Production of 68Ga-labeled ᴅ-peptides
To perform 68Ga-radiolabeling and concurrently preserve their affinity and specificity, the modification of ᴅ-peptides with polyethylene glycol linkers and NOTA was designed and explored. Several NOTA-ᴅ-peptides were synthesized, radiolabeled with 68Ga and screened for TIGIT imaging (Fig. 1a and Fig. S1-4). 68Ga-labeled ᴅ-peptides were obtained after Sep-Pak purification with a radiosynthesis time of 40-60 min (n = 8). As shown in Fig. 1b, the overall radiochemical yields were 43.5-83.3 % (n = 5) and their molar activities at end-of-synthesis were calculated as 30.6-57.3 GBq/μmol, respectively (n = 3). The final products were verified by their non-radioactive standards (Fig. S5), and the radiochemical purities were > 99 % (n = 5). The partition coefficients (Log P) at pH 7.4 were determined to be -3.31- -1.56 (n = 6), indicating their hydrophilicity. In vitro stabilities of 68Ga-labeled ᴅ-peptides were confirmed in saline and serum within 4 h (Fig. S6).
In vitro assays
The significant upregulation of TIGIT on human PBMCs after stimulation was demonstrated (Fig. S7). Saturation binding experiments were performed in activated PBMCs. Among them, 68Ga-GP12 displayed a much higher affinity for TIGIT protein with a dissociation constant KD of 37.28 nM (Fig. 1b, 1c and Fig. S8). Time-dependent cellular uptake of 68Ga-labeled ᴅ-peptides was studied in non-activated and activated PBMCs, respectively (Fig. S9). It was found that cellular uptakes of 68Ga-labeled ᴅ-peptides increased with time and were saturated after 60 min of incubation. With an exception of 68Ga-LA12, the uptakes were significantly decreased after blocking with an excess of unlabeled ᴅ-peptides (Fig. 2a), indicating their specific uptake in activated PBMCs. 68Ga-GP12 uptake (45.88 ± 4.98 %) in the activated PBMCs was substantially higher than that of 68Ga-LA12 (8.08 ± 1.48 %, P < 0.001), 68Ga-GS12 (16.29 ± 3.20 %, P < 0.001) and 68Ga-SP12 (12.66 ± 2.47 %, P < 0.001) (Fig. S10). Furthermore, the activated-to-nonactivated ratios and the activated-to-blocking ratios were determined and compared, which indicates the excellent specificity of 68Ga-GP12 in vitro (Fig. 2b). In addition, through docking analysis, the optimized steric complementarity of 68Ga-GP12 with binding sites of TIGIT was formed with a binding energy of -10.35 kcal/mol (Fig. 1d). It revealed that 68Ga-GP12 might interact with TIGIT through the binding interface of TIGIT/PVR, owing to the shared key residues of Asn-58 and Thr-117.
Small animal PET imaging
The TIGIT-targeting ability of 68Ga-labeled ᴅ-peptides was compared by PET imaging in B16F10 xenograft models. Tumor uptake of 68Ga-GP12 (3.76 ± 0.68 % ID/g) was observed after 60 min of injection, which is fully distinguished from other radiotracers (Fig. 2c and 2d). The potential of 68Ga-GP12 for imaging of TIGIT was further evaluated by whole-body dynamic imaging. As shown in Fig. 3a, tumors were visualized rapidly at 10 min p.i. (1.58 ± 0.26 % ID/g) and the optimized images were obtained at 60 min p.i. (4.22 ± 0.68 % ID/g) after injection, with the highest tumor/muscle ratio of 12.94 ± 2.64 (Fig. 3b and 3c). The blocking study pretreated with an excess of GP12 showed no tumor uptake during PET acquisition. At 60 min p.i., the accumulation of 68Ga-GP12 in tumor was decreased to 0.78 ± 0.16 % ID/g (P < 0.001) with a tumor/muscle ratio of 1.66 ± 0.35 (P < 0.01). This demonstrated the specificity of 68Ga-GP12 for imaging of TIGIT in vivo. However, tumor uptake of 68Ga-GP12 was not blocked by the pretreatment with anti-TIGIT mAb at any time points (4.18 ± 0.23 % ID/g, tumor/muscle ratio 10.40 ± 0.14 at 60 min p.i.). These results were also demonstrated by ex vivo autoradiography of tumors (Fig. S11a and S11b). The expression of TIGIT in tumors was determined by IHC (Fig. S11d).
In addition, 68Ga-GP12 PET imaging of TIGIT was verified by other types of tumor-bearing mice. In the B16F10 melanoma pulmonary metastasis models, diffuse bilateral lung abnormal uptake of 68Ga-GP12 was detected, which was manifested as focal asymmetry uptake in 18F-FDG PET/CT imaging (Fig. S12). This result indicated the heterogeneity of TIGIT expression, further confirmed by IHC. As expected, the capacity of 68Ga-GP12 for PET imaging was also confirmed in Panc02 and MC38 tumor models (Fig. S13 and S14).
Flow cytometry and correlation with tumor uptake
The relevance between tumor uptake of 68Ga-GP12 on PET images and TIGIT expression in tumor microenvironment was thoroughly investigated (Fig. S15). As measured in flow cytometry, the expression of TIGIT on CD45+ cell, CD4+ T cell, CD8+ T cell, NK cell and Treg cell was determined to be 5.21 ± 1.90 %, 13.39 ± 5.00 %, 6.12 ± 2.20 %, 9.73 ± 4.39 % and 3.34 ± 1.30 % (Fig. S16). It was found that a positive correlation occurs for CD4+ T cell (R2 = 0.5686, P = 0.0307), CD8+ T cell (R2 = 0. 593, P = 0.0305), NK cell (R2 = 0.5413, P = 0.0375) and Treg cell (R2 = 0. 5102, P = 0.0465), but not for CD45+ cell (R2 = 0.4562, P = 0.0660) (Fig. 4).
Biodistribution, pharmacokinetics and radiation dosimetry
The biodistribution of 68Ga-GP12 in B16F10 melanoma-bearing mice was investigated (Fig. 5 and Table S1). The radiotracer displayed a rapid and broad distribution in tissues, predominantly in the kidney with subsequent elimination through the urinary system. At 60 min p.i., the kidney (42.33 ± 3.52 % ID/g) had relatively higher uptake of radioactivity compared with the spleen (1.99 ± 0.48 % ID/g) and other organs (< 1.00 % ID/g). The accumulation of 68Ga-GP12 in tumors reached a plateau (5.00 ± 1.24 % ID/g), resulting in the optimized tumor/muscle and tumor/blood ratios (11.14 ± 2.18 and 5.59 ± 0.83) (Fig. S17a). Tumor uptake of 68Ga-GP12 was decreased to 0.66 ± 0.17 % ID/g in GP12 blocking group with absent tumor/muscle and tumor/blood ratios (1.09 ± 0.30 and 0.53 ± 0.12) (Fig. S17b). Conversely, the anti-TIGIT mAb blocking group demonstrated a slight decline in tumor uptake (4.56 ± 1.15 % ID/g) and tumor/muscle and tumor/blood ratios (8.06 ± 1.90 and 4.89 ± 0.89). The pharmacokinetics study revealed that 68Ga-GP12 was quickly cleared from the blood with a half-life of 27.02 min (Fig. S18). In addition, in vivo metabolic stability of 68Ga-GP12 was verified by radio-HPLC analysis, which reveals more than 90 % of intact radiotracer in blood, the liver and urine within 1 h after injection (Fig. S19).
To assess the safety of human use with 68Ga-GP12, a rodent dosimetry study was conducted to estimate human-equivalent absorbed doses of organs and effective doses (Table S2). Owing to urinary excretion of 68Ga-GP12, the organs that received the highest absorbed dose were kidneys and urinary bladder wall. The effective dose was calculated to be 1.28E-02 mSv/MBq for adult females and 1.02E-02 mSv/MBq for adult males, which is comparable to that of 18F-FDG as previously reported [26].
First-in-human PET/CT imaging
Two patients with advanced NSCLC received the intravenous injection of 68Ga-GP12 (203.5 and 233.1 MBq, respectively) for PET/CT imaging. No adverse or clinically detectable pharmacologic effects were observed. There were no significant changes in vital signs or the results of laboratory studies or electrocardiograms.
The biodistribution of 68Ga-GP12 in patients was mainly observed in the kidney, ureter and bladder, followed by moderate accumulation in tumor, blood pool, liver and spleen (Fig. S20). Other tissues or organs such as the brain, muscle, intestine and thyroid showed weak uptake of radioactivity. The tracer was rapidly eliminated from the blood pool, resulting in high tumor/muscle and tumor/blood ratios (4.06 and 1.39 at 41 min, 3.89 and 1.28 at 50.5 min). The optimized time-point for image acquisition was 40 min after injection of 68Ga-GP12.
Patient 1 (a 72-year-old man) was diagnosed with primary bronchogenic adenocarcinoma. The primary tumor in the right lung (white arrow) showed focal uptake of 68Ga-GP12 (SUVmax = 4.82, Fig. 6a), and 18F-FDG (SUVmax = 9.45, Fig. 6c). Furthermore, a metastatic lesion on the right femur was detected in both 68Ga-GP12 PET/CT (SUVmax = 2.80, Fig. 6b) and 18F-FDG PET/CT imaging (SUVmax = 7.75, Fig. 6d). The expression of TIGIT in the primary tumor was confirmed by immunohistochemistry (Fig. S21).
Patient 2 (a 57-year-old man) was diagnosed with primary bronchogenic adenocarcinoma. A large tumor on the right lung (white arrow) showed the diffuse uptake in 68Ga-GP12 PET/CT imaging (SUVmax = 2.95, Fig. 7a) and intensive uptake in 18F-FDG PET/CT imaging (SUVmax = 18.56, Fig. 7b), indicating the heterogeneity of TIGIT expression in the large tumor.