In Vivo Imaging of the Programmed Death Ligand 1 by ¹⁸F PET

Production of ZPD L1_1

E. coli T7E2 cells (GeneBridges GmBH) were transformed with plasmids containing the gene fragment encoding the PD-L1–targeting Affibody ligand with an N terminal 6 × histidine tag, referred to as His-ZPD L1_1, or a C terminal cysteine residue, referred to as ZPD L1_1. Cells were cultivated at 37°C in TSB-YE medium and protein expression was induced with IPTG.

ZPD L1_1 was purified by anion exchange (Q Sepharose Fast Flow resin; GE Healthcare) followed by reversed-phase chromatography (SOURCE 15 RPC; GE Healthcare) and the disulfides were reduced with 20 mM DTT for 1 h at room temperature prior to each purification step. Buffer exchange to 20 mM HEPES, 1 mM EDTA (pH 7.2) was carried out using HiPrep 26/10 columns (GE Healthcare). Finally, ZPD-L1_1 was purified on EndoTrap® red columns (Hyglos) to ensure low endotoxin content.

His-ZPD L1_1 was purified by Immobilized Metal ion Affinity Chromatography using a His-GraviTrap IMAC column (GE Healthcare). Buffer exchange to phosphate-buffered saline (PBS) (10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4) was performed using PD-10 desalting columns (GE Healthcare). The thermal stability of ZPD L1_1 was analyzed by circular dichroism spectroscopy.

DOTA Conjugation of MEB037.22C3.138 (22C3)

Two milligrams of the human PD-L1–specific murine mAb 22C3 (Merck & Co., Inc.) and 0.11 mg (0.13 μmol) of DOTA-NHS were stirred together for 18 h at 4°C in Na2HPO4 (0.1 M, pH 8.5). The reaction was then purified via a Centricon YM-100 Centrifugal Filter (Millipore Corp.), with the purified DOTA-22C3 collected in ammonium citrate and stored in 100-μL aliquots at −70°C.

⁶⁴Cu-DOTA-22C3

Two microliters of a ⁶⁴CuCl2 solution in 0.1 M HC were added to 25 μg of DOTA-22C3 in 100 μL of ammonium citrate (0.1M, pH 5.5). After the reaction mixture was incubated at 37°C for 30 min and allowed to cool for 5 min, diethylenetriaminepentaacetic acid (5 μL, 10 mM) was added. The ⁶⁴Cu-DOTA-22C3 was purified via a Bio-Spin 30 column (Bio-Rad), with radiochemical purity determined using a Waters 2795 HPLC system equipped with a Waters 2998 UV detector and β-RAM Model 4 radio-HPLC detector using a TSKgel G3000SWXL size-exclusion column.

Homogenate Binding Studies

Tissue homogenates at 6–8 mg/mL of wet tissue weight were preincubated in Skatron tube strips (SK15776) with assay buffer, or unlabeled 22C3 antibody (0.5 μM) at room temperature for 30 min in assay buffer (PBS, pH 7.5, 1:1,000 protease inhibitor, 0.1% bovine serum albumin). Nondisplaceable binding of ⁶⁴Cu-DOTA- 22C3 was defined using self-block (0.5 μM unlabeled affibody). ⁶⁴Cu-DOTA- 22C3 concentrations were tested from 0.1 nM to 10 μM. The assay tubes were incubated at room temperature for 195 min. Following incubation, the reaction mixtures were transferred by a Skatron Combi cell harvester onto Skatron GF/C filters (SK11731) soaked in 0.3% PEI for 30 min at 4°C before use. The filters were promptly washed with ice-cold wash buffer (PBS, pH 7.5) 3 times, punched into Pico Pro vials, and then counted in a gamma counter using the ⁶⁴Cu 30-s protocol. All data were analyzed by nonlinear fit (1-site binding) model using Prism software (GraphPad Software).

Immunohistochemistry

Slides containing sections adjacent to those used for ex vivo autoradiography were fixed in acetone/ethanol (3:1). After incubations with a monoclonal anti–PD-L1 antibody (22C3) followed by anti-mouse IgG polymer (IH-8061; ImmunoBioScience Corp.) and DAB chromogen (Dako Corp.), the slides were counterstained with hematoxylin. 22C3 anti–PD-L1 immunohistochemical staining of SUDHL6 and LOX xenograft tumor samples showed absence of signal in the negative control tumor, whereas clear membranous and some cytoplasmic cell staining could be observed in the LOX sample (Supplemental Fig. 1; supplemental materials are available at http://jnm.snmjournals.org).

Cell Lines

The LOX malignant melanoma cell line was selected because it expresses PD-L1 constitutively (20). The SUDHL6 lymphoma cell line was chosen as a negative control because it is reported not to express PD-L1 (21). The mixed tumor model was intended to demonstrate the specificity of tracer distribution ex vivo, within the same subject in a tumor mass with a common blood supply. The expression level of PD-L1 by the LOX cell line was within the median of the expression levels evaluated from melanoma tumor samples (data not shown).

NOTA Conjugation of Z_{PD-L1_1}

To a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–ethylenediaminetetraacetic acid (EDTA) solution containing 5 mg of Z_{PD-L1_1} were added 3 equivalents of tris(2-carboxyethyl)phosphine in 0.5 mL of 0.2 M ammonium acetate buffer (pH 7.0), which had been degassed 3 times. The reaction was kept at room temperature for 60 min before being transferred to an Ultracel 3K Centrifugal Filter and centrifuged at 4,000 rpm for 90 min. The solution was then rinsed with 1 mL of 0.2 M ammonium acetate buffer and the reduced Affibody molecule transferred to a second reaction vessel in 2 mL of oxygen-free 0.2 M ammonium acetate buffer (pH 7.0). NOTA-maleimide (4 mg) in 0.5 mL of 0.2 M ammonium acetate buffer (pH 7.0) was added and the reaction vessel purged with argon and heated to 40°C for 3 h, at which point the reaction mixture was transferred to a Ultracel 3K Centrifugal Filter and centrifuged for 90 min at 4,000 rpm. After the flow-through was discarded and 2 mL of milliQ water were added, the mixture was again centrifuged for 90 min and the flow-through discarded. Purified NOTA- Z_{PD-L1_1} was collected in 1 mL of milliQ water, lyophilized and stored at −70°C before use. The purity of the final product was determined via LC/MS.

¹⁸F-AlF-NOTA-Z_{PD-L1_1} Labeling

A cartridge containing ¹⁸F-fluoride was first washed with 1.5 mL of ultrapure water and then ¹⁸F-fluoride was eluted with 1.0 mL of 0.4 M KHCO₃. One hundred microliters of the eluted ¹⁸F-fluoride solution were added to a stem vial charged with 10 μL of acetic acid, 50 μL of AlCl₃ (2 mM in 0.1 M NaOAc buffer, pH 4), and 125 μL of NaOAc (0.1 M, pH 4). The solution was incubated for 2 min at room temperature before 1 mg of NOTA-Z_{PD-L1_1} in 400 μL of a 1:1 solution of acetonitrile and NaOAc (0.1 M, pH 4) was added and then heated to 100°C for 15 min. It was then transferred to a vial containing 0.7 mL of 0.1% formic acid, mixed and purified by high-performance liquid chromatography (Xselect CSH C18 [Waters]; 250 × 10 mm, 130 μm) using a gradient of 10%–30% MeCN in 0.1% formic acid over 15 min at a flow rate of 5 mL/min. The peak corresponding to ¹⁸F-AlF-NOTA-Z_{PD-L1_1} was collected and the MeCN was removed in vacuo and transferred to a sterile vial using physiologic saline as a rinse to yield ¹⁸F-AlF-NOTA-Z_{PD-L1_1}. Specific activity and radiochemical purity were determined via a Waters Acquity LC/MS system and a β-RAM Model 4 radio–high-performance liquid chromatography (HPLC) detector (IN/US Systems).

Animal Models

All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee at Merck. Female severe combined immunodeficiency Beige mice (6–8 wk old) were ordered from Charles River Laboratories and housed in a temperature- and humidity-controlled room and on regular diet. LOX-IMVI (LOX) or SUDHL6 (American Type Culture Collection) cells were cultured in complete growth medium containing RPMI 1640 with 10% fetal bovine serum at 37°C with 5% CO₂. The growth medium was changed 2 or 3 times per week, and the cells were subcultured at a ratio of 1:10 when needed. Tumors were implanted at the right shoulder with a subcutaneous injection of 1 × 10⁶ LOX cells in 100 μL of phosphate-buffered saline or 10 × 10⁶ SUDHL6 cells in 100 μL of phosphate-buffered saline plus growth factor–reduced Matrigel (1:1). The mice were used for small-animal PET and ex vivo studies about 5–7 d and 3 wk after the injection of LOX and SUDHL6 cells, respectively, when the tumors reached a mass of 100–400 mg.

For the development of the mixture of LOX and SUDHL6 tumors, the LOX cells were injected into the SUDHL6 tumors, which were inoculated about 2 wk early. About 1 wk after implantation of LOX cells, mice were imaged and the tumors were harvested for histologic and autoradiographic studies.

PET Data Acquisition and Ex Vivo Biodistribution Measurements

Mice were anesthetized with isoflurane (4%–5% induction, 1%–3% maintenance), prepared with tail vein catheters, and placed in a dedicated small-animal PET scanner (microPET Focus220; Siemens Preclinical Solutions). A 20-min transmission scan with a ⁵⁷Co source was obtained to correct for photon attenuation and scatter. Then 0.2–0.6 MBq of ¹⁸F-AlF-NOTA-Z_{PD-L1_1} were administered via the tail vein, and PET list-mode data were collected for 90 min. Immediately after PET acquisition, the mice were euthanized, and the tumor, heart, lung, spleen, liver, kidneys, blood, plasma, and muscle were collected and measured using a γ-counter (PerkinElmer). For each mouse, biodistribution measurements were converted into units of percentage injected dose per gram (%ID/g).

Ex Vivo Autoradiography

The SUDHL6/LOX xenograft tumors removed after imaging were bisected and immediately frozen. Replicate sets of 14-μm-thick cryosections were prepared and adhered to glass slides. For autoradiography, slides were air dried at room temperature, apposed to a phosphor imaging plate (TR 2025; Fujifilm Medical Systems), and exposed overnight. The plates were scanned on a BAS-5000 phosphor image plate scanner (Fujifilm Medical Systems), and the resulting images were processed with MCID software (Imaging Research, Inc.).