WO2025105948A1 - [99mtc]tc-ipd-l1 as a tetradecapeptide radiopharmaceutical for detecting and monitoring the overexpression of the pd-l1 protein - Google Patents

Abstract

The present invention relates to a cyclic tetradecapeptide containing a hydrazine group in the side chain of the lysine for use as a novel PD-L1 protein inhibitor (iPD-L1). The hydrazine as a side chain in the macrocycle of the tetradecapeptide to be patented, referred to as iPD-L1 [cyclo(Trp-Ser-Trp-Leu-Leu-Lys(hydrazine-nicotinoyl)-Cys-Tyr-Ala-Asn-Pro-His-Leu-Pro)], generates a simultaneous dual interaction (ionic/salt bridge interaction and hydrogen bond) between the carboxylate ion (-COO-) of the Glu-58 and an ammonium ion (-NH_{3 +}) of the hydrazine present in the iPD-L1, the hydrogen of the - NH_{3 +} forming strong hydrogen bonds with the oxygen of -COO-. Said interaction allows a rigid structure to be obtained with sufficient anchorage to and recognition of the PD-L1 ligand. The iPD-L1 ligand (-6.7 kcal/mol, AutoDock affinity) labelled with ^99mTc ([^99mTc]Tc-iPD-L1) is obtained with >95% radiochemical purity and is capable of detecting the PD-L1 protein in vivo by means of single photon emission computerised tomography (SPECT). The purpose of this invention is to provide a new specific SPECT radiopharmaceutical (molecular target radiopharmaceutical) with high sensitivity for the detection of tumours with PD-L1 protein overexpression.

Description

[ ^99m Tc]Tc-iPD-L1 as a tetradecapeptide radiopharmaceutical for the detection and monitoring of PD-L1 protein overexpression

DESCRIPTION

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a cyclic tetradecapeptide containing a hydrazine group on the lysine side chain for use as a novel inhibitor of the PD-L1 protein (PD-L1), which, upon labeling with ^99mTc ( ^99mTc -¡PD-L1), functions as a novel radiopharmaceutical for imaging PD-L1 expression. The ¡PD-L1 ligand (-6.7 kcal/mol, AutoDock affinity) labeled with " ^mTc is obtained with a radiochemical purity >95%. The radiopharmaceutical [ ^99mTc ]Tc-¡PD-L1 can detect PD-L1 positive tumor lesions ¡n vivo in patients undergoing whole-body scanning by single-photon emission computed tomography (SPECT) in molecular nuclear medicine.

BACKGROUND

Immune checkpoint inhibitor (ICI) therapy has emerged as an important therapeutic option for the treatment of various cancer types [1-5]. Among immune checkpoints, programmed death receptor 1 (PD-1) and its ligand (PD-L1) are the key molecular targets for ICI treatment. PD-L1 has been shown to be a reproducible biomarker. It can be used to guide therapeutic decisions and monitor response [1]. However, PD-L1 expression is not only heterogeneous between and within tumor lesions, but is also highly dynamic, with accelerated changes in relatively short times. Therefore, the detection of PD-L1 by immunohistochemical techniques should be complemented by other diagnostic modalities. On the other hand, molecular imaging techniques such as single photon emission computed tomography (SPECT) and positron emission tomography (PET) have the advantage of obtaining noninvasive, in vivo images of the whole body, with high sensitivity, adequate spatial resolution and image acquisition times in minutes. Therefore, the heterogeneous expression of the PD-L1 gene among all tumor lesions of patients over time can be visualized [6-14]. The preclinical development of PD-L1 inhibitory radiopharmaceuticals for SPECT and PET is based on three different types of molecules: antibodies, peptides, and small non-peptide molecules [6]. At preclinical and clinical levels, a high tissue accumulation of various types of radiolabeled antibodies (minibodies, aphibiods, and nanobodies) has been observed [7,8,12-14]. Furthermore, these molecules have a relatively high degree of immunogenicity and can even induce potentially adverse immunological effects, such as cytokine storms [6]. In contrast, small peptide and non-peptide molecules accumulate more rapidly in tumor tissue, allowing for same-day imaging of patients [16-19]. Furthermore, the elimination of radiopharmaceuticals is rapid and occurs within a favorable timeframe of minutes to hours. They also have the advantage of being manufactured following Good Manufacturing Practices (GMP) protocols. Furthermore, small molecules have the advantage of high tissue penetration and cell membrane permeability to reach defined molecular targets [16-19], In recent years, several important peptide- and small-molecule-based inhibitors have been developed for PD-1/PD-L1 blockade [6,10,11,13,16-19], However, only one peptide has been used in humans for PET imaging [15] and none for SPECT imaging.

In 2022, the first human PET image of PD-L1 expression in lung cancer patients was published, with excellent uptake and specificity [15], The peptide used was a cyclopeptide (14 amino acids) called WL12 (peptide containing a thiol within a macrocycle and patented by Bristol-Myers Squibb Company, US Pat. No. 9,879,046) [9], with four mediated amino acids [Trp(Me), NMeAla and two NMeNle] and a side chain of the Orn residue conjugated to ⁶⁸ Ga-NOTA for PET imaging.

Our invention is based on the use of hydrazine (pyridine-hydrazine) outside of a peptide macrocycle, different from WL12 as it does not contain an -S- group in the macrocycle, the fundamental basis of the Bristol-Myers Squibb Company, US patent (Pat. No. 9,879,046). Hydrazine as a side chain in the peptide macrocycle to be patented (PD-L1), generates a simultaneous double interaction (ionic interaction/salt bridge and hydrogen bond) between the carboxylate ion (-COO-) of Glu-58 and an ammonium ion (- NHs ⁺ ) of the hydrazine present in the PD-L1, where the hydrogen of the -NHs ⁺ forms strong hydrogen bonds with the oxygen of -COO. It is common knowledge that the stabilization energy due to ionic interactions (salt bridge) is many times higher than that of hydrogen bonding interactions, which add up to a simultaneous interaction such as that generated in the PD-L1 molecule (Figure 1). This interaction allowed obtaining a more rigid structure with anchoring and recognition. sufficient to the PD-L1 ligand without having to use, in the new peptide macrocycle ¡PD- L1 to be patented, methylation sites in the amino acids, or -S- bonds as in the cyclic chain, as occurs in WL12. In addition, Orn was replaced by Lys to obtain a longer side chain that would not interfere with the recognition site, thus improving the affinity of the PD-L1 inhibitory peptide ¡PD-L1 ) (Table 1). Even after ^99m Tc-HYNIC conjugation (SPECT image), the advantage of hydrazine is its double amino group, so the interaction of the non-terminal amino with Glu-58 (PD-L1 ) exists given the proximity to Glu-58 (4.88 nm) (Figure 1 ).

Table 1. Affinity and inhibition constant of macrocyclic peptide derivatives determined by molecular docking (AutoDock).

Peptide Inhibitory Binding Site Affinity Constant (kcal/mol)

(pM)

Lys25, Lys41, Leu94, Leu27, ¡PD-L1 -

Phe42, Val23. Glu39, Lys124, macrocycle no -6.7 12.26

Pro24, Thr22, Val44 conjugate

Gln107, Arg125, Alai 32, hydrazine- Leu106, Valí 11, lie 126, Glu60, nicotinoyl-iPD- -7.2 5.27 Alai 09, Met5, Thr127, Arg1 13,

L1 (PD-L1 ) Lys129, Tyr1 12

WL12- -5.1 82.51 Ser34, Glu39, Glu31, Asn35 non-conjugated macrocycle hydrazine-

Leu94, Leu88, Glu31, Gln100, nicotinoyl - -6.0 39.95

Thr102, Ser34

WL12 References

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Gabrielson M, Pomper MG, Gabelli SB, Nimmagadda S. Rapid PD-L1 detection in tumors with PET using a highly specific peptide. Biochem Biophys Res Commun. 2017 Jan 29;483(1):258-263.

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Rao, K.; Mease, R.; Dannals, R.F.; Pomper, M.G.; Nimmagadda, S. Peptide-based 68Ga-PET radiotracer for imaging PD-L1 expression in cancer. Molecular Pharmaceutics 2018, 15, 3946-3952.

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DETAILED DESCRIPTION OF THE INVENTION

For the purposes of the invention, a cyclic tetradecapeptide containing a hydrazine group on the lysine side chain is presented for use as a novel inhibitor of the PD-L1 protein (PD-L1), which, after labeling with ^mTc ( ^99mTc -¡PD-L1), functions as a novel radiopharmaceutical for imaging PD-L1 expression. The ¡PD-L1 ligand (-6.7 kcal/mol, AutoDock affinity) labeled with ^99mTc is obtained with a radiochemical purity >90%. The radiopharmaceutical [ ^99mTc ]Tc-¡PD-L1 can detect PD-L1-positive tumor lesions in vivo in patients undergoing whole-body scanning by single-photon emission computed tomography (SPECT) in molecular nuclear medicine.

Our invention is based on the use of the hydrazine group (-NH2-NH2) outside a peptide macrocycle, different from WL12 [cyclo(AcTyr-MeAla-Asn-Pro-His-Leu-Hyp-Trp- Ser-Trp(Me)-MeNle-MeNle-Orn-Cys)-Gly-NH2 (thioether bridge -S- between Cys ¹⁴ and Ac-Tyr)] by not containing a -S- group within the peptide sequence of the macrocycle, the fundamental basis of the Bristol-Myers Squibb Company, US patent (Pat. No. 9,879,046). Hydrazine as a side chain in the tetradecapeptide macrocycle to be patented called hydrazine-nicotinoyl-PD-L1 [cyclo(Trp-Ser-Trp-Leu-Leu-Lys(hydrazine-nicotinoyl)-Cys-Tyr-Ala-Asn-Pro-H¡s-Leu-Pro)] (PD-L1 ), generates a simultaneous double interaction (ionic interaction/salt bridge and hydrogen bond) between the carboxylate ion (-COO-) of Glu-58 and an ammonium ion (-NHs ⁺ ) of the hydrazine present in the ¡PD-L1 , where the hydrogen of the -NHs ⁺ forms strong hydrogen bonds with the oxygen of -COO . It is well known that the stabilization energy due to ionic interactions (salt bridge) is many times higher than that of hydrogen bonding interactions, which add up in a simultaneous interaction such as that generated in the PD-L1 molecule (Figure 1). This interaction made it possible to obtain a more rigid structure with sufficient anchoring and recognition to the PD-L1 ligand without having to use, in the new PD-L1 peptide macrocycle to be patented, mediation sites in the amino acids, or -S- bonds as in the cyclic chain, as occurs in WL12. In addition, Lys provides a long side chain that does not interfere with the recognition site, thus improving the affinity of the PD-L1 inhibitory peptide (PD-L1) with respect to WL-12 (Table 1). Even after ^99m Tc-HYNIC conjugation (SPECT image), the advantage of hydrazine is its double amino group, so the interaction of the non-terminal amino with Glu-58 (PD-L1) exists given the proximity to Glu-58 (4.88 nm) (Figure 1). Method of design and preparation of the radiopeptide of the invention

AutoDock software was used to perform molecular docking for affinity calculations. The chemical synthesis was based on the 6-hydroxybenzoic acid coupling reaction. hydrazinylpyridine-3-carboxylic acid with a 14 amino acid cyclic peptide. ¡PD-L1 was prepared for labeling with ^99m Tc. Radio-HPLC was used to verify radiochemical purity. The stability of the radiopeptide in human serum was assessed by HPLC. The affinity of [ ^99m Tc]Tc-¡PD-L1 was evaluated by radio-SDS-PAGE. Cellular uptake of [ ^99m Tc]Tc-¡PD-L1 in PD-L1 positive cells (HCC827 and HCT116) and biodistribution in tumor-induced mice were also performed. A patient with advanced plantar malignant melanoma received [ ^99m Tc]Tc-¡PD-L1 and underwent a whole-body scan to differentiate between PD-L1-positive (with [ ^99m Tc]Tc-¡PD- L1 uptake) tumor lesions (visualized by their high metabolic activity with [ ¹⁸ F]FDG) and PD-L1-negative (without [ ^99m Tc]Tc-¡PD-L1 uptake) tumor lesions. Molecular docking (Docking)

The unconjugated PD-L1 ligands and hydrazine-nicotinoyl-iPD-L1 , as well as WL12 (positive control) and hydrazine-nicotinoyl-WL12 (positive control), were modeled in two dimensions (2D) using ChemDraw to obtain the basic structures. The three-dimensional structure of each ligand was exported in mol2 format using Chem3D to optimize the molecular geometry using the MMFF94 force field via the OpenBabel chemistry toolbox. Human programmed death-1 protein was used as the receptor and its ligand (PD-L1 ) obtained from the protein database (PDB ID: 4ZQK), which was edited to remove water molecules, ions, and structures unrelated to PD-L1 in the X-ray crystallography process. For virtual screening, each of the structures shown in Figure 1 was used as the ligand.

The receptor as a macromolecule and the corresponding ligands were prepared using the graphical package AutoDock Tools 1.5.7, with the search box cubically fixed 80 Á on the x, y, and z axes, centered on the macromolecule. Semi-rigid docking was performed with AutoDock vina 1.1.2 around the entire receptor surface, generating 20 poses for each ligand. Finally, the inhibition constants were generated (Table 1).

Synthesis and chemical characterization

Succinimidyl 6-Boc-hydrazinopyridine-3-carboxylate (NHS-HYNIC-Boc) was purchased from Synchem UG & Co (Felsberg, Germany). Cyclo(Trp-Ser-Trp-Leu-Leu-Lys-Cys-Tyr-Ala- Asn-Pro-His-Leu-Pro) (unconjugated PD-L1) was ordered to be custom synthesized according to our design and exclusively for our laboratory by Shanghai Yaxian Chemical Co, Ltd (Jiading, Shanghai, China). All other reagents were purchased from Merck (Burlington, MA, USA).

The hydrazine-nicotinoyl-iPD-L1 peptide (Figure 2) (6 mg; 3.5 pmol) was dissolved in 0.5 mL of dimethylformamide (DMF), followed by the addition of N,N-diisopropylethylamine (20 pL). NHS-HYNIC-Boc (2 mg; 5.7 pmol) was dissolved in 100 µl of DMF and added to the peptide solution for 24 h at room temperature. Unreacted NHS-HYNIC-Boc was removed from the reaction mixture by dialysis (Tube-O-DIALYZER™ mini dialysis system, 1 kDa MWCO; Merck; Burlington, MA, USA) for 24 h using injectable water. To the resulting cloudy aqueous solution (0.5 mL), 1 mL of trifluoroacetic acid (TFA) was added to deprotect HYNIC (Boc removal), and the mixture was kept at room temperature for 2 h. The dialysis process was then repeated. Acetonitrile was added to the obtained cloudy solution in a final ratio of 40:60 acetonitrile:water to completely dissolve the conjugate. Finally, the conjugate was purified by HPLC (Discovery® C18 HPLC column, 5 pm particle size, L x Dl 25 cm x 10 mm) (Merck; Burlington, MA, USA) using a linear gradient of 0.1% TFA-water/0.1% TFA-acetonitrile from 100 to 20% of the aqueous phase in 20 min at a flow rate of 4 mL/min. The fraction collected between 13.5 and 15 min was lyophilized.

The resulting powder was analyzed by FT-IR vibrational spectroscopy (400-4000 cm-1 , 50 scans at 0.4 cm-1 ; FT-IR 660 spectrometer, Agilent Technologies). The peptide hydrazine-nicotinoyl-iPD-L1 dissolved in 40:60 ethane-water was used for characterization by UPLC mass spectroscopy (ADQUITY UPLC H-Class with QDa mass detector; Waters Corporation, Milford, USA) and UV-vis spectroscopy in the range of 200-400 nm (PerkinElmer LambdaBio spectrometer; Waltham, Massachusetts, USA). The IR and mass spectra showed characteristic vibrational bands and molecular masses corresponding to the peptide hydrazine-nicotinoyl-iPD-L1 (Figure 3). Radiolabeled

The hydrazine-nicotinoyl-iPD-L1 peptide was dissolved in a 1:1 ethanol:water solution at a concentration of 1 mg/mL. For labeling with " ^m Tc (formation of the complex [ ^99m Tc]Tc- ¡PD-L1 ), 100 pL of peptide solution, 500 pL of ethylenediamine-N,N'-diacetic acid (EDDA)/tricine solution (30 mg of EDDA in 1 .5 mL of 0.1 M NaOH / 60 mg of tricine in 1 .5 mL of 0.2 M phosphate buffer at a concentration of 1 mg / mL. 2 M at pH 7) and 500 pL of ^99m TcÜ4Na (GETEC, ININ; Mexico) with an activity of 11 10 MBq were dissolved, followed by 20 pL of SnCh (10 mg/10 pL of conc. HCl in 10 mL of water). The solution was stirred for 1 min, then incubated in a dry bath at 95°C for 30 min and allowed to cool to room temperature over 10 min. A batch of the lyophilized formulation containing EDDA, tricine, SnCls and mannitol was also prepared in an aseptic area under good manufacturing practices (GMP certified area) for reconstitution with sodium pertechnetate-99m solution. Quality control

For quality control of the [ ^99m Tc]Tc-¡PD-L1 formulation, parameters such as appearance (clear solution), pH (neutral), sterility, absence of bacterial endotoxins, and radiochemical purity (radio-HPLC; Waters Corporation, Milford, USA; 3.9 mm x 30 cm pBondapak™, C18 column; linear gradient system; solvent A: 0.1% TFA-acetonitrile and solvent B: 0.1% TFA-water, from 100 to 30% of A in 20 min) were evaluated according to the Mexican Pharmacopoeia [16], in its "General Methods of Analysis" (MGA) section. The retention time of [ ^99m Tc]Tc-¡PD-L1 was 16.5 ± 0.3 min (Figure 3), while the retention times of ^99m TcO4 ^_ and [ ^99m Tc]Tc-EDDA/tricin were 3.5 ± 0.2 min and 4.5 ± 0.2 min, respectively, confirming a radiochemical purity greater than 95% for [ ^99m Tc]Tc-¡PD-L1 (Figure 3). Stability

The stability of [ ^99m Tc]Tc-PD-L1 was confirmed by diluting radiocomplex samples in human serum (5x). Conjugates (n = 3) were incubated at 37°C, then samples were taken at 0.5, 3 and 24 h, and radiochemical purity was assessed by size exclusion radio-HPLC (ProteinPak 300SW, Waters Corporation, Milford, USA). In vitro stability studies of [ ^99m Tc]Tc-PD-L1 in human serum showed serum protein binding of 1.3 ± 0.8% at 30 min, 3.2 ± 1.3% at 3 h and 7.6 ± 1.9% at 24 h, as well as high radiochemical stability (> 90% at 24 h). Molecular recognition by the PD-L1 protein: radio-SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. The distribution of radioactivity was detected using a radio-TLC scanner (Mini-Gita, 60-150 keV, BGO-Crystal, window 25 x 2 mm, thickness 5 mm, resolution optimized for Tc-99m; RayTest, Munich, Germany) to detect the binding of [ ^99m Tc]Tc-IPD-L1 to the PD-L1 protein.

Briefly, a solution of ^99m Tc-labeled PD-L1 (100 µL) (1 mg/mL peptide) was incubated with recombinant human PD-L1 protein (100 µL) (200 pg/mL; FineTest; Cat: P6857;)(radiopeptide-PD-L1 interaction) or human integrin αVp3 protein (100 µL) (negative control; 200 pg/mL) (Chemicom, Merck; Burlington, MA, USA) (radiopeptide-integrin interaction) for 30 min at 37°C, after which the samples were diluted with Laemmli sample buffer (containing 10% SDS, 125 mM Tris-Cl, pH = 6.8, 12.5% glycerol and 0.005% bromophenol blue after dye) to give final protein concentrations. of 25 pg/mL. Samples ( ^99m Tc-¡PD-L1, PD-L1 protein, integrin, ^99m Tc-¡PD-L1 protein -PD-L1 and ^99m Tc-¡PD-L1 -integrin) were loaded onto SDS-PAGE gel at room temperature. 30 pL (30 pg) of each sample were loaded into 1.5 mm thick wells of 15% polyacrylamide gels. The experiments were performed on a single gel. In all cases, electrophoresis was run until just before the bromophenol dye reached the bottom of the gel (total gel distance of 100 mm). Pre-established molecular weight standards (BLUE stainTM Protein Ladder 11-245 kDa Cat: P007-500) were also used on each gel. The gels were stained with Coomassie blue for 30 to 45 minutes at room temperature and allowed to destain for 4 to 20 hours before being photographed. Finally, the distribution of radioactivity in the gels was scanned using a radio-TLC detector to determine the percentage of [ ^99m Tc]Tc-¡PD-L1 radioactivity associated with proteins. The results showed 30% of the [ ^99m Tc]Tc-¡PD-L1 radioactivity associated with molecular recognition of PD-L1 (Figure 4).

Immunofluorescence

Cellular PD-L1 expression was confirmed by immunofluorescence. HCC827, HCT116, and C6 cell lines were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.5% TritonX-100, and blocked with 1% bovine serum albumin. Cells were then incubated overnight with an anti-PD-L1 antibody according to the supplier's instructions (FineTest, Wuhan Fine Biotech Co., China). Cells were then incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) (Invitrogen Cat. No. A32731) for 1 h. DAPI was used to observe the fluorescence intensity in cells for nuclear staining. Fluorescence was observed by microscopy (Meiji Techno; model MT6200; Saitama, Japan).

Cellular uptake and internalization

Cells diluted in phosphate-buffered saline (PBS) (pH 7.4) (1 x 106 cells/tube) received two different treatments: a) [ ^99m Tc]Tc-¡PD-L1 (7.4 kBq) (n = 3), and b) ^99m TcO4Na (7.4 kBq) (n = 3). Cells were incubated with each treatment at 37 °C for 1 h. After incubation, tubes were measured in a Nal(TI) detector (NML Inc., Houston, Texas, USA) to determine the initial activity (100%). The tubes were centrifuged at 500 g for 10 minutes, washed once with PBS, and centrifuged again. The liquid was removed, and the button activity, corresponding to the percentage of cellular uptake (surface uptake) with respect to the initial activity, was measured. A mixture of acetic acid/0.5 M NaCl was added, and centrifugation was repeated, followed by activity measurement. The liquid was removed, and the button activity, corresponding to the percentage of activity internalized by the cells with respect to the uptake activity (fraction of internalization of surface uptake), was measured. The percentage of cellular uptake of [ ^99m Tc]Tc-¡PD-L1 correlated with the expression of Cellular PD-L1 was determined by immunofluorescence in each of the cell lines analyzed. Uptake was zero for ^99m TcO4Na (negative control) in all cells analyzed.

Biodistribution

All animal procedures were performed in accordance with the Ethical Regulations for the Management of Laboratory Animals (NOM-062-ZOO-1999) and the requirements of the Institutional Committee for the Care and Use of Animals under an approved protocol (No. 09-2018-2022). Male Nu/Nu mice (CINVESTAV, I.PN., Mexico City), 6-8 weeks old, were maintained in an aseptic barrier environment. Mice were inoculated by subcutaneous injection of 1 x106 HCC827 cells/0.1 mL PBS into the upper back. Injection sites were periodically monitored for tumor progression. On day 10 after tumor cell inoculation, animals were injected into the tail vein with 18.5 MBq (50 pL) of [ ^99m Tc]Tc-ÍPD-L1. Animals were sacrificed at 1, 3, and 24 h (n=3). Samples of kidneys, lungs, heart, liver, spleen, pancreas, small intestine, stomach, muscle, blood, and tumor were dissected. Radioactivity was measured using a Nal(TI) detector. Results were expressed as percentage of the injected dose per gram of tissue (%ID/g), showing uptake of [ ^99m Tc]Tc-IPD-L1 in the ipD-positive tumors (Figure 5).

Clinical image

A study of a 62-year-old man (weight 60 kg; height 168 cm) diagnosed with advanced plantar malignant melanoma is shown (Ethics Committee: Protocol n ^e 2023-MN02). The study was approved based on the microdose concept and proof-of-concept studies. In addition, the ethical standards of INCan Hospital based on the Declaration of Helsinki regarding human experimentation and the GMP certificate granted to INCan Hospital by the Mexican Ministry of Health were taken into account. The patient signed an informed consent after receiving detailed information about the purpose of the study, which could help decide on the treatment and monitor disease progression. The patient underwent SPECT/CT (Symbia TruePoint, Siemens) 2 hours after intravenous administration of [ ^99m Tc]Tc-IPD-L1 (740 MBq) and had had a previous [ ¹⁸ F]FDG PET/CT scan (Excel 20; Siemens Medical Solutions) 5 days earlier. A whole-body scan was performed to differentiate between PD-L1-positive ([ ^99m Tc]Tc-IPD-L1 uptake) tumor lesions (all those visualized by their high metabolic activity with [ ¹⁸ F]FDG) and PD-L1-negative (no [ ^99m Tc]Tc-IPD-L1 uptake) tumor lesions (Figure 6). In conclusion, the [ ^99m Tc]Tc-iPD-L1 is obtained with the following characteristics:

• Radiochemical purity greater than 95%

• Ability of the radiopharmaceutical to detect in vivo and specifically tumors that overexpress the immune checkpoint protein PD-L1 by single photon emission computed tomography (SPECT) in nuclear medicine.

• In addition to the molecular recognition of the ^99mTc -labeled tetradecapeptide amino acid sequence, [ ^99mTc ]Tc-¡PD-L1 has the ability to significantly uptake and detect with high sensitivity tumors and metastases with PD-L1 expression, due to a simultaneous double interaction (ionic/salt bridge interaction and hydrogen bonding) between the carboxylate ion (-COO-) of Glu-58 and an ammonium ion (-NHs ⁺ ) of the hydrazine present in ¡PD-L1 , where the hydrogen of -NHs ⁺ forms strong hydrogen bonds with the oxygen of -COO . It is general knowledge that the stabilization energy due to ionic interactions (salt bridge) is many times higher than that of hydrogen bonding interactions, which add up to a simultaneous interaction such as that generated in the ¡PD-L1 molecule.

Claims

Having sufficiently described my invention, I consider as a novelty and therefore claim as my exclusive property, the contents of the following clauses: 1 A compound having the structure hydrazine-nicotinoyl-PD-L1 [cyclo(Trp-Ser-Trp-Leu-Leu-Lys(hydrazine-nicotinoyl)-Cys-Tyr-Ala-Asn-Pro-His-Leu-Pro)] (PD-L1):

2.- A compound having the structure claimed in claim 1 in which Lys(hydrazine-nicotinoyl) is replaced by any molecule dedicated to chelating a radionuclide. 3.- A radiopharmaceutical with the formula: [ ^99m Tc]Tc-PD-L1 having the following structure:

4.- A radiopharmaceutical as claimed in claim 3 to be used 410 in the detection of tumors expressing the PD-L1 protein.