Treatment of advanced stage cancers is extremely challenging, and more effective systemic therapy is needed. Oncolytic adenoviruses (OAds) are one of the most promising anti-cancer agents. However, systemic delivery of OAd is challenging due to the low transduction in tumor cells caused by non-selective distribution and sequestration by non-target organs. To overcome this issue, we have previously generated a mesothelin (MSLN)-targeted OAd (AdML-VTIN). Here, we are reporting the potential of MSLN-targeted OAd as an agent for novel systemic treatment using MSLN-expressing lung and pancreatic cancer models. The in vivo biodistribution of AdML-VTIN after intravenous injection showed significantly lower liver sequestration compared to the wild type of OAd (AdML-5WT). By day 7, the intratumoral viral copy number of AdML-VTIN was significantly higher than that of AdML-5WT. For therapeutic efficacy, systemically injected AdML-VTIN exhibited statistically significant anti-tumor effects in both lung and pancreatic cancer xenograft tumor models. In addition, we tested the effect of preexisting immunity using human serum. In a neutralization assay, AdML-VTIN was more resistant to preexisting antibodies, compared to Ad5-WT. Interestingly, the hemagglutination profile of AdML-VTIN was also changed. Our results indicate that MSLN-targeted OAd has great potential to facilitate systemic therapy of advanced cancers.

Epithelial ovarian cancer is the most prevalent gynecological malignancy, characterized by high mortality rates due to its late-stage diagnosis and frequent recurrence. The current standard of care for ovarian cancer is a combination of debulking surgery followed by the conventional mode of chemotherapy. Despite significant advances in therapeutic modalities, the overall survival rate of EOC continues to be poor, mainly because low concentrations of the chemotherapeutics reach the peritoneum, which is the primary site of ovarian cancer, leading to disease relapse. Here, intraperitoneal chemotherapy gains advantage due to its ability to deliver the drug molecules directly to the peritoneal cavity and provide localized and sustained effects. This is facilitated by the use of diverse kinds of nano or micron sized delivery systems, which help in transporting drugs, vaccines, antibodies and genes appropriately to the peritoneum for its desired function. This review article delves on how intraperitoneal delivery impacts the therapy of epithelial ovarian cancer spanning the conventional therapeutic modes to the recent nanoinnovations in chemotherapy, immunotherapy and gene therapy.

Endocrine cancer, a relatively rare and heterogeneous tumor with diverse clinical features. The facile synthesis of hormones further complicates endocrine cancer treatment. Thus, the development of safe and effective systemic treatment approaches, such as chimeric antigen receptor (CAR) T cell therapy, is imperative to enhance the prognosis of patients with endocrine cancer. Although this therapy has achieved good results in the treatment of hematological malignancies, it encounters diverse complications and challenges in the context of endocrine cancer. This review delineates the generation of CAR-T cells, examines the potential of CAR-T cell therapy for endocrine cancer, enumerates pivotal antigens linked to endocrine cancer, encapsulates the challenges confronted with CAR-T cell therapy for endocrine cancer, and expounds upon strategies to overcome these limitations. The primary objective is to provide insightful perspectives that can contribute to the advancement of CAR-T cell therapy in the field of endocrine cancer.

Cell surface mesothelin (MSLN) can be solubilized and released into the systemic circulation. The resulting soluble MSLN (sMSLN) may interfere with therapies targeting surface MSLN. We investigated the effects of sMSLN on anetumab, an antibody-based therapy against MSLN, anetumab ravtansine, an antibody drug conjugate, and mechanisms to decrease sMSLN. Whole blood samples were collected before and after one plasma volume of therapeutic plasma exchange (TPE). sMSLN levels were measured with ELISA assays in matched pre- and post-TPE plasma samples, and anetumab-immunoprecipitated samples. We also used protease inhibitors (PIs) as a mechanism to stabilize surface MSLN, then evaluated the cytotoxic effects of anetumab ravtansine. Our findings indicate that sMSLN sequesters and may impair the efficacy of this anti-MSLN antibody based on results showing that anetumab decreases the concentration of MSLN in plasma (p < 0.05) and reduced cytotoxicity of anetumab ravtansine in the presence of recombinant MSLN in cell lines, a surrogate for sMSLN. TPE consistently reduced sMSLN (p < 0.05) with an average decrease of 43.6% (15.4 ng/mL). Surface MSLN stabilization was inconsistently observed with PIs. Overall, sMSLN could represent a predictive biomarker for MSLN directed therapies. TPE may be more reliable than PIs to reduce sMSLN and ultimately restore sensitivity to these therapies in patients with high sMSLN.

The glycosylphosphatidylinositol-anchored cell surface protein mesothelin (MSLN) shows elevated expression in many malignancies and is an established clinical-stage target for antibody-directed therapeutic strategies. Of these, the harnessing of autologous patient T cells via engineered anti-MSLN chimeric antigen receptors (CAR-T) is an approach garnering considerable interest. Although generally shown to target tumor MSLN safely, CAR-T trials have failed to deliver the impressive curative or response metrics achieved for hematological malignancies using the same technology. A need exists, therefore, for improved anti-MSLN molecules and/or more optimal ways to leverage immune effector cells.

We performed ELISA, label-free kinetic binding assays, FACS, Western blotting, and transient recombinant MSLN expression to characterize the recognition properties of a novel CAR-active human scFv clone, LABC-13F08. To investigate T cell redirection, we conducted kinetic IncuCyte co-culture killing assays using transduced primary T cells and MSLN+ target cell lines and assessed levels of activation markers and effector cytokines. The antitumor potential of LABC-13F08 formatted as a bispecific engager (BiTE) was evaluated in vivo using transduced human primary T cells and immunocompromised NSG mice xenografted with ovarian, mesothelioma, and pancreatic MSLN+ tumor cell lines.

The LABC-13F08 scFv is highly unusual and distinct from existing (pre)clinical anti-MSLN antibody fragments, exhibiting an absolute requirement for divalent cations to drive MSLN recognition. As a monovalent BiTE, LABC-13F08 demonstrates robust in vitro potency. Additionally, primary human T cells engineered for constitutive secretion of the 13F08 BiTE exhibit strong antitumor activity toward in vivo ovarian and mesothelioma xenograft models and show encouraging levels of monotherapy control in a challenging pancreatic model. LABC-13F08 BiTE secreted from engineered T cells (BiTE-T) can both recruit non-engineered bystander T cells and also induce activation-dependent MSLN-independent bystander killing of cells lacking cognate antigen. To address safety concerns, 13F08 BiTE-T cells can be rapidly targeted for clearance via a molecular "off" switch.

The novel LABC-13F08 scFv exhibits a mode of binding to MSLN which is not observed in typical anti-MSLN antibodies. Efficacious targeting by a T cell secreted engager would represent a clinically differentiated approach for the treatment of MSLN+ tumors.

Cholangiocarcinoma (CCA) is a highly fatal malignancy with an increasing prevalence, a high mortality rate, poor overall survival, and limited responsiveness to conventional chemoradiotherapy. Targeted therapies addressing specific gene mutations have expanded treatment options for some patient populations. The introduction of chimeric antigen receptor-modified T-cell (CAR-T) immunotherapy and personalized vaccines have opened up a new avenue for managing various cancers. Considerable efforts have been dedicated to preclinical research and ongoing clinical trials of immunotherapeutic approaches including CAR-T therapy, vaccines, and antibody-based therapies such as antibody drug conjugates. However, the potential of CAR-T therapy and vaccines in treating advanced unresectable/metastatic cholangiocarcinoma remains largely unexplored. This article offers an overview of the current landscape of antibody-based immunotherapy, particularly CAR-T therapy and vaccines in the context of cholangiocarcinoma treatment. It outlines a framework for selecting CAR-T and vaccine targets and delves into the biology of promising targetable antigens, as well as potential future therapeutic targets.

High-grade serous ovarian cancer (HGSOC) is a highly aggressive malignancy marked by an immunosuppressive tumor microenvironment that hinders effective immune responses. A key feature of this environment is the extensive infiltration of myeloid cells, which contributes to immune evasion. This study explored how mesothelin (MSLN), a tumor-associated antigen, modulates the expression of CD24, an emerging target for immune modulation, and its role in promoting immune evasion in HGSOC. Understanding these underlying mechanisms is crucial for enhancing the efficacy of immune checkpoint blockade (ICB) therapies and improving outcomes in patients with HGSOC.

We analyzed the expression of MSLN in HGSOC samples and examined its correlation with clinical outcome. In vitro and in vivo models were used to explore how MSLN influences CD24 expression and the polarization of tumor-associated macrophages (TAMs). We also investigated the role of MSLN in the activation of Wnt/β-catenin signaling and its impact on T-cell function and antitumor immunity. The effects of Msln knockdown on CD24 expression and the response to anti-programmed cell death protein-1 (PD-1) therapy were evaluated in syngeneic mouse models.

MSLN expression was found to be significantly elevated in HGSOC, with high MSLN levels correlating with poor prognosis and resistance to ICB. MSLN upregulated CD24 and promoted the protumorigenic polarization of TAMs, contributing to T-cell dysfunction. Mechanistically, MSLN activated Wnt/β-catenin signaling, which in turn enhanced CD24 expression. This activation forms a positive feedback loop that further promotes MSLN transcription. In contrast, Msln knockdown reduced CD24 expression, relieved cytotoxic T-cell suppression, and significantly improved the efficacy of anti-PD-1 therapy in syngeneic models.

This study elucidates the critical role of MSLN in immune evasion in HGSOC and its underlying mechanisms. Targeting MSLN in combination with ICB is a promising strategy to enhance the efficacy of immunotherapy and improve patient outcomes in HGSOC.

The omentum is a common site of peritoneal metastasis in various cancers, including gastric cancer. It contains immune cell aggregates known as milky spots, which provide a microenvironment for peritoneal immunity by regulating innate and adaptive immune responses. In this study, we investigated gene expression profiles in cells from omental milky spots of patients with gastric cancer (n = 37) by RNA sequencing analysis and classified the patients into four groups (G1-4). Notably, significant differences were observed between the groups in terms of macroscopic type, lymphatic invasion, venous invasion, and pathological stage (pStage). G3, which was enriched in genes related to acquired immunity, showed earlier tumor stages (macroscopic type 0, Ly0, V0, and pStage I) and a better prognosis. In contrast, G4 showed enrichment of genes related to neutrophils and innate immunity; G1 and G2 showed no enrichment of innate or adaptive immune-related genes, suggesting an immune desert microenvironment. Cytometric analysis revealed significantly more T and B cells and fewer neutrophils in G3. Accordingly, the immune microenvironment in omental milky spots may vary depending on the stage of gastric cancer progression. When univariate Cox proportional hazards regression models were used to search for prognostically relevant genes specific to G3, 23 potential prognostic genes were identified as common genes associated with relapse-free survival and overall survival. In addition, the multivariate Cox proportional hazards model using these prognostic genes and clinicopathological information showed that combining the B cell marker CD19 and Ly had a high predictive accuracy for prognosis. Based on this study's results, it is possible that tumor progression, such as lymphatic and/or venous infiltration of tumor cells, may affect the immune cell composition and proportions in omental milky spots of patients with gastric cancer and analysis of gene expression in omental milky spots may help to predict gastric cancer prognosis.

Mesothelin (MSLN) is an antigen that is overexpressed in various cancers, and its interaction with tumor-associated cancer antigen 125 plays a multifaceted role in tumor metastasis. The serum MSLN expression level can be detected using enzyme-linked immunosorbent assay; however, non-invasive visualization of its expression at the tumor site is currently lacking. Therefore, the aim of this study was to develop a molecular probe for imaging MSLN expression through positron emission tomography (PET).

VHH 269-H4 was obtained via immunization of llama using a fragment of MSLN from residue 360 to residue 597. S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) was conjugated to VHH 269-H4 to yield precursor NOTA 269-H4 for radiolabeling. The chelator-to-VHH ratio was determined by mass spectrometry. The binding kinetics of VHH 269-H4 and NOTA 269-H4 were measured by surface plasmon resonance. Flow cytometry was carried out using the anti-mesothelin monoclonal antibody Anetumab to select MSLN-positive and MSLN-negative cell lines. After radiolabeling, the radiochemical purity and in vitro stability were tested by radio-thin-layer chromatography and size exclusion chromatography, respectively. A saturation binding assay was conducted to measure the dissociation constant (Kd) of [68Ga]Ga-NOTA-269-H4. By mircoPET/CT imaging and biodistribution studies, the in vivo performances of the novel tracer were investigated in NCG mice bearing OVCAR-8, SKOV-3, or patient-derived xenografts.

VHH 269-H4 targeting MSLN was obtained with a Kd value of 0.3 nM. After conjugation, approximately 27% and 3.2% of VHH were coupled to one and two NOTA chelators, respectively. This yielded precursor NOTA 269-H4 with a Kd value of 1.1 nM. The radiochemistry was accomplished with moderate radiochemical yields (34 ± 14%, n = 9, decay-corrected). [68Ga]Ga-NOTA-269-H4 was obtained with high radiochemical purity (> 99%), and was stable after 90 min incubation at room temperature. The binding affinity of the radioligand towards MSLN was kept in the nanomolar range. Flow cytometry revealed that OVCAR-8 cells possess a high level of MSLN expression, while MSLN expression on SKOV-3 cells was negligible. Consistently, in microPET/CT imaging, [68Ga]Ga-NOTA-269-H4 demonstrated clear tumor visualization using NCG mice bearing OVCAR-8 xenografts, but no radioactivity accumulation was observed in SKOV-3 xenografts, suggesting a high specificity of the tracer in vivo. In biodistribution studies, [68Ga]Ga-NOTA-269-H4 displayed radioactivity accumulation of 2.93 ± 0.39%ID/g in OVCAR-8 xenografts at 30 min post-injection, and the highest tumor-to-blood ratio (~ 3) was achieved at 90 min post-injection. In NCG mice bearing patient-derived xenografts, [68Ga]Ga-NOTA-269-H4 was able to noninvasively detect MSLN expression via microPET/CT imaging.

To our knowledge, our studies achieved the first-time to non-invasively detect MSLN expression clearly using a single domain antibody fragment. To sum up, [68Ga]Ga-NOTA-269-H4 is a highly promising PET probe to visualize MSLN expression in vivo and holds great potential to monitor MSLN expression during tumor development.

Resistance to the currently available treatment paradigms is one of the main factors that contributes to poor outcomes in patients with advanced cervical cancer. Novel targeted therapy approaches might enhance the patient's treatment outcome and are urgently needed for this malignancy. While chimeric-antigen receptor (CAR)-based adoptive immunotherapy displays a promising treatment strategy for liquid cancers, their use against cervical cancer is largely unexplored. This study used alpharetroviral SIN vectors to equip natural killer (NK) cells with a third-generation CAR (including CD28 and 4-1BB co-stimulatory domains) targeting Mesothelin, which was identified to be highly expressed on primary human cervical cancer tissues and cervical cancer cell lines in this and other studies. Anti-Mesothelin CAR-NK cells demonstrated high cytotoxicity against cervical cancer cells in 2D and 3D culture models, which corresponded to increased degranulation of CAR-NK-92 cells upon exposure to Mesothelin+ target cells. Mesothelin- cervical cancer cells were generated by CRISPR-Cas9-mediated knockout and used to show target antigen specificity of anti-Mesothelin CAR-NK-92 cells and primary NK cells derived from different healthy donors in co-culture experiments. Combination of anti-Mesothelin CAR-NK-92 cells with chemotherapy revealed increased elimination of cancer cells as compared to monotherapy settings. Our findings indicate the promise of anti-Mesothelin CAR-NK cells as a potential treatment option against cervical cancer, as well as other Mesothelin+ malignancies.

The application of chimeric antigen receptor (CAR) T cell therapy in solid tumors is hindered by life-threatening toxicities resulting from on-target, off-tumor killing of nonmalignant cells that express low levels of the target antigen. Mesothelin (MSLN) has been identified as a target antigen for CAR T cell treatment of mesothelioma, lung, ovarian, and other cancers because of its high expression on tumor cells and limited expression on mesothelial cells. However, fatal off-tumor toxicity of high-affinity MSLN-targeting CAR T cells has been reported in multiple clinical trials. In this study, we constructed CARs using mutant variants of a single-domain nanobody that bind both human and mouse MSLN with a wide range of affinities and examined tumor responses and their toxicities from on-target, off-tumor interactions in mouse models. CAR T cells with low nanomolar affinity (equilibrium dissociation constant, KD) exhibited profound systemic expansion with no apparent infiltration into the tumor. With a gradual reduction of CAR affinity toward the micromolar KD, the expansion of CAR T cells became more restricted to tumors. Our preclinical studies demonstrated that high-affinity MSLN CARs were associated with fatal on-target, off-tumor toxicity and that affinity-tuned CARs rendered T cells more selective for MSLN-high tumors.

Intraperitoneal cellular immunotherapy with CAR-redirected lymphocytes is an intriguing approach to target peritoneal carcinomatosis (PC) from ovarian cancer (OC), which is currently evaluated in clinical trials. PC displays a composite structure with floating tumor cells within ascites and solid-like masses invading the peritoneum. Therefore, a comprehensive experimental model is crucial to optimize CAR-cell therapies in such a peculiar environment. Here, we explored the activity of cytokine-induced killer lymphocytes (CIK), redirected by CAR against mesothelin (MSLN-CAR.CIK), within reductionistic 3D models resembling the structural complexity of both liquid and solid components of PC. MSLN-CAR.CIK effectively killed and were functionally efficient against OC targets. In a "floating-like" 3D context with floating OC spheroids, both tumor localization and killing by MSLN-CAR.CIK were significantly boosted by fluid flow. In a "solid-like" context, MSLN-CAR.CIK were recruited through the extracellular matrix on embedded tumor aggregates, with variable kinetics depending on the effector-target distance. Furthermore, MSLN-CAR.CIK penetrated the inner levels of OC spheroids exerting effective tumor killing. Our findings provide currently unknown therapeutically relevant information on intraperitoneal approaches with CAR.CIK, supporting further developments and improvements for clinical studies in the context of locoregional cell therapy approaches for patients with PC from OC.

Patients with advanced gastric and colorectal cancers have limited treatment options. Since mesothelin is highly expressed in these tumour types, we evaluated the therapeutic benefits of anti-mesothelin hYP218 CAR T cells alone, and in combination with anti-PD1 antibody, pembrolizumab. GEPIA analysis was performed using human gastric (n = 408) and colon cancer tumours (n = 275) in TCGA database, to evaluate mRNA expression of mesothelin, compared to normal tissues. Mesothelin expression in gastric and colorectal cancer cell-lines (n = 5) was analysed using flow cytometry. In vitro efficacy by hYP218 CAR T cells was tested by cytotoxicity and cytokine release assays. In vivo anti-tumour efficacy of hYP218 CAR T cells alone, and in combination with pembrolizumab, was evaluated in NSG mice bearing human gastric (HGC27) and colorectal (SW48) tumour xenografts. Additionally, hYP218 CAR-T cell persistence, activation and exhaustion marker-expression were studied. Mesothelin expression was significantly higher in gastric and colon cancer biopsies compared to normal tissues (p < .005). Mesothelin expression in gastric and colon cancer cell lines ranged from 10 000 to 70 000 molecules per cell. hYP218 CAR T cells demonstrated strong cytotoxic activity at low effector to target ratio, ranging from 0.24 to 1.0. In NSG mouse-models, hYP218 CAR T cells demonstrated anti-tumour efficacy and persisted in the tumour microenvironment in a functional state at day 40 posttreatment with expression of activation markers CD39 and CD69, increased production of IFN-γ and TNF-α and ability to kill tumour cells in vitro when isolated from tumours. There was increased PD1 expression. In combination with pembrolizumab, hYP218 CAR T cells led to slower tumour growth in NSG mice bearing large but not small HGC27 tumours. Anti-tumour efficacy of hYP218 CAR T cells is due to increased accumulation of activated CAR T cells in the tumour and combination with pembrolizumab resulted in improvement in anti-tumour activity of large established tumours. HIGHLIGHTS: Mesothelin expression is significantly higher in gastric and colorectal cancers than normal tissues. hYP218 CAR T cells demonstrate strong anti-tumour activity against mesothelin-positive gastric and colorectal carcinomas. Activated hYP218 CAR T cells persist in the tumour microenvironment and retain their cytotoxic activity. Addition of pembrolizumab in larger tumours enhance CAR T cell efficacy.

T cell engaging bispecific antibodies (TCBs) have recently become significant in cancer treatment. In this study we developed MSLN490, a novel TCB designed to target mesothelin (MSLN), a glycosylphosphatidylinositol (GPI)-linked glycoprotein highly expressed in various cancers, and evaluated its efficacy against solid tumors. CDR walking and phage display techniques were used to improve affinity of the parental antibody M912, resulting in a pool of antibodies with different affinities to MSLN. From this pool, various bispecific antibodies (BsAbs) were assembled. Notably, MSLN490 with its IgG-[L]-scFv structure displayed remarkable anti-tumor activity against MSLN-expressing tumors (EC50: 0.16 pM in HT-29-hMSLN cells). Furthermore, MSLN490 remained effective even in the presence of non-membrane-anchored MSLN (soluble MSLN). Moreover, the anti-tumor activity of MSLN490 was enhanced when combined with either Atezolizumab or TAA × CD28 BsAbs. Notably, a synergistic effect was observed between MSLN490 and paclitaxel, as paclitaxel disrupted the immunosuppressive microenvironment within solid tumors, enhancing immune cells infiltration and improved anti-tumor efficacy. Overall, MSLN490 exhibits robust anti-tumor activity, resilience to soluble MSLN interference, and enhanced anti-tumor effects when combined with other therapies, offering a promising future for the treatment of a variety of solid tumors. This study provides a strong foundation for further exploration of MSLN490's clinical potential.

Malignant peritoneal mesothelioma (MPeM) is a rare primary malignant tumor originating from peritoneal mesothelial cells. Insufficient specificity of the symptoms and their frequent reappearance following surgery make it challenging to diagnose, creating a need for more efficient treatment options. Natural killer cells (NK cells) are part of the innate immune system and are classified as lymphoid cells. Under the regulation of activating and inhibiting receptors, NK cells secrete various cytokines to exert cytotoxic effects and participate in antiforeign body, antiviral, and antitumor activities. This review provides a comprehensive summary of the specific alterations observed in NK cells following MPeM treatment, including changes in cell number, subpopulation distribution, active receptors, and cytotoxicity. In addition, we summarize the impact of various therapeutic interventions, such as chemotherapy, immunotherapy, and targeted therapy, on NK cell function post-MPeM treatment.

Ovarian cancer is the most common gynecological malignancy worldwide with the highest mortality. This low survival rate can be attributed to the fact that symptoms arise only at an advanced disease stage, characterized by a (micro)metastatic spread across the peritoneal cavity. Radiopharmaceuticals, composed of a targeting moiety coupled with either a diagnostic or therapeutic radionuclide, constitute a relatively underexplored theranostic approach that may improve the current standard of care. Efficient patient stratification, follow-up and treatment are several caveats that could be addressed with theranostics to improve patient outcomes. So far, the bulk of research is situated and often halted at the preclinical level, employing murine models of primary and metastatic peritoneal disease that do not necessarily provide an accurate representation of the disease heterogeneity, (intrinsic) drug resistance or the complex physiological interactions with the tumor microenvironment. Radioimmunoconjugates with therapeutic α- and electron-emitting radionuclides have been the prevailing standard, targeting a myriad of cell-membrane markers that are expressed in the various heterogeneous histological subtypes of ovarian cancer. Evidently, several hurdles exist within preclinical research that are potentially withholding these agents from advancing into clinical practice. On the other hand, the field of nuclear medicine has also seen significant innovation to address shortcomings related to target/ligand identification, preclinical research models, radiochemistry, radiopharmacy and dosimetry, as outlined in this review. Altogether, theranostics hold great promise to answer an unmet medical need for ovarian cancer.

Canine mesotheliomas are uncommon malignant tumors typically detected late. Minimally invasive diagnostic biomarkers would facilitate diagnosis at earlier stages, thereby improving clinical outcomes. We hypothesized that mesothelin could be used as a reliable diagnostic biomarker for canine mesotheliomas since it has been used as a cancer biomarker for human mesothelioma. We aimed to explore and characterize mesothelin gene expression in dogs and assess its use as a diagnostic biomarker for canine mesotheliomas.

We quantified expressed canine mesothelin transcripts via reverse transcription polymerase chain reaction (RT-PCR) and sequenced them using ribonucleic acid (RNA) extracted from a canine mesothelioma cell line. After confirming mesothelin expression, we assessed its levels in major organ tissues and compared them with those in the mesothelioma tissues using quantitative PCR (qPCR). Mesothelin overexpression in mesotheliomas was detected, and we further compared its levels using qPCR between mesotheliomas and non-mesotheliomas using tumor tissues and clinical sample effusions, confirming its significance as a diagnostic biomarker for canine mesothelioma.

Mesothelin complementary deoxyribonucleic acid (cDNA) was amplified via RT-PCR, yielding a single band of expected upon DNA electrophoresis. Sequence analyses confirmed it as a predicted canine mesothelin transcript from the genome sequence database. Comparative sequence analysis of the deduced amino acid sequence of the expressed canine mesothelin demonstrated molecular signature similarities with the human mesothelin. However, the pre-sequence of canine mesothelin lacks the mature megakaryocyte potentiating factor (MPF) portion, which is typically cleaved post-translationally with furin. Mesothelin expression was quantified via qPCR revealing low levels in the mesothelial and lung tissues, with negligible expression in the other major organs. Canine mesothelin exhibited significantly higher expression in the canine mesotheliomas than in the noncancerous tissues. Moreover, analysis of clinical samples using qPCR demonstrated markedly elevated mesothelin expression in canine mesotheliomas compared to non-mesothelioma cases.

Canine mesothelin exhibits molecular and biological characteristics akin to human mesothelin. It could serve as a vital biomarker for diagnosing canine mesotheliomas, applicable to both tissue- and effusion-based samples.

Brain tumors pose formidable challenges in oncology due to the intricate biology and the scarcity of effective treatment modalities. The emergence of immunotherapy has opened new avenues for innovative therapeutic strategies. Chimeric antigen receptor, originally investigated in T cell-based therapy, has now expanded to encompass macrophages, presenting a compelling avenue for augmenting anti-tumor immune surveillance. This emerging frontier holds promise for advancing the repertoire of therapeutic options against brain tumors, offering potential breakthroughs in combating the formidable malignancies of the central nervous system. Tumor-associated macrophages constitute a substantial portion, ranging from 30% to 50%, of the tumor tissue and exhibit tumor-promoting phenotypes within the immune-compromised microenvironment. Constructing CAR-macrophages can effectively repolarize M2-type macrophages towards an M1-type phenotype, thereby eliciting potent anti-tumor effects. CAR-macrophages can recruit T cells to the brain tumor site, thereby orchestrating a remodeling of the immune niche to effectively inhibit tumor growth. In this review, we explore the potential limitations as well as strategies for optimizing CAR-M therapy, offering insights into the future direction of this innovative therapeutic approach.

The mesothelin-targeting antibody-drug conjugate anetumab ravtansine was evaluated in combination with the programmed cell death-1 (PD-1) inhibitor pembrolizumab based on the common expression of mesothelin and reports of activity in mesothelioma.

A phase 1 safety run-in of the combination of anetumab ravtansine (6.5 mg/kg iv q3weeks) and pembrolizumab (200 mg, IV q3weeks) was conducted, followed by a phase 2 randomization to the combination or pembrolizumab alone at medical centers across the United States and Canada in the National Cancer Institute's Experimental Therapeutics Clinical Trials Network. Patients with pleural mesothelioma that expressed mesothelin and had previously received platinum-based therapy were eligible.

In phase 1 (n = 12) only one dose limiting toxicity was observed and the rules for dose reduction were not met. In phase 2, there was no difference in the confirmed response rates between the combination group (n = 18, 2 partial responses [PR], 11 %) and the pembrolizumab group (n = 17, 1 PR, 6 %; z = -0.5523, p = 0.29116). The median PFS was 12.2 months (95 % CI 5.1-not evaluable [NE]) for the combination, and 3.9 months for pembrolizumab (95 % CI 2.1-NE)(HR=0.55, p = 0.20). Patients with high baseline levels of soluble mesothelin who received anetumab ravtansine had a median PFS of 5 months.

The numeric difference in PFS between treatment groups was not statistically significant, likely related to a smaller than planned sample size. High levels of soluble mesothelin should potentially be considered to select against the use of mesothelin-targeting therapies in development that are neutralized by soluble mesothelin.

Ranked high in worldwide growing health issues, pleural diseases affect approximately one million people globally per year and are often correlated with a poor prognosis. Among these pleural diseases, malignant pleural mesothelioma (PM), a neoplastic disease mainly due to asbestos exposure, still remains a diagnostic challenge. Timely diagnosis is imperative to define the most suitable therapeutic approach for the patient, but the choice of diagnostic modalities depends on operator experience and local facilities while bearing in mind the yield of each diagnostic procedure. Since the analysis of pleural fluid cytology is not sufficient in differentiating historical features in PM, histopathological and morphological features obtained via tissue biopsies are fundamental. The quality of biopsy samples is crucial and often requires highly qualified expertise. Since adequate tissue biopsy is essential, medical or video-assisted thoracoscopy (MT or VATS) is proposed as the most suitable approach, with the former being a physician-led procedure. Indeed, MT is the diagnostic gold standard for malignant pleural pathologies. Moreover, this medical or surgical approach can allow diagnostic and therapeutic procedures: it provides the possibility of video-assisted biopsies, the drainage of high volumes of pleural fluid and the administration of sterile calibrated talcum powder under visual control in order to achieve pleurodesis, placement of indwelling pleural catheters if required and in a near future potential intrapleural therapy. In this context, dedicated diagnostic pathways remain a crucial need, especially to quickly and properly diagnose PM. Lastly, the interdisciplinary approach and multidisciplinary collaboration should always be implemented in order to direct the patient to the best customised diagnostic and therapeutic pathway. At the present time, the diagnosis of PM remains an unsolved problem despite MDT (multidisciplinary team) meetings, mainly because of the lack of standardised diagnostic work-up. This review aims to provide an overview of diagnostic procedures in order to propose a clear strategy.

Therapeutic cancer vaccines are valuable tools for educating the immune system to fight tumors precisely. Cancer cells are characterized with genetic instability and abundant somatic mutations, leading to the production of tumor specific antigens (TSA) called neoantigens. The main goal of neoantigen-based cancer vaccines is to activate the immune system and elicit effective tumor-specific T-cell responses. There have been no reports of advanced esophageal squamous cell carcinoma (ESCC) cases achieving partial remission after personalized mRNA (messenger RNA) vaccine treatment. As personalized neoantigen-based immunotherapies are emerging, here we report a 67-year-old male patient diagnosed with ESCC and multiple enlarged mediastinal lymph nodes, where mRNA vaccines were used for the first time. Tissue samples from the recurrence focus in the esophagus were subjected to whole transcriptome sequencing. The neoantigens were identified by bioinformatics analyses. The top 20 neoantigens were selected to compose the polyneoantigen vaccine, which were administered at 1 mg every 3 weeks for 4 cycles in combination with a PD-1 (programmed death-1) inhibitor. The patient was boosted with a single dose of the PD-1 inhibitor 8 weeks after the 4th cycle. In addition, immune responses were evaluated before and after the 4 cycles of vaccine therapy, and the lesions were evaluated by imaging examination. Our results revealed that neoantigen-based vaccines significantly activated the tumour-specific immune response. TCR (T cell receptor) V-J pairing analysis showed an increase in the abundance of oligoclonal TCRs, indicating improved homogeneity. No grade 3 or higher drug-related adverse events were observed, except for grade 4 thrombocytopenia caused by PD-1 inhibitor treatment. The patient achieved a partial response (PR), with a progression-free survival (PFS) time of 457 days, the OS (overall survival) time of 457 days, and DOR (duration of response) of 377 days. Our report suggests that combining the personalized mRNA vaccine therapy with PD-1 blockade therapy may be an effective treatment strategy for patient with advanced esophageal cancer. However, further clinical trials are necessary to confirm the efficacy and safety of personalized neoantigen-based immunotherapies in the treatment of advanced ESCC. This trial is registered with ClinicalTrials.gov, NCT03468244 on March 16, 2018, and is now complete.

Chimeric antigen receptor (CAR)-T-cell therapy is one of the most effective immunotherapies. CAR-T-cell therapy has achieved great success in the treatment of hematological malignancies. However, due to the characteristics of solid malignant tumors, such as on-target effects, off-tumor toxicity, an immunosuppressive tumor microenvironment (TME), and insufficient trafficking, CAR-T-cell therapy for solid tumors is still in the exploration stage. Mesothelin (MSLN) is a molecule expressed on the surface of various solid malignant tumor cells that is suitable as a target of tumor cells with high MSLN expression for CAR-T-cell therapy. This paper briefly described the development of CAR-T cell therapy and the structural features of MSLN, and especially summarized the strategies of structure optimization of MSLN-targeting CAR-T-cells and the enhancement methods of MSLN-targeting CAR-T cell anti-tumor efficacy by summarizing some preclinical experiment and clinical trials. When considering MSLN-targeting CAR-T-cell therapy as an example, this paper summarizes the efforts made by researchers in CAR-T-cell therapy for solid tumors and summarizes feasible treatment plans by integrating the existing research results.

Ovarian carcinoma is usually diagnosed at an advanced stage with peritoneal dissemination and/or lymph node metastasis, and the prognosis for such advanced carcinoma is very poor. Therefore, new biomarkers to predict patient prognosis are needed. Miyamoto et al. previously showed that keratan sulfate (KS) detected by the 5D4 monoclonal antibody was expressed in ovarian carcinoma. However, the detailed structure of such KS was not determined, and the biological significance of this finding remained to be clarified. We previously generated the 297-11A monoclonal antibody, which recognizes galactose (Gal)-6-O-sulfated N-acetyllactosamine (LacNAc) located at the nonreducing terminus. Because the 297-11A epitope overlaps with that of 5D4, here we chose to use the 297-11A antibody as a tool to analyze KS and related structures. We conducted immunohistochemical analysis of 98 ovarian carcinoma cases with 297-11A antibody combined with a series of glycosidases and performed mass spectrometry analysis of the human serous ovarian carcinoma cell line OVCAR-3 to deduce the glycan structure of 297-11A-sulfated glycans. We also performed western blot analysis to assess a potential association of 297-11A-sulfated glycans with the mucin core protein mucin 16 (MUC16; also known as cancer antigen 125 (CA125)). Finally, we examined the relationship between 297-11A expression and patient prognosis. Consequently, 297-11A-sulfated glycans were primarily expressed in serous and endometrioid carcinomas and poorly expressed in mucinous and clear cell carcinomas. We reveal that structurally, 297-11A-sulfated glycans expressed in ovarian carcinoma are O-glycans carrying partially sialylated, Gal-6-O-sulfated LacNAc and that these glycans are likely displayed on MUC16 mucin core proteins. Of clinical importance is that expression of 297-11A-sulfated glycans correlated with shorter progression-free survival in patients. Thus, 297-11A-sulfated glycans may serve as a predictor of ovarian carcinoma recurrence.

Mesothelioma (MM) is an aggressive and lethal disease with few therapeutic opportunities. Platinum-pemetrexed chemotherapy is the backbone of first-line treatment for MM. The introduction of immunotherapy (IO) has been the only novelty of the last decades, allowing an increase in survival compared to standard chemotherapy (CT). However, IO is not approved for epithelioid histology in many countries. Therefore, therapy for relapsed MM remains an unmet clinical need, and the prognosis of MM remains poor, with an average survival of only 18 months. Increasing evidence reveals MM complexity and heterogeneity, of which histological classification fails to explain. Thus, scientific focus on possibly new molecular markers or cellular targets is increasing, together with the search for target therapies directed towards them. The molecular landscape of MM is characterized by inactivating tumor suppressor alterations, the most common of which is found in CDKN2A, BAP1, MTAP, and NF2. In addition, cellular targets such as mesothelin or metabolic enzymes such as ASS1 could be potentially amenable to specific therapies. This review examines the major targets and relative attempts of therapeutic approaches to provide an overview of the potential prospects for treating this rare neoplasm.

Chimeric antigen receptor (CAR)-modified T cell therapy has achieved remarkable efficacy in treating hematological malignancies, but it confronts many challenges in treating solid tumors, such as the immunosuppressive microenvironment of the solid tumors. These factors reduce the antitumor activity of CAR-T cells in clinical trials. Therefore, we used the immunocytokine interleukin-12 (IL-12) to enhance the efficacy of CAR-T cell therapy. In this study, we engineered CAR-IL12R54 T cells that targeted mesothelin (MSLN) and secreted a single-chain IL-12 fused to a scFv fragment R54 that recognized a different epitope on mesothelin. The evaluation of the anti-tumor activity of the CAR-IL12R54 T cells alone or in combination with anti-PD-1 antibody in vitro and in vivo was followed by the exploration of the functional mechanism by which the immunocytokine IL-12 enhanced the antitumor activity. CAR-IL12R54 T cells had potency to lyse mesothelin positive tumor cells in vitro. In vivo studies demonstrated that CAR-IL12R54 T cells were effective in controlling the growth of established tumors in a xenograft mouse model with fewer side effects than CAR-T cells that secreted naked IL-12. Furthermore, combination of PD-1 blockade antibody with CAR-IL12R54 T cells elicited durable anti-tumor responses. Mechanistic studies showed that IL12R54 enhanced Interferon-γ (IFN-γ) production and dampened the activity of regulatory T cells (Tregs). IL12R54 also upregulated CXCR6 expression in the T cells through the NF-κB pathway, which facilitated T cell infiltration and persistence in the tumor tissues. In summary, the studies provide a good therapeutic option for the clinical treatment of solid tumors.

Mesothelin (MSLN) is a cell-surface protein that is expressed on many cancers, which makes it a popular target for antibody-based cancer therapy. However, MSLN is shed from cancer cells at high levels via proteases that cleave at its membrane-proximal C-terminal region. Shed MSLN accumulates in patient fluids and tumors and can block antibody-based MSLN-targeting drugs from killing cancer cells. A previously established monoclonal antibody (mAb), 15B6, binds MSLN at its protease-sensitive C-terminal region and does not bind shed MSLN. 15B6 variable fragment (Fv)-derived chimeric antigen receptor (CAR) T cells are not inhibited by shed MSLN and kill tumors in mice more effectively than mAb SS1 Fv-derived CAR T cells, which bind an epitope retained in shed MSLN. Here, we have established 15B6 Fv-derived MSLN x CD3 bispecific antibodies (BsAbs) that target MSLN-expressing cancers. We identified our lead candidate, BsAb 5, after screening multiple 15B6-derived BsAb formats in vitro for cytotoxic activity. BsAb 5 activates T cells to kill various cancer cell lines in a MSLN-specific manner. MSLN 296-591 His, a recombinant protein mimicking shed MSLN, does not inhibit 15B6-derived BsAb 5 but completely inhibits humanized SS1-derived BsAb 7. Furthermore, BsAb 5 inhibits and delays tumor growth and is not inhibited by MSLN 296-585 His in mice. Our findings indicate that by targeting the protease-sensitive region of MSLN, BsAb 5 has high MSLN-specific anticancer activity that is not inhibited by shed MSLN. BsAb 5 may be a promising immunotherapy candidate for MSLN-expressing cancers.

Canonical α/β 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/β 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.

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.

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.

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.

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.

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.

As the most lethal gynecologic oncological indication, carcinoma of the ovary has been ranked as the 5th cause of cancer-related mortality in women, with a high percentage of the patients being diagnosed at late stages of the disease and a five-year survival of ~ 30%. Ovarian cancer patients conventionally undergo surgery for tumor removal followed by platinum- or taxane-based chemotherapy; however, a high percentage of patients experience tumor relapse. Cancer immunotherapy has been regarded as a silver lining in the treatment of patients with various immunological or oncological indications; however, mirvetuximab soravtansine (a folate receptor α-specific mAb) and bevacizumab (a VEGF-A-specific mAb) are the only immunotherapeutics approved for the treatment of ovarian cancer patients. Chimeric antigen receptor T-cell (CAR-T) therapy has achieved tremendous clinical success in the treatment of patients with certain B-cell lymphomas and leukemias, as well as multiple myeloma. In the context of solid tumors, CAR-T therapies face serious obstacles that limit their therapeutic benefit. Such hindrances include the immunosuppressive nature of solid tumors, impaired tumor infiltration, lack of qualified tumor-associated antigens, and compromised stimulation and persistence of CAR-Ts following administration. Over the past years, researchers have made arduous attempts to apply CAR-T therapy to ovarian cancer. In this review, we outline the principles of CAR-T therapy and then highlight its limitations in the context of solid tumors. Ultimately, we focus on preclinical and clinical findings achieved in CAR-T-mediated targeting of different ovarian cancer-associated target antigens.

Cancer is the primary and one of the most prominent causes of the rising global mortality rate, accounting for nearly 10 million deaths annually. Specific methods have been devised to cure cancerous tumours. Effective therapeutic approaches must be developed, both at the cellular and genetic level. Immunotherapy offers promising results by providing sustained remission to patients with refractory malignancies. Genetically modified T‑lymphocytic cells have emerged as a novel therapeutic approach for the treatment of solid tumours, haematological malignancies, and relapsed/refractory B‑lymphocyte malignancies as a result of recent clinical trial findings; the treatment is referred to as chimeric antigen receptor T‑cell therapy (CAR T‑cell therapy). Leukapheresis is used to remove T‑lymphocytes from the leukocytes, and CARs are created through genetic engineering. Without the aid of a major histocompatibility complex, these genetically modified receptors lyse malignant tissues by interacting directly with the carcinogen. Additionally, the outcomes of preclinical and clinical studies reveal that CAR T‑cell therapy has proven to be a potential therapeutic contender against metastatic breast cancer (BCa), triple‑negative, and HER 2+ve BCa. Nevertheless, unique toxicities, including (cytokine release syndrome, on/off‑target tumour recognition, neurotoxicities, anaphylaxis, antigen escape in BCa, and the immunosuppressive tumour microenvironment in solid tumours, negatively impact the mechanism of action of these receptors. In this review, the potential of CAR T‑cell immunotherapy and its method of destroying tumour cells is explored using data from preclinical and clinical trials, as well as providing an update on the approaches used to reduce toxicities, which may improve or broaden the effectiveness of the therapies used in BCa.

Mesothelin (MSLN) is a tumor-associated antigen found in a variety of cancers and is a target for imaging and therapeutic applications in MSLN-expressing tumors. We have developed high affinity anti-MSLN human VH domain antibodies, providing alternative targeting vectors to conventional IgG antibodies that are associated with long-circulating half-lives and poor penetration of tumors, limiting antitumor activity in clinical trials. Based on two newly identified anti-MSLN VH binders (3C9, 2A10), we generated VH-Fc fusion proteins and modified them for zirconium-89 radiolabeling to create anti-MSLN VH-Fc PET tracers. The focus of this study was to assess the ability of PET-imaging to compare the in vivo performance of anti-MSLN VH-Fc fusion proteins (2A10, 3C9) targeting different epitopes of MSLN vs IgG1 (m912; a clinical benchmark antibody with an overlapped epitope as 2A10) for PET imaging in a mouse model of colorectal cancer (CRC). The anti-MSLN VH-Fc fusion proteins were successfully modified and radiolabeled with zirconium-89. The resulting MSLN-targeted PET-imaging agents demonstrated specific uptake in the MSLN-expressing HCT116 tumors. The in vivo performance of the MSLN-targeted PET-imaging agents utilizing VH-Fc showed more rapid and greater accumulation and deeper penetration within the tumor than the full-length IgG1 m912-based PET-imaging agent. Furthermore, PET imaging allowed us to compare the pharmacokinetics of epitope-specific VH domain-based PET tracers. Overall, these data are encouraging for the incorporation of PET imaging to assess modified VH domain structures to develop novel anti-MSLN VH domain-based therapeutics in MSLN-positive cancers as well as their companion PET imaging agents.

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.

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.

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.

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.

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.