^86/90Y-Labeled Monoclonal Antibody Targeting Tissue Factor for Pancreatic Cancer Theranostics

Abstract

Pancreatic cancer is highly aggressive, with a median survival time of less than 6 months and a 5-year overall survival rate of around 7%. The poor prognosis of PaCa is largely due to its advanced stage at diagnosis and the lack of efficient therapeutic options. Thus, the development of an efficient, multifunctional PaCa theranostic system is urgently needed. Overexpression of tissue factor (TF) has been associated with increased tumor growth, angiogenesis, and metastasis in many malignancies, including pancreatic cancer. Herein, we propose the use of a TF-targeted monoclonal antibody (ALT836) conjugated with the pair ^86/90Y as atheranostic agent against pancreatic cancer. For methods, serial PET imaging with ⁸⁶Y-DTPA-ALT836 was conducted to map the biodistribution the tracer in BXPC-3 tumor-bearing mice. ⁹⁰Y-DTPA-ALT836 was employed as a therapeutic agent that also allowed tumor burden monitoring through Cherenkov luminescence imaging. The results were that the uptake of ⁸⁶Y-DTPA-ALT836 in BXPC-3 xenograft tumors was high and increased over time up to 48 h postinjection (p.i.), corroborated through ex vivo biodistribution studies and further confirmed by Cherenkov luminescence Imaging. In therapeutic studies, ⁹⁰Y-DTPA-ALT836 was found to slow tumor growth relative to the control groups and had significantly smaller (p < 0.05) tumor volumes 1 day p.i. Histological analysis of ex vivo tissues revealed significant damage to the treated tumors. The conclusion is that the use of the ^86/90Y theranostic pair allows PET imaging with excellent tumor-to-background contrast and treatment of TF-expressing pancreatic tumors with promising therapeutic outcomes.

Graphical Abstract

INTRODUCTION

Pancreatic cancer (PaCa) accounts for about 50 000 new cancer cases per year in the U.S.¹ and is considered to be one of the most difficult to treat. As such, it is the fourth leading cause of cancer-related deaths in both sexes, with the lowest 5-year survival rate among all major cancers of around 7%.² Pancreatic adenocarcinomas, which account for over 90% of PaCa malignancies, have a strong tendency toward invasion and metastasis and, due to a lack of adequate screening strategies, patients are often diagnosed presenting with locally advanced or metastatic disease.³ The mechanisms of PaCa development and progression are still not fully understood, limiting the success of disease management. The standard of care for PaCa is surgical resection followed by adjunctive therapy.⁴ However, this treatment is not available to the majority of patients, since only 10–15% of patients are diagnosed early enough for surgery to be a viable option.⁵ Data also demonstrate that ~30% of resections have positive margins leading to recurrence. Moreover, traditional therapies, such as gemcitabine and radiotherapy, have very low success rates among these patients and their prognosis is consequentially poor.⁴ Unfortunately, this pattern is not expected to change and the death rate of PaCa patients is predicted to increase by a further 28% by 2026.⁶ While surgery currently offers the only chance to eradicate this type of tumor, postsurgery survival rates are still disappointing, often measured in months, even after margin-negative pancreatectomy.⁷ Therefore, the survival rate for PaCa can be improved solely if early diagnosis is achieved or if better PaCa therapeutic agents are developed. The development of an efficient, PaCa targeted theranostic system is urgently needed to improve PaCa patient management.

With advancements in our understanding of cancer biology, targeted therapeutic agents are being developed for many malignancies. Overexpression of tissue factor (TF) has been associated with increased tumor growth, tumor angiogenesis, and metastatic potential in many malignancies,⁸ including pancreatic cancer.⁹ TF expression has been correlated with VEGF expression and microvessel density, symptomatic thromboembolism, and decreased survival in PaCa patients.¹⁰ Additionally, in PaCa it has been correlated with high tumor grade, extent of the primary disease, and local and distant metastatic invasion. TF is expressed on 77% of pancreatic intraepithelial neoplasms, with no expression on health adjacent ductal tissue, and on 91% of intraductal papillary mucinous neoplasms, with higher expression indicating more extensive dysplasia.^11–13 Our group has previously demonstrated that ALT836, a chimeric monoclonal antibody (mAb) that has been subject to clinical trials¹⁴ and proven to target human TF,¹⁵ selectively accumulates in pancreatic tumor tissue with high affinity as well as great correlation between tumor uptake and TF expression levels.^16,17 Herein, we propose the use of ALT836 as the main targeting agent for pancreatic cancer theranostics. By use of identical radiochemistry, the same monoclonal antibody targeting TF can utilize the ^86/90Ytheranostic pair for PET imaging-guided radiation dose estimation for subsequent therapy.

EXPERIMENTAL SECTION

Cell Culture and Animal Models.

All experiments involving animals were conducted under an approved protocol by the University of Wisconsin—Madison Institutional Animal Care and Use Committee. BXPC-3 human pancreatic tumor cells were acquired from the ATCC (American Type Culture Collection). Cell culture was performed using RPMI 1640 medium supplemented with 10% FBS at 37 °C and 5% CO₂. 4–5 week old female Athymic Nude-Foxn1nu mice (Envigo) were inoculated, in the lower flank, with a 1:1 mixture of BXPC-3 cells (approximately 2 × 10⁶ cells) and Matrigel (BD Biosciences). At 7 days after tumor implantation, in vivo studies were carried out once tumors were palpable and reached approximately 10 mm in diameter.

Flow Cytometry.

Tissue factor binding activities of ALT836, DTPA-ALT836, and IgG to BXPC-3 and PANC-1 cells were analyzed by flow cytometry following methods previously described.¹⁶ Briefly, pancreatic cells were collected and resuspended in PBS with 1% bovine serum albumin (BSA). The cells (100 μL/test) were incubated for 30 min at room temperature with PBS (control), second antibody alone, ALT836 (5 μg/mL), DTPA-ALT836 (5 μg/mL), or IgG (5 μg/mL). The cells were then thoroughly washed with PBS incubated with AlexaFluor488-labeled secondary antibody (5 μg/mL). The samples were measured using The MACSQuant cytometer (Miltenyi Biotech, Bergisch Gladbach, Germany), and data were processed using FlowJo analysis software (Tree Star, Inc.).

⁸⁶Y Production and Antibody Radiolabeling.

⁸⁶Y (t_1/2 = 14.7 h, 31.9% β⁺, E_β+ave = 660 keV) was produced according to a previously described method using a 16 MeV GE PETtrace cyclotron.¹⁸ Briefly, a transmutation reaction ⁸⁶Sr-(p,n)⁸⁶Y from enriched ⁸⁶SrCO₃ targets of pressed powder was achieved. ⁸⁶Y was then isolated from the irradiated ⁸⁶SrCO₃.

For the radiolabeling with yttrium isotopes, diethylenetriaminepentaacetic acid (DTPA) was selected as the chelator and was conjugated to the antibodies (ALT836 or IgG) according to a previously described procedure.¹⁹ DTPA-ALT836 or DTPA-IgG was then incubated for 1 h at 37 °C with ⁸⁶YCl₃ in a sodium acetate buffer, under constant agitation. The radiolabeled antibodies were purified using a PD-10 column (GE Healthcare). Radiolabeling yields of both Y-86 and Y-90 were determined using TLC plates at different time points. The yields of Y-labeled ALT836 and IgG were above 90% (Figure S2).

PET Imaging and Biodistribution Studies.

An amount of 3–6 MBq of ⁸⁶Y-DTPA-ALT836 or ⁸⁶Y-DTPA-IgG was intravenously injected in mice bearing BXPC-3 xenografts. PET scans were performed on an Inveon PET/CT scanner (Siemens) and collected using 100 million coincidence events. Serial PET scans were taken at 4, 12, 24, and 48 h postinjection (p.i.). Regions-of-interest (ROI) were calculated using the Inveon Research Workspace (Siemens). Following the last PET scan, mice were euthanized via CO₂ asphyxiation. The major organs were collected, and their radioactive contents were measured using a γ counter (PerkinElmer). Results of PET ROI analysis and biodistribution studies are presented as percent injected dose per gram of tissue (%ID/g).

Therapeutic Administration.

ALT836 and IgG radiolabeling with the ⁹⁰Y therapeutic isotope were carried out using the same methodology used to prepare the PET agents. DTPA-ALT836 and DTPA-IgG were incubated with 100–150 MBq of ⁹⁰YCl₃ in sodium acetate buffer for 1 h at 37 °C and then purified via PD-10 columns using sterile PBS. Each treatment group, outlined in Table 1, was composed of five BXPC-3 tumor bearing mice with therapeutic agent dosing determined from previous studies and literature.^18,20 Therapeutic administration was given after the implanted tumors were palpable and reached around 10 mm in diameter. Tumor volume data are presented as relative tumor volumes (tumor volume found for that time point divided by the initial tumor volume found on day 0). Day 0 refers to the first day therapeutic administration was given 7 days after tumor cells were implanted.

Table 1.

Groups and Doses for the Radioimmunotherapy Study

treatment group	therapeutic agent
90Y-DTPA-ALT836	~5.5 MBq 90Y-DTPA-ALT836
90Y-DTPA-IgG	~5.5 MBq 90Y-DTPA-IgG (human isotype IgG control)
ALT836 only	45 μg of ALT836 in 50 μL of PBS
PBS only	50 μL of PBS

Treatment Monitoring and Cherenkov Imaging.

Throughout the course of the study, mice were monitored every other day. Tumor dimensions were measured using digital calipers, and body weight measurements were taken. Tumor volume and relative tumor volume as V/V₀ (V₀ = tumor volume at day 0) were calculated. Cherenkov luminescence imaging of ⁹⁰Y-labeled tracers was also carried out every other day using an IVIS optical imaging system (PerkinElmer) with open filter and 60 s exposure. The humane end point criterion was selected to be a tumor volume increase of more than 300%, body weight drop of more than 20%, or if general health was deemed to be too poor to continue. The duration of the study was 15 days, corresponding to approximately 5.6 half-lives of the therapeutic isotope (64 h).

Blood and Tissue Analysis.

Blood samples were collected from mice of all groups via retro-orbital bleed prior to the therapeutic study and at day 15 p.i. Complete blood count measurements were undertaken in an Abaxis VetScan HM5 hematology analyzer. At the end of the study, aliquots of 200 μL of blood serum were collected and submitted for liver and kidney function analysis. For histological assessment, heart, lungs, kidney, spleen, liver, and tumor were excised from mice of all groups after the end of the study. Each organ was hematoxylin and eosin (H&E) stained to monitor histological changes.

Radiation Dosimetry.

PET ROI analysis data of ⁸⁶Y-DTPA-ALT836 and ⁹⁰Y-DTPA-IgG injected mice were used to extrapolate the doses of radiation absorbed by the major organs of an adult human female. Tumor doses were calculated using dose-to-sphere modeling. All the aforementioned activities were carried out using OLINDA/EXM software.²¹

Statistical Analysis.

All quantitative data are presented as the mean ± standard deviation. Direct comparisons between groups were made using a two-sided Student’s t test, where p < 0.05 was considered statistically significant.

RESULTS

In Vitro Assay and Radiochemistry.

Flow cytometry results are shown in Figure S1 and demonstrated high TF expression in BXPC-3 cells in comparison with the PANC-1 pancreatic cancer cell line. In addition, no differences between ALT836 and DTPA-ALT836 were observed. For PANC-1 cells, which are considered TF-negative, neither ALT836 nor DTPA-ALT836 bound to the cells. Binding affinity of IgG to either BXPC-3 or PANC-1 was also tested and found to have no specificity. These results confirmed that DTPA conjugation did not affect binding affinity or specificity of ALT836. Additionally, the IgG antibody would be a good negative control in future experiments. These results are in accordance with similar experiments described elsewhere.^16,17

Radiolabeling with ⁸⁶Y was attained with a yield above 90% for all time points studied with radiochemical purity of >95% (Figure S2). Similar results were found for ⁹⁰Y-radiolabeling of DTPA-ALT836 and DTPA-IgG as well as ⁸⁶Y-labeling of IgG (data not shown due to identical radiochemistry).

PET Imaging.

After administration of 3–6 MBq of ⁸⁶Y-DTPA-ALT836, serial PET images were acquired at 4, 12, 24, and 48 h p.i. of BXPC-3 tumor-bearing mice (Figure 1). The tumor was apparent after the first time point (4 h p.i.), in which tumor uptake was 8.6 ± 2.4 %ID/g (n = 4) (Figure 1A). Tumor uptake increased over time and peaked at 27.3 ± 11.6 %ID/g at 48 h p.i. (Figure 1B). The tracer was slowly cleared from circulation, with 8.8 ± 1.2 %ID/g in the blood pool at 4 h p.i. and 3.8 ± 0.9 %ID/g remaining at 48 h p.i. The nontargeted organ with the highest radiotracer accumulation was the liver, with an uptake of 12.4 ± 1.5 %ID/g at 4 h p.i., ascribed to typical antibody clearance patterns.²² Similar studies were conducted using ⁸⁶Y-DTPA-IgG, an isotype control antibody (Figure 1). Tumor uptake in this group reached a maximum of 5.3 ± 0.9 %ID/g at 48 h p.i. Similar clearance patterns were observed with this tracer, with liver accumulation of 7.6 ± 1.1 %ID/g observed at the first time point. Tumor uptake was higher for the group injected with the ALT836-labeled antibody and was statistically significant at all time points when compared to the IgG group (Figure 2A). In addition, tumor-to-blood and tumor-to-muscle ratios were calculated for both groups and were statistically significant at all time points investigated (Figure 2B,C). Tumor-to-muscle ratios peaked at 40.8 ± 20.7 for ⁸⁶Y-DTPA-ALT836 and 8.5 ± 5.9 for ⁸⁶Y-DTPA-IgG, both at 48 h p.i. Similarly, the tumor-to-blood ratio reach maximums at 48 h p.i. for ⁸⁶Y-DTPA-ALT836 of 7.3 ± 2.7 (Figure 2B) and 1.3 ± 0.3 of ⁸⁶Y-DTPAIgG (Figure 2C).

Figure 1.

In vivo PET and ex vivo biodistribution results of mice injected with ⁸⁶Y-DTPA-ALT836 (left column) or ⁸⁶Y-DTPA-IgG (right column) (n = 4). (A) Representative serial PET MIP images at different time points postinjection. Tumor site is shown by red arrows. (B) ROI analysis of blood pool and major organs. (C) Ex vivo biodistribution results at 48 h postinjection.

Figure 2.

(A) Direct comparison of tumor uptake, (B) tumor-to-blood, and (C) tumor-to-muscle ratios of the mice injected with ⁸⁶Y-ALT836 or ⁸⁶Y-IgG, based on the quantitative data obtained from the analysis of PET images: *p < 0.05 and **p < 0.005; n = 4.

Ex Vivo Studies.

Following the last imaging time point, mice were euthanized, and main organs were excised and wet-weighed, and their radioactive contents were measured using a γ counter (Figure 1C). Tumor uptake of ⁸⁶Y-DTPA-ALT836 was 34.8 ± 14.9 %ID/g and 3.8 ± 0.9 %ID/g for ⁸⁶Y-DTPAIgG, both similar to the PET ROI analysis.

Treatment Monitoring and Therapeutic Efficacy.

Cherenkov luminescence imaging of mice injected with ⁹⁰Y-DTPA-ALT836 or ⁹⁰Y-DTPA-IgG allowed a more broad visualization of the tracer biodistribution. The luminescent signal in the tumors of each group was measured for at least 5 d p.i.(Figure 3). Relatively higher signal levels that persisted slightly longer in the tumor were found for the specific antibody compared to the nonspecific group (Figure S3).

Figure 3.

Long-term Cherenkov luminescence imaging of mice injected with (A) ⁹⁰Y-DTPA-IgG and (B) ⁹⁰Y-DTPA-ALT836.

Therapeutic studies were carried out by injecting ~5.5 MBq of ⁹⁰Y-DTPA-ALT836 or ⁹⁰Y-DTPA-IgG. Additionally, PBS and ALT836 groups were used as controls. The tumor volume of ⁹⁰Y-DTPA-ALT836 was found to be statistically smaller than PBS treated mice at 1 d p.i., ⁹⁰Y-DTPA-IgG treated mice at 3 d p.i., and ALT836 treated mice at 7 d p.i., and all time points thereafter (p < 0.05, n = 5) (Figure 4A). The overall survival curve demonstrated a significantly longer median survival for mice treated with ⁹⁰Y-DTPA-ALT836 (Figure 4B). None of the mice in the ⁹⁰Y-DTPA-ALT836 group had to be removed from the study from reaching the tumor volume end point criteria (>300% of initial) throughout the entire study, while 5/5 of the mice treated with PBS or ALT836 only and 4/5 mice treated with ⁹⁰Y-DTPA-IgG either died or reached the end point value (Figure 4B). These results substantiate the antitumor effect from radioimmunotherapy for pancreatic cancer using ⁹⁰Y and ALT836 to target TF expressed on tumor tissue.

Figure 4.

Therapeutic studies with 5 mice per group. (A) Tumor growth curve. Significantly smaller relative tumor volumes found for ⁹⁰Y-DTPA-ALT836 treated group in comparison with assigned groups in the graph. (B) Overall survival. Extension in median survival was observed in the group treated with ⁹⁰Y-DTPA-ALT836.

Toxicity Evaluation.

Several studies were performed to investigate potential acute toxicities associated with the ⁹⁰Y-labeled antibodies used for therapy. H&E staining of the major organs revealed no abnormal histopathological changes in any of the organs other than the tumor for all groups (Figure 5). Tumor tissues of mice injected with ⁹⁰Y-DTPA-IgG had some signs of necrosis, but that was not observed for either PBS or ALT836 treated mice. Importantly, tumor tissues of mice injected with ⁹⁰Y-DTPA-ALT836 presented significant damage and large areas of necrosis (Figure S4). Throughout the study, no groups presented body weights close to the weight-loss end point (below 80% of initial), indicating low toxicity of the treatment agents (Figure S5A). As shown in Figure 6, kidney and liver function tests demonstrated no statistical difference between PBS treated mice and the other groups. However, data from complete blood count revealed certain toxicity from the radioimmunotherapy treatments (Figure S5B). Decreased white blood cell, lymphocyte, monocyte, neutrophils, and platelet counts were found in those groups administered Y-90 compared with the baseline values attained prior to treatment.

Figure 5.

Histological evaluation of different tissues from all therapeutic groups at 15 days postinjection. Scale bar = 100 nm.

Figure 6.

Kidney and liver function tests. Serum values of (A) urea nitrogen, (B) creatinine, (C) alkaline phosphatase (ALP), (D) aspartate amino transferase (AST), (E) alanine amino transferase (ALT), and (F) bilirubin collected at 15 days postinjection for all treatment groups. n = 5.

Dosimetric Extrapolation Using PET Data.

Dosimetric extrapolation of ⁹⁰Y-DTPA-ALT836 and ⁹⁰Y-DTPA-IgG organ doses to an adult human female was performed using OLINDA/EXM software (Figure 7). As seen in Figure 7A, the nontargeted organ with the highest dose for both groups was the liver, at 3.48 ± 0.59 mSv/MBq for radiolabeled ALT836 group and 1.46 ± 0.59 mSv/MBq for ⁹⁰Y-DTPA-IgG injected group. Dose received by tumor tissue was also estimated (Figure 7B), and for a 1 g tumor mass, a dose of 106 ± 30 mGy/MBq was calculated for the group injected with ⁹⁰Y-DTPA-ALT836, significantly higher than a dose of 11 ± 3 mGy/MBq found for the group injected with ⁹⁰Y-DTPA-IgG.

Figure 7.

Dosimetry of ⁹⁰Y-DTPA-ALT836 and ⁹⁰Y-DTPA-IgG. (A) Doses to major organs. (B) Tumor doses calculated through dose-to-sphere modeling. n = 4.

DISCUSSION

Targeted radionuclide therapy (TRT) is an approach that combines a targeting molecule with a radioisotope to deliver a therapeutic level of radiation to tumor sites.²³ TRT is similar to chemotherapy in that it is administered systemically but differs by being able to exert a crossfire effect and killing adjacent tumor cells even if the specific tumor-associated antigen is not present. Another major advantage of TRT is its potential to simultaneously act on both primary tumor and metastatic sites, even if the metastatic site is undetectable by traditional diagnostic imaging.²⁴ Several clinical trials of TRT of pancreatic cancer have demonstrated these agents to be safe and to potentially have a therapeutic effect.²⁵ Even though recent TRT developments seem promising, especially those combined with chemotherapeutics,²⁶ the treatment of PaCa is still a challenge. That is mostly due to late diagnosis, chemotherapy and/or radiotherapy resistance, tumor heterogeneity, and lack of personalized therapeutics. Thus, the development of new TRT approaches and also the diagnostic visualization of targets to determine whether patients will benefit from a particular treatment will tremendously contribute to personalized medicine efforts for PaCa management.²⁷ The use of a theranostic approach, by first performing diagnostic and predictive PET scans followed by a personalized treatment decision, could significantly increase therapeutic efficacy. This approach can be quite complicated since the radionuclides are rarely the same for both diagnosis and therapy. While the nuclide for imaging must emit short path length radiation (such as positrons), those for therapy must penetrate a higher depth of tissue (γ rays).²⁴ For example, ⁹⁰Y is a very promising nuclide for targeted radionuclide therapy based on its conjugation with antibodies or peptides, and many of them have been/are currently part of clinical trials.^28–31 However, since ⁹⁰Y is a pure β⁻ emitter, its in vivo pharmacokinetics cannot be imaged or quantified for patient-specific dosimetry quantification. The use of a theranostic isotopic pair that utilizes two radionuclides of the same element allows for equivalent radiochemistry to be used for PET and internal radiotherapy. Although ⁸⁶Y is not yet clinically available, its decay characteristics (33% positron emission and t_1/2 of 14.7 h) are compatible with nuclear medicine imaging.³² More importantly, due to the chemical similarity of ⁸⁶Y to ⁹⁰Y, ⁸⁶Y-labeled probes have identical biodistribution of ⁹⁰Y-probes and therefore should inherently enable more accurate absorbed dose estimates for ⁹⁰Y-based therapy.³³ As such, a pancreatic cancer-specific target coupled with ^86/90Y pair through the same radiochemistry is an ideal diagnostic agent with predictive value for TRT as well as the TRT agent itself.

Tissue factor is overexpressed in many types of tumors and has been suggested to have a key role in the development of cancer-associated thrombosis, tumor growth, tumor angiogenesis, and tumor metastasis. ALT836, a recombinant human-chimeric monoclonal antibody, is an antagonist of tissue factor and exhibits antitumor, antithrombotic, and anti-inflammatory activities with a remarkable safety profile in preclinical studies.^15,34,35 ALT836 has also been investigated in clinical trials as a therapeutic agent in coronary artery disease (CAD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS).¹⁴ Our group previously demonstrated that radiolabeled ALT836 antibody specifically accumulates in pancreatic tumor bearing mice,^16,17 with good correlation between radiotracer accumulation and TF expression levels. In addition, PaCa tissue staining revealed TF expression prevalence,¹⁶ demonstrating the promise of TF-targeting for imaging and therapy of this disease. These encouraging results warranted further exploration of radiolabeled ALT836 as a PaCa-targeted theranostic agent. Even though the use of ¹⁷⁷Lu-Dotatate has been recently approved for the treatment of pancreatic neuroendocrine tumors,³⁶ we still believe ⁹⁰Y-DTPA-ALT836 is clinically relevant. First, there are intrinsic differences between ¹⁷⁷Lu and ⁹⁰Y emission characteristics. It has been reported that the mean absorbed dose to tumors is much higher for ⁹⁰Y than ¹⁷⁷Lu-labeled conjugates after administration of the same level of radioactivity, which can lead to improved therapeutic efficacy and survival rates.³⁷ Second and most importantly, the therapeutic agents act within distinct targets. While ¹⁷⁷Lu-Dotatate targets somatostatin receptors (SSRT), ALT836 targets tissue factor (TF). Although it is definitely hard to compare targets in regards to pancreatic cancer, tumor heterogeneity is well-known and not all pancreatic tumors overexpress SSRTs or TFs. Therefore, the ability to have a different therapeutic option could be extremely relevant in the clinical settings, especially for pancreatic cancer patients with limited therapeutic options.

Herein, we investigated ^86/90Y-labeled ALT836 to act as a diagnostic, predictive, and therapeutic agent for pancreatic cancer. We first confirmed TF-expression using the ALT836 antibody (with and without the chelator) and found high binding specificity to the PaCa cell line BXPC-3, as found in previous reports.^16,17 Imaging using ⁸⁶Y or ⁹⁰Y allowed us to visualize and monitor PaCa tumors for up to 48 h through PET imaging, solving the tissue penetration depth limitation of Cherenkov luminescence imaging of ⁹⁰Y.³⁸ Tumor tissue was clearly visualized by ⁸⁶Y-DTPA-ALT836 at all time points with impressive tumor-to-muscle ratios and with lower background than usually found for ⁸⁶Y PET Imaging.³⁹ Tumor uptake was significantly higher for ⁹⁰Y-DTPA-ALT836 compared to ⁹⁰YDTPA-IgG, demonstrating the specificity of ALT836 to accumulate in PaCa tumor tissues. In the therapeutic studies, some antitumor effect with the nonspecific tracer was observed, probably due to EPR effect-based tumor accumulation and the high energy of ⁹⁰Y emission. Nonetheless, the therapeutic efficacy of the Y-90 labeled IgG was still significantly less than that found for the specific tracer. Since it has been previously demonstrated that the ALT836 antibody has antitumor effects, we hypothesized that combining those effects with targeted radiotherapy could produce a synergistic effect in suppressing tumor growth. Indeed, ALT836 alone slowed tumor growth compared to the PBS treated group, but ⁹⁰Y-DTPA-ALT836 treated mice presented significantly smaller tumor volumes at the majority of time points investigated. Those antitumor effects were also evidenced and confirmed by histological assessment of tumor tissue. Although tumor growth inhibition was achieved, complete tumor regression was not achieved using ⁹⁰Y-DTPA-ALT836. However, given that PaCa is very hard to treat because of the late diagnosis that makes surgical resection impossible, we believe that the theranostic ALT836 agent investigated is valuable for PaCa treatment management by enabling earlier diagnosis and patient stratification as well as diminishing tumor burden and increasing the possibility of surgical resection. Furthermore, the group treated with ⁹⁰Y-DTPA-ALT836 presented increased survival, as 80% of the mice were still alive at the end of the study while only 20% of the mice injected with the nonspecific tracer were still alive. Moreover, all the mice from the other groups were found deceased or were removed from the study for reaching end points. Importantly, no apparent toxicity for any of the groups was revealed by histological tissue analysis of major organs. However, radiation-induced toxicity can be a long-term phenomenon and further studies are warranted. Kidney and liver function tests demonstrated no significant differences between any treatment groups. Blood toxicity analysis revealed that mice injected with radiolabeled tracers had lower levels of blood components including platelets, indicating some acute toxicty. Future studies could optimize the TRT to reduce the observed toxicity. A few strategies that can be used to overcome the apparent radiotoxicity are the use of fractionated doses, the use of different antibody approaches such as the use of fragmented antibody⁴⁰ or pretargeting,⁴¹ and the use of a different theranostic isotopic pair.

It is known that one of the biggest limitations of internal radiotherapy is the quantification of radiation dose that reaches major organs, especially because of inaccurate external body measurements of internal dose.³² By extrapolating data from PET, it was possible to estimate the radioactive dose of ⁹⁰Y received by all the major organs and the tumor. With this approach, it will be possible to predict the amount of radioactivity that the tumor tissue of each patient will receive, as well as off-target doses. This will allow for truly precise and personalized plans. To the best of our knowledge, this is the first study reporting the use of a PaCa targeting agent radiolabeled with ^86/90Y for cancer theranostics.

CONCLUSION

Herein, we have employed a theranostic approach using the isotopic pair ⁸⁶Y/⁹⁰Y targeting tissue factor in a pancreatic cancer model. We demonstrated that the anti-TF monoclonal antibody, when radiolabeled with ⁸⁶Y, was selectively taken up by tumors cells and was clearly visualized through PET imaging at all time points investigated. This was further corroborated using ex vivo biodistribution studies. When radiolabeled with ⁹⁰Y, ALT836 allowed long-term tumor burden monitoring through Cherenkov luminescence imaging. In addition, ⁹⁰Y-DTPA-ALT836 exhibited promising therapeutic results by significantly slowing tumor growth when compared to all the other groups investigated. Even though antitumor effects were not sufficient to eradicate the tumors, a significantly increased average survival was achieved, as 80% of mice were still alive at the last time point of the study (compared to 20% for those injected with radiolabeled IgG and 0% for the other groups). These findings can potentially improve and contribute to a better PaCa management. Early diagnosis together with decreased tumor growth rate can possibly support surgical treatment and potentially provide a synergistic effect with other treatment options. Future investigation is necessary to conceivably improve antitumor efficacy and fully assess how ^86/90Y-DTPA-ALT836 can supplement or improve the current standard of care for pancreatic cancer.