better binding affinity (kd = 10.2 nM) than P2SC8 (kd = 106.8 nM) (Table 2). A recent affinity maturation study showed that changing S61 to R61 within the HV4 in nurse shark NAR V contributed to increased binding affinity resulting from more van der Waals' contacts on the mature sdAb . The change of K61 to E61 within HV4 in P1SD3-3 might exhibit a similar effect and perhaps resulted in observed higher binding affinity and specificity of P1SD3-3 compared to P2SC8 (Fig. 2B, Figs. 3D and 3E). P2SC8 and P4BF7-3 had identical sequences for most regions except CDR3 and amino acid 48 in HV2. The resulting differential binding reactivity to all tested toxins suggests that CDR3 alone may play a major role to determine the binding specificity in these two clones.
Thermal stability determinations
Of the two, P4BF7-3 appeared to be more stable than P4BH8 (Fig. 4C), suggesting that it might have a more stable CDR conformation. The conformational stability may partially result from the formation of an intra-loop disulfide bridge within CDR3 (Fig. 2A). Interestingly, P4BH8, showed 30% more than initial binding reactivity to target, upon heating for 5 min (Fig. 4C). This phenomenon is often seen in sdAbs, possibly due to better binding of the refolded structure that results from a short period of heating and rapid cooling; this same phenomenon may also be the cause for improvements in specificity [12, 13, 20].
Both SEB binders exhibited better thermal stability than tested conventional antibodies (Fig. 4F). They showed similar thermal stability profiles, suggesting both share a similar CDR conformation resulting from similar sequences (Fig. 2B).
Use of sdAb as reporter reagents in sandwich assays
The sdAbs selected from the new randomized naïve shark library exhibit serviceable specificity and excellent thermal stability, but their binding affinity was weaker than the conventional antibodies tested. Often, affinity maturation is required to obtain high affinity binders. Besides the common approaches described in the background section, 3D structural modeling also provides a powerful tool to reveal more detailed information regarding the change of amino acids within the constant regions and HVs . However, the typical increase in binding affinity has been only 10-fold using these approaches, which would still not produce binders comparable to the best conventional antibodies . Alternatively, multiple copies of sdAbs against the same target can be linked together to generate high avidity structures [2, 25, 26], which can result in three orders of magnitude higher affinity than the monomeric sdAb. This may be a more attractive way to improve binding reactivity for our sdAb (Table 2).
In the future, we may need to introduce more variation in the CDR3 loop length in combination with the preferred insertion of Cys into defined CDR3 positions. Construction of a more diverse library, containing a greater percentage of conventional type 1 and 2 NAR sequences may allow us to obtain specific binders for each BoNT subtype for detection purpose. Nevertheless, our results indicated that the new semi-synthetic shark library had better CDR3 diversity and better utility than our previously established naturally occurring NAR V display library, suggesting this may be the correct path towards obtaining a limitless source of sdAbs against a variety of toxins for sensor applications.
DNA template was isolated from the naturally occurring shark library using the plasmidpure DNA miniprep kit (Sigma, St. Louis, MO) and was used to amplify randomized CDR3 fragments with forward primer mix and randomized reverse primers (table 1). PCR condition was as follows: 94°C for 30s, 55°C for 90s, and 70°C for 30s, and 35 amplification cycles. The resulting 400 bp PCR products were then used as templates to amplify the full length NAR V fragments flanked with SfiI and NotI restriction sites on 5' and 3' respectively using forward and reverse primer mixes . The full length NAR V fragments and pHen2 plasmids were then cut with SfiI and NotI, followed by overnight ligation at 15°C. The ligation mixture was cleaned by Qiagen PCR kit (Qiagen, Valencia, CA) and subjected to electroporation using XL 1 Blue cells (Strategene, La Jolla, CA). The cells were plated on big Nunc Bio-assay dishes containing LB agar, 2% glucose, and 100 μg/mL ampicillin and grown overnight. The transformed bacteria were scrapped and stored at -80°C the next day. The resulting semi-synthetic library was named SPSL1.
Toxin binder selection by panning and ELISA
Panning and ELISA assays were carried out essentially as described previously . Ricin, SEB and BoNT/A complex toxoid at concentrations of 10 μg/mL were passively immobilized on high binding 96 well plates and used to mine the semi-synthetic shark NAR V display library, SPSL1, in 3 rounds of panning. Polyclonal phage ELISAs on target and control antigens were used to identify rounds in which clones had been successfully enriched. Monoclonal ELISAs were subsequently used to identify monoclonal sdAbs . Typically, 15 antigen positive clones were subjected to DNA sequencing to identify unique clones.
Expressing and purifying sdAb
NAR V fragments isolated from potential binders were cut with SfiI and NotI. The resulting fragments were inserted into pEcan 22 vectors and transformed into the E. coli Tuner strain for protein expression. Expressed protein was purified by osmotic shock from Tuner cultures, immobilized metal affinity chromatography, and gel filtration [20, 27].
Characterizing purified sdAb by Luminex 100 immunoassays
For direct binding assay, Luminex microspheres coated with relevant and irrelevant toxins were incubated with purified sdAbs at the concentrations of 0.01–2500 nM for 30 min, followed by the addition of fluorescent tracer, Ni-streptavidin-phycoerythrin (Ni-SA-PE). For thermal stability testing, purified sdAb proteins and control antibodies were heated to 85°C for 60 min with samples removed to cooling at various time points and analyzed for antigen binding activity. In all analyses, sdAb binding to irrelevant antigens was also monitored to ensure signal was not due to non-specific binding of unfolded sticky protein. For sandwich assay, Luminex microspheres with antibodies immobilized were mixed with target antigens. Then a second sdAb, and Ni-SA-PE were added into the reaction to measure the binding of captured antigens. The preparation of immuno-reagents was described previously .
We would like to thank Dr. Andrew Hayhurst for his expression vector, pEcan22. This work was supported by DTRA JSTO CBD Physical Science and Technology, DTRA JSTO CBD Medical Science and Technology, and the Office of Naval Research. The views expressed here are those of the authors and do not represent the opinions of the U.S. Navy, the U.S. Department of Defense, or the U.S. Government.
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