Recombinant molecules and antibodies

All antibodies used in this study are listed in Additional file 4: Table S2.

Cell lines culturing

Chinese hamster ovary (CHO)-K1 cells were obtained from the American Type Culture Collection (ATCC). Cells were quarantined until screening for microbial contamination, and mycoplasma was performed and proved to be negative. Cells were grown in DMEM/F-12, GlutaMAX™ Supplement + 5% FCS + 25 mM HEPES and incubated in a humidified atmosphere with 5% CO2 at 37 °C.

CD103/β7 expressing CHO clones were previously reported [17] and generated by nonliposomal transfection (FuGENE) of pcDNA3.1+_Hygro encoding ITGB7_HUMAN (uniProtKB #P26010) and pCI-neo encoding ITGAE_HUMAN (uniProtKB #P38570) plasmids (GeneArt/ThermoFisher Regensburg, Germany).

Generation of hCD103 Fab fragment

Anti-hCD103 Fab candidates were produced by ImmunoPrecise. Synthetic vectors encoding for the DNA sequences of the VH and VL domains of previously described antibody hCD103.01A was synthesized and subsequently cloned into ImmunoPrecise’s human IgG1-Fab-K vector and human kappa light chain vector, respectively, followed by transfection of HEK293 cells. Fab fragments from harvested supernatants were purified by endotoxin-free purification using CaptureSelect IgG-CH1 affinity matrix. The molecular weight (MW) of the hCD103.01A is 50 kDa. Fab concentrations were quantified using spectrophotometry and Fab purity was assessed by SDS-PAGE and HP-SEC. Endotoxin levels were determined by LAL assay. Previous work on anti-CD103 mAb tracer development [17] showed that among 6 mAb candidates, clone 01A have the strong binding to the CD103+ CD8+ T cell subpopulation in tumor digests, nonoverlapping binding epitopes and differential CD103 blocking properties. Therefore, for further Fab tracer development we selected Fab 01A.

[89Zr]Zr-hCD103.Fab01A and [68Ga]Ga-hCD103.Fab01A tracer development and quality control

TFP-N-Suc-desferal-Fe (Df, ABX GmbH, Hamburg, Germany) was conjugated with a fourfold molar excess to lysine residues of hCD103.Fab01A based on the previous study [17]. The identity and purity of Df-Fab conjugate was determined by size exclusion ultra-performance liquid chromatography (SE-UPLC). The Waters ACQUITY SE-UPLC system was equipped with a dual wavelength absorbance detector, in-line radioactivity detector and UPLC column: TSK-GEL G3000SWXL column (JSB, Eindhoven, Netherlands) or BioSep SEC-s3000, (LC column 300 × 7.8 mm, Phenomenex, Netherlands). The conjugate was aliquoted and frozen.

89Zr-labeling was performed as described earlier [31] using clinical-grade [89Zr]Zr- oxalate (PerkinElmer, Amsterdam, The Netherlands). Radiochemical purity (RCP) was checked by trichloroacetic acid (TCA) precipitation test. The quality control concerned with aggregation and fragmentation of both conjugates and tracer was checked by size exclusion ultra-performance liquid chromatography (SE-UPLC). Radioactivity during the labeling was measured by the dose calibrator (VDC-505, Comecer, Netherlands).

For TCA test, one microliter (1 μL) of the final tracer products was added to 1 ml of 0.5% human serum albumin (HSA). Additionally, 1 mL of TCA was added and mixed, centrifuged the mixture at 3000 rpm for 10 min. After centrifugation, 1 mL of the supernatant was carefully transferred to another tube, leaving 1 ml of the supernatant and the pellet in the original tube. The radioactivity in both tubes was then measured using a 2480 Wizard Detector Gamma Counter (PerkinElmer, Netherlands).

68Ga was obtained by fractionated elution of the < 9 month old GMP 68Ge/68Ga generator (Eckert and Ziegler, Berlin, Germany) with 0.1 M HCl. The eluate with the highest radioactivity concentration was used for labeling. Briefly, the [68Ga]GaCl3 (5–10 mL, 600–1000 MBq) was concentrated on a PS-H+ cartridge and eluted with a 200 μL 5 M NaCl solution. 80 MBq of [68Ga]GaCl3 (100–200 μL) was added to into a reaction vial containing N-Suc-Df‐hCD103.Fab01 solution (30–60 μg, 5 mg/mL) mixed with 100 μL of HEPES buffer (1 M, pH 8). The reaction mixture was allowed to react for 15 min at room temperature while being thoroughly vortexed. If RCP did not reach 90%, purification was performed with pre-conditioned (8 ml of PBS) PD10 miniTrap G-25 Sephadex resin size exclusion column (Cytiva, MA, USA) by PBS fractioned elution (100 μL). The labeling yield and radiochemical purity of labeled conjugates were measured using the instant thin-layer chromatography (ITLC) (ITLC-SG, Agilent Technologies, Santa Clara, CA, USA). The strips were developed with 0.05 M citric acid. Distribution of the radioactivity among the strips was measured on GE Amersham Typhoon Scanner using phosphorus plates and ImageQuant TL 1D software for data processing (both GE Healthcare Life Science, USA).

Cell based ELISA

To compare the binding ability of conjugate and the antibody, the following experiment was conducted. One day before the experiment, CD103/β7 transfected CHO cells (30 000 cells/well in 50 μL) were seeded in 96-well plates. Subsequently, serial dilutions of CD103 Fabs, CD103 Fabs-N-Suc-Df conjugate and isotype controls were added to each well of a 96-well plate and incubated for 1 h at 37 °C. The final total volume in each well was 100 μL. Wells were washed with PBS and incubated with rabbit anti-mouse/IgG-HRP (1:4000, Dako) for 1 h at 37 °C. Next wells were washed with 200 μL PBS and 100 μL of TMB substrate (KPL) was added. The color reaction was stopped by adding 1 M HCl solution and the absorbance was measured by a microplate reader (Thermo Scientific).

In vitro and ex vivo stability of the tracers

In vitro stability of [89Zr]Zr-hCD103.Fab01A and [68Ga]Ga-hCD103.Fab01A was tested in PBS and in the presence of 1000-fold molar excess of EDTA (ethylenediaminetetraacetic acid), whereas ex vivo stability in human serum. After purification, samples of freshly labeled conjugate (5.9 µg, 50 µL) were mixed with EDTA (64 µg, 2.5 mg/mL in 50 µLPBS). For PBS and serum stability, samples were mixed with equal volumes (100 µL). For [89Zr]Zr-hCD103.Fab01A samples were measured after 1, 3, and 24 h. For [68Ga]Ga-hCD103.Fab01A samples were measured after 0.5, 1, 2, 3, and 4 h. RCP of samples submitted for stability was quantified using iTLC strips. The strips were developed and measured as described previously. The experiment was performed in triplicate.

In vitro binding ability of the tracers

To test the in vitro binding ability of both tracers to CHO.CD103 cell and CHO.K1 cell, the following experiment was conducted. CHO.CD103 and CHO.K1 cells were transferred on the day of experiment to 4 ml plastic tubes (250 000 cells/tube in 100 µL of 2% FCS in PBS). A serial dilution of tracer was added to tubes to achieve 10 different concentrations (0–5000 ng/mL). After 1-h incubation at 37 °C, cells were washed thrice with PBS + 2% FBS (2 ml) and bound [68Ga]Ga- or [89Zr]Zr-hCD103.Fab01A was measured in Wizard Detector Gamma Counter. For blocking studies, CHO.CD103 cells were transferred on the day of experiment to 4-ml plastic tubes (250 000 cells/tube in 100 µL of 2% FCS in PBS) and set of samples were blocked using 1µL CD103 antibody (10 µg/mL, mouse antihuman CD103, clone Ber-ACT8, BD Bioscience) for 1 h before experiment. Afterward, tracer, in 10 different concentrations (0–2500 ng/mL), was added to the samples, incubated for 1 h at 37 °C, and cells were washed thrice with PBS + 2% FBS. Bound [68Ga]Ga/ [89Zr]Zr-hCD103.Fab01A was measured in Wizard Detector Gamma Counter.

Animal study

Animal experiments were planned and performed under approval of the Institutional Animal Care and Use Committee of the University of Groningen in agreement with EU Directive 2010/63/EU (IvD: 16395–01-019).

In vivo studies were performed with male nude mice (BALB/cOlaHsd-Foxn1nu, Envigo, The Netherlands, 6–8 weeks) inoculated subcutaneously with CHO.K1 or CHO.CD103 xenografts (5*106 in 300 µL 1:1 PBS and high growth factor Matrigel (BD Biosciences, Breda, The Netherlands)). In order to be visible under PET scans, tracer injection should be performed at least when the xenograft is at least 200 mm3.

For microPET imaging, mice (n = 4) were injected intravenously (iv) with [89Zr]Zr-hCD103.Fab01A and [68Ga]Ga-hCD103.Fab01A, respectively. For mice injected with [89Zr]Zr-hCD103.Fab01A, scans were made 3, 6 and 24 h post-injection using a Focus 220 PET scanner (CTI Siemens), followed by ex vivo biodistribution analysis after the final scan. For mice injected with [68Ga]Ga-hCD103.Fab01A, scan was only made at 3 h post-injection (pi) due to the short half-life of the 68Ga (t1/2 = 68 min). During the scan, anesthesia was induced and maintained by the administration of a mixture of isoflurane (2.5–3.5%), oxygen, and medical air.

PET data were reconstructed into a static image, and in vivo quantification was performed using AMIDE (v1.0.4, Stanford University, Stanford, CA, USA). MicroPET data are presented as mean standardized uptake value (SUVmean) and xenograft-to-blood ratio of SUVmean. Regions of interest (ROI) were drawn for xenograft based upon ex vivo weight, assuming 1 g/ml tissue density. For blood pool measurements, a fixed-sized sphere was drawn in the center of the heart. After the final scan, mice were killed and organs of interest collected for biodistribution studies. Organs and standards of the injected tracer were counted in a calibrated well-type LKB-1282-Compu-gamma system (LKB WALLAC) and weighed. After decay correction, ex vivo tissue activity was expressed as the percentage of injected dose per gram tissue (%ID/g).

Immunohistochemistry staining

For human CD103 IHC, previously formalin-fixed, paraffin-embedded tissue slices were deparaffinized in xylene and rehydrated. Heat-induced antigen retrieval was performed in 10 mM TRIS/EDTA (pH 9.0) at 100 °C for 15 min, endogenous peroxidase was blocked by 10-min incubation with 3% H2O2 in PBS and nonspecific binding of antibodies was blocked using 1% human serum albumin + 1% bovine serum albumin in PBS for 30 min. Next slides were incubated with rabbit anti-mouse CD103 antibody (1:500, ab224202, Abcam) for 60 min at room temperature. Incubation with secondary antibody (EnVision System, Dako HRP; Dako) was performed for 30 min, followed by application of diaminobenzidine chromogen for 10 min. Hematoxylin counterstaining was applied routinely. Digital scans of slides were acquired by a NanoZoomer 2.0-HT multi-slide scanner (Hamamatsu) and analyzed with NanoZoomer Digital Pathology viewer software.

Statistics

Data are expressed as mean ± SD unless stated otherwise. Statistical analyses were performed in GraphPad Prism version 8.4.2 (GraphPad Software) using the Mann–Whitney test (2 groups, nonparametric).