Cell culture
SUP-T1 cells were purchased from ATCC (Wesel, Germany). The MC38 cell line was kindly provided by Massimiliano Mazzone (VIB-KU Leuven, Belgium). Primary PBMCs of healthy volunteers were kindly provided by Karine Breckpot (Vrije Universiteit Brussels, Belgium). All cells were grown at 5% CO2 and 37°C. SUP-T1 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with 1% Penicillin/Streptomycin (Gibco, Thermo Fisher Scientific) and 10% Fetal Bovine Serum (FBS, Serana, Pessin, Germany). MC38 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Thermo Fisher Scientific) supplemented with 1% Penicillin/Streptomycin and 10% FBS. Primary PBMCs were grown in Iscove's Modified Dulbecco's Medium (IMDM, Gibco, Thermo Fisher Scientific) supplemented with 1% Penicillin/Streptomycin, 10% human AB serum (ZenBio, Durham, NC, USA).
Animal models
Male and female wild type C57BL6/J mice, nu(ncr)-foxn1nu and human CD8 transgenic mice (B6;SJL-Tg(CD8αCD8β)57Scr/J) were purchased from Charles River (Ecully, France) and Jackson laboratory (Bar Harbor, ME, USA), respectively. In the case of imaging of tumor-bearing mice, mice were subcutaneously injected with 1 million MC38 or 5 million SUP-T1 cells in the flank. In the case of SUP-T1 cells, cells were resuspended in 50% Matrigel (Corning, Somerville, MA, USA) prior to inoculation. Mice were examined daily and tumor growth was measured using a caliper. Tumor volume was calculated using the formula (length × width2)/2. All mouse experiments were approved by the Ethical Committee for laboratory animals of the Vrije Universiteit Brussel and executed in accordance with the European guidelines for animal experimentation (ethical dossier number 21-272-1).
Two young adult male cynomolgus macaques (Macaca fascicularis), aged 4 and 5 years, F2 generation originating from Mauritian AAALAC certified breeding centers, were used in this study. Cynomolgus macaques were housed at the IDMIT infrastructure facilities (CEA, Fontenay-aux-roses, France) under BSL-3 containment (Animal facility authorization #D92-032-02, Préfecture des Hauts de Seine, France) and in compliance with European Directive 2010/63/EU, French regulations, and the Standards for Humane Care and Use of Laboratory Animals of the Office for Laboratory Animal Welfare (OLAW, assurance number #A5826-01, US). The protocols were approved by the institutional ethical committee ‘Comité d’Ethique en Expérimentation Animale du Commissariat à l’Energie Atomique et aux Energies Alternatives’ (CEtEA number 44) under statement number A23-057. The study was authorized by the ‘‘Research, Innovation and Education Ministry’’ under registration number APAFIS #46283-202312131546674 v1.
Nanobody generation, selection and production
Two llamas were subcutaneously injected 6 times with 100 µg recombinant human (h)CD8β-Avi-His6 (U-Protein Express BV, Utrecht, The Netherlands) and 100 µg recombinant human CD8β-hIgG1 Fc (Sino Biological, Eschborn, Germany) mixed with Gerbu adjuvant P (Gerbu Biotechnik, Heidelberg, Germany) on a weekly basis. After immunizations, peripheral blood of both llamas was collected and peripheral blood mononuclear cells were isolated using lymphoprep tubes (Greiner Bio-one, Kremsmunster, Austria). RNA was isolated from peripheral blood lymphocytes using an RNA extraction kit (Qiagen, Hilden, Germany) and reverse transcribed into cDNA. Next, genes coding for the variable domain of the heavy-chain only antibodies were amplified and ligated into the pMECS phage vector[15] resulting in 2 separate phage display libraries. Subsequent biopanning was performed by infection of the libraries with M13K07 helper phages, resulting in phage production. For each library, 3 rounds of panning in solution were performed using in-house site-specifically biotinylated hCD8β-Avi-His6 protein. For rounds 1 and 2, 100 nM antigen was used while 10 nM antigen was used during the final round of panning. In total, 190 unique clones (95 from round 2 and 95 from round 3) were randomly selected and screened for their ability to specifically bind to hCD8β via ELISA. Specific binding was determined via ELISA using site-specifically biotinylated hCD8β-Avi-His6 protein, immobilized on a streptavidin-coated 96-well plate (Thermo Fisher Scientific). Positive hits were sent for sequencing (Eurofins genomics, Ebersberg, Germany) and grouped into different B cell lineages based on the CDR3 sequence. Nanobodies were produced and purified as previously described [16].
Surface plasmon resonance
The affinity of purified anti-hCD8β nanobody to recombinant hCD8αβ protein (Sino Biological) was determined using a BIACORE-T200 device (Cytiva, Freiburg, Germany). The CD8αβ protein was immobilized on a CM5 chip (Cytiva) in 10 mM sodium acetate pH 4.5 via amine coupling chemistry to reach a final change in response units (RU) of 600 RU. Surface plasmon resonance measurements were performed at 25°C with HEPES buffered saline (HBS, 10 mM of HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween-20) running buffer. The nanobody was injected sequentially, in a 2-fold serial dilution, starting from 0.98 to 250 nM at 30 µL/min. For each concentration cycle, an association step of 120 s was followed by a dissociation step of 600 s, a regeneration pulse of 60 s, using 100 mM glycine at pH 2.0, and a stabilization time of 180 s. For the single cycle kinetics measurements, increasing concentrations of a 4-fold dilution of the nanobody, ranging from 0.49 to 125 nM, were consecutively injected for 180 s each, followed by a single dissociation phase of 600 s. After each binding cycle an identical regeneration pulse was performed followed by a stabilization period of 180 s. Local curve fitting analysis was performed using the BIACORE evaluation software (Cytiva) by fitting the obtained sensorgrams to theoretical curves, assuming 1–1 binding geometries. For the determination of the equilibrium dissociation constant, the ratio of the association and dissociation rate constants was determined.
Affinity determination via flow cytometry
Serial dilutions of the anti-hCD8β nanobody were incubated with 500.000 SUP-T1 cells in FACS buffer (HBSS (Gibco, Thermo Fisher Scientific) supplemented with 1% FBS and 2mM EDTA (Duchefa Biochemie, Haarlem, The Netherlands)) for 1 h at 4°C. Cells were washed once with FACS buffer. Next, nanobody binding was detected by incubation of the cells with an Alexa Fluor®-488 tagged anti-HA antibody (1:1000 in FACS buffer, clone 16B12, Biolegend, San Diego, CA, USA) or PE-conjugated rabbit anti-camelid VHH cocktail (1:500 in FACS buffer, Genscript, Piscataway, New Jersey, USA) for 30 min at 4°C. Again, cells were washed once with FACS buffer. Nanobody binding was determined using the FACS CANTO II analyser (BD Biosciences, Franklin Lakes, NJ, USA). The mean fluorescence intensity of nanobody binding was determined using FlowJo version 10.
Affinity determination via ELISA
Wells of a 96 well MicroWell MaxiSorp flat bottom plate (Thermo Fisher Scientific) were coated with 0.2µg of recombinant hCD8αβ protein, 0.2µg of cynomolgus CD8β-Fc protein (Sino Biologicals) or PBS overnight at 4°C. The next day, wells were washed 3 times with PBS-T (PBS + 0.05% Tween20 (Merck-Millipore, Burlington, MA, USA). Next, wells were blocked with blocking buffer (2% skimmed milk powder (Régilait) in PBS) for 1 h at room temperature (RT). Different concentrations of nanobodies, diluted in blocking buffer, were added to the wells and incubated for 1 h at RT. Nanobody binding was detected using a mouse-anti-HA antibody (1:2000, clone 16B12, Biolegend) and alkaline-phosphatase conjugated goat-anti-mouse antibody (1:2000, clone A90-116AP, Bethyl Laboratories, Montgomery, TX, USA). Wells were washed 5 times with PBS-T between all incubation steps. Binding was determined using p-nitrophenyl phosphate (2mg/mL resuspended in AP blot buffer (12.12 g/L Trizma base, 10.17g/L MgCl2.6H20, 5.84g/L NaCl, pH 9.5); Thermo Fisher Scientific). Absorbance at 405 nm was measured via a VersaMax ELISA Microplate Reader, using the SoftMax® Pro software (Molecular Devices, San Jose, CA, USA).
Thermal shift assay
The anti-hCD8β nanobody (concentration 0.2 mg/mL) was mixed with 1x SYPRO™ Orange Protein Gel Stain (Thermo Fisher Scientific) in PBS and added to white 96-well PCRs plates (Bio Rad, Pleasanton, CA, USA). Fluorescence signal was measured during increasing temperature steps ranging from 20 to 95°C, with stepwise increments of 0.5°C, using CFX connect™ Real-Time PCR (Bio Rad). The melting temperature of the nanobody was calculated using the Boltzmann equation.
Nanobody binding to primary PBMCs
One day prior to the analysis, primary peripheral blood mononuclear cells were thawed and taken into culture. The next day, cells were resuspended in HBSS and 500.000 cells were taken for each sample. Cells were stained with eBioscience™ Fixable Viability Dye eFluor™ 506 (1:1000 in HBSS; Thermo Fisher Scientific) for 30 min at 4°C. Cells were washed once with FACS buffer. Next, samples were incubated with human FcR blocking agent (Miltenyi Biotec, Bergisch Gladbach, Germany) diluted in FACS buffer according to manufacturer’s protocol for 10min at 4°C. Next, 100 nM of anti-hCD8α (clone R3HCD27, patent US20190071500A1), anti-hCD8β or irrelevant nanobody were added for 1 h at 4°C. Cells were washed once with FACS buffer and incubated with a mix of fluorescent antibodies (Table 1) for 30 min at 4°C. Cells were washed once again with FACS buffer before nanobody binding was determined using the FACS CANTO II analyser. Analysis of the nanobody binding was performed using FlowJo version 10.
Immunofluorescence staining of non-human primate tissue
Non-human primate lymph node tissues were fixed in Gerner buffer (0.1 M L-Lysine, 2 mg/mL NaIO4, 4% formaldehyde and 0.05 M Phosphate buffer) overnight at 4°C followed by dehydration in 30% sucrose for 24h. Next, samples were embedded in Optimal Compound Temperature mounting medium (OCT; VWR International, Radnor, PA, USA) and frozen in liquid nitrogen cooled iso-pentane. Tissue sections of 7 µm were cut using a Cryostat (Leica Biosystems) and mounted on Superfrost®Plus Gold glass slides (VWR International). Tissues were washed in wash buffer (0.05% Tween-20 in PBS) followed by incubation in permeabilization buffer (0.3% Triton X-100 in PBS) for 30 min at RT. Next, tissues were washed with PBS and incubated in blocking buffer (bovine serum albumin (BSA) in PBS) for 30 min at RT. Following another rinse step with PBS, tissues were incubated with saturation buffer (0.2% BSA in PBS) before overnight incubation at 4°C with Alexa Fluor®-647 conjugated anti-hCD8β (10 µg/mL) or irrelevant nanobody (10 µg/mL) and primary CD3 antibody (5 µg/mL; Clone SP34.2, BD Biosciences) in Discovery Antibody diluent (Ventana, Roche). Subsequently, following a rinse with PBS and wash buffer, tissues were incubated with a Alexa Fluor®-594-conjugated goat anti-mouse IgG1 secondary antibody (1 µg/mL diluted in Discovery Ab diluent; Invitrogen) for 4 hours at RT. Next, tissues were washed with wash buffer and fixed with 4% formaldehyde for 15 min at RT. Fixed tissues were repeatedly washed with PBS and stained with 4′,6-diamidino-2-phenylindole (DAPI, 1:50.000 in PBS; Invitrogen) for 20 min at RT. Stained tissues were washed to remove excess dye and mounted with anti-fade mounting medium (ProLong™ Gold Antifade Mountant, Thermo Fisher) before being imaged using an automatic wide-field microscope (AxioScan Series 7, Zeiss).
T-cell activation assay
One day prior to the analysis, primary peripheral blood mononuclear cells were thawed and taken into culture. The next day, 1 million cells were incubated with anti-CD3/CD28 dynabeads (Thermo Fisher) or 300 nM of GLP-grade anti-hCD8β or irrelevant nanobody for 24 h. The next day, cells were spun down and culture medium was collected. Secreted IFN-γ levels in the medium were determined using the human IFN-γ DUOset ELISA (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. Cells were stained with fluorescent antibodies (Table 4) for flow cytometry analysis as described above. Flow cytometry was performed using the FACS CANTO II analyser. Analysis was performed using FlowJo version 10.
Dendritic cell/T cell restimulation experiments
Immunogenicity of the nanobodies was determined via a dendritic cell/T cell restimulation assay and was outsourced to Lonza (Basel, Switzerland). The assay was performed using PBMCs of 30 pre-HLA-typed healthy donors as described previously [17].
99mTc-radiolabeling of nanobodies
Nanobodies were labeled with 99mTc as previously described [18]. Briefly, 99mTc-tricarbonyl was generated via the addition of 150 mCi 99mTcO4− to the Isolink® labelling kit (Paul Scherrer Institute, Villigen, Switzerland) for 20 min at 100°C. Next, 50 µg of His-tagged nanobody was added and incubated for 90 min at 50°C. 99mTc-labeled nanobodies were purified via gel filtration from the unbound [99mTc(H2O)3(CO)3] + via a NAP-5 column (Cytiva) and filtered through a Millex 0.22 µm filter (Millipore, Haren, Belgium). The radiochemical purity of radiolabeled nanobodies was evaluated by instant thin layer chromatography (iTLC, Pall Corporation, Hoegaarden, Belgium)
SPECT-CT imaging and image analysis
Mice were injected i.v. with 5 µg of radiolabeled (± 37 MBq) nanobody. One hour post injection, mice were anesthetized with 75 mg/kg ketamine and 1 mg/kg medetomidine (Ketamidor, Richter Pharma AG, Weis, Austria) via intraperitoneal injection and SPECT/micro-CT imaging was performed using a Vector+ scanner (MiLABS, Houten, The Netherlands). Imaging set-up consisted of a 1.5 mm 75-pinhole general-purpose collimator, in spiral mode with 6 bed positions. Total SPECT scanning time was 15 minutes with 150 seconds per position and CT scanning (60 kV and 615 mA) was 2 minutes. After imaging, mice were euthanized and organs were collected. Radioactivity in each organ was determined using a Wizard2 γ-counter (Perkin-Elmer, Waltham, MA, USA). Uptake in each organ was corrected for radioactive decay and calculated as percentage of injected activity per gram of organ. SPECT/CT image analysis was performed using AMIDE (UCLA, CA, USA) and OsiriX (Pixmea, Geneva, Switzerland) software.
Alphafold nanobody binding prediction
Nanobody binding models to human CD8 were generated using Colabfold (patch v1.5.2) [19]. The input query sequence included the extracellular part of the human CD8α and human CD8β chain and the amino acid sequence of the anti-hCD8β Nb. The number of recycles were set to 6 while all other standard parameters were unchanged. Analysis of the Alphafold model was done using pyMOL.
NOTA-conjugation of nanobodies
The conjugation of the anti-hCD8β nanobody to p-SCN-Bn-NOTA (NOTA-NCS, Macrocyclics, Inc., Plano, TX, USA) was based on the standard protocol previously described with some adaptations [20]. The nanobody was first buffer-exchanged to 0.25 M sodium carbonate adjusted to pH 9.25 (sodium carbonate anhydrous; sodium hydrogen carbonate; sodium chloride, VWR Chemicals, Leuven, Belgium) using a PD-10 size exclusion column (Cytiva). A 20-fold molar excess of NOTA-NCS was added to the nanobody solution and incubated for 2h30 at RT. After incubation, the NOTA-nanobody was purified via size exclusion chromatography (SEC) on a Hiload™ 16/600 Superdex™ 30 pg column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) with 0.1 M NaOAc as the mobile phase (0.8 mL/min) to separate the conjugated nanobody from excess NOTA-NCS. The concentrations of the collected NOTA-nanobody fractions were measured spectrophotometrically using a Nanodrop 2000 by UV absorption at 280 nm. In addition, SEC with a Superdex Peptide 10/300 GL column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) was performed for quality control of the NOTA-nanobody. The number of chelates per nanobody was determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS). After determining the chelator-to-nanobody ratio, anion exchange chromatography (AEX) was performed using an ENrich Q 5 × 50 column (Bio-Rad Laboratories, Inc., California, CA, USA) with 0.02 M Tris (VWR Chemicals, Leuven, Belgium) adjusted to pH 7.5 as solvent A and 0.02 M Tris with 0.31 M NaCl as solvent B (1.5 mL/min) to determine the fractions with different chelator-to-nanobody ratios. Based on these results, a 1:1 chelator-to-nanobody ratio was used for further radiolabeling.
68Ga-radiolabeling of nanobodies
The NOTA-conjugated nanobody (7.8 nmol for anti-hCD8β nanobody and 7.2 nmol for the irrelevant nanobody) was added to 1 mL of 1 M NaOAc buffer pH 5 and 1 mL of Gallium-68 (68Ga) eluate (424–636 MBq) eluted from a 68Ge/68Ga generator in 0.1 M HCl (Galli Eo™, IRE ELiT, Fleurus, Belgium) and incubated for 10 min at RT. Purification was performed on a PD-10 desalting column pre-equilibrated with 1x PBS in case of the test-labeling or 0.9% NaCl containing 5 mg/mL vitamin C pH 5.8–6.1 (injection buffer) for stability and in vivo studies. After purification, the radioactive nanobody solution was filtered through a 0.22 µm filter (Millipore, Belgium). The radiochemical purity was assessed before and after purification by radio-iTLC ([68Ga]Ga-NOTA-nanobody Rf = 0, [68Ga]Ga-citrate Rf = 1). Radiometal chelation stability of the radiolabeled nanobody was assessed in different conditions (injection buffer (0.9%NaCl + 5 mg/mL Vitamin C) at RT, 37°C; human serum 37°C) at 30min, 60min, 120min and 180min after labeling. Stability of the radiolabeled compound was analyzed via radio-iTLC and radio-SEC at these timepoints.
PET-CT imaging and image analysis in mice
Mice were injected (i.v.) with 5 µg of radiolabeled nanobody (15.5 ± 0.34 MBq). One hour post injection, mice were anesthetized with 75 mg/kg ketamine and 1 mg/kg medetomidine via intraperitoneal injection or isoflurane (5% induction, 2.5% maintenance, oxygen flow rate between 0.3 and 1.5 L/min; Virbac, Nice, France) via inhalation and PET/CT Imaging was performed (MoleCubes, Gent, Belgium). PET scans of 12–20 min were performed followed by a CT scan. After imaging, mice were euthanized and organs were collected. Radioactivity in each organ was measured using a Wizard2 γ-counter (Perkin-Elmer). Uptake in each organ was corrected for radioactive decay and calculated as percentage of injected activity per gram of organ. PET/CT image analysis was performed using VivoQuant software (Invicro, Needham, MA, USA).
Processing organs and flow cytometry analysis
Single cell preparations of MC38 tumors were prepared as described previously [21]. Antibodies used for staining of single cell preparations can be found in Table 1. Delta median fluorescence intensity (ΔMFI) was determined via subtraction of the MFI of the staining and the MFI of the isotype control. Data were acquired using the FACS CANTO II or FACS CELESTA analyser and analyzed using FlowJo software.
64 Cu-radiolabeling of nanobodies
Copper-64 (64Cu) in 1 M HCl (1 GBq, 375 µL, ARRONAX, Nantes, France) was concentrated at 90°C under an argon stream to dryness. NaOAc buffer 0.1 M was prepared and the pH was adjusted to 6.5 using HCl. Then, 365 µL of sodium acetate buffer was added to solubilized 64CuCl2 and this solution was transferred to NOTA-hCD8β Nb. The resulting mixture was stirred at 500 rpm in a thermoshaker at 37°C. Radio-TLC was performed, using 50 mM citric acid as eluent, to monitor the reaction. Full conversion was observed after 1 h (Rf = 0.05) as no residual free 64Cu was observed (Rf = 0.9). In the meantime, a PD-10 column (GE Healthcare, USA) was rinsed with 20 mL of PBS. The reaction mixture was loaded on the column and the flow-through was discarded. PBS was used as eluent and the flow-through was collected in 500 µL fractions. Radioactivities were measured in a dose calibrator (Capintec®, Berthold, France), fractions showing the highest activities were pooled together and analyzed by SEC chromatography (Alliance e2695 system, Waters, USA). Radiolabeled nanobodies were identified as radioactive peak detected by a gamma detector (Berthold, France).
PET-CT imaging and image analysis of macaques
All imaging acquisition was performed using the Digital Photon Counting (DPC) PET-CT system (Vereos-Ingenuity, Philips). Animals were first anesthetized with 10mg/kg ketamine and 0.05mg/kg medetomidine, intubated, and then maintained under 0.5-1% isoflurane and placed in a supine position on a warming blanket (Bear Hugger, 3M) on the machine bed with monitoring of the cardiac rate, oxygen saturation, and temperature. The CT detector collimation used was 64 × 0.6 mm, the tube voltage was 120 kV, and the intensity was approximately 150 mA. Whole-body CT images were reconstructed with a slice thickness of 1.5 mm and an interval of 0.75 mm. A whole-body PET scan (5 bed positions, 1 min/bed position) was performed approximately 60 min post-injection of 500 µg of 64Cu-radiolabeled nanobodies via the saphenous vein (230 ± 23 MBq, 5 mL). PET images were reconstructed onto a 256 x 256 matrix using OSEM (3 iterations, 15 subsets). PET and CT images were analyzed using INTELLISPACE PORTAL 8 (Philips Healthcare) and 3DSlicer (open-source tool) software. For segmentation, various regions of interest were semi-automatically contoured according to anatomical information and PET signal. A 3D volume of interest (VOI) was interpolated from several ROIs in different image slices to cover the entire organ or anatomical structure. Mean radioactive signal in each VOI was expressed in mean standardized uptake value (SUVmean ± SD).
Table 1
Overview of antibodies used for flow cytometry
Target | Fluorophore | Species reactivity | Provider | Clone |
CD11b | PE/Cyanine7 | Human, Mouse | Biolegend | M1/70 |
His-tag | APC | / | Miltenyi Biotec | GG11-8F3.5.1 |
CD8 | APC | Human | BD Bioscience | 2ST8.5H7 |
CD8 Isotype control | APC | / | BD Bioscience | G155-178 |
CD69 | APC | Human | Biolegend | FN50 |
CD4 | PerCP/Cyanine5.5 | Human | Biolegend | RPA-T4 |
CD4 | PerCP/Cyanine5.5 | Mouse | Biolegend | GK1.5 |
CD56 | PerCP/Cyanine5.5 | Human | Biolegend | QA18A21 |
CD3 | FITC | Human | eBioscience | SK7 |
TCR beta | FITC | Mouse | eBioscience | H57-597 |
CD19 | PE | Human | Biolegend | SJ25C1 |
CD19 | PE | Mouse | eBioscience | 1D3 |
CD45 | APC/Cyanine7 | Human | Biolegend | HI30 |
CD45 | APC/Cyanine7 | Mouse | Biolegend | 30-F11 |
CD8 | Brilliant violet 421 | Human | Biolegend | RPA-T8 |
CD8 | Brilliant violet 421 | Mouse | Biolegend | 53 − 6.7 |