Preclinical and Exploratory Clinical Studies of Novel 68Ga-labeled ᴅ-Peptide Antagonist for PET Imaging of TIGIT Expression in Cancers

doi:10.21203/rs.3.rs-1022828/v1

PDF

Research Article

Preclinical and Exploratory Clinical Studies of Novel ⁶⁸Ga-labeled ᴅ-Peptide Antagonist for PET Imaging of TIGIT Expression in Cancers

https://doi.org/10.21203/rs.3.rs-1022828/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

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 stratification is critical for this therapy, which could benefit from a whole-body, non-invasive and quantitative evaluation of TIGIT expression in cancers. In this study, a ⁶⁸Ga-labeled ᴅ-peptide antagonist, ⁶⁸Ga-GP12, was developed and validated for PET imaging of TIGIT expression in vitro, in vivo, and first-in-human pilot study.

Methods

The ᴅ-enantiomer peptide antagonists were modified and radiolabeled with ⁶⁸Ga. In vitro binding assays were performed in human peripheral blood mononuclear cells (PBMCs) to assess their affinity and specificity. The imaging capacity, biodistribution, pharmacokinetics, and radiation dosimetry were investigated in vivo. Flow cytometry, autoradiography, and immunohistochemical staining were used to confirm the expression of TIGIT ex vivo. The safety and potential of ⁶⁸Ga-GP12 for PET/CT imaging of TIGIT expression were further evaluated in a first-in-human pilot study with advanced NSCLC.

Results

⁶⁸Ga-labeled ᴅ-peptides were conveniently produced with high radiochemical yields,radiochemical purity and molar activities. In vitro binding assays demonstrated ⁶⁸Ga-GP12 has favorable affinity and specificity for TIGIT with a K_D of 37.28 nM. In vivo and ex vivo studies demonstrated the favorable pharmacokinetics of ⁶⁸Ga-GP12 for PET imaging of TIGIT expression with high tumor uptake of 4.22 ± 0.68 %ID/g and the tumor-to-muscle ratio of 12.94 ± 2.64 at 60 min post-injection. The primary and metastatic lesions found in the first-in-human studies of ⁶⁸Ga-GP12 PET/CT imaging were comparable to that in ¹⁸F-FDG PET/CT imaging. Moreover, the inhomogenous intra-and-inter-tumoral uptake of ⁶⁸Ga-GP12 was presented, reflecting the heterogeneity of TIGIT expression levels.

Conclusion

⁶⁸Ga-GP12 is a promising radiotracer for PET imaging of TIGIT expression in cancers, indicating its potential as a potential companion diagnostic for anti-TIGIT therapies.

Nuclear Medicine & Medical Imaging

PET imaging

TIGIT

Immune checkpoints

ᴅ-Peptide antagonist

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 benefit 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 benefits 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 [7–9]. The upregulation of TIGIT has been observed in many malignant tumors and correlates with dismal clinical outcomes [10–12], 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 efficacy 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 efficacy 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 benefit 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 stratification 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 exemplified 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 [19–22]. This instigated research in PET imaging of TIGIT expression for the stratification of patients. Most recently, Shaffer et. al. validated ⁶⁴Cu and ⁸⁹Zr-labeled anti-TIGIT mAb as radiotracers for PET imaging of TIGIT status on tumor-infiltrating lymphocytes and screening patients for anti-TIGIT therapy [23]. However, the long blood circulation and slow clearance from the body make it difficult 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 affinity and specificity, favorable pharmacokinetics, and easy synthesis and modification [24].

Following this tendency, the first ᴅ-enantiomer peptide antagonists were identified by mirror-image phage display biopanning and blocked the interaction of TIGIT/PVR with high affinity and specificity [25], which motivates our design of TIGIT-targeting small molecule PET tracers. In this study, we developed and validated ⁶⁸Ga-labeled ᴅ-peptide antagonist, ⁶⁸Ga-GP12, for PET imaging of TIGIT expression in the tumor. Furthermore, the safety and potential of ⁶⁸Ga-GP12 for PET/CT imaging of TIGIT expression were evaluated in a first-in-human pilot study with advanced NSCLC.

Production of ⁶⁸Ga-labeled ᴅ-peptides

NOTA-ᴅ-peptides with polyethylene glycol linkers were custom synthesized by GL Biochem Co., Ltd. (Shanghai, China) and fully characterized by mass spectrometry (Fig. S1-4). Details of ⁶⁸Ga-labeled ᴅ-peptides preparation were described in Supplemental Information following the general methods [21,24].

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 ⁶⁸Ga-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 ⁶⁸Ga-labeled ᴅ-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 ⁶⁸Ga-GP12 on PET images (% ID/g) with TIGIT expression, flow 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 × 10⁶ 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 specific fluorochrome-conjugated antibodies in PBS buffer for 30 min at 4 °C in the dark. The flow cytometry analysis was conducted using a BD influx 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 ⁶⁸Ga-GP12. At the indicated time points (30, 60, 120 min), each mouse was sacrificed 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 first-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 low-dose CT scan, the patients underwent whole-body dynamic imaging for approximately 60 min after injection of ⁶⁸Ga-GP12 (3.7-5.2 MBq/kg) on a GE Discovery PET/CT 690 Elite scanner (Waukesha, USA). The ¹⁸F-FDG PET/CT imaging was acquired within one week. ⁶⁸Ga-GP12 and ¹⁸F-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 findings of ⁶⁸Ga-GP12 PET/CT were compared with that of ¹⁸F-FDG PET scans and further confirmed 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 significance.

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 K_D 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 (SUV_max = 4.82, Fig. 6a), and ¹⁸F-FDG (SUV_max = 9.45, Fig. 6c). Furthermore, a metastatic lesion on the right femur was detected in both ⁶⁸Ga-GP12 PET/CT (SUV_max = 2.80, Fig. 6b) and ¹⁸F-FDG PET/CT imaging (SUV_max = 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 (SUV_max = 2.95, Fig. 7a) and intensive uptake in ¹⁸F-FDG PET/CT imaging (SUV_max = 18.56, Fig. 7b), indicating the heterogeneity of TIGIT expression in the large tumor.

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 stratification is critical for this therapy, which could benefit from a whole-body, non-invasive and quantitative evaluation of TIGIT expression in cancer. In this study, we developed and validated a novel ⁶⁸Ga-labeled ᴅ-peptide antagonist, ⁶⁸Ga-GP12, for PET imaging of TIGIT expression in cancers. Furthermore, the safety and potential of ⁶⁸Ga-GP12 for PET/CT imaging of TIGIT expression were evaluated in a first-in-human pilot study with advanced NSCLC.

Owing to the considerable potential in the design of PET imaging tracers, the first ᴅ-enantiomer peptide antagonists were modified, radiolabeled with ⁶⁸Ga and screened for TIGIT imaging. ⁶⁸Ga-labeled ᴅ-peptides were conveniently produced with high radiochemical yields and molar activities. In vitro binding assays demonstrated ⁶⁸Ga-GP12 has favorable affinity and specificity for TIGIT, which were further confirmed by preliminary PET/CT imaging in B16F10 xenograft models. The peptide-protein docking revealed that ⁶⁸Ga-GP12 might interact with TIGIT through the binding interface of TIGIT/PVR. These results encouraged us to further investigate the potential of ⁶⁸Ga-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 ⁶⁴Cu and ⁸⁹Zr-labeled anti-TIGIT mAb [23]. The in vivo specificity of ⁶⁸Ga-GP12 for TIGIT was identified in a blocking study pretreated with an excess of GP12. Interestingly, tumor uptakes of ⁶⁸Ga-GP12 were not blocked by pretreatment of anti-TIGIT mAb. This finding indicated the different binding epitopes of ⁶⁸Ga-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 ⁶⁸Ga-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 ⁶⁸Ga-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 ¹⁸F-FDG, highlighting the safety of ⁶⁸Ga-GP12 as a PET tracer. All these results indicate ⁶⁸Ga-GP12 as a promising radiotracer for PET imaging of TIGIT expression in vivo.

To the best of our knowledge, ⁶⁸Ga-GP12 is the first 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 ⁶⁸Ga-GP12. Impressively, the primary and metastatic lesions found in ⁶⁸Ga-GP12 PET/CT imaging were comparable to that in ¹⁸F-FDG PET/CT imaging. Moreover, the inhomogenous intra-and-inter-tumoral uptake of ⁶⁸Ga-GP12 was presented in PET/CT imaging, reflecting the heterogeneity of TIGIT expression levels. However, the tumor uptake of ¹⁸F-FDG was intensive in the lesions regardless of TIGIT expression. In addition, the biodistribution of ⁶⁸Ga-GP12 in patients was similar to that observed in preclinical studies. No adverse effect was found in patients receiving the injection of ⁶⁸Ga-GP12. Although promising preliminary results of this pilot study, the potential of ⁶⁸Ga-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 COVID-19). More patients are being recruited in clinical trials for evaluating ⁶⁸Ga-GP12 PET/CT as a potential companion diagnostic for anti-TIGIT therapies.

A novel ⁶⁸Ga-labeled ᴅ-peptide antagonist, ⁶⁸Ga-GP12, was developed and validated for PET imaging of TIGIT expression in vitro and in vivo. The high affinity and specificity, favorable pharmacokinetics and excellent imaging capacity indicated that ⁶⁸Ga-GP12 is a promising radiotracer for PET imaging of TIGIT expression in cancers. Furthermore, the encouraging preliminary results of this first-in-human pilot study support the continued clinical development of ⁶⁸Ga-GP12 PET/CT as a potential companion diagnostic for anti-TIGIT therapies.

Acknowledgements

The authors acknowledge the staff of the Department of PET Center, Xiangya Hospital for the help in the first-in-human study. The authors thank Dr. Chao Chen (Xiangya Hospital) for FACS analysis, Prof. Jie Dong (Central South University) and Dr. Xin Yan (Sun Yat-sen University) for the assistance in peptide-protein docking.

Funding

This study was financially supported by the Joint Fund of National Natural Science Foundation of China - China National Nuclear Corporation for Nuclear Technology Innovation (U1967222), National Natural Science Foundation of China (81801762 and 91859207) and Natural Science Foundation of Hunan Province of China (2020JJ5956).

Author information

Affiliations

Center for Molecular Imaging and Translational Medicine, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361102, China

Xiaobo Wang, Huanhuan Liu, Jianyang Fang & Xianzhong Zhang

Department of PET Center, Xiangya Hospital, Central South University, Changsha 410008, China

Ming Zhou, Bei Chen, Shijun Xiang & Shuo Hu

Contributions

Conceptualization: XZ and SH; Methodology: XW, MZ, XZ and SH; Data collection and analysis: XW, MZ, BC, HL, JF and SX; Patient recruitment, PET imaging and image analysis: MZ, BC, SX, XW and SH; Manuscript writing, review and editing: XW, XZ and SH.

Corresponding authors

Correspondence to Shuo Hu or Xianzhong Zhang.

Ethics declarations

Ethics approval

All procedures involving human participants were approved by the Medical Ethics Committee of Xiangya Hospital, Central South University (Ethics Approval No. 202106115). All animal studies were performed according to the guidelines of the Animal Care Committee of Xiamen University.

Conflict of interest

The authors declare no competing interests.

Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012; 12:252–264.
Sharma P, Hu-Lieskovan S, Wargo JA, et al. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell. 2017;168:707–723.
Andrews LP, Yano H, Vignali DAA. Inhibitory receptors and ligands beyond PD-1, PD-L1 and CTLA-4: breakthroughs or backups. Nat Immunol. 2019;20:1425–1434.
Meric-Bernstam F, Larkin J, Tabernero J, et al. Enhancing anti-tumour efficacy with immunotherapy combinations. Lancet. 2021;397:1010–1022.
Chauvin JM, Zarour HM. TIGIT in cancer immunotherapy. J Immunother Cancer. 2020;8:e000957.
Stengel KF, Harden-Bowles K, Yu X, et al. Structure of TIGIT immunoreceptor bound to poliovirus receptor reveals a cell-cell adhesion and signaling mechanism that requires cis-trans receptor clustering. Proc Natl Acad Sci U S A. 2012;109:5399–404.
Manieri NA, Chiang EY, Grogan JL. TIGIT: A key inhibitor of the cancer immunity cycle. Trends Immunol. 2017;38:20–28.
Zhang Q, Bi J, Zheng X, et al. Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity. Nat Immunol. 2018;19:723–732.
Yu X, Harden K, Gonzalez LC, et al. The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells. Nat Immunol. 2009;10:48–57.
Yang ZZ, Kim HJ, Wu H, et al. TIGIT expression is associated with T-cell suppression and exhaustion and predicts clinical outcome and anti-PD-1 response in follicular lymphoma. Clin Cancer Res. 2020;26:5217–5231.
Liu Z, Zhou Q, Wang Z, et al. Intratumoral TIGIT⁺CD8⁺ T-cell infiltration determines poor prognosis and immune evasion in patients with muscle-invasive bladder cancer. J Immunother Cancer. 2020;8:e000978.
Chiu DK, Yuen VW, Cheu JW, et al. Hepatocellular carcinoma cells up-regulate PVRL1, stabilizing PVR and inhibiting the cytotoxic T-cell response via TIGIT to mediate tumor resistance to PD1 inhibitors in mice. Gastroenterology. 2020;159:609–623.
Harjunpää H, Guillerey C. TIGIT as an emerging immune checkpoint. Clin Exp Immunol. 2020;200:108–119.
Yeo J, Ko M, Lee DH, et al. TIGIT/CD226 axis regulates anti-tumor immunity. Pharmaceuticals (Basel). 2021;14:200.
Anonymous. Tiragolumab Impresses in multiple trials. Cancer Discov. 2020;10: 1086–1087.
Topalian SL, Taube JM, Anders RA, et al. Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy. Nat Rev Cancer. 2016;16:275–287.
van der Leun AM, Thommen DS, Schumacher TN. CD8(+) T cell states in human cancer: insights from single-cell analysis. Nat Rev Cancer. 2020;20:218–232.
Teng MWL, Ngiow SF, Ribas A, et al. Classifying cancers based on T-cell infiltration and PD-L1. Cancer Res. 2015;75:2139–2145.
Bensch F, van der Veen EL, Lub-de Hooge MN, et al. ⁸⁹Zr-atezolizumab imaging as a non-invasive approach to assess clinical response to PD-L1 blockade in cancer. Nat Med. 2018;24:1852–1858.
Niemeijer AN, Leung D, Huisman MC, et al. Whole-body PD-1 and PD-L1 positron emission tomography in patients with non-small-cell lung cancer. Nat Commun. 2018;9:4664.
Zhou X, Jiang J, Yang X, et al. First-in-human evaluation of a PD-L1-binding peptide radiotracer in non-small cell lung cancer patients with PET. J Nucl Med. 2021; doi: 10.2967/jnumed.121.262045.
Xing Y, Chand G, Liu C, et al. Early phase I study of a ^99mTc-labeled anti-programmed death ligand-1 (PD-L1) single-domain antibody in SPECT/CT assessment of PD-L1 expression in non-small cell lung cancer. J Nucl Med. 2019;60:1213–1220.
Shaffer T, Natarajan A, Gambhir SS. PET Imaging of TIGIT expression on tumor-infiltrating lymphocytes. Clin Cancer Res. 2021;27:1932–1940.
Kumar D, Lisok A, Dahmane E, et al. Peptide-based PET quantifies target engagement of PD-L1 therapeutics. J Clin Invest. 2019;129:616–630.
Zhou X, Zuo C, Li W, et al. A novel d-peptide identified by mirror-image phage display blocks TIGIT/PVR for cancer immunotherapy. Angew Chem Int Ed Engl. 2020;59:15114–15118.
Boellaard R, O'Doherty MJ, Weber WA, et al. FDG PET and PET/CT: EANM procedure guidelines for tumour PET imaging: version 1.0. Eur J Nucl Med Mol Imaging. 2010;37:181–200.

PDF

Reviews received at journal
31 Oct, 2021
Reviewers invited by journal
28 Oct, 2021
Editor assigned by journal
27 Oct, 2021
First submitted to journal
26 Oct, 2021

You are reading this latest preprint version

Preclinical and Exploratory Clinical Studies of Novel ⁶⁸Ga-labeled ᴅ-Peptide Antagonist for PET Imaging of TIGIT Expression in Cancers

Status:

Version 1

Abstract

Purpose

Methods

Results

Conclusion

Figures

Introduction

Materials And Methods

Results

Discussion

Conclusion

Declarations

References

Supplementary Files

Status:

Version 1