For the generation of hOX40-specific Nbs, two alpacas (Vicugna pacos) were immunized with the extracellular domain of hOX40, which contains the amino acid residues from Leu29 to Ala216. A positive immune response in both animals was confirmed on day 63 by serum ELISA ( Supplementary Figure S1 ). Starting from peripheral blood lymphocytes (PBLs), we established a Nb-phagemid library representing the VHH repertoire of both animals (size: ~3 x 10⁷ clones), from which hOX40-Nbs were enriched against recombinant hOX40 in two consecutive rounds of phage display. The selective binding of individual clones was tested in a whole-cell phage ELISA with U2OS cells stably expressing hOX40 (U2OS-hOX40). Subsequent sequencing revealed four unique hOX40-Nbs, namely O7, O12, O18 and O19, which exhibited highly diverse complementarity determining regions (CDRs) 3 ( Figure 1A , Supplementary Table S1 ). All selected Nbs were expressed in Escherichia coli (E. coli) and purified by immobilized metal ion affinity chromatography (IMAC) followed by size exclusion chromatography (SEC), yielding high purity binding molecules ( Figure 1B ). To initially assess their binding affinities, we performed biolayer interferometry (BLI) and determined K_D values in the pico- to low nanomolar range (0.2 - 3.4 nM), while O7 showed a substantially weaker affinity as reflected by a K_D of ~ 150 nM ( Figure 1C , Supplementary Figure S2A ). In addition, we determined the folding stability of the selected candidates using differential scanning fluorimetry (nanoDSF). All Nbs showed high thermal stabilities with melting temperatures (T_M) between 50°C and 74°C without aggregation. Notably, this was not affected by an accelerated aging period of 10 days at 37°C ( Figure 1D , Supplementary Figure S2B ).

For the fluorescent functionalization of hOX40-Nbs, we took advantage of a sortase-based approach to selectively attach an azide-group to the C-termini of the Nbs, which served as chemical handle for the addition of a AlexaFluor647(AF647)-conjugated dibenzocyclooctyne (DBCO-AF647) group utilizing click chemistry (16). As a result, we obtained Nbs comprising a C-terminal fluorophore with a defined labeling ratio of 1:1. The fluorescent Nbs were used to determine corresponding K_D (half-maximum binding at equilibrium) values on U2OS-hOX40 cells by flow cytometry. In accordance with the BLI-determined affinities, a strong functional binding for O12 and O18 with K_D values in the subnanomolar range (~ 0.1 nM for O12; ~ 0.3 nM for O18) was determined, whereas O7 and O19 displayed slightly weaker affinities ( Figure 2A , Supplementary Figure S3A ). To further confirm specific binding of the selected Nbs to hOX40 localized at the plasma membrane of mammalian cells, we used the fluorescent Nbs for live cell staining of U2OS-hOX40 cells in comparison to wild-type U2OS (U2OS-WT) cells. The images displayed intense signals localized at the cellular surface for all tested binders, which was comparable to the staining with a commercially available anti-hOX40 mAb, while none of the tested Nbs showed non-specific binding to U2OS-WT cells ( Figure 2B ). In addition, we used this approach to test a potential cross-reactivity of the Nbs to murine OX40 (mOX40) which has a ~63% sequence homology and performed live cell imaging on U2OS cells transiently expressing mOX40. Only O7 bound to mOX40, while all other candidates showed no staining of mOX40 expressing U2OS cells ( Supplementary Figure S3B ). In summary, we identified four hOX40-Nbs that bind recombinant as well as cell-resident hOX40. With respect to O12, O18 and O19 we selected high-affinity binders with K_D values in the pico- to low nanomolar range that exhibited strong specific binding to membrane-exposed hOX40. Notably, only O7 was less affine to recombinant hOX40 but showed additional cross-reactivity towards mOX40.

To localize the binding sites of the selected hOX40-Nbs within the natively folded hOX40, we generated cellular expression constructs comprising domain-deletion mutants of hOX40 domains 1-3, which we transiently expressed in U2OS cells. Nb binding to truncated versions of hOX40 was visualized by immunofluorescence imaging of live cells. An anti-hOX40 mAb directed against domain 4 was used as a positive control ( Figure 3A ). Based on these results, we allocated binding of O7 and O19 to domain 3 and of O12 and O18 to domain 1 of hOX40 ( Figure 3B ). To examine a potential combinatorial binding of the different hOX40-Nbs, we further performed epitope binning analysis by BLI ( Figure 3C ). As expected from the domain mapping, O12 and O18, both targeting domain 1, simultaneously bound hOX40 in complex with O7 or O19, each targeting domain 3. However, only weak combinatorial binding was observed for Nbs addressing the same domain, suggesting that O12 and O18, as well as O7 and O19 address identical or at least overlapping epitopes ( Figure 3D , Supplementary Figure S4 ).

Having demonstrated that all selected Nbs recognize recombinant and exogenously overexpressed cellular hOX40, we next investigated their specificity for binding to endogenous hOX40 on activated T cells. Therefore, human peripheral blood mononuclear cells (hPBMCs) from three healthy donors (K025, K029 and K034) were either left untreated or incubated for 24 h with phytohaemagglutinin L (PHA-L) as a pan T cell stimulus and interleukin 2 (IL-2) to induce expression of OX40 (49) ( Figure 4A ). Subsequently, hPBMCs were double-stained with fluorescently labeled hOX40-Nbs, the non-binding PEP-Nb (50) (Nb Ctrl.) or a phycoerythrin (PE)-labeled anti-hOX40 mAb in combination with a T cell-specific anti-CD3 mAb, and the percentage of OX40⁺ positive T cells (CD3⁺) was analyzed by flow cytometry ( Figure 4C , gating: Supplementary Figure S5A ). The obtained data show that all hOX40-Nbs except O7 bound specifically to T cells upon PHA-L-/IL-2 mediated activation, comparable to the anti-hOX40 mAb. Notably, no binding prior to stimulation was observed ( Figures 4B, C , Supplementary Figure S5B ). Determination of mean fluorescence intensities (MFI) normalized to the negative control (PEP-Nb) revealed differences that reflect the affinities of the Nbs previously determined with BLI ( Supplementary Figure S5C ). To further investigate the binding of hOX40-Nbs to different T cell subpopulations, we performed flow cytometry analysis of activated hPBMCs with Nbs O12, O18, O19, non-binding PEP-Nb or the anti-hOX40-mAb in combination with antibodies indicative for main T cell populations including anti-CD4, anti-CD8 as well as anti-CD25 and anti-FoxP3 antibodies for the assessment of Tregs ( Supplementary Figure S6A ). As shown in Figure 4D , the results indicated that the selected hOX40-Nbs bind to OX40 on all three analyzed T cell subpopulations upon activation, comparable to the anti-hOX40 mAb. Furthermore, differences in OX40 expression were found for the various T cell populations and the donors analyzed. While we observed a higher percentage of OX40-expressing cells in non Treg CD4⁺ T cells and Tregs, lower expression of OX40 on CD8⁺ T cells was observed for all three tested donors ( Figure 4D ). In summary, the flow cytometric analysis confirmed the specificity of three of the selected hOX40 Nbs for their antigen and their functionality to bind endogenous hOX40 exclusively on activated T cells.

Targeting OX40, can trigger strong immune responses (51). Therefore, we next investigated whether binding of the Nbs exerts agonistic or antagonistic effects on OX40-mediated signaling by using a genetically engineered Jurkat T cell-based bioassay. These effector cells express hOX40 and contain a luciferase reporter driven by a response element downstream of the OX40 signaling axis. Non-stimulated OX40 effector cells exhibited a weak luminescent signal, which was not further enhanced by addition of increasing concentrations of Nbs O12, O18, and O19. However, when using OX40L as positive control, we observed a strong concentration-dependent induction of NF-kB promotor activity, reflected by an increasing luminescence signal, as expected ( Figure 5A ). Next, we used this assay to analyze a possible competition between OX40L and Nbs for hOX40 binding. Therefore, we preincubated the OX40 effector cells with serial dilutions of Nbs ranging from 0.13 µM to 0.002 nM before adding OX40L at the saturation concentration of 0.12 µM. In this setting, we observed a reduction in luminescence in the presence of Nb O12 and, to a minor extent, of O18, whereas pre-incubation with O19 did not have any effect on OX40L-mediated induction of OX40 signaling ( Figure 5B ). These results were consistent with a BLI-based competition assay in which we monitored the binding of OX40L to recombinant hOX40 in the presence or absence of Nbs ( Supplementary Figure S7 ). From these findings, we concluded that none of the Nbs augment OX40-mediated signaling. However, O12 and, to a minor degree, O18, which both target domain 1 of hOX40, competed with the binding of the natural ligand OX40L and could therefore potentially exert an antagonistic effect.

To further explore possible effects of hOX40-Nb binding on T cells, we next investigated its influence on proliferation and cytokine release in hPBMCs. Therefore, hPBMCs from three donors (K025, K029 and K034) were labeled with carboxyfluorescein succinimidyl ester (CFSE) followed by induction of hOX40 expression by PHA-L and IL-2 stimulation for 24 h. After confirming successful activation and hOX40 expression by flow cytometry ( Supplementary Figures S8A, B ), hPBMCs were either left untreated or incubated with Nbs O12, O18, O19 or the non-binding PEP-Nb as a negative control (each 0.5 µM). For targeted hOX40 stimulation 0.5 µM OX40L was used ( Figure 5C ). Cell proliferation was monitored on days 4, 6, 8 and 12 by flow cytometry ( Figure 5D ). The results revealed similar proliferation profiles in the hPBMC samples from the same donor upon Nb treatment compared to the untreated samples, while treatment with OX40L induced a ~10% increase at day 6 and 8 in proliferation ( Figure 5D , Supplementary Figures S8C, D ). In addition, we investigated effects of hOX40-Nbs on the release of cytokines. Therefore, we determined the concentration of a panel of pro- and anti-inflammatory cytokines ( Supplementary Table S2 ) in the supernatant of samples collected after 24, 72 and 168 hours post Nb treatment using a previously reported microsphere-based sandwich immunoassay (16). While Nb O18 and O19 showed only minor effects on cytokine release compared to the untreated control or samples incubated with a non-binding PEP-Nb, a significant increase of the NFκ-driven cytokines TNF and IL-6 and of the Th2 cytokines IL-4, and IL-10 upon treatment with O12 was observed. Interestingly, the cytokine release of O12-treated samples differed from samples treated with OX40L, in which only elevated levels of IL-13 were observed ( Figure 5E , Supplementary Figure S9 ).

For in vivo OI, we chose the fluorescently-labeled Nb O18 (O18_AF647) as it showed the strongest binding to cellular exposed hOX40 with only minor blocking effect on OX40L and did not modulate T cell function or cytokine expression. CD1 nude mice with subcutaneous HT1080-hOX40 or HT1080-WT tumors were intravenously (i.v.) injected with 5 μg of O18_AF647 and non-invasively in vivo investigated by OI over 6 h. The signal intensity (SI) of O18_AF647 in both the HT1080-hOX40 and the HT1080-WT tumors peaked within 5 min after injection. While the SI continuously decreased over time in the HT1080-WT tumors, it remained stable in the HT1080-hOX40 tumors between 3 h and 6 h post injection, indicating target-specific accumulation after the initial clearing phase of O18_AF647. Importantly, we determined an increased O18_AF647-related SI in the HT1080-hOX40 tumors compared to HT1080-WT tumors at all imaging time points with the greatest difference 6 h post injections of ~7-fold ( Figures 6A, B ). Finally, mice were sacrificed, and the presence of O18_AF647 within the explanted tumors was analyzed by ex vivo OI. Consistent with the in vivo data, HT1080-hOX40 tumors exhibited a significantly higher uptake (SI) when compared to HT1080-WT control tumors, indicating a specific binding of the Nb O18 to its target antigen and a favorable signal-to-background ratio for this Nb-derived immunoprobe ( Figure 6C ). These findings demonstrated a specific binding for O18_AF647 in vivo, highlighting its potential as a promising tool for noninvasive monitoring of OX40 expression in immune diagnostics.

hOX40-encoding DNA (GenBank accession: NM_003327.3) was synthesized and cloned into NheI and EcoRI site of pcDNA3.1(+) by GenScript Biotech. The vector backbone was changed by cutting with the restriction enzymes EcoRI and BstBI into a backbone comprising an internal ribosomal entry site (IRES) and genes for eGFP as reporter and Blasticidin S deaminase for antibiotics resistance from the expression construct as described previously (16). For the generation of hOX40 domain deletion mutant expression constructs hOX40ΔD1 (aa 66-277), hOX40ΔD1-2 (aa 108-277), hOX40ΔD1-3 (aa 127-277) of UniProtKB P43489, respective fragments were amplified ( Supplementary Table S3 ) and genetically fused N-terminally to a SPOT-Tag (69). DNA encoding for murine OX40 (mOX40) was purchased from Sino Biological (Catalog Number MG50808-NM).

U2OS cells (ATCC) were cultured according to standard protocols in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (v/v) FCS and 1% (v/v) penicillin/streptomycin (all Thermo Fisher Scientific). Cultivation conditions were 37°C and 5% CO₂ atmosphere in a humidified incubator and passaged using 0.05% trypsin-EDTA (Thermo Fisher Scientific). Transfection of plasmid DNA (0.8 µg per 24 well plate well) was performed using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. To generate U2OS cells stably overexpressing hOX40 on their surface (U2OS-hOX40), 24 h after transfection, selection pressure by 5 µg/mL Blasticidine S (Sigma Aldrich) was applied for a period of two weeks. After single cell separation, monoclonal cells were analyzed for hOX40 expression using live-cell fluorescence microscopy.

The alpaca immunization was performed with the approval of the Government of Upper Bavaria (approval number: 55.2-1-54-2532.0-80-14). Two alpacas (Vicugna pacos) were immunized using the extracellular part of recombinant hOX40 (hOX40 AA Leu 29 – Ala 216) produced in human HEK293 cells (Acrobiosystems). During a period of 91 days, the animals were vaccinated six times at day 0, 21, 28, 35, 49, 87. The initial vaccination was performed with 560 µg followed by five booster injections each consisting of 280 µg hOX40 with Adjuvant F (Gebru). After the 91-day period, lymphocytes were isolated from ~200 mL of blood performing Ficoll gradient centrifugation with lymphocyte separation medium (Carl Roth) and total RNA was extracted by NucleoSpin^® RNA II (Macherey&Nagel). The mRNA was subsequently transcribed into cDNA by the First Strand cDNA Synthesis Kit (GE Healthcare). The Nb repertoire was isolated as described in three subsequent PCR reactions using the following primer combinations: (1) CALL001 and CALL002, (2) forward primers FR1-1, FR1-2, FR1-3, FR1-4, and reverse primer CALL002, and (3) forward primers FR1-ext1 and FR1-ext2 and reverse primers FR4-1, FR4-2, FR4-3, FR4-4, FR4-5, and FR4-6 introducing SfiI and NotI restriction sites (16) ( Supplementary Table S3 ). This enables subcloning of Nb library into the pHEN4 phagemid vector (70).

The selection of hOX40-specific Nbs was performed by two consecutive rounds of phage display against immobilized recombinant antigen. For this purpose, electrocompetent TG1 E. coli bacteria were transformed with the hOX40-Nb library in pHEN4 and infected with M13K07 helper phages leading to the generation of hOX40-Nb presenting phages. 1 x 10¹¹ phages were enriched by adsorption to streptavidin or neutravidin plates (Thermo Fisher Scientific) coated with hOX40 (5 µg/mL), biotinylated by Sulfo-NHS-LC-LC-Biotin (Thermo Fisher Scientific) in 5 molar excess at ambient temperature for 30 min and purified using Zeba™ Spin Desalting Columns 7 K MWCo 0.5 mL (Thermo Fisher Scientific) according to manufacturer’s protocol. Antigen and phage blocking was performed with 5% milk in PBS-T during the first round or BSA in the second. Washing stringency was increased with each panning round and elution of bound phages was performed by 100 mM triethylamine pH 10 (TEA, Roth), followed by neutralization with 1 M Tris/HCl pH 7.4. For phage rescue, TG1 bacteria were infected with the eluted phages during their exponential growth phase, spread on selection plates for subsequent selection rounds and incubated at 37°C overnight. Enrichment of antigen-specific phages was monitored by counting colony forming units (CFUs).

Monoclonal phage ELISA was executed in a whole cell setting. Individual clones were picked, and phage production was induced as described above. For antigen presentation U2OS-hOX40 cells or wild type (wt) U2OS for background determination were seeded in a density of 2 × 10⁴ cells per well in 100 µL in 96-well cell culture plates (Corning) coated with poly-L-lysine (Sigma Aldrich) and grown overnight. The next day, 70 µL of phage supernatant was added to each cell type and incubated at 4°C for 3 h. Cells were washed 5 × with 5% FCS in PBS, followed by incubation with M13-HRP-labeled detection antibody (Progen, 1:2000 Dilution) for 1 h and washed again 3 × with 5% FCS in PBS. For the final detection, Onestep ultra TMB 32048 ELISA substrate (Thermo Fisher Scientific) was added to each well and incubated until the color changed. The reaction was stopped with 100 µL of 1 M H₂SO₄ and the signal was detected with the Pherastar plate reader at 450 nm. Phage ELISA-positive clones were defined by a 2-fold signal above U2OS-WT control cells.

For production, hOX40-Nbs were cloned into pHEN6 vector (70), expressed in XL-1 and purified using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography according to standard procedures as previously described (16). Sortase A pentamutant (eSrtA) in pET29 was a gift from David Liu (Addgene plasmid # 75144) and expressed and purified as published (71). The quality of all purified proteins was analyzed via standard SDS-PAGE under denaturizing and reducing conditions (5 min, 95°C in 2x SDS-sample buffer containing 100 mM Tris/HCl, pH 6.8; 2% (w/v) SDS; 5% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol, 0.02% bromphenole blue). Proteins were visualized by InstantBlue Coomassie (Expedeon) staining or alternatively by immunoblotting transferring proteins to nitrocellulose membrane (GE Healthcare, Chicago, IL, USA) and detection using a primary anti-Penta-His antibody (Qiagen) and secondary donkey anti-mouse AF647 antibody (Invitrogen) on a Typhoon Trio scanner (GE-Healthcare, excitation 633 nm, emission filter settings 670 nm BP30).

The binding kinetics analysis of hOX40-Nbs was performed using the Octet RED96e system (Sartorius) applying manufacturer’s recommendations. Therefore, 5 µg/mL of biotinylated hOX40-Nbs diluted in Octet buffer (PBS, 0.1% BSA, 0.02% Tween20) were immobilized on streptavidin coated biosensor tips (SA, Sartorius) for 30 s and unbound Nb was washed away. For the association step, a dilution series of hOX40 ranging from 0.2 nM – 320 nM were applied for 300 s followed by dissociation in Octet buffer for 720 s. Each concentration was normalized to a reference applying Octet buffer only for association. Data were analyzed using the Octet Data Analysis HT 12.0 software applying the 1:1 ligand-binding model and global fitting. For epitope binning 5 µg/mL of each Nb, except O7 due to its inappropriate dissociation behavior, was immobilized to SA tips and the association was performed with a premixture of OX40 (100 nM) and an excess of unbiotinylated second Nb (1000 nM). By analyzing the binding behavior of the premixture, conclusions about shared epitopes were drawn. To determine a potential competition of Nbs with the natural OX40 ligand OX40L, OX40L was biotinylated and 10 µg/mL were immobilized to the SA tips. Premixture of hOX40 with a ten-time molar excess of each Nb was applied for the association step.

U2OS-hOX40 cells, U2OS-WT or U2OS cells transiently expressing hOX40 domain deletion mutants or murine OX40 were plated at a density of 1 x 10⁴ cells per well in 100 µL of a µClear 96-well plate (Greiner Bio One, cat. #655090) and cultivated overnight at standard conditions. The next day, cells were stained with 2 µg/mL Hoechst33258 (Sigma Aldrich) for nuclear staining in live-cell visualization medium DMEMgfp-2 (Evrogen, cat. #MC102) supplemented with 10% FCS for 30 min at 37°C. Afterwards 10 -1000 nM fluorescently labeled hOX40-Nbs, a non-binding PEP-Nb (Nb Ctrl.) or an OX40 antibody (positive control) were added and incubated for 30 min at 4°C. Staining solution was replaced by live-cell visualization medium DMEMgfp-2 with 10% FCS and images were acquired with a MetaXpress Micro XL system (Molecular Devices) at 20 x magnification.

For cell-based affinity determination, U2OS-hOX40 cells were detached using enzyme-free cell dissociation buffer (Gibco) and resuspended in FACS buffer (PBS containing 0.02% sodium azide, 2 mM EDTA, 2% FBS). For each staining condition, 200,000 cells were incubated with suitable dilution series (O12 and O18: starting from 2.5 µM; O7 and O19 starting from 10 µM) of AF647-labeled hOX40-Nbs at 4°C for 30 min. After two washing steps, samples were resuspended in 200 μL FACS buffer and analyzed on the same day using a LSRFortessa™ flow cytometer (Becton Dickinson) equipped with the DIVA Software (Becton Dickinson). Final data analysis was performed using the FlowJo10^® software (Becton Dickinson).

To assess the thermal stability of the Nbs, nanoscale differential scanning fluorimetry (nanoDSF) with the Prometheus NT.48 device (Nanotemper) was performed. Freshly thawed hOX40-Nbs were diluted to 0.25 mg/mL in PBS and measured at time point d0 and after an incubation period of ten days at 37°C (d10) using standard capillaries. A thermal gradient ramping from 20°C to 95°C was applied while measuring fluorescence ratios (F350/F330) and light scattering. Using PR. ThermControl v2.0.4 the melting (T_M) and aggregation (T_Agg) temperatures were determined.

For sortase A based coupling of 50 μM Nb were added to 250 μM sortase peptide (H-Gly-Gly-Gly-propyl-azide synthesized by Intavis AG) and 10 μM sortase A both dissolved in sortase buffer (50 mM Tris, and 150 mM NaCl, pH 7.4 at 4°C) and reaction was started by adding 10 mM CaCl₂ for 4 h at 4°C. To avoid reverse sortase reaction, sortase A and uncoupled Nb were removed by Ni-NTA affinity chromatography. The coupled Nbs were concentrated and residual peptide was depleted using Amicon ultra-centrifugal filters MWCO 3 kDa. Taking advantage of SPAAC (strain-promoted azide-alkyne cycloaddition) click chemistry reaction fluorescent labeling was performed by incubating azide-coupled Nbs with 2-fold molar excess of DBCO-AF647 (Jena Bioscience) for 2 h at room temperature. Subsequent dialysis (GeBAflex-tube, 6-8 kDa, Scienova) led to removal of excess of DBCO-AF647. As final polishing step a hydrophobic interaction chromatography (HIC, HiTrap Butyl-S FF, Cytiva) was performed to deplete unlabeled Nb. The final products were analyzed via SDS-PAGE and spectrophotometry.

The OX40 bioassay kit (Promega) was used to determine a potential agonistic activity of Nbs O12, O18 and O19 according to manufacturer´s instructions. On the day before assay, thaw-and-use OX40 effector cells (Promega) were thawed and seeded into the inner 60 wells of two white 96 well assay plates cultured in assay buffer (RPMI1640 with 5% FBS) at standard conditions overnight. The next day OX40L and Nbs were serially diluted (OX40L: 50 – 0.0008 nM; Nbs: 2000-0.3 nM) in assay buffer, and 20 μL of the diluted recombinant proteins were added to the assay plate. The assay plate was incubated at 37°C, 5% CO₂ for 5 h. Afterwards, the assay plate was equilibrated to ambient temperature for 10 min. For detection of agonistic function, 75 µL of Bio-GloReagent was added to all wells. The assay plate was incubated at room temperature for 5 min, and luminescence was measured using a Tecan M2000 plate reader. The average relative luminescence unit (RLU) was calculated for each dilution. The average RLU data were plotted against the different concentrations of OX40L and hOX40-Nbs Nbs. To test antagonistic properties of the Nbs, the OX40 bioassay was transformed into a competition assay. For this purpose, the cells were pre-incubated with a serial dilution of hOX40-Nbs (0.13 µM to 0.002 nM) for one hour, followed by a 5 h incubation period with 0.12 µM OX40L. All experiments were performed in three independent replicates.

Human peripheral blood mononuclear cells (hPBMCs) were isolated as described in (16). In brief, fresh mononuclear blood cell concentrates were obtained from healthy volunteers at the ZKT Tübingen gGmbH. Participants gave informed written consent and the studies were approved by the ethical review committee of the University of Tübingen, projects 156/2012BO1 and 713/2018BO2. Blood products were diluted with PBS 1x (homemade from 10x stock solution, Lonza, Switzerland) and PBMCs were isolated by density gradient centrifugation with Biocoll separation solution (Biochrom, Germany). PBMCs were washed twice with PBS 1x, counted with a NC-250 cell counter (Chemometec, Denmark), resuspended in heat-inactivated (h.i.) fetal bovine serum (FBS) (Capricorn Scientific, Germany) containing 10% DMSO (Merck) and frozen in aliquots using a freezing container before transfer to nitrogen for long term storage. For the experiments, cells were thawed in Iscove´s Modified Dulbecco´s Medium (IMDM + L-Glutamin + 25 mM HEPES; Thermo Fisher Scientific) supplemented with 2.5% h.i. FBS (Thermo Fisher Scientific), 1% P/S (Sigma-Aldrich), and 50 μM β-mercaptoethanol (β-ME; Merck), washed once, counted, and rested for 1 h at 37°C 5% CO₂ in T cell medium (TCM, IMDM + 2% h.i. FBS + 1x P/S + 50 µM β-ME) supplemented with 1 µg/mL DNase I (Sigma-Aldrich). After resting, cells were washed once again, counted and used for subsequent analysis.

For the validation of Nb binding to activated T cells, hPBMCs were stained before and after stimulation for 24 h with 5 µg/mL PHA-L and 50 U/mL IL-2 in TCM at 37°C 5% CO₂. For flow cytometry analysis 2x10⁵ cells per staining condition in FACS buffer (PBS containing 0.02% sodium azide, 2 mM EDTA, 2% h.i. FBS) were used. Extracellular staining was performed with AF647-labeled hOX40-Nbs or the non-binding PEP-Nb [Nb Ctrl (50)] (each 200 nM), phycoerythrin (PE)-labeled anti-hOX40 mAb (Ber-Act35, BioLegend), CD3 Ab APC-Cy7 (HIT3a, BioLegend), dead cell marker Zombie Violet (BioLegend) and isotype control Abs (BioLegend) each in pretested optimal concentrations by incubation for 30 min at 4°C. Cells were washed twice with FACS buffer and acquired on the same day using a LSRFortessa™ flow cytometer (Becton Dickinson) equipped with the DIVA Software (Becton Dickinson). Final data analysis was performed using the FlowJo10^® software (Becton Dickinson). Geometric mean fluorescence intensity (MFI) for the four Nbs was determined using the FlowJo10^® software (Becton Dickinson) and normalized to the non-binding PEP-Nb control.

To assess Nb binding to T cell subpopulations, hPBMCs were stained after stimulation for 24 h with 5 μg/mL PHA-L and 50 U/mL IL-2 in TCM at 37°C 5% CO₂. For flow cytometry analysis, 4x10⁵ cells per staining condition in FACS buffer were used. Extracellular staining was performed with AF647-labeled hOX40-Nbs or the non-binding PEP-Nb [Nb Ctrl (50)] (each 400 nM), phycoerythrin (PE)-labeled anti-hOX40 mAb (Ber-Act35, BioLegend), CD3 Ab PE-Cy5.5 (SK7, eBioscience), CD4 Ab APC-Cy7 (RPA-T4, BD), CD8 Ab BV605 (RPA-T8, BioLegend), CD25 Ab or isotype control PE-Cy7 (BC96 or MOPC-21, BioLegend), dead cell marker Zombie aqua (BioLegend) each at pretested optimal concentrations by incubation for 20 min at 4°C and washed. Afterwards, cells were fixed and permeabilized (Intracellular Fixation & Permeabilization Buffer Set, eBioscience) and intracellular staining with anti-FoxP3 Ab FITC or isotype control Ab (PCH101 or eBR2a, respectively, both from eBioscience) was performed according to the manufacturer´s instructions for 20 min at RT. Cells were washed twice with permeabilization buffer, resuspended in FACS buffer and acquired on the same day using a LSRFortessa™ flow cytometer (Becton Dickinson) equipped with the DIVA Software (Becton Dickinson). Final data analysis was performed using the FlowJo10^® software (Becton Dickinson).

The proliferation behavior of T cells was assessed using a carboxyfluorescein succinimidyl ester (CFSE) based approach. Up to 1×10⁸ cells were labeled with 2.5 µM CFSE (BioLegend) in 1 ml PBS for 20 min according to the manufacturer’s protocol. The cells were washed twice in medium containing 10% h.i. FBS to stop CFSE labeling and stimulated for 24 h with 5 µg/mL PHA-L in TCM at 37°C 5% CO₂ in a 48-well cell culture plate with 1.6–2.5×10⁶ cells/well. Induced OX40 expression was validated via flow cytometry. Subsequent to the stimulation, hPBMCs were treated with 0.5 µM of OX40 specific Nbs O12, O18 and O19, a non-binding PEP-Nb (Ctrl. Nb), OX40L or left untreated and cultured at 37°C and 5% CO₂. Concentrations were chosen in a large excess than the expected concentration during clinical application. On days 3, 5 and 7, 2 ng/mL recombinant human IL-2 (R&D, USA) were added. One-third of the culture on day 4, one half of the culture on days 6 and 8, and the remaining cells on day 12 were harvested and counted. Cells from each condition were washed twice with FACS buffer (PBS containing 0.02% sodium azide, 2 mM EDTA, 5% h.i. FBS). Extracellular staining was performed with CD3 Ab APC-Cy7 (HIT3a, BioLegend), dead cell marker Zombie Violet (BioLegend) and isotype control Abs (BioLegend) each in pretested optimal concentrations by incubation for 30 min at 4°C. Cells were washed two times with FACS buffer and acquired on the same day using a LSRFortessaTM flow cytometer (Becton Dickinson) equipped with the DIVA Software (Becton Dickinson). Final data analysis was performed using the FlowJo10^® software (Becton Dickinson). The percentage of proliferating T cells was determined by assessment of CFSE negative cells.

For cytokine release analysis, a set of in-house developed Luminex-based sandwich immunoassays was used. Supernatants after 24, 72 and 168 hours post Nb treatment of the proliferation assay were frozen at -80°C until cytokine measurements. Levels of IL-1b, IL-1Rα, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ, macrophage chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1b, TNFα, and vascular endothelial growth factor (VEGF) were determined using a those immunoassays each consisting of commercially available capture and detection antibodies and calibrator proteins. All assays were thoroughly validated ahead of the study with respect to accuracy, precision, parallelism, robustness, specificity, and sensitivity (72, 73). Samples were diluted at least 1:4 or higher. After incubation of the prediluted samples or calibrator protein with the capture coated microspheres, beads were washed and incubated with biotinylated detection antibodies. Streptavidin-phycoerythrin was added after an additional washing step for visualization. For control purposes, calibrators and quality control samples were included on each microtiter plate. All measurements were performed on a Luminex FlexMap^® 3D analyzer system using Luminex xPONENT^® 4.2 software (Luminex, USA). For data analysis, MasterPlex QT, version 5.0, was employed. Standard curve and quality control samples were evaluated according to internal criteria adapted to the Westgard Rules (74) to ensure proper assay performance.

For optical in vivo imaging, we labeled the Nb O18 with the fluorophore AlexaFluor647 (O18_AF647) by sortase-mediated attachment of an azide group followed by click-chemistry addition of DBCO-AF647. To establish hOX40⁺ expressing tumors, 5 x 10⁶ HT1080 cells stably expressing human OX40 (HT1080-hOX40) or 5 x 10⁶ wild type HT1080 (HT1080-WT) cells serving as negative control were resuspended in 50% Matrigel (BD) and 50% PBS and subcutaneously injected into the right upper flank of 7-week-old CD1 nude mice (Charles River Laboratories). When the tumors reached a size of 50 - 100 mm³, HT1080-hOX40 (n=3) or HT1080-WT (n=3) bearing mice were i.v. injected with 5 µg of Nb O18_AF647 and noninvasively in vivo investigated by optical imaging (OI). For the in vivo measurements, the mice were anaesthetized with 1.5% isoflurane and the body temperature was kept constant at 37°C using a heating mat. The mice were imaged over the course of 6 h and were sacrificed after the last imaging time point before tumors were explanted for ex vivo OI analysis. A bright field image and an image of the fluorescence signal (excitation 640 nm/emission 680 nm) were recorded using an IVIS Spectrum OI System (PerkinElmer, Waltham, MA, USA). The fluorescence intensities were quantified by drawing regions of interest around the tumor borders and were expressed as average radiant efficiency (photons/s)/(μW/cm²) subtracted by the background fluorescence signal using the Living Image software 4.4 (Perkin Elmer). Statistical analyses using an unpaired t-test, corrected for multiple comparisons using the Holm-Sidak method, were conducted with graph pad prism, Version 10. All mouse experiments were performed according to the German Animal Protection Law and were approved by the local authorities (Regierungspräsidium Tübingen).