Application of 99mTc-Labeled WL12 Peptides as a Tumor PD-L1-Targeted SPECT Imaging Agent: Kit Formulation, Preclinical Evaluation, and Study on the Influence of Coligands

With the development of PD-1/PD-L1 immune checkpoint inhibitor therapy, the ability to monitor PD-L1 expression in the tumor microenvironment is important for guiding therapy. This study was performed to develop a novel radiotracer with optimal pharmacokinetic properties to reflect PD-L1 expression in vivo via single-photon emission computed tomography (SPECT) imaging. [99mTc]Tc-HYNIC-WL12-tricine/M (M = TPPTS, PDA, ISONIC, 4-PSA) complexes with high radiochemical purity (>97%) and suitable molar activity (from 100.5 GBq/μmol to 300 GBq/μmol) were prepared through a kit preparation process. All 99mTc-labeled HYNIC-WL12 radiotracers displayed good in vitro stability for 4 h. The affinity and specificity of the four radiotracers for PD-L1 were demonstrated both in vitro and in vivo. The results of biodistribution studies displayed that the pharmacokinetics of the 99mTc-HYNIC-conjugated radiotracers were significantly influenced by the coligands of the radiotracers. Among them, [99mTc]Tc-HYNIC-WL12-tricine/ISONIC exhibited the optimal pharmacokinetic properties (t1/2α = 8.55 min, t1/2β = 54.05 min), including the fastest clearance in nontarget tissues, highest tumor-to-background contrast (e.g., tumor-to-muscle ratio, tumor-to-blood ratio: 40.42 ± 1.59, 14.72 ± 2.77 at 4 h p.i., respectively), and the lowest estimated radiation absorbed dose, highlighting its potential as a clinical SPECT imaging probe for tumor PD-L1 detection.


Introduction
Since immunotherapy began in 2011, immune checkpoint inhibitor (ICI) therapy based on the programmed death protein 1 (PD-1)/programmed death protein ligand 1 (PD-L1) signaling pathway has played an increasingly important role in cancer treatment [1].Recent clinical studies have demonstrated that PD-L1 expression has a significant influence on therapeutic efficacy, and positive responses to PD-1/PD-L1 ICI therapy are only possible in patients with tumors that contain high levels of expressed PD-L1 [2,3].The main method used in the clinic to assess PD-L1 expression in the tumor microenvironment is invasive biopsy in conjunction with immunohistochemistry (IHC).However, due to the high heterogeneity of PD-L1 expression within both primary tumors and metastases, the ability of IHC detection to accurately evaluate the PD-L1 expression status in realtime and predict treatment response is limited, especially for patients with metastatic diseases [4].Compared with IHC, nuclear medicine techniques allow real-time, noninvasive visualization of tumor PD-L1 expression in the whole body, which can overcome the shortcomings of IHC methods.
Currently, developing a novel WL12-based radiotracer with optimal excretion kinetics is highly desirable to obtain a high target-to-background ratio for PD-L1-positive tumor imaging.In this study, we focused on developing 99m Tc-labeled HYNIC-conjugated WL12 for SPECT imaging, which is expected to provide a simple, convenient, and inexpensive diagnostic tool for assessing the status of PD-L1 in cancer patients.99m Tc is the most widely used radionuclide due to its excellent nuclide properties, low cost, and high availability through a 99 Mo/ 99m Tc generator.Herein, 6-hydrazino nicotinamide (HYNIC) was used as the bifunctional chelating group to conjugate the -Orn of WL12 for the following reasons: (1) A high 99m Tc-labeling efficiency can be achieved at very low concentrations of HYNICconjugated biomolecules [44], which is beneficial for obtaining radiotracers with high molar activity through a kit preparation process while following the requirements of good manufacturing practice (GMP).(2) During the 99m Tc-HYNIC radiolabeling process, coligands are essential because they occupy the remaining sites of the 99m Tc coordination sphere to form 99m Tc-HYNIC complexes with good stability.Due to the significant effect of coligands on their physicochemical properties, such as hydrophilicity, charge, and stability [44][45][46][47][48], a strategy is convenient for optimizing the pharmacokinetic properties of 99m Tc-HYNIC complexes via the selection of coligands.In this study, lyophilized kits containing different coligands, tricine/M (M = triphenylphosphine-3,3 ′ ,3 ′′ -trisulfonic acid trisodium salt (TPPTS), isonicotinic acid (ISONIC), 3,5-pyridine dicarboxylic acid (PDA), or 4-pyridinesulfonic acid (4-PSA), respectively), were developed to prepare 99m Tc-WL12 complexes with high radiochemical purity and suitable molar activity.In vitro and in vivo evaluations of these radiotracers were performed, and the results were compared to develop a favorable PD-L1-targeted tumor imaging agent with optimal pharmacokinetic properties for SPECT imaging.

Peptide Synthesis
First, the peptide WL12 was synthesized via solid-phase synthesis.The HYNIC moiety was introduced into the -Orn of the WL12 peptide to produce the HYNIC-WL12 peptide (Scheme 1).Both the WL12 and HYNIC-WL12 peptides were purified by high-performance liquid chromatography (HPLC).The identities of the final peptides were confirmed by electrospray ionization mass spectrometry (ESI-MS).The chemical purity of each sample was analyzed by HPLC.The ESI-MS and HPLC results obtained for the final peptides are shown in the Supplemental Information (Figures S1-S4). in vivo evaluations of these radiotracers were performed, and the results were compared to develop a favorable PD-L1-targeted tumor imaging agent with optimal pharmacokinetic properties for SPECT imaging.

Peptide Synthesis
First, the peptide WL12 was synthesized via solid-phase synthesis.The HYNIC moiety was introduced into the -Orn of the WL12 peptide to produce the HYNIC-WL12 peptide (Scheme 1).Both the WL12 and HYNIC-WL12 peptides were purified by high-performance liquid chromatography (HPLC).The identities of the final peptides were confirmed by electrospray ionization mass spectrometry (ESI-MS).The chemical purity of each sample was analyzed by HPLC.The ESI-MS and HPLC results obtained for the final peptides are shown in the supplemental information (Figures S1-S4).

Radiochemistry
As shown in Scheme 1, the radiochemical synthesis of [ 99m Tc]Tc-HYNIC-WL12-tricine/M (M = TPPTS or PDA or ISONIC or 4-PSA, respectively) was performed by adding 99m Tc eluent into the kit containing the HYNIC-WL12 peptide and coligands and reacting the substances at 100 °C.[ 99m Tc]Tc-HYNIC-WL12-tricine/M (M = TPPTS or PDA or ISONIC or 4-PSA, respectively) was obtained with a high labeling yield (>98%) under the optimized reaction conditions.The molar activity (Am) of the obtained radiotracer ranged from 1.5 GBq/µmol to 300 GBq/µmol according to the added radioactivity.
The octanol-water partition coefficient (log D) of the four radiotracers was determined in a mixture of phosphate-buffered saline (PBS, 0.1 M, pH = 7.4) and n-octanol.As shown in Table 1, all four radiotracers were hydrophilic.Among them, [ 99m Tc]Tc-HYNIC-WL12-tricine/TPPTS was the most hydrophilic, with a Log D of −1.71 ± 0.09, followed by

Radiochemistry
As shown in Scheme 1, the radiochemical synthesis of [ 99m Tc]Tc-HYNIC-WL12tricine/M (M = TPPTS or PDA or ISONIC or 4-PSA, respectively) was performed by adding 99m Tc eluent into the kit containing the HYNIC-WL12 peptide and coligands and reacting the substances at 100 • C. [ 99m Tc]Tc-HYNIC-WL12-tricine/M (M = TPPTS or PDA or ISONIC or 4-PSA, respectively) was obtained with a high labeling yield (>98%) under the optimized reaction conditions.The molar activity (A m ) of the obtained radiotracer ranged from 1.5 GBq/µmol to 300 GBq/µmol according to the added radioactivity.
To evaluate the stability, the radiotracers were incubated in saline at room temperature or mouse serum at 37 • C for 4 h, and the [ 99m Tc]Tc-HYNIC-WL12-tricine/M samples were analyzed by radio-HPLC.As shown in Figure 1, the RCP of all the samples remained above 99%, which demonstrated the good stability of [ 99m Tc]Tc-HYNIC-WL12-tricine/M in vitro.To evaluate the stability, the radiotracers were incubated in saline at room temperature or mouse serum at 37 °C for 4 h, and the [ 99m Tc]Tc-HYNIC-WL12-tricine/M samples were analyzed by radio-HPLC.As shown in Figure 1, the RCP of all the samples remained above 99%, which demonstrated the good stability of [ 99m Tc]Tc-HYNIC-WL12-tricine/M in vitro.

Cellular Uptake and Blocking Assays
To evaluate the ability of [ 99m Tc]Tc-HYNIC-WL12-tricine/M (M = TPPTS, ISONIC, PDA, and 4-PSA) to target PD-L1, mouse colon cancer MC38 and human PD-L1 genetransfected MC38 cells (MC38-B7H1) were used.The PD-L1 expression levels in the MC38-B7H1 and MC38 cell lines were evaluated by flow cytometry.As shown in the supplemental information (Figure S6), PD-L1 expression was lower in MC38 cells than in MC38-B7H1 cells.Therefore, the affinity and specificity of 99m Tc-labeled WL12 radiotracers for PD-L1 were evaluated in the MC38-B7H1 and MC38 cell lines as positive and negative models, respectively.
In cellular uptake studies, the RCP of 99m Tc-labeled WL12 radiotracers was greater than 97%, with Am values ranging from 30 to 60 GBq/µmol.As shown in Figure 2A-D, the cellular uptake of the four 99m Tc-HYNIC-WL12 radiotracers in PD-L1-positive MC38-B7H1 cells was significantly greater than that in MC38 cells at each time point, indicating that the uptake of the radiotracers in the cells was dependent on PD-L1 expression.

Cellular Uptake and Blocking Assays
To evaluate the ability of [ 99m Tc]Tc-HYNIC-WL12-tricine/M (M = TPPTS, ISONIC, PDA, and 4-PSA) to target PD-L1, mouse colon cancer MC38 and human PD-L1 genetransfected MC38 cells (MC38-B7H1) were used.The PD-L1 expression levels in the MC38-B7H1 and MC38 cell lines were evaluated by flow cytometry.As shown in the Supplemental Information (Figure S6), PD-L1 expression was lower in MC38 cells than in MC38-B7H1 cells.Therefore, the affinity and specificity of 99m Tc-labeled WL12 radiotracers for PD-L1 were evaluated in the MC38-B7H1 and MC38 cell lines as positive and negative models, respectively.
In cellular uptake studies, the RCP of 99m Tc-labeled WL12 radiotracers was greater than 97%, with A m values ranging from 30 to 60 GBq/µmol.As shown in Figure 2A-D, the cellular uptake of the four 99m Tc-HYNIC-WL12 radiotracers in PD-L1-positive MC38-B7H1 cells was significantly greater than that in MC38 cells at each time point, indicating that the uptake of the radiotracers in the cells was dependent on PD-L1 expression.The PD-L1 specificity of 99m Tc-labeled WL12 radiotracers was further confirmed by blocking studies (Figure 2E-H).The uptake of the four radiotracers in MC38-B7H1 cells was clearly blocked by the WL12 peptide (** p < 0.01, *** p < 0.001).In the presence of 11 µM WL12, the cellular uptake decreased by approximately 94%, 68%, 82%, and 87% for coligands as tricine/M (M = TPPTS or PDA or ISONIC or 4-PSA), respectively.

Effect of Molar Activity on Biodistribution
To determine the suitable molar activity of the radiotracer for radiolabeling with a kit formulation, we first investigated the effect of excess ligands on the biological properties of [ 99m Tc]Tc-HYNIC-WL12-tricine/TPPTS using female C57BL/6N mice bearing MC38-B7H1 tumors, which were confirmed to be PD-L1-positive tumor models by immunohistochemistry (Supplementary Information, Figure S7).All animal experiments were approved by the Institutional Animal Care and Use Committee of Beijing Normal University and were carried out in accordance with the Principles of Laboratory Animal Care and the guidelines of the Ethics Committee.

Ex Vivo and In Vivo Studies 2.4.1. Effect of Molar Activity on Biodistribution
To determine the suitable molar activity of the radiotracer for radiolabeling with a kit formulation, we first investigated the effect of excess ligands on the biological properties of [ 99m Tc]Tc-HYNIC-WL12-tricine/TPPTS using female C57BL/6N mice bearing MC38-B7H1 tumors, which were confirmed to be PD-L1-positive tumor models by immunohistochemistry (Supplementary Information, Figure S7).All animal experiments were approved by the Institutional Animal Care and Use Committee of Beijing Normal University and were carried out in accordance with the Principles of Laboratory Animal Care and the guidelines of the Ethics Committee.

Dosimetry Estimation
Based on the biodistribution results, time-activity curve fitting and subsequent dose calculation were performed using OLINDA/EXM, version 1.1.The nonlinear curve fitting parameters were applied to derive the best curve fit for the residence time of activity in the source organ.The derived organ residence times were entered in the assumed human model data to derive the absorbed doses to all the organs and the whole-body effective dose, which were generated in mSv/MBq.As shown in Table 8, the effective doses of [ 99m Tc]Tc-HYNIC-WL12-tricine/M (M = TPPTS, PDA, ISONIC, or 4-PSA) were 2.90 × 10 −3 , 2.12 × 10 −3 , 2.24 × 10 −3 , and 2.53 × 10 −3 mSv/MBq, respectively.The high effective dose of [ 99m Tc]Tc-HYNIC-WL12-tricine/TPPTS was attributed to the high absorbed radiation dose in the kidneys.Among them, [ 99m Tc]Tc-HYNIC-WL12-tricine/ISONIC displayed the lowest organ doses for the liver and kidneys and the lowest effective dose for the whole body.

Discussion
A simple, efficient, and reproducible kit-based radiolabeling process is essential for the clinical application of 99m Tc-radiolabeled radiopharmaceuticals.In this study, a kit formulation was developed for the routine preparation of [ 99m Tc]Tc-HYNIC-WL12-tricine/M (M = TPPTS, PDA, ISONIC, and 4-PSA).During the process of optimizing the kit formulation, a high radiolabeling yield (>97%) of the radiotracers could be obtained with 5 µg of the cold peptide HYNIC-WL12 (the lowest amount tested, Figure S5).However, significant glass surface absorption of these radiotracers was also observed under these low levels of cold peptide.After all the [ 99m Tc]Tc-HYNIC-WL12-tricine/TPPTS solution was removed from the common glass vial, more than 80% of the radioactivity remained on the glass wall.The glass-surface absorption of the radiotracer could be effectively reduced by utilizing silanized glass vials and adding more HYNIC-WL12 peptide as a carrier.However, the addition of cold ligands decreased the molar activity of radiotracers.Generally, high molar radioactivity is needed for receptor-targeted probes due to the limited binding sites and low concentration of biomarkers (usually at the nanomolar level).To evaluate

Discussion
A simple, efficient, and reproducible kit-based radiolabeling process is essential for the clinical application of 99m Tc-radiolabeled radiopharmaceuticals.In this study, a kit formulation was developed for the routine preparation of [ 99m Tc]Tc-HYNIC-WL12-tricine/M (M = TPPTS, PDA, ISONIC, and 4-PSA).During the process of optimizing the kit formulation, a high radiolabeling yield (>97%) of the radiotracers could be obtained with 5 µg of the cold peptide HYNIC-WL12 (the lowest amount tested, Figure S5).However, significant glass surface absorption of these radiotracers was also observed under these low levels of cold peptide.After all the [ 99m Tc]Tc-HYNIC-WL12-tricine/TPPTS solution was removed from the common glass vial, more than 80% of the radioactivity remained on the glass wall.The glass-surface absorption of the radiotracer could be effectively reduced by utilizing silanized glass vials and adding more HYNIC-WL12 peptide as a carrier.However, the addition of cold ligands decreased the molar activity of radiotracers.Generally, high molar radioactivity is needed for receptor-targeted probes due to the limited binding sites and low concentration of biomarkers (usually at the nanomolar level).To evaluate the impact of excessive cold HYNIC-WL12 ligand, a comparative biodistribution experiment was conducted between [ 99m Tc]Tc-HYNIC-WL12-tricine/TPPTS injection with or without excess cold ligand.The results showed that both tumor uptake and tumor-to-background ratios were significantly reduced when excess cold HYNIC-WL12 was removed by further HPLC purification.This result suggested that excessive mass of cold HYNIC-WL12 exerts a positive effect on [ 99m Tc]Tc-HYNIC-WL12-tricine/TPPTS sensitivity in PD-L1-positive tumors.As shown in the biodistribution data of [ 99m Tc]Tc-HYNIC-WL12-tricine/TPPTS with different molar activities (Max: >3 TBq/µmol by HPLC purification, Min: 1.5 GBq/µmol), tumor uptake exhibited a bell-shaped trend with decreasing molar activity.A similar phenomenon was also found for several reported peptide-based radiotracers [49][50][51] and PD-L1-targeted radiolabeled antibodies [52,53].This was probably because cold HYNIC-WL12 could occupy nonspecific or PD-L1 binding sites in nontarget tissues [8,54], allowing more "free state" radiotracers to accumulate in tumors with high PD-L1 expression.We concluded that a radiotracer with an A m ranging from 100.5 GBq/µmol to 300 GBq/µmol yielded the best tumor uptake and tumor-to-background contrast.At the typical radiopharmaceutical dose (740-1110 MBq), a kit containing 15 µg of HYNIC-WL12 peptide in a silanized glass vial would be suitable for routine clinical 99m Tc radiolabeling.
The results of the IC 50 determination (as shown in Table S1) displayed that the introduction of the HYNIC moiety in the -Orn of WL12 has little influence on the affinity of HYNIC-WL12 for the PD-L1 protein.The results of in vitro cellular assays further demonstrated that four 99m Tc-labeled HYNIC-WL12 radiotracers bind to tumor cells in a PD-L1 expression-dependent manner.The cellular uptake of the four radiotracers in MC38-B7H1 cells (PD-L1-positive) was approximately 2.38-6.73-foldhigher than that in MC38 cells (PD-L1-negative), which could also be significantly blocked by the addition of the WL12 peptide (p < 0.01).The uptake of [ 99m Tc]Tc-HYNIC-WL12-tricine/M (M = TPPTS or PDA or ISONIC or 4-PSA) in MC38-B7H1 tumors was 18.22 ± 4.57, 4.61 ± 1.32, 6.63 ± 0.80, and 6.96 ± 1.15%ID/g at 2 h p.i., respectively, which was 3.11-6.93-foldgreater than that in MC38 tumors at the same time points (Figure 3, 2.63 ± 0.98, 1.48 ± 0.55, 1.70 ± 0.27, and 1.22 ± 0.19%ID/g, respectively).The difference in radioactive uptake between the two tumor models was consistent with the IHC staining results, in which PD-L1 expression in the MC38-B7H1 tumors was higher than that in the MC38 tumors (Figure S7).In addition, radioactive accumulation in MC38-B7H1 tumors was reduced by approximately 76.44-89.44% in the blocking group (Figure 3, p < 0.01).These results suggested that the tumor uptake of [ 99m Tc]Tc-HYNIC-WL12-tricine/M was PD-L1-specific and associated with the expression level of PD-L1.

General Information
99m Tc was obtained from a 99 Mo/ 99m Tc generator (Guangzhou Diqi Trading Co., Ltd., Guangzhou, China) and eluted with saline.All chemical reagents and solvents performed using the Student's t-test for unpaired data to determine the significance of differences.Differences at the 95% confidence level (p < 0.05) were considered statistically significant.

Funding:
This work was supported by the National Natural Science Foundation of China [21976019] and the National HighLevel Hospital Clinical Research Funding [2023-PUMCH-E-007].Institutional Review Board Statement: Animal studies were carried out in accordance with the principles of laboratory animal care and the guidelines of the Ethics Committee of Beijing Normal University (permit no.BNUCC-EAW-2023-002). Informed Consent Statement: Not applicable.Data Availability Statement: Data are contained within the article.