Mesothelin targeted cancer immunotherapy

Mesothelin is a tumour differentiation antigen that is normally present on the mesothelial cells lining the pleura, peritoneum and pericardium. It is, however, highly expressed in several human cancers including malignant mesothelioma, pancreatic, ovarian and lung adenocarcinoma. The normal biologic function of mesothelin is unknown but recent studies have shown that it binds to CA-125 and may play a role in the peritoneal spread of ovarian cancer. The limited mesothelin expression in normal tissues and high expression in many cancers makes it an attractive candidate for cancer therapy. Three mesothelin targeted agents are in various stages of clinical evaluation in patients. These include SS1P (CAT-5001) a recombinant immunotoxin targeting mesothelin, MORAb-009 a chimeric anti-mesothelin monoclonal antibody and CRS-207 a live-attenuated Listeria monocytogenes vector encoding human mesothelin. These ongoing clinical trials will help define the utility of mesothelin as a target for cancer therapy.     

Cytotoxic activity of immunotoxin SS1P is modulated by TACE-dependent mesothelin shedding

Mesothelin is a cell-surface tumor-associated antigen expressed in several human cancers. The limited expression of mesothelin on normal tissues and its high expression in many cancers make it an attractive candidate for targeted therapies using monoclonal antibodies, immunoconjugates, and immunotoxins. Mesothelin is actively shed from the cell surface and is present in the serum of patients with malignant mesothelioma, which could negatively affect the response to these therapies. We have found that mesothelin sheddase activity is mediated by a TNF-α converting enzyme (TACE), a member of the matrix metalloproteinase/a disintegrin and metalloprotease family. We showed that EGF and TIMP-3 act through TACE as endogenous regulators of mesothelin shedding. We also found that reducing shedding significantly improved the in vitro cytotoxicity of immunotoxin SS1P, which targets mesothelin and is currently in clinical trials for the treatment of patients with mesothelioma and lung cancer. Our findings provide a mechanistic understanding of mesothelin shedding and could help improve mesothelin-based targeted therapies.     

Identification of novel human CTL epitopes and their agonist epitopes of mesothelin.

PURPOSE: Mesothelin is overexpressed in many pancreatic and ovarian cancers, mesotheliomas, and other tumor types. Clinical trials are ongoing using immunotoxins to target mesothelin, and patients immunized with allogeneic pancreatic tumor cell lines have shown immune responses to previously defined mesothelin epitopes. The purpose of this study was to define novel mesothelin CTL epitopes and, more importantly, agonist epitopes that would more efficiently activate human T cells to more efficiently lyse human tumors. EXPERIMENTAL DESIGN AND RESULTS: Two novel mesothelin HLA-A2 epitopes were defined. T-cell lines generated from one of these epitopes were shown to lyse pancreatic and ovarian tumor cells. Several agonist epitopes were defined and were shown to (a) have higher affinity and avidity for HLA-A2, (b) activate mesothelin-specific T cells from normal individuals or cancer patients to a greater degree than the native epitope in terms of induction of higher levels of IFN-gamma and the chemokine lymphotactin, and (c) lyse several mesothelin-expressing tumor types in a MHC-restricted manner more effectively than T cells generated using the native peptide. External beam radiation of tumor cells at nontoxic levels was shown to enhance the expression of mesothelin and other accessory molecules, resulting in a modest but statistically significant increase in tumor cell lysis by mesothelin-specific T cells. CONCLUSIONS: The identification of novel CTL agonist epitopes supports and extends observations that mesothelin is a potential target for immunotherapy of pancreatic and ovarian cancers, as well as mesotheliomas.     

Detection and quantitation of serum mesothelin, a tumor marker for patients with mesothelioma and ovarian cancer.

PURPOSE: To determine whether mesothelin, a cell surface protein highly expressed in mesothelioma and ovarian cancer, is shed into serum and if so to accurately measure it. EXPERIMENTAL DESIGN: We developed a sandwich ELISA using antibodies reacting with two different epitopes on human mesothelin. To quantitate serum mesothelin levels, a standard curve was generated using a mesothelin-Fc fusion protein. Sera from 24 healthy volunteers, 95 random hospital patients, 56 patients with mesothelioma, and 21 patients with ovarian cancer were analyzed. Serum mesothelin levels were also measured before and after surgical cytoreduction in six patients with peritoneal mesothelioma. RESULTS: Elevated serum mesothelin levels were noted in 40 of 56 (71%) patients with mesothelioma and in 14 of 21 (67%) patients with ovarian cancer. Serum mesothelin levels were increased in 80% and 75% of the cases of mesothelioma and ovarian cancer, respectively, in which the tumors expressed mesothelin by immunohistochemistry. Out of the six patients with peritoneal mesothelioma who underwent surgery, four had elevated serum mesothelin levels before surgery. Out of these four patients, three had cytoreductive surgery and the serum mesothelin level decreased by 71% on postoperative day 1 and was undetectable by postoperative day 7. CONCLUSIONS: We developed a serum mesothelin assay that shows that mesothelin is elevated in patients with mesothelioma and ovarian cancer. The rapid decrease in mesothelin levels after surgery in patients with peritoneal mesothelioma suggests that serum mesothelin may be a useful test to monitor treatment response in mesothelin-expressing cancers.     

Mesothelin-related predictive and prognostic factors in malignant mesothelioma: a nested case-control study

Soluble mesothelin-related protein (SMRP) in serum is potentially a sensitive marker of malignant mesothelioma (MM) diagnosis and progression, and may be useful as screening marker. Mesothelin expression in tumors is regarded as a sensitive marker for diagnosis and disease progression, and is a candidate prognostic marker. Levels of SMRP, CA125 and CYFRA 21-1 in pre-diagnostic (1-30 years) serum samples from 47 mesothelioma cases and 141 matched controls were analysed. Mesothelin expression in tumors was assessed. The association between biomarker level and mesothelioma risk and survival was analysed, adjusting for asbestos exposure. Survival related to tumor mesothelin expression, age, sex, histological type, location, asbestos exposure and pre-clinical SMRP was analysed. There was no significant association between biomarker levels and mesothelioma risk when analysed as continuous variables or as tertiles. Biomarker levels <10, 10-19 and >or=20 years before diagnosis were not significantly associated to mesothelioma risk. Mesothelin expressed in >50% of tumor cells was seen in 36 of 47 (77%) tumors. Mesothelin expression in <50% of tumor cells was a significant negative prognostic marker in all cases of malignant mesothelioma (median survival=6 months vs. 12 months, hazard ratio (HR)=2.49, 95%CI 1.17-5.27), and also when only epithelial mesothelioma was analysed (median=6 months vs. 14 months, HR=2.36, 95%CI 1.07-5.22). When adjusted for age and gender, the prognosis was still dismal, but non-significant (HR=1.85, 95%CI 0.85-4.05). High age (>65 years) was an independent negative prognostic factor that was related to both mesothelin expression and asbestos exposure. Mesothelioma of the epithelial type of the peritoneum had a significantly longer survival than epithelial type in pleura and was also related to mesothelin expression.     

Anti-mesothelin antibodies and circulating mesothelin relate to the clinical state in ovarian cancer patients.

Most human ovarian carcinomas express mesothelin, which is shed as a diagnostically useful biomarker. We applied an ELISA to measure antibodies to native mesothelin in serum from a series of patients with divergent clinical outcomes. The level of anti-mesothelin antibodies determined as OD(450 nm) and referred to as absorption units (AU) for 1:20 diluted serum was higher in patients who remained disease-free after therapy [no evidence of disease (NED); n = 14] than in patients whose disease recurred [clinical evidence of disease (CED); n = 21; P < 0.01]. Applying AU > or = 0.5 at a serum dilution of 1:20 as cutoff, 10 of 14 (71%) ovarian carcinoma patients with NED and 9 of 21 (43%) patients with CED had antibodies to mesothelin compared with 6 of 23 (26%) healthy women (P < 0.008) and 5 of 24 (21%) women with other benign gynecologic diseases (P < 0.003), whereas 7 of 9 (78%) of women with pelvic inflammatory disease were positive. Three of the 14 (21%) NED patients had circulating mesothelin detected as an AU > or = 0.2 at a serum dilution of 1:40 (P < 0.005) compared with 15 of 21 (71%) CED patients, and 9 of 14 (64%) NED patients (P < 0.0002) were positive for antibodies and negative for antigen compared with 1 of 21 (5%) CED patients. Although our data indicate that an antibody response to mesothelin is an important correlate of ovarian carcinoma, prospective studies are needed to show whether the measurement of such antibodies (alone or together with antigen) aids the diagnosis and monitoring of patients.     

Antigen shedding may improve efficiencies for delivery of antibody-based anticancer agents in solid tumors

Recombinant immunotoxins (RIT) are targeted anticancer agents that are composed of a targeting antibody fragment and a protein toxin fragment. SS1P is a RIT that targets mesothelin on the surface of cancer cells and is being evaluated in patients with mesothelioma. Mesothelin, like many other target antigens, is shed from the cell surface. However, whether antigen shedding positively or negatively affects the delivery of RIT remains unknown. In this study, we used experimental data with SS1P to develop a mathematical model that describes the relationship between tumor volume changes and the dose level of the administered RIT, while accounting for the potential effects of antigen shedding.     

A novel high‐affinity human monoclonal antibody to mesothelin

Mesothelin is a glycosylphosphatidylinositol‐anchored glycoprotein that is highly expressed on the cell surface of mesothelioma, ovarian cancer and other malignant tumors. The interaction between mesothelin and CA125 (also called MUC16) may facilitate the implantation and metastasis of tumors in the peritoneal cavity. A desirable therapeutic agent involves finding a fully human monoclonal antibody (mAb) that binds to mesothelin or CA125 and inhibits their interaction. Here, we report the identification of a novel human mAb to mesothelin. HN1, a human single‐chain Fv specific for mesothelin, was isolated from a naïve human single‐chain variable fragment (scFv) phage display library. To investigate HN1 as a potential therapeutic, we generated a fully human IgG with the γ 1 heavy chain and the κ light chain and an immuntoxin by fusing the HN1 scFv to a truncated Pseudomonas exotoxin A. The HN1 IgG kills cancer cells with very strong antibody‐dependent cell‐mediated cytotoxicity. HN1 binds a conformation‐sensitive epitope in human mesothelin with high affinity (K_D = 3 nM). The HN1 epitope is different from that of SS1, a mouse Fv used to develop therapeutic antibodies that are currently in clinical trials. HN1 binds to cell surface‐associated mesothelin on human mesothelioma, ovarian cancer, lung adenocarcinoma and pancreatic cancer cells. In addition, HN1 can functionally block the interaction of mesothelin and CA125 on cancer cells. Most importantly, because the HN1 immuntoxin kills mesothelin‐expressing cancer cells with high cytotoxic activity, we believe that it has significant potential for mesothelin‐expressing cancer treatment and diagnosis. Published 2010 UICC. This article is a US Government work and, as such, is in the public domain of the United States of America.     

Mesothelin, a novel immunotherapy target for triple negative breast cancer

Mesothelin is a cell-surface glycoprotein present on mesothelial cells and elicits T cell responses in a variety of cancers including pancreatic, biliary and ovarian cancer. Breast cancer is not known to express mesothelin. We postulated that mesothelin may be a unique tumor-associated antigen in triple negative breast cancer (TNBC), a less common breast cancer subtype which may have been under-represented in prior studies that characterized mesothelin expression. Therefore, we screened 99 primary breast cancer samples by immunohistochemistry analysis using formalin-fixed paraffin-embedded archival tumor tissues and confirmed that mesothelin was overexpressed in the majority of TNBC (67 %) but only rarely in <5 % ER(+) or Her2-neu(+) breast cancer, respectively. To determine whether mesothelin may be exploited as a novel immunotherapy target in breast cancer, an in vitro cell killing assay was performed to compare the ability of genetically modified T cells expressing a chimeric antibody receptor (CAR) specific for mesothelin (mesoCAR T cells) or non-transduced T cells to kill mesothelin-expressing primary breast cancer cells. A significantly higher anti-tumor cytotoxicity by mesoCAR T cells was observed (31.7 vs. 8.7 %, p < 0.001). Our results suggest that mesothelin has promise as a novel immunotherapy target for TNBC for which effective targeted therapy is lacking to date.     

Effects of personal characteristics on serum CA125, mesothelin, and HE4 levels in healthy postmenopausal women at high-risk for ovarian cancer

OBJECTIVE: To evaluate if serum levels of candidate ovarian cancer biomarkers vary with individual characteristics of healthy women who are likely candidates for an ovarian cancer screening program. METHODS: We analyzed serum CA125, mesothelin, and HE4 levels in a sample of 155 healthy postmenopausal women at increased risk for developing ovarian cancer based on personal and family cancer history. Information on reproductive, family and medical histories, lifestyle factors, and anthropometry was collected by self-report. Twenty-two factors were examined using univariate and multiple linear regression models for the three biomarker levels. RESULTS: In the multivariate models, CA125 levels were significantly higher in women who had used talcum powder (P = 0.02) and were lower in women who were parous (P = 0.05). Mesothelin levels were significantly higher in older women (P = 0.01) and lower in heavier women (P = 0.03). HE4 levels were higher in older women (P = 0.001) and in women who began menstruating at an older age (P = 0.03). CONCLUSIONS: CA125, mesothelin, and HE4 levels in healthy, postmenopausal women at increased risk for ovarian cancer are significantly associated with a few ovarian cancer risk factors. Since the effects of these personal characteristics on these serum markers are not large, their incorporation in screening algorithms may be unnecessary. This is true especially if a longitudinal algorithm is used because the marker level at the previous screen reflects personal characteristics such as age, body mass index, and age of menarche. Understanding the influence of personal factors on levels of novel early detection markers in healthy, unaffected women may have clinical utility in interpreting biomarker levels.     

血清Mesothelin和CA125联合检测在上皮性卵巢癌诊断中的意义

目的：研究联合检测血清Mesothelin和CA125在上皮性卵巢癌诊断中的临床意义。方法：采用定量酶联免疫吸附试验（ELISA）检测上皮性卵巢癌患者31例,良性卵巢肿瘤32例和正常妇女30例血清中的Me-sothelin和CA125水平。结果：上皮性卵巢癌患者的Mesothelin水平明显高于良性卵巢肿瘤患者和正常对照组（P〈0.01）,良性卵巢肿瘤组患者与正常对照组的差异无显著性（P〉0.05）。不同类型的上皮性卵巢癌的Mesothelin的差异无显著性（P〉0.05）。Mesothelin和CA125联合检测对上皮性卵巢癌诊断的灵敏度和正确率明显高于单项检测CA125或Mesothelin（P值均〈0.05）。结论：血清Mesothelin和CA125联合检测可提高上皮性卵巢癌的早期诊断率。     

血清Mesothelin和CA125联合检测在上皮性卵巢癌诊断中的意义

目的:研究联合检测血清Mesothelin和CA125在上皮性卵巢癌诊断中的临床意义.方法:采用定量酶联免疫吸附试验(ELISA)检测上皮性卵巢癌患者31例,良性卵巢肿瘤32例和正常妇女30例血清中的Mesothelin和CA125水平.结果:上皮性卵巢癌患者的Mesothelin水平明显高于良性卵巢肿瘤患者和正常对照组(P<0.01),良性卵巢肿瘤组患者与正常对照组的差异无显著性(P>0.05).不同类型的上皮性卵巢癌的Mesothelin的差异无显著性(P>0.05).Mesothelin和CA125联合检测对上皮性卵巢癌诊断的灵敏度和正确率明显高于单项检测CA125或Mesothelin(P值均<0.05).结论:血清Mesothelin和CA125联合检测可提高上皮性卵巢癌的早期诊断率.     

New monoclonal antibodies to mesothelin useful for immunohistochemistry, fluorescence-activated cell sorting, Western blotting, and ELISA.

PURPOSE: Mesothelin is a cell surface protein that is highly expressed in some malignant tumors, and is a promising target for immunotherapy. Recent data suggests that mesothelin is an adhesive protein and may have a role in the metastases of ovarian cancer. Although a few monoclonal antibodies (MAb) to mesothelin have been produced, they have limitations for the study of expression of native mesothelin because of their low affinity or reactivity only with denatured mesothelin protein. We have produced novel MAbs to mesothelin to help study mesothelin function and to develop improved diagnosis and immunotherapy of mesothelin-expressing tumors. EXPERIMENTAL DESIGN: Mesothelin-deficient mice were immunized with plasmid cDNA encoding mesothelin, and boosted with a mesothelin-rabbit IgG Fc fusion protein prior to cell fusion. Hybridomas were screened by an ELISA using plates coated with mesothelin-Fc protein. RESULTS: Seventeen hybridomas producing anti-mesothelin antibodies were established and shown to react with two epitopes on mesothelin. One group reacts with the same epitope as the low affinity antibody K1 that was originally used to identify mesothelin. The other is a new group that reacts with a new epitope. One antibody from each group was chosen for further study and shown to react strongly on ELISA, on immunohistochemistry, and by fluorescence-activated cell sorting on living cells. CONCLUSION: Our two newly established MAbs, MN and MB, have different and useful properties compared with current antibodies used for the detection of mesothelin by immunohistochemistry, fluorescence-activated cell sorting, ELISA, and Western blotting.     

Mesothelin variant 1 is released from tumor cells as a diagnostic marker.

The mesothelin family comprises (at least) three variants and includes the precursor for megakaryocyte potentiating factor (MPF). Assaying soluble mesothelin-related protein (SMRP) molecules in serum and other body fluids from patients with certain cancers can provide diagnostically useful information. We have constructed fusion proteins of mesothelin variants 1, 2, and 3, made monoclonal antibodies, and investigated the binding specificity of these and three previously generated monoclonal antibodies to each of the three mesothelin variants. According to flow cytometry, the molecule that is most frequently expressed at the surface of cells from ovarian carcinomas and certain other tumors is mesothelin variant 1. Similarly, SMRP released into ascites from a patient with ovarian carcinoma was shown to have a molecular weight of approximately 40 kDa and, according to sequencing, to be variant 1. A published sandwich ELISA was shown to detect variants 1 and 3 and to be much more sensitive than a newly constructed ELISA, which detects only variant 3, the former being positive in 28 of 41 (68%) sera from patients with ovarian cancer as compared with 6 of 41 sera (15%). A standard curve was constructed to measure SMRP with a limit of detection of 200 pg/mL to facilitate future quantitative studies.     

A novel cancer/testis antigen KP-OVA-52 identified by SEREX in human ovarian cancer is regulated by DNA methylation

SEREX has proven to be a powerful method that takes advantage of the presence of spontaneous humoral immune response in some cancer patients. In this study, immunoscreening of normal testis and two ovarian cancer cell line cDNA expression libraries with sera from ovarian cancer patients led to the isolation of 75 independent antigens, designated KP-OVA-1 through KP-OVA-75. Of these, RT-PCR showed KP-OVA-52 to be expressed strongly in normal testis, in ovarian cancer cell lines (3/9) and in ovarian cancer tissues (1/17). The expression of KP-OVA-52 in cancer cells is also induced by the demethylating agent 5?aza?2'?deoxycytidine (ADC). To test immunogenicity, we used the Serum Antibody Detection Assay (SADA) to analyze anti-IgG antibodies against the 75 antigens that were initially isolated by SEREX. Four of the 75 antigens (KP?OVA?25, KP?OVA?35, KP?OVA?68 and KP?OVA?73) reacted exclusively with sera from cancer patients. However, KP?OVA?52 reacted with 1 of 20 ovarian cancer sera. These data suggest that the KP-OVA-52 can be considered a novel CT antigen that is regulated by DNA methylation.     

Overexpression and potential targeting of the oncofoetal antigen 5T4 in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is resistant to conventional treatments. Novel, targeted treatments are hampered by the relative lack of MPM-associated tumour antigens. The aim of this study was to evaluate the level of expression and the relevance of 5T4 as a tumour-associated antigen in MPM. 5T4 expression was assessed by Western blotting, flow cytometry, immuno-cytochemistry and -histochemistry in 11 mesothelioma cell lines, 21 tumour biopsies, and ex vivo tumour cells obtained from the pleural fluid (PF) of 10 patients. 5T4 antibody levels were also determined in the plasma of patients and healthy donors. The susceptibility of MPM cells to 5T4-specific T-cell-mediated killing was determined using an HLA-A2(+), CD8(+) T-cell line, developed against the 5T4(17-25) peptide. We report here that cell surface 5T4 expression was detected in all mesothelioma cell lines and PF cell samples. Mesothelin and CD200, a suggested mesothelioma marker, were co-expressed with 5T4 on tumour cells in PF. Immunohistochemistry confirmed overexpression of 5T4, similar to mesothelin, on tumour cells but not on reactive stroma in all tissue sections tested. Median 5T4 antibody levels were 46% higher in patient than in healthy donor plasma, indicating immune recognition. Importantly, 5T4-specific CD8(+) T-cells were able to kill four out of six HLA-A2(+) MPM cell lines but not an HLA-A2(-) cell line, demonstrating immune recognition of MPM-associated 5T4 antigen at the effector T-cell level. We conclude that 5T4 is a potential new antigen for targeted therapies such as immunotherapy in MPM, as it is overexpressed on mesothelioma cells and recognised by 5T4-specific cytotoxic T-cells. Our findings have been translated into a Phase II clinical trial applying 5T4-targeted therapies in MPM patients.     

A fiber-modified mesothelin promoter-based conditionally replicating adenovirus for treatment of ovarian cancer

PURPOSE: Recently, virotherapy has been proposed as a new therapeutic approach for ovarian cancer. Conditionally replicative adenoviruses (CRAd) may contain tumor-specific promoters that restrict virus replication to cancer cells. Mesothelin, a cell surface glycoprotein, is overexpressed in ovarian cancer but not in normal ovarian tissues. The purpose of this study was to explore the therapeutic utility of a mesothelin promoter-based CRAd in a murine model of ovarian cancer, using noninvasive in vivo imaging. EXPERIMENTAL DESIGN: We constructed a mesothelin promoter-based CRAd with a chimeric Ad5/3 fiber (AdMSLNCRAd5/3) that contains an Ad5 tail, Ad5 shaft, and an Ad3 knob. Previously, a chimeric Ad5/3 fiber has shown improved infectivity in many ovarian cancer cells. Viral replication and oncolysis were assessed in a panel of ovarian cancer cell lines. To test the oncolytic efficacy of AdMSLNCRAd5/3 in a murine model, bioluminescence imaging of tumor luciferase activity and survival analysis were done. RESULTS: AdMSLNCRAd5/3 achieved up to a 10,000-fold higher cell killing effect and up to 120-fold higher levels of viral replication in all human ovarian cancer cells, compared with wild-type Ad5. AdMSLNCRAd5/3 significantly inhibited tumor growth as confirmed by in vivo imaging (P < 0.05). Survival with AdMSLNCRAd5/3 was significantly enhanced when compared with no virus or with a wild-type Ad5-treated group (P < 0.05). CONCLUSIONS: The robust replication, oncolysis, and in vivo therapeutic efficacy of AdMSLNCRAd5/3 showed that this CRAd is a promising candidate for treating ovarian cancer. Importantly, we have applied in vivo imaging that has allowed repeated and longitudinal measurements of tumor growth after CRAd treatment.     

Novel ELISA system for detection of N-ERC/mesothelin in the sera of mesothelioma patients

We have developed a novel enzyme-linked immunosorbent assay (ELISA) system for the detection of N-ERC/mesothelin in the serum of mesothelioma patients and have begun to examine its clinical usefulness. N-ERC/mesothelin is a 31-kDa protein that forms the N-terminal fragment of the full-length 71-kDa ERC/mesothelin protein, and is physiologically secreted into the blood of mesothelioma patients where it can be detected using our sandwich ELISA containing two antibodies (rabbit polyclonal anti-ERC/mesothelin antibody-282 and mouse monoclonal antibody 7E7). Our ELISA system has thus far detected much higher serum levels of N-ERC/mesothelin in mesothelioma patients than in healthy controls or patients with other lung or pleural diseases. In conclusion, N-ERC/mesothelin is a promising candidate tumor marker for mesothelioma.