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. 2024 Nov 26;9:79. doi: 10.1186/s41181-024-00313-8

SPECT/CT imaging of EGFR-positive head and neck squamous cell carcinoma patient-derived xenografts with 203Pb-PSC-panitumumab in NRG mice

Nasim Sarrami 1, Bryce Nelson 2, Samantha Leier 2, John Wilson 2, Conrad Chan 3, Jalna Meens 4, Teesha Komal 5, Laurie Ailles 4,6, Melinda Wuest 2, Michael Schultz 7, Afsaneh Lavasanifar 1, Raymond M Reilly 3,4, Frank Wuest 1,2,8,9,✉
PMCID: PMC11599518  PMID: 39589608

Abstract

Background

The objective of this research was the development and evaluation of 203Pb-labelled panitumumab (203Pb-PSC-panitumumab) as an immuno-SPECT radioligand for the detection of EGFR + head and neck squamous cell carcinoma (HNSCC) in a patient-derived xenograft (PDX) mouse model. The 51.9 h physical half-life and favourable γ-emission (279 keV; 81%) of 203Pb offer an excellent opportunity for developing immuno-SPECT radioligands. Moreover, 203Pb has a complementary therapeutic radionuclide (212Pb), making 203Pb and 212Pb an ideal matched radiotheranostic pair.

Results

Radiolabeling of panitumumab was performed at a pH of 5.0 and room temperature for 5–10 min with [203Pb]Pb(OAc)2, and the incorporation efficiency was determined using radio-TLC. 203Pb-PSC-panitumumab (~ 10 MBq, 140 μl of saline) was injected into the tail vein of NRG mice bearing subcutaneous (s.c.) HNSCC patient-derived xenografts (PDX). SPECT/CT images were acquired at 48 and 120 h post-injection. For biodistribution studies, mice were euthanized five days after 203Pb-panitumumab injection. The tumour and normal tissues were collected and weighed, and uptake of 203Pb was measured in a γ-counter. The uptake was calculated as the percent injected dose per gram of each tissue (ID%/g). Blocking experiments were performed by pretreating a group of mice (n = 5) with 1 mg of panitumumab 1 h before administering 203Pb-PSC-panitumumab. 4–5 chelators of a new lead-specific chelator (PSC) were attached per antibody; radiolabeling efficiency was 99.2 ± 0.7%. The isolated radiochemical yield of 203Pb-PSC-panitumumab was 41.4 ± 8% (n = 5), and the molar activity was 1.2 ± 0.35 GB/mg. SPECT imaging and biodistribution confirmed high accumulation and retention of 203Pb-PSC-panitumumab in the tumour (26% ID/g) at 120 h post-injection (p.i.), which could be reduced to 6.2%ID/g at 120 h p.i. by predosing with panitumumab (1 mg) confirming EGFR specificity of 203Pb-PSC-panitumumab uptake.

Conclusions

Panitumumab was successfully and reproducibly labelled with 203Pb in high radiochemical purity using the chelator PSC-NCS. 203Pb-PSC-panitumumab was specifically accumulated and retained in EGFR + tumours in NRG mice with s.c. HNSCC PDX. 203Pb-PSC-panitumumab is a suitable immuno-SPECT radioligand for imaging EGFR + tumours and has great potential for combining with 212Pb-PSC-panitumumab in a radiotheranostic strategy for imaging and treating HNSCC.

Keywords: Panitumumab, Pb-203, Single-Photon Emission Computed Tomography (SPECT), Epidermal growth factor receptor (EGFR), Radiotheranostics, Pb-212

Introduction

Targeted radionuclide therapy (TRT) is a widely used cancer treatment option that employs radiopharmaceuticals to target and deliver ionizing radiation to kill cancer cells (Li et al. 2020; Sun et al. 2022; Chan et al. 2020). TRTs have been used for cancer therapy, demonstrating increased overall survival as exemplified in patients with thyroid cancer, prostate cancer, and neuroendocrine tumours (Kerr et al. 2022). As a promising type of current cancer therapy, TRT delivers a therapeutic dose of radiation to cancer cells using radioactive drugs (radiopharmaceuticals) labelled with alpha(α)- or beta(β−)-emitting radioisotopes (Chan et al. 2020; Gill et al. 2017). TRT with α emitters (α-TRT) offers several advantages compared to β− emitters, mainly due to the delivery of high-energy α-particles (5–9 MeV) to the tumour with a short pathlength (50–100 μm) and high linear energy transfer (LET) (80 keV/μm), causing less toxicity to neighbouring healthy tissues (Pandit-Taskar 2019). However, α-TRT radiopharmaceuticals cannot be used directly for imaging applications for assessing biodistribution and target binding capacity as crucial criteria for patient selection and dose calculation in the clinical setting due to the lack of photons suitable for in vivo imaging (Gallivanone 2017). The development and application of radiopharmaceuticals combining targeted imaging and therapy, also called radiotheranostics, represent a rapidly evolving field in oncologic nuclear medicine (Srivastava 2012; Qaim et al. 2018). Ideal radiotheranostics use different radioisotopes of the same chemical element for imaging and therapy to ensure similar pharmacokinetics, metabolism and biodistribution patterns (Li et al. 2020; Srivastava 2012). Typical examples of ideal radionuclide pairs for radiotheranostics reported in the literature include 64Cu/67Cu, 86Y/90Y, 124I/131I, 152 Tb/161 Tb, 133La/135La and 203Pb/212Pb (Nelson et al. 2020; Nelson et al. 2023; Kokov et al. 2022; McNeil et al. 2021). In addition, physical half-life, availability, and production costs also require special consideration in the design and development of radiotheranostics (Srivastava 2013).

The 203Pb/212Pb radionuclide pair has recently gained much attention for developing radiotheranostics for TRT (Nelson et al. 2023; Kokov et al. 2022). 203Pb emits γ-photons through electron capture, allowing detection with single-photon emission computed tomography (SPECT) for diagnostic imaging, whereas 212Pb decays by emitting β−-particles and α-particles suitable for delivering therapeutic doses of radiation to cancer cells (Kokov et al. 2022; McNeil et al. 2021).

Targeting vectors in radiotheranostics for TRT encompass small molecules, peptides, antibodies, and nanoparticles (Kerr et al. 2022). Among the targeting vector landscape, monoclonal antibodies (mAb) display exceptional target specificity, making mAb excellent candidates for TRT.

However, their relatively high molecular weight (~ 150 kDa) results in a long biological half-life (slow distribution and elimination profile), which must be matched with a compatible physical half-life of the radionuclide (Mammatas et al. xxxx). Several radiolabelled mAb and antibody fragments are tested in preclinical studies for clinical translation of radioimmunotherapy (RIT), demonstrating promising results for targeting cancer biomarkers epidermal growth factor receptor (EGFR), prostate-specific membrane antigen (PSMA), and human epidermal growth factor receptor 2 (HER2) (Parakh et al. 2022; Chamarthy et al. 2011). The relatively short 10.5 h physical half-life of 212Pb represents particular challenges for targeted RIT, which can be addressed by pretargeting concepts or using smaller immunoconjugates with shorter biological half-lives like antibody fragments (Jiao et al. 2023). However, the efficacy of RIT with 212Pb using full-length antibodies was recently demonstrated with a 212Pb-labeled antibody targeting melanin in a preclinical melanoma model (Bauer et al. 2024).

Panitumumab is an FDA-approved human monoclonal antibody specific to EGFR used as a single drug or in combination with other drugs to treat certain types of colorectal cancer (CRC), especially for the treatment of metastatic colorectal carcinoma with disease progression. Panitumumab binds to the extracellular domain of the EGFR, preventing EGFR dimerization, thus, halting ligand-induced receptor autophosphorylation and intracellular signalling pathway activation (Gemmete and Mukherji 2011). EGFR is overexpressed in many solid tumour cancers, including CRC, head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC) and breast cancer (Thomas and Weihua 2019; Rogers et al. 2005). Several studies demonstrated the relationship between EGFR overexpression and survival rate in these cancers: as EGFR expression increased, survival decreased (Braun et al. 2018), making EGFR a promising target for TRT of solid tumours.

Herein we describe the radiolabelling of panitumumab with 203Pb using novel bifuntional chelating agent 2,2'-(4-(2-amino-2-oxoethyl)-10-(2-((4-isothiocyanatobenzyl)amino)-2-oxoethyl)-1,4,7,10 tetraazacyclododecane-1,7-diyl) diacetic acid (PSC-NCS) (Nelson et al. 2023). Immuno-SPECT radioligand 203Pb-PSC-panitumumab was evaluated using SPECT and biodistribution studies in NRG mice bearing subcutaneous (s.c.) HNSCC patient-derived xenografts (PDX). Our work using 203Pb-PSC-panitumumab provides critical data for developing and testing 212Pb-PSC-panitumumab for future TRT applications using 203/212Pb-PSC-panitumumab radiotheranostics.

Materials and methods

Production of 203Pb

203Pb was produced using a recently published procedure (Nelson et al. 2023). Briefly, 205Tl metal (99.9% isotopic enrichment) targets were irradiated at 23.3 MeV on a TR-24 cyclotron at currents up to 60 μA to produce 203Pb via the 205Tl(p,3n)203Pb nuclear reaction. Following a cool-down period of > 12 h, targets were removed and irradiated 205Tl dissolved in 4 M HNO3. A NEPTIS Mosaic-LC synthesis unit performed automated separation using Eichrom Pb resin, and 203Pb was eluted using 8 M HCl or 1 M NH4OAc. Purified 203Pb yields of up to 12 GBq were attained (15.8 GBq at EOB). The [203Pb]PbCl2 and [203Pb]Pb(OAc)2 products contained no detectable radionuclidic impurities besides 201Pb (< 0.1%), and < 0.4 ppm stable Pb. 205Tl metal was recovered with a 92% batch yield (Nelson et al. xxxx).

Preparation of 203Pb-PSC-panitumumab radioimmunoconjugate

General

All glassware was rinsed with ultra-pure HCl (Fisherbrand, A508-P500). Trace metal-based ultra-pure chemicals for buffer preparations were purchased from Sigma Aldrich. All buffer solutions were treated with biotechnology-grade Chelex 100 (Bio-Rad, 143–2832).

PSC functionalization of panitumumab and radiolabeling with [203Pb]Pb(OAc)2

2,2'-(4-(2-amino-2-oxoethyl)-10-(2-((4-isothiocyanatobenzyl)amino)-2-oxoethyl)-1,4,7,10-tetra-azacyclododecane-1,7-diyl) diacetic acid (PSC-NCS) chelator (Li et al. 2023) (200 μg) was dissolved in 50 μL of 0.1 M NaHCO3 (pH = 9.0) and added to 400 μL of panitumumab (Vectibex ® 20 mg/mL). The pH was adjusted to 8.0 and left on a thermoshaker set at 800 rpm and 30 °C for 2.5 h.

The samples were purified via size exclusion chromatography (Bio-Rad 10DG desalting column, USA), which was pre-equilibrated and eluted with 0.025 M NaOAc buffer (pH = 5.5). The antibody concentration of each fraction was measured using a nanodrop spectrophotometer (Thermo Scientific, NanoDrop OneC), and the fraction with the highest protein concentration was submitted for analysis via matrix-assisted laser desorption/ionization (MALDI) to assess the number of PCS chelators per antibody (~ 5 chelators per antibody). [203Pb]Pb(OAc)2 (150–200 MBq) was added to PSC-panitumumab (200 μg), and the reaction was kept at room temperature for 5–10 min at pH = 5.

Radio-thin layer chromatography (radio-TLC) analysis (AR-2000, Eckert and Ziegler) was used to determine 203Pb incorporation efficiency by spotting samples on silica plates and using 20 mM EDTA and 0.2 M NaOAc as the mobile phase; in this system, the Rf for [203Pb]Pb(OAc)2 was1.0 and for 203Pb-PSC-panitumumab will be zero (McNeil et al. 2021). 203Pb-PSC-panitumumab was purified on an Econo-Pac 10DG desalting column pre-equilibrated with 0.25 M sodium acetate, pH 5.5 used as the eluant.

Elution fractions (300 μL) were collected from the column, and the radioactivity was measured using an Atomlab 400 dose calibrator (Biodex, Shirley, NY, USA). Laemmli buffer (Bio-Rad, USA) was added to 203Pb-PSC-panitumumab, and the samples (15 μL) were incubated at 95 °C for 5 min. Then, the samples were loaded on SDS-PAGE (Bio-Rad, Mini-PROTEAN® TGX™ Precast Protein Gels) and ran at 120 V. The gel was imprinted on film and evaluated by autoradiography on a BAS-5000 phosphor imager (Fujifilm).

Functional characterization of 203Pb-PSC-panitumumab

Cell uptake studies

EGFR-expressing neck and neck cancer FaDu cells were cultured in a 5% CO2 incubator at 37 °C in Gibco DMEM media supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin with media renewal 2–3 times per week. For cellular uptake studies, 200,000 cells per well were seeded in 6-well plates and left in the incubator overnight. The media was removed, and 500 μL of Kerbs-Ringer buffer solution (Bailey and Ong 1978) was added to each well, followed by the addition of 0.2 MBq of 203Pb-PSC-panitumumab, and the plate was kept in the incubator at 37 °C at 5% CO2. Cell uptake was terminated at 1, 5, 15, 30, 45, 60, and 90 min by adding ice-cold Krebs buffer (120 mM NaCl, 4 mM KCl, 1.2 mM KH2PO4, 2.5 mM, MgSO4, 25 mM NaHCO3, 70 μM CaCl2, pH 7.4) and rinsing the wells twice to wash away unbound radioimmunoconjugates before lysing the cells with RIPA buffer. The cell lysates were transferred to scintillation vials and measured for radioactivity using a γ-counter (Wizard2® 2480 Automatic Gamma Counter, Perkin-Elmer, Canada). In addition, according to the manufacturer's recommendations, protein levels in each sample was quantified using a Pierce™ BCA protein assay kit. For blocking studies, the FaDu cells were co-treated with different amounts of unlabeled panitumumab (0.5–10 µg) and 0.2 MBq of 203Pb-PSC-panitumumab. Cell uptake levels were normalized to percent of the total amount of radioactivity per milligram of protein (% radioactivity/mg protein) and plotted as a function of time. All experiments were performed in triplicates.

Immunoreactivity

The immunoreactivity of 203Pb-PSC-panitumumab was assessed by a cell binding assay with EGFR-expressing FaDu cells using the Lindmo et al. method (Lindmo et al. 1984). 0.25, 0.5, 1, 2, 3, 4, and 5 × 106 FaDu cells were disperesed in 500 μL of PBS supplemented with 1% bovine serum albumin (Sigma, USA). Then, 20,000 cpm of 203Pb-PSC-panitumumab was added to each cell concentration. The samples were placed on a rocker for one hour at room temperature. After triplicate PBS washes, the samples were transferred to 1.5 mL LoBind® tubes (Eppendorf, USA), and by using a γ-counter (Wizard2® 2480 Automatic Gamma Counter, Perkin-Elmer, Canada), the counts of each sample were determined.

The radioactivity data obtained was corrected in the background and compared to counts from the total activity added to control samples in the experiments. The immunoreactive fraction was calculated by performing a linear regression analysis on a double-inverse plot of (total/bound) activity versus normalized cell concentration.

In vivo experiments

Xenograft models

A primary tumour specimen (#391) was surgically obtained from a patient with HNSCC under a protocol approved by the Research Ethics Board at the University Health Network (Protocol No. 12–5639). This tumour was dissected into small fragments (~ 1 mm3) and engrafted subcutaneously (s.c.) on the right flank of NOD Rag2 γc (NRG) immunodeficient male mice (3–4 months old). These patient-derived tumour xenografts (PDX) were serially propagated in NRG mice following an Animal Care Protocol (No. 1542.28) approved by the Animal Care Committee at the University Health Network and following Canadian Council on Animal Care guidelines. The PDX used in this study was between the 3rd to 5th passage from the initial engraftment of the HNSCC tumour in NRG mice.

SPECT/CT imaging

NRG mice were anesthetized using 2% isoflurane in O2, and microSPECT/CT images were acquired in a supine position at 48 h and 120 h on a NanoScan® SPECT/CT/PET system (Mediso). Either 203Pb-PSC-panitumumab or 203Pb-DOTA-panitumumab-NPs (~ 10 MBq; 140 μL) were injected into the tail vein of NRG mice engrafted with subcutaneous HNSCC PDX. Mice were divided into two study groups: A group of mice (n = 3), was only injected with 203Pb-PSC-panitumumab, and the blocking group (n = 3) was injected with 1 mg of panitumumab 1 h before administration of 203Pb-PSC-panitumumab to block EGFR. SPECT/CT images were acquired 48 and 120 h post-injection (p.i.) of the radioligand. Images were acquired in a 256 × 256 matrix.

A Mediso APT62 collimator (WB-HS standard) was affixed to each of the four detector NaI (TI) detector heads. Images were reconstructed by Monte Carlo methods with three subsets of data undergoing 48 iterations using the Mediso Nucline NanoScan acquisition and reconstruction software (ver 3.00.020.0000). Before SPECT imaging, CT images were acquired with 50 kVp X-rays, 980 μA and a 300 ms exposure time. CT scans were reconstructed using the medium voxel and slice thickness settings, resulting in an isotropic voxel size of 250 μm. SPECT and CT were co-registered by the Mediso Nucline acquisition/reconstruction software. All animal studies were conducted under a protocol (AUP 2843.14) approved by the Animal Care Committee at the University Health Network following the Canadian Council on Animal Care guidelines.

Biodistribution studies

Mice were euthanized five days (120 h) after the injection of 203Pb-PSC-panitumumab, and tissues and organs were collected, weighed, and radioactivity was counted in a γ-counter. The uptake was calculated as injected dose percentage per gram of each tissue (ID%/g). Two groups were studied for biodistribution experiments: (1) Mice (n = 5) injected with 203Pb-PSC-panitumumab, and (2) Mice (n = 3) were injected with 1 mg of panitumumab one hour before the injection of 203Pb-PSC-panitumumab to block EGFR.

Statistical analysis

All data are expressed as means ± SEM. Graphs were constructed using GraphPad Prism 4.0 (GraphPad Software). Where applicable, statistical differences were tested by unpaired Student's t-test and were considered significant for p < 0.05.

Results

Radiochemistry

MALDI analysis confirmed the conjugation of 4–5 PSC chelators per antibody by reacting the isothiocyanate group in PSC-NCS with lysine residues in panitumumab (Fig. 1A). PSC-conjugated panitumumab was used for radiolabeling with [203Pb]Pb(OAc)2, and the radiolabelling efficiency was measured with radio-TLC, indicating 99.2 ± 0.7% incorporation of 203Pb2+ (Fig. 1B and 1C) which is comparable to results obtained by Nelson et al. (Nelson et al. 2023). SDS-PAGE analysis further confirmed incorporation of 203Pb to panitumumab. Reductive SDS-PAGE conditions resulted in the formation of panitumumab light and heavy chains, which were visible at 25 and 50 kDa, respectively, indicating that both antibody portions were modified with the PSC chelator and labelled with 203Pb (Fig. 1D).

Fig. 1.

Fig. 1

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Synthesis and radiolabeling of PSC-conjugated panitumumab with 203Pb (A and B) and measurement of 203Pb2+ incorporation efficiency and radiochemical purity of purified 203Pb-PSC-panitumumab by radio-TLC (C) and SDS-PAGE (D)

203Pb-labeling and purification of radioimmunoconjugate 203Pb-PSC-panitumumab provided isolated radiochemical yields of 41.5 ± 8% (n = 5) at a molar activity of 1.2 ± 0.35 GBq/mg. The radiochemical purity of the isolated radioimmunoconjugates was greater than 99%, as analyzed by radio-TLC. Purified radioimmunoconjugates were > 95% stable in human serum over 48 h.

Cellular uptake of 203Pb-PSC-panitumumab

EGFR-expressing FaDu cells were incubated with 203Pb-PSC-panitumumab, and cell uptake was measured at different time points. 203Pb-PSC-panitumumab uptake in FaDu cells increased until it reached a plateau at 60 min (Fig. 2A). EGFR specificity of cellular uptake was confirmed with blocking studies using different amounts of panitumumab (0.5 µg–10), demonstrating 70%, 85%, and 95% blocking at 0.5 µg, 1 µg, 5 µg and 10 µg, respectively (Fig. 2B). The immunoreactive fraction of 203Pb-PSC-panitumumab was found to be ~ 30%, which is lower than the reported 68% for 89Zr-labelled panitumumab measured in MDA-MB-468 cells (Bhattacharyya et al. 2013) (Fig. 2C).

Fig. 2.

Fig. 2

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Cellular uptake of 203Pb-PSC-panitumumab uptake in EGFR + FaDu cells (A) and studies of 203Pb-PSC-panitumumab binding competed with increasing amounts of panitumumab (B). Representative double inverse plot from Lindmo assay performed in FaDu cells with 203Pb-PSC-panitumumab (C)

In vivo imaging (microSPECT/CT)

MicroSPECT/CT images were acquired to visualize the biodistribution of 203Pb-PSC-panitumumab in NRG mice bearing subcutaneous EGFR + HNSCC PDX tumours (Fig. 3). EGFR specificity was tested with in vivo blocking studies by administering 1 mg of panitumumab before 203Pb-PSC-panitumumab injection to block EGFR (n = 3). SPECT images were taken at two time points of 48 and 120 h p.i. of 203Pb-PSC-panitumumab to monitor changes in tumour uptake and distribution of 203Pb-PSC-panitumumab over time. The tumours were clearly visible in SPECT/CT images at both time points without significant radioactivity in other organs and tissues (Fig. 3).

Fig. 3.

Fig. 3

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Representative SPECT/CT images of NRG mice engrafted with EGFR + HNCC PDX at 48 and 120 h p.i. under control and blocking conditions. Tumours are highlighted with an arrow

Under EGFR blocking conditions, SPECT/CT images after 48 h and 120 h indicated lower tumour uptake and somewhat increased radioactivity in the heart and blood pool. The remaining radioactivity observed in the tumour under blocking conditions at 48 and 120 h is presumably due to the EPR effect, as typically found in immunoPET and immunoSPECT experiments with solid tumours (Dewulf et al. 2020).

Biodistribution studies

NRG mice bearing EGFR + PDX HNCC tumours were euthanized at 120 h p.i. either after 203Pb-PSC-panitumumab (n = 5) or 1 mg of panitumumab pre-injection with 203Pb-PSC-panitumumab (n = 5). The results (Table 1) indicated that baseline tumour uptake at 120 h p.i. was significantly higher (26 ± 07 ID%/g) than in all other organs reaching tumour-to-blood (T/B) and tumour-to-muscle (T/M) radios of 4.2 and 37.3, respectively.

Table 1.

Biodistribution of 203Pb-PSC-panitumumab at 120 h p.i

Tissue Percent injected dose per gram (mean ± SEM)
203Pb-PSC panitumumab 203Pb-PSC panitumumab + panitumumab (1 mg)
Blood 6.2 ± 1.2 6.6 ± 2.4
Heart 1.8 ± 0.6 1.7 ± 0.6
Lung 2.5 ± 0.8 3.2 ± 1.7
Liver 2.5 ± 0.8 2.1 ± 0.3
Kidney 1.8 ± 0.4 2.1 ± 0.7
Spleen 3.1 ± 0.5 3.2 ± 2.2
Pancreas 0.7 ± 0.2 0.5 ± 0.2
Stomach 0.7 ± 0.2 0.6 ± 0.3
Intestine 0.5 ± 0.1 0.5 ± 0.3
Muscle 0.7 ± 0.3 0.7 ± 0.3
Bone 1.1 ± 0.3 0.9 ± 0.4
Skin 1.3 ± 0.5 1.4 ± 0.4
Brain 0.1 ± 0.0 0.2 ± 0.1
Tumour 26.1 ± 1.2 6.2 ± 1.0
T/B ratio 4.2 0.94
T/M ratio 37.3 8.8
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Tumour uptake could be significantly reduced (6.2 ± 1.0 ID%/g (p < 0.05)) in mice pretreated with an excess panitumumab (1 mg) one hour before radiotracer administration. In mice pretreated with panitumumab, tumour uptake was comparable to that of the blood at 120 min p.i. as represented by a T/B ratio of 0.94 at 120 h p.i. The remaining radioactivity in the tumour under EGFR-blocking conditions can be attributed to the EPR effect.

Discussion

EGFR + HNCC accounts for almost 90% of HNCC cases diagnosed in the clinic (Fasano et al. 2021). This expression offers EGFR as a suitable receptor to target head and neck cancer cells when developing radiotheranostics for HNCC. Creating a radiotheranostic pair using radionuclides of the same chemical element that targets EGFR provides a probe for imaging and treatment with similar pharmacokinetics, leading to similar biodistribution in EGFR + tumours (Li et al. 2020; Srivastava 2012).

As in the presented work, the uptake and biodistribution of 203Pb-PSC-panitumumab was studied in vitro on EGFR + HNCC FaDu cells as well as in mice bearing patient-derived xenografts which were taken from patients with EGFR + HNCC.

Immuno-SPECT probe 203Pb-PSC-panitumumab displayed EGFR-mediated uptake in vitro and in vivo with high tumour retention, enabling the detection of tumours at 48 h and 120 h p.i.. Previous reports in the literature for radiolabelling panitumumab with SPECT-detectable radioisotopes were labelling with 111In (Facca et al. 2023) and 177Lu (Aghevlian et al. 2018); also, the Fab fragment of panitumumab was labelled with 177Lu (Ku et al. 2021) and 99mTc (Ku et al. 2019); however, no previous studies describing labelling panitumumab with 203Pb were reported. Antibody-based compounds such as trastuzumab (Herceptin) via DOTA (Garmestani et al. 2005), c8C3 via TCMC (Jiao et al. 2023), and peptides such as α-melanocyte-stimulating hormone (Miao et al. 2008), and low molecular weight PSMA ligands (Banerjee et al. 2020) have been labelled with 203Pb using DOTA coordination chemistry. Also, the conditions required for labelling panitumumab with 111In using diethylenetriamine-pentaacetic acid (DTPA) and/or 89Zr using p-isothiocyanatobenzyl-desferrioxamine B included heating to 37 °C, and depending on the chelator administered, different reaction times of up to 4 h were required (Ray et al. 2009; Nayak et al. 2012). Moreover, high temperatures (60–75 °C) were reported for the radiolabelling of peptides with 203Pb to achieve high incorporation efficiency with shorter incubation times (Liu et al. 2014; Nayak et al. 2010). However, in the presented work, the PSC-NCS chelator labelling process did not require any elevated temperatures, and 203Pb2+ incorporation proceeded with high efficiency (> 99%) at short reaction times of 5–10 min at room temperature. The observed high labelling efficiency > 99 aligns with previously reported data using PSC-NCS as a lead-specific bifunctional chelating agent for rapid coordination chemistry with 203Pb2+ under mild conditions (Nelson et al. 2023).

Figure 1D indicates that both light and heavy chains of panitumumab are labelled with 203Pb, confirming the bioconjugation of lysine residues through thiourea formation with the PSC-NCS chelator being present in both light and heavy chains of panitumumab (Ho 2023). Modifying both heavy and light chains of panitumumab can also explain the only moderate immunoreactivity of ~ 30% (Fig. 2C). However, 203Pb-PSC-panitumumab still provided clear SPECT images for detecting EGFR + HNSCC PDX tumours.

The specific binding to EGFR was confirmed by blocking studies by pre-administering an excess of panitumumab. Also, 203Pb-PSC-panitumumab allowed for imaging to be conducted at several time points for SPECT/CT imaging by remaining in the tumour up to 120 h post-injection in tumour-bearing mice. Our findings indicate that 203Pb-PSC-panitumumab is a suitable SPECT probe with desirable biodistribution for EGFR + HNCC. 203Pb-PSC-panitumumab was taken up by FaDu cells, and the uptake could be reduced under blocking conditions in a concentration-dependent manner using panitumumab. Indicating that the cell uptake of 203Pb-PSC-panitumumab is receptor mediated and not affected by PSC chelators. SPECT/CT images acquired from tumour-bearing mice after the injection of 203Pb-PSC-panitumumab allowed clear delineation of the tumour tissue, confirming high tumour uptake and retention of the radioligand in the EGFR + HNSCC PDX model.

Our work aligns with reported PET and SPECT data in mice using 89Zr-, 177Lu- and 111In-labelled panitumumab immunoconjugates (Ray et al. 2009; Nayak et al. 2012; Liu et al. 2014), confirming that radioligand 203Pb-PSC-panitumumab is a suitable immuno-SPECT probe for detecting EGFR + tumours. The long physical half-life of 203Pb (t1/2 = 51.9 h) allows SPECT imaging protocols for several days at a high image quality.

The biodistribution data (%ID/g) in tissues and organs confirmed the SPECT/CT data in mice injected with 203Pb-PSC-panitumumab. The highest radioactivity was measured in tumours (26.1 ± 1.2) after 120 p.i. of 203Pb-PSC-panitumumab, which is comparable to the radioactivity accumulation and retention in reported for LS-174 T, SHAW and SKOV-3 xenografts using 111In-CHX-A”-DTPA-panitumumab (Ray et al. 2009; Chopra 2004) and MDA-MB-468 xenografts using 89Zr-labelled panitumumab (Nayak et al. 2012). The observed biodistribution pattern was also similar to a study using 86Y-CHX-A”-DTPA-panitumumab in EGFR + human colorectal, prostate, and epidermoid tumour xenografts (Nayak et al. 2010). Radioactivity uptake in EGFR + HNSCC PDX could be reduced by ~ 75% by predosing mice with panitumumab (1 mg), confirming EGFR-mediated uptake of radioligand 203Pb-PSC-panitumumab. As panitumumab is a human antibody against human EGFR it does not bind to mouse EGFR; therefore, administering an excess of panitumumab before injecting 203Pb-PSC-panitumumab will only block EGFR on the tumour. The remaining radioactivity amount (6.24 ± 0.97%ID/g) can be attributed to the EPR effect commonly observed during SPECT and PET imaging in solid tumours using radiolabeled immunoconjugates (Sharma et al. 2014).

Our work with 203Pb-PSC-panitumumab as a novel immuno-SPECT probe highlights the opportunity for developing 203/212Pb-PSC-panitumumab as ideal radiotheranostics for combined SPECT imaging and targeted alpha therapy of EGFR-expressing cancers. 203/212Pb radiotheranostics represent an attractive alternative to currently used 225Ac-based radiotheranostics for targeted alpha therapy.

The availability of 212Pb through the emerging 224Ra/212Pb generator technology and the ideal radionuclide matching pair characteristics of radiometals 203Pb and 212Pb ensure identical coordination chemistry and identical biodistributions profiles of 203/212Pb radiotheranostics. This represents a significant advantage to currently used 225Ac-based radiotheranostics for targeted alpha therapy, which rely on chemically different imaging surrogates, such as 133La and 134Ce, as no Ac isotopes are available suitable for imaging (Nelson et al. 2023).

Conclusion

In this study, panitumumab was successfully modified with PSC-NCS as a novel Pb-specific bifunctional chelating agent. PSC-decorated human antibody panitumumab was rapidly and reproducibly radiolabelled with 203Pb(OAc)2 in good radiochemical yields under mild reaction conditions compatible with the structural and functional integrity of an antibody. Novel immunoSPECT probe 203Pb-PSC-panitumumab showed EGFR-mediated uptake in FaDu cells and high uptake and retention in an EGFR + HNSCC PDX model. EGFR-mediated uptake was confirmed by SPECT/CT and biodistribution studies using blocking experiments with panitumumab. Our work introduces 203Pb-PSC-panitumumab as a novel immuno-SPECT probe for imaging EGFR + tumours and an opportunity to develop 203/212Pb-PSC-panitumumab radiotheranostics for combined SPECT imaging and targeted alpha therapy of EGFR-expressing cancers.

Acknowledgements

Nasim Sarrami expresses gratitude for the NSERC-CREATE “Polymer Nanoparticles for Drug Delivery (PoND)” program, Alberta Innovates, and Graduate scholarships from the Faculty of Pharmacy and Pharmaceutical Sciences of the University of Alberta. The authors thank the Cross Cancer Institute pharmacy for generously gifting Vectibex®.

Author contributions

NS was responsible for the synthesis of 203Pb-PSC-panitumumab, cell-based experiments and SPECT imaging and writing the manuscript; BN, SM, JW, and MS were responsible for preparing 203Pb and PSC-NCS; CC, JM, TK, LA contributed to the PDX-model experiments, SPECT imaging and biodistribution; MW contributed to data analysis, AL, RR, LA and FW edited and revised the manuscript. FW and RR were responsible for the design of the study and critically reviewed the manuscript; and all authors read and approved the final manuscript and agreed to be accountable for the integrity of the work.

Funding

The work was supported by a New Frontiers in Research Fund-Transformation grant with funds from the Government of Canada and the Dianne and Irving Kipnes Foundation.

Availability of data and materials

Not applicable.

Declarations

Ethics approval and consent to participate

All experimental protocols were approved by the Research Ethics Board at the University Health Network (Protocol No. 12–5639). All methods were performed per the ethical standards laid down in the Declaration of Helsinki and its later amendments or comparable ethical standards.

Consent for publication

Not applicable.

Competing interests

All authors declare no competing financial or non-financial interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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