Mesothelin promotes brain metastasis of non-small cell lung cancer by activating MET.

BACKGROUND: Brain metastasis (BM) is common among cases of advanced non-small cell lung cancer (NSCLC) and is the leading cause of death for these patients. Mesothelin (MSLN), a tumor-associated antigen expressed in many solid tumors, has been reported to be involved in the progression of multiple tumors. However, its potential involvement in BM of NSCLC and the underlying mechanism remain unknown. METHODS: The expression of MSLN was validated in clinical tissue and serum samples using immunohistochemistry and enzyme-linked immunosorbent assay. The ability of NSCLC cells to penetrate the blood-brain barrier (BBB) was examined using an in vitro Transwell model and an ex vivo multi-organ microfluidic bionic chip. Immunofluorescence staining and western blotting were used to detect the disruption of tight junctions. In vivo BBB leakiness assay was performed to assess the barrier integrity. MET expression and activation was detected by western blotting. The therapeutic efficacy of drugs targeting MSLN (anetumab) and MET (crizotinib/capmatinib) on BM was evaluated in animal studies. RESULTS: MSLN expression was significantly elevated in both serum and tumor tissue samples from NSCLC patients with BM and correlated with a poor clinical prognosis. MSLN significantly enhanced the brain metastatic abilities of NSCLC cells, especially BBB extravasation. Mechanistically, MSLN facilitated the expression and activation of MET through the c-Jun N-terminal kinase (JNK) signaling pathway, which allowed tumor cells to disrupt tight junctions and the integrity of the BBB and thereby penetrate the barrier. Drugs targeting MSLN (anetumab) and MET (crizotinib/capmatinib) effectively blocked the development of BM and prolonged the survival of mice. CONCLUSIONS: Our results demonstrate that MSLN plays a critical role in BM of NSCLC by modulating the JNK/MET signaling network and thus, provides a potential novel therapeutic target for preventing BM in NSCLC patients.

Mesothelin-based CAR-T cells exhibit potent antitumor activity against ovarian cancer.

BACKGROUND: Ovarian cancer (OC) is characterized by its rapid growth and spread which, accompanied by a low 5-year survival rate, necessitates the development of improved treatments. In ovarian cancer, the selective overexpression of Mucin-16 (MUC16, CA125) in tumor cells highlights its potential as a promising target for developing anti-tumor therapies. However, the potential effectiveness of CAR-T cell therapy that targets MUC16 in ovarian cancer cells is unknown. METHODS: The expression of MUC16 in viable OC cells was detected using immunofluorescence and flow cytometry techniques. A MSLN-CAR construct, comprising the MUC16-binding polypeptide region of mesothelin (MSLN), a CD8 hinge spacer and transmembrane domain, 4-1BB, and CD3ζ endo-domains; was synthesized and introduced into T cells using lentiviral particles. The cytotoxicity of the resultant CAR-T cells was evaluated in vitro using luciferase assays. Cytokine release by CAR-T cells was measured using enzyme-linked immunosorbent assays. The anti-tumor efficacy of the CAR-T cells was subsequently assessed in mice through both systemic and local administration protocols. RESULTS: MSLN-CAR T cells exhibited potent cytotoxicity towards OVCAR3 cells and their stem-like cells that express high levels of MUC16. Also, MSLN-CAR T cells were inefficient at killing SKOV3 cells that express low levels of MUC16, but were potently cytotoxic to such cells overexpressing MUC16. Moreover, MSLN-CAR T cells delivered via tail vein or peritoneal injection could shrink OVCAR3 xenograft tumors in vivo, with sustained remission observed following peritoneal delivery of MSLN-CAR T cells. CONCLUSIONS: Collectively, these results suggested that MSLN-CAR T cells could potently eliminate MUC16- positive ovarian cancer tumor cells both in vitro and in vivo, thereby providing a promising therapeutic intervention for MUC16-positive patients.

Selection, engineering, and in vivo testing of a human leukocyte antigen-independent T-cell receptor recognizing human mesothelin.

BACKGROUND: Canonical α/ß T-cell receptors (TCRs) bind to human leukocyte antigen (HLA) displaying antigenic peptides to elicit T cell-mediated cytotoxicity. TCR-engineered T-cell immunotherapies targeting cancer-specific peptide-HLA complexes (pHLA) are generating exciting clinical responses, but owing to HLA restriction they are only able to target a subset of antigen-positive patients. More recently, evidence has been published indicating that naturally occurring α/ß TCRs can target cell surface proteins other than pHLA, which would address the challenges of HLA restriction. In this proof-of-concept study, we sought to identify and engineer so-called HLA-independent TCRs (HiTs) against the tumor-associated antigen mesothelin. METHODS: Using phage display, we identified a HiT that bound well to mesothelin, which when expressed in primary T cells, caused activation and cytotoxicity. We subsequently engineered this HiT to modulate the T-cell response to varying levels of mesothelin on the cell surface. RESULTS: The isolated HiT shows cytotoxic activity and demonstrates killing of both mesothelin-expressing cell lines and patient-derived xenograft models. Additionally, we demonstrated that HiT-transduced T cells do not require CD4 or CD8 co-receptors and, unlike a TCR fusion construct, are not inhibited by soluble mesothelin. Finally, we showed that HiT-transduced T cells are highly efficacious in vivo, completely eradicating xenografted human solid tumors. CONCLUSION: HiTs can be isolated from fully human TCR-displaying phage libraries against cell surface-expressed antigens. HiTs are able to fully activate primary T cells both in vivo and in vitro. HiTs may enable the efficacy seen with pHLA-targeting TCRs in solid tumors to be translated to cell surface antigens.

Mesothelin antigen density influences anti-mesothelin chimeric antigen receptor T cell cytotoxicity.

BACKGROUND AIMS: Several anti-mesothelin (MSLN) chimeric antigen receptor (CAR) T cells are in phase 1/2 clinical trials to treat solid-organ malignancies. The effect of MSLN antigen density on MSLN CAR cytotoxicity against tumor cells has not been examined previously, nor are there data regarding the effect of agents that increase MSLN antigen density on anti-MSLN CAR T cell efficacy. METHODS: MSLN antigen density was measured on a panel of pancreatic cancer and mesothelioma cell lines by flow cytometry. In parallel, the cytotoxicity and specificity of two anti-MSLN CAR T cells (m912 and SS1) were compared against these cell lines using a real-time impedance-based assay. The effect of two MSLN 'sheddase' inhibitors (lanabecestat and TMI-1) that increase MSLN surface expression was also tested in combination with CAR T cells. RESULTS: SS1 CAR T cells were more cytotoxic compared with m912 CAR T cells against cell lines that expressed fewer than ∼170 000 MSLN molecules/cell. A comparison of the m912 and amatuximab (humanized SS1) antibodies identified that amatuximab could detect and bind to lower levels of MSLN on pancreatic cancer and mesothelioma cell lines, suggesting that superior antibody/scFv affinity was the reason for the SS1 CAR's superior cytotoxicity. The cytotoxicity of m912 CAR T cells was improved in the presence of sheddase inhibitors, which increased MSLN antigen density. CONCLUSIONS: These data highlight the value of assessing CAR constructs against a panel of cells expressing varying degrees of target tumor antigen as occurs in human tumors. Furthermore, the problem of low antigen density may be overcome by concomitant administration of drugs that inhibit enzymatic shedding of MSLN.

Macrophages Under the Influence of Tumor Mesothelin Weaken Host Defenses against Pancreatic Cancer Metastasis.

Although pancreatic cancer is a systemic disease that metastasizes early in its course, the signaling systems that promote this behavior remain incompletely understood. In this issue of Cancer Research, Luckett and colleagues identify a paracrine signaling pathway between cancer cells and macrophages that promotes pancreatic cancer metastasis. The authors used immunocompetent murine pancreatic cancer models with high versus low metastatic potential, genetic knockout and complementation strategies, and The Cancer Genome Atlas human data to demonstrate that tumor-secreted mesothelin repolarizes tumor and lung macrophages to a tumor-supportive phenotype. The repolarized macrophages increase secretion of VEGF and S100A9, raising local concentrations. In turn, VEGF enhances colony formation of cancer cells, while S100A9 promotes the recruitment of neutrophils to the lungs and the formation of neutrophil extracellular traps that support tumor metastasis. Together, these findings reveal a systemic signaling pathway that promotes pancreatic cancer metastasis by co-opting macrophages typically protective against cancer to instead promote its spread. See related article by Luckett et al., p. 527.

Mesothelin Secretion by Pancreatic Cancer Cells Co-opts Macrophages and Promotes Metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease, yet effective treatments to inhibit PDAC metastasis are lacking. The rich PDAC tumor microenvironment plays a major role in disease progression. Macrophages are the most abundant immune cell population in PDAC tumors and can acquire a range of functions that either hinder or promote tumor growth and metastasis. Here, we identified that mesothelin secretion by pancreatic cancer cells co-opts macrophages to support tumor growth and metastasis of cancer cells to the lungs, liver, and lymph nodes. Mechanistically, secretion of high levels of mesothelin by metastatic cancer cells induced the expression of VEGF alpha (VEGFA) and S100A9 in macrophages. Macrophage-derived VEGFA fed back to cancer cells to support tumor growth, and S100A9 increased neutrophil lung infiltration and formation of neutrophil extracellular traps. These results reveal a role for mesothelin in regulating macrophage functions and interaction with neutrophils to support PDAC metastasis. SIGNIFICANCE: Mesothelin secretion by cancer cells supports pancreatic cancer metastasis by inducing macrophage secretion of VEGFA and S100A9 to support cancer cell proliferation and survival, recruit neutrophils, and stimulate neutrophil extracellular trap formation. See related commentary by Alewine, p. 513.

ZFP36 disruption is insufficient to enhance the function of mesothelin-targeting human CAR-T cells.

Loss of inflammatory effector function, such as cytokine production and proliferation, is a fundamental driver of failure in T cell therapies against solid tumors. Here, we used CRISPR/Cas9 to genetically disrupt ZFP36, an RNA binding protein that regulates the stability of mRNAs involved in T cell inflammatory function, such as the cytokines IL2 and IFNγ, in human T cells engineered with a clinical-stage mesothelin-targeting CAR to determine whether its disruption could enhance antitumor responses. ZFP36 disruption slightly increased antigen-independent activation and cytokine responses but did not enhance overall performance in vitro or in vivo in a xenograft tumor model with NSG mice. While ZFP36 disruption does not reduce the function of CAR-T cells, these results suggest that singular disruption of ZFP36 is not sufficient to improve their function and may benefit from a multiplexed approach.

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.

Specific Targeting of Mesothelin-Expressing Malignant Cells Using Nanobody-Functionalized Magneto-Fluorescent Nanoassemblies.

Introduction: Most current anti-cancer therapies are associated with major side effects due to a lack of tumor specificity. Appropriate vectorization of drugs using engineered nanovectors is known to increase local concentration of therapeutic molecules in tumors while minimizing their side effects. Mesothelin (MSLN) is a well-known tumor associated antigen overexpressed in many malignancies, in particular in malignant pleural mesothelioma (MPM), and various MSLN-targeting anticancer therapies are currently evaluated in preclinical and clinical assays. In this study, we described, for the first time, the functionalization of fluorescent organic nanoassemblies (NA) with a nanobody (Nb) targeting MSLN for the specific targeting of MSLN expressing MPM cancer cells. Methods: Cell lines from different cancer origin expressing or not MSLN were used. An Nb directed against MSLN was coupled to fluorescent NA using click chemistry. A panel of endocytosis inhibitors was used to study targeted NA internalization by cells. Cancer cells were grown in 2D or 3D and under a flow to evaluate the specificity of the targeted NA. Binding and internalization of the targeted NA were studied using flow cytometry, confocal microscopy and transmission electron microscopy. Results: We show that the targeted NA specifically bind to MSLN-expressing tumor cells. Moreover, such functionalized NA appear to be internalized more rapidly and in significantly larger proportions compared to naked ones in MSLN+ MPM cells, thereby demonstrating both the functionality and interest of the active targeting strategy. We demonstrated that targeted NA are mainly internalized through a clathrin-independent/dynamin-dependent endocytosis pathway and are directed to lysosomes for degradation. A 3D cell culture model based on MSLN-expressing multicellular tumor spheroids reveals NA penetration in the first superficial layers. Conclusion: Altogether, these results open the path to novel anticancer strategies based on MSLN-activated internalization of NA incorporating drugs to promote specific accumulation of active treatments in tumors.

Tumor resistance to anti-mesothelin CAR-T cells caused by binding to shed mesothelin is overcome by targeting a juxtamembrane epitope.

Despite many clinical trials, CAR-T cells are not yet approved for human solid tumor therapy. One popular target is mesothelin (MSLN) which is highly expressed on the surface of about 30% of cancers including mesothelioma and cancers of the ovary, pancreas, and lung. MSLN is shed by proteases that cleave near the C terminus, leaving a short peptide attached to the cell. Most anti-MSLN antibodies bind to shed MSLN, which can prevent their binding to target cells. To overcome this limitation, we developed an antibody (15B6) that binds next to the membrane at the protease-sensitive region, does not bind to shed MSLN, and makes CAR-T cells that have much higher anti-tumor activity than a CAR-T that binds to shed MSLN. We have now humanized the Fv (h15B6), so the CAR-T can be used to treat patients and show that h15B6 CAR-T produces complete regressions in a hard-to-treat pancreatic cancer patient derived xenograft model, whereas CAR-T targeting a shed epitope (SS1) have no anti-tumor activity. In these pancreatic cancers, the h15B6 CAR-T replicates and replaces the cancer cells, whereas there are no CAR-T cells in the tumors receiving SS1 CAR-T. To determine the mechanism accounting for high activity, we used an OVCAR-8 intraperitoneal model to show that poorly active SS1-CAR-T cells are bound to shed MSLN, whereas highly active h15B6 CAR-T do not contain bound MSLN enabling them to bind to and kill cancer cells.

A predictive and prognostic model for surgical outcome and prognosis in ovarian cancer computed by clinico-pathological and serological parameters (CA125, HE4, mesothelin).

OBJECTIVES: Numerous prognostic models have been proposed for ovarian cancer, extending from single serological factors to complex gene-expression signatures. Nonetheless, these models have not been routinely translated into clinical practice. We constructed a robust and readily calculable model for predicting surgical outcome and prognosis of ovarian cancer patients by exploiting commonly available clinico-pathological factors and three selected serum parameters. METHODS: Serum CA125, human epididymis protein 4 (HE4) and mesothelin (MSL) were quantified by Lumipulse® G chemiluminescent enzyme immunoassay (Fujirebio) in a total of 342 serum samples from 190 ovarian cancer patients, including 152 paired pre- and post-operative samples. RESULTS: Detection of pre-operative HE4 and CA125 was the optimal marker combination for blood-based prediction of surgical outcome (AUC=0.86). We constructed a prognostic model, computed by serum levels of pre-operative CA125, post-operative HE4, post-operative MSL and surgical outcome. Prognostic performance of our model was superior to any of these parameters alone and was independent from BRCA1/2 mutational status. We subsequently transformed our model into a prognostic risk index, stratifying patients as "lower risk" or "higher risk". In "higher risk" patients, relapse or death was predicted with an AUC of 0.89 and they had a significantly shorter progression free survival (HR: 9.74; 95 % CI: 5.95-15.93; p<0.0001) and overall survival (HR: 5.62; 95 % CI: 3.16-9.99; p<0.0001) compared to "lower risk" patients. CONCLUSIONS: We present a robust predictive/prognostic model for ovarian cancer, which could readily be implemented into routine diagnostics in order to identify ovarian cancer patients at high risk of recurrence.

M9657 Is a Bispecific Tumor-Targeted Anti-CD137 Agonist That Induces MSLN-Dependent Antitumor Immunity without Liver Inflammation.

The costimulatory receptor CD137 (also known as TNFRSF9 or 4-1BB) sustains effective cytotoxic T-cell responses. Agonistic anti-CD137 cancer immunotherapies are being investigated in clinical trials. Development of the first-generation CD137-agonist monotherapies utomilumab and urelumab was unsuccessful due to low antitumor efficacy mediated by the epitope recognized on CD137 or hepatotoxicity mediated by Fcγ receptors (FcγR) ligand-dependent CD137 activation, respectively. M9657 was engineered as a tetravalent bispecific antibody (mAb2) in a human IgG1 backbone with LALA mutations to reduce binding to FCγRs. Here, we report that M9657 selectively binds to mesothelin (MSLN) and CD137 with similar affinity in humans and cynomolgus monkeys. In a cellular functional assay, M9657 enhanced CD8+ T cell-mediated cytotoxicity and cytokine release in the presence of tumor cells, which was dependent on both MSLN expression and T-cell receptor/CD3 activation. Both FS122m, a murine surrogate with the same protein structure as M9657, and chimeric M9657, a modified M9657 antibody with the Fab portion replaced with an anti-murine MSLN motif, demonstrated in vivo antitumor efficacy against various tumors in wild-type and human CD137 knock-in mice, and this was accompanied by activated CD8+ T-cell infiltration in the tumor microenvironment. The antitumor immunity of M9657 and FS122m depended on MSLN expression density and the mAb2 structure. Compared with 3H3, a murine surrogate of urelumab, FS122m and chimeric M9657 displayed significantly lower on-target/off-tumor toxicity. Taken together, M9657 exhibits a promising profile for development as a tumor-targeting immune agonist with potent anticancer activity without systemic immune activation and associated hepatotoxicity.

Novel Anti-Mesothelin Nanobodies and Recombinant Immunotoxins with Pseudomonas Exotoxin Catalytic Domain for Cancer Therapeutics.

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli, achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC50 values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.

INFLUENCE OF PROBIOTICS ON THE MESOTHELIN LEVEL IN WOMEN WITH ENDOMETRIOSIS ASSOCIATED WITH INFERTILITY IN COMPLEX PREPARATION FOR ASSISTED REPRODUCTIVE TECHNOLOGIES.

OBJECTIVE: The aim: To study the determination of Mesothelin level in women with endometriosis associated with infertility and estimate influence of probiotic on endometriosis according of Mesothelin level in complex preparation before assisted reproductive technologies. PATIENTS AND METHODS: Materials and methods: In this study, we conducted a retrospective analysis of the medical records of 40 infertile women who underwent assisted reproductive technologies while also using the probiotic "Femina Probiz." We divided the participants into two groups. The control group comprised 11 women who had tubal infertility due to a previous inflammatory condition but were otherwise found to be in good health through comprehensive clinical and laboratory assessments. These women, aged between 21 and 42 with an average age of 29.75 years, did not use the probiotic "Femina Probiz." The main group consisted of 29 women diagnosed with external genital endometriosis who were undergoing assisted reproductive technologies. Women in the main group received the probiotic "Femina Probiz" from Unic Biotech Ltd, India. They took one tablet twice a day for one month as part of their overall treatment before undergoing assisted reproductive technologies. We measured the Mesothelin levels before and after this preparation phase. This study was conducted at Bukovinian State Medical University and Centre of Reproductive Medicine. It's worth noting that the primary infertility incidence was significantly higher in the main group of patients. RESULTS: Results: In the main group, we observed that the Mesothelin level was 0.73±0.01, which was significantly higher than the post-preparation level (0.59±0.01). In contrast, the control group had a Mesothelin level of 0.49±0.01. Interestingly, we noted that the Mesothelin level in patients increased approximately twofold before preparation compared to those who had undergone preparation. This suggests that the use of the probiotic led to a sharp reduction in the elevated Mesothelin levels. Consequently, the significant decrease in Mesothelin levels after using the probiotic indicates its effectiveness and potential utility in the preparation phase of assisted reproductive technologies programs. CONCLUSION: Conclusions: The elevated Mesothelin levels indicate a strong association between the pathogenesis of endometriosis and inflammation, as well as damage to the peritoneum. The incorporation of a probiotic as part of a comprehensive preparation regimen prior to assisted reproductive technologies notably enhances the overall health of patients and leads to a reduction in Mesothelin levels. Based on our findings, we highly recommend the inclusion of this probiotic preparation in clinical practice.

Pleural inflammatory response, mesothelin content and DNA damage in mice at one-year after intra-pleural carbon nanotube administration.

Many in vitro and in vivo studies have shown that exposure to carbon nanotubes (CNTs) is associated with inflammation, oxidative stress and genotoxicity, although there is a paucity of studies on these effects in the pleural cavity. In the present study, we investigated adverse outcomes of pleural exposure to multi-walled CNTs (MWCNT-7, NM-401 and NM-403) and single-walled CNTs (NM-411). Female C57BL/6 mice were exposed to 0.2 or 5 µg of CNTs by intra-pleural injection and sacrificed one-year post-exposure. Exposure to long and straight types of MWCNTs (i.e. MWCNT-7 and NM-401) was associated with decreased number of macrophages and increased number of neutrophils and eosinophils in pleural lavage fluid. Increased protein content in the pleural lavage fluid was also observed in mice exposed to MWCNT-7 and NM-401. The concentration of mesothelin was increased in mice exposed to MWCNT-7 and NM-411. Levels of DNA strand breaks and DNA oxidation damage, measured by the comet assay, were unaltered in cells from pleural scrape. Extra-pleural effects were seen in CNT exposed mice, including enlarged and pigmented mediastinal lymph nodes (all four types of CNTs), pericardial plaques (MWCNT-7 and NM-401), macroscopic abnormalities on the liver (MWCNT-7) and ovaries/uterus (NM-411). In conclusion, the results demonstrate that intra-pleural exposure to long and straight MWCNTs is associated with adverse outcomes. Certain observations such as increased content of mesothelin in pleural lavage fluid and ovarian/uterine abnormalities in mice exposed to NM-411 suggests that exposure to SWCNTs may also be associated with some adverse outcomes.

Mesothelin expression remodeled the immune-matrix tumor microenvironment predicting the risk of death in patients with malignant pleural mesothelioma.

Background: The combination of immunobiological agents with immune checkpoint proteins is a promising treatment for malignant pleural mesothelioma (MPM). Mesothelin and anti-PD-L1 antibody-drug conjugates specifically target malignant neoplastic cells, inhibit the migration and invasion of neoplastic cells, and restore the immune landscape. In this study, we confirmed the importance of mesothelin and examined the relationship between mesothelin and the immune landscape of the tumor microenvironment (TME) in two MPM cohorts. Methods: The discovery cohort included 82 MPM cases. Tissue microarray slides were generated, and samples were processed for hematoxylin & eosin staining, immunohistochemistry, and immunofluorescence assays. The relationship between mesothelin, biomarkers of histogenesis, histological aggressiveness, PD-L1, immune cells (CD4, CD8, CD20, CD68), and collagen type I and type V fibers was evaluated by quantitative digital analyses. The outcome was the survival time until death from disease recurrence. The exploratory cohort included 87 malignant mesothelioma (MESO) patients from The Cancer Genome Atlas database. Results: Most patients were male (70.7%) with a history of asbestos exposure (53.7%) and with the epithelioid subtype (89%). Surgical resection was performed in 85.4% of patients, and 14.6% received chemotherapy; 59.8% of patients died from disease extension to the mediastinum. Low tumor mesothelin expression was associated with tumor necrosis and nuclear grade 1, whereas high mesothelin expression was significantly associated with the epithelioid histotype and high density of T cells CD8+, macrophages CD68+, and collagen type I fibers. Cox multivariate analysis showed a high risk of death for non-operated patients [hazard ratio (HR), 3.42 (1.15-10.16)] with low tumor mesothelin levels [HR, 2.58 (1.09-6.10)] and high PD-L1 and low infiltration of T cells CD4+ [HR, 3.81 (1.58-9.18)]. In the exploratory cohort, low mesothelin and high COL1A1 and COL5A1 expression were associated with poor overall survival. Conclusion: Tumor mesothelin expression associated with the TME immune landscape predicts the risk of death for patients with MPM and could be a new target for immunotherapy in MPM.

Functional enhancement of mesothelin-targeted TRuC-T cells by a PD1-CD28 chimeric switch receptor.

T cells expressing a mesothelin (MSLN)-specific T cell receptor fusion construct (TRuC®), called TC-210, have demonstrated robust antitumor activity in preclinical models of mesothelioma, ovarian cancer, and lung cancer. However, they are susceptible to suppression by the programmed cell death protein 1 (PD-1)/programmed cell death protein ligand 1 (PD-L1) axis and lack intrinsic costimulatory signaling elements. To enhance the function of anti-MSLN TRuC-T cells, chimeric switch receptors (CSRs) have been designed to co-opt the immunosuppressive PD-1/PD-L1 axis and to deliver a CD28-mediated costimulatory signal. Here, we report that coexpression of the PD1-CD28 CSR in TRuC-T cells enhanced T cell receptor signaling, increased proinflammatory effector cytokines, decreased anti-inflammatory cytokines, and sustained effector function in the presence of PD-L1 when compared with TC-210. Anti-MSLN TRuC-T cells engineered to coexpress PD1-CD28 CSRs comprising the ectodomain of PD-1 and the intracellular domain of CD28 linked by the transmembrane domain of PD-1 were selected for integration into an anti-MSLN TRuC-T cell therapy product called TC-510. In vitro, TC-510 showed significant improvements in persistence and resistance to exhaustion upon chronic stimulation by tumor cells expressing MSLN and PD-L1 when compared with TC-210. In vivo, TC-510 showed a superior ability to provide durable protection following tumor rechallenge, versus TC-210. These data demonstrate that integration of a PD1-CD28 CSR into TRuC-T cells improves effector function, resistance to exhaustion, and prolongs persistence. Based on these findings, TC-510 is currently being evaluated in patients with MSLN-expressing solid tumors.

Novel mesothelin-targeted chimeric antigen receptor-modified UNKT cells are highly effective in inhibiting tumor progression.

The design of chimeric antigen receptors (CAR) significantly enhances the antitumor efficacy of T cells. Although some CAR-T products have been approved by FDA in treating hematological tumors, adoptive immune therapy still faces many difficulties and challenges in the treatment of solid tumors. In this study, we reported a new strategy to treat solid tumors using a natural killer-like T (NKT) cell line which showed strong cytotoxicity to lyse 15 cancer cell lines, safe to normal cells and had low or no Graft-versus-host activity. We thus named it as universal NKT (UNKT). In both direct and indirect 3D tumor-like organ model, UNKT showed efficient tumor-killing properties, indicating that it could penetrate the microenvironment of solid tumors. In mesothelin (MSLN)-positive tumor cells (SKOV-3 and MCF-7), MSLN targeting CAR modified-UNKT cells had enhanced killing potential against MSLN positive ovarian cancer compared with the wild type UNKT, as well as MSLN-CAR-T cells. Compared with CAR-T, Single-cell microarray 32-plex proteomics revealed CAR-UNKT cells express more effector cytokines, such as perforin and granzyme B, and less interleukin-6 after activation. Moreover, our CAR-UNKT cells featured in more multifunctionality than CAR-T cells. CAR-UNKT cells also demonstrated strong antitumor activity in mouse models of ovarian cancer, with the ability to migrate and infiltrate the tumor without inducing immune memory. The fast-in and -out, enhanced and prolonged tumor killing properties of CAR-UNKT suggested a novel cure option of cellular immunotherapy in the treatment of MSLN-positive solid tumors.

Ligand-based adoptive T cell targeting CA125 in ovarian cancer.

BACKGROUND: Ovarian cancer (OC) is a highly aggressive gynecological malignancy prevalent worldwide. Most OC cases are typically diagnosed at advanced stages, which has led to a 5-year overall survival rate of less than 35% following conventional treatment. Furthermore, immune checkpoint inhibitor therapy has shown limited efficacy in the treatment of patients with OC, and CAR-T therapy has also demonstrated modest results owing to inadequate T cell infiltration. Therefore, novel strategies must be developed to enhance T cell persistence and trafficking within the OC tumor microenvironment. METHODS: In this study, we developed a novel adoptive T-cell therapy for ovarian cancer based on a chimeric antigen receptor structure. We used a ligand-receptor binding motif to enhance the therapeutic effect of targeting CA125. Since mesothelin can naturally bind to CA125 with high affinity, we concatenated the core-binding fragment of mesothelin with the 4-1BB and CD3ζ signal fragments to assemble a novel CA125-targeting chimeric receptor (CR). The CAR structure targeting CA125 derived from the 4H11 antibody was also constructed. CR- and CAR-encoding RNA were electroporated into T cells to evaluate their antitumor activity both in vitro and in vivo. RESULTS: While CR-T or CAR-T cells exhibited moderate activity against two ovarian cancer cell lines, T cells co-expressing CR and CAR exhibited a superior killing effect compared to T cells expressing either CR or CAR alone. Furthermore, upon interaction with ovarian tumors, the ability of CR and CAR T cells to release activation markers and functional cytokines increased significantly. Similarly, CR and CAR co-expressing T cells persistently controlled the growth of transplanted ovarian cancer tumors in NSG mice and significantly prolonged the overall survival of tumor-challenged mice. Transcriptome sequencing revealed that the survival and cytotoxicity of T cells co-expressing CR and CAR were significantly altered compared with those of T cells expressing either CR or CAR. CONCLUSION: Our findings demonstrate that CA125 targeting CR and CAR can synergistically kill ovarian cancer cells, indicating that CA125 targeting by the two binding motifs simultaneously in tumors may improve the therapeutic outcomes of ovarian cancer treatment.

Mesothelin-targeted CAR-T therapy combined with irinotecan for the treatment of solid cancer.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has shown promising results in treating blood cancers, but it has limited efficacy against solid tumors that express mesothelin (MSLN). One of the reasons is the immunosuppressive tumor microenvironment, which consists of physical barriers, multiple mechanisms of immune evasion, and various biochemical factors that favor tumor growth and survival. These factors reduce the antitumor activity of MSLN-targeted CAR T cells in clinical trials. Therefore, new therapeutic strategies are needed to enhance the effectiveness of MSLN-targeted CAR T cell therapy. METHODS: To investigate whether the antitumor efficacy of anti-MSLN CAR-T cells depends on the epitopes they recognize, we generated MSLN-targeted CAR T cells that bind to different regions of MSLN (Region I, II, III and Full length). We then evaluated the antitumor activity of MSLN-targeted CAR T cells alone or in combination with the chemotherapeutic drug irinotecan or an anti-PD-1 antibody in vitro and in vivo. RESULTS: We found that MSLN-targeted CAR T cells effectively killed MSLN-positive cancer cells (H9, H226 and Panc-1), but not MSLN-negative cells (A431) in vitro. In a mouse model of H9 tumor xenografts, all CAR T cells showed similar tumor suppression, but an MSLN-targeted scFv with Region I epitope, R47, performed slightly better. Combining irinotecan with CAR_R47 T cells enhanced tumor control synergistically in both H9 xenograft mice and patient-derived xenograft mice. CONCLUSIONS: Our results suggest that irinotecan can enhance the antitumor activity of MSLN-targeted CAR T cells, and offer a promising combination therapy strategy for MSLN-positive solid tumors.