New insight and future perspective of mesothelin-targeted agents in nuclear medicine

The aim of this review is to summarize the main applications of mesothelin-targeting agents in the diagnosis of different types of cancers with a brief mention of nuclear magnetic resonance. The articles taken into account were selected from PubMed, Scopus, and Web of Sciences, including research articles and abstracts that deal with radioimmunotherapy and new tracers for nuclear medicine and radiodiagnosis. Articles that are not in English have been excluded. Mesothelin-targeting agents were the subject of the selected articles in which tracers as 64Cu-DOTA-11-25mAb anti MSLN, 111In-MORAb-009-CHX-A″, 89Zr-MMOT0530A, 111In-amatuximab, 99mTc-A1, 89Zr-AMA, 89Zr-amatuximab, 64Cu-amatuximab, 89Zr-labeled MMOT0530A and 89Zr-B3 found application in detection of malignancies that overexpressed mesothelin. Only one article approached magnetic resonance imaging (MRI) diagnosis using superparamagnetic iron oxide nanoparticles linked to anti-mesothelin antibodies. The tracers proved to be highly sensitive in detecting mesothelin positive cells. 89Zr-labeled MMOT0530A could also be used to predict the suitability of patients to radioimmunotherapy. Radiolabeled anti-mesothelin antibodies could be crucial as a treatment tool and for predicting the eligibility and the response of the patient to radioimmunotherapy through the study of the expression grade of mesothelin. They can be a relevant tool for pancreatic adenocarcinoma, lung cancer, human epidermoid carcinoma, ovarian cancer, malignant mesothelioma in which mesothelin is widely expressed.


Definition and functions of mesothelin
Mesothelin (MSLN) is a glycoprotein of 40 KDa located on the surface cellular membrane through a glycosylphosphatidylinositol (GPI) linkage. The precursor of MSLN is a protein of 71 KDa encoded by 2138-bp-long cDNA and it is characterized by 628 amino acids. It is cleaved into two molecules: a 31 kDa N-terminal region called megakaryocyte potentiating factor (MPF) and a 40 kDa protein that is MSLN, the most frequent form present on the cellular membrane [3]. Different malignancies express MSLN on their membranes, while its expression is severely limited in healthy cells. Only low levels were described in pleura cells, pericardium and peritoneum cells [18] but also on the epithelium of the trachea [5], on Fallopian tubes epithelial cells [39], on the surface epithelium of the ovary, on the tunica vaginalis, the rete testis and tonsil [8,39]. Conversely, there are many examples of cancers that overexpress MSLN: gastrointestinal cancers such as adenocarcinoma of the pancreas, ampulla of Vater adenocarcinoma, cholangiocarcinoma, pancreatic ductal adenocarcinoma; also genital cancer are mesothelin-positive as endometrium adenocarcinoma and ovarian non-mucinous carcinoma [9]. Other malignancies as adenocarcinoma of the liver and lungs, the epithelial component of biphasic synovial sarcomas and mesothelioma also express MSLN [39,49]. The biological function of MSLN is not well understood. It is hypnotized that MSLN was involved in the metastatic spread of mesothelin-bearing cancer cells via its interaction with the protein carbohydrate antigen CA125/mucin 16 1 3 (MUC16) [22]. CA125 is a tumor antigen used for diagnosis and follow-up of ovarian cancer [4,43,50,59]. High levels of CA125 is expressed not only on the cell surface but it can be released in extracellular space in soluble proteolytic fragments form. As CA125 belongs to the mucin family of glycoproteins, it is also called mucin 16 or MUC16 [59]. In particular, the binding between CA125 and MSLN promotes ovarian cancer metastases facilitating their heterotypic cellular adhesion (CA125 itself inhibits the human natural killer cells cytolytic responses), [26,42], pancreatic cancer cell motility and then invasion into other tissues via matrix metalloproteinase (MMP)-7 activation [6,53]. Blocking the interaction between MSLN and CA125 with antibodies anti-MSLN, the heterotypic cell adhesion has been hindered [50]. Several studies (Table 1) indicate that MSLN expression is correlated with chemoresistance and shorter progressionfree survival and overall survival [7] given by the upregulation of anti-apoptotic proteins as Bcl-2 and Mcl-1 [53]. Its high expression, as recently observed, is correlated with poor prognosis in patients suffering from advanced serous ovarian cancer (SOD) [58]. The review aims to summarize the main uses of labeled MSLN antibodies in diagnosis also briefly citing the recent application in magnetic resonance imaging.

Diagnosis with radiolabeled anti-mesothelin antibodies: an overall view
Clinical evidence suggests that MSLN could be considered a crucial target for the cancer treatment of MSLN positive tumors. Numerous clinical trials for MSLN-targeting methods gave promising results, as targeting MSLN with antibodies, antibody derivatives, antibody-drug conjugates, immunotoxin and CAR-T cells for therapy (Table 2). To give some examples, in a phase I trial of SS1P, an anti-MSLN immunotoxin, patients with mesotheliomas, ovarian and pancreatic cancer were enrolled and among the 33 treated patients, minor responses were seen in four patients, 19 had a stable disease whereas the progressive disease was seen in ten patients [20,53]. Analogously, a vaccination with autologous mesothelin-positive tumor cells induced a cytotoxic T cell response to mesothelin epitopes HLA-A2, A3, A2A in three patients who developed a delayed-type hypersensitivity after vaccination. It was evident that a cross-presentation by immunotherapy induced CD8 T cell responses that are tumor-specific and capable of eradicating cancer [57]. To give another example, it has been proposed to use MSLN -targeted thorium-227 ( 227 Th) to evaluate survival benefit of alpha therapy for MSLN positive cancers. BAY2287411, a human IgG1 antibody BAY86-1903 (Anetumab) covalently bound to 227 Th complexing 3,2-HOPO chelator was used. The agent had a potent in vitro and in vivo activity in models of breast, lung, ovarian and pancreatic cancer derived from cellular patient xenograft (PDX) [15,17]. Studies like these show how the detection of MSLN positive cancer cells is crucial to develop targeting tailored therapies and to gain more personalized treatments [10].

Mesothelin and pancreatic cancer
MSLN is also over-expressed in up to 86-100% of pancreatic ductal adenocarcinoma (PDAC) [35]. The differences of distribution of MSLN between PDAC and normal tissue were highlighted in a latest study through a standardized immunohistochemical detection with two anti-MSLN antibodies (EPR4509 and EPR19025-42). MSLN seems to be widely distributed on PDAC cell membranes while paracancer tissues had no expression of MSLN with a statistically significant difference (χ 2 = 40.615, p = 0.000). PDAC represents the fourth most frequent cause of death caused by cancer worldwide and has a 5-years overall survival that is less than 8%. Recent projections show a twofold increase in the PDAC incidence in the U.S. and Europe within the next ten years [40,47,48,52]. New treatments and the development of new sensitive approaches in diagnosis could therefore be an increasingly urgent need. In a clinical phase I trial the MSLN antibody MMOT0530A has been labeled with 89 Zr and administered to 11 IV stage patients. Among these, seven had PDAC and four ovarian cancer. In all patients, this method allowed the visualization of at least one lesion. The mean standardized uptake value (SUVmax) of the radiopharmaceutical in 37 lesions was 13.1 (± 7.5) on Positron Emission Tomography (PET) 4 days post-injection. In particular, 11.5 (± 7.5) in (N = 17) pancreatic and 14.5 (± 8.7) in (N = 20) ovarian cancer lesions [31]. Moreover, a single photon emission computed tomography (SPECT) radiopharmaceutical developed has been 111 In-labeled amatuximab. Of six patients enrolled in the trial, two had PDAC and four malignant mesothelioma. At the imaging, it was possible to recognize at least one tumor lesion from the background. In all patients, the tumor to background ratios (TBR) were 1.2 or superior (range 1.2-62.0), with higher TBR in mesothelioma than pancreatic adenocarcinoma. The uptake was evident in primary tumors but also in metastases [2,24,29,30,34,41]. Montemagno et al. proved that high expression of MSLN gene in PDAC patients correlates with a decreased overall survival in comparison with patients with lower expression [38]. In this study, Technetium-99 m ( 99m Tc)-A1, an anti-MSLN single domain antibody labeled with 99m Tc, was evaluated in vitro and in vivo. They used high-mesothelin-expressing AsPC-1 cells and moderatemesothelin-expressing SW1990 cells and developed a mouse model of PDAC (AsPC-1). They observed a 2.1-fold higher uptake in AsPC-1 cells than in SW1990 cells. As previously stated, the normal tissue exhibits the glycoprotein MSLN too but it is limited in healthy cells [18,28,39]. For this  89 Zr-AMA uptake was also evident in ex vivo studies conducted on tumoral cell lines. 89 Zr-AMA had a specific binding and provided a good tumor-to-background ratio, so giving important information on the accessibility of the tumor by antibodies and the availability of the target in tumors. It could be proposed as the Antibody-drug conjugates treatment tool, as this therapeutic efficacy is dependent on the accessibility, the representation and the internalization in the tumor target [55].

Radiolabeled MORAb-009
MORAb-009 (amatuximab) is an IgG1κ mAb (monoclonal antibody) against MSLN with an affinity of 1.5 nM for human MSLN [22]. Moreover, as shown in the paper by Shin et al. it has been linked to 2-(pisothiocyanatobenzyl)cyclohexyl-diethylenetriamine-pentaacetic acid (CHX-A″) and labeled with 111 In [51]. The distribution of the compound has been studied using a mouse bearing A431/K5 In another study, after the treatment with MORAb-009, an increase in serum CA-125 was evident. The increase of CA-125 was not due to a progression disease. After stopping the MORAb-009 treatment, the serum levels of CA-125 decreased. The increase of CA-125 during the treatment seemed produced by the inhibiting capacity of MORAb-009 towards the binding of tumor shed CA-125 to MSLN on mesothelial cellular membranes. The inhibition of the interaction between MSLN and CA-125 could also be an interesting therapy tool to hinder tumor metastases in mesotheliomas and ovarian malignancies [25]. In addition, it was demonstrated that MORAb-009 is well tolerated with a maximum tolerated weekly dose (MTD) of 200 mg/ m 2 [21]. A significant study was conducted by Lindenberg et al. that studied the biodistribution and dosimetry of amatuximab radiolabeled with 111 In in MSLN positive cancers [23,34]. Amatuximab is also used in mesothelioma cancer therapy. They enrolled seven patients, four with malignant mesothelioma and two with pancreatic adenocarcinoma. All the patients underwent SPECT/CT after administration of 111 In amatuximab except for one patient, unable to lie on the scanner. The SPECT/CT images were obtained after 2-4 h, 24-48 h and 96-168 h after the injection and TBR was of 1.2 (range 1.2-62.0). 111 In-amatuximab was taken up by both primary tumors and metastatic sites but this uptake was higher in mesothelioma than in pancreatic adenocarcinoma. Heart, liver, kidneys, and spleen were the physiological sites of uptake [54]. 111 In amatuximab reached the maximum TBR at 96-168 h after the injection. Before 96 h, the non-specific uptake of the background hinders the visualization of tumor uptake.

Diagnosis for antibody-drug conjugates treatment eligibility
An exciting field is represented by antibody-drug conjugate (ADC). ADC is a specific mAb linked with a cytotoxic drug. The acting mechanism is its release after internalization in the intracellular environment acting as a carrier to permit the internalization of potent cytotoxic drugs to tumor cells expressing the substrate for the specific antibody. Recently Hassan et al. [19] conducted a multicenter phase I study to establish the safety and the tolerability of anetumab ravtansine (BAY 94-9343), an ADC conjugated to the human immunoglobulin G1 anti-MSLN monoclonal antibody linked to the maytansine derivative tubulin inhibitor DM4. They enrolled 148 adult patients with multiple solid tumor types. Among these, ten patients were included in a dose-escalation study receiving a dose of 0.15-7.5 mg/kg anetumab ravtansine once every 3 weeks. They administered to the six patients with an advanced, recurrent ovarian cancer or a malignant mesothelioma a dose of 1.8 mg/kg once per week and 2.2 mg/kg once per week anetumab ravtansine once every 3 weeks. The study highlighted that the maximum tolerated dose of the ADC was 6.5 mg/kg once every 3 weeks or 2.2 mg/kg once per week. The side effects were fatigue, nausea, diarrhea, anorexia, vomiting, peripheral sensory neuropathy, and keratitis/keratopathy while related-drug deaths were not evidenced. These results demonstrated BAY 94-9343 safety, tolerability, and manageability in patients with advanced or metastatic solid tumors, including mesothelioma and ovarian cancer that was refractory to standard therapy. Common adverse events moreover were manageable with treatment interruptions or dose reductions. However, even though the encouraging results, the authors specified that all patients who responded to ADC had high MSLN expression tumors, some patients had at least 60% tumor MSLN expression but did not respond to BAY 94-9343. Further investigations are required to explore the correlation between MSLN expression and the response to the treatment also including other malignancies. Two other examples of a molecule that acts in this way are monomethyl auristatin E (MMAE) and maytansinoid drug conjugates, which is brentuximab vedotin for CD30-positive lymphomas and trastuzumabemtansine (TDM1) for metastatic breast cancer. An anti-MSLN antibody, MMOT0530A, was used to compare its effectiveness as ADC. It has been labeled with 89 Zr to evaluate imaging tumor absorption [13,56]. The inhibition of MSLN -targeted tumor growth after the administration of MMAE, ADC AMA-MMAE (DMOT4039A), was measured in mouse xenograft cancer models: OVCAR-3 X2.1 ovarian cancer, pancreatic cancers Capan-2, HPAC, AsPC-1, HPAF-II and mesothelioma MSTO-211H. The humanized IgG1 5B6, an antibody to HSV-1 viral coat protein gD, was used as nonbinding isotype control (anti-gD) and was linked to MMAE (anti-gD-MMAE). Tumor growth inhibition after the administration of ADC was compared with the tumor antibody uptake using the 89 Zr-labeled AMA. AMA-MMAE determined the greatest growth inhibition in OVCAR-3X2.1, Capan-2 and HPAC tumors. These isotypes had target-specific tumor uptake of 89 Zr-AMA. The less responsive xenografts (AsPC-1, HPAF-II, and MSTO-211H) did not have 89 Zr-AMA uptake even if they expressed MSLN. It was an interesting result giving the basis for a new perspective of treatment effects of the AMA-MMAE therapeutic ADC prediction. Nevertheless, absolute tumor absorption of 89 Zr-AMA is not necessarily predictive for the effectiveness of ADC. Many factors are important for the outcome. Examples are cellular sensitivity to the drug, the operation of drug efflux pumps or other resistance mechanisms, changes in vascular density and permeability, the nature of the extracellular matrix and the presence of non-internalizing soluble MSLN (Fig. 1) [56]. Lamberts et al. selected a study population of seven patients with unresectable metastatic pancreatic cancer that was histologically confirmed and four with platinum-resistant ovarian cancer. These patients had been included in the phase I study with DMOT4039A, an ADC formed by the humanized IgG1 mAb MMOT0530A and the mitotic agent monomethyl auristatin MMAE. The study aimed to identify tumor antibody uptake and the relation between the tracer uptake and MSLN response to the ADC treatment. 89 Zr-labeled anti-MSLN antibody MMOT0530A exhibited yet a specific tumor uptake on microPET at 1, 3 and 6 days after the injection of tracer yet in human MSLN positive tumorbearing mice [31,55]. In the study of Lamberts et al. 37 MBq 89 Zr-labeled anti-MSLN MMOT0530A was administered in all patients and after 2, 4, and 7 days post-injection the patients underwent to PET/CT imaging and the uptake was expressed as SUV. The convenient dose was 10 mg MMOT0530A with an improved visualization in the imaging at 4 and 7 days postinjection. 37 lesions has a mean SUVmax of 13.1 (± 7.5) on 4 days post-injection PET, with 11.5 (± 7.5) in (N = 17) pancreatic and 14.5 (± 8.7) in (N = 20) ovarian cancer lesions. The uptake visualized in blood, liver, kidneys, spleen, and intestine was attributable to the normal antibody distribution. The best response to DMOT4039A was in one patient that showed a partial response. They concluded that this tracer could give information in early drug development on tracer-antibody distribution in the whole body and their uptake in different tumor lesions to estimate which patients are eligible for the antibodybased treatment. The ZEPHIR study [16] demonstrated the predictive value of pretreatment with 89 Zr-trastuzumab PET in patients with metastatic breast cancer before they received 1 3 ADC TDM1 treatment. Otherwise, using 89 Zr-trastuzumab PET in combination with an early 18 F-FDG PET demonstrated a negative predictive value for RECIST response of 100%. This proved that combining these two techniques could be a potentially helpful tool to select the patient that could respond to ado-trastuzumab emtansine (T-DM1). Analogously, 89 Zr-MMOT0530A PET may be a promising tracer in combination with DMOT4039A to identify a patient that responds to the treatment with DMOT4039A [31].

Pitfalls of mesothelin use: effects of shed mesothelin on antibodies binding capacity
A limit of MSLN use is the presence in the bloodstream of high levels of shed antigen that hamper the capability of the mAb in biding tumor antigen [27,44]. Pimm et al. in particular, noticed that the immune complex formed by mAb and tumor antigen in mice with colon carcinoma xenografts had a hepatic localization of the radioiodinated monoclonal antibody due to its hepatic clearance [44,45]. To identify the effects of shed Ag on tumor uptake, two antibodies, anti-MSLN mAb amatuximab, and control anti-Lewis-Y mAb B3, were compared [33]. The population study has been constituted by a mouse model bearing A431/H9 tumor expressing both shed MSLN and noshed Lewis-Y antigen. At 24 h the tumor, hepatic, splenic uptake and the presence in bloodstream of 89 Zr-amatuximab was directly correlated with dose levels. Oppositely, the uptake of anti-Lewis-Y antibody 89 Zr-B3 in these districts was dose-independent. 89 Zr-amatuximab tumor uptake was evident in PET at 3 h post-injection (p.i.) at 10 and 60 µg dose levels. During the 3-48 h period, 89 Zr-amatuximab was washed out from the blood at the 10 µg dose while at the 60 µg dose 89 Zramatuximab gradually cleared from the bloodstream and a drastic increase in tumor uptake (%ID/g) was evident at 24 h and 48 h. 89 Zr-B3 had tumor uptake at 3 h p.i. at 15 and 1 3 60 µg doses. But, opposite to 89 Zr-amatuximab PET images, the uptake and the clearance of 89 Zr-B3 was not influenced by dose effects. These differences in biodistribution characteristics may be explained by the shed MSLN presence in blood and also by the tumor tissue that influences the serum half-life and the Ag-specific tumor uptake of radiolabeled amatuximab [33]. The effect of shed MSLN in blood circulation has already been previously defined in a study conducted on nude mice bearing the A431/K5 tumor: the tumor targeting of amatuximab labeled with 111 In or 64 Cu decreased in the presence of shed MSLN in the bloodstream. The spleen and liver uptakes increased. The blood retention and tumor uptake diminished when the dose was unable to saturate the shed MSLN. It was noted that the MORAb-009 dose affected the tumor uptake: a 30-μg dose was correlated to a higher tumor uptake than the 0.2-μg and 2-μg doses but it determined lower liver and spleen uptakes than the 0.2μg dose [51]. The injection of 30-60 μg amatuximab dose could increase the tumor uptake, maintaining lower liver and spleen uptake and allowing an easier penetration in the tumor core [32,51].

SPIONs and mesothelin
A recent prospective in diagnosis is using superparamagnetic iron oxide nanoparticles (SPIONs), 20-150 nm crystals of iron oxide with a functionalized shell that gains for the particles more stability in water and different biochemical properties for many uses in the biochemical field [14]. Shao et al. thought to bind SPIONs with Ab anti-mesothelin (A-MSF) to detect pancreatic carcinoma cells using mouse xenografts. The nanoparticle shell was constituted by silica that gave good biocompatibility to the compound, called Fe 3 O 4 @ SiO 2 (FS). A-MSF has a T2 relaxation rate of 59.435 nM/s and T1 of 0.549 nM/s. After the injection of the probe FS linked to A-MSF, a decrease of the tumor signal was observed demonstrating that the compound could be a valid T2 targeting agent in pancreatic cancer of nude mice [35].
There are different types of SPIONs based on dimension differences: oral SPIONs have a diameter between 300 nm and 3.5 μm, standard SPIONs size is 50-150 nm and ultra-small SPIONs have diameter smaller than 50 nm [1,12,14]. In a study of Deng et al. ultrasmall superparamagnetic iron oxides (USPIOs) were also linked to antimesothelin antibody-conjugated PEGlyated liposomal doxorubicin (M-PLDU). M-PLDUs obtained were spherical with a diameter about ∼ 180 nm and a zeta potential of about − 28 ∼ − 30 mV. Panc-1 human pancreatic cancer carcinoma cell line for in vitro antitumor activity evaluation was used. The half-maximal inhibitory concentration values were 0.53 µM for free DOX, 1.95 for M-PLDUs, and 3.5 for PEGlyated nanoimmunoliposome without antibody conjugation (PLDUs). These results showed that M-PLDU has an antitumor activity 1.8 times higher than PLDU. No significant cytotoxicity was determined by USPIOs. To establish the antitumor activity in vivo and to evaluate the anticancer effect of M-PLDU in the animal model, BALB/c nu/nu mice bearing Panc-1 xenografts (∼ 50-60 mm 3 ) were injected intravenously with FD, PLDUs and M-PLDUs once a week. The total DOX dosage administered was equal to 6 mg/kg (2 mg/kg). The antitumor effect of M-PLDUs was confirmed by the final tumor weigh in the animals: for M-PLDUs it was 0.06 g versus 0.10 g in the mice treated with PLDUs and 0.18 g in FD treatment. After 4 h from the injection, RMN showed the dropping of the tumor signal intensity (SI), where the lower SI demonstrated the higher concentration of iron oxide in the tissue. M-PLDUs demonstrated to be an affective delivery system to vehicle anticancer drugs but also a promising contrast agents to pancreatic cancer cells [11]. Another solution for immunotargeting against tumor antigens has been binding Biobodies (Bb) with SPIONs. Biobodies are fragments of antibodies that are biotinylated by yeast and secreted into the culture environment. In the research of Prantner et al. Bb against MSLN were obtained and linked to streptavidin-labeled nanoparticles (SA-SPIONs). Chemical-based strategies used for bioconjugating antibodies to nanoparticles have the deal to produce heterogeneous populations of antibodynanoparticle conjugates with different number and site of covalent bond formation. It can lead to a damage in the site of antibody binding conformation and inhibit the recognition of its antigen. SA-SPIONs, on the contrary, permit a selected and rapid self-assembly of immunotargeted nanoparticles in the yeast culture supernatant thanks to the high sensibility and specificity of the binding between Bb and SA-SPIONs. Even if it is necessary to conduct further investigation, Bb-SA-SPIONs represent a step forward in research of more and more specific therapies that can spare normal tissue limiting the systemic side-effects of nowadays chemotherapies [46]. Recently, dual-modality SPECT/MR imaging agents for SPECT/MRI scanners have been proposed with the aim to achieve a dual modality molecular imaging bioprobes for MSLN positive cancer. 111In labelled anti-mesothelin antibody mAbMB (111In-mAbMB), yet proposed as SPECT probes [37], has been conjugated with SPIONs. 74 kBq of 111InmAbMB-SPIONs were administered to three SCID mice bearing A431K5 tumor xenografts. A431K5 cells are human epidermoid A431 cells transfected with the plasmid pcD3CAK1-9 which encodes and produces the MSLN. After 24 and 72 h the mice were underwent 7 T MR imaging. The results demonstrated specific uptake into A431K5 tumors and a change of the SI was seen in MRI, as a hypointensity area, in correspondence with cancer localization and confirmed through autoradiographs [36].

3
Conclusions MSLN represents an intriguing subject of research. Different oncological applications have been explored, such as mesothelioma, ovarian adenocarcinoma, lung cancer, human epidermoid carcinoma to PDA and colon adenocarcinoma, as a consequence of the variety of MSLN expression in tumors. 64 Cu-DOTA-11-25mAb anti-MSLN, 111 In-MORAb-009-CHX-A, 89 Zr-MMOT0530A, 111 In-amatuximab, 99m Tc-A1, 89 Zr-AMA, 89 Zr-amatuximab, 64 Cu-amatuximab, 89 Zrlabeled MMOT0530A, and 89 Zr-B3 are the main compounds designed. SPIONs with A-MSF, instead, have been proposed as the T2 targeting agent in pancreatic cancer in MRI. 111 InmAbMB-SPIONs, indeed, was an exciting proposal as SPECT/MRI probe to detect MSLN positive cancers. Bb-SA-SPIONs are recent applications of nanotechnology developed to obtain a chemical-based strategy that does not interfere with the antibody site of binding of immunotargeted nanoparticles. All the tracers showed specific tumor uptake, while 89 Zr-labeled MMOT0530A could be used also to predict if a selected patient suits the DMOT4039A ADC treatment. Thus, radiolabeled Ab anti-MSLN could be crucial not only as a treatment tool but it could also be convenient in radioimmunotherapy. An example is M-PLDUs that demonstrated to be a valid vehicle anticancer drugs and a promising contrast agents for pancreatic cancer cells. The assessment of the expression grade of MSLN malignancies gives the possibility to predict the eligibility and the response of a single patient to radioimmunotherapy. It could be of primary importance for developing target therapy and, as a consequence, to be more specific in properly selecting the eligible patients for antibody-drug conjugate treatment.