Mesothelin Protein Research Articles

Mesothelin expression correlates with elevated inhibitory immune activity in patients with colorectal cancer

The expression of the protein Mesothelin (MSLN) is highly variable in several malignancies, including colorectal cancer (CRC), and high levels are associated with aggressive clinicopathological features and worse patient survival. Colorectal cancer is both a common and deadly cancer; being the third most common in incidence and second most common cause of cancer-related death. While systemic therapy remains the primary therapeutic option for most patients with stage IV (metastatic; m) CRC, their disease eventually becomes treatment refractory, and 85% succumb within 5 years. Microsatellite-stable (MSS) CRC tumors, which constitute more than 90% of patients with mCRC, are generally refractory to immunotherapeutic interventions. In our current work, we characterize MSLN levels in CRC, specifically correlating expression with clinical outcomes in relevant CRC subtypes, and explore how MSLN expression impacts the status of immune activation and suppression in the peritumoral microenvironment. Higher MSLN expression is prevalent in CMS1 and CMS4 CRC subtypes and correlates with higher gene mutation rates across the patient cohorts. Further, MSLN-high patients exhibit increased M1/M2 macrophage infiltration, PD-L1 staining, immune-inhibitory gene expression, enrichment in inflammatory, TGF-β, IL6/JAK/STAT3, IL2/STAT5 signaling pathways, and mutation in KRAS and FBXW7. Together, these results suggest that MSLN protein is a potential target for antigen-specific therapy and supports investigation into its tumorigenic effects to identify possible therapeutic interventions for patients with high MSLN expressing MSS CRC.

Enhanced Expression of Glycolytic Enzymes and Succinate Dehydrogenase Complex Flavoprotein Subunit A by Mesothelin Promotes Glycolysis and Mitochondrial Respiration in Myeloblasts of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive malignancy characterized by rapid growth and uncontrolled proliferation of undifferentiated myeloid cells. Metabolic reprogramming is commonly observed in the bone marrow of AML patients, as leukemia cells require increased ATP supply to support disease progression. In this study, we examined the potential role of mesothelin as a metabolic modulator in myeloid cells in AML. Mesothelin is a well-known marker of solid tumors that promotes cancer cell proliferation and survival. We initially analyzed alterations in mesothelin expression in the myeloblast subpopulations, defined as SSC-Alow/CD45dim, obtained from the bone marrow of AML patients using flow cytometry. Our results showed overexpression of mesothelin in 34.8% of AML patients. Subsequently, metabolic changes in leukemia cells were evaluated by comparing the oxygen consumption rates (OCR) of bone marrow samples derived from adult AML patients. Notably, a higher OCR was observed in the mesothelin-positive compared to the mesothelin-low and non-expressing groups. Treatment with recombinant human mesothelin protein enhanced OCR and increased the mRNA expression of glycolytic enzymes and mitochondrial complex II in KG1α AML cells. Notably, siRNA targeting mesothelin in KG1α cells led to the reduction of glycolysis-related gene expression but had no effect on the mitochondrial complex gene. The collective results demonstrate that mesothelin induces metabolic changes in leukemia cells, facilitating the acquisition of a rapid supply of ATP for proliferation in AML. Therefore, the targeting of mesothelin presents a potentially promising approach to mitigating the progression of AML through the inhibition of glycolysis and mitochondrial respiration in myeloid cells.

One Third of Malignant Pleural Mesothelioma Shows High Immunohistochemical Expression of MSLN or CXCR4 Which Indicates Potent Candidates for Endo-Radiotherapy

Malignant pleural mesothelioma (MPM) is a mainly asbestos-related tumour associated with a very poor prognosis. Therapeutic approaches include multimodal therapy and chemotherapeutics, with cisplatin being the drug of choice, but response rates of only up to 14% indicate very poor outcomes. Effective treatment options are lacking. Besides the diagnostic usage of radioligands in positron emission tomography (PET)/computed tomography (CT), the endo-radioligand therapy with Lu177 has been proven as a powerful tool in cancer therapy. Mesothelin (MSLN) and C-XC chemokine receptor 4 (CXCR4) are membrane-bound proteins, expressed in certain cancers, and thus are promising targets for endo-radiotherapy. A significant portion of high MSLN- or CXCR4-expressing tumors within the MPM may open the field for this sophisticated treatment approach in the near future. Formalin-fixed, paraffin-embedded (FFPE) tumour specimens from 105 patients suffering from MPM and treated at the Lung Cancer Centre of Essen and at the Helios Klinikum Emil von Behring Berlin were screened. The tumour samples were arranged in tissue microarrays. We immunohistochemically stained the tumour samples against MSLN and CXCR4. The protein expressions of the stainings were scored by a pathologist by using a semiquantitative method. The data obtained were correlated with the clinical outcome. Overall, 77.1% of the analysed tumours showed CXCR4 protein expression (25.7% of them at high expression level (Score 3)). 48.6% of all samples showed an overall strong staining (Score ≥ 2), 59% of the investigated tumours showed MSLN protein expression (10.5% of them at high expression (Score 3)), and 36.2% of all samples showed an overall strong staining (Score ≥ 2). Our results show significant tissue expression levels, for both CXCR4 and MSLN protein, in a major portion of clinical MPM samples. One-third of patients showed outstanding immunoexpression of at least one of these markers, making them interesting candidates for radioligand-based PET/CT diagnostics and follow-up and furthermore may profit from endo-radiotherapy.

Identification of a new potential plasmatic biomarker panel for the diagnosis of malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare highly aggressive tumor strongly associated with asbestos exposure and characterized by poor prognosis. Currently, diagnosis is based on invasive techniques, thus there is a need of identifying non-invasive biomarkers for early detection of the disease among asbestos-exposed subjects. In the present study, we measured the plasmatic concentrations of Mesothelin, Fibulin-3, and HMGB1 protein biomarkers, and of hsa-miR-30e-3p and hsa-miR-103a-3p Extracellular-Vesicles- embedded micro RNAs (EV-miRNAs). We tested the ability of these biomarkers to discriminate between MPM and PAE subjects alone and in combination. the study was conducted on a population of 26 patients with MPM and 54 healthy subjects with previous asbestos exposure (PAE). Mesothelin, Fibulin-3, and HMGB1 protein biomarkers were measured by the enzyme-linked immunosorbent assay (ELISA) technique; the levels of hsa-miR-30e-3p and hsa-miR-103a-3p EV-miRNAs was assessed by quantitative real-time PCR (qPCR). the most discriminating single biomarker resulted to be Fibulin-3 (AUC 0.94 CI 95% 0.88-1.0; Sensitivity 88%; Specificity 87%). After investigating the different possible combinations, the best performance was obtained by the three protein biomarkers Mesothelin, Fibulin-3, and HMGB1 (AUC 0.99 CI 95% 0.97-1.0; Sensitivity 96%; Specificity 93%). the results obtained contribute to identifying new potential non-invasive biomarkers for the early diagnosis of MPM in high-risk asbestos-exposed subjects. Further studies are needed to validate the evidence obtained, in order to assess the reliability of the proposed biomarker panel.

Identification of Potential Prognostic Biomarkers Associated with Monocyte Infiltration in Lung Squamous Cell Carcinoma

The five-year survival rate of lung squamous cell carcinoma is significantly lower than that of other cancer types. It is therefore urgent to discover novel prognosis biomarkers and therapeutic targets and understand their correction with infiltrating immune cells to improve the prognosis of patients with lung squamous cell carcinoma. In this study, we employed robust rank aggregation algorithms to overcome the shortcomings of small sizes and potential bias in each Gene Expression Omnibus dataset of lung squamous cell carcinoma and identified 513 robust differentially expressed genes including 220 upregulated and 293 downregulated genes from six microarray datasets. Functional enrichment analysis showed that these robust differentially expressed genes were obviously involved in the extracellular matrix and structure organization, epidermis development, cell adhesion molecule binding, p53 signaling pathway, and interleukin-17 signaling pathway to affect the progress of lung squamous cell carcinoma. We further identified six hub genes from 513 robust differentially expressed genes by protein-protein interaction network and 10 topological analyses. Moreover, the results of immune cell infiltration analysis from six integrated Gene Expression Omnibus datasets and our sequencing transcriptome data demonstrated that the abundance of monocytes was significantly lower in lung squamous cell carcinoma compared to controls. Immune correlation analysis and survival analysis of hub genes suggested that three hub genes, collagen alpha-1(VII) chain, mesothelin, and chordin-like protein 1, significantly correlated with tumor-infiltrating monocytes as well as may be potential prognostic biomarkers and therapy targets in lung squamous cell carcinoma. The investigation of the correlation of hub gene markers and infiltrating monocytes can also improve to well understand the molecular mechanisms of lung squamous cell carcinoma development.

Characterization of Mesothelin Glycosylation in Pancreatic Cancer: Decreased Core Fucosylated Glycoforms in Pancreatic Cancer Patients' Sera.

Currently, there are no reliable biomarkers for the diagnosis of pancreatic cancer (PaC). Glycoproteomic approaches that analyze the glycan determinants on specific glycoproteins have proven useful to develop more specific cancer biomarkers than the corresponding protein levels. In PaC, mesothelin (MSLN) is a neo-expressed glycoprotein. MSLN glycosylation has not been described and could be altered in PaC. In this work, we aimed to characterize MSLN glycans from PaC cells and serum samples to assess their potential usefulness as PaC biomarkers. First, we analyzed MSLN glycans from PaC cell lines and then we developed an enzyme-linked lectin assay to measure core fucosylated-MSLN (Cf-MSLN) glycoforms. MSLN glycans from PaC cells were analyzed by glycan sequencing and through Western blotting with lectins. All of the cell lines secreted MSLN, with its three N-glycosylation sites occupied by complex-type N-glycans, which were mainly α2,3-sialylated, core fucosylated and highly branched. The Cf-MSLN glycoforms were quantified on PaC serum samples, and compared with MSLN protein levels. The Cf-MSLN was significantly decreased in PaC patients compared to control sera, while no differences were detected by using MSLN protein levels. In conclusion, Cf-MSLN glycoforms were differently expressed in PaC, which opens the way to further investigate their usefulness as PaC biomarkers.

Construction of a 124I-Labeled Specific Antibody for the Noninvasive Detection of Mesothelin-Overexpressing Tumors.

Mesothelin (MSLN) is a molecular biomarker of many types of solid tumors, such as mesothelioma, pancreatic cancer, and colon cancer. Owing to the significant difference in expression between cancer cells and normal cells, mesothelin has been widely used as a key target in cancer immunotherapy. In this study, we used iodine isotope (nat/124/125I)-labeled mesothelin antibodies to noninvasively detect MSLN expression in mice with LS174T colon cancer. The 124I-labeled MSLN antibody showed a high radiochemical purity (RCP, >99%) and specific activity (20.8-67.8 GBq/μmol) after purification and was stable in 5% HSA and PBS (>95% RCP at 8 days). Western blot analysis indicated that the LS174T cells showed a higher MSLN protein level than the HepG2 cells. The half maximal effective concentration (EC50) values of the MSLN antibody and natI-anti-MSLN were 34.77 ± 3.72 ng/mL and 32.60 ± 2.52 ng/mL (P = 0.63), respectively. The dissociation constant of 124I-anti-MSLN binding to MSLN protein was 16.0 nM. The radiotracer showed a significantly higher uptake in LS174T cells than in HepG2 tumor cells (1.56 ± 0.09 vs 0.81 ± 0.03, P = 0.0016) 2 days postinjection. The LS174T mouse models showed extremely low organ uptake and high tumor uptake 96 h after the injection of 124I-anti-MSLN, and the T/M values were much higher than those of the other imaging groups (10.56 ± 1.20 for 124I-anti-MSLN in LS174T mice vs 3.27 ± 0.20 for 124I-anti-MSLN in HepG2 mice vs 3.53 ± 0.2 for 124I-IgG in LS174T mice). The immunochemical histology results showed that LS174T tumors were strongly positive (+++) for MSLN, while those in the HepG2 group showed slight expression (+). The dosimetry estimation study showed that the effective dose of 124I-anti-MSLN was 0.185 mSv/MBq, which is within the range of acceptable doses for further nuclear medicine translational research. Taken together, these results suggest that this radiotracer has the potential for detecting mesothelin-overexpressing tumors.

Defining a prognostic risk score in ovarian cancer patients by integrated analysis of serum mesothelin, HE4, CA125 and surgical outcome.

e17611 Background: In ovarian cancer, serum CA125 is currently the only biomarker used in routine clinical practice and has several limitations, e.g. it is not entirely specific for ovarian malignancy. Alternative serum biomarkers have been proposed for ovarian cancer, such as mesothelin or human epididymis protein 4 (HE4). Although it is known that single marker analysis is often insufficient for diagnostic purposes or relapse prediction, no comprehensive studies on the combination of these markers have been conducted, yet. Using a multi-marker prediction model, we herein investigated the clinical usefulness of mesothelin and HE4, detected in a sequence of pre- and post-operative serum samples, in complementing the standard marker CA125. Methods: Serum mesothelin, HE4 and CA125 were quantified by Lumipulse G chemiluminescent enzyme immunoassay (Fujirebio) in a total of 432 serum samples from 90 healthy controls and 192 ovarian cancer patients (thereof 81.8% with FIGO III/IV), including 151 paired pre- and post-operative samples. Results: Pre-operative serum mesothelin and HE4 were significantly elevated compared to healthy controls with a median ratio (IQR) to controls of 2.6 and 9.6, respectively (p < 0.0001). High pre-operative levels of mesothelin or HE4 alone were significantly correlated with advanced FIGO-stage (p < 0.0001) and predicted surgical outcome (p < 0.0001). We retrospectively compiled mesothelin and HE4 levels with CA125, BRCA mutational status and surgical outcome, in order to define a multi-marker setting. Both, mesothelin and HE4 levels were independent from BRCA mutational status, however they positively correlated with CA125. Detection of pre-operative HE4 > 498pM (OR = 4.1, p < 0.0001) combined with pre-operative CA125 > 516U/mL (OR = 2.2, p = 0.001) was the best combination to predict surgical outcome (AUC = 0.815). Finally, we defined a prognostic signature, including pre-operative CA125, post-operative mesothelin, HE4 and surgical outcome, which was superior to the individual prognostic capacity of all included parameters. Our model allowed the calculation of a weighted risk score, stratifying patient at “lower risk” (score 0-4) or at “higher risk” (score 5-10). Patients with higher risk had significantly shorter PFS (19.0 months (95%CI = 16.1-21.8) vs 64.1 months (95%CI = 56.8-71.3) p < 0.0001) and OS (34.0 months (95%CI = 28.6-39.3) vs 71.2 months (95%CI = 64.6-77.7) p < 0.0001). Conclusions: This is the first study, proposing an integrated prognostic risk score for ovarian cancer patients, which could easily be implemented into routine diagnostics and may guide personalized treatment decisions. Patients at “higher risk”, as identified by this model, could potentially benefit from an intensified therapy regime, whereas patients at “lower risk” might be suitable for treatment de-escalation.

Clinical utility of diagnostic biomarkers in malignant pleural mesothelioma: a systematic review and meta-analysis.

Malignant pleural mesothelioma (MPM) is characterised by late-stage diagnosis and poor prognosis. Currently, no screening tool is advocated and diagnosis is based on invasive techniques, which are not well tolerated. Non-invasive diagnostic biomarkers have shown potential and could have a huge clinical benefit. However, despite extensive research, there is no consensus yet on their clinical use, with many articles reporting contradicting results, limiting their clinical implementation. The aim of this systematic review is therefore to explore the different semi- and non-invasive diagnostic markers in several human matrices and identify those that might clinically be relevant. A total of 100 articles were selected through Web of Science and PubMed, with 56 articles included in the quantitative analysis. Although many studies have reported on the diagnostic accuracy of MPM biomarkers such as serum mesothelin and high-mobility group box protein 1 and plasma fibulin-3, none have resulted in a validated test for early detection. Future research should focus on external validation, combinations into biomarker panels, the inclusion of early stage MPM patients and a combination of different biomarker matrices, as well as new markers.

Abstract PO079: Effects of the lung tumor microenvironment on T cell therapy

Abstract Immune checkpoint therapy (ICT) is a front-line treatment for lung cancer; however, low mutational burden and ‘non-T cell inflamed’ signatures predict poor responses to ICT in ~50% of patients. Adoptive cellular therapy (ACT) with T cells engineered to express T cell receptors (TCRs) specific for tumor-associated antigens (TAAs; native proteins that are overexpressed by cancers) is an approach that circumvents the need for endogenous T cell responses. TCR-ACT has been effective against hematologic cancers, but ACT against solid tumors is still in the early stages of exploration. A deeper understanding of the complex interaction between therapeutic T cells and the tumor microenvironment (TME) will be important for identifying successful strategies to enhance function and mitigate toxicity. Genetically engineered mouse models (GEMMs) achieve in situ tumor development alongside competent immune systems, recapitulating the native TME over the full spectrum of disease progression in a preclinical setting. Targeting TAAs naturally overexpressed by GEMM tumors, and expressed at native levels in healthy adult tissues, allows for study of the factors dictating the efficacy and toxicity of TCR-ACT in experimentally tractable systems. GEMM lung tumors driven by oncogenic Kras and deletion of the tumor suppressor p53 (KP) harbor few mutations, are poorly infiltrated by T cells, and are refractory to ICT, modeling treatment-recalcitrant patient disease. Using CD8+ T cells transduced with a TCR against the mesothelin (msln) protein, a TAA commonly overexpressed across many cancer types, including lung cancers, we showed that TAA-specific T cells recognize KP lung cancer cells and efficiently home to tumors. However, therapeutic T cells rapidly lose function in the lung TME compared to those recovered from the spleen, or from KP pancreatic tumors, which bear similar driver mutations and also overexpress the msln TAA. Repetitive TCR-ACT can extend survival of animals with lung tumors, dependent on treatment timing, but animals eventually succumb to disease. These results show promise for TCR-ACT against lung cancer but highlight the presence of tissue-specific obstacles that must be overcome to enhance efficacy. For example, TAA-specific T cells accumulate less efficiently in KP tumors engineered to express strong model T cell antigens, indicating acquired immune suppressive TME phenotypes. Preliminary studies indicate that engineered therapeutic CD8+ T cells modified to express costimulatory immunomodulatory fusion proteins (IFPs) or accompanied by engineered TAA-specific CD4+ helper T cells exhibit increased function in lung tumors, indicating that increased costimulation signaling or T cell help might partially overcome T cell suppression in the lung TME. As we continue to leverage these models to advance our understanding of tissue-specific effects on therapeutic outcomes, these findings offer inroads to uncovering more effective therapies, with the ultimate goal of benefitting cancer patients. Citation Format: Leah M. Schmidt, Oanh Tran, Shannon Oda, Quintin Inman, Sasha Tan, Cody Jenkins, Philip Greenberg. Effects of the lung tumor microenvironment on T cell therapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO079.

Mesothelin Expression Is Associated with Extramedullary Disease and Promotes In Vivo Leukemic Growth in Acute Myeloid Leukemia

The mesothelin (MSLN) protein is overexpressed in many solid tumors and is considered a viable immunotherapeutic target. We recently demonstrated that MSLN is aberrantly expressed on the blast surface in approximately 1/3 of AML cases and lacks expression in normal hematopoiesis, making it an attractive target in AML. While the exact function of MLSN is unknown, multiple studies, including its recently described role in the development of surgical adhesions, implicate it in cell adhesion in both healthy and malignant cells. We hypothesized that MLSN expression in AML may be involved in extramedulllary disease (EMD). We correlated MLSN transcription expression with EMD status and utilized xenograft models to evaluate MSLN's role in AML in vivo. A total of 1,038 patients with de novo AML (age 0-29 years) enrolled on COG AAML1031 with available transcriptome, clinical, and biologic data were included for analysis. Transcriptome sequencing was performed on diagnostic bone marrow or peripheral blood specimens and gene expression was quantified as transcripts per million (TPM). The cohort with MLSN positive (MSLN+) AML (n=359) was defined as patients with MSLN overexpression, 5 TPM, and <5 TPM was considered MSLN-negative (MSLN-; n=679). EMD was defined as having non-bone marrow sites of disease and/or CNS positive (CNS3) disease (CSF WBC 5/ml with blasts or clinical signs of CNS leukemia). Xenograft experiments were performed in NSG mice using cell lines (CDX) or patient leukemias (PDX). Parental Nomo-1, which express endogenous MSLN, and Kasumi-1 (MSLN-) and their engineered Nomo-1 MSLN knockout (Nomo-1KO-MSLN) or MSLN-transduced (Kasumi-1-MSLN+) counterparts and we developed the MSLN+ PDX NTPL-146 for use in CDX and PDX studies respectively. Engraftment was measured using non-invasive bioluminescent (IVIS) imaging. Our analysis demonstrated a striking association between MSLN expression and EMD. Among the MSLN+ cohort, the rate of EMD was significantly higher compared to the MSLN- cohort (27.8% vs. 18.8%, p=0.001; Table 1). When stratified by EMD type, we observed a similar pattern with higher rates of both non-CNS and CNS EMD in MSLN+ AML. Non-CNS EMD occurred in 17% of MSLN+ vs. 12.4% of MSLN- AML (p=0.042). The rate of CNS positive (CNS3) disease was significantly higher in the MSLN+ cohort compared to the MSLN- cohort (13.2% vs. 7.6%, p=0.004; Table 1). While MSLN+ cases made up 35% of the cohort, they accounted for 47% of the CNS positive cases. Of interest, MSLN+ disease was also associated with significantly higher occurrence of CNS2 disease compared to MSLN- (26.4% vs. 19.1% respectively, p=0.008; Table 1). In patients with very high MSLN expression (100 TPM, n=112), 29% (n=32) of patients had EMD (n=23 non-CNS, n=9 CNS). Notably, among the cohort in this very high expressing group that lacked EMD at diagnosis but who experienced relapse (n=35), 38% (n=13) had EMD at relapse. Among patients with relapsed/refractory (R/R) AML (n=486), MSLN overexpression was also highly associated with EMD. Although MSLN+ cases comprised 35% of R/R cases, they accounted for 53% of EMD cases. The rate of EMD was 40.8% in MSLN+ cases vs. 18.9% of MSLN- cases (p<0.001). We evaluated the role of MLSN in EMD and its potential function in engraftment in vivo with xenograft models. A higher incidence of EMD was detected in MSLN+ xenografts; 90% (9/10) of Nomo-1 xenografts developed abdominal myeloid sarcomas compared to 30% of Nomo-1KO-MSLN (p<0.005; Fig 1A). The MSLN+ PDX showed EMD and associated renal sarcomas and enlargement of the kidneys (Fig 1B). Evaluation of MSLN+ CDX demonstrated more rapid and higher levels of tumor burden compared to their MSLN- counterparts. At 6 weeks post injection, tumor load of Nomo-1 CDX was 3-fold higher than Nomo-1KO-MSLN (p<0.005; Fig 1C,D) and was 8-fold higher in Kasumi-1-MSLN+ compared to parental Kasumi-1 (p<0.05; Fig 1E,F). Our findings demonstrate that MSLN is highly implicated in EMD and our xenograft studies further support the hypothesis that MSLN may be involved in cell adhesion and establishing sites of disease. EMD can be very challenging to treat, and in the setting of recurrence often indicates incurable disease, thus novel therapeutic strategies for this group of patients are urgently needed. Our findings support additional studies of how MSLN may be functionally implicated in the development of EMD as well as clinical trials that evaluate MSLN targeting in AML. Disclosures No relevant conflicts of interest to declare.

Correlation of mesothelin (MSLN) expression with future peritoneal metastases (PM) in colorectal cancer (CRC).

176 Background: CRC patients with PM have a significantly worse median overall survival (OS) when compared to those with liver and lung metastases. We studied MSLN protein expression using immunohistochemistry (IHC) on primary resected CRC (PRC), its correlation with future development of PM and its effect on OS. Our hypothesis is that PRC tumors from patients who later developed PM will have an increased proportion of positive MSLN scores based on IHC. Methods: We performed a retrospective case control study of all localized CRC (Stage I, II, III) patients who underwent curative resection from 2000- 2017 at the University of Oklahoma. The target population consisted of PRC patients who had distant recurrence during surveillance. Control group comprised of patients who never recurred after a minimum of 5 years of surveillance. MSLN score was calculated by the sum of staining percentage and intensity scores on each MSLN stained slide. Kaplan-Meier method was used to estimate OS and differences in survival were estimated using log-rank test. Results: Among 100 patients with recurrent CRC, 19 patients had PM, 51 had solid organ only metastases (SOM) and 66 are in the control group. MSLN staining results by IHC were satisfactory in 87 patients (Table). 91%, 46% and 21% of the patients in PM, SOM, and control groups had a positive MSLN score on PRC respectively. A significant association between MSLN score and future development of PM was observed (p<0.001) based on Fisher’s exact test. Sensitivity of MSLN as a marker for future PM was 93% and specificity was 58%. Patients with a positive MSLN score had a poor OS when compared to those with a negative MSLN score on univariate analysis (95% CI: 4.1-NR (not reached), p <0.001), which lost significance on multi-variable cox proportional analyses. Conclusions: A higher proportion of patients in the PM group had a positive MSLN score on PRC when compared to the SOM and control groups. MSLN was found to be a highly sensitive marker for future PM in CRC. We intend to expand this work by examining an additional cohort to validate these findings. [Table: see text]

Abstract 2557: Assessing the therapeutic efficacy of disease-specific T-cell biofactories

Abstract Current protein-based cancer therapies have several disadvantages, which include general toxicity, lack of response at the administered dose and differing responses from patients to patient. To circumvent these issues, we propose a platform in which engineered T-cells specifically recognize tumors and secrete therapeutic peptides directly at the disease site leading to targeted cancer cell killing. This approach will increase the specificity of the therapy and decrease toxicity to healthy cells. We transformed acute T-cell lymphoma cells into biofactories for site-specific synthesis of therapeutic proteins upon stimulation by antigen-presenting disease cells. The effector T-cell line was engineered by stably introducing a chimeric T-cell receptor to recognize Folate-Receptor alpha (FRα) or Mesothelin (MSLN) protein. Upon binding of the effector T-cells to the receptor, an intracellular cascade directed expression of non-human proteins is induced. Specific T-cell binding to human ovarian cancer cell lines and signaling was measured by in vitro co-culture using luciferase production as a surrogate for therapeutic peptide secretion. We demonstrated that T-cells can be genetically programed to synthesize and secrete proportionate amounts of engineered proteins upon engaging the tumor-associated antigens (FRα or MSLN) on a human ovarian cancer cell line, OVCAR3 (FRα+MSLN+). A FRα-MSLN- ovarian cancer cell line, A2780cis, was used as the non-targeted negative control. The difference in protein secretion following stimulation by the two cell lines, as measured by luciferase activity, was statistically significant within 1 hour. It reached ~35-fold within 1 to 3 days, and we observed stable expression for at least 10 days. The luminescent signal was proportionate to the number of OVCAR3 cells. To further validate the specificity of target engagement, we generated A2780cis-FRα positive and A2780cis-MSLN positive cell lines and demonstrated selective binding and activation of the corresponding effector cells in co-culture assays. No binding was detected to the A2780cis-vector control cells. In vivo results for T-cell biofactories targeting OVCAR3 tumors 24 hours post-stimulation validated the in vitro results. Currently, we are engineering T-cell biofactories to release cytotoxic peptides and are assessing their therapeutic efficacy against cancer cells in vitro using co-culture assays and supernatant transfer. Our results show that T-cells can be genetically reprogrammed to serve as biofactories for the synthesis of therapeutic proteins upon stimulation by antigen-presenting disease cells. Importantly, these studies demonstrate the feasibility of developing the next generation of adoptively transferred T-cell therapies to target tumors that express FRα (e.g., ovarian, breast, lung) and/or MSLN (e.g. ovarian, lung, pancreatic) on their cell surfaces for cancer therapy. Citation Format: Claire E. Repellin, Puja Patel, Lucia Beviglia, Harold Javitz, Lidia C. Sambucetti, Parijat Bhatnagar. Assessing the therapeutic efficacy of disease-specific T-cell biofactories [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2557.

Abstract 5921: Systemic treatment with mesothelin-targeted oncolytic adenovirus shows efficacy patient-derived xenograft of pancreatic cancer

Abstract Pancreatic cancer is an aggressive malignant disease. Despite extensive efforts, systemic therapies have provided only limited efficacy for patients with this disease. Oncolytic Adenovirus (OAd) is a promising therapeutics, and it is also known for its efficient in vivo gene delivery. However, when adenovirus vectors are injected intravenously into mice, most of the virus goes to the liver and can lead to liver toxicity at high dosage. One of the reason for liver tropism is that hepatocytes express high levels of the primary adenovirus receptor, and non-parenchymal liver cells, such as Kupffer cell and epithelial cell, also capture the viral particle. As a consequence of large sequestration of adenovirus by liver, the tumor transduction rate is low and the in vivo efficacy is limited. Therefore, the improvement of cancer selective transduction and vector distribution to avoid liver sequestration would overcome the obstacles for systemic delivery required for efficient systemic treatment of spread and/or metastatic lesions of pancreatic cancer with OAd. To improve the tumor transduction, we have generated the pancreatic cancer-targeted OAd by high-throughput screening of Ad-fiber library in mesothelin (MSLN) expressing cells. The pancreatic cancer-targeted OAd binds to MSLN protein, which is overexpressed on the surface of pancreatic cancer. MSLN-targeted OAd showed selective and powerful anti-tumor effect against Panc-1 xenograft tumor model in both intratumoral (i.t.) and intravenous (i.v.) injection. Importantly, when we assessed viral distribution after i.v. injection, the liver sequestration of MSLN-targeted OAd was lower than untargeted OAd (Ad5 WT virus) at 48 hrs after injection. By day 7, the viral copy number of MSLN-targeted OAd in the tumor was significantly higher than Ad5 WT virus. These results suggest that systemic injection of the tumor targeted-OAd showed significantly lower liver sequestration and better tumor accumulation. Additionally, we performed multiple time point injection of MSLN-targeted OAd against regrown tumors. Four out of six tumors were controlled with repeated injection. Next, antitumor effect of MSLN-targeted OAd was assessed in patient-derived xenograft (PDX) model. After intravenous administration, only the MSLN-targeted OAd showed significant antitumor effect compared to the untreated group (p<0.05), while the growth of Ad5 WT virus injected group was same as untreated group. In this study, systemic injection of MSLN-targeted OAd showed remarkable anti-tumor effect in both systemic and intratumoral injections at low dose. Our results indicated that tumor targeted-OAd can embody efficient systemic treatment for pancreatic cancer which are mostly found with spread or metastatic lesions. Citation Format: Mizuho Sato-Dahlman, Yoshiaki Miura, Praveensingh Hajeri, Hideki Yoshida, Kari Jacobsen, Chikako Yanagiba, Masato Yamamoto. Systemic treatment with mesothelin-targeted oncolytic adenovirus shows efficacy patient-derived xenograft of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5921.

Development of a mesothelin-based prophylactic vaccine against ovarian cancer

Abstract Mesothelin is a tumor-associated antigen overexpressed in ovarian cancer and normally expressed by mesothelial linings. While mesothelin function remains to be fully elucidated, the lack of phenotype of mesothelin-knockout mice makes it plausible that mesothelin can safely serve as a vaccine target. We designed a new vaccination strategy based on mesothelin targeting with activation of type I IFN signaling via cyclic dinucleotides (CDN). Vaccines first combined human mesothelin protein with alum- vs. CDN-based adjuvants. C57Bl/6 mice received a priming immunization followed by 2 boosts; two weeks later, immunized mice were injected orthotopically with luciferase-transduced mouse ovarian cancer cells (Luc-ID8). More than 2/3 of the mesothelin/CDN-immunized mice were protected against invasive cancer and presented increased cytotoxic T cells (CTL) and plasmacytoid dendritic cells in spleens. Effector memory T cells were also increased and MDSC were profoundly decreased in peritoneal lavages after immunization with mesothelin +/− CDN compared with all the other groups. IFNγ ELISPOT assays performed with protected animal splenocytes stimulated with mesothelin peptides identified 2 immunogenic regions. We next tested a peptide-based subunit vaccine derived from mouse mesothelin sequence. Immunization and 2 boosts of 25-mer peptides with CDN were sufficient to mount an immune response against 2 overlapping mouse mesothelin peptides. Immunizations with peptide pools mapping in the immunogenic regions are currently assessed for protection against Luc-ID8 growth and progression. Peptide-based prophylactic vaccination against ovarian cancer may be possible in combination with adjuvants that trigger cellular immunity.

Molecular Imaging of Mesothelin-Expressing Ovarian Cancer with a Human and Mouse Cross-Reactive Nanobody.

Mesothelin is an epithelial marker highly expressed at the cell surface of cancer cells from diverse origins, including ovarian and pancreatic adenocarcinomas and mesotheliomas. Previously, we identified and characterized an antimesothelin nanobody (NbG3a) for in vitro diagnostic applications. The main goal of this research was to establish the potential of NbG3a as a molecular imaging agent. Site-specific biotinylated NbG3a (bNbG3a) was bound to streptavidin-conjugated reagents for in vitro and in vivo assays. Initially, we performed microscale thermophoresis to determine the binding affinity between bNbG3a and human ( Kd = 46 ± 8 nM) or mouse ( Kd = 4.8 ± 0.4 nM) mesothelin protein. The human and mouse cross-reactivity was confirmed by in vivo optical imaging using bNbG3a bound to fluorescent streptavidin. We also localized the binding site of nNbG3a on human mesothelin using overlapping peptide scan. NbG3a recognized an epitope within residues 21-65 of the mature membrane bound form of human mesothelin, which is part of the N-terminal region of mesothelin that is important for interactions between mesothelin on peritoneal cells and CA125 on tumor cells. Next, the bNbG3a in vivo half-life after intravenous injection in healthy mice was estimated by ELISA assay to be 5.3 ± 1.3 min. In tumor-bearing animals, fluorescent bNbG3a accumulated in a subcutaneous ovarian xenograft (A1847) and in two syngeneic, orthotopic ovarian tumors (intraovary and intraperitoneal ID8) within an hour of intravenous injection that peaked by 4 h and persisted up to 48 h. MRI analysis of bNbG3a-targeted streptavidin-labeled iron oxides showed that the MRI signal intensity decreased 1 h after injection for a subcutaneous xenograft model of ovarian cancer for bNbG3a-labeled iron oxides compared to unlabeled iron oxides. The signal intensity differences continued up to the final time point at 24 h post injection. Finally, in vivo immunofluorescence 24 or 48 h after bNbG3a intravenous injection showed bNbG3a diffuse distribution of both xenograft and syngeneic ovarian tumors, with local areas of high concentration throughout A1847 human tumor. The data support the use of NbG3a for continued preclinical development and translation to human applications for cancers that overexpress mesothelin.

Abstract B029: Deletion of mesothelin impairs intraperitoneal growth of pancreatic cancer

Abstract The mesothelin (MSLN) protein is expressed in at least 85% of pancreatic adenocarcinomas but not on cells of the healthy pancreas nor in the parenchyma of other vital organs. Due to this differential expression, MSLN has been used as a target for various antibody-based treatments and cancer vaccines. The physiologic role of MSLN is unknown. The MSLN gene encodes for a precursor protein, which is cleaved to produce the mature MSLN protein. Previous reports have suggested that overexpression of MSLN precursor protein may increase tumorigenicity and metastatic potential of pancreatic adenocarcinoma. Here, we have used CRISPR-Cas9 gene editing to delete MSLN from the KLM-1 pancreatic cancer cell line (ΔMSLN) and have examined the growth of these cells compared to mock deleted cells (Mock). The ΔMSLN and Mock cells grew at the same rate both in cell culture and as subcutaneous tumors in nude mice, but had significantly impaired growth when inoculated into the mouse intraperitoneal cavity. Lentivirus transduction was used to restore expression of MSLN in ΔMSLN cells. Transduction of full-length WT MSLN, but not a Y318A mutant, restored intraperitoneal growth. Interestingly, reintroduction of mature MSLN instead of the precursor protein did not restore growth even though it was appropriately expressed on the cell surface. In summary, we have demonstrated that MSLN enhances the growth of pancreatic cancer within the peritoneal cavity and defined key regions of MSLN important for this activity. Citation Format: Christine Campo Alewine, Michael Rudloff, Danielle Arons, Salma El-Behaedi, Xianyu Zhang, Leela Avula. Deletion of mesothelin impairs intraperitoneal growth of pancreatic cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B029.

Better Predictive Value of Cancer Antigen125 (CA125) as Biomarker in Ovary and Breast Tumors and its Correlation with the Histopathological Type/Grade of the Disease.

Both ovarian/breast cancers are the most prevalent hormone-associated gynecological-cancers, where uncontrolled cellular proliferations/genetic-errors are noticed. The cancer-antigen125 (CA125), which we assessed presently is an important biomarker in the early detection/monitoring of this disease-pathogenesis. Serum/tissue CA125 was measured by solid-phase-Enzyme-linked-immunosorbentassay in women with ovarian/breast tumors of different types (epithelial/non-epithelial; benign/ borderline/ malignant)/stages. Breast-tumor tissues were employed for histoarchitecture/DNAstability (comet) assay. Extensive protein-protein(CA125) interactions were studied by the STRING (Search-Tool-for-the-Retrieval-of-Interacting Genes/Proteins) Bioinformatics-software. Serum CA125 levels were <35 U/ml in 94% of benign epithelial-cases, 35-65 U/ml in 100% of borderline-tumors and >100 U/ml in the 41.17% of patients with malignant-tumors. The malignant epithelial tumor showed significantly higher (>100U/ml) CA125 than the non-epithelial malignant-tumor (<35-65 U/ml). Highly enhanced cellularity/histo-architectural impairment/unstable- DNA-materials/CA125 was found in advanced breast-cancers. The CA125 is highly metabolically interactive, especially with mesothelin impairing cell-cell adhesion and enhancing tissue anti-establishment. CA125 is a predictive-marker in ovarian/breast carcinoma depending on disease nature/stages. CA125, in combination with other tests like mesothelin/estradiol can be of great use in the better diagnosis of this disease. Not only as a detection-marker, has the CA125 played an interactive role in the disease processes. The Bioinformatics study revealed an important role of mesothelin and other mucin like proteins. Early detection and proper diagnosis are important determinants for the greater possibility of the relief from the disease like cancers. Selection of suitable biomarker combinations may increase the better diagnosis of the types and severity of this disease. Our present result has an impact on these aspects.

Mesothelin as a novel biomarker and immunotherapeutic target in human glioblastoma.

Glioblastoma multiforme (GBM) presents the most malignant form of glioma, with a 5-year survival rate below 3% despite standard therapy. Novel immune-based therapies in improving treatment outcomes in GBM are therefore warranted. Several molecularly defined targets have been identified mediating anti-GBM cellular immune responses. Mesothelin is a tumor-associated antigen (TAA) which is expressed in several solid tumors with different histology. Here, we report the immunological significance of mesothelin in human malignant glioma. Expression of mature, surface-bound mesothelin protein was found to bein human GBM defined by immunofluorescence microscopy, and on freshly isolated, single cell suspension of GBM tumor cells and GBM tumor cell lines, determined by based on flow cytometric analysis. Peripheral blood (PB) from patients with GBM, stimulated with mesothelin peptides and IL-2, IL-15 and IL-21, exhibited increased antigen-specific IFN-γ and TNF-α production. Anti-mesothelin directed T-cell responses could also be detected in tumor - infiltrating lymphocytes (TIL) isolated from GBM speciments. Furthermore, T cells cultured in the presence of IL-2, IL-15 and IL-21 displayed enhanced mesothelin-specific CD4+ and CD8+ subset proliferation, based on ELISA and flow cytometric readouts. Mesothelin-specific IgG antibodies as well as (shed) mature mesothelin protein were detected in plasma samples from patients with GBM by indirect ELISA. Finally yet importantly, we identified distinct immune recognition hotspots within the mature mesothelin component, defined by peptide-specific IFN-γ responses from peripheral T-cells from patients with GBM. Mesothelin may therefore qualify as a viable target for immunotherapeutic approaches for patients with GBM.

Abstract 3813: MPF as a monitoring biomarker for a mesothelin-targeted therapy against malignant mesothelioma and other cancer

Abstract Introduction: Tumor antigen mesothelin had been shown to be elevated in mesothelioma, ovarian, pancreatic, and lung cancer. The mesothelin gene encodes a precursor protein of 69 kDa, which is cleaved into a 31 kDa soluble megakaryocyte potential factor (MPF), and a cell membrane-bound 40 kDa mesothelin. There is a high degree of interest in targeting mesothelin for mesothelioma and other cancers with various antibody-based agents, and some successes in early stage clinical trials with some of the agents. For the development of these targeted therapeutics, specific biomarkers would be needed for monitoring treatment responses, and detecting and evaluating tumor progression. The current test that detects mesothelin is not suitable due to the antigen-binding from the therapeutic antibodies. There is an urgent need to develop and validate an alternative test to support these clinical trials and for disease management. Methods: Using ECLIA technology and proprietary antibody reagents, we developed a new test for the cleaved MPF polypeptide. We further conducted comprehensive analytical and clinical validation of the test according to the guidelines for tumor antigen tests. Using samples from a retrospective clinical trial with an anti-mesothelin immunotoxin and the standard 1st line chemotherapeutic agents for newly diagnosed malignant mesothelioma patients, we examined the effectiveness of the test for treatment monitoring of the investigational therapy. Results: Our data gives a strong validation for the analytical and clinical performances of MPF test. While no other current test has been proven to be able to monitor response of mesothelioma to systemic therapies, our data provides the proof of clinical effectiveness for MPF test. In the patients with the elevated tumor antigen, there is a specific and highly significant reduction of MPF at the end of each treatment cycle for a total of 6 cycles in the patients with objective response. Later, about half of these patients with objective response and MPF reduction regained the levels of MPF at the time of disease progression. Conclusion: The study indicates that MPF test is effective in monitoring the treatment response to systemic therapies for mesothelioma patients with elevated MPF. It may also provide pivotal information on the expression of target mesothelin protein when tumors progress. Citation Format: Yunkai Yu, Raffit Hassan, Jingli Zhang, Masanori Onda, Ira Pastan, Liang Cao. MPF as a monitoring biomarker for a mesothelin-targeted therapy against malignant mesothelioma and other cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3813. doi:10.1158/1538-7445.AM2017-3813