Preclinical and Exploratory Clinical Studies of Novel 68 Ga-labeled (cid:0)-Peptide Antagonist for PET Imaging of TIGIT Expression in Cancers

Purpose While TIGIT has been propelled under the spotlight as a next-generation target in cancer immunotherapy, anti-TIGIT therapy seems to be promising for a fraction of patients in clinical trials. Therefore, patient stratication is critical for this therapy, which could benet from a whole-body, non-invasive and quantitative evaluation of TIGIT expression in cancers. In this study, a 68 Ga-labeled (cid:0)-peptide antagonist, 68 Ga-GP12, was developed and validated for PET imaging of TIGIT expression in vitro, in vivo, and rst-in-human pilot study. tumor-bearing C57BL/6 intravenously injected with 7.4-11.1 MBq of 68 Ga-GP12 and the subsequent acquisition was performed for 120 min with an Inveon PET scanner under anesthesia. For static imaging, a 10 min PET scan was obtained after 60 min post-injection (p.i.) of 3.7-7.4 MBq 68 Ga-labeled (cid:0)-peptides. The images were reconstructed and regions of interest (ROIs) were drawn to obtain time-radioactivity curves and tumor-to-muscle ratios. The quantitative data were indicated as the percentage injected dose per gram of tissue (% ID/g). The blocking experiments were further conducted by either intravenous injection of GP12 (2 mg/kg) 1 h before the injection of radiotracers or intraperitoneal administration of anti-TIGIT mAb (5 mg/kg) 24 hours in advance.


Introduction
Undoubtedly, cancer immunotherapy has been boosted by the discovery of inhibitory immune checkpoints such as CTLA-4 and PD-1/PD-L1 in the past decade [1]. While blockade of CTLA-4 or PD-1/PD-L1 has manifested compelling responses against certain cancer types in the clinic, only a subset of cancer patients could bene t from these inhibitors, and primary/adaptive resistance is often observed [2]. Therefore, alternative immune checkpoints are intensely pursued as therapeutic targets to modulate the immune responses [3,4], which may bring additional clinical bene ts for cancer patients.
One such immune checkpoint is the T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) receptor [5]. TIGIT is an inhibitory receptor expressed on CD4 + T cells, CD8 + T cells, natural killer (NK) cells and regulatory T cells (Treg), which could compete with costimulatory receptor CD226 for binding of the ligand Poliovirus Receptor (PVR) to deliver immunosuppressing signals [6]. Furthermore, TIGIT can inhibit NK cell-mediated tumor killing, induce immunosuppressive dendritic cells, impair T cell priming and differentiation and suppress cell killing by CD8 + T cells in the cancer immunity cycle, subsequently leading to tumor cell immune escape [7][8][9]. The upregulation of TIGIT has been observed in many malignant tumors and correlates with dismal clinical outcomes [10][11][12], which makes it a promising therapeutic target with the chance of clinical application.
A growing number of studies have reported the blockade of TIGIT with antagonistic monoclonal antibodies (mAbs), which produce favorable therapeutic e cacy in preclinical and clinical trials [13]. To date, there are approximate ten human anti-TIGIT mAbs of IgG isotypes that have entered clinical trials for evaluating their e cacy and safety either as a monotherapy or in combination with anti-PD-1/PD-L1 mAbs or chemotherapeutics [14]. In a phase I trial, anti-TIGIT MK-7684 used as monotherapy and in combination with pembrolizumab in patients with advanced solid tumors was evaluated (NCT02964013) [13]. The partial response rate was 3 % (n = 1/34) and 19 % (n = 8/43) in two groups, respectively.
Recently, anti-TIGIT tiragolumab was granted Breakthrough Therapy Designation (BTD) and promoted to phase III trials based on the phase II CITYSCAPE trial (NCT03563716) [15]. This randomized study revealed an objective response rate (ORR) of 37 % in PD-L1-positive non-small cell lung cancer with tiragolumab plus atezolizumab treatment. In the subgroup with high PD-L1 expression, the combined treatment group had an ORR of 66 %. It was observed that a subset of patients could bene t from anti-TIGIT therapy owing to generally high but variable TIGIT expression between individuals. Currently, there are no companion diagnostics of TIGIT expression available for patient strati cation in clinical trials.
While immunohistochemistry (IHC) is the clinical gold standard for pathological diagnosis, the heterogeneous and dynamic expression of immune checkpoints within the tumor microenvironment makes it inaccurate or imprecise presentation of clinical results [16,17]. This is exempli ed by the observation that PD-L1 negative tumors have responded to anti-PD-1/PD-L1 treatment [18]. Fortunately, the use of positron emission tomography (PET) as an in vivo imaging method to quantify PD-1/PD-L1 expression has demonstrated a better relevance with therapy response than IHC in clinical trials [19][20][21][22].
This instigated research in PET imaging of TIGIT expression for the strati cation of patients. Most recently, Shaffer et. al. validated 64 Cu and 89 Zr-labeled anti-TIGIT mAb as radiotracers for PET imaging of TIGIT status on tumor-in ltrating lymphocytes and screening patients for anti-TIGIT therapy [23].
However, the long blood circulation and slow clearance from the body make it di cult to obtain the optimal target-to-background ratio in a short time. Small molecule candidates, such as peptides, have attracted considerable attention for their potentials in the design of PET imaging tracers with the advantages of comparable a nity and speci city, favorable pharmacokinetics, and easy synthesis and modi cation [24].
Following this tendency, the rst -enantiomer peptide antagonists were identi ed by mirror-image phage display biopanning and blocked the interaction of TIGIT/PVR with high a nity and speci city [25], which motivates our design of TIGIT-targeting small molecule PET tracers. In this study, we developed and validated 68 Ga-labeled -peptide antagonist, 68 Ga-GP12, for PET imaging of TIGIT expression in the tumor. Furthermore, the safety and potential of 68 Ga-GP12 for PET/CT imaging of TIGIT expression were evaluated in a rst-in-human pilot study with advanced NSCLC.

Materials And Methods
Production of 68 Ga-labeled -peptides NOTA--peptides with polyethylene glycol linkers were custom synthesized by GL Biochem Co., Ltd.

In vitro assays
Human PBMCs were isolated from fresh peripheral blood of healthy donors by centrifugation over Ficoll-Paque and activated by 5 μg/mL of phytohemagglutinin (PHA-M). The procedures of saturation binding assays and cell uptake studies in activated PBMCs were described in Supplemental Information.

Small animal PET imaging
All animal studies were performed according to the guidelines of the Animal Care Committee of Xiamen University. The establishment of B16F10, Panc02 and MC38 subcutaneous xenograft models and B16F10 pulmonary metastases models were described in Supplemental Information. For dynamic imaging, the tumor-bearing C57BL/6 mice were intravenously injected with 7.4-11.1 MBq of 68 Ga-GP12 and the subsequent acquisition was performed for 120 min with an Inveon PET scanner under anesthesia. For static imaging, a 10 min PET scan was obtained after 60 min post-injection (p.i.) of 3.7-7.4 MBq 68 Ga-labeled -peptides. The images were reconstructed and regions of interest (ROIs) were drawn to obtain time-radioactivity curves and tumor-to-muscle ratios. The quantitative data were indicated as the percentage injected dose per gram of tissue (% ID/g). The blocking experiments were further conducted by either intravenous injection of GP12 (2 mg/kg) 1 h before the injection of radiotracers or intraperitoneal administration of anti-TIGIT mAb (5 mg/kg) 24 hours in advance.

Flow cytometry
To explore the relationship of tumor uptake of 68 Ga-GP12 on PET images (% ID/g) with TIGIT expression, ow cytometry was employed to determine the TIGIT expression on CD45 + cell, CD4 + T cell, CD8 + T cell, NK cell, and regulatory T cell (Treg), respectively. Single-cell suspensions of mononuclear cells were isolated from tumor samples after PET imaging by centrifugation with 40 % Percoll as described in Supplemental Information. 1 × 10 6 mononuclear cells were resuspended in PBS buffer containing 0.2 % BSA and co-cultured with CD16/CD32 Fc block antibodies (Biolegend) for 20 min at 4 °C. Then, the cells were stained for surface phenotypic markers using speci c uorochrome-conjugated antibodies in PBS buffer for 30 min at 4 °C in the dark. The ow cytometry analysis was conducted using a BD in ux cell sorter (BD Biosciences) according to the strategy shown in Fig. S15. The acquired data were analyzed with the FlowJo Analysis Software (Version 10).

Biodistribution
The biodistribution was conducted in B16F10 xenograft models after intravenous injection of 1.85 MBq of 68 Ga-GP12. At the indicated time points (30, 60, 120 min), each mouse was sacri ced and dissected, and organs/tissues of interest were collected and weighed. The radioactivity in each sample was measured by a γ-counter to calculate the percentage of injected dose per gram (% ID/g, mean ± SD).

First-in-human study
This rst-in-human pilot study was approved by the Medical Ethics Committee of Xiangya Hospital, Central South University (Ethics Approval No. 202106115) and conducted according to the guidelines of the Declaration of Helsinki. Written informed consent was provided by all the participants. After a lowdose CT scan, the patients underwent whole-body dynamic imaging for approximately 60 min after injection of 68 Ga-GP12 (3.7-5.2 MBq/kg) on a GE Discovery PET/CT 690 Elite scanner (Waukesha, USA).
The 18 F-FDG PET/CT imaging was acquired within one week. 68 Ga-GP12 and 18 F-FDG PET/CT scans were processed by two independent, experienced nuclear medicine physicians. The standardized uptake value (SUV) in the volume ROIs was obtained using a GE AW 4.6 workstation. The imaging ndings of 68 Ga-GP12 PET/CT were compared with that of 18 F-FDG PET scans and further con rmed by histopathology.

Statistical analysis
The quantitative data were indicated as mean ± SD and statistically analyzed by GraphPad Prism 7.0 software. The differences within groups and between groups were compared by using two-tailed paired, unpaired Student's t-tests and one-way ANOVA, respectively. Differences of P < 0.05 indicate statistical signi cance.

Results
Production of 68 Ga-labeled -peptides To perform 68 Ga-radiolabeling and concurrently preserve their a nity and speci city, the modi cation of -peptides with polyethylene glycol linkers and NOTA was designed and explored. Several NOTA-peptides were synthesized, radiolabeled with 68 Ga and screened for TIGIT imaging ( Fig. 1a and Fig. S1-4). 68 Ga-labeled -peptides were obtained after Sep-Pak puri cation 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 nal products were veri ed by their non-radioactive standards (Fig. S5), and the radiochemical purities were > 99 % (n = 5). The partition coe cients (Log P) at pH 7.4 were determined to be -3.31--1.56 (n = 6), indicating their hydrophilicity. In vitro stabilities of 68 Ga-labeled -peptides were con rmed in saline and serum within 4 h (Fig. S6).

In vitro assays
The signi cant upregulation of TIGIT on human PBMCs after stimulation was demonstrated (Fig. S7).
Saturation binding experiments were performed in activated PBMCs. Among them, 68 Ga-GP12 displayed a much higher a nity for TIGIT protein with a dissociation constant K D of 37.28 nM (Fig. 1b, 1c and Fig.   S8). Time-dependent cellular uptake of 68 Ga-labeled -peptides was studied in non-activated and activated PBMCs, respectively (Fig. S9). It was found that cellular uptakes of 68 Ga-labeled -peptides increased with time and were saturated after 60 min of incubation. With an exception of 68 Ga-LA12, the uptakes were signi cantly decreased after blocking with an excess of unlabeled -peptides (Fig. 2a), indicating their speci c uptake in activated PBMCs. 68 Ga-GP12 uptake (45.88 ± 4.98 %) in the activated PBMCs was substantially higher than that of 68 Ga-LA12 (8.08 ± 1.48 %, P < 0.001), 68 Ga-GS12 (16.29 ± 3.20 %, P < 0.001) and 68 Ga-SP12 (12.66 ± 2.47 %, P < 0.001) (Fig. S10). Furthermore, the activated-tononactivated ratios and the activated-to-blocking ratios were determined and compared, which indicates the excellent speci city of 68 Ga-GP12 in vitro (Fig. 2b). In addition, through docking analysis, the optimized steric complementarity of 68 Ga-GP12 with binding sites of TIGIT was formed with a binding energy of -10.35 kcal/mol (Fig. 1d). It revealed that 68 Ga-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 68 Ga-labeled -peptides was compared by PET imaging in B16F10 xenograft models. Tumor uptake of 68 Ga-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 68 Ga-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 68 Ga-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 speci city of 68 Ga-GP12 for imaging of TIGIT in vivo. However, tumor uptake of 68 Ga-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, 68 Ga-GP12 PET imaging of TIGIT was veri ed by other types of tumor-bearing mice. In the B16F10 melanoma pulmonary metastasis models, diffuse bilateral lung abnormal uptake of 68 Ga-GP12 was detected, which was manifested as focal asymmetry uptake in 18 F-FDG PET/CT imaging (Fig. S12). This result indicated the heterogeneity of TIGIT expression, further con rmed by IHC. As expected, the capacity of 68 Ga-GP12 for PET imaging was also con rmed in Panc02 and MC38 tumor models ( Fig.  S13 and S14).

Biodistribution, pharmacokinetics and radiation dosimetry
The biodistribution of 68 Ga-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 68 Ga-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 68 Ga-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 68 Ga-GP12 was quickly cleared from the blood with a half-life of 27.02 min (Fig. S18). In addition, in vivo metabolic stability of 68 Ga-GP12 was veri ed 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 68 Ga-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 68 Ga-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 18 F-FDG as previously reported [26].
First-in-human PET/CT imaging Two patients with advanced NSCLC received the intravenous injection of 68 Ga-GP12 (203.5 and 233.1 MBq, respectively) for PET/CT imaging. No adverse or clinically detectable pharmacologic effects were observed. There were no signi cant changes in vital signs or the results of laboratory studies or electrocardiograms.
The biodistribution of 68 Ga-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 68 Ga-GP12.
The expression of TIGIT in the primary tumor was con rmed by immunohistochemistry (Fig. S21).

Discussion
While TIGIT has been propelled under the spotlight as a next-generation target in cancer immunotherapy, anti-TIGIT therapy seems to be promising for a fraction of patients in clinical trials [14,15,23]. Therefore, patient strati cation is critical for this therapy, which could bene t from a whole-body, non-invasive and quantitative evaluation of TIGIT expression in cancer. In this study, we developed and validated a novel 68 Ga-labeled -peptide antagonist, 68 Ga-GP12, for PET imaging of TIGIT expression in cancers.
Furthermore, the safety and potential of 68 Ga-GP12 for PET/CT imaging of TIGIT expression were evaluated in a rst-in-human pilot study with advanced NSCLC.
Owing to the considerable potential in the design of PET imaging tracers, the rst -enantiomer peptide antagonists were modi ed, radiolabeled with 68 Ga and screened for TIGIT imaging. 68 Ga-labeledpeptides were conveniently produced with high radiochemical yields and molar activities. In vitro binding assays demonstrated 68 Ga-GP12 has favorable a nity and speci city for TIGIT, which were further con rmed by preliminary PET/CT imaging in B16F10 xenograft models. The peptide-protein docking revealed that 68 Ga-GP12 might interact with TIGIT through the binding interface of TIGIT/PVR. These results encouraged us to further investigate the potential of 68 Ga-GP12 for in vivo imaging of TIGIT. In four tumor models, the tumor could be visualized rapidly with the optimized tumor-to-muscle ratio at 60 min p.i., which is superior to that of 64 Cu and 89 Zr-labeled anti-TIGIT mAb [23]. The in vivo speci city of 68 Ga-GP12 for TIGIT was identi ed in a blocking study pretreated with an excess of GP12. Interestingly, tumor uptakes of 68 Ga-GP12 were not blocked by pretreatment of anti-TIGIT mAb. This nding indicated the different binding epitopes of 68 Ga-GP12 and anti-TIGIT mAb to TIGIT protein, which endows it with the capacity of detecting TIGIT expression and evaluating prognosis in the course of anti-TIGIT therapy.
As expected, tumor uptake of 68 Ga-GP12 was positively associated with TIGIT expression on CD4 + T cell, CD8 + T cell, NK cell and Treg cell, respectively. The considerable tumor accumulation and rapid blood clearance of 68 Ga-GP12 contributed to the optimized PET imaging with high tumor/muscle and tumor/blood ratios in a short time. The uptake of radioactivity was also detected in the spleen because of abundant lymphocytes [8]. The radiotracer showed a broad distribution in tissues, predominantly in the kidney with subsequent elimination through the urinary system, owing to its hydrophilicity. The kidneys and urinary bladder wall received the highest absorbed dose. The effective dose was comparable to that of 18 F-FDG, highlighting the safety of 68 Ga-GP12 as a PET tracer. All these results indicate 68 Ga-GP12 as a promising radiotracer for PET imaging of TIGIT expression in vivo.
To the best of our knowledge, 68 Ga-GP12 is the rst TIGIT-targeting PET tracer that was tested in patients with advanced NSCLC. The optimized time-point for image acquisition is 40 min after injection of 68 Ga-GP12. Impressively, the primary and metastatic lesions found in 68 Ga-GP12 PET/CT imaging were comparable to that in 18 F-FDG PET/CT imaging. Moreover, the inhomogenous intra-and-inter-tumoral uptake of 68 Ga-GP12 was presented in PET/CT imaging, re ecting the heterogeneity of TIGIT expression levels. However, the tumor uptake of 18 F-FDG was intensive in the lesions regardless of TIGIT expression.
In addition, the biodistribution of 68 Ga-GP12 in patients was similar to that observed in preclinical studies. No adverse effect was found in patients receiving the injection of 68 Ga-GP12. Although promising preliminary results of this pilot study, the potential of 68 Ga-GP12 for PET imaging of TIGIT expression in cancer was not well characterized in the clinical setting because of the unavailability of enough patient samples (affected by . More patients are being recruited in clinical trials for evaluating 68 Ga-GP12 PET/CT as a potential companion diagnostic for anti-TIGIT therapies.

Conclusion
A novel 68 Ga-labeled -peptide antagonist, 68 Ga-GP12, was developed and validated for PET imaging of TIGIT expression in vitro and in vivo. The high a nity and speci city, favorable pharmacokinetics and excellent imaging capacity indicated that 68 Ga-GP12 is a promising radiotracer for PET imaging of TIGIT expression in cancers. Furthermore, the encouraging preliminary results of this rst-in-human pilot study support the continued clinical development of 68 Ga-GP12 PET/CT as a potential companion diagnostic for anti-TIGIT therapies. Figure 1 Design and development of 68Ga-labeled -peptide antagonists. (a) Radiosynthesis of 68Ga-labeledpeptide antagonists. (b) Radiochemical characterization and binding a nity of 68Ga-labeled -peptide antagonists. aRCY, radiochemical yield, n = 5. bRCP, radiochemical purity, n = 5. cMA, molar activity, n = 3. dn = 6. (c) The saturation binding assay of 68Ga-GP12 (en = 3). (d) Peptide-protein docking. The binding mode and key interactions between 68Ga-GP12 (cyan) and TIGIT (pale yellow).

Figure 2
Discovery and validation of 68Ga-GP12 for TIGIT imaging. (a) In vitro binding assay in human PBMCs. The cell uptake of 68Ga-labeled -peptide antagonists after 60 min of incubation (n = 4) in activated and non-activated PBMCs. (b) The ratios of activated to non-activated cells and activated to blocking cells were derived from above data. (c) PET imaging of 68Ga-labeled -peptide antagonists in B16F10 xenograft models (n = 5). (d) The tumor uptakes (% ID/g) were determined from PET images. The data are shown as mean ± SD, ****P < 0.0001 ***P < 0.001, **P < 0.01; n.s., not signi cant.

Figure 3
In vivo PET imaging of 68Ga-GP12 (n = 3). (a) Dynamic PET scanning of B16F10 xenograft models with or without pretreatment with GP12 or anti-TIGIT mAb over 0-120 min after injection of 68Ga-GP12. The time-activity curves (b) and tumor/muscle ratios (c) were derived from the PET images. Correlation between the TIGIT expression analyzed by ex vivo ow cytometry and tumor uptake (%ID/g) of 68Ga-GP12 on PET images in B16F10 xenograft models (n = 8).

Figure 6
First-in-human study of 68Ga-GP12. A 72-year-old male with lung adenocarcinoma showed focal uptake of (a) 68Ga-GP12 (SUVmax = 4.82) and (c) 18F-FDG (SUVmax = 9.45) in the primary tumor of the right lung (white arrow). A metastatic lesion on the right femur was detected in both (b) 68Ga-GP12 PET/CT

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