Specific Imaging of CD8+ T-Cell Dynamics with a Nanobody Radiotracer against Human CD8β

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De Groof, Yoline Lauwers, Tessa De Pauw, Mohit Saxena, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4322357/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose While immunotherapy has revolutionized the oncology field, variations in therapy responsiveness limit the broad applicability of these therapies. Diagnostic imaging of immune cell, and specifically CD8 + T cell, dynamics could allow early patient stratification and result in improved therapy efficacy and safety. In this study, we report the development of a nanobody-based immunotracer for non-invasive SPECT and PET imaging of human CD8 + T-cell dynamics. Methods Nanobodies targeting human CD8β were generated via llama immunizations and subsequent biopanning. The lead anti-human CD8β nanobody was characterized in vitro on binding, specificity, stability and toxicity. The lead nanobody was labelled with 99m Tc and 68 Ga for non-invasive imaging of human T-cell lymphomas and CD8 + T cells in human CD8 transgenic mice and non-human primates via SPECT or PET/CT. Repeated imaging of CD8 + T cells in MC38 tumor-bearing mice was performed to visualize CD8 + T-cell dynamics. Results The nanobody-based immunotracer showed high affinity and specific binding to human CD8 without unwanted immune activation. CD8 + T cells were non-invasively visualized via SPECT and PET imaging in naïve and tumor-bearing mice and in naïve non-human primates with high sensitivity. The nanobody-based immunotracer showed enhanced specificity for CD8 + T cells and/or faster in vivo pharmacokinetics compared to previous human CD8-targeting immunotracers, allowing us to follow human CD8 + T-cell dynamics already at early timepoints. Conclusion Overall, this study describes the development of a more specific human CD8 + T-cell-targeting immunotracer, allowing follow up of immunotherapy responses via non-invasive imaging of human CD8 + T-cell dynamics. Nuclear Medicine & Medical Imaging Immuno-imaging nuclear imaging nanobodies T-cell dynamics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction In the last decade, immunotherapy has revolutionized the oncology field and has become the standard of care for some cancer types [ 1 ]. However, variations in therapy responsiveness and side effects between patients limit the broad applicability and effectiveness of these therapies [ 2 ]. Diagnostic imaging of immune cell dynamics during immunotherapy could allow early patient stratification and, as such, result in improvements regarding therapy efficacy, safety and therapy costs [ 3 ]. Consequently, the development of diagnostic imaging tracers, capable of non-invasively visualizing distinct immune cell populations, has gained a lot of interest over the last years. Among the key players in immunotherapy responses, cytotoxic CD8 + T cells have received particular attention [ 4 , 5 ]. As a result, several PET imaging tracers have been developed to target murine or human CD8 + T cells, facilitating the monitoring and/or prediction of immunotherapy responses [ 5 – 8 ]. However, most of these tracers suffer from suboptimal pharmacokinetics (i.e. long circulation time), necessitating the use of long-lived radionuclides and potentially raising concerns regarding radiotoxicity [ 3 , 9 ]. Furthermore, all of these tracers bind the CD8α-chain, which is also expressed on other types of immune cells, decreasing the tracer's specificity for CD8 + T cells [ 10 , 11 ]. Nanobodies (Nbs), the variable region fragments of camelidae heavy chain-only antibodies, have emerged as interesting scaffolds for the development of diagnostic tracers [ 12 ]. Nbs are smaller (15 kDa) than conventional monoclonal antibodies, while still maintaining their ability to bind their target with high affinity and specificity [ 13 ]. This results in rapid tumor uptake and blood clearance, allowing fast and specific imaging of immune cells within the tumor microenvironment and a lower radiation burden [ 14 ]. In this study, we describe the development of a radiolabeled anti-human (h)CD8β Nb, that allows the non-invasive imaging of human CD8 + T cells via SPECT and PET imaging. These Nb-based tracers show high affinity and specific binding to CD8 + T cells and ideal in vivo pharmacokinetics. Furthermore, we show the ability of these Nb-based tracers to follow up immune cell dynamics during tumor growth. Overall, this study describes the development of a more specific hCD8 + T-cell-targeting tracers, allowing the follow-up of hCD8 + T-cell dynamics via non-invasive imaging. Materials and Methods 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% CO 2 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 × width 2 )/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-His 6 (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-His 6 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-His 6 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 NaIO 4 , 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 ]. 99m Tc-radiolabeling of nanobodies Nanobodies were labeled with 99m Tc as previously described [ 18 ]. Briefly, 99m Tc-tricarbonyl was generated via the addition of 150 mCi 99m TcO 4 − 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. 99m Tc-labeled nanobodies were purified via gel filtration from the unbound [ 99m Tc(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 Wizard 2 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. 68 Ga-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 ( 68 Ga) eluate (424–636 MBq) eluted from a 68 Ge/ 68 Ga 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 ([ 68 Ga]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 Wizard 2 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 ( 64 Cu) 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 64 CuCl2 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 64 Cu 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 64 Cu-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 Results The anti-human CD8β nanobody binds human CD8 with high affinity and has a good safety profile To identify Nbs against the extracellular part of the hCD8β chain, two llamas were immunized with recombinant hCD8β protein. Subsequent phage display panning and screening resulted in 175 unique Nbs, belonging to 33 different B-cell lineages. Further characterization of these Nbs resulted in the selection of a lead anti-hCD8β Nb. The anti-hCD8β Nb displayed low nanomolar binding affinity to the hCD8αβ protein as shown via surface plasmon resonance (Fig. 1 a and Table 2 ). In line with this, a similar binding affinity to SUP-T1 cells, a T-cell lymphoma cell line endogenously expressing hCD8αβ, was seen in flow cytometry, while no binding was observed with an irrelevant Nb, binding the 5T2 multiple myeloma M protein (Fig. 1 b and Table 2 ). Next, we assessed the cross-reactive binding of the anti-hCD8β Nb (Supplemental Fig. 1 and Table 2 ). While no Nb binding to murine CD8β protein was observed (Supplemental Fig. 1a), equilibrium binding ELISA with the anti-hCD8β Nb did indicate similar low nanomolar binding affinities to the human and cynomolgus CD8β proteins (Supplemental Fig. 1b). This result is in line with the poor homology between the human and mouse proteins (50%), and the very high homology between human and cynomolgus CD8β proteins (93%). Subsequently, the thermostability of the Nb was determined, considering that the Nb will be incubated at higher temperatures during radiolabeling (Fig. 1 c and Table 2 ). The anti-hCD8β Nb displayed a melting temperature of 68.1 ± 0.3°C. Finally, the binding epitope of the anti-hCD8β Nb was modelled using Alphafold (Fig. 1 d). Although the predicted local distance difference test (pLDDT) score of the model indicated that the exact interaction between the CDR regions of the Nbs and CD8β could only be estimated (Supplemental Fig. 2), the Alphafold model nicely predicted binding to the β chain of human CD8. Table 2 Overview of the in vitro characteristics of the anti-human CD8β nanobody (hCD8β Nb). Data are presented as mean ± S.D of at least 3 independent experiments. Nb K D hCD8αβ protein (nM) SPR K D SUP-T1 cells (nM) Flow cytometry K D hCD8αβ protein ELISA (nM) K D cynomolgus CD8β protein ELISA (nM) Melting temperature (°C) hCD8β Nb 1.0 ± 0.1 16.0 ± 9.0 2.4 ± 0.2 7.2 ± 1.4 68.1 ± 0.3 SPR: Surface plasmon resonance. Next, we determined the ability of the anti-hCD8β Nb to bind primary CD8 + T cells ex vivo (Fig. 2 a-b). While no binding was seen for the irrelevant Nb, the anti-hCD8β Nb bound the CD8 + T cell population within a pool of primary T cells. Moreover, the anti-hCD8β Nb did not show any binding to NK or myeloid cells, known to express hCD8α, but not hCD8β (Fig. 2 c and Supplemental Fig. 3). In contrast, binding of an anti-hCD8α Nb (WO2017134306A1) to NK or myeloid cells was observed. These data illustrate the superior specificity of the anti-hCD8β Nb for CD8 + T cells, as compared to the currently used hCD8α-targeting compounds. As the anti-hCD8β Nb showed cross-reactive binding to the cynomolgus CD8β protein, in vitro binding to non-human primate lymph node tissue was determined via fluorescence immunohistochemistry (Fig. 2 d). While no binding of the irrelevant Nb was observed, the anti-hCD8β Nb bound to CD3 + T cells present in the lymph node tissue. As binding of the anti-hCD8β Nb should not induce unwanted cytotoxicity, we next assessed the effect of Nb binding on T-cell activation. Hereto, primary human peripheral blood mononuclear cells (PBMCs) were incubated overnight with the anti-hCD8β Nb, irrelevant Nb or anti-CD3/CD28 dynabeads. The next day, T-cell activation was assessed via human CD69 expression, an early activation marker (Fig. 3 a-b), and via the secretion of IFN-γ (Fig. 3 c). Stimulation with anti-CD3/CD28 dynabeads resulted in a significant increase of CD69 expression and IFN-γ secretion by CD8 + T cells. In contrast, no changes in CD69 expression nor secreted IFN- γ levels were observed upon incubation with the anti-hCD8β Nb or the irrelevant Nb. Finally, the immunogenicity of the Nbs was determined via a dendritic cell-T cell co-culture assay using PBMCs of 30 independent healthy donors (Fig. 3 d-e). To this end, monocytes of the PBMC fractions were differentiated toward monocyte-derived dendritic cells (moDCs) followed by loading with the Nbs and maturation. Afterwards, CD4 + T cells were co-cultured with the loaded moDCs for 6 days. Freshly isolated monocytes were challenged again with the Nbs and added to the co-culture for an additional 2 days. Afterwards, IFN-γ (Th1 cytokine) and IL-5 (Th2 cytokine) levels were assessed. As negative and positive controls, we took along clinically benchmarked Bevacizumab and immunogenic KLH protein. In contrast to KLH, neither of the Nbs showed any sign of immunogenicity, as no significant increase of IFN-γ nor IL-5 levels was observed. 99m Tc-labeled anti-human CD8β nanobody non-invasively images CD8 + T cells in naïve and tumor-bearing mice Next, we determined the potential of the anti-hCD8β Nb to target and visualize hCD8 + T cells in vivo . To this end, the irrelevant Nb and anti-hCD8β Nb were site-specifically radiolabeled with Technetium-99m ( 99m Tc) via their C-terminal His-tag [ 18 ]. Both Nbs were successfully labeled with radiochemical purities above 90% after labeling and 99% after purification (Table 3 ). First, the targeting potential of the 99m Tc-radiolabeled Nbs was assessed in CD8 + SUP-T1 tumor-bearing nude mice, which lack endogenous T cells, by SPECT/CT imaging 1 hour post intravenous (i.v.) injection (Fig. 4 a). Both 99m Tc-radiolabeled Nbs showed a high uptake in the kidneys and the bladder, due to rapid blood clearance [ 12 ]. However, the 99m Tc-radiolabeled anti-hCD8β Nb showed a significantly higher uptake in the SUP-T1 tumors as compared to the 99m Tc-radiolabeled irrelevant Nb. Specific uptake of the 99m Tc-labeled anti-hCD8β Nb in tumors was further confirmed via ex vivo γ-counting and subsequent tumor-to-blood ratio calculation of isolated organs from SUP-T1 tumor-bearing mice (Fig. 4 b-c and Supplemental Fig. 4). These data suggest the ability of the 99m Tc-labeled anti-hCD8β Nb to visualize endogenously expressed hCD8. The in vivo specificity of the 99m Tc-labeled anti-hCD8β Nb was further determined by SPECT/CT imaging in C57BL/6 wild type (WT) and hCD8 transgenic mice (Fig. 4 d-e). The 99m Tc-labeled irrelevant Nb did not show any specific accumulation in both mice. In contrast, the 99m Tc-labeled anti-hCD8β Nb showed uptake in T-cell-rich organs such as lymph nodes, spleen, intestines and appendix of hCD8 transgenic, but not WT, mice. Again, these data were confirmed via ex vivo γ-counting of the isolated organs (Fig. 4 f-i and Supplemental Fig. 5). Finally, the ability of the 99m Tc-labeled anti-hCD8β Nb to target intratumoral T cells was assessed. To this end, hCD8 transgenic mice were subcutaneously inoculated with MC38 tumor cells. When the tumor size reached approximately 500 mm 3 , mice were injected i.v. with the 99m Tc-labeled Nbs and uptake in the different organs was assessed via SPECT/CT and ex vivo γ-counting. The 99m Tc-labeled anti-hCD8β Nb showed a significantly higher uptake in the tumor compared to the 99m Tc-labeled Irr Nb (Fig. 4 j-k). Moreover, the 99m Tc-labeled anti-hCD8β Nb showed a significantly higher tumor-to-blood ratio compared to the 99m Tc-labeled irrelevant Nb (Fig. 4 l) indicating that the anti-hCD8β Nb binds to intratumoral hCD8 + T cells. Table 3 Radiochemical purity of radiolabeled anti-human CD8β or irrelevant nanobody. The percentage of 99m Tc- or 68 Ga-labeled human CD8β-targeting nanobody (hCD8β Nb) or irrelevant nanobody (Irr Nb) after labeling (before purification) with 99m Tc or 68 Ga and after purification. Data are presented as mean ± S.D of at least 3 independent experiments. Nb 99m Tc RCP before purification (%) 99m Tc RCP after purification (%) 68 Ga RCP before purification (%) 68 Ga RCP after purification (%) hCD8β Nb 90.5 ± 6.5 99.8 ± 0.4 99.2 ± 0.3 99.4 ± 0.3 Irr Nb 92.7 ± 6.8 99.9 ± 0.1 98.6 ± 0.7 98.9 ± 0.8 RCP: Radiochemical purity The anti-human CD8β PET tracer visualizes human CD8 + T-cell dynamics in vivo As PET imaging enables a higher spatial resolution and is more commonly used in the clinic, the anti-hCD8β Nb was converted to a PET tracer. To this end, the Nb was first conjugated to NOTA on its lysines and subsequently radiolabeled with Gallium-68 ( 68 Ga). A radiochemical purity above 98% was observed even before purification (Table 3 ). In addition, the radiolabeled anti-hCD8β Nb remained stable after incubation in injection buffer and human serum, at room temperature or at 37°C (Supplemental Fig. 6). To ensure that NOTA-coupling did not interfere with Nb binding to hCD8, the binding affinity of the NOTA-coupled anti-hCD8β Nb was determined via surface plasmon resonance and flow cytometry (Fig. 5 a-b and Table 4 ). NOTA-coupling did not have a large impact on the binding of anti-hCD8β Nb (Fig. 5 a). In line with the surface plasmon resonance results, NOTA-coupling of the anti-hCD8β Nb did not impact binding to SUP-T1 cells (Fig. 5 b). Table 4 Overview of the in vitro characteristics of the NOTA-conjugated anti-human CD8β nanobody (hCD8β Nb). Data are presented as mean ± S.D of at least 3 independent experiments. Nb K D hCD8αβ protein (nM) SPR K D SUP-T1 cells (nM) Flow cytometry NOTA-hCD8β Nb 6.4 ± 2.1 9.3 ± 0.5 Next, the ability of 68 Ga-labeled anti-hCD8β Nb to target hCD8 + T cells in vivo was determined in naïve hCD8 transgenic mice. Similar to the 99m Tc-labeled variant, specific uptake of 68 Ga-labeled anti-hCD8β Nb in T-cell-rich organs was observed (Fig. 5 c-d) via PET/CT imaging and ex vivo γ-counting. Then, we assessed whether 68 Ga-labeled anti-hCD8β Nb was able to visualize T-cell dynamics. To this end, hCD8 transgenic animals were subcutaneously inoculated with MC38 tumor cells and were imaged over time (Fig. 6 a). Tumor growth and CD8 + T-cell dynamics were followed over a period of 18 days. Individual differences in tumor growth could be observed as of day 9 post inoculation (Fig. 6 b). Interestingly, PET imaging with the 68 Ga-labeled anti-hCD8β Nb at endpoint (day 15 or day 18) allowed, despite overall low levels of T-cell infiltration, the differentiation of mice with lower or higher levels of human CD8 + T-cell infiltration in the MC38 tumors (Fig. 6 c). These findings were quantified via analysis of the individual PET images, ex vivo γ-counting and flow cytometric immunophenotyping of the dissected tumors. Quantification of radioactive uptake in the tumor determined via the PET images nicely correlated with the radioactive uptake measured via ex vivo γ-counting of the dissected tumors (Fig. 6 d). In addition, a good correlation was observed between the radioactive uptake and the amount of intratumoral human CD8 + T cells (Fig. 6 e). Overall, these results show that, despite the low levels of intratumoral human CD8 + T cells, the differences in T-cell tumor infiltration can be visualized using the 68 Ga-labeled anti-hCD8β Nb. In addition to visualizing intratumoral T cells, we could also follow up T-cell dynamics in the lymph nodes. Baseline PET scans revealed already a significantly higher level of CD8 + T cells in the tumor-draining lymph node compared to the opposing inguinal lymph node (Fig. 6 f). To determine whether this difference was tumor specific, we analyzed the ratio of radioactive uptake between these lymph nodes and compared it to the ratio between opposing cervical lymph nodes (Fig. 6 g). Repeated imaging showed that a consistent significantly higher ratio of radioactive uptake was observed in the tumor-draining lymph node relative to the contralateral inguinal lymph node, compared to that between the cervical lymph nodes during the whole experiment. Finally, we assessed the prognostic value of the 68 Ga-labeled anti-hCD8β Nb (Fig. 6 h). To this end, we correlated the amount of intratumoral human CD8 + T cells present at baseline, as measured via PET imaging, and the increase of tumor growth, as determined via the ratio of tumor size at day 15 and baseline. First, mice showing higher radioactive uptake levels at baseline also showed higher uptake levels at endpoint (Fig. 6 d-e-h). Interestingly, a significant correlation was observed between the radioactive uptake measured at baseline and the tumor growth, indicating that 68 Ga-labeled anti-hCD8β Nb may hold prognostic value to differentiate the speed of tumor growth based on the amount of intratumoral hCD8 + T cells at early timepoints. Finally, we performed a proof-of-concept study to investigate the biodistribution profile of the anti-hCD8β Nb in healthy non-human primates. To this end, the NOTA-conjugated anti-hCD8β Nb was first radiolabeled with 64 Cu. Next, 500 µg of the 64 Cu-labeled anti-hCD8β Nb was injected intravenously in 2 cynomolgus monkeys followed by PET/CT imaging 1 hour post injection (Fig. 7 a). Similar to the mice, a quick renal clearance of the 64 Cu-labeled anti-hCD8β Nb was observed with a high uptake in the kidneys and bladder while no signal was observed in the blood (Fig. 7 b-c). Furthermore, only minor uptake in the liver was observed. In both animals, the 64 Cu-labeled anti-hCD8β Nb showed a similar uptake pattern with the ability to visualize both primary and secondary lymphoid organs, such as thymus, spleen, tonsils and a multitude of lymph nodes including the cervical, inguinal, axillary and tracheobronchial lymph nodes. Interestingly, both cynomolgus monkeys showed a high uptake around the mouth area. Furthermore, the dose of 500 µg of the 64 Cu-labeled anti-hCD8β Nb was well-tolerated. In conclusion, these results indicated the possibility to visualize CD8 + T cells in bigger animals. Discussion Here, we report the development of a novel immunotracer for the non-invasive imaging of hCD8 + T-cell dynamics via SPECT and/or PET. In-depth in vitro and in vivo characterization shows high affinity and specificity of these tracers towards human CD8 + T cells and their ability to non-invasively visualize T-cell dynamics with high sensitivity. As T-cell-based immunotherapies have been a major point of focus in anti-cancer therapies, non-invasive imaging of T-cell dynamics has gained quite some interest in recent years. To this end, several tracers targeting different T-cell markers (e.g. CD3, CD4, CTLA-4, PD-1, LAG-3, TIGIT,etc.) have been reported [ 3 ]. In particular, imaging of cytotoxic CD8 + T cells has received attention as this T-cell population is believed to hold predictive and/or prognostic value [ 3 , 9 ]. In this study, we show the ability of our novel nanobody-based immunotracer to detect human CD8 + T cells using SPECT and PET scanning. Nanobodies have emerged as an interesting targeting scaffold for diagnostic imaging due to their in vivo characteristics, resulting in a fast tissue-to-background contrast, enabling same-day imaging of patients with short-lived isotopes [ 12 ]. Our study also shows the ability of nanobody-based immunotracers to visualize CD8 + T cells already 1h post injection via the use of short-lived isotopes such as 99m Tc (for SPECT imaging) and 68 Ga or 64 Cu (for PET imaging). Both isotopes are currently being employed as part of clinically tested tracers, showing translatability of our developed immunotracers [ 22 – 25 ]. Furthermore, the use of these short-lived isotopes results in a lower radiation burden for patients compared to long-lived isotopes, such as 89 Zr, which are used for bigger antibody-(fragment-)based tracers [ 26 , 27 ]. All in vitro results show a high affinity and specific binding of the anti-human CD8β Nb. Furthermore, the human CD8β-targeting Nb does not cause unwanted T-cell activation. That is important, since CD8 is an important mediator of T-cell activation and previous studies indicated that binding of CD8-targeting antibodies may induce T-cell activation [ 28 , 29 ]. Furthermore, no immunogenic responses towards the Nb were observed in our T cell/DC co-culture assays. Although Nbs are assumed to be non-immunogenic due to their small size, high homology to human VH fragments and their fast half-life time [ 30 , 31 ], patients developing anti-drug antibodies after Nb treatments have been reported and the immunogenic profiles of the Nbs have to be evaluated on an individual basis [ 32 , 33 ]. In the future, immunogenic responses towards the NOTA-conjugated immunotracers will also need to be assessed during clinical testing. However, immunogenic responses are expected to be low, since diagnostic tracers can be microdosed [ 26 , 34 ]. This notion is corroborated by previous findings, showing that a HER2-targeted Nb-based tracer used in a phase I clinical trial had a low immunogenicity [ 30 ]. Together, these are important parameters for future clinical translatability of this tracer. To date, a few hCD8-targeting tracers have been reported and are being tested clinically. This includes the 89 Zr-labeled minibody Df-IAB22M2C, 89 Zr-labeled antibody ZED88082A, 68 Ga-labeled Nb SNA006 and 18 F-labeled Nb VHH5v2 [ 6 , 8 , 35 , 36 ]. While these tracers have shown promising results so far, they still suffer from some disadvantages with respect to pharmacokinetics and specificity. Df-IAB22M2C and ZED88082A are bulky proteins (≥ 80kDa), making them unsuitable for early point and/or fast repeated imaging due to their long in vivo half-life time and slower tumor penetration. For this reason, nanobody-based tracers could be complementary to these tracers, or could be preferred altogether thanks to the characteristics mentioned previously. In our study, we show the ability of our tracer to detect CD8 + T cells already 1h post injection and to follow T-cell dynamics via repeated imaging over a time period of 18 days. These characteristics could potentially be essential for the prediction and/or follow-up of immunotherapy responses as a recent study of Kist de Ruijter et al. indicated the need for CD8-targeting tracers that allow (early) sequential imaging timepoints in order to follow spatio-temporal changes of CD8 + T cells [ 35 ]. In line with these findings, we were able to correlate early baseline levels of intratumoral hCD8 + T cells to subsequent tumor growth, suggesting that early timepoint imaging of hCD8 + T cells may indeed hold a prognostic value. All of the current (pre-)clinically-tested tracers against CD8 + T cells (including the Nb-based SNA006 and VHH5v2) target the α-chain of the CD8 protein. While CD8α is indeed expressed on CD8 + T cells, several studies have reported the presence of CD8α on other immune cell populations, including NK cells and monocytes [ 10 , 11 ]. In contrast, CD8β is exclusively expressed on CD8 + T cells, making it a more selective target for visualizing T-cell dynamics. Indeed, our results show the enhanced specificity of our anti-CD8β Nb for CD8 + T cells, as no binding to NK or myeloid cells was observed, while an anti-CD8α Nb strongly stains NK and myeloid cells. While it remains to be seen whether this enhanced specificity also results in an enhanced predictive or prognostic value, it would not be surprising as tumor-associated monocytes can encompass a major immune cell population in the tumor microenvironment and give rise to immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells [ 37 ]. The PET imaging results in cynomolgus monkeys also showed the feasibility to image CD8 + T cells in bigger animals. However, it is important to note that this was a proof-of-concept study. In the future, a dose escalation study and imaging at multiple timepoints could give additional information for the clinical translation. To enable these future studies, we decided to radiolabel our anti-hCD8β Nb with 64 Cu instead of 68 Ga. As 64 Cu has a half-life time of 12.7h, this would enable us to assess the uptake of the anti-hCD8β Nb for a time period of multiple hours to day(s) and give additional information on the pharmacokinetics of this Nb. While we saw a similar biodistribution profile in cynomolgus monkeys as in our mouse imaging experiments, we also noticed uptake of the radiolabeled human CD8β-targeting Nb in the mouth area of both monkeys. The exact reason for this radioactive uptake in the mouth area is still unclear. However, it has been reported that CD8 + T cells are present in the oral tissues of non-human primates [ 38 ]. Moreover, while no acute inflammation could be observed upon visual inspection, a local inflammation cannot be excluded. Interestingly, we did not observe much liver uptake while high liver and gallbladder uptake has been reported for 64 Cu-labeled compounds [ 39 , 40 ]. Finally, the non-invasive imaging of CD8 + T-cell dynamics could be interesting for multiple immune-related diseases. The presence of CD8 + T cells may be indicative of viral infections or chronic inflammatory and autoimmune diseases, including immune thrombocytopenia, rheumatoid arthritis and giant cell arteritis. Furthermore, previous preclinical research has focused on the non-invasive imaging of T-cell populations in graft-versus-host disease and multiple sclerosis, conditions for which imaging of CD8 + T cells might also be interesting [ 41 – 46 ]. Therefore, it is not unlikely that these tracers could be widely applied in multiple disease settings in the future. In conclusion, we have developed a novel SPECT- and PET-tracer to visualize hCD8 + T-cell dynamics at early timepoints with high specificity and sensitivity. These tracers could potentially be widely applied in multiple immune-related diseases to follow-up and/or predict immunotherapy responses. Declarations Funding This work has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 831514 (Immune-Image). The JU receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA. This work was further funded by the Strategic Research Programme and Wetenschappelijk Fonds Willy Gepts from the Vrije Universiteit Brussel. This work is also supported by Kom op Tegen Kanker and Research Foundation Flanders (FWO) research projects G087524N I005622N and I001618N. Timo W.M. De Groof is funded by a post-doctoral fellowship (12ZO723N) from the Research Foundation Flanders (FWO), Belgium. he Infectious Disease Models and Innovative Therapies (IDMIT) research infrastructure is supported by the “Programme Investissements d’Avenir”, managed by the ANR under reference ANR-11-INBS-0008. Competing Interests Timo W.M. De Groof, Yoline Lauwers, Cécile Vincke, Geert Raes, Jo A.Van Ginderachter and Nick Devoogdt are co-inventors on a pending patent application (EP23153689.7), which covers the use of the described CD8-targeting immunotracer Author Contributions Timo W.M. De Groof, Yoline Lauwers, Jo A.Van Ginderachter and Nick Devoogdt designed the study; Timo W.M. De Groof performed the in vitro characterization assays with the help from Yoline Lauwers, Tessa De Pauw and Cécile Vincke; Timo W.M. De Groof and Yoline Lauwers performed the SPECT and PET imaging experiments with the help from Tessa De Pauw and Jolien Van Craenenbroeck; Mohit Saxena, Thibault Naninck, Roger Le Grand and Catherine Chapon performed the immunostaining and PET imaging experiments on non-human primate tissue; Timo W.M. De Groof analyzed the Alphafold binding prediction models; Timo W.M. De Groof, Yoline Lauwers and M.S. analyzed the experimental data; Geert Raes, Jo A.Van Ginderachter and Nick Devoogdt supervised the study; Timo W.M. De Groof wrote the paper with the help from all the authors Data availability The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval 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). 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. Acknowledgments We would like to thank Kevin De Jonghe (MITH, VUB), Steve Huvelle (Biomaps Unit, CEA), VIB nanobody core facility, VIB protein core facility, VIB proteomics core facility, VUB In vivo Cellular and Molecular Imaging (ICMI) core facility and Roche for the technical assistance. We also would like to thank ARRONAX (Nantes) for the delivery of 64 Cu. This work has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 831514 (Immune-Image). The JU receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA. This work was further funded by the Strategic Research Programme and Wetenschappelijk Fonds Willy Gepts from the Vrije Universiteit Brussel. This work is also supported by Kom op Tegen Kanker and Research Foundation Flanders (FWO) research projects G087524N, I005622N and I001618N. Timo W.M. 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J Clin Immunol. 2013;33:1192-203. doi:10.1007/s10875-013-9915-0. Barakat S, Berksoz M, Zahedimaram P, Piepoli S, Erman B. Nanobodies as molecular imaging probes. Free Radic Biol Med. 2022;182:260-75. doi:10.1016/j.freeradbiomed.2022.02.031. Kist de Ruijter L, van de Donk PP, Hooiveld-Noeken JS, Giesen D, Elias SG, Lub-de Hooge MN, et al. Whole-body CD8(+) T cell visualization before and during cancer immunotherapy: a phase 1/2 trial. Nat Med. 2022;28:2601-10. doi:10.1038/s41591-022-02084-8. Sriraman SK, Davies CW, Gill H, Kiefer JR, Yin J, Ogasawara A, et al. Development of an (18)F-labeled anti-human CD8 VHH for same-day immunoPET imaging. Eur J Nucl Med Mol Imaging. 2023;50:679-91. doi:10.1007/s00259-022-05998-0. Ugel S, Cane S, De Sanctis F, Bronte V. Monocytes in the Tumor Microenvironment. Annu Rev Pathol. 2021;16:93-122. doi:10.1146/annurev-pathmechdis-012418-013058. Hernandez JL, Park J, Hughes SM, Hladik F, Woodrow KA. Characterization of Immune Cells in Oral Tissues of Non-human Primates. Front Oral Health. 2021;2:821812. doi:10.3389/froh.2021.821812. Lee CH, Lim I, Woo SK, Kim KI, Lee KC, Song K, et al. The Feasibility of (64)Cu-PSMA I&T PET for Prostate Cancer. Cancer Biother Radiopharm. 2022;37:417-23. doi:10.1089/cbr.2020.4189. Lee I, Kim MH, Lee K, Oh K, Lim H, Ahn JH, et al. Comparison of the Effects of DOTA and NOTA Chelators on (64)Cu-Cudotadipep and (64)Cu-Cunotadipep for Prostate Cancer. Diagnostics (Basel). 2023;13. doi:10.3390/diagnostics13162649. Rothlauf PW, Li Z, Pishesha N, Xie YJ, Woodham AW, Bousbaine D, et al. Noninvasive Immuno-PET Imaging of CD8(+) T Cell Behavior in Influenza A Virus-Infected Mice. Front Immunol. 2021;12:777739. doi:10.3389/fimmu.2021.777739. Audia S, Samson M, Mahevas M, Ferrand C, Trad M, Ciudad M, et al. Preferential splenic CD8(+) T-cell activation in rituximab-nonresponder patients with immune thrombocytopenia. Blood. 2013;122:2477-86. doi:10.1182/blood-2013-03-491415. Samson M, Ly KH, Tournier B, Janikashvili N, Trad M, Ciudad M, et al. Involvement and prognosis value of CD8(+) T cells in giant cell arteritis. J Autoimmun. 2016;72:73-83. doi:10.1016/j.jaut.2016.05.008. Carvalheiro H, da Silva JA, Souto-Carneiro MM. Potential roles for CD8(+) T cells in rheumatoid arthritis. Autoimmun Rev. 2013;12:401-9. doi:10.1016/j.autrev.2012.07.011. Cheng HM, Honda T, Asahina R, Miyake T, Chow Z, Tomura M, et al. In Vivo Imaging of CD8(+) T-Cell‒Mediated Keratinocyte Apoptosis in Graft-Versus-Host Disease‒Like Dermatitis in Involucrin Membrane-Bound Ovalbumin Mice. J Invest Dermatol. 2022;142:2827-31 e3. doi:10.1016/j.jid.2022.03.010. Salou M, Nicol B, Garcia A, Laplaud DA. Involvement of CD8(+) T Cells in Multiple Sclerosis. Front Immunol. 2015;6:604. doi:10.3389/fimmu.2015.00604. Additional Declarations The authors declare potential competing interests as follows: Timo W.M. De Groof, Yoline Lauwers, Cécile Vincke, Geert Raes, Jo A.Van Ginderachter and Nick Devoogdt are co-inventors on a pending patent application (EP23153689.7), which covers the use of the described CD8-targeting immunotracer Supplementary Files SupplementalFiguresEJNMMIDeGroofLauwersetal.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4322357","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":295310530,"identity":"ffb79971-5c00-4082-9094-78e9e54629cd","order_by":0,"name":"Timo W.M. De Groof","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYDACZhDBBuVUVDAwGDDw4NfBg6LlzBlitDAgaznbRoQWe3buxAcMZTYM8tGHDz44OO9w4nYG3oMP8DuMd7MBw7k0BsNzackGB7cdTtzZwJdsQEDLNgnGtsMMhj08ZtIft6UlbjjAYyZBhJb/YC0SB+eAtZj/IELLAQZ5HpCWBhuwLfh0MPAcBvol4VwyjwEPW7LBgWM2xjub+ZLxOoy9/+zGBx/K7OTke5gPPjhQIyG7nb334Ae81oBAAtA2gwMwHjNB9VAg30CsylEwCkbBKBhxAAAv1kLnxjp0FwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-6918-2955","institution":"Vrije Universiteit Brussel","correspondingAuthor":true,"prefix":"","firstName":"Timo","middleName":"W.M.","lastName":"De Groof","suffix":""},{"id":295310644,"identity":"6a79fa98-6542-4777-9ad7-9d1e2e9c7239","order_by":1,"name":"Yoline Lauwers","email":"","orcid":"https://orcid.org/0000-0002-6619-7403","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Yoline","middleName":"","lastName":"Lauwers","suffix":""},{"id":295310883,"identity":"28fb40dd-e539-4491-8c65-673edd6931cf","order_by":2,"name":"Tessa De Pauw","email":"","orcid":"https://orcid.org/0000-0001-7359-1751","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Tessa","middleName":"","lastName":"De Pauw","suffix":""},{"id":295311075,"identity":"1917b83e-64d0-4da0-901a-6abaf896ffd9","order_by":3,"name":"Mohit Saxena","email":"","orcid":"","institution":"Université Paris-Saclay","correspondingAuthor":false,"prefix":"","firstName":"Mohit","middleName":"","lastName":"Saxena","suffix":""},{"id":295311374,"identity":"5dbfd232-ca59-4e9a-a05e-6fa3f86ba5ee","order_by":4,"name":"Cécile Vincke","email":"","orcid":"https://orcid.org/0000-0003-0681-7444","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Cécile","middleName":"","lastName":"Vincke","suffix":""},{"id":295311375,"identity":"831788f3-ccae-4527-bf95-e052e273fea3","order_by":5,"name":"Jolien Van Craenenbroeck","email":"","orcid":"https://orcid.org/0009-0000-1795-3957","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Jolien","middleName":"Van","lastName":"Craenenbroeck","suffix":""},{"id":295311707,"identity":"b5dda0fb-e6da-4edd-8282-e85b1eabc54f","order_by":6,"name":"Catherine Chapon","email":"","orcid":"https://orcid.org/0000-0002-3210-8468","institution":"Université Paris-Saclay","correspondingAuthor":false,"prefix":"","firstName":"Catherine","middleName":"","lastName":"Chapon","suffix":""},{"id":295311708,"identity":"31127205-8add-4a62-857e-01117a1d7c6f","order_by":7,"name":"Roger Le Grand","email":"","orcid":"","institution":"Université Paris-Saclay","correspondingAuthor":false,"prefix":"","firstName":"Roger","middleName":"Le","lastName":"Grand","suffix":""},{"id":295311709,"identity":"148950e5-4a1d-44af-9763-94ba3a426d40","order_by":8,"name":"Geert Raes","email":"","orcid":"https://orcid.org/0000-0002-9065-1549","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Geert","middleName":"","lastName":"Raes","suffix":""},{"id":295311948,"identity":"08c17ef2-d1aa-4bf2-8758-573e52ff12e2","order_by":9,"name":"Thibaut Naninck","email":"","orcid":"","institution":"Université Paris-Saclay","correspondingAuthor":false,"prefix":"","firstName":"Thibaut","middleName":"","lastName":"Naninck","suffix":""},{"id":295311949,"identity":"cf8cff0d-46a8-410e-a59b-518b6d6bf1b2","order_by":10,"name":"Jo A. 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Van","lastName":"Ginderachter","suffix":""},{"id":295311950,"identity":"23cc7e7f-ce28-4076-9292-7bbd22ac3e35","order_by":11,"name":"Nick Devoogdt","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYDACZiBkYIOwJRgqGBgMGHhI0XLgDDFaGJC1HGwjQos5O/NjA4Yym8R+9vaLtz/OO5y4nYH34AN8Wiyb2YwTGM6lJc7sOVNscXDb4cSdDXzJBvi0GBzmYT7A2HbY2OBGTprEwW1piRsO8JhJEKHlP1TLHLAW8x+EtCQwth2QM7iRfkziYIMN2BZ8OoBa2IwNEs4ly0n2nGG2OHPMxnhnM18yfoedP/xY4kOZHQ8/e/vDGxU1ErLb2XsPfsBrDQgkgEkeaDgxE1QPB+wPiFc7CkbBKBgFIwoAAGccSbDdMkH9AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-9220-4833","institution":"Vrije Universiteit Brussel","correspondingAuthor":true,"prefix":"","firstName":"Nick","middleName":"","lastName":"Devoogdt","suffix":""}],"badges":[],"createdAt":"2024-04-25 07:34:42","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-4322357/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4322357/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55508381,"identity":"dc7be157-a979-41c3-9202-316ab4f5d23b","added_by":"auto","created_at":"2024-04-29 12:13:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":563116,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThe anti-human CD8β nanobody binds to human CD8 with high affinity.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Surface plasmon resonance plots of the binding of different nanobody concentrations to the human CD8αβ protein. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Binding of different human CD8β-targeting nanobody (hCD8β Nb) or irrelevant nanobody (Irr Nb) concentrations to T-cell-lymphoma SUP-T1 cells. Binding was detected as mean fluorescence intensity (MFI) via the C-terminal HA-tag and a fluorescently labeled anti-HA antibody using flow cytometry. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Thermostability of the hCD8β Nb was measured via a Thermofluor Assay using Sypro Orange dye. The percentage of unfolded protein was determined at increasing temperatures to calculate the melting temperature (50% unfolded protein). Data is presented as mean ± S.D. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eAlphafold prediction model of the binding between the anti-hCD8β Nb and human CD8.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4322357/v1/59472e47b2e8b14405e53656.png"},{"id":55508383,"identity":"342460c8-241f-4a7f-bf55-1399971b315f","added_by":"auto","created_at":"2024-04-29 12:13:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1661431,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThe anti-human CD8β nanobody binds specifically to primary CD8\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003e T cells.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Binding of an irrelevant nanobody (Irr Nb) or the human CD8β-targeting nanobody (hCD8β Nb) to primary T cells obtained from a healthy donor. CD3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e T cells (CD45\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, CD11b\u003c/em\u003e\u003csup\u003e\u003cem\u003e-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, CD19\u003c/em\u003e\u003csup\u003e\u003cem\u003e-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, CD3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e) were gated and anti-CD4 mAb staining was plotted against Nb binding. Nb binding was compared to a sample without any Nb (No Nb) and detected via the C-terminal HA-tag by a fluorescently labeled anti-HA antibody. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Quantification of Nb binding to CD8\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e T cells. Nanobody binding on primary T cells of 3 independent donors was detected as mean fluorescence intensity (MFI). \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Binding of an Irr Nb, anti-human CD8α Nb or hCD8β Nb to primary T cells, NK cells and myeloid cells obtained from 3 independent healthy donors. T cells (CD45\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, CD11b\u003c/em\u003e\u003csup\u003e\u003cem\u003e-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, CD19\u003c/em\u003e\u003csup\u003e\u003cem\u003e-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, CD3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e), NK cells (CD45\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, CD11b\u003c/em\u003e\u003csup\u003e\u003cem\u003e-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, CD19\u003c/em\u003e\u003csup\u003e\u003cem\u003e-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, CD3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, CD56\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e) or myeloid cells (CD45\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, CD19\u003c/em\u003e\u003csup\u003e\u003cem\u003e-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, CD3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, CD56\u003c/em\u003e\u003csup\u003e\u003cem\u003e-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e) were gated. Nb binding was compared to a sample without any Nb (No Nb) and detected via the C-terminal HA-tag by a fluorescently labeled anti-HA antibody. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Fluorescence immunohistochemistry staining of non-human primate lymph node tissue with a fluorescently labeled Irr Nb or hCD8β Nb. Nuclei were stained using DAPI and CD3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e T cells were detected using a fluorescently labeled anti-CD3 antibody. All data are presented as mean ± S.D.. Statistical analyses were performed using one-way ANOVA with Dunnett's post hoc test or two-way ANOVA with Šídák's multiple comparisons test. ns, p\u0026nbsp;\u0026gt; 0.05; ****, p\u0026nbsp;\u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4322357/v1/8ce67952851b54008d72514c.png"},{"id":55509274,"identity":"6007acb3-db78-446f-9068-5548d52b158e","added_by":"auto","created_at":"2024-04-29 12:21:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":232769,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThe anti-human CD8β nanobody does not induce T-cell activation.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Histogram plots of human CD69 expression on CD8\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e T cells without treatment (No Nb) or after overnight incubation with anti-CD3/CD28 dynabeads, GLP-grade irrelevant nanobody (Irr Nb) or human CD8β-targeting nanobody (hCD8β Nb) to human primary peripheral blood mononuclear cells. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Quantification of human CD69\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e CD8\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eT cells without treatment (No Nb) or after overnight incubation with anti-CD3/CD28 dynabeads, GLP-grade Irr Nb or hCD8β Nb. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e ELISA quantification of secreted IFN-γ levels in the supernatant of human primary peripheral blood mononuclear without treatment (No Nb) or after overnight incubation with anti-CD3/CD28 dynabeads, GLP-grade Irr Nb or hCD8β Nb. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed-e)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Stimulation indices of IFN-γ (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) and IL-5 (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) in the DC/CD4\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e T cell (from 30 healthy donors) restimulation assays after incubation with non-immunogenic Bevacizumab (BVZ), immunogenic keyhole limpet hemocyanin (KLH), GLP-grade Irr Nb or hCD8β Nb. Stimulation index indicates the amount of positive cells compared to untreated cells. All data are presented as mean ± S.D.. Statistical analyses were performed using one-way ANOVA with Dunnett's post hoc test. ns, p\u0026nbsp;\u0026gt; 0.05; ***, p\u0026nbsp;\u0026lt; 0.001; ****, p\u0026nbsp;\u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4322357/v1/2a8a6ed524076d703d342785.png"},{"id":55508380,"identity":"97bf1ed6-99f3-4274-8b58-fc8bd4a53f13","added_by":"auto","created_at":"2024-04-29 12:13:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1309235,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSPECT/CT imaging with the \u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e99m\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003eTc-labeled anti-human CD8β nanobody.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Representative SPECT/CT images of SUP-T1 tumor-bearing mice (tumor outlined in yellow), intravenously injected with \u003c/em\u003e\u003csup\u003e\u003cem\u003e99m\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eTc-labeled irrelevant nanobody (Irr Nb) or human CD8β-targeting nanobody (hCD8β Nb). \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Ex vivo γ-counting of the isolated tumors 80min after injection with\u0026nbsp;\u003c/em\u003e\u003csup\u003e\u003cem\u003e99m\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eTc-labeled Nbs. Uptake of the Nbs is expressed as injected activity per gram of tumor (%IA/g). \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Tumor-to-blood ratios of the \u003c/em\u003e\u003csup\u003e\u003cem\u003e99m\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eTc-labeled Nbs, calculated by dividing the %IA/g tumor by the %IA/g blood. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Schematic cartoon of mouse (m) and human (h) CD8 expression on mouse CD8\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e T cells of C57BL/6 wild type and human CD8 transgenic mice.\u003c/em\u003e \u003cem\u003e\u003cstrong\u003ee)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Representative SPECT/CT images of wild type and human (h)CD8 transgenic mice intravenously injected with \u003c/em\u003e\u003csup\u003e\u003cem\u003e99m\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eTc-labeled Nbs. Lymph nodes (LN), spleen (Spl) and small intestines (I) are highlighted. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ef-i)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Ex vivo γ-counting of the isolated lymph nodes (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e), spleen (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eg\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e), small intestines (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eh\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) and appendix (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ei\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) 80min after injection with\u0026nbsp;\u003c/em\u003e\u003csup\u003e\u003cem\u003e99m\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eTc-labeled Nbs. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ej)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Representative coronal section of a MC38 tumor-bearing hCD8 transgenic mouse intravenously injected with \u003c/em\u003e\u003csup\u003e\u003cem\u003e99m\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eTc-labeled Nbs. The MC38 tumor is delineated. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ek)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Ex vivo γ-counting of the isolated MC38 tumors 80min after injection with\u0026nbsp;\u003c/em\u003e\u003csup\u003e\u003cem\u003e99m\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eTc-labeled Nbs. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003el)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Tumor-to-blood ratios of the \u003c/em\u003e\u003csup\u003e\u003cem\u003e99m\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eTc-labeled Nbs in MC38 tumor-bearing mice. All data are presented as mean ± S.D. Statistical analyses were performed using a two-tailed unpaired t test. ns, p\u0026nbsp;\u0026gt; 0.05; **, p\u0026nbsp;\u0026lt; 0.01; ***, p\u0026nbsp;\u0026lt; 0.001; ****, p\u0026nbsp;\u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4322357/v1/614bfbaddd23b6998f6e56b5.png"},{"id":55508385,"identity":"1d48508c-6ef2-4ae7-9de8-9feb98ae310f","added_by":"auto","created_at":"2024-04-29 12:13:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":731262,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePET/CT imaging with \u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e68\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003eGa-labeled anti-human CD8β nanobody.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Surface plasmon resonance plots of the kinetic titration of increasing concentrations of human CD8β-targeting nanobody (hCD8β Nb) and NOTA-conjugated hCD8β Nb concentrations to human CD8αβ protein. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Binding of different concentrations of the NOTA-conjugated hCD8β Nb or an irrelevant nanobody (Irr Nb) to T-cell-lymphoma SUP-T1 cells. Binding was detected as mean fluorescence intensity (MFI) via a fluorescently-labeled anti-VHH antibody using flow cytometry. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Representative PET/CT image of human CD8 transgenic mice intravenously injected with \u003c/em\u003e\u003csup\u003e\u003cem\u003e68\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eGa-labeled Irr Nb or hCD8β Nb. Lymph nodes (LN), spleen (Spl) and thymus (T) are highlighted. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Ex vivo γ-counting of the organs 80min after injection with\u0026nbsp;\u003c/em\u003e\u003csup\u003e\u003cem\u003e68\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eGa-labeled Nbs. Biodistribution of the nanobodies in three mice is shown and uptake of the Nbs is expressed as injected activity per gram (%IA/g). All data are presented as mean ± S.D.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4322357/v1/84511153385bb1d1f7f29f66.png"},{"id":55508386,"identity":"44837cf6-fe3e-46c0-a207-2a5f995fc95e","added_by":"auto","created_at":"2024-04-29 12:13:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1034134,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePET/CT imaging of hCD8\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003e T-cell dynamics with the \u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e68\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003eGa-labeled anti-human CD8β nanobody.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Schematic overview of the imaging regimen of MC38 tumor-bearing human (h) CD8 transgenic mice. Mice were inoculated with MC38 cells at day 0. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e MC38 tumor growth curves in hCD8 transgenic mice. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eRepresentative PET/CT image (coronal, sagittal and transverse section) of a MC38 tumor-bearing hCD8 transgenic mouse with low and high hCD8\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e intratumoral T-cell infiltration at endpoint (day 15 or 18). The tumor is delineated. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed-e) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eCorrelation plots of ex vivo measured radioactive uptake of the tumor versus the radioactive uptake measured via PET image analysis using a region of interest (ROI) (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) or hCD8\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e T cells quantified via flow cytometry (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) at endpoint (day 15 or 18). Each individual mouse is highlighted in a different color. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ef)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Radioactive uptake measured in the inguinal lymph node (LN) or inguinal tumor-draining lymph node (TdLN) via PET image analysis using a ROI at day 5. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eg) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eRatio of radioactive uptake measured in inguinal lymph nodes (TdLN/LN) and cervical lymph nodes (cLN1/cLN2) via PET image analysis using ROIs. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eh)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Correlation plot of the intratumoral hCD8\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e T cells at baseline (day 5) versus tumor growth. Intratumoral hCD8\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e T cells were determined via the radioactive uptake of \u003c/em\u003e\u003csup\u003e\u003cem\u003e68\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eGa-hCD8β Nb in the tumor measured via PET image analysis using a ROI. Tumor growth is presented as the ratio of increase of tumor size at day 15 compared to baseline (day 5). Each individual mouse is highlighted in a different color. All data are presented as mean ± S.D. Correlations were determined via Pearson correlation and 95% confidence bars are presented. Statistical analyses were performed using unpaired two-tailed t-test or one way ANOVA with Dunnett's post hoc test. *, p\u0026nbsp;\u0026lt; 0.05; ***, p\u0026nbsp;\u0026lt; 0.001, ****p\u0026lt;0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4322357/v1/319a49263071d1de06e2a4b7.png"},{"id":55508387,"identity":"2e3ccd67-86b9-4a98-917a-a0db4afad87b","added_by":"auto","created_at":"2024-04-29 12:13:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1198300,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePET/CT imaging of CD8\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003e T cells in naïve cynomolgus monkeys with the \u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e64\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003eCu-labeled anti-human CD8β nanobody.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Schematic overview of the imaging experiment. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e PET/CT images of 2 naïve cynomolgus monkeys intravenously injected with \u003c/em\u003e\u003csup\u003e\u003cem\u003e64\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCu-labeled anti-human CD8β Nb. Different regions of uptake are highlighted including cervical (cLN), axillary (Ax LN), tracheobronchial (Tr LN) lymph nodes, spleen (Spl) and thymus. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eQuantification of the radioactive uptake (SUV\u003c/em\u003e\u003csub\u003e\u003cem\u003emean\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e) in different organs via PET image analysis.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4322357/v1/93d950665a2a70d8e42669ec.png"},{"id":55510595,"identity":"5cad5176-cbc7-410b-af81-63b0f999818b","added_by":"auto","created_at":"2024-04-29 12:29:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3654251,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4322357/v1/6e2fec19-17e0-4386-b08a-e4c6940dc298.pdf"},{"id":55509276,"identity":"3fa71a68-2227-42d8-bc9d-bf4a08d71b55","added_by":"auto","created_at":"2024-04-29 12:21:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1116675,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFiguresEJNMMIDeGroofLauwersetal.docx","url":"https://assets-eu.researchsquare.com/files/rs-4322357/v1/41aa56fda935d6b92894a2ba.docx"}],"financialInterests":"The authors declare potential competing interests as follows: Timo W.M. De Groof, Yoline Lauwers, Cécile Vincke, Geert Raes, Jo A.Van Ginderachter and Nick Devoogdt are co-inventors on a pending patent application (EP23153689.7), which covers the use of the described CD8-targeting immunotracer","formattedTitle":"\u003cp\u003eSpecific Imaging of CD8\u003csup\u003e+\u003c/sup\u003e T-Cell Dynamics with a Nanobody Radiotracer against Human CD8β\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the last decade, immunotherapy has revolutionized the oncology field and has become the standard of care for some cancer types [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, variations in therapy responsiveness and side effects between patients limit the broad applicability and effectiveness of these therapies [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Diagnostic imaging of immune cell dynamics during immunotherapy could allow early patient stratification and, as such, result in improvements regarding therapy efficacy, safety and therapy costs [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Consequently, the development of diagnostic imaging tracers, capable of non-invasively visualizing distinct immune cell populations, has gained a lot of interest over the last years.\u003c/p\u003e \u003cp\u003eAmong the key players in immunotherapy responses, cytotoxic CD8\u003csup\u003e+\u003c/sup\u003e T cells have received particular attention [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. As a result, several PET imaging tracers have been developed to target murine or human CD8\u003csup\u003e+\u003c/sup\u003e T cells, facilitating the monitoring and/or prediction of immunotherapy responses [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, most of these tracers suffer from suboptimal pharmacokinetics (i.e. long circulation time), necessitating the use of long-lived radionuclides and potentially raising concerns regarding radiotoxicity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Furthermore, all of these tracers bind the CD8α-chain, which is also expressed on other types of immune cells, decreasing the tracer's specificity for CD8\u003csup\u003e+\u003c/sup\u003e T cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNanobodies (Nbs), the variable region fragments of \u003cem\u003ecamelidae\u003c/em\u003e heavy chain-only antibodies, have emerged as interesting scaffolds for the development of diagnostic tracers [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Nbs are smaller (15 kDa) than conventional monoclonal antibodies, while still maintaining their ability to bind their target with high affinity and specificity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This results in rapid tumor uptake and blood clearance, allowing fast and specific imaging of immune cells within the tumor microenvironment and a lower radiation burden [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we describe the development of a radiolabeled anti-human (h)CD8β Nb, that allows the non-invasive imaging of human CD8\u003csup\u003e+\u003c/sup\u003e T cells via SPECT and PET imaging. These Nb-based tracers show high affinity and specific binding to CD8\u003csup\u003e+\u003c/sup\u003e T cells and ideal \u003cem\u003ein vivo\u003c/em\u003e pharmacokinetics. Furthermore, we show the ability of these Nb-based tracers to follow up immune cell dynamics during tumor growth. Overall, this study describes the development of a more specific hCD8\u003csup\u003e+\u003c/sup\u003e T-cell-targeting tracers, allowing the follow-up of hCD8\u003csup\u003e+\u003c/sup\u003e T-cell dynamics via non-invasive imaging.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eSUP-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% CO\u003csub\u003e2\u003c/sub\u003e and 37\u0026deg;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\u0026rsquo;s Modified Eagle\u0026rsquo;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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAnimal models\u003c/h2\u003e \u003cp\u003eMale 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\u0026nbsp;million MC38 or 5\u0026nbsp;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 \u0026times; width\u003csup\u003e2\u003c/sup\u003e)/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).\u003c/p\u003e \u003cp\u003eTwo young adult male cynomolgus macaques (\u003cem\u003eMacaca fascicularis\u003c/em\u003e), 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, Pr\u0026eacute;fecture 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 \u0026lsquo;Comit\u0026eacute; d\u0026rsquo;Ethique en Exp\u0026eacute;rimentation Animale du Commissariat \u0026agrave; l\u0026rsquo;Energie Atomique et aux Energies Alternatives\u0026rsquo; (CEtEA number 44) under statement number A23-057. The study was authorized by the \u0026lsquo;\u0026lsquo;Research, Innovation and Education Ministry\u0026rsquo;\u0026rsquo; under registration number APAFIS #46283-202312131546674 v1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eNanobody generation, selection and production\u003c/h2\u003e \u003cp\u003eTwo llamas were subcutaneously injected 6 times with 100 \u0026micro;g recombinant human (h)CD8β-Avi-His\u003csub\u003e6\u003c/sub\u003e (U-Protein Express BV, Utrecht, The Netherlands) and 100 \u0026micro;g 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[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] 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-His\u003csub\u003e6\u003c/sub\u003e 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-His\u003csub\u003e6\u003c/sub\u003e 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 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSurface plasmon resonance\u003c/h2\u003e \u003cp\u003eThe 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\u0026deg;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 \u0026micro;L/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\u0026ndash;1 binding geometries. For the determination of the equilibrium dissociation constant, the ratio of the association and dissociation rate constants was determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAffinity determination via flow cytometry\u003c/h2\u003e \u003cp\u003eSerial 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\u0026deg;C. Cells were washed once with FACS buffer. Next, nanobody binding was detected by incubation of the cells with an Alexa Fluor\u0026reg;-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\u0026deg;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAffinity determination via ELISA\u003c/h2\u003e \u003cp\u003eWells of a 96 well MicroWell MaxiSorp flat bottom plate (Thermo Fisher Scientific) were coated with 0.2\u0026micro;g of recombinant hCD8αβ protein, 0.2\u0026micro;g of cynomolgus CD8β-Fc protein (Sino Biologicals) or PBS overnight at 4\u0026deg;C. The next day, wells were washed 3 times with PBS-T (PBS\u0026thinsp;+\u0026thinsp;0.05% Tween20 (Merck-Millipore, Burlington, MA, USA). Next, wells were blocked with blocking buffer (2% skimmed milk powder (R\u0026eacute;gilait) 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\u0026reg; Pro software (Molecular Devices, San Jose, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eThermal shift assay\u003c/h2\u003e \u003cp\u003eThe anti-hCD8β nanobody (concentration 0.2 mg/mL) was mixed with 1x SYPRO\u0026trade; 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\u0026deg;C, with stepwise increments of 0.5\u0026deg;C, using CFX connect\u0026trade; Real-Time PCR (Bio Rad). The melting temperature of the nanobody was calculated using the Boltzmann equation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eNanobody binding to primary PBMCs\u003c/h2\u003e \u003cp\u003eOne 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\u0026trade; Fixable Viability Dye eFluor\u0026trade; 506 (1:1000 in HBSS; Thermo Fisher Scientific) for 30 min at 4\u0026deg;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\u0026rsquo;s protocol for 10min at 4\u0026deg;C. Next, 100 nM of anti-hCD8α (clone R3HCD27, patent US20190071500A1), anti-hCD8β or irrelevant nanobody were added for 1 h at 4\u0026deg;C. Cells were washed once with FACS buffer and incubated with a mix of fluorescent antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) for 30 min at 4\u0026deg;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining of non-human primate tissue\u003c/h2\u003e \u003cp\u003eNon-human primate lymph node tissues were fixed in Gerner buffer (0.1 M L-Lysine, 2 mg/mL NaIO\u003csub\u003e4\u003c/sub\u003e, 4% formaldehyde and 0.05 M Phosphate buffer) overnight at 4\u0026deg;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 \u0026micro;m were cut using a Cryostat (Leica Biosystems) and mounted on Superfrost\u0026reg;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\u0026deg;C with Alexa Fluor\u0026reg;-647 conjugated anti-hCD8β (10 \u0026micro;g/mL) or irrelevant nanobody (10 \u0026micro;g/mL) and primary CD3 antibody (5 \u0026micro;g/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\u0026reg;-594-conjugated goat anti-mouse IgG1 secondary antibody (1 \u0026micro;g/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\u0026prime;,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\u0026trade; Gold Antifade Mountant, Thermo Fisher) before being imaged using an automatic wide-field microscope (AxioScan Series 7, Zeiss).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eT-cell activation assay\u003c/h2\u003e \u003cp\u003eOne day prior to the analysis, primary peripheral blood mononuclear cells were thawed and taken into culture. The next day, 1\u0026nbsp;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\u0026amp;D systems, Minneapolis, MN, USA) according to the manufacturer\u0026rsquo;s protocol. Cells were stained with fluorescent antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) for flow cytometry analysis as described above. Flow cytometry was performed using the FACS CANTO II analyser. Analysis was performed using FlowJo version 10.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDendritic cell/T cell restimulation experiments\u003c/h2\u003e \u003cp\u003eImmunogenicity 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 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e99m\u003c/span\u003e\u003c/sup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTc-radiolabeling of nanobodies\u003c/span\u003e \u003c/p\u003e \u003cp\u003eNanobodies were labeled with \u003csup\u003e99m\u003c/sup\u003eTc as previously described [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Briefly, \u003csup\u003e99m\u003c/sup\u003eTc-tricarbonyl was generated via the addition of 150 mCi \u003csup\u003e99m\u003c/sup\u003eTcO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e to the Isolink\u0026reg; labelling kit (Paul Scherrer Institute, Villigen, Switzerland) for 20 min at 100\u0026deg;C. Next, 50 \u0026micro;g of His-tagged nanobody was added and incubated for 90 min at 50\u0026deg;C. \u003csup\u003e99m\u003c/sup\u003eTc-labeled nanobodies were purified via gel filtration from the unbound [\u003csup\u003e99m\u003c/sup\u003eTc(H2O)\u003csub\u003e3\u003c/sub\u003e(CO)\u003csub\u003e3\u003c/sub\u003e]\u0026thinsp;+\u0026thinsp;via a NAP-5 column (Cytiva) and filtered through a Millex 0.22 \u0026micro;m filter (Millipore, Haren, Belgium). The radiochemical purity of radiolabeled nanobodies was evaluated by instant thin layer chromatography (iTLC, Pall Corporation, Hoegaarden, Belgium)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSPECT-CT imaging and image analysis\u003c/h2\u003e \u003cp\u003eMice were injected i.v. with 5 \u0026micro;g of radiolabeled (\u0026plusmn;\u0026thinsp;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\u003csup\u003e+\u003c/sup\u003e 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 Wizard\u003csup\u003e2\u003c/sup\u003e 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAlphafold nanobody binding prediction\u003c/h2\u003e \u003cp\u003eNanobody binding models to human CD8 were generated using Colabfold (patch v1.5.2) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eNOTA-conjugation of nanobodies\u003c/h2\u003e \u003cp\u003eThe 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 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. 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\u0026trade; 16/600 Superdex\u0026trade; 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 \u0026times; 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.\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e68\u003c/span\u003e\u003c/sup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGa-radiolabeling of nanobodies\u003c/span\u003e \u003c/p\u003e \u003cp\u003eThe 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 (\u003csup\u003e68\u003c/sup\u003eGa) eluate (424\u0026ndash;636 MBq) eluted from a \u003csup\u003e68\u003c/sup\u003eGe/\u003csup\u003e68\u003c/sup\u003eGa generator in 0.1 M HCl (Galli Eo\u0026trade;, 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\u0026ndash;6.1 (injection buffer) for stability and \u003cem\u003ein vivo\u003c/em\u003e studies. After purification, the radioactive nanobody solution was filtered through a 0.22 \u0026micro;m filter (Millipore, Belgium). The radiochemical purity was assessed before and after purification by radio-iTLC ([\u003csup\u003e68\u003c/sup\u003eGa]Ga-NOTA-nanobody Rf\u0026thinsp;=\u0026thinsp;0, [68Ga]Ga-citrate Rf\u0026thinsp;=\u0026thinsp;1). Radiometal chelation stability of the radiolabeled nanobody was assessed in different conditions (injection buffer (0.9%NaCl\u0026thinsp;+\u0026thinsp;5 mg/mL Vitamin C) at RT, 37\u0026deg;C; human serum 37\u0026deg;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePET-CT imaging and image analysis in mice\u003c/h2\u003e \u003cp\u003eMice were injected (i.v.) with 5 \u0026micro;g of radiolabeled nanobody (15.5\u0026thinsp;\u0026plusmn;\u0026thinsp;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\u0026ndash;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 Wizard\u003csup\u003e2\u003c/sup\u003e 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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eProcessing organs and flow cytometry analysis\u003c/h2\u003e \u003cp\u003eSingle cell preparations of MC38 tumors were prepared as described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Antibodies used for staining of single cell preparations can be found in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. 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.\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e64\u003c/span\u003e \u003c/sup\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCu-radiolabeling of nanobodies\u003c/span\u003e \u003c/p\u003e \u003cp\u003eCopper-64 (\u003csup\u003e64\u003c/sup\u003eCu) in 1 M HCl (1 GBq, 375 \u0026micro;L, ARRONAX, Nantes, France) was concentrated at 90\u0026deg;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 \u0026micro;L of sodium acetate buffer was added to solubilized \u003csup\u003e64\u003c/sup\u003eCuCl2 and this solution was transferred to NOTA-hCD8β Nb. The resulting mixture was stirred at 500 rpm in a thermoshaker at 37\u0026deg;C. Radio-TLC was performed, using 50 mM citric acid as eluent, to monitor the reaction. Full conversion was observed after 1 h (Rf\u0026thinsp;=\u0026thinsp;0.05) as no residual free \u003csup\u003e64\u003c/sup\u003eCu was observed (Rf\u0026thinsp;=\u0026thinsp;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 \u0026micro;L fractions. Radioactivities were measured in a dose calibrator (Capintec\u0026reg;, 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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePET-CT imaging and image analysis of macaques\u003c/h2\u003e \u003cp\u003eAll 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 \u0026times; 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 \u0026micro;g of \u003csup\u003e64\u003c/sup\u003eCu-radiolabeled nanobodies via the saphenous vein (230\u0026thinsp;\u0026plusmn;\u0026thinsp;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\u0026thinsp;\u0026plusmn;\u0026thinsp;SD).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOverview of antibodies used for flow cytometry\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarget\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFluorophore\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpecies reactivity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProvider\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eClone\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD11b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePE/Cyanine7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman, Mouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eM1/70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHis-tag\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMiltenyi Biotec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGG11-8F3.5.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBD Bioscience\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2ST8.5H7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD8 Isotype control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBD Bioscience\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eG155-178\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFN50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePerCP/Cyanine5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRPA-T4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePerCP/Cyanine5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGK1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePerCP/Cyanine5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eQA18A21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFITC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eeBioscience\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSK7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTCR beta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFITC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eeBioscience\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eH57-597\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSJ25C1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eeBioscience\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1D3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAPC/Cyanine7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHI30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAPC/Cyanine7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30-F11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrilliant violet 421\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRPA-T8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrilliant violet 421\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53\u0026thinsp;\u0026minus;\u0026thinsp;6.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cspan class=\"BoldUnderline\"\u003eThe anti-human CD8\u0026beta; nanobody binds human CD8 with high affinity and has a good safety profile\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eTo identify Nbs against the extracellular part of the hCD8\u0026beta; chain, two llamas were immunized with recombinant hCD8\u0026beta; protein. Subsequent phage display panning and screening resulted in 175 unique Nbs, belonging to 33 different B-cell lineages. Further characterization of these Nbs resulted in the selection of a lead anti-hCD8\u0026beta; Nb. The anti-hCD8\u0026beta; Nb displayed low nanomolar binding affinity to the hCD8\u0026alpha;\u0026beta; protein as shown via surface plasmon resonance (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). In line with this, a similar binding affinity to SUP-T1 cells, a T-cell lymphoma cell line endogenously expressing hCD8\u0026alpha;\u0026beta;, was seen in flow cytometry, while no binding was observed with an irrelevant Nb, binding the 5T2 multiple myeloma M protein (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Next, we assessed the cross-reactive binding of the anti-hCD8\u0026beta; Nb (Supplemental Fig.\u0026nbsp;1 and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). While no Nb binding to murine CD8\u0026beta; protein was observed (Supplemental Fig.\u0026nbsp;1a), equilibrium binding ELISA with the anti-hCD8\u0026beta; Nb did indicate similar low nanomolar binding affinities to the human and cynomolgus CD8\u0026beta; proteins (Supplemental Fig.\u0026nbsp;1b). This result is in line with the poor homology between the human and mouse proteins (50%), and the very high homology between human and cynomolgus CD8\u0026beta; proteins (93%). Subsequently, the thermostability of the Nb was determined, considering that the Nb will be incubated at higher temperatures during radiolabeling (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The anti-hCD8\u0026beta; Nb displayed a melting temperature of 68.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026deg;C. Finally, the binding epitope of the anti-hCD8\u0026beta; Nb was modelled using Alphafold (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed). Although the predicted local distance difference test (pLDDT) score of the model indicated that the exact interaction between the CDR regions of the Nbs and CD8\u0026beta; could only be estimated (Supplemental Fig.\u0026nbsp;2), the Alphafold model nicely predicted binding to the \u0026beta; chain of human CD8.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003e\u003cstrong\u003eOverview of the in vitro characteristics of the anti-human CD8\u0026beta; nanobody (hCD8\u0026beta; Nb).\u003c/strong\u003e Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D of at least 3 independent experiments.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNb\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eK\u003csub\u003eD\u003c/sub\u003e hCD8\u0026alpha;\u0026beta; protein (nM) SPR\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eK\u003csub\u003eD\u003c/sub\u003e SUP-T1 cells (nM)\u003c/p\u003e\n\u003cp\u003eFlow cytometry\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eK\u003csub\u003eD\u003c/sub\u003e hCD8\u0026alpha;\u0026beta; protein ELISA (nM)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eK\u003csub\u003eD\u003c/sub\u003e cynomolgus CD8\u0026beta; protein ELISA (nM)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMelting temperature (\u0026deg;C)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehCD8\u0026beta; Nb\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16.0\u0026thinsp;\u0026plusmn;\u0026thinsp;9.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e68.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eSPR: Surface plasmon resonance.\u003c/p\u003e\n\u003cp\u003eNext, we determined the ability of the anti-hCD8\u0026beta; Nb to bind primary CD8\u003csup\u003e+\u003c/sup\u003e T cells \u003cem\u003eex vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea-b). While no binding was seen for the irrelevant Nb, the anti-hCD8\u0026beta; Nb bound the CD8\u003csup\u003e+\u003c/sup\u003e T cell population within a pool of primary T cells. Moreover, the anti-hCD8\u0026beta; Nb did not show any binding to NK or myeloid cells, known to express hCD8\u0026alpha;, but not hCD8\u0026beta; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec and Supplemental Fig.\u0026nbsp;3). In contrast, binding of an anti-hCD8\u0026alpha; Nb (WO2017134306A1) to NK or myeloid cells was observed. These data illustrate the superior specificity of the anti-hCD8\u0026beta; Nb for CD8\u003csup\u003e+\u003c/sup\u003e T cells, as compared to the currently used hCD8\u0026alpha;-targeting compounds.\u003c/p\u003e\n\u003cp\u003eAs the anti-hCD8\u0026beta; Nb showed cross-reactive binding to the cynomolgus CD8\u0026beta; protein, \u003cem\u003ein vitro\u003c/em\u003e binding to non-human primate lymph node tissue was determined via fluorescence immunohistochemistry (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). While no binding of the irrelevant Nb was observed, the anti-hCD8\u0026beta; Nb bound to CD3\u003csup\u003e+\u003c/sup\u003e T cells present in the lymph node tissue.\u003c/p\u003e\n\u003cp\u003eAs binding of the anti-hCD8\u0026beta; Nb should not induce unwanted cytotoxicity, we next assessed the effect of Nb binding on T-cell activation. Hereto, primary human peripheral blood mononuclear cells (PBMCs) were incubated overnight with the anti-hCD8\u0026beta; Nb, irrelevant Nb or anti-CD3/CD28 dynabeads. The next day, T-cell activation was assessed via human CD69 expression, an early activation marker (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea-b), and via the secretion of IFN-\u0026gamma; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec). Stimulation with anti-CD3/CD28 dynabeads resulted in a significant increase of CD69 expression and IFN-\u0026gamma; secretion by CD8\u003csup\u003e+\u003c/sup\u003e T cells. In contrast, no changes in CD69 expression nor secreted IFN- \u0026gamma; levels were observed upon incubation with the anti-hCD8\u0026beta; Nb or the irrelevant Nb. Finally, the immunogenicity of the Nbs was determined via a dendritic cell-T cell co-culture assay using PBMCs of 30 independent healthy donors (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed-e). To this end, monocytes of the PBMC fractions were differentiated toward monocyte-derived dendritic cells (moDCs) followed by loading with the Nbs and maturation. Afterwards, CD4\u003csup\u003e+\u003c/sup\u003e T cells were co-cultured with the loaded moDCs for 6 days. Freshly isolated monocytes were challenged again with the Nbs and added to the co-culture for an additional 2 days. Afterwards, IFN-\u0026gamma; (Th1 cytokine) and IL-5 (Th2 cytokine) levels were assessed. As negative and positive controls, we took along clinically benchmarked Bevacizumab and immunogenic KLH protein. In contrast to KLH, neither of the Nbs showed any sign of immunogenicity, as no significant increase of IFN-\u0026gamma; nor IL-5 levels was observed.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e \u003cspan class=\"BoldUnderline\"\u003e99m\u003c/span\u003e\u003c/sup\u003e\u003cspan class=\"BoldUnderline\"\u003eTc-labeled anti-human CD8\u0026beta; nanobody non-invasively images CD8\u003c/span\u003e\u003csup\u003e\u003cspan class=\"BoldUnderline\"\u003e+\u003c/span\u003e \u003c/sup\u003e \u003cspan class=\"BoldUnderline\"\u003eT cells in na\u0026iuml;ve and tumor-bearing mice\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eNext, we determined the potential of the anti-hCD8\u0026beta; Nb to target and visualize hCD8\u003csup\u003e+\u003c/sup\u003e T cells \u003cem\u003ein vivo\u003c/em\u003e. To this end, the irrelevant Nb and anti-hCD8\u0026beta; Nb were site-specifically radiolabeled with Technetium-99m (\u003csup\u003e99m\u003c/sup\u003eTc) via their C-terminal His-tag [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. Both Nbs were successfully labeled with radiochemical purities above 90% after labeling and 99% after purification (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). First, the targeting potential of the \u003csup\u003e99m\u003c/sup\u003eTc-radiolabeled Nbs was assessed in CD8\u003csup\u003e+\u003c/sup\u003e SUP-T1 tumor-bearing nude mice, which lack endogenous T cells, by SPECT/CT imaging 1 hour post intravenous (i.v.) injection (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). Both \u003csup\u003e99m\u003c/sup\u003eTc-radiolabeled Nbs showed a high uptake in the kidneys and the bladder, due to rapid blood clearance [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the \u003csup\u003e99m\u003c/sup\u003eTc-radiolabeled anti-hCD8\u0026beta; Nb showed a significantly higher uptake in the SUP-T1 tumors as compared to the \u003csup\u003e99m\u003c/sup\u003eTc-radiolabeled irrelevant Nb. Specific uptake of the \u003csup\u003e99m\u003c/sup\u003eTc-labeled anti-hCD8\u0026beta; Nb in tumors was further confirmed via \u003cem\u003eex vivo\u003c/em\u003e \u0026gamma;-counting and subsequent tumor-to-blood ratio calculation of isolated organs from SUP-T1 tumor-bearing mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb-c and Supplemental Fig.\u0026nbsp;4). These data suggest the ability of the \u003csup\u003e99m\u003c/sup\u003eTc-labeled anti-hCD8\u0026beta; Nb to visualize endogenously expressed hCD8. The \u003cem\u003ein vivo\u003c/em\u003e specificity of the \u003csup\u003e99m\u003c/sup\u003eTc-labeled anti-hCD8\u0026beta; Nb was further determined by SPECT/CT imaging in C57BL/6 wild type (WT) and hCD8 transgenic mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed-e). The \u003csup\u003e99m\u003c/sup\u003eTc-labeled irrelevant Nb did not show any specific accumulation in both mice. In contrast, the \u003csup\u003e99m\u003c/sup\u003eTc-labeled anti-hCD8\u0026beta; Nb showed uptake in T-cell-rich organs such as lymph nodes, spleen, intestines and appendix of hCD8 transgenic, but not WT, mice. Again, these data were confirmed via \u003cem\u003eex vivo\u003c/em\u003e \u0026gamma;-counting of the isolated organs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef-i and Supplemental Fig.\u0026nbsp;5). Finally, the ability of the \u003csup\u003e99m\u003c/sup\u003eTc-labeled anti-hCD8\u0026beta; Nb to target intratumoral T cells was assessed. To this end, hCD8 transgenic mice were subcutaneously inoculated with MC38 tumor cells. When the tumor size reached approximately 500 mm\u003csup\u003e3\u003c/sup\u003e, mice were injected i.v. with the \u003csup\u003e99m\u003c/sup\u003eTc-labeled Nbs and uptake in the different organs was assessed via SPECT/CT and \u003cem\u003eex vivo\u003c/em\u003e \u0026gamma;-counting. The \u003csup\u003e99m\u003c/sup\u003eTc-labeled anti-hCD8\u0026beta; Nb showed a significantly higher uptake in the tumor compared to the \u003csup\u003e99m\u003c/sup\u003eTc-labeled Irr Nb (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ej-k). Moreover, the \u003csup\u003e99m\u003c/sup\u003eTc-labeled anti-hCD8\u0026beta; Nb showed a significantly higher tumor-to-blood ratio compared to the \u003csup\u003e99m\u003c/sup\u003eTc-labeled irrelevant Nb (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003el) indicating that the anti-hCD8\u0026beta; Nb binds to intratumoral hCD8\u0026thinsp;+\u0026thinsp;T cells.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003e\u003cstrong\u003eRadiochemical purity of radiolabeled anti-human CD8\u0026beta; or irrelevant nanobody.\u003c/strong\u003e The percentage of \u003csup\u003e99m\u003c/sup\u003eTc- or \u003csup\u003e68\u003c/sup\u003eGa-labeled human CD8\u0026beta;-targeting nanobody (hCD8\u0026beta; Nb) or irrelevant nanobody (Irr Nb) after labeling (before purification) with \u003csup\u003e99m\u003c/sup\u003eTc or \u003csup\u003e68\u003c/sup\u003eGa and after purification. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D of at least 3 independent experiments.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNb\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003csup\u003e99m\u003c/sup\u003eTc RCP before purification (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003csup\u003e99m\u003c/sup\u003eTc RCP after\u003c/p\u003e\n\u003cp\u003epurification (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003csup\u003e68\u003c/sup\u003eGa RCP before\u003c/p\u003e\n\u003cp\u003epurification (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003csup\u003e68\u003c/sup\u003eGa RCP after\u003c/p\u003e\n\u003cp\u003epurification (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ehCD8\u0026beta; Nb\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e90.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e99.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e99.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e99.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eIrr Nb\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e92.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e99.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e98.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e98.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eRCP: Radiochemical purity\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldUnderline\"\u003eThe anti-human CD8\u0026beta; PET tracer visualizes human CD8\u003c/span\u003e\u003csup\u003e\u003cspan class=\"BoldUnderline\"\u003e+\u003c/span\u003e \u003c/sup\u003e \u003cspan class=\"BoldUnderline\"\u003eT-cell dynamics\u003c/span\u003e \u003cspan class=\"BoldItalicUnderline\"\u003ein vivo\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eAs PET imaging enables a higher spatial resolution and is more commonly used in the clinic, the anti-hCD8\u0026beta; Nb was converted to a PET tracer. To this end, the Nb was first conjugated to NOTA on its lysines and subsequently radiolabeled with Gallium-68 (\u003csup\u003e68\u003c/sup\u003eGa). A radiochemical purity above 98% was observed even before purification (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In addition, the radiolabeled anti-hCD8\u0026beta; Nb remained stable after incubation in injection buffer and human serum, at room temperature or at 37\u0026deg;C (Supplemental Fig.\u0026nbsp;6). To ensure that NOTA-coupling did not interfere with Nb binding to hCD8, the binding affinity of the NOTA-coupled anti-hCD8\u0026beta; Nb was determined via surface plasmon resonance and flow cytometry (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea-b and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). NOTA-coupling did not have a large impact on the binding of anti-hCD8\u0026beta; Nb (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). In line with the surface plasmon resonance results, NOTA-coupling of the anti-hCD8\u0026beta; Nb did not impact binding to SUP-T1 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab4\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003e\u003cstrong\u003eOverview of the in vitro characteristics of the NOTA-conjugated anti-human CD8\u0026beta; nanobody (hCD8\u0026beta; Nb).\u003c/strong\u003e Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D of at least 3 independent experiments.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNb\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eK\u003csub\u003eD\u003c/sub\u003e hCD8\u0026alpha;\u0026beta; protein (nM)\u003c/p\u003e\n\u003cp\u003eSPR\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eK\u003csub\u003eD\u003c/sub\u003e SUP-T1 cells (nM)\u003c/p\u003e\n\u003cp\u003eFlow cytometry\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eNOTA-hCD8\u0026beta; Nb\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNext, the ability of \u003csup\u003e68\u003c/sup\u003eGa-labeled anti-hCD8\u0026beta; Nb to target hCD8\u003csup\u003e+\u003c/sup\u003e T cells \u003cem\u003ein vivo\u003c/em\u003e was determined in na\u0026iuml;ve hCD8 transgenic mice. Similar to the \u003csup\u003e99m\u003c/sup\u003eTc-labeled variant, specific uptake of \u003csup\u003e68\u003c/sup\u003eGa-labeled anti-hCD8\u0026beta; Nb in T-cell-rich organs was observed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec-d) via PET/CT imaging and \u003cem\u003eex vivo\u003c/em\u003e \u0026gamma;-counting. Then, we assessed whether \u003csup\u003e68\u003c/sup\u003eGa-labeled anti-hCD8\u0026beta; Nb was able to visualize T-cell dynamics. To this end, hCD8 transgenic animals were subcutaneously inoculated with MC38 tumor cells and were imaged over time (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). Tumor growth and CD8\u003csup\u003e+\u003c/sup\u003e T-cell dynamics were followed over a period of 18 days. Individual differences in tumor growth could be observed as of day 9 post inoculation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). Interestingly, PET imaging with the \u003csup\u003e68\u003c/sup\u003eGa-labeled anti-hCD8\u0026beta; Nb at endpoint (day 15 or day 18) allowed, despite overall low levels of T-cell infiltration, the differentiation of mice with lower or higher levels of human CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration in the MC38 tumors (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec). These findings were quantified via analysis of the individual PET images, \u003cem\u003eex vivo\u003c/em\u003e \u0026gamma;-counting and flow cytometric immunophenotyping of the dissected tumors. Quantification of radioactive uptake in the tumor determined via the PET images nicely correlated with the radioactive uptake measured via \u003cem\u003eex vivo\u003c/em\u003e \u0026gamma;-counting of the dissected tumors (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed). In addition, a good correlation was observed between the radioactive uptake and the amount of intratumoral human CD8\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ee). Overall, these results show that, despite the low levels of intratumoral human CD8\u003csup\u003e+\u003c/sup\u003e T cells, the differences in T-cell tumor infiltration can be visualized using the \u003csup\u003e68\u003c/sup\u003eGa-labeled anti-hCD8\u0026beta; Nb. In addition to visualizing intratumoral T cells, we could also follow up T-cell dynamics in the lymph nodes. Baseline PET scans revealed already a significantly higher level of CD8\u003csup\u003e+\u003c/sup\u003e T cells in the tumor-draining lymph node compared to the opposing inguinal lymph node (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ef). To determine whether this difference was tumor specific, we analyzed the ratio of radioactive uptake between these lymph nodes and compared it to the ratio between opposing cervical lymph nodes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eg). Repeated imaging showed that a consistent significantly higher ratio of radioactive uptake was observed in the tumor-draining lymph node relative to the contralateral inguinal lymph node, compared to that between the cervical lymph nodes during the whole experiment. Finally, we assessed the prognostic value of the \u003csup\u003e68\u003c/sup\u003eGa-labeled anti-hCD8\u0026beta; Nb (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eh). To this end, we correlated the amount of intratumoral human CD8\u003csup\u003e+\u003c/sup\u003e T cells present at baseline, as measured via PET imaging, and the increase of tumor growth, as determined via the ratio of tumor size at day 15 and baseline. First, mice showing higher radioactive uptake levels at baseline also showed higher uptake levels at endpoint (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed-e-h). Interestingly, a significant correlation was observed between the radioactive uptake measured at baseline and the tumor growth, indicating that \u003csup\u003e68\u003c/sup\u003eGa-labeled anti-hCD8\u0026beta; Nb may hold prognostic value to differentiate the speed of tumor growth based on the amount of intratumoral hCD8\u003csup\u003e+\u003c/sup\u003e T cells at early timepoints.\u003c/p\u003e\n\u003cp\u003eFinally, we performed a proof-of-concept study to investigate the biodistribution profile of the anti-hCD8\u0026beta; Nb in healthy non-human primates. To this end, the NOTA-conjugated anti-hCD8\u0026beta; Nb was first radiolabeled with \u003csup\u003e64\u003c/sup\u003eCu. Next, 500 \u0026micro;g of the \u003csup\u003e64\u003c/sup\u003eCu-labeled anti-hCD8\u0026beta; Nb was injected intravenously in 2 cynomolgus monkeys followed by PET/CT imaging 1 hour post injection (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea). Similar to the mice, a quick renal clearance of the \u003csup\u003e64\u003c/sup\u003eCu-labeled anti-hCD8\u0026beta; Nb was observed with a high uptake in the kidneys and bladder while no signal was observed in the blood (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb-c). Furthermore, only minor uptake in the liver was observed. In both animals, the \u003csup\u003e64\u003c/sup\u003eCu-labeled anti-hCD8\u0026beta; Nb showed a similar uptake pattern with the ability to visualize both primary and secondary lymphoid organs, such as thymus, spleen, tonsils and a multitude of lymph nodes including the cervical, inguinal, axillary and tracheobronchial lymph nodes. Interestingly, both cynomolgus monkeys showed a high uptake around the mouth area. Furthermore, the dose of 500 \u0026micro;g of the \u003csup\u003e64\u003c/sup\u003eCu-labeled anti-hCD8\u0026beta; Nb was well-tolerated. In conclusion, these results indicated the possibility to visualize CD8\u003csup\u003e+\u003c/sup\u003e T cells in bigger animals.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we report the development of a novel immunotracer for the non-invasive imaging of hCD8\u003csup\u003e+\u003c/sup\u003e T-cell dynamics via SPECT and/or PET. In-depth \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e characterization shows high affinity and specificity of these tracers towards human CD8\u003csup\u003e+\u003c/sup\u003e T cells and their ability to non-invasively visualize T-cell dynamics with high sensitivity.\u003c/p\u003e \u003cp\u003eAs T-cell-based immunotherapies have been a major point of focus in anti-cancer therapies, non-invasive imaging of T-cell dynamics has gained quite some interest in recent years. To this end, several tracers targeting different T-cell markers (e.g. CD3, CD4, CTLA-4, PD-1, LAG-3, TIGIT,etc.) have been reported [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In particular, imaging of cytotoxic CD8\u003csup\u003e+\u003c/sup\u003e T cells has received attention as this T-cell population is believed to hold predictive and/or prognostic value [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we show the ability of our novel nanobody-based immunotracer to detect human CD8\u003csup\u003e+\u003c/sup\u003e T cells using SPECT and PET scanning. Nanobodies have emerged as an interesting targeting scaffold for diagnostic imaging due to their \u003cem\u003ein vivo\u003c/em\u003e characteristics, resulting in a fast tissue-to-background contrast, enabling same-day imaging of patients with short-lived isotopes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Our study also shows the ability of nanobody-based immunotracers to visualize CD8\u003csup\u003e+\u003c/sup\u003e T cells already 1h post injection via the use of short-lived isotopes such as \u003csup\u003e99m\u003c/sup\u003eTc (for SPECT imaging) and \u003csup\u003e68\u003c/sup\u003eGa or \u003csup\u003e64\u003c/sup\u003eCu (for PET imaging). Both isotopes are currently being employed as part of clinically tested tracers, showing translatability of our developed immunotracers [\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Furthermore, the use of these short-lived isotopes results in a lower radiation burden for patients compared to long-lived isotopes, such as \u003csup\u003e89\u003c/sup\u003eZr, which are used for bigger antibody-(fragment-)based tracers [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAll \u003cem\u003ein vitro\u003c/em\u003e results show a high affinity and specific binding of the anti-human CD8β Nb. Furthermore, the human CD8β-targeting Nb does not cause unwanted T-cell activation. That is important, since CD8 is an important mediator of T-cell activation and previous studies indicated that binding of CD8-targeting antibodies may induce T-cell activation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Furthermore, no immunogenic responses towards the Nb were observed in our T cell/DC co-culture assays. Although Nbs are assumed to be non-immunogenic due to their small size, high homology to human VH fragments and their fast half-life time [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], patients developing anti-drug antibodies after Nb treatments have been reported and the immunogenic profiles of the Nbs have to be evaluated on an individual basis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In the future, immunogenic responses towards the NOTA-conjugated immunotracers will also need to be assessed during clinical testing. However, immunogenic responses are expected to be low, since diagnostic tracers can be microdosed [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This notion is corroborated by previous findings, showing that a HER2-targeted Nb-based tracer used in a phase I clinical trial had a low immunogenicity [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Together, these are important parameters for future clinical translatability of this tracer.\u003c/p\u003e \u003cp\u003eTo date, a few hCD8-targeting tracers have been reported and are being tested clinically. This includes the \u003csup\u003e89\u003c/sup\u003eZr-labeled minibody Df-IAB22M2C, \u003csup\u003e89\u003c/sup\u003eZr-labeled antibody ZED88082A, \u003csup\u003e68\u003c/sup\u003eGa-labeled Nb SNA006 and \u003csup\u003e18\u003c/sup\u003eF-labeled Nb VHH5v2 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. While these tracers have shown promising results so far, they still suffer from some disadvantages with respect to pharmacokinetics and specificity. Df-IAB22M2C and ZED88082A are bulky proteins (\u0026ge;\u0026thinsp;80kDa), making them unsuitable for early point and/or fast repeated imaging due to their long \u003cem\u003ein vivo\u003c/em\u003e half-life time and slower tumor penetration. For this reason, nanobody-based tracers could be complementary to these tracers, or could be preferred altogether thanks to the characteristics mentioned previously. In our study, we show the ability of our tracer to detect CD8\u003csup\u003e+\u003c/sup\u003e T cells already 1h post injection and to follow T-cell dynamics via repeated imaging over a time period of 18 days. These characteristics could potentially be essential for the prediction and/or follow-up of immunotherapy responses as a recent study of Kist de Ruijter \u003cem\u003eet al.\u003c/em\u003e indicated the need for CD8-targeting tracers that allow (early) sequential imaging timepoints in order to follow spatio-temporal changes of CD8\u003csup\u003e+\u003c/sup\u003e T cells [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In line with these findings, we were able to correlate early baseline levels of intratumoral hCD8\u003csup\u003e+\u003c/sup\u003e T cells to subsequent tumor growth, suggesting that early timepoint imaging of hCD8\u003csup\u003e+\u003c/sup\u003e T cells may indeed hold a prognostic value.\u003c/p\u003e \u003cp\u003eAll of the current (pre-)clinically-tested tracers against CD8\u003csup\u003e+\u003c/sup\u003e T cells (including the Nb-based SNA006 and VHH5v2) target the α-chain of the CD8 protein. While CD8α is indeed expressed on CD8\u003csup\u003e+\u003c/sup\u003e T cells, several studies have reported the presence of CD8α on other immune cell populations, including NK cells and monocytes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In contrast, CD8β is exclusively expressed on CD8\u003csup\u003e+\u003c/sup\u003e T cells, making it a more selective target for visualizing T-cell dynamics. Indeed, our results show the enhanced specificity of our anti-CD8β Nb for CD8\u003csup\u003e+\u003c/sup\u003e T cells, as no binding to NK or myeloid cells was observed, while an anti-CD8α Nb strongly stains NK and myeloid cells. While it remains to be seen whether this enhanced specificity also results in an enhanced predictive or prognostic value, it would not be surprising as tumor-associated monocytes can encompass a major immune cell population in the tumor microenvironment and give rise to immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe PET imaging results in cynomolgus monkeys also showed the feasibility to image CD8\u003csup\u003e+\u003c/sup\u003e T cells in bigger animals. However, it is important to note that this was a proof-of-concept study. In the future, a dose escalation study and imaging at multiple timepoints could give additional information for the clinical translation. To enable these future studies, we decided to radiolabel our anti-hCD8β Nb with \u003csup\u003e64\u003c/sup\u003eCu instead of \u003csup\u003e68\u003c/sup\u003eGa. As \u003csup\u003e64\u003c/sup\u003eCu has a half-life time of 12.7h, this would enable us to assess the uptake of the anti-hCD8β Nb for a time period of multiple hours to day(s) and give additional information on the pharmacokinetics of this Nb. While we saw a similar biodistribution profile in cynomolgus monkeys as in our mouse imaging experiments, we also noticed uptake of the radiolabeled human CD8β-targeting Nb in the mouth area of both monkeys. The exact reason for this radioactive uptake in the mouth area is still unclear. However, it has been reported that CD8\u003csup\u003e+\u003c/sup\u003e T cells are present in the oral tissues of non-human primates [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Moreover, while no acute inflammation could be observed upon visual inspection, a local inflammation cannot be excluded. Interestingly, we did not observe much liver uptake while high liver and gallbladder uptake has been reported for \u003csup\u003e64\u003c/sup\u003eCu-labeled compounds [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFinally, the non-invasive imaging of CD8\u003csup\u003e+\u003c/sup\u003e T-cell dynamics could be interesting for multiple immune-related diseases. The presence of CD8\u003csup\u003e+\u003c/sup\u003e T cells may be indicative of viral infections or chronic inflammatory and autoimmune diseases, including immune thrombocytopenia, rheumatoid arthritis and giant cell arteritis. Furthermore, previous preclinical research has focused on the non-invasive imaging of T-cell populations in graft-versus-host disease and multiple sclerosis, conditions for which imaging of CD8\u003csup\u003e+\u003c/sup\u003e T cells might also be interesting [\u003cspan additionalcitationids=\"CR42 CR43 CR44 CR45\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Therefore, it is not unlikely that these tracers could be widely applied in multiple disease settings in the future.\u003c/p\u003e \u003cp\u003eIn conclusion, we have developed a novel SPECT- and PET-tracer to visualize hCD8\u0026thinsp;+\u0026thinsp;T-cell dynamics at early timepoints with high specificity and sensitivity. These tracers could potentially be widely applied in multiple immune-related diseases to follow-up and/or predict immunotherapy responses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 831514 (Immune-Image). The JU receives support from the European Union\u0026rsquo;s Horizon 2020 research and innovation programme and EFPIA. This work was further funded by the Strategic Research Programme and Wetenschappelijk Fonds Willy Gepts from the Vrije Universiteit Brussel. This work is also supported by Kom op Tegen Kanker and Research Foundation Flanders (FWO) research projects G087524N I005622N and I001618N. Timo W.M. De Groof is funded by a post-doctoral fellowship (12ZO723N) from the Research Foundation Flanders (FWO), Belgium. he Infectious Disease Models and Innovative Therapies (IDMIT) research infrastructure is supported by the \u0026ldquo;Programme Investissements d\u0026rsquo;Avenir\u0026rdquo;, managed by the ANR under reference ANR-11-INBS-0008.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTimo W.M. De Groof, Yoline Lauwers, C\u0026eacute;cile Vincke, Geert Raes, Jo A.Van Ginderachter and Nick Devoogdt are co-inventors on a pending patent application (EP23153689.7), which covers the use of the described CD8-targeting immunotracer\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTimo W.M. De Groof, Yoline Lauwers, Jo A.Van Ginderachter and Nick Devoogdt designed the study; Timo W.M. De Groof performed the \u003cem\u003ein vitro\u003c/em\u003e characterization assays with the help from Yoline Lauwers, Tessa De Pauw and C\u0026eacute;cile Vincke; Timo W.M. De Groof and Yoline Lauwers performed the SPECT and PET imaging experiments with the help from Tessa De Pauw and Jolien Van Craenenbroeck; Mohit Saxena, Thibault Naninck, Roger Le Grand and Catherine Chapon performed the immunostaining and PET imaging experiments on non-human primate tissue; Timo W.M. De Groof analyzed the Alphafold binding prediction models; Timo W.M. De Groof, Yoline Lauwers and M.S. analyzed the experimental data; Geert Raes, Jo A.Van Ginderachter and Nick Devoogdt supervised the study; Timo W.M. De Groof wrote the paper with the help from all the authors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 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).\u003c/p\u003e\n\u003cp\u003eCynomolgus macaques were housed at the IDMIT infrastructure facilities (CEA, Fontenay-aux-roses, France) under BSL-3 containment (Animal facility authorization #D92-032-02, Pr\u0026eacute;fecture 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 \u0026lsquo;Comit\u0026eacute; d\u0026rsquo;Ethique en Exp\u0026eacute;rimentation Animale du Commissariat \u0026agrave; l\u0026rsquo;Energie Atomique et aux Energies Alternatives\u0026rsquo; (CEtEA number 44) under statement number A23-057. The study was authorized by the \u0026lsquo;\u0026lsquo;Research, Innovation and Education Ministry\u0026rsquo;\u0026rsquo; under registration number APAFIS #46283-202312131546674 v1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Kevin De Jonghe (MITH, VUB), Steve Huvelle (Biomaps Unit, CEA), VIB nanobody core facility, VIB protein core facility, VIB proteomics core facility, VUB In vivo Cellular and Molecular Imaging (ICMI) core facility and Roche for the technical assistance. We also would like to thank ARRONAX (Nantes) for the delivery of \u003csup\u003e64\u003c/sup\u003eCu. This work has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 831514 (Immune-Image). The JU receives support from the European Union\u0026rsquo;s Horizon 2020 research and innovation programme and EFPIA. This work was further funded by the Strategic Research Programme and Wetenschappelijk Fonds Willy Gepts from the Vrije Universiteit Brussel. This work is also supported by Kom op Tegen Kanker and Research Foundation Flanders (FWO) research projects G087524N, I005622N and I001618N. Timo W.M. De Groof is funded by a post-doctoral fellowship (12ZO723N) from the Research Foundation Flanders (FWO), Belgium. he Infectious Disease Models and Innovative Therapies (IDMIT) research infrastructure is supported by the \u0026ldquo;Programme Investissements d\u0026rsquo;Avenir\u0026rdquo;, managed by the ANR under reference ANR-11-INBS-0008.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEsfahani K, Roudaia L, Buhlaiga N, Del Rincon SV, Papneja N, Miller WH, Jr. A review of cancer immunotherapy: from the past, to the present, to the future. Curr Oncol. 2020;27:S87-S97. doi:10.3747/co.27.5223.\u003c/li\u003e\n\u003cli\u003eKourie HR, Klastersky J. Immune checkpoint inhibitors side effects and management. Immunotherapy. 2016;8:799-807. doi:10.2217/imt-2016-0029.\u003c/li\u003e\n\u003cli\u003eArnouk S, De Groof TWM, Van Ginderachter JA. Imaging and therapeutic targeting of the tumor immune microenvironment with biologics. 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Front Immunol. 2015;6:604. doi:10.3389/fimmu.2015.00604.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Vrije Universiteit Brussel","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Immuno-imaging, nuclear imaging, nanobodies, T-cell dynamics","lastPublishedDoi":"10.21203/rs.3.rs-4322357/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4322357/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eWhile immunotherapy has revolutionized the oncology field, variations in therapy responsiveness limit the broad applicability of these therapies. Diagnostic imaging of immune cell, and specifically CD8\u003csup\u003e+\u003c/sup\u003e T cell, dynamics could allow early patient stratification and result in improved therapy efficacy and safety. In this study, we report the development of a nanobody-based immunotracer for non-invasive SPECT and PET imaging of human CD8\u003csup\u003e+\u003c/sup\u003e T-cell dynamics.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eNanobodies targeting human CD8β were generated via llama immunizations and subsequent biopanning. The lead anti-human CD8β nanobody was characterized \u003cem\u003ein vitro\u003c/em\u003e on binding, specificity, stability and toxicity. The lead nanobody was labelled with \u003csup\u003e99m\u003c/sup\u003eTc and \u003csup\u003e68\u003c/sup\u003eGa for non-invasive imaging of human T-cell lymphomas and CD8\u003csup\u003e+\u003c/sup\u003e T cells in human CD8 transgenic mice and non-human primates via SPECT or PET/CT. Repeated imaging of CD8\u003csup\u003e+\u003c/sup\u003e T cells in MC38 tumor-bearing mice was performed to visualize CD8\u003csup\u003e+\u003c/sup\u003e T-cell dynamics.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe nanobody-based immunotracer showed high affinity and specific binding to human CD8 without unwanted immune activation. CD8\u003csup\u003e+\u003c/sup\u003e T cells were non-invasively visualized via SPECT and PET imaging in na\u0026iuml;ve and tumor-bearing mice and in na\u0026iuml;ve non-human primates with high sensitivity. The nanobody-based immunotracer showed enhanced specificity for CD8\u003csup\u003e+\u003c/sup\u003e T cells and/or faster \u003cem\u003ein vivo\u003c/em\u003e pharmacokinetics compared to previous human CD8-targeting immunotracers, allowing us to follow human CD8\u003csup\u003e+\u003c/sup\u003e T-cell dynamics already at early timepoints.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOverall, this study describes the development of a more specific human CD8\u003csup\u003e+\u003c/sup\u003e T-cell-targeting immunotracer, allowing follow up of immunotherapy responses via non-invasive imaging of human CD8\u003csup\u003e+\u003c/sup\u003e T-cell dynamics.\u003c/p\u003e","manuscriptTitle":"Specific Imaging of CD8+ T-Cell Dynamics with a Nanobody Radiotracer against Human CD8β","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-29 12:13:41","doi":"10.21203/rs.3.rs-4322357/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1c2b503f-e995-405d-9ad0-0a8c11d9512c","owner":[],"postedDate":"April 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":31138157,"name":"Nuclear Medicine \u0026 Medical Imaging"}],"tags":[],"updatedAt":"2024-04-29T12:13:41+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-29 12:13:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4322357","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4322357","identity":"rs-4322357","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}