Lutetium-177 labeled iPD-L1 as a novel immunomodulator for cancer-targeted radiotherapy

Abstract

Background

Cancer immunotherapy is a relatively new approach to cancer treatment. Peptides that target specific pathways and cells involved in immunomodulation can potentially improve the efficacy of cancer therapy. Recently, we reported iPD-L1 as a novel inhibitor peptide that specifically targets the cancer cell ligand PD-L1 (programmed death ligand 1). PD-L1 is responsible for inhibiting the immune checkpoint protein PD-1 expressed by regulatory T cells. On the other hand, anti-PD-L1 immunotherapy in combination with external beam radiotherapy has shown improved outcomes in the treatment of breast and lung cancer. The aim of this research was to prepare ¹⁷⁷Lu-labeled iPD-L1 and to preclinically evaluate its radiotherapeutic potential and role as a tumor immunomodulator by measuring macrophage activation, IL-10, TGFβ, and PD-L1 expression in 4T1 triple-negative breast cancer cells and murine 4T1 tumors after treatment with ¹⁷⁷Lu-iPD-L1.

Results

The iPD-L1 ligand, characterized by UPLC mass, UV-Vis, and FT-IR spectroscopies, showed a chemical purity of 99%. The ¹⁷⁷Lu-iPD-L1 radiochemical purity was 98.9 ± 1.1%. In vitro and in vivo studies demonstrated radiotracer stability in human serum (> 97% after 24 h evaluated by radio-HPLC), adequate affinity by the PDL1 protein (IC₅₀ = 4.21 nM), and specific detection for PD-L1 assessed in 4T1, HCT116, and AR42J cancer cells, in which PD-L1 expression was verified by immunofluorescence and Western Blot assays. After treatment with ¹⁷⁷Lu-iPD-L1 (0.4 Bq/cell), flow cytometry results showed a significant decrease in cell viability of 4T1 cells (dead 56.2%) compared to ¹⁷⁷LuCl₃ (dead 34.2%) and untreated cells (dead 9.4%). With high tumor uptake (6.97 ± 1.04%ID) and hepatobiliary and renal clearance, lutetium-177-labeled iPD-L1 delivered a tumor dose of 27 Gy/37 MBq and less than 0.36 Gy/37 MBq to non-source organs. PD-L1 positive tumors showed a significant increase in activated macrophages, PD-L1, IL-10, and TGFβ expression levels after ¹⁷⁷Lu-iPD-L1 treatment as evaluated by ELISA assay and immunohistochemistry.

Conclusions

Therefore, this study warrants further dosimetric and clinical studies to determine the immunomodulatory effect and therapeutic efficacy of ¹⁷⁷Lu-iPD-L1 in treating PD-L1-positive tumors in combination with anti-PD-1/PD-L1 immunotherapy protocols.

Introduction

It is well-established that cancer is a complex disease that shares several biological characteristics common to all oncological pathologies, most notably the capacity of cancer cells to evade the immune response (Hanahan and Weinberg 2011) (Hanahan 2022).

Genetic and epigenetic alterations provide antigens that the immune system utilizes to distinguish tumor cells from their normal counterparts (Hanahan 2022; Hanahan and Weinberg 2011). Likewise, immune checkpoint proteins (ICP) regulate the immune response, maintaining self-tolerance and protecting tissues when the immune system responds to a pathogen (He and Xu 2020). However, tumors can evade natural immunity by regulating the ICP, for example, through the cancer cell expression of the programmed death ligand 1 (PD-L1); this ligand binds to the programmed cell death protein 1 (PD-1) expressed on natural killer (NK), T, and B cells, thereby inhibiting, in this way, the ICP activation (Morad et al. 2021). Consequently, antagonists of inhibitory signals (e.g., anti-PD-L1) have been shown to enhance the antitumor immune response, representing a topic of significant research interest across a range of oncological pathologies that exhibit diverse ICP profiles (Bullock and Richmond 2024; Wang et al. 2021; Yoon et al. 2024; Zhang et al. 2022a, b). Therapies targeting ICP with specific antibodies (Cui et al. 2024; Qu et al. 2024; Quintana et al. 2024) or peptides (Bojko et al. 2024; Chen et al. 2019) against PD1 or PD-L1 have demonstrated significant clinical responses in patients with advanced-stage breast and lung cancer (Bojko et al. 2024; Bullock and Richmond 2024; Cui et al. 2024; Qu et al. 2024; Quintana et al. 2024; Zhang et al. 2022a, b).

Radiation therapy represents a fundamental aspect of the treatment of multiple solid tumors. Radiotherapy results in direct damage to the DNA of tumor cells and generates a change in the tumor microenvironment (Jagodinsky et al. 2020). This is achieved by inducing the release of tumor antigens, enhancing tumor cell immunogenicity, activating immune cells, and thus effectively activating the antitumor immune response (Wen et al. 2021). Furthermore, evidence suggests that external beam radiation therapy produces a proinflammatory environment, enhancing the expression of PD-1 in T cells and PD-L1 in tumor cells (Jagodinsky et al. 2020; M. Zhang et al. 2022a, b; Z. Zhang et al. 2023). This can result in the inactivation and suppression of the immune response, which may contribute to the development of tolerance to radiotherapy. Consequently, the combination of radiotherapy with immunotherapy using anti-PD1 or anti-PD-L1 monoclonal antibodies has been employed with the objective of maximizing the immunogenic effects of radiotherapy while minimizing its immunosuppressive effects (Theelen et al. 2020).

Given that radiotherapy induces PD1/PD-L1 upregulation, there has been an effort to immune-activate tumor cells from cancer patients with the aim of increasing the efficacy of immunotherapy (Liu et al. 2023; Theelen et al. 2020). The preliminary results of the combination treatment with lutetium-177 (¹⁷⁷Lu) and immunotherapy in prostate cancer are encouraging, suggesting the potential for this approach to be applied in clinical practice (Aggarwal et al. 2023).

In a recent publication, we described iPD-L1 as a novel peptide that specifically binds the cancer cell ligand PD-L1 (Ferro-Flores et al. 2023). The aim of this research was to prepare ¹⁷⁷Lu-labeled iPD-L1 and to preclinically evaluate its radiotherapeutic potential and role as a tumor immunomodulator by measuring macrophage activation, interleukin-10 (IL-10), transforming growth factor beta (TGFβ), and PD-L1 expression in 4T1 triple-negative breast cancer cells and tumors after treatment of mice with ¹⁷⁷Lu-iPD-L1.

Materials y methods

¹⁷⁷Lu-iPD-L1 preparation and characterization

The cyclic peptide WL12 is a PD-L1 inhibitor composed of fourteen amino acids, a –S– bond as part of the cycle, and four methylated amino acids [two NMeNle, NMeAla, and Trp(Me)] (Fig. 1) (Miller et al. 2019). The WL12 peptide has previously been conjugated to various chelators for labeling with ⁶⁴Cu, ¹⁸F, ⁶⁸Ga and ¹²³I and ^99mTc for cancer immunoimaging (Du et al. 2025; Fan et al. 2024). Our group recently reported a cyclic peptide, iPD-L1 (Fig. 1), lacking the –S– bond and without methylated amino acids, which showed high uptake and internalization in PD-L1-positive cancer cells (Ferro-Flores et al. 2023). In this research, the molecular design consisted of conjugating the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to the amino-terminal group of the Lys residue in iPD-L1 to be labeled with ¹⁷⁷Lu. For the scale-up of DOTA-iPD-L1 synthesis, support was provided by Yaxian Chemical (Zhejiang, China). DOTA-iPD-L1 was chemically characterized by molecular docking, mass spectroscopy (UPLC QDa mass detector, Waters, CT, USA), FT-Infrared (ATR 400–4000 cm^− 1; PerkinElmer, MA, USA) and UV-Vis (200–400 nm; LambdaBio PerkinElmer, MA, USA) spectroscopies, and HPLC (Shimadzu LC-40DxR, C18 reversed-phase column, Kyoto, Japan). DOTA-WL12 was also synthesized by conjugating DOTA to the Orn residue. After labeling, ¹⁷⁷Lu-DOTA-WL12 (¹⁷⁷Lu-WL12) was used as a positive control for comparison with ¹⁷⁷Lu-DOTA-iPD-L1 (¹⁷⁷Lu-iPD-L1) in all in vitro evaluations.

Fig. 1.

(a) Molecular docking of DOTA-iPD-L1 to PD-L1 (left): affinity score of − 6.1 kcal/mol and Ki = 33.75 µM; the chemical interactions of DOTA-iPD-L1 with the amino acids of the target protein PD-L1 are shown on the right (distances are shown in Å scale). (b) Molecular docking of DOTA-WL12 to PD-L1 (left): affinity score of -5.7 kcal/mol and Ki = 66.3 µM. The chemical interactions of DOTA-WL12 with the amino acids of the target protein PD-L1 are shown on the right (distances are shown in Å scale)

Docking

DOTA-iPD-L1 and DOTA-WL12 structures were created in ChemOffice and generated as 3D-pdb files, pre-optimized with an MMFF94 force field in Chem3D, and fully optimized using the semi-empirical PM7 method (Mopac2016). For molecular docking, the receptor (human programmed death-1 ligand: PDB ID: 4ZQK) and PD-L1 inhibitor peptides (DOTA-iPDL1 and DOTA-WL12) were configured using the AutoDock Tools 1.5.7 graphics (The Scripps Research Institute, CA, USA). The search box had a cubic size of 80 Å on the x, y,z axis, centered on the receptor. Semi-rigid docking was executed using AutoDock vina 1.1.2 (The Scripps Research Institute, CA, USA) over the whole surface of the receptor. Twenty poses were generated for each PD-L1 inhibitor. The inhibition constants (Ki) were calculated taking into account the affinity score, the universal gas constant (R, kcal/mol K,) and the temperature (kelvin scale) as follows:

1

Radiolabeling

DOTA-iPD-L1 or DOTA-WL12 (1.2 mg) and 160 mg of ascorbic acid (USP grade, Sigma-Aldrich, MA, USA) were dissolved in 1.2 mL of 1 M sodium acetate buffer pH 5, and sterilized by membrane filtration (0.22 μm pore size, mixed cellulose esters, Merck Millipore, Darmstadt, Germany). Next, 150 µg of the solution was mixed with 7.4 GBq of non-carrier added ¹⁷⁷LuCl₃ (EndolucinBeta 40 GBq/mL, ITM, Munich, Germany) diluted to 0.4 mL with 1 M sodium acetate buffer, pH 5, in a sterile glass vial, which was incubated in a dry bath at 95 °C for 30 min. Finally, the solution was diluted with injectable grade water according to the activity required by the protocols described below, including, in some cases, the addition of cold peptide for the blocking studies (iPD-L1 or WL12) as described below.

Radiochemical purity evaluation

High-performance liquid chromatography (HPLC) with radioactivity and UV-Vis detectors (Shimadzu LC-40DxR, Kyoto, Japan) was used to assess both the purity of the DOTA-iPD-L1 peptide and the radiochemical purity of ¹⁷⁷Lu-iPD-L1 under the following conditions: (1) stationary phase C18 column (Shim-pack GIST C18, 4.6 mm x 25 cm, 5 μm particle size; Shimadzu, Kyoto, Japan); (2) mobile phases (phase A: 0.1% CF₃COOH solution in CH₃CN; phase B: 0.1% CF₃COOH solution in H₂O); (3) flow rate 1 mL/min. The following gradient system used 100% mobile phase A from 0 to 3 min, changed to 50% A after 10 min and remained constant until 20 min, then changed to 30% A for 5 min and ended with 100% A from 25 to 30 min. The retention time of the DOTA-iPD-L1 peptide was 13.2 ± 0.2 min, while that of the ¹⁷⁷Lu-iPD-L1 radiopharmaceutical was 14.0 ± 0.2 min and that of ¹⁷⁷LuCl₃ was 4.0 ± 0.2 min. DOTA-WL12 was also labeled with lutetium-177 under the conditions described above. The chemical and radiochemical purity of DOTA-WL12 and ¹⁷⁷Lu-WL12 was also verified.

Serum stability

The serum stability of ¹⁷⁷Lu-iPD-L1 and ¹⁷⁷Lu-WL12 was determined by protein precipitation. In brief, to 800 µL of mouse serum (n = 6), 200 µL of the radiopharmaceutical was added and incubated for 24 h at 37 °C. Then 200 µL of a methanol/acetonitrile solution (1:1) was added, and the solution was centrifuged at 500 x g for 10 min. The radiochemical purity analysis of the supernatant was assessed by radio-HPLC, as described above.

In vitro biological studies

Cell culture

American Cell Type Collection (ATCC, MD, USA) 4T1 (CRL-2539; triple-negative breast cancer; murine), HCT116 (CCL-247; colorectal; human), and AR42J (CRL-1492; pancreas cancer; murine) cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Gibco, Thermo Fisher Scientific, MA, USA) in a 5% CO₂ atmosphere at 37 °C.

In vitro cell treatments

A sterile 0.25% trypsin solution in PBS (Gibco, Life Technologies, NY, USA) was used to detach the cells from the culture bottle to obtain cells in suspension. Cell viability was verified to be greater than 85% by trypan blue exclusion assay, which also allowed the determination of the number of cells in one milliliter of cell suspension. Subsequently, cells were suspended in the RPMI medium. For viability, clonogenicity, and protein expression assays, 1 × 10⁶ cells suspended in RPMI medium were incubated with ¹⁷⁷LuCl₃ (0.4 MBq), PD-L1 (25 µg), WL12 (25 µg), ¹⁷⁷Lu-PD-L1 (0.4 MBq/25 µg), or ¹⁷⁷Lu-WL12 (0.4 MBq/25 µg) for one hour before washing with PBS to remove excess treatment and to determine the different biological effects.

Affinity assay

A competition assay determined the affinity of iPD-L1 (DOTA- iPD-L1) and WL12 (DOTA-WL12) in the 4T1 cell line (PD-L1 positive). Cells adhered in 96-well plates were incubated at 37 °C for 1 h with different concentrations (eight dilutions ranging from 10,000 to 0.01 nM) of unlabeled iPD-L1 or WL12 peptides (n = 3). The concentration of ¹⁷⁷Lu-iPD-L1 or ¹⁷⁷Lu-WL12 (20 kBq/1 ng/50 µL) was kept constant. Plates were washed with buffer containing 0.1% BSA, 1 mM CaCl₂, 25 mM Tris-HCl, and 120 mM NaCl, pH 7.4. The activity (% of binding) was measured in each well using a NaI(Tl) detector (NLM, TX, USA). Binding percentages for each well were corrected using the unspecific binding values obtained from a curve of an independent assay using ¹⁷⁷Lu-DOTA (20 kBq/1 ng/50 µL) and different concentrations of cold peptide under the same conditions described above. Using GraphPad Prism software (v. 10.4.0), the percentage uptake in each well relative to the initial activity was fitted as a competition curve to obtain the IC₅₀.

Uptake and internalization assay

Microtubes were prepared with 1 × 10⁵ cells (4T1, HCT116, or AR42J) (n = 6), of which the blocked cells were treated with the corresponding cold peptide (iPD-L1 or WL12, 25 µg/25 µL/tube) for 1 h before the addition of treatments (¹⁷⁷Lu-iPD-L1 or ¹⁷⁷Lu-WL12, 370 kBq). After ¹⁷⁷Lu-radiopharmaceutical treatments were added (blocked and unblocked cells), the microtubes were incubated for one hour at room temperature. At the end of the incubation period, the radioactivity present in the cell suspension (initial activity) was measured. After removing excess treatment by centrifugation at 500 x g for 5 min, the remaining activity in the cell pellet was resuspended in 1 mL of PBS, and the radioactivity was measured (cellular uptake fraction). Cells were centrifuged, and the pellet was treated with 1 mL of 0.2 M acetic acid solution in 0.5 M NaCl, followed by centrifugation and decantation. Finally, the bottom radioactivity, which corresponded to the internalized fraction, was measured. Radioactivity measurements were performed by triplicate in a well counter NaI(Tl) detector. Percent uptake was calculated by considering the initial activity as 100%.

Immunofluorescence assay

Into immunodetection chambers (chamber slide system, Nunc Merck, Darmstadt, Germany), 1.5 × 10⁴ cells (4T1, HCT116, and AR42J) were seeded for 24 h under standard culture conditions. Once adhered, the cells were fixed with 4% paraformaldehyde solution in PBS for 30 min, washed and blocked with 2% BSA solution containing 0.5% Triton x-100 for 30 min before incubation with 3 µg/mL anti-PD-L1 antibody (PD-L1 antibody - BSA-free; Novus Biologicals, Wuhan Fine Biotech., Wuhan, China) overnight at 4°C. For protein localization, goat anti-mouse secondary antibody was used and then conjugated with secondary antibody (A32731, goat anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor™ Plus 488; Invitrogen Thermo Scientific, MA, USA) at 1:1000 dilution and incubated for 1 h at room temperature. After, the slides were allowed to dry and mounted using Vectashield with DAPI (4’,6-diamidino-2-phenylindole) (Vector Laboratories, CA, USA). The slides were then visualized with a 40X objective on an MT-6200 microscope (Meiji Techno., Saitama, Japan) to verify the expression and subcellular localization of the PD-L1 molecular target by the cell lines studied.

Evaluation of protein expression by western blot

The concentration of protein in total lysates obtained from semi-confluent cultures with RIPA buffer (89901; Pierce, Pierce, Thermo Scientific, MA, USA) containing protease inhibitors (A32953; Pierce, Pierce, Thermo Scientific, MA, USA) was determined using a Epoch microplate spectrophotometer (Thermo Scientific, MA, USA). Before electrophoretic separation, samples were mixed in a ratio of 1:1 with 2x Laemlli sample buffer (1610737; Bio-Rad Laboratories, CA, USA) and incubated in a dry bath (IVYX Scientific, WA, USA) at 95 °C for 5 min before loading on a 4–15% polyacrylamide gel (4561083; mini-PROTEAN TGX-10 well comb, Bio-Rad Laboratories, CA, USA) mounted in a mini Protean II run chamber (Bio-Rad Laboratories, CA, USA) using freshly prepared 1X run buffer (161–0732; 10x Tris/Glycine/SDS buffer, Bio-Rad Laboratories, CA, USA) while maintaining a voltage of 100 –80 V for approximately 2 h generated by the Consort E122 power supply (ALYS Technologies, Aclens, Switzerland). To have a reference for the weight of each protein, a protein ladder (10 µL) with a size range of 10–250 kDa (1610373; Precision Plus Protein™ All Blue Prestained Protein Standards, Bio-Rad Laboratories, CA, USA) was also loaded. Proteins were transferred to a 0.45 μm pore PVDF Immobilon-P transfer membrane (Merck Millipore, DUB, IRL) by applying 100 V current for 1 h. The membrane was washed with PBS-Tween buffer (PBST) and prepared with PBS-TWEEN tablets (Calbiochem, EMD Biosciences, CA, USA). The membrane was blocked with 4% bovine albumin fraction V (BSA) in PBST and then incubated overnight at 4 °C with the primary antibody anti-PD-L1 antibody [CAL10] - mouse IgG1 at a concentration of 1:500 in 5% BSA (ab279292; Abcam Limited, CA, USA) followed by the secondary antibody goat anti-mouse IgG H&L coupled to peroxidase at a dilution of 1:5000 in PBST for 1 h at room temperature. To visualize the bands, the membrane was treated with 1 mL of chemiluminescent HRP Substrate (WBKLS0500; Millipore Corp., MA, USA). The bands were then developed in the darkroom using a Medical X-ray Blue/MXB Film radiographic plaque, GBX developer, and fixer solutions (8225526, 5158597, 5158605; Carestream Health, NY, USA). The membrane was then washed with PBST and stripped with a solution containing 1.5% glycine, 0.5% SDS, 1% Tween-20 at pH 4.0 before incubation with the antibody against β-actin (A3854, anti-β-actin peroxidase antibody, mouse monoclonal clone AC-15, purified from hybridoma cell culture, Sigma Aldrich. Hamburg, Germany) used as loading control.

Evaluation of cytotoxicity by flow cytometry

The number of live and dead cells (cellular viability evaluation) in 4T1 cell cultures treated with ¹⁷⁷LuCl₃ or ¹⁷⁷Lu-iPD-L1 (0.4 Bq/cell was incubated for 30 min and then washed with PBS) (n = 3) was determined at 24 h, 48 h, and 72 h by flow cytometry (Muse Cell Analyzer, Merck KGaA, Darmstadt, Germany) (n = 1000 cells/measurement). Cells in a monodisperse suspension (50 µL) were treated with 450 µL of the Muse™ Count & Viability Reagent (MCH100102, Merck Millipore, Darmstadt, Germany) according to the manufacturer’s instructions. For analysis, the detection window was previously established using a healthy group of live 4T1 cells and a group of dead 4T1 cells treated with hydrogen peroxide (0.5 mM for 30 min). Induction of cell death was verified in all cases by visual inspection under a microscope using the trypan blue exclusion assay.

Clonogenic assay

4T1 cells (1 × 10⁶) in culture medium suspension were treated with 0.4 MBq of ¹⁷⁷LuCl₃, ¹⁷⁷Lu-iPD-L1 (added with 25 µg of iPD-L1), or ¹⁷⁷Lu-WL12 (added with 25 µg of WL12) or with 25 µg of PD-L1 or WL12 peptide for 1 h (n = 3). Excess treatment was removed before plating 3 × 10³ cells in a six-well plate containing 2 mL of fresh RPMI medium. The cells were incubated for 7 days until macroscopic colony formation was observed. The medium was removed, and the colonies were fixed with 4.5% paraformaldehyde in PBS for 30 min. The paraformaldehyde was removed and washed off, and the plate was allowed to dry before 5% crystal violet in water was added for 30 min. The crystal violet was collected and washed with distilled water to remove excess dye. The colonies were then photographed, and the number of colonies in each condition was determined using the free Image J software. An average of three independent experiments was used to calculate the percentage of clonogenicity.

Determination of cytokines by ELISA assay

To determine the levels of IL-10 and TGFβ secreted by 4T1 cells, 5 × 10³ colonies were seeded in each condition. 5 × 10³ 4T1 cells were seeded in 96-well plates in serum-free media for 24 h before treatment with iPD-L1 or WL12 (25 µg) or ¹⁷⁷Lu-iPD-L1 or ¹⁷⁷Lu-WL12 (25 µg/0.4 MBq) for 1 h. Cultures were incubated for five days before supernatant (SN) collection; SN samples were kept on ice and centrifuged at 2000 x g 4 °C for 7 min to remove cell debris.

SN samples used for TGFβ detection (20 µL) were mixed with 180 µL buffer assay (BA) and 20 µL of 1 M HCl and then incubated for 1 h at RT; finally, 20 µL of 1 M NaOH was added to neutralize the reaction.

Standard curves were generated for both cases. In the case of IL-10, the preloaded standard curve (39-2500 pg/mL) was hydrated with 100 µL of distilled water. For the TGFβ standard curve, the standard was dissolved in water, and after washing the required wells with Washing Buffer (WB), 100 µL of BA was added to each well, and 100 µL of TGFβ standard was added to the first well. The contents of these wells were gently mixed, and sequential dilutions were made in subsequent wells.

Samples (40 µL) were added to the remaining TGFβ assay wells to make up to 100 µL. The wells were incubated for 2 h at room temperature with moderate shaking in the dark. At the end of the incubation, the wells were washed five times. After drying, 100 µL of biotin was added and incubated for 1 h at room temperature with shaking, followed by incubation with 100 µL of streptavidin for 30 min. After washing the wells, 100 µL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added and incubated at room temperature for at least 30 min in the dark until the absorbance at 620 nm of the peak of the standard curve was between 0.9 and 0.95. The reaction was stopped by adding 100 µL of Stop Solution (SS) and reading the absorbance at 450 nm.

For samples used to determine IL-10, 50 µL were collected and added to wells containing 100 µL of distilled water and incubated for 3 h at room temperature with gentle rocking. After washing the wells, 100 µL of TMB substrate was added and incubated at RT for at least 1 h. The reaction was stopped by adding 100 µL of SS, and the absorbance was read at 450 nm.

The mean value of the readings was obtained, and standard curves were constructed for both analytes to estimate the concentration of cytokines in the samples, taking into account the dilution factors for each assay of 1:2 for IL-10 and 1:30 for TGFβ.

In vivo biological studies

Tumor induction in mice

Tumor formation was induced by subcutaneous injection of 1 × 10⁶ 4T1 cells in 6-week-old female Balb-c mice, obtained from the Laboratory Animal Production and Experimentation Unit (UPEAL) of CINVESTAV and treated as described in protocol No. 12-2018-2022, approved by the Internal Committee for Laboratory Animal Care of ININ (CICUAL-ININ) under the official standard (NOM-062-ZOO-1999). Tumor growth was monitored by visual inspection, and when tumors reached 0.4–0.5 cm in diameter (0.03–0.07 g, considering a tumor density of 1 g/cm³), they were treated for the biological experiments described below.

Biodistribution

Mice with induced tumors were injected with 3.7 MBq/100 µL of ¹⁷⁷Lu-iPD-L1 (0.02 µg/MBq) into the tail vein and sacrificed at 1, 4, 24, 24, 72, and 96 h (n = 3) after radiopharmaceutical administration. Whole organs (heart, lung, liver, spleen, kidney, intestine, and Tumor) were removed, rinsed with isotonic saline solution, placed on absorbent gauze to remove excess fluid, and finally placed in glass vials for radioactivity quantification using a thallium-activated sodium iodide detector (NaI(Tl)). The percentage of activity in each organ (%ID/organ) was calculated with respect to a standard containing an aliquot of the initial activity administered to each mouse, which represented 100% of the injected activity. One group of mice (n = 3) was initially inoculated with 100 µL (1 mg/mL) of non-radiolabeled iPD-L1 and 30 min later injected with 3.7 MBq/100 µL of ¹⁷⁷Lu-iPD-L1. Next, mice were sacrificed 4 h after radiopharmaceutical administration, and each organ’s radioactivity percentage was assessed as described above. The %ID/tumor of this latter group was considered the blocked receptor tumor group to help determine if the uptake observed in the tumors of the unblocked mice was specific.

Treatments in mice

Mice with induced tumors were divided into four groups (n = 3) and treated as follows: (Group 1) 37 MBq/100 µL ¹⁷⁷Lu-iPD-L1 (0.02 µg/MBq) followed 15 min after injection of 25 µL (1 mg/mL) non-radioactive iPD-L1 (1.25 mg/kg); (Group 2) 37 MBq/100 µL ¹⁷⁷LuCl₃; (Group 3) 25 µL (1 mg/mL) non-radioactive iPD-L1; (Group 4) 25 µL (1 mg/mL) non-radioactive WL12. A fifth group of mice (n = 3) received no treatment and served as the control group. Seven days after acute treatment (37 MBq of ¹⁷⁷Lu-iPD-L1 to produce a therapeutic tumor dose between 20 and 30 Gy in agreement with the biodistribution data), the mice were sacrificed, and the tumors and spleens were removed. The tumor of each mouse was divided into two parts, one to be used for immunohistochemistry studies and the other part and spleen to be used for TGFβ and IL-10 evaluation, as described below.

To calculate the absorbed dose at 7 days post-administration of ¹⁷⁷Lu-iPD-L1 administration, %ID/organ versus time curves were constructed from the biodistribution data at 5 distinct time points to obtain biexponential biokinetic models for each organ (activity as a function of time: A(t)). By integrating A(t) over 7 days, the number of nuclear transformations (N) that occurred in the organs and tumors by the administered activity was calculated as follows (Eq. 2):

2

The dose factors (DF) (Gy/MBq-s) for Lu-177 in the organs were obtained for the murine models reported in the OLINDA 2.0 software. Finally, the average absorbed radiation dose (D)(Gy) to organs and tumors was calculated as follows (Eq. 3):

3

The sphere model included in the OLINDA software was used to calculate the tumor radiation dose.

Immunohistochemistry

Tumor tissue was washed with cold PBS and stored in 4.5% paraformaldehyde for one week, followed by incubation in 30% sucrose at 4 °C for three days. Upon completion of the incubation period, the tissue was subjected to a graded alcohol dehydration series until it was cleared in xylol. Subsequently, 2 cm wide and 3 mm thick sections of tumor tissue were embedded in liquid paraffin and cut into 3–4 μm thick sections using a Reichert-Jung type microtome (Leica Biosystems, IL, USA). The sections were then mounted on previously silanized glass slides and allowed to dry at 60 °C for one hour. To expose the epitope, the slides were incubated in citrate-EDTA buffer at 97 °C for 30 min before blocking endogenous peroxidases by incubation with 2% hydrogen peroxide for 5 min, followed by incubation with 5% BSA for 10 min. Subsequently, the sections were then incubated with primary antibodies against PD-L1, Ki67 and mature macrophages (ab279292, anti-PD-L1 antibody [CAL10] - mouse IgG1; ab16667, anti-Ki67 antibody, Abcam Limited, CA, US and NB119885, macrophage marker antibody (25F9) - mature mouse monoclonal antibody, Novus Biologicals, CO, US) diluted in 0. 05 M Tris solution with pH between 6.2 and 7.3 at 1:100, 1:200, and 1:50, respectively, for one hour at room temperature. Donkey anti-rabbit secondary antibody (406401, BioLegend, CA, US) coupled to peroxidase at 1:100 dilution and Dako solution (K3468, Dako Liquid DAB + Substrate Chromogen System, Agilent Technologies, CA, US) were used for antigen detection and chromogenic reaction, which was counterstained with Harris hematoxylin and observed under an inverted microscope (Axiostar microscope, Zeiss Group, Stuttgart, DE) using a 40X objective. To determine the relative amount of antigen present in each slide, photomicrographs captured with the INFINITY X-32 MP camera (Lumenera Corp., ON, Canada) were analyzed using Image-Pro Plus software v. 5.1 (DC Imaging, OH, USA) to compare the intensity of the signal produced by diaminobenzidine oxidation.

TGFβ and IL-10 evaluation in tissues

Tissue samples (0.2 g tumor and spleen) from 4T1 cell-induced tumor-bearing mice were snap-frozen in liquid nitrogen and pulverized. The powder was dissolved in 1 mL RIPA lysis buffer (89901; Pierce, Thermo Scientific, MA, USA) containing protease inhibitors (A32953; Pierce, Thermo Scientific, MA, USA) and incubated on ice for 10 min before clarification by centrifugation at 2000 x g for 5 min at 4 °C. Total protein extracts from these tissues were used for the determination of the cytokines TGFβ and IL-10 by ELISA, as described above.

Results

Molecular docking results showed an appropriate scoring function (kcal/mol) in terms of affinity for both DOTA-iPD-L1 (-6.1 kcal/mol) and DOTA-WL12 (-5.7 kcal/mol). As shown in Fig. 1, DOTA-iPD-L1 interacts primarily with amino acids Lys25, Val30, Tyr28, Asp26, Lys41, Thr22, Pro24, Leu94, Thr37, and Val23 of the PD-L1 protein, whereas WL12 interacts through residues Lys124, Glu39, Val30, Lys41, Thr20, Pro43, Pro24, Phe42, Thr22, and Glu31. The relevant structural difference between the two inhibitory peptides is observed in the formed cycle, where iPD-L1, being structurally more restricted by the absence of the -S- bond, tends to form more intermolecular hydrogen bonds. In addition, the presence of carbon-hydrogen bonds (CH^….O) was observed for DOTA-iPD-L1 but not for DOTA-WL12. The carbon-hydrogen bonds are as strong as those formed by conventional oxygen and nitrogen donors such as NH^….N, NH^….O, OH^….N, and OH^….O(Horowitz and Trievel 2012), which may explain the relative higher molecular binding energy score function observed for DOTA-iPD-L1 (-6.1 kcal/mol) compared to DOTA-WL12 (-5.7 kcal/mol). At this point, it is relevant to mention that in previous results obtained with the same molecular docking methodology, the value related to the initial affinity of WL12 without the DOTA molecule was − 5.1 kcal/mol (Ferro-Flores et al. 2023), confirming that the addition of the DOTA chelator does not reduce its biological recognition capacity by PD-L1. Therefore, DOTA-WL12 labeled with lutetium-177 was used as a positive control in all in vitro evaluations.

Mass spectrometry (Fig. 2a) revealed the two major fragments of DOTA-iPD-L1 (2096 Da), the ions M + 2 H/2 (1049 m/z, Da) and M + 3 H/3 (699 m/z, Da).

Fig. 2.

Chemical characterization of DOTA-iPD-L1 by (a) UPLC mass, (b) FIT-Infrared, and (c) UV-Vis spectroscopies. (d) Chemical purity (99%) of DOTA-iPD-L1 and radiochemical purity (> 98%) of ¹⁷⁷Lu-iPD-L1 evaluated by reversed-phase HPLC and radio-HPLC, respectively

FT-IR spectroscopy of DOTA-iPD-L1 (Fig. 2b) showed the presence of the carboxylate contribution from the DOTA chelating motif, observed at 3303 cm^− 1. The band at 1654 cm^− 1 was attributed to the stretching of carbonyl bonds (-C = O). In addition, C-O stretching and O-H bending vibrations were identified at 1340 cm^− 1, while the vibrations associated with the N-H group appeared at 1455 cm^− 1 and 1436 cm^− 1. The C-S stretching vibration of the cysteine residue was observed at 669 cm^− 1. The vibration associated with the S-H bond, which typically appears at 2550 cm^− 1, was not detected in the spectrum, which is mainly attributed to the intramolecular geometry (orientation of the cysteine) and the conformational dynamics and interactions within cyclic structure (Deniz et al. 2022).

The vibrational signals for the in-plane bending vibrations of indole were found in the range of 1300–1500 cm^− 1, specifically at 1342 cm^− 1, 1438 cm^− 1, and 1455 cm^− 1. In addition, a contribution from the C-H stretching of aromatic hydrogen atoms in the indole ring was observed at 3073 cm^− 1. The C-H stretching vibrations are generally found in the 3000–3100 cm^− 1 range. These modes are associated with the aromatic hydrogen atoms in the indole ring (Majoube and Vergoten 1992).

The UV-Vis spectra of the DOTA-iPD-L1 compound (Fig. 2c) show two main absorption bands at 219 nm and 278 nm. The absorption band at 219 nm was assigned to the electronic transitions of the sulfur present in the structure. The absorption band at 278 nm was assigned to -C = C- and -C = N- from the peptide.

After radiolabeling, the radiochemical purity of ¹⁷⁷Lu-iPD-L1 was 98.9 ± 1.1% (n = 12) and > 97.5% for ¹⁷⁷Lu-WL12, as determined by reversed-phase radio-HPLC. Figure 2d shows the radiochromatogram where the retention time for ¹⁷⁷Lu-iPD-L1 was 14.1 min. After 24 h at 37 °C, ¹⁷⁷Lu-iPD-L1 and ¹⁷⁷Lu-WL12 (t_R=14.6 min) remained essentially unaltered in the blood serum medium (Fig. 3).

Fig. 3.

Stability in serum at 24 h of ¹⁷⁷Lu-iPD-L1 and ¹⁷⁷Lu-WL12. In both cases, radiochemical purity remained above 97%

DOTA-iPD-L1 showed an IC50 in 4T1 cells of 4.21 nM (95% CI 2.49 nM-5.32 nM), while DOTA-WL12 showed an affinity of 7.31 nM (95% CI 4.39 nM-11.89 nM) (Fig. 4). These results correlated with those obtained in the molecular docking procedure, which also showed an improved scoring function for DOTA-iPD-L1 (-6.1 kcal/mol) regarding the positive control peptide DOTA-WL12 (-5.7 kcal/mol).

Fig. 4.

Competition binding assay in 4T1 cells of ¹⁷⁷Lu-iPD-L1 and ¹⁷⁷Lu-WL12

The uptake and internalization of ¹⁷⁷Lu-iPD-L1 in 4T1 and HCT116 cells was found to be somewhat higher than that of ¹⁷⁷Lu-WL12, with a significant decrease (p < 0.05, t˗student) in the cellular uptake and internalization of both radiopharmaceuticals when the PD-L1 receptors of the cells were blocked with an excess of cold peptide, indicating that the cellular uptake and internalization of ¹⁷⁷Lu-iPD-L1 and ¹⁷⁷Lu-WL12 are specific (Fig. 5a and c, and Fig. 5e). It is important to note that 4T1 and HCT116 cells are highly positive for PD-L1 expression, as confirmed by immunofluorescence (Fig. 5b and d) and Western blot (Fig. 6). The negligible uptake and, consequently, internalization levels of ¹⁷⁷Lu-iPD-L1 and ¹⁷⁷Lu-WL12 in AR42J cells can be attributed to the negligible presence of PD-L1 protein on the cell surface, despite its presence in the cytoplasm (Fig. 5f), which indeed explains the presence of a certain amount of PD-L1 protein in AR42J cells observed in the Western blot assay (Fig. 6). These results may be related to the intracellular presence of PD-L1, which acts as an RNA-binding protein, as discussed below. Therefore, PD-L1 expression in the order 4T1 > HCT116 > AR42J directly correlated with the cellular uptake and internalization levels observed for the ¹⁷⁷Lu-labeled PD-L1 inhibitor peptides (Figs. 5 and 6).

Fig. 5.

Cellular uptake and relative internalization (percentage of cellular uptake that was internalized) of ¹⁷⁷Lu-iPD-L1 and ¹⁷⁷Lu-WL12 in (a) 4T1, (c) HCT116, and (e) AR42J cell lines. Blocking (B): 25 µg of unlabeled iPD-L1 or WL12 peptides. * Statistically significant difference (p < 0.05, t-student test). Micrographs of immunofluorescence staining for PD-L1 in (b) 4T1, (d) HCT116, and (f) AR42J cells showing a clear positive PD-L1 expression in 4T1 and HCT116 cells and low PD-L1 expression in AR42J (PD-L1 stained in green with anti-PD-L1 and cell nuclei stained in blue with DAPI)

Fig. 6.

Western blot assay showing PD-L1 expression in 4T1, HCT116, and AR42J cell lines (left) and graphical representation of the PD-L1/β-actin optical density (O.D.) ratio in 4T1, HCT116, and AR42J (right), which corroborates the low PD-L1 expression in AR42J cells. The molecular weight of PD-L1 and β-actin proteins correlated with the protein ladder used as a reference for the molecular weight markers

A decrease in the viability of the 4T1 cells was observed as a clear function of the target-specific radiation therapy, mediated by the recognition of PD-L1 (Fig. 7). Cells treated at different times (24 h, 48 h, and 72 h) and doses showed that reaching 3.39 Gy with ¹⁷⁷Lu-iPD-L1 (cells incubated with ¹⁷⁷Lu-iPD-L1 for 72 h; considering a 4T1 cellular uptake of 0.1 Bq/cell) resulted in a net decrease in viability of 53.4 ± 3.01%, with a statistically significant difference (p < 0.05, Student’s t-test) compared to the ¹⁷⁷LuCl₃-treated group (decrease in viability to 35.8 ± 2.7%) (Fig. 7). Consistent with the cytotoxicity results, the clonogenic assay revealed that the ability of 4T1 cells to proliferate and form colonies after cytotoxic treatments resulted in the following clonogenicity (%) data: PD-L1 (91.62 ± 1.45%), WL12 (93.13 ± 2.03%), ¹⁷⁷LuCl₃ (78.19 ± 2.39%), ¹⁷⁷Lu-iPD-L1 (39.74 ± 2.85%), and ¹⁷⁷Lu-WL12 (42.86 ± 3.24%), with a statistically significant difference (p < 0.05, Student’s t-test) between the effect produced by ¹⁷⁷Lu-iPD-L1 or ¹⁷⁷Lu-WL12 and that of ¹⁷⁷LuCl₃.

Fig. 7.

Representative flow cytometry histograms of 4T1 cell viability assay at different times after treatment with ¹⁷⁷LuCl₃ or ¹⁷⁷Lu-iPD-L1 or no treatment (control)

Two-way ANOVA and Dunnett’s multiple comparison analyses of the amount of IL-10 and TGFβ secreted by PD-L1-positive 4T1 and HCT116 cancer cells after treatment with ¹⁷⁷LuCl₃, ¹⁷⁷Lu-iPD-L1, ¹⁷⁷Lu-WL12, iPD-L1 or WL12, revealed a significant increase in cytokine levels after the targeted treatment with ¹⁷⁷Lu-labeled PD-L1 peptide inhibitors compared to the cold peptides and the control group (Figs. 8 and 9). Local radiotherapy can produce systemic immune stimulation effects. It has previously been reported that external radiation therapy induces immunosuppressive effects by the activation of immune inhibitory components such as TGFβ and the production of anti-inflammatory cytokines such as IL-10 (Zhang et al. 2023). However, this research also revealed that TGFβ and IL-10 production are significantly higher (p < 0.001, ANOVA) with a targeted radiotherapy treatment (¹⁷⁷Lu-iPD-L1) than with a non-molecularly targeted radio-treatment (¹⁷⁷LuCl₃) in PD-L1-positive 4T1 and HCT116 cancer cells (Figs. 8 and 9). In addition, the highest cytokine production was observed in cells with the highest PD-L1 expression (4T1 cells), which correlated with the highest cellular uptake and internalization observed for ¹⁷⁷Lu-iPD-L1 and, therefore, with the highest targeted radiation dose delivered to the cancer cells.

Fig. 8.

Quantitation of IL-10 secreted by 4T1, HCT116, and AR42J cancer cells determined by ELISA assay after treatment with ¹⁷⁷LuCl₃, ¹⁷⁷Lu-iPD-L1, ¹⁷⁷Lu-WL12, iPD-L1 or WL12. *Significant difference (p < 0.05, two-way ANOVA and Dunnett’s multiple comparison analyses)

Fig. 9.

Quantitation of TGFβ secreted by 4T1, HCT116, and AR42J cancer cells determined by ELISA assay after treatment with ¹⁷⁷LuCl₃, ¹⁷⁷Lu-iPD-L1, ¹⁷⁷Lu-WL12, iPD-L1 or WL12. *Significant difference (p < 0.05, two-way ANOVA and Dunnett’s multiple comparison analyses)

As shown above, AR42J cells presented a negligible uptake of ¹⁷⁷Lu-iPD-L1, suggesting an absence of PD-L1 in the cell membrane, so the observed effect on the increase in cytokine levels may be related to cross-dose (cross-fire) from the presence of the radiotracers in the medium, which would also explain the lack of a significant difference in cytokine levels between the ¹⁷⁷Lu -labeled PD-LI inhibitor peptides and ¹⁷⁷LuCl₃.

In the case of ¹⁷⁷Lu-iPD-L1 in vivo evaluation, only 4T1 triple-negative breast cancer tumors were used. Results of the ¹⁷⁷Lu-iPD-L1 biodistribution profile showed predominantly hepatobiliary elimination followed by renal clearance (Table 1). ¹⁷⁷Lu-iPD-L1 is mainly eliminated by the hepatobiliary system due to the lipophilic nature of iPD-L1 with a ClogP of 6.3 (Ferro-Flores et al. 2023).

Table 1.

Biodistribution of ¹⁷⁷Lu-iPD-L1 in mice bearing 4T1 tumors (%ID/organ). Total nuclear transformations (N)(MBq-s) in source organs and radiation absorbed dose (gy) seven days after administration of 37 MBq

Organ/tissue	Time (h)	% ID	A(t)dt (MBq-s)	Radiation Absorbed Dose at 7 days (Gy)
Heart	1	0.10 ± 0.12	26.67	0.10
	4	0.08 ± 0.05
	24 72 96	0.02 ± 0.03 0.00 ± 0.00 0.00 ± 0.00
Liver	1	20.41 ± 4.82	9053.94	4.46
	4	14.27 ± 0.3.58
	24 72 96	5.46 ± 1.86 2.45 ± 1.02 1.90 ± 0.51
Kidney	1	11.20 ± 1.77	2877.61	7.99
	4	8.23 ± 1.47
	24 72 96	2.17 ± 0.71 0.36 ± 0.11 0.16 ± 0.05
Spleen	1	0.11 ± 0.09	49.13	0.36
	4	0.08 ± 0.04
	24 72 96	0.04 ± 0.03 0.01 ± 0.01 0.00 ± 0.00
Lung	1	0.14 ± 0.02	36.49
	4	0.08 ± 0.03
	24 72 96	0.03 ± 0.02 0.00 ± 0.00 0.00 ± 0.00		0.28
Intestine	1	0.34 ± 0.12	317.59
	4	2.18 ± 0.72
	24 72 96	1.71 ± 0.38 1.02 ± 0.20 0.81 ± 0.13		0.16
4T1 Tumor (unblocking)	1	6.97 ± 1.04	15984.77
	4	6.58 ± 0.98*
	24 72 96	5.74 ± 1.01 5.08 ± 0.88 4.79 ± 0.92		27.23
4T1 Tumor (blocking)	4	4.87 ± 1.01*	-	-

*Statistically significant difference (p < 0.05, t-student)

The uptake of ¹⁷⁷Lu-iPD-L1 in tumors was relevant (6.97% at 1 h) and with adequate tumor retention after 96 h (4.79%) (Table 1). Although the tumor uptake of ¹⁷⁷Lu-iPD-L1 cannot be directly compared with other PD-L1 inhibitor radiopeptides due to the heterogeneity of PD-L1 protein expression in different tumors, the % tumor uptake at 1 h post-administration correlated in a range similar to that of WL12 labeled with Lu-177 (10% in lungs invaded by HCC827 cancer cells)(Luna-Gutierrez et al. 2023) or Tc-99 m (6.8% in MC38-B7H1 tumors) (Fan et al. 2024). Specificity was demonstrated by a significant reduction (p < 0.05, t-Student) in tumors from mice that received a blocking dose by administration of 100 µg of unlabeled iPD-L1 peptide. Tumors were the tissues receiving the highest number of nuclear transformations (N) and, consequently, the highest radiation doses (27.23 Gy), followed by kidney (7.99 Gy) and liver (4.46 Gy), which are well below the established maximum tolerated doses (< 30 Gy), indicating the potential of ¹⁷⁷Lu-iPD-L1 for clinical use in targeted radiotherapy protocols (Table 1).

The ex vivo assessment of IL-10 and TGF-β levels in tumors and spleens revealed an increase in cytokine levels compared to those observed in untreated mice (Fig. 10). These results not only demonstrate that ¹⁷⁷Lu-iPD-L1 can induce immunosuppressive and anti-inflammatory effects in vivo, but also suggest that the immune response observed in the spleen may be linked to a systemic response, indicating an activation of the immune system modulated by ¹⁷⁷Lu-iPD-L1 (Fig. 10).

Fig. 10.

Quantitation of IL-10 and TGFβ present in 4T1 tumors and spleens of mice treated with ¹⁷⁷Lu-iPD-L1. *Significant difference (p < 0.05, t-student)

Immunohistochemistry analysis of tumors after the acute radiation treatment with ¹⁷⁷Lu-iPD-L1 showed a significant difference (p < 0.05, two-way ANOVA and Dunnett’s multiple comparisons) in cell proliferation (8.85 ± 0.71% Ki-67 positive), induction of PD-L1 expression (64.76 ± 0.49% PD-L1 positive) and infiltration of activated macrophages (22.56 ± 0. 69% mature macrophage positive) compared to the untreated control group (42.86 ± 0.57% Ki-67; 46.01 ± 0.66% PD-L1; 6.087 ± 0.62% mature macrophages) and ¹⁷⁷LuCl₃ (30.44 ± 0.66% Ki-67; 53. 65 ± 0. 64% PD-L1; 4.86 ± 0.65% mature macrophages), iPD-L1 (25.45 ± 0.45% Ki-67; 29.40 ± 0.72% PD-L1; 4.94 ± 0.58% mature macrophages) and WL12 (24.48 ± 0.55% Ki-67; 30.52 ± 0.74% PD-L1; 5.90 ± 0.61% mature macrophages) treatments as determined with the Image-Pro Plus software (Fig. 11). Radiotherapy has been shown to have immunomodulatory effects. In this study, the results demonstrated the potential immunomodulatory effect of ¹⁷⁷Lu-iPD-L1, which may be related to the observed macrophage activation, production of anti-inflammatory and immune inhibitory cytokines (e.g., IL-10 and TGFβ, respectively), and significant decrease in proliferation markers (Ki-67) in tumors overexpressing PD-L1.

Fig. 11.

Micrographs of tumor immunohistochemistry after the acute radiation treatment with ¹⁷⁷Lu-iPD-L1 showing a significant difference (p < 0.05, two-way ANOVA and Dunnett’s multiple comparisons) in cell proliferation (Ki-67 expression), infiltration of activated macrophages (indicated by black arrows) and PD-L1 expression compared to the control group and ¹⁷⁷LuCl₃, iPD-L1, and WL12 treatments as determined with the Image-Pro Plus software. Black bar: 200 μm scale

Discussion

PD-L1 is a 33 kDa protein found on the cell membrane or cytoplasm. Membrane-localized PD-L1 can inhibit T-cell proliferation and enhance NK cytolytic activity to help cancer cells escape immune surveillance by interacting with its receptor (PD-1) on immune cells. In recent years, ICP blockade therapy has been a focus of cancer research and treatment. Immunotherapies targeting PD-1/PD-L1 have demonstrated substantial clinical benefits in different tumor types. In addition to its well-established function as an immune checkpoint molecule, intracellular PD-L1 also acts as an RNA-binding protein and competes with RNA exosomes to increase RNA stability globally. Specifically, PD-L1 in the cytoplasm binds to messenger RNAs (mRNAs) of several DNA damage repair (DDR)-related genes and protects them from degradation, which facilitates DDR and enhances tumor resistance to DNA-damaging therapy (Zhang et al. 2022a, b). Radiation therapy (RT) is an ideal candidate for combinator immunotherapy strategies, given the immunosuppressive and DDR-facilitating role of PD-L1. In addition to reducing tumor mass and releasing tumor antigens, RT has well-established immunomodulatory effects. These interdependent and overlapping effects include increased effector and homing function of tumor-infiltrating lymphocytes (TILs), including T and NK cells, increased diversity of the T-cell response, destruction of immunosuppressive cells in the tumor microenvironment, induction of immunogenic cell death, increase of dendritic cells (DCs) migration, presentation of tumor antigens, positive regulation of immunogenic cell surface receptors and stress ligands (Monjazeb 2016). In this research, the combination of iPD-L1 with RT, given by ¹⁷⁷Lu, substantially reduced PD-L1-positive cancer cell viability compared to the treatments alone. Furthermore, the immune system activation in vivo mediated by ¹⁷⁷Lu-iPD-L1 was investigated by looking for activated macrophages in the tumor microenvironment and PD-L1 expression (Fig. 12). The immunomodulatory effects of RT are complex, from the induction of an antitumor immune response triggered by chemokines such as CCL3, 4, and 5 that enhance the efficacy of checkpoint blockade immunotherapy to some suppressive effects, such as those conducted by TGFβ induction, immunosuppressive cells accumulation, PD-L1 upregulation, and indolamine upregulation.

Fig. 12.

Schematic representation of the immunomodulatory effect of ¹⁷⁷Lu-iPD-L1 on triple-negative breast tumor cells. Radiation emission of ¹⁷⁷Lu-iPD-L1 promotes PD-L1 expression, in addition to macrophage activation, while inhibition of the PD-1/PD-L1 complex by iPD-L1/¹⁷⁷Lu-iPD-L1 promotes T lymphocyte activation

Local RT also produces systemic effects on immune stimulation. Radiation induces tumor cell death and the release of proinflammatory cytokines, chemokines, tumor antigens, and damage-associated molecular patterns (DAMPs). These signals can enhance the functions of antigen-presenting cells (APCs) to activate tumor-specific T-cell immunity. They stimulate APC recruitment, promote major histocompatibility complex (MHC) expression by APCs and tumor antigen loading on APCs, drive migration to draining lymph nodes, and enhance tumor proliferation and recognition by T cells. On the other hand, RT can also induce immunosuppressive effects at the tumor site. It can activate immune inhibitory components such as TGFβ, regulatory T cells (Treg), tumor-associated macrophages (TAM), and myeloid-derived suppressor cells (MDSC), and positively regulate the expression of PD-L1 on tumor cells, which is related to immunosuppression in cancer tumors (Zhang et al. 2023). Indeed, radiation produced by ¹⁷⁷Lu induced a local inflammatory response that could increase tumor-specific T-cell infiltration and simultaneously induce PD-L1 expression in the tumor microenvironment, which markedly weakens radiation-induced antitumor immunity. Therefore, the concept of ¹⁷⁷Lu-iPD-L1 (radiation)-induced PD-L1 expression and simultaneous and/or subsequent blockade by application of iPD-L1 or anti-PD-L1/PD-1 may be a potent therapy against e.g. triple-negative breast cancer (Fig. 12). In addition, macrophages contribute to tumorigenesis by producing stimulatory or inhibitory molecules that affect tumor cell growth, blood vessel formation, cell adhesion, and tissue architecture. Tumor susceptibility is strongly influenced by genetic makeup, and in model systems, part of the genetic component lies in differences in inflammatory responses, including differences in the genetics of macrophage function. Therefore, macrophages are likely to be major determinants of changes in the tumor microenvironment (Coates et al. 2008).

In our study, the immunomodulatory effect of ¹⁷⁷Lu-iPD-L1 can be observed in macrophage activation, production of anti-inflammatory cytokines such as IL-10, and significant decrease of proliferation (Ki-67) in tumors overexpressing PD-L1.

Conclusions

¹⁷⁷Lu-iPD-L1 was prepared as a stable radiopharmaceutical with specific recognition by the PD-L1 protein. The biodistribution and biokinetic profile showed the potential of ¹⁷⁷Lu-iPD-L1 for clinical use in targeted radiotherapy protocols.

Results of this research demonstrated the potential immunomodulatory effect of ¹⁷⁷Lu-iPD-L1, which may be related to the observed macrophage activation, production of anti-inflammatory and immune inhibitory cytokines (e.g., IL-10 and TGFβ, respectively), and significant decrease in proliferation (Ki-67) in tumors overexpressing PD-L1. Therefore, this study warrants further dosimetric and clinical studies to determine the immunomodulatory effect and therapeutic efficacy of ¹⁷⁷Lu-iPD-L1 in the treatment of PD-L1-positive tumors in combination with anti-PD-1/PD-L1 immunotherapy protocols.

Abbreviations

APCs

Antigen-presenting cells

DAMPs

Damage-associated molecular patterns

DAPI

4’,6-diamidino-2-phenylindole

ICP

Immune checkpoint proteins

IL-10

Interleukin-10

DCs

Dendritic cells

DDR

DNA damage repair

DOTA

1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid

MDSC

Myeloid-derived suppressor cells

MHC

Major histocompatibility complex

NK

Natural killer cells

PD-1

Programmed cell death protein 1

iPD-L1

Programmed death-ligand 1 inhibitor peptide

PD-L1

Programmed death-ligand 1

RT

Radiotherapy

TAM

Tumor-associated macrophages

TGFβ

Transforming growth factor beta

TILs

Tumor-infiltrating lymphocytes

Treg

Regulatory T cells

Author contributions

Conceptualization – GF-F, EA-V, ML-G,; Methodology – ML-G, EA-V, RO-P, BO-G, PC-N, CS-C, NJ-M, GF-F, GB-V; Formal analysis – GF-F, EA-V, BO-G, ML-G, LM-A; Writing – Original Draft – ML-G, EA-V, PC-N. GF-F; Writing – Review & Editing – GF-F, LM-A; Funding acquisition, GF-F, ML-G, LM-A.

Funding

This research was funded by the “Consejo Mexiquense de Ciencia y Tecnología” (COMECyT) through the program collaboration networks EDOMEX, grant number FICDTEM-2023-152. This study was also partially supported by the International Atomic Energy Agency (CRP-F22078) and by the Italian Ministry of Health No. 5 × 1000 (2019) “BRIDGE” project “DECURTA” granted to L.M.-A.

Data availability

Data is contained within the article.

Declarations

Ethical approval

All applicable institutional and international guidelines for the use and care of animals were followed. This research was approved by the Ethics Internal Committee of Use and Care of Laboratory Animals (CICUAL-ININ) of the National Institute of Nuclear Research, Approval No. 12-2018-2022.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.