Nurse sharks (Ginglymostoma cirratum) were placed in containers filled with artificial sea water containing 0.1% (w/v) tricaine methanesulfonate (MS-222). When the desired level of narcosis was reached, they were removed for immunization or bleeding. Recombinant in-house CHO-expressed mouse and human ICOSL-flag-His (200 μg/shark) emulsified in complete Freund’s adjuvant (CFA) were injected using a 20 gauge needle into the lateral fin of the shark. Four weeks later, antigens (200 μg/shark) emulsified in incomplete Freund’s adjuvant were similarly administered to the shark. Three immunization boosts (100 μg/shark) were given at 4-week intervals intravenously into the caudal vein as soluble antigen in PBS (sample 0.45 µM sterile filtered). Between 3 and 5 ml of blood samples were collected from the caudal vein into a 30 ml syringe containing 200 µl porcine heparin (1,000 U/ml in PBS) at weeks 0 (pre-immunization bleed), 10, 14, 18 and 22. Blood samples were spun at 1,000 × g for 10 min to separate blood cells from plasma. The plasma supernatant fraction was carefully removed into a sterile tube with RNA stabilization buffer and stored at −80°C.

Immunoplates (Nunc, Thermo Scientific) were coated overnight at 4°C with 1 µg/ml human or murine ICOSL and then blocked with 4% (w/v) milk PBS (MPBS) for 2 h at 37°C. Shark serum was diluted 1:30 and then a 1:3 dilution series set up on each plate and incubated for 1 h at room temperature following incubation for 1 h with anti-nurse shark IgNAR mouse monoclonal antibody GA8 (28) (1:500 in PBST). The plates were incubated for a final time with anti-mouse IgG-HRP (SIGMA) diluted 1:1,000 in PBST. After each step, the ELISA wells were washed three times with 200 µl/well PBST. Plates were developed by adding 100 μl/well TMB substrate (Thermo Scientific) and the reaction stopped with 50 μl/well 1 M H₂SO₄.

Peripheral blood lymphocytes (PBLs) were harvested from the plasma of the bleed with the best IgNAR response (titer) and total RNA isolated using a QIAGEN kit following the manufacturer’s instructions. cDNA was synthesized with IgNAR transmembrane- (Tm 5′-TACAAATGTGGTGTACAGCAT-3′) and secretory- (Sec 5′-TAGTACGACCTGAAACATTA AC-3′)-specific primers. Using the NEB Phusion HF PCR Master Mix protocol, VNAR DNA was amplified with framework (FW) nurse shark-specific primer combinations FW1/FW4r1 or FW1/FW4r2:

FW15′-GAGGAGGAGGAGAGGCCCAGGCGGCCGCTCGAGTGGACCAAACACCG-3′

Amplicons were cloned into an in-house phage display vector (pEDV1) with in-frame 6xHis-tag and c-myc-tag via SfiI restriction sites. The library was transformed into electrocompetent TG1 cells (Lucigen), and the library size was calculated as described by Müller et al. (29).

To rescue phage to be used in library selections, cultures from library glycerol stocks were grown at 37°C and 250 rpm, in 2xTY, 2% glucose, 100 µg/ml ampicillin to an OD₆₀₀ of 0.5. Cells were superinfected with 10⁹ M13K07 helper phage (NEB) and then incubated overnight in 2xTY, 100 µg/ml ampicillin, 50 µg/ml kanamycin at 25°C and 250 rpm. The cultures were PEG-precipitated (20% (w/v) PEG/2.5 M NaCl) twice, and the resulting phage pellets were resuspended in 1 ml PBS. To establish antigen “decorated” bead selections, recombinant ICOSL-flag-His protein was biotinylated with Sulfo-NHS-LC-Biotin as per manufacturer’s instructions (21327, Thermo Scientific). Two hundred microliters of magnetic Dynabeads M-280 Streptavidin (11205D, Invitrogen), pre-blocked with 2% (w/v) MPBS, were coated with 50–200 nM biotinylated material by rotating at 20 rpm, at room temperature for 1 h. Library phage was incubated first with Dynabeads for 1 h rotating at room temperature to remove phage specific to the beads and added then to the antigen-coated beads. After 1 h incubation at room temperature at 20 rpm, beads with bound phage were washed 5–10 times with PBST and 5–10 times with PBS, eluted by rotating for 8 min in 400 µl 100 mM TEA and neutralized by the addition of 200 µl 1 M Tris-HCl pH 7.5. Escherichia coli TG1 cells (10 ml) were infected with 300 µl of eluted phage for 30 min at 37°C and grown overnight at 37°C on TYE agar plates containing 2% (w/v) glucose and 100 µg/ml ampicillin. Two further rounds of selection were conducted, and outputs were screened for antigen-specific binding by monoclonal phage and periplasmic extract ELISAs against human or mouse ICOSL. Phage binders were detected using HRP-conjugated anti-M13 antibody (27942101, GE Healthcare), and periplasmic protein was detected using HRP-conjugated anti-c-Myc antibody (118 141 50 001, Roche).

To express monomeric VNARs, non-amber-suppressor HB2151 E. coli cells were used. VNARs were isolated from periplasm by osmotic shock with 50 mM Tris/HCl pH8.0, 1 mM EDTA, 20% sucrose and purified via His-tag capture on NiNTA resin. VNARs were eluted with 0.5 M imidazole pH 8.0 followed by dialysis in PBS. Selected positive monomeric VNAR domains were PCR amplified and subcloned into an in-house Fc-fusion mammalian expression vector (pEEE2A), which facilitated Protein A affinity purification of expressed protein post transient expression in a HEK 293 suspension culture. HEK cells at ~10⁶ cells/ml in GIBCO FreeStyle 293 media were transiently transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Twenty four hours post transfection, tryptone (0.5% w/v) in PBS was added to the culture to enhance expression and the cells incubated for 5 days. Cells were pelleted at 1,000 × g for 15 min and the supernatants sterile filtered before adding PROSEP A resin (Millipore). After washing with PBS, fractions of purified VNAR Fc were eluted with 0.1 M glycine pH 3.0 (Severn Biotech Ltd.) and neutralized by adding 1 M Tris-HCl pH 8.0. Samples were dialyzed using Slide-A-Lyser dialysis cassettes (Thermo Scientific) in PBS pH 7.4. Expression levels of VNAR Fc-fusion proteins were generally in the range of 50–70 mg per liter using serum-free media. Electrophoresis of purified protein samples was performed on NuPAGE 4–12% Bis-Tris gels using a MES buffer system (Invitrogen) in accordance with the manufacturer’s instructions.

CHO cells expressing human or mouse ICOSL were grown to 90% confluency in DMEM/F12 + 5% FBS media, in 96-well cell culture plates (Greiner, Bio-One). Anti-human or murine VNARs in DMEM/F12 + 2% FBS were added to the corresponding CHO cells. Following 1 h incubation at 16°C, cells were gently washed three times with DMEM/F12 + 2% FBS and incubated for 40 min at 16°C with anti-His-HRP (SIGMA) diluted 1:1000 in the same media. Cells were washed and developed as described previously.

CHO cells expressing human ICOS receptor were grown to 90% confluency. A total of 20 µl at 450 ng/ml of ICOSL-hFc (rhB7-H2/Fc—165-B7, R&D Systems or rmB7-H2/Fc—158-B7, R&D Systems) was preincubated for 1 h with 40 µl of serially diluted anti-ICOSL-VNAR-Fc in DMEM/F12 + 2% FBS and then added to the cells. Following 1 h incubation at 16°C, cells were gently washed three times with DMEM/F12 + 2% FBS and incubated for 40 min at 16°C with goat anti-human Fc-HRP (SIGMA) diluted 1:10,000 in the same media. Cells were washed and developed as described previously. The absorbance was measured at 450 nm wavelength using a microplate reader and data were plotted using Sigma Plot software.

96-well flat bottom Maxisorp Nunc Immuno plates (Thermo Scientific) were coated at 4°C overnight with 1 µg/ml of the antigen of interest: for monomeric VNAR binding—ICOSL-hFc (rhB7-H2/Fc—165-B7, R&D Systems or rmB7-H2/Fc—158-B7, R&D Systems), human ICOSL-IgV, mouse ICOSL-IgV, human ICOSL-IgC, or mouse ICOSL-IgC (all ICOSL-Igs were produced in-house) and for VNAR-Fc binding—human or mouse recombinant ICOSL-flag-His (produced in-house). The plates were washed three times with 200 µl/well PBS before blocking with 200 µl of 4% (w/v) MPBS/well and incubated at 37°C for 1 h. The blocked plates were washed three times with PBS and serial dilutions of VNAR proteins were then added per designated well and the plates incubated at room temperature for 1 h. Plates were washed three times with PBST before 100 µl of 1:1,000 dilution HRP-conjugated anti-His or goat anti-human IgG antibody (for Fc-fused VNAR detection) was added to the plates and incubated for 1 h at room temperature. The plates were washed and developed by adding 100 µl TMB substrate solution and stopped using 1 M H₂SO₄ as previously described. The absorbance was measured at 450 nm wavelength using microplate reader and data were plotted using Sigma Plot software.

The kinetic constants of VNAR-Fc were determined by surface plasmon resonance using Biacore T200 and 2000 biosensors (GE Healthcare). Anti-human IgG1 antibody diluted in 10 mM sodium acetate buffer was immobilized on a CM5 chip, and VNAR-Fcs were captured via their Fc region. HuICOSL-flag-His (100 nM) was serially diluted two-fold in HEPES running buffer (HBS EP+, BR-1006-69, GE Healthcare) with 150 mM NaCl, 3 mM EDTA and 0.05% v/v Surfactant P20 pH 7.4, and was injected for 2 min at a flow rate of 60 µl/min. Dissociation phase was monitored for 5 min followed by two 10 µl regeneration pulses using 10 mM glycine pH 1.5, at a flow rate of 100 µl/min. Association and dissociation rates were calculated using the 1:1 global Langmuir binding model fit analysis (Biacore Evaluation Software).

Adult female C57BL/6 mice were randomly allocated to experimental groups and allowed to acclimatize for 1 week. Treatments were administered according to the schedule below (Table 1). Test articles were administered in PBS. On Day 0, animals were administered with an emulsion containing 500 µg of IRBP peptide 1-20 (IRBP p1-20) in CFA supplemented with 2.5 mg/ml Mycobacterium tuberculosis H37 Ra by subcutaneous injection. Also on Day 0, animals were administered with 1.5 µg Bordetella pertussis toxin by intraperitoneal injection.

From Day 7 until the end of the experiment on Day 28, animals were monitored once per week for clinical signs of uveitis using topical endoscopic fundal imaging (TEFI) (Table 2). Animals were also monitored twice weekly for signs of ill-health, weighed and any abnormalities recorded. At termination on Day 28, eyes were removed into tissue fixative for histopathology.

Wild-type mice BALB/C were divided into three groups of two animals each. All procedures were performed on anesthetized animals. Corneal epithelium of the right eye was scratched and 20 µg/3 μl of VNAR, VNAR Fc, or mAb AF158 (R&D) was applied four times topically to the right eye at 5-minute intervals. The eye was then washed with saline solution and 2 µl of anterior fluid sampled. All animal studies were carried out under the Animals (Scientific Procedures) Act 1986 regulations (Home Office UK). For ELISA, 96-well flat bottom Maxisorp Nunc Immuno plates (Thermo Scientific) were coated with 1 µg/ml of rmB7-H2/Fc or mICOSL-flag-His at 4°C overnight. The plates were washed three times with 200 µl/well PBS before blocking with 200 µl of 4% (w/v) MPBS per well and incubated at 37°C for 1 h. The blocked plates were washed three times with PBS, 100 µl of anterior fluid (start dilution 1:50 in PBS, then serial dilutions 1:2) was then added and incubated at room temperature for 1 h. Plates were washed three times with PBST and 100 µl of 1:1,000 dilution HRP-conjugated anti-His (for VNAR detection) or goat anti-human IgG antibody (for Fc-fused VNAR and mAb detection) was added to the plate and incubated for 1 h at room temperature. The plates were washed and developed by adding 100 µl TMB substrate solution and stopped using 1 M H₂SO₄ as previously described.

Two nurse sharks were immunized with both recombinant human and mouse ICOSL. An antigen-specific IgNAR immune response was observed and confirmed after 18 weeks through the analysis of post-immunized sera. The response was exemplified by a gradual increase in titer values from early to late bleeds (data not shown). The VNAR repertoire was amplified from isolated PBLs, cloned into a pEDV1 which contained an in-frame coat protein pIII of the bacteriophage M13 gene and transformed into E. coli TG1 cells. The library size was calculated to be 10⁸ transformants.

Domains were isolated following three rounds of selection utilizing biotinylated antigen immobilized on streptavidin-coated beads to maximize the chance of delivering potent ICOSL binders (30). huICOSL-specific VNARs were obtained after the first selection round and enriched further after round three with >90% of domains specific for antigen (Figure 1A). As blockade of the ICOS–ICOSL interaction was the desired functional outcome of the selection process, a cell-based assay designed to detect domains that can block receptor/ligand binding was introduced into the screen. Positive hits from selections against huICOSL were assessed for their ability to block huICOSL binding to ICOS expressing cells (Figure 1C). Signals that decreased by 50% or more were considered positive for receptor–ligand binding inhibition. In total, six unique (based on CDR3 sequence differentiation) anti-human ICOSL VNAR clones were identified. Cell surface antigen-target selectivity was assessed by FACS analysis utilizing CHO cells overexpressing human or murine ICOSL (data not shown) as well as in a binding ELISA format (Figure 2A). All domains were found to be strong huICOSL binders but with no species cross-reactivity to the mouse ligand. The affinity of huICOSL binders was in the range 1–9 nM (Table 3). ICOSL is a two-domain protein, where receptor binding is mediated solely by the membrane distal IgV domain but requires the membrane proximal IgC domain to maintain the structural integrity of the protein (31). All of the isolated blocking huICOSL VNARs cross-reacted strongly with the ICOSL-IgV domain with weaker or negligible binding to the ICOSL-IgC domain (Figure 2B). As the percentage homology between the human and murine IgV region of ICOSL is only 43%, it was not surprising that the screen did not isolate species cross-reactive VNARs that block receptor/ligand interaction.

Anti-mouse ICOSL VNAR domains were isolated using the same method as for the anti-human domains and lead clone isolation determined following their performance in in vitro (Figure 1B) and cell-based binding assays. Four unique binders, all within an EC50 (effective concentration) range from 1.4 to 11.4 nM (Figure 3A), were taken forward for further study. Three of these anti-mICOSL clones (A5, A7, and B8) could block ligand/receptor binding in a CHO-cell-based blocking assay, whereas clone F11 lacks this blocking activity and was used here as a non-blocking control (Figure 3B). The naïve VNAR domain 2V was also included as an isotype control in ligand/receptor binding assays. This clone originally isolated from the dogfish (Squalus acanthias) is part of a sequence database from this species and has no known target, making it an ideal negative control for these and other studies (11).

Unlike VNARs obtained from the human ICOSL selection campaign, domains isolated from mouse ICOSL selections were species cross-reactive. Three of the four clones recognize both mouse and human ligand in an ELISA with clear binding to IgC domains (Figures 3C,D). Interestingly, clone A5, which was the strongest ICOS/ICOSL blocker (Figure 3B), bound only to the full length mouse protein, but not to the individual IgV and IgC domains implying that it may recognize an interdomain or linking region between them (Figure 3C).

A key aim of this work was to determine the efficacy of VNAR domains in an in vivo mouse model. As VNAR domains alone are cleared rapidly from the systemic circulation (11), all VNAR clones were first converted into a fusion format with a human Fc (Figure 4A) to facilitate an extension of serum half-life. All VNAR Fcs retained binding to mICOSL with improved, presumably through avidity, EC50s in the range 0.6 to 3 nM (Figure 4B). In cell-based ICOS/ICOSL blocking assays conducted with Fc-reformatted VNAR domains, clone A7, which performed well as a monomeric VNAR, lost this ability after Fc-fusion (Figure 4C). The reason for the loss of activity was not investigated further at this time. In summary, A5-Fc and B8-Fc, both blocked receptor/ligand interaction, did not bind human ICOS ligand but did bind mouse and rat protein. F11-Fc did not block ICOS/ICOSL binding, and A7-Fc, which lost its blocking ability after reformatting, bound ligands from all three species (Figure 4D).

The lead domain A5-Fc was assessed in a murine model of EAU by KWS (Bristol, UK). Adult female C57BL/6 mice were randomly allocated to experimental groups and allowed to acclimatize for 1 week. Treatments were administered according to the protocol described in Section “Materials and Methods” from Day 1 to Day 14 or Day 1 to Day 28 for the corticosteroid control (five mice per group) and from Day 1 to Day 14 for the A5-Fc protein (six mice per group). On Day 0, animals were administered with IRBP p1-20 in CFA supplemented with M. tuberculosis H37 Ra to induce uveitis. All animals were weighed three times a week and also monitored twice weekly for signs of ill-health and any abnormalities recorded. The disease-induction procedure (subcutaneous administration of IRBP/CFA and intraperitoneal injections of pertussis toxin) caused a mild bodyweight loss on Day 2 and Day 5, as expected for this model. However, from day 7 until the end of the experiment, there was no further treatment-induced bodyweight loss in any of the experimental groups (Figure 5A). From day 7 until the end of the experiment, animals were monitored once a week for clinical signs of uveitis using TEFI. A significant increase in TEFI scores was observed on Day 21 when compared with the untreated Day 7 control group. By day 21, both the Cyclosporin A and the anti-mICOSL VNAR domain A5-Fc groups showed a significant reduction in recorded clinical scores and a marked lag in the onset of any disease when compared to the untreated control animals (Figure 5B). Histopathology analyses of eyes, removed at the end of the experimental period (Day 28), confirmed that this observed reduction in inflammation translated into differences at the tissue level too (Figure 5C). Furthermore, detailed cellular observation clearly showed that animals treated with either Cyclosporin A or with anti-mICOSL VNAR domain A5-Fc presented with mild disease pathology compared to the untreated control. The control animals exhibited extensive cellular inflammation with a high number of neutrophilic cells accumulating in the vitreous, the drainage angle, the anterior chamber, the ciliary body and also in the surrounding blood vessels (Figure 5D).

The influence of molecular size on the ability of biologics to enter the eye if applied topically was assessed in a scratched corneal mouse model that goes some way to mimicking the situation seen in severe inflammatory eye conditions. It was hoped that the smaller size of the VNAR domains may provide a delivery option for site-specific targeting of ocular disorders. Three formats of increasing molecular weight were tested: VNAR single domains (11 kDa), VNAR Fc (80 kDa) and mAb (150 kDa). All three were applied dropwise onto cornea without an epithelium and anterior fluid was collected after 20 min of treatment. The presence of VNAR was clearly observed in the anterior fluid compared to VNAR-Fc and mAb (Figure 6).