Preclinical imaging of the co-stimulatory molecules CD80 and CD86 with indium-111-labeled belatacept in atherosclerosis

Background The inflammatory nature of atherosclerosis provides a broad range of potential molecular targets for atherosclerosis imaging. Growing interest is focused on targets related to plaque vulnerability such as the co-stimulatory molecules CD80 and CD86. We investigated in this preclinical proof-of-concept study the applicability of the CD80/CD86-binding fusion protein belatacept as a probe for atherosclerosis imaging. Methods Belatacept was labeled with indium-111, and the binding affinity was determined with CD80/CD86-positive Raji cells. In vivo distribution was investigated in Raji xenograft-bearing mice in single-photon emission computed tomography (SPECT)/CT scans, biodistribution, and ex vivo autoradiography studies. Ex vivo SPECT/CT experiments were performed with aortas and carotids of ApoE KO mice. Accumulation in human carotid atherosclerotic plaques was investigated by in vitro autoradiography. Results 111In-DOTA-belatacept was obtained in >70 % yield, >99 % radiochemical purity, and ~40 GBq/μmol specific activity. The labeled belatacept bound with high affinity to Raji cells. In vivo, 111In-DOTA-belatacept accumulated specifically in Raji xenografts, lymph nodes, and salivary glands. Ex vivo SPECT experiments revealed displaceable accumulation in atherosclerotic plaques of ApoE KO mice fed an atherosclerosis-promoting diet. In human plaques, binding correlated with the infiltration by immune cells and the presence of a large lipid and necrotic core. Conclusions 111In-DOTA-belatacept accumulates in CD80/CD86-positive tissues in vivo and in vitro rendering it a research tool for the assessment of inflammatory activity in atherosclerosis and possibly other diseases. The tracer is suitable for preclinical imaging of co-stimulatory molecules of both human and murine origin. Radiolabeled belatacept could serve as a benchmark for future CD80/CD86-specific imaging agents. Electronic supplementary material The online version of this article (doi:10.1186/s13550-015-0157-4) contains supplementary material, which is available to authorized users.


Supplementary Tables
Supplementary    Table 4 Overview of all human endarterectomy samples and their respective score according to Supplementary Table 3. In some cases, no scoring of the sample was possible due to fragmentary plaque tissue e.g. no cap present hampering the morphological assessment of the plaques (indicated as n.d., non-determined).

Conjugation, radiolabeling and quality control
Chemicals and solvents were purchased in trace analysis grade from Fluka or Sigma-Aldrich (Buchs, Switzerland) and were used without further purification. Briefly, conjugation of 92.3 µM belatacept was performed under metal-free conditions using a 20-fold molar excess of chelating agent. The reaction mixture was incubated overnight at 4°C yielding after purification in 1.4 mL protein conjugate solution with a concentration of 5.56 ± 0.02 mg/mL determined by a NanoDrop® ND-1000 spectrophotometer (Witec AG, Littau, Switzerland). Aliquots were stored at -20°C.
After radiolabeling (see main manuscript) the product (500 µL) was purified by FPLC on a Shimadzu SCL-10A VP system with a superose 12 column (GE Healthcare). Isocratic conditions with PBS (pH 7.4) as mobile phase and a flow rate of 0.5 mL/min were applied. Quality control of the purified product was performed under isocratic conditions (0.3 M NaCl, 0.05 M NaH 2 PO 4 , pH 6.2) with a TSKgel G3000SWxl column (7.8 mm x 30 cm, 5 µm; Tosoh Bioscience LLC). HPLC data were analyzed with the software RadioStar.

LC-MS analysis
LC-MS analysis was performed on a Waters LCT Premier mass spectrometer. Prior to analysis, conjugated belatacept was deglycosylated using a Protein Deglycosylation Mix (P6039, New England BioLabs Inc.). G7 reaction buffer (2.5 µL 10x concentrated) and 2.5 µL deglycosylation enzyme cocktail were added to 20 µL of 1.1 mg/mL belatacept in H 2 O and the mixture was incubated at 37°C overnight.

Stability evaluation of 111 In-DOTA-belatacept
The in vitro stability of 111 In-DOTA-belatacept was evaluated in PBS (pH 7.4), human and murine (male NMRI nu/nu mice) blood plasma, respectively. Approximately 1.4 MBq radiotracer (20 µL, 76.5 nM) was added to 500 µL PBS or plasma and the samples were incubated at 37°C. Fifty-microliter samples were analyzed after 0, 24, 48 and 72 h by size-exclusion chromatography on a SCL-10A VP system (Shimadzu) equipped with a UV/Vis and a radiodetector. Chromatographic analysis corresponded with the conditions applied for quality control of the radiolabed product.

Cell culture
Raji cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium with GlutaMAX (Gibco, Life Technologies) supplemented with 10% heat-inactivated fetal calf serum and penicillinstreptomycin. NCI-H69 cells were cultured in RPMI 1640 medium with GlutaMAX and 25 mM HEPES (Gibco, Life Technologies) supplemented with 10% heat-inactivated fetal calf serum, penicillinstreptomycin and 1 mM sodium pyruvate. Cells were cultured in suspension at 37°C in a humidified atmosphere containing 5% CO 2 . Routine culture treatment was performed twice a week.

Cell immunofluorescence microscopy
All incubation steps were performed at room temperature unless otherwise stated. Raji cells were Feldbach, Switzerland). Images were acquired using the AxioVision software (Carl Zeiss AG).

Tissue immunofluorescence microscopy
Raji or NCI-H69 xenografts were dissected from euthanized mice and embedded in Tissue-Tek (Thermo Scientific). Frozen sections (7 µm) were cut on a cryostat, thaw-mounted onto coated glass slides (Superfrost plus, Menzel) and stored at −20°C un l further use. Sections were fixed with ice cold methanol/aceton (1:1; v/v) for 5 min following a washing step with TBS for 5 min. The following blocking, incubation and washing procedures were performed under the same conditions as mentioned above for immunocytochemistry as well as the microscopic analysis.

Binding studies
Saturation binding and Lindmo assays with 111 In-DOTA-belatacept were performed with Raji and NCI-H69 cells. For the saturation binding assay, the cells (approx. 8.5 × 10 6 cells per tube) were washed twice with PBS and incubated for 90 min on ice with increasing concentrations of radiotracer (0.03 to 330 nM) in triplicates. The total volume per tube was adjusted with RPMI medium to 200 µL. Nonspecific binding was determined by adding 100 µM unlabeled belatacept as displacer. After 2 h incubation, the cells were washed three times with 2 mL ice-cold PBS (pH 7.4) and centrifuged at 1260 x g for 5 min at 4°C (Sorvall RC-5C centrifuge, Thermo Fisher). Samples were analyzed in a γ-counter (Packard Cobra II Auto Gamma, PerkinElmer). The dissociation constant (K d ) was calculated by fitting the specific radiotracer binding with non-linear regression analysis assuming a 1:1 binding (GraphPad Prism Software). In the Lindmo assay a fixed amount of 111 In-DOTA-belatacept (0.5 nM) was incubated with a standard dilution (dilution factor 2) of Raji and NCI-H69 cells, respectively, in RPMI medium. The highest cell number for the Raji cells was approx. 2.9 × 10 7 cells/tube and for the NCI-H69 approx. 1.6 × 10 7 cells/tube. The total volume per tube was adjusted with RPMI medium to 200 µL. To assess the non-specific binding, 100 µM unlabeled belatacept was used as displacer. The samples were incubated for 2 h at 4°C, thereafter washed three times with 2 mL ice-cold PBS and centrifuged at 1260 x g for 5 min at 4°C. Samples were analyzed in a γ-counter (Packard Cobra II Auto Gamma, PerkinElmer).

Oil red o tissue staining
Lipid staining of the aorta and the carotids was accomplished with the dye oil red o at room temperature according to a previously published procedure [ref. S1]. In brief, the tissue was fixed in 4% PFA for 10 min, thereafter washed twice with PBS and preincubated in 60% 2-propanol for 5 min.
Tissues were stained with 0.3% (w/v) oil red o (Sigma-Aldrich) in 60% 2-propanol for 30 min on a shaking platform. Finally, the tissue was washed under running tap water for 2 min. Images of the stained tissues were obtained with a Nikon SMZ1000 microscope equipped with a Nikon 1 J3 digital camera (Nikon, Melville, NY, USA).