Identification of anti-canine CTLA4 Nbs
Nbs targeting canine CTLA4 were identified using a yeast surface display platform following the published protocol (35). The recombinant canine CTLA4 protein (Sino Biologicals) was labeled either with Alexa fluor-647 (AF 647) or biotin using microscale protein labeling kits (ThermoFisher). The labeled canine CTLA4 protein was used for biopanning, and CTLA4 binders were enriched by magnetic-assisted cell sorting (MACS) using anti-AF647 or anti-biotin microbeads (Miltenyi). For each round of MACS selection, yeast was grown for 48–72 hours in galactose supplemented tryptophan dropout media (Sigma) to induce Nb expression. Initially, 2.5 × 109 induced yeast cells were washed, resuspended, and incubated at 4°C for 30 min with anti-Alexa Fluor 647 microbeads in selection buffer (20 mM HEPES, pH 7.5, 150 mM sodium chloride, 0.1% (w/v) ovalbumin, 1 mM EDTA). The yeast cells were then passed through the LD column (Miltenyi) to remove yeast-expressing Nbs that bound nonspecifically to magnetic beads. After pre-clearing, yeast cells were incubated with labeled canine CTLA-4 protein (1 µM final concentration) at 4°C for 30 min. Yeast cells were then incubated with anti-AF647 microbeads at 4°C for 30 min and passed through the LS column to enrich for yeast-expressing Nbs specific to canine CTLA4 protein. The enriched yeast was collected, grown, induced, and used for the second round of MACS selection using biotin-labeled canine CTLA4 protein. A total of three rounds of MACS selection were performed. We also used successively lower concentrations (1 µM for 1st round; 500 nM for 2nd round; and 75 nM for 3rd round) of CTLA4 protein to enrich high-affinity binders. After the 3rd round of MACS selection, yeast cells were grown, induced, and labeled with AF647-labeled canine CTLA4 protein, anti-HA AF488 antibody, and propidium iodide. Yeast cells positive for AF647, AF488, and negative for propidium iodide were sorted into single clones in 96-well plates using a flow sorter (Daiko).
Purification of Nbs. Three yeast clones (cNb6, cNb13 & cNb17) with a strong affinity for canine CTLA-4 protein were selected for in-depth characterization (35). The DNA sequences coding for Nbs were amplified by polymerase chain reaction (PCR) and cloned into a periplasmic expression vector pET22b. The Nb genes were tagged with 6xHis and Strep Tag II on the C-terminus and expressed in BL21 (DE3) E. coli. For each purification, E. coli were cultured in 500 ml of terrific broth (MP Biosciences) and induced after reaching OD600 0.6–0.8 with 1 mM Isopropyl β- d-1-thiogalactopyranoside (IPTG, Zymo research) at 25°C for 16–18 hours. Induced cells were pelleted and resuspended in 25 mL of TSE buffer (0.2 M Tris-HCL, pH 8, 200 g/L sucrose, 0.5 mM EDTA). 50 mL of cold, sterile water was added to the cells and stirred for 45 minutes @ 4°C to provide an osmotic shock to release periplasmic Nbs. The periplasmic fraction containing the tagged Nbs was centrifuged at 16,000 x g for 10 minutes @ 4°C. The supernatant was collected and dialyzed against 2X PBS buffer overnight @ 4°C using a 3 kDa cutoff dialysis cassette (ThermoFisher). The dialyzed proteins were centrifuged at 10,000 x g for 15 minutes @ 4°C, and the supernatant was loaded onto a 5 mL StrepTrap column (GE Healthcare) using an AKTA Explorer (GE Healthcare). The column was washed with 10 column volumes (CV) of 2X PBS buffer and bound Nbs were eluted with 2X PBS buffer containing 2.5 mM desthiobiotin. The eluted Nbs were concentrated using a protein concentrator (Cytiva) with a molecular weight cutoff (MWCO) of 5kDa. The concentrated Nbs were applied to a superdex 200 10/300 GL column to further purify Nbs using size exclusion chromatography. The recombinant protein was concentrated to 1.0 mg/ml using 3 kDa protein concentrators (Pierce™), filter sterilized (0.2 µM), snap-frozen in liquid nitrogen, and stored at -80oC. Purified Nbs were analyzed by SDS-PAGE to confirm purity.
Expression and purification of chimeric heavy chain only antibodies (cHcAbs): The nucleotide sequences coding for cNb6 were fused in silico with the hinge and Fc domain of subclass B of canine IgG (36). A secretion signal from the V-J2-C region of the mouse Ig Kappa-chain followed by a Strep II Tag were also cloned at the N-terminus for efficient secretion and purification, respectively. The fused sequence was synthesized (Gene Universal) and cloned into a mammalian expression vector pCDNA3.1/Hygro (+). The recombinant plasmid was transiently transfected into ExpiCHO-S cells using ExpiFectamine™ CHO transfection kit (ThermoFisher). The ExpiCHO-S cells and conditioned media were harvested after 7 days. Following harvest, the supernatant containing the cHcAbs was clarified by centrifugation at 4000 x g for 30 minutes. The conditioned media was diluted (1:2) with binding buffer (20mM sodium phosphate) and applied to HiTrap protein A HP column (Cytiva) using AKTA explorer. The resin was washed with 10 column volumes of wash buffer (10mM sodium phosphate [pH 7.0]). Proteins were eluted with 100 mM sodium citrate (pH 3.0) directly into 1M Tris-Hcl (pH 8; 0.25 ml/ml elution). The purified cHcAbs were buffer exchanged into 1X PBS and applied to Superdex200 inrease10/300 GL column for size exclusion chromatography. The purified cHcAbs were concentrated using 10kDa protein concentrators (Pierce™) to a final concentration of 1.0 mg/ml, filter-sterilized (0.2 µM), snap-frozen in liquid nitrogen, and stored at -80°C. Endotoxin levels were measured using the ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (Genscript), and all cHcAbs contained less than five endotoxin units per milligram of protein.
Purification of canine peripheral blood mononuclear cells: 30 ml of peripheral blood was collected from three healthy dogs in heparinized tubes. The blood sample was diluted 1:1 with Dulbecco's phosphate buffer saline (DPBS). Two Sepmate™ PBMC isolation tubes were filled with 15 ml of histopaque. The diluted blood sample was carefully layered over the Sepmate™ insert. The layered blood was centrifuged at 800 x g for 10 minutes. The PBMCs layer was carefully collected and diluted with DPBS in a new 15ml conical tube. The cell suspension was mixed well and centrifuged at 800 x g for 10 minutes. The supernatant was discarded, and the cell pellet was resuspended and washed again with DPBS. After the supernatant was discarded, the cells were resuspended in complete RPMI-1640 media containing 10% fetal bovine serum (FBS) plus penicillin (200U/ml) and streptomycin (100 µg/ml).
SDS-PAGE and Western blotting
The expression and purity of eluted Nbs and chimeric HcAbs were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For western blotting, conditioned media containing cHcAbs was separated under reducing (beta-mercaptoethanol) and non-reducing conditions and transferred to the nitrocellulose membrane. The cHcAb6 band was detected with anti-Strep Tag II and rabbit anti-canine IgG Fc antibodies (Novus Biologicals). Secondary antibodies conjugated with IRDye680RD (LI-COR Biosciences) were used to detect the primary antibodies. Blots were scanned on LI-COR Odyssey 9120 digital scanning system.
Cell Culture and in vitro cell line generation
Stable cell lines were generated to assess the binding of anti-CTLA4 Nbs and cHcAbs. Briefly, MDCK cells were transfected with recombinant plasmids expressing canine FcγRI or CTLA4 using Lipofectamine 3000 (Life Technologies) according to the manufacturer’s directions. Cells were selected with hygromycin and flow-sorted to obtain single-cell clones. Clones expressing a high level of CTLA4 and FcγRI were used for flow cytometry. MDCK, MDCK cell expressing FcγRI (MDCK-FcγRI) or CTLA4 (MDCK-CTLA4) were maintained in DMEM media supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO2.
MDCK, MDCK-FcγRI and MDCK-CTLA4 cells were trypsinized and washed with FACS buffer (1X PBS containing 1% bovine serum albumin (BSA), 2mM EDTA, 0.02% sodium azide, and HEPES). Cells were incubated in blocking buffer (PBS containing 4% goat or rabbit serum, 1% BSA, 0.02% sodium azide) and stained with anti-CTLA4 Nbs and cHcAbs. Bound Nbs or cHcAbs were detected by using anti-His 647 or anti-canine IgG Fc 647 or 750 secondary antibodies and analyzed by flow cytometry. Canine PBMC were cultured overnight in RPMI media. PBMCs were stimulated with various concentrations (50, 100 & 150 ng/ml) of PMA and Ionomycin (1µM/ml final concentration) for 6 hours. Stimulated PBMCs were blocked and stained with anti-CTLA4 Nbs and cHcAbs. Cells were also stained with anti-CD3FITC (clone CA17.2A12), anti-CD4RPE (clone YKIX 302.9), and anti-CD8 647 (clone YCATE55.9) antibodies to characterize subpopulations expressing canine CTLA4. For intracellular FOXP3 staining, cells were fixed and permeabilized using the fix & perm cell fixation and cell permeabilization kit from Life Technologies and stained with FOXP3 monoclonal antibody (FJK-16s [ThermoFisher]).
In vitro functional assays
Canine PBMCs isolated from three healthy dogs were stimulated with anti-CD3 antibody (1µg/ml) in the presence or absence of cHcAb6 (100 nM). After three days, total RNA was isolated, reverse-transcribed, and quantified for IFN-γ mRNA levels using TaqMan assays (Cf02623316_m1). All assays were performed in triplicates, and HPRT1 (Cf02690456_g1) was used as endogenous control. Canine IgG was used as isotype control.
Protein structure modeling
The complementarity determining regions (CDRs) of the nanobodies cNb6, cNb13, and cNb17 were determined from their genetic sequences using the multiple sequence alignment tool Clustal Omega(37), EMBL-EBI (38). The structures of the three anti-CTLA4 Nbs, the extracellular domain of canine CTLA4, and the entirety of canine CTLA4 were predicted from their genetic sequences using I-Tasser (39), Robetta (40), and trRosetta (41) (15 total predicted structures). As the computational methods are predictions, multiple methods were utilized to provide insight into whether there was consensus or differences among varying approaches. The ZDOCK server (42) was used to globally dock the individual Nbs predicted by each method with the corresponding canine CTLA4 extracellular domains. 30 results were generated for each of the protein prediction methods (10 per Nb per method, 90 total predicted complexes). These complexes were observed using the molecular visualization tool UCSF Chimera (43).
All experiments were performed in triplicates. The quantitative PCR data was analysed using a two-samples t-test. All statistical analysis was performed with SAS. All statistical tests were two-sided, and a P-value of < 0.05 was regarded as statically significant.