Dosimetry Prediction for Clinical Translation of 64Cu-Pembrolizumab ImmunoPET Targeting Human PD-1 Expression

The immune checkpoint programmed death 1 receptor (PD-1) expressed on some tumor-infiltrating lymphocytes, and its ligand (PD-L1) expressed on tumor cells, enable cancers to evade the immune system. Blocking PD-1 with the monoclonal antibody pembrolizumab is a promising immunotherapy strategy. Thus, noninvasively quantifying the presence of PD-1 expression in the tumor microenvironment prior to initiation of immune checkpoint blockade may identify the patients likely to respond to therapy. We have developed a 64Cu-pembrolizumab radiotracer and evaluated human dosimetry. The tracer was utilized to image hPD-1 levels in two subcutaneous mouse models: (a) 293 T/hPD-1 cells xenografted into NOD-scid IL-2Rγnull mice (NSG/293 T/hPD-1) and (b) human peripheral blood mononuclear cells engrafted into NSG bearing A375 human melanoma tumors (hNSG/A375). In each mouse model two cohorts were evaluated (hPD-1 blockade with pembrolizumab [blk] and non-blocked [nblk]), for a total of four groups (n = 3–5/group). The xenograft-to-muscle ratio in the NSG/293 T/hPD-1 model at 24 h was significantly increased in the nblk group (7.0 ± 0.5) compared to the blk group (3.4 ± 0.9), p = 0.01. The radiotracer dosimetry evaluation (PET/CT ROI-based and ex vivo) in the hNSG/A375 model revealed the highest radiation burden to the liver. In summary, we validated the 64Cu-pembrolizumab tracer’s specific hPD-1 receptor targeting and predicted human dosimetry.

The DOTA-pembrolizumab reaction mixtures were purified from excess DOTA-NHS by SEC2000 HPLC using 0.1 M ammonium acetate buffer (pH 5.5) as the mobile phase eluted at 1 mL/min. The immunoconjugate was concentrated to achieve ~2.5 mg/mL using a Vivaspin, 30 kDa cut off centrifugal filter and stored in 200 µL aliquots in 0.1 M ammonium acetate buffer (pH 5.5) at -20 °C.

Cell Assay
In each well, 100 µL of pembrolizumab or DOTA-pembrolizumab was mixed with 100 µL of cell suspension containing 5×10 5 293T/hPD-1 cells and incubated for 1 h on ice. Cells were washed three times with FACS buffer. After washing, 100 µL of biotin-anti-human antibody (eBioscience, Inc., San Diego, CA) pre-diluted to 1:100 in FACS-buffer was added to each well.
The ELISA plate containing cells was kept on ice and incubated for 45 min; afterwards incubated cells were washed three times. Finally, the cells were stained with 100 µL of streptavidin-APC

Immunoreactivity
Briefly, 293T/hPD-1 cells were suspended in microcentrifuge tubes (200 µL) at seven concentrations from 1 to 24 million cells per mL in 1% (w/v) bovine serum albumin in PBS (pH 7.4). Each tube received 100 µL of 64 Cu-pembrolizumab (stock solution: 5 kBq/22.5 µg/mL) tracer (n = 2). After addition of the tracer (final volume 300 mL), the cells were gently vortexed and incubated at 37 °C for 2 h. Following incubation, two 100-µL aliquots were removed from each tube to measure for total activity as well as cell-bound activity. Tubes marked for cell-bound activity were centrifuged (300 g for 3 min), and the supernatant was removed to measure cell pellet activity. The blocking experiment was performed simultaneously with the same procedure except with the addition of a 100-fold excess of non-radioactive pembrolizumab 1 h prior to the addition of the tracer. Radioactivity associated with cells was measured with a gamma counter (1470 WIZARD Automatic Gamma Counter; Perkin Elmer, Walthem, MA). The counts per minute (cpm) data were background-corrected and compared with the total activity versus bound activity

MicroPET/CT System
This system is capable of operating both the PET and CT scanners independently, or in combination, with excellent radial, tangential, and axial resolutions higher than 1.5 mm at the center of the field of view of the PET module. CT imaging was performed at 80 kVp and 500 µA, second bed position, half scan 220° of rotation, and 120 projections per bed position with a cone beam micro-x-ray source (50 µm focal spot size) and a 40 64 × 40 64 -pixel X-ray detector. The data were reconstructed using Shepp-Logan filtering and cone-beam filtered back-projection.

Supplementary Video S3
Three-dimensional microPET-CT visualization of hPD-1-expressing tumor-infiltrating lymphocytes in the tumor microenvironment of hNSG/A375 melanoma-bearing mice, at 24 hours post-injection of 64 Cu-pembrolizumab. (a) Pre-blocking with non-radioactive pembrolizumab (b) No pre-blocking with non-radioactive pembrolizumab. Yellow arrows indicate the location of the