# Mesothelin biology and the evolving landscape of targeted immunotherapy

Remy Boisgard, Patrick Chames, Brigitte Kerfelec

► To cite this version:

Remy Boisgard, Patrick Chames, Brigitte Kerfelec. Mesothelin biology and the evolving landscape of targeted immunotherapy. Molecular Therapy: Oncology, 2026, 34 (1), pp.201148. 10.1016/j.omton.2026.201148. hal-05530530

HAL Id: hal-05530530

https://hal.science/hal-05530530v1

Submitted on 27 Feb 2026

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
# Mesothelin biology and the evolving landscape of targeted immunotherapy

Remy Boisgard,¹ Patrick Chames,¹ and Brigitte Kerfelec¹

¹Aix Marseille University, CNRS, INSERM, Institut Paoli-Calmettes, CRCM, Marseille, France

Due to its restricted expression in normal tissues, its frequent overexpression in various aggressive malignancies, and its involvement in several pro-tumorigenic signaling pathways, mesothelin, a glycosylphosphatidylinositol-anchored cell surface protein, has been identified as a promising tumor-associated antigen. This review provides an up-to-date comprehensive overview regarding the role of mesothelin in cancer. It encompasses the protein structural characteristics, expression profiles, glycosylation patterns, shedding mechanisms, prognostic significance, and functional roles. Subsequently, therapeutic strategies targeting mesothelin that have advanced to clinical trial stage are discussed, including vaccine-based approaches, antibody-mediated immunotherapies, and cell therapies. The remaining challenges of mesothelin targeted treatments are highlighted, along with the ongoing options aimed at overcoming these limitations.

## INTRODUCTION

Discovered in 1992 by Chang and Pastan, mesothelin (MSLN) is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein predominantly expressed on the mesothelial cells that line the pleural, peritoneal, and pericardial cavities.¹,² A recent immunohistochemical study on 76 healthy organs has revealed a broader expression of mesothelin, primarily observed in non-vital tissues in adults such as the tongue, thymus, gallbladder, fallopian tubes, uterus, and placenta.³

The biological role of mesothelin in healthy tissues remains to be elucidated. Its function appears non-essential, as MSLN gene invalidation experiments in mice did not result in any significant abnormalities in development, fertility, or platelet count compared with wild-type mice.⁴

It has been hypothesized that MSLN, akin to several other GPI-anchored proteins, plays a role in cell adhesion processes and signaling pathways. Sathyanarayana et al. proposed that MSLN contributes to the modeling of the extracellular matrix through its interaction with matrix glycoproteins.⁵

The only described partner of mesothelin is the glycosylated trans-membrane protein mucin 16 (MUC16) (~3–5 MDa), also known as CA125, expressed at the surface of various epithelial cells but over-

expressed in several cancer types.⁶⁻⁸ Notably, in healthy tissues, MSLN and MUC16 are expressed at low levels and in distinct cell types, suggesting that no physiological interaction occurs under normal conditions.³,⁸

The mesothelin gene is located on chromosome 16 (16p13.3) and encodes the mesothelin precursor protein (~70 kDa).² Three MSLN transcript variants have been described.⁹ Variant 1 is regarded as the canonical form because it is the predominant form expressed in both healthy and tumor tissues (Figure 1A).¹⁰

The MSLN precursor is transported and anchored to the cell membrane through a GPI anchor (Figure 1B). Subsequently, the MSLN precursor is cleaved by proteolysis, releasing the N-terminal soluble fragment, designated as megakaryocyte potentiating factor (MPF) (~30 kDa). This process leaves the mature form of MSLN (~40 kDa) anchored to the membrane.¹¹ A furin cleavage site (RPRFRR) located between amino acids 286 and 295 has been identified as the primary proteolytic processing site. However, several recent studies have shown that alternative proteolytic sites are also present in this region.¹²,¹³

The membrane-bound mature MSLN can be shed from cell surface under normal conditions, contributing to serum MSLN levels (Figure 1C).¹⁴,¹⁵ However, the rate, the specific protease profile, the regulatory pathways as well as its functional significance are not documented.

The three-dimensional structure of mature MSLN has been recently determined in complex with therapeutic antibodies.¹⁶,¹⁷ In accordance with the predicted structure, MSLN exhibits a curved superhelical structure comprising a series of ARM (three helices) and HEAT (two helices) repeats.⁵ MSLN is organized in four domains: A (296–359), B (360–427), C (428–502), and D (503–598) (Figure 1A).¹⁶

https://doi.org/10.1016/j.omton.2026.201148.

**Correspondence:** Remy Boisgard, Aix Marseille University, CNRS, INSERM, Institut Paoli-Calmettes, CRCM, Marseille, France.

E-mail: remy.boisgard@inserm.fr

**Correspondence:** Brigitte Kerfelec, Aix Marseille University, CNRS, INSERM, Institut Paoli-Calmettes, CRCM, Marseille, France.

E-mail: brigitte.kerfelec@inserm.fr

<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /><figcaption>**Figure 1. Mesothelin structure and processing**</figcaption></figure>

(A) Mesothelin precursor is a 622-amino-acid protein with four glycosylation sites (purple) and five regions: signal peptide (SP, orange); megakaryocyte potentiating factor (MPF, green); proteolytic cleavage site (red); mature mesothelin (MSLN, blue); and the GPI anchor (GA, gray). (B) Schematic representation of the mesothelin processing from precursor to soluble mesothelin. (C) Three-dimensional AlphaFold ribbon representation of mature MSLN with major cleavage site (MCS, red) locations leading to soluble mesothelin (yellow).

<figure><img src="image_4.png" /></figure>

# MESOTHELIN IN CANCER

## Expression

Mesothelin overexpression has been demonstrated in a multitude of tumor types, as evidenced by an extensive immunohistochemical analysis of 15,050 tumors of 122 different types. The analysis revealed that cancers with the highest prevalence and expression levels of MSLN include all types of ovarian cancer, endometrial carcinoma, pancreatic adenocarcinoma, malignant mesothelioma of the epithelioid type, and adenocarcinoma of the lung, stomach, esophagus, and colorectum.³ Further immunohistochemical analysis revealed a predominant membranous localization, with a less frequent cytoplasmic localization, which is likely due to the detection of the MSLN precursor not yet anchored to the membrane.¹⁸,¹⁹ Weidemann's study corroborates previous results obtained in a transcriptomic study involving 19,746 samples covering 41 tumor types.²⁰ Recently, a transcriptomic study of 2051 pediatric and young adult patients with acute myeloid leukemia (AML) revealed that MSLN overexpression is not exclusive to solid cancer.²¹ The authors demonstrated that MSLN is overexpressed in approximately 36% of AML cases, despite its absence in normal bone marrow.

The mechanisms leading to MSLN overexpression in tumors have yet to be elucidated, but several potential mechanisms have been postulated. As demonstrated in the literature, mesothelioma, lung, ovarian, and pancreatic cancers with constitutive activation of the Wnt signaling pathway exhibit high mesothelin expression.¹² The presence of an 18-base-pair enhancer sequence, CanScript, that drives high levels of gene expression has been described in the

MSLN promoter region in different MSLN-overexpressing cancer lines.²² However, the co-factor involved in the regulation of MSLN expression remains to be identified. The hypothesis of an epigenetic regulation has been postulated subsequent to the observation of the hypomethylation of the MSLN promoter in pancreatic tumor samples.²³ However, this phenomenon was not observed in other tumors overexpressing MSLN, such as ovarian cancer. In the context of ovarian cancer, Park and Kim have demonstrated an upregulation of MSLN and MUC16 expression through the stimulation of TLR5 and TLR7 (Toll-like receptors). This upregulation is mediated by the activation of WAVE3, a member of the Wiskott-Aldrich syndrome protein (WASP) family. WAVE3 has been identified as a significant driver of the invasive and metastatic phenotypes in multiple cancer types.²⁴

## Mesothelin binding partners

The only well-described partner of mesothelin is MUC16, overexpressed in several cancer types.⁶⁻⁸ MSLN/MUC16 interaction is a feature specific to malignant cells linked to the co-expression of MUC16 and MSLN.

Mesothelin-MUC16 binding is mediated by protein-protein interaction between domain A (296-359) of MSLN and MUC16 SEA (sea urchin sperm protein, enterokinase, and agrin) modules.⁷,²⁵ Involvement of the SEA modules suggests that MUC16 might engage MSLN molecules, thereby generating an avidity phenomenon at the cell surface. However, enzymatic removal of MUC16 N-glycans disrupts MUC16/MSLN interaction, indicating that glycans, by maintaining MUC16 stability, are structurally critical for the binding.²⁶

Recently, it has been shown that, on portal fibroblasts, MSLN can also bind to Thy-1 (thymocyte differentiation antigen 1, CD90) and form an MSLN/MUC16/Thy-1 complex in response to profibrotic signals, such as transforming growth factor β1 (TGF-β1). This complex by modulating TGF-β1/TGF-βR1 signaling promotes fibroblast activation and the transcription of fibrogenic and
<figure><img src="image_5.png" /><figcaption>**Figure 2. Prognostic value regarding MSLN overexpression in cancers**</figcaption></figure>

The cancers presented are those for which at least three studies have demonstrated the prognostic value of MSLN. Cancers are listed in descending order of publication demonstrating a correlation between MSLN level expression and bad prognosis: pancreatic, ³⁶⁻³⁸,⁴⁴⁻⁴⁷ colorectal, ⁴⁸⁻⁵² breast, ⁴²,⁵³⁻⁵⁸ ovarian, ⁵⁹⁻⁶³ gastric, ⁶⁴⁻⁶⁷ and mesothelioma, ¹⁸,⁶⁸⁻⁷²

pro-tumorigenic genes via canonical TGF-β1/Smad2/3 signaling, thereby contributing to desmoplasia and tumor progression.²⁷⁻²⁹

## Glycosylation

While glycosylation in general has been demonstrated to play critical roles in cancer progression, including cellular adhesion, resistance to cell death, and metastatic potential, there is currently a lack of knowledge regarding mesothelin glycosylation.³⁰,³¹ To address this knowledge gap, Duran et al. conducted a comprehensive analysis of the N-glycan structures of MSLN from pancreatic ductal adenocarcinoma (PDAC) cell lines and tissues. Their findings revealed full occupancy of the three N-glycosylation sites (N388, N488, and N515) and a structure predominantly corresponding to a complex core fucosylated-N-glycan with terminal sialylation on MSLN from the cell-conditioned media.³² By contrast, a decrease of core-fucosylated glycoforms of MSLN was observed in both serum and tissues from PDAC patients. This observation was replicated by Zhan et al. who demonstrated that N-glycan fucosylation was predominant on soluble MSLN (sMSLN) from a pancreatic cancer cell line, while in ascites, sMSLN exhibited heavy sialylation and moderate fucosylation.¹⁶ These two studies indicate a shift in the glycoforms of MSLN during the progression of pancreatic cancer, with a decrease in core-fucosylated glycoforms and an increase in highly sialylated and moderately core-fucosylated glycoforms.

Fujihira and colleagues have demonstrated that the glycosylation profile of MSLN in epithelial mesothelioma differs from that of MSLN in normal mesothelial cells, thereby supporting a potential role for glycosylation in the malignant transformation of cells.³³

## Shedding

In cancer, MSLN shedding is mediated by upregulated and activated enzymes, such as members of the ADAM, MMP, TACE, and BACE families.¹⁵ These enzymes cleave MSLN at different sites in the C-terminal region close to the cell surface (Figure 1C). The action of these proteases contributes to the elevated shedding rate that appears to vary according to the cancer type and the tumor stage.³⁴ The presence of sMSLN in the bloodstream has been demonstrated in patients diagnosed with mesothelioma and ovarian cancer. Conversely, patients diagnosed with pancreatic adenocarcinoma exhibit minimal or no increase in serum mesothelin concentration. A recent study has demonstrated, employing PDAC models, that shed mesothelin is sequestered within the tumor microenvironment (TME). The authors of the study postulated that this phenomenon is associated with collagen density and elevated MUC16 expression.³⁴

## Prognostic value

The sMSLN and the membrane-bound MSLN (mbMSLN) have been the focus of studies exploring their potential as biomarkers for various cancers, particularly for mesothelioma, pancreatic cancer, and ovarian cancer. However, as highlighted in an extensive review by Hagerty and Takabe, the heterogeneity of study designs, outcomes, and protocols complicates the interpretation of their clinical relevance.³⁵ Furthermore, the co-expression status of the ligand MUC16 is rarely addressed in studies examining the prognostic value of MSLN. This information could offer valuable insights into clinicopathological implications that could influence prognosis, as MUC16 co-expression has been linked to accelerated tumor growth, metastasis, and poor prognosis. Recently MSLN-MUC16 co-expression has been associated to tumor aggressiveness and poor prognosis in patients with PDAC,³⁶⁻³⁸ endometrial serous carcinoma,³⁹,⁴⁰ perihiilar bile duct carcinoma,⁴¹ and luminal-type breast cancer.⁴² Regarding the use of MSLN as a diagnostic biomarker, it has been demonstrated that neither elevated levels of circulating mesothelin nor the expression of membrane-bound mesothelin, in isolation, are adequate for establishing a reliable diagnosis of cancer. However, when considered in conjunction with other markers, MSLN can serve as an indication of cancer pathology and provide a compelling rationale for further investigations.⁴³

Several prognostic survival studies have been conducted on various MSLN-positive cancers, with the aim of determining the prognostic significance of soluble and/or membrane MSLN (Figure 2). A significant number of these studies have validated the correlation between elevated MSLN expression in immunohistochemistry and unfavorable prognosis in multiple cancers, including malignant pleural mesothelioma (MPM),⁶⁸ ovarian cancer,⁵⁹,⁶⁰ and triple-negative breast cancer (TNBC).⁵³
<figure><img src="image_6.png" /><figcaption>**Figure 3.** Schematic representation of MSLN-related metabolic pathways in cancer</figcaption></figure>

Regarding apoptosis pathways, under PI3K activation, mesothelin induces AKT phosphorylation and regulates RARG and TNK2 (encoding RARγ and ACK1), known to phosphorylate and activate AKT, promoting cell survival and disease progression in pancreatic and hepatocellular carcinoma.⁷⁸,⁷⁹ AKT activation, through RARγ, ACK1, and PDK1, leads to the inhibition of the transcription factor Forkhead box O3a (FoxO3a) through mTOR.⁸⁰ This inhibition is emphasized by MUC16-MSLN interaction leading to SGK3/FOXO3 pathway activation, as DDK1 is inhibited stopping SGK3 kinase inhibition, a FoxO3a inhibitor.⁸¹ Phosphorylated FoxO3a is translocated to the cytoplasm, ubiquitinated and degraded.⁸² Thus, two BH3-only sensitizers are inhibited: Bad, by AKT and mTOR, and Puma, no longer activated by FoxO3a.⁵³ In healthy condition, Bad and Puma inhibit anti-apoptotic multidomain Bcl-2 proteins (e.g., Bcl-2, Bcl-xL, and Mcl-1). In MSLN-positive cancers, these proteins are no longer inhibited. Furthermore, anti-apoptotic Bcl-2 proteins are activated by NF-κB and Stat3 both overactivated in MSLN⁺ cancers. In normal condition, the BH3-only activator family, made up of Bim, Bid, and Puma, promote Bax/Bak heterodimerization, leading to death cell.⁸³ In MSLN-positive cancers, Bim is partly activated by FoxO3a, which is inhibited by MSLN, and the BH3-only activator family is inhibited by the now activated anti-apoptotic Bcl-2 proteins.⁸³ Thus, MSLN in cancer decreases apoptosis, thereby promoting cell survival. Furthermore, the expression level of MSLN has been demonstrated to be correlated with chemotherapy resistance.⁴⁸,⁵⁹,⁶¹,⁸⁴ This phenomenon can be explained by the inhibition of the apoptosis pathway. Regarding cell proliferation pathways, AKT and mTOR activate NF-κB pathway, leading to the production of IL-6 known to activate AKT through PI3K, generating previously discussed events.⁸⁵,⁸⁶ In pancreatic cancers, mesothelin expression level correlates with IL-6 expression.⁸⁷ NF-κB also generates a self-activation loop through TNF-α and promote anti-apoptotic Bcl-2 proteins.⁸⁸ Despite TNF-α overexpression, a protein considered as inducing death cells, tumor cells might survive using various mechanisms and may acquire resistance.⁸⁹ Autocrine IL-6 binds its receptor, inducing JAK phosphorylation, leading to Stat3 activation through homodimerization mediated by subunit phosphorylation.⁸⁵,⁹⁰ The IL-6/Stat3 pathway is renowned in cancer for promoting cell survival, proliferation, and migration.⁹¹,⁹² Stat3 acts as a transcription factor for oncogenes like IL-6, anti-apoptotic Bcl-2 proteins, and cyclin E, which, through the cyclin E/CDK2 complex, induces cell proliferation.⁹³ Under normal condition, cyclin E/CDK2 complex is closely regulated by p21 and p27, both activated by FoxO3a, inhibited in MSLN-positive cancers.⁹⁴ Cell proliferation, cell migration, and cell invasion pathways appear closely related to MMP-7 and MMP-9.⁳⁷,⁹⁵ Mesothelin directly activates MAPK cascades and indirectly through IL-6 and

(legend continued on next page)
The cellular localization of MSLN has been demonstrated to possess potential prognostic value, with luminal/membranous localization of MSLN being associated with a more unfavorable prognosis in pancreatic cancers and extrahepatic bile duct cancers when compared with cytoplasmic localization.¹⁹ A recent observation by a Japanese research team has revealed a similar finding, demonstrating that membrane MSLN positivity in luminal-type breast cancers appears to be associated with a poor prognosis and an elevated risk of recurrence.⁴²,⁵⁴

In the context of colorectal cancer, elevated MSLN expression has been associated with chemoresistance, peritoneal metastases, and a lower 3-year overall survival rate.⁴⁸,⁴⁹

The presence of sMSLN in the bloodstream has not yielded satisfactory prognostic results for pancreatic cancers. This is due to the fact that serum levels of mesothelin do not differentiate between stages of the disease nor reflect tumor burden.⁷³

Conversely, a systematic review of 20 studies regarding MPM demonstrated that a decrease in serum MSLN levels correlates with a positive response to treatment. This suggests that measuring serum MSLN could enable the monitoring of disease progression, risk of recurrence after surgery, and response to systemic therapy.⁷⁴ Data from a clinical trial for a specific CAR-T cell -MSLN immunotherapy approach in malignant pleural mesothelioma (NTC02414269) presented at the American Society of Clinical Oncology (ASCO) and the American association for cancer research (AACR) in 2019 point in the same direction. The observed decrease in serum sMSLN levels is associated with tumor regression and persistence of CAR-T cells.⁷⁵

It is noteworthy that a recent publication reported the potential predictive value of MSLN regarding therapies.⁷⁶ The study demonstrated a significant correlation between MSLN and both cetuximab and gefitinib sensitivity in pancreatic cancer.

## Role in cancer

Preclinical and clinical data suggest an active role of MSLN overexpression in the invasive and aggressive phenotypes of MSLN-positive tumors. It is generally accepted that MSLN overexpression promotes

tumor cell survival, proliferation, and migratory and invasive capacities and contributes to chemoresistance. However, the precise mechanisms by which MSLN exerts its effects and its interactions with the TME are not yet fully understood. One hypothesis suggests that MSLN may interact with various signaling transmembrane proteins within lipid rafts, a phenomenon observed with GPI-anchored proteins.⁷⁷ It appears that MSLN can function in a MUC16-dependent or MUC16-independent manner (Figure 3). However, distinguishing between these two modes of action is challenging due to the lack of systematic assessment of MUC16 status in studies. Additionally, the distinction between the soluble and membrane forms of mesothelin is not always clear in existing literature.

MSLN overexpression has been demonstrated to regulate multiple signaling pathways identified as contributing to cancer progression (Figure 3). This review does not aim to provide an exhaustive overview of all these pathways. Instead, it will offer an overview of some of the pathophysiological implications observed in different types of cancer.

In pancreatic cancer, MSLN-dependent nuclear factor κB (NF-κB) pathway activation has been observed to result in elevated levels of interleukin-6 (IL-6), which, in turn, have been found to be responsible for the activation of anti-apoptotic factors, such as the Bcl-2 family.⁸⁷,⁸⁹,⁹³ In mesothelioma and pancreatic cancer, mitogen-activated protein kinase (MAPK) pathway activation through MSLN has been shown to promote cell migration and invasion by facilitating the expression and activation of matrix metalloproteinase (MMP)-7 and MMP-9, which are metalloproteinases implicated in the degradation of the extracellular matrix.³⁷,⁹⁵ In pancreatic cancer, independently of MUC16, MSLN contributes to the development of peritoneal metastasis by enhancing angiogenesis, cell proliferation, and invasion.⁹⁹ In breast cancer cell lines, ectopic expression of MSLN facilitates anchorage-independent cell growth in vitro and induces resistance to anoikis.¹⁰⁴

Recent studies have demonstrated that the inactivation of MSLN in various cancer cell lines disrupts the expression of several genes associated with the epithelial-mesenchymal transition (EMT). These results suggest that MSLN promotes the EMT and stem cell properties by enhancing the expression of EMT regulators (snail, twist, slug,

TNF-α, leading to ERK, JNK, and p38 activation.⁸⁸,⁹⁶,⁹⁷ These three proteins, along with Stat3, promote MMP-7, leading to cell migration and invasion.³⁷,⁹⁸ Additionally, cases of increased angiogenesis through MSLN have been reported, notably in PDAC.⁹⁰ IL-6 seems to play a role in angiogenesis mediated by MSLN as a correlation between IL-6 and vascular endothelial growth factor (VEGF) expression in cancers is well established.¹⁰⁰ While no study has clearly proven a pathway involving MSLN, IL-6, Stat3, HIF-1α, and VEGF, the Stat3, HIF-1α, and VEGF correlation is well established in many cancers, suggesting MSLN could regulate angiogenesis through this same pathway.¹⁰¹ Moreover, MMP-7 is also increasing angiogenesis, at least in part, by stimulating the proliferation of VEGF.⁹⁸ Finally, MSLN has been demonstrated to induce EMT by activating EMT transcription factors.⁸⁴,¹⁰² This mechanism is known in cancer to be activated through AKT, which inhibits GSK3β and activates NF-κB, and through ERK, JNK, and p38.¹⁰³ ACK1, activated CDC42-associated kinase 1; Bad, Bcl-2-associated agonist of cell death; Bak, Bcl-2 antagonist/killer; Bax, Bcl-2-associated X-protein; Bcl-2, B cell lymphoma-2; Bcl-xL, B cell lymphoma-extra large; Bid, BH3-interacting domain death agonist; Bim, Bcl-2-interacting mediator of cell death; CDK2, cyclin-dependent kinase 2; DKK1, Dickkopf 1; EMT, epithelial-mesenchymal transition; ERK, extracellular-signal-regulated kinase; FoxO3a, Forkhead box class O 3a; GSK3β, glycogen synthase kinase-3 β; IL-6, interleukin 6; JAK, Janus kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; Mcl-1, myeloid cell leukemia-1; MMP-7/9, matrix metalloproteinase-7/matrix metalloproteinase-9; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PDK1, phosphoinositide-dependent protein kinase-1; PI3K, phosphoinositide 3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PTEN, phosphatase and tensin homolog; Puma, p53 upregulated modulator of apoptosis; RARγ, retinoic acid receptor γ; SGK3, serum and glucocorticoid-inducible protein kinase 3; Stat3, signal transducer and activator of transcription 3; TNF-α, tumor necrosis factor alpha.
ABCG2) and reducing the expression of mesenchymal markers (E-cadherin, caveolin-2, cytokeratins, and claudins).<sup>84,102</sup> *In vivo*, genetic knockdown of MSLN has been demonstrated to decrease tumor growth and metastasis, while mesothelin overexpression in a non-cancerous cell line has been shown to induce a malignant phenotype, arguing in favor of an oncogenic role for mesothelin.<sup>102</sup>

In ovarian cancers, particularly in high-grade serous carcinoma, a correlation has been observed between MSLN expression levels and chemoresistance.<sup>59,61</sup> A close association has also been established between chemotherapy resistance and the level of MSLN expression in malignant pleural mesothelioma and pancreatic cancers.<sup>68,84</sup>

MSLN has also been identified as a potential contributor to the progression of lung cancer, particularly through its role in facilitating the disruption of the blood-brain barrier (BBB) in non-small cell lung cancer (NSCLC).<sup>105</sup>

Beyond its oncogenic roles, recent studies have demonstrated a correlation between high MSLN expression and the remodeling of the tumor immune landscape.<sup>106</sup> Bioinformatics approaches have demonstrated a positive correlation between high MSLN expression and immune infiltration in ovarian cancers, including Th17, dendritic cells, and natural killer (NK) cells.<sup>61</sup> In microsatellite-stable colorectal tumors, MSLN overexpression promotes immune checkpoint expression (PD-1, LAG-3, TIM-3), immune-related marker expression (PD-L1), immune cell infiltration, and the activation of pro-tumoral pathways (TGF-β and IL-6/JAK/Stat3).<sup>107</sup> In PDAC, MSLN expression has been associated with reduced cytotoxic T cell infiltration, increased PD-L1 expression in the stroma, and transcriptional features of T cell exhaustion, suggesting an immune-excluded phenotype.<sup>108</sup> Still in PDAC, sMSLN promotes the polarization of macrophages into an immunosuppressive phenotype (CD106<sup>+</sup>), which supports tumor cell growth, neutrophil recruitment, and the spread of metastasis to the lungs.<sup>109</sup> Most studies indicate that MSLN expression negatively contributes to the remodeling of the TME, by promoting immunosuppressive features in several cancers. However, a recent study reports that the role of MSLN may be context-dependent, as in MPM its expression appears to reshape the immune landscape of the TME by promoting the recruitment of CD8<sup>+</sup> T cells and CD68<sup>+</sup> macrophages, while increasing the abundance of type I collagen fibers.<sup>110</sup>

## STRATEGIES IN CLINICAL TRIALS TARGETING MESOTHELIN

As described above, MSLN has emerged as a promising tumor-associated antigen (TAA). The field of mesothelin-targeted therapies has undergone continuous evolution over the past 25 years, encompassing the following three categories: vaccine, antibody-based immunotherapy, and cell therapy. However, despite the extensive research conducted in this field, no MSLN-targeted treatment has yet received the approval for clinical utilization from the Food and Drug Administration (FDA). In the following section, recent clinical trial data related to these approaches are compiled.

## Vaccines

### *Listeria monocytogenes*-based vaccines: CRS-207 and JNJ-757

CRS-207, developed by Aduro Biotech, is an attenuated strain of *Listeria monocytogenes* engineered to express the human mesothelin within the cytosol of infected antigen-presenting cells (APCs).<sup>12,111</sup> This strain has been genetically modified through the deletion of two virulence genes, significantly reducing its pathogenicity while preserving its capacity to elicit robust immune responses.<sup>112</sup>

Since the first trial in 2007 with CRS-207, 11 clinical trials have been registered on [ClinicalTrials.gov](https://ClinicalTrials.gov): eight phase 2, one phase 1/2, and two phase 1 (Table 1).<sup>116</sup> CRS-207 has been evaluated both as monotherapy and in combination with different therapies such as immune checkpoint inhibitors (ICIs) (ipilimumab, nivolumab, and pembrolizumab), chemotherapies (epacadostat, an oral inhibitor of the IDO1 enzyme, cisplatin, cyclophosphamide, and pemetrexed), tadalafil, or vaccine (GVAX pancreas vaccine). The drug has been studied in patients with various types of cancer. In a clinical trial for patients with advanced MPM (NCT01675765), the combination treatment of CRS-207 and chemotherapy demonstrated a higher overall response rate compared to chemotherapy alone.<sup>117</sup> By contrast, the combination treatment of CRS-207 and pembrolizumab in patients with MPM exhibited no clinical activity, resulting in the cessation of the clinical development of CRS-207 for this indication.<sup>115</sup> Despite this discontinuation, mesothelin-targeted CRS-207 in combination with immune checkpoint inhibitors, dioxygenase (IDO) inhibitors, and CY/GVAX is currently being evaluated in other indications, notably in patients with pancreatic cancers.<sup>118</sup>

A similar vaccine (JNJ-757), developed by Janssen, a Johnson & Johnson subsidiary, has undergone early phase trials, but the absence of clinical activity prompted its discontinuation.<sup>119</sup>

### Neoantigen DNA and peptide vaccines

The Washington University School of Medicine has developed personalized cancer DNA- and peptide-based vaccines that incorporate prioritized neoantigens and mesothelin epitopes for patients with pancreatic cancers.<sup>120</sup>

Both approaches reached phase 1 clinical trials, demonstrating an acceptable toxicity and a neoantigen-specific immune response in all patients analyzed by ELISPOT (Table 1).<sup>120</sup> However, despite the encouraging preliminary results, both trials were terminated due to insufficient funding.

### Antibody-based immunotherapies

Currently, 17 antibody-based immunotherapies have advanced to the clinical research stage: two immunotoxins (iTOXs), two antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) mediators, five antibody-drug conjugates (ADCs), two immunostimulators, one targeted radionuclide therapy (TRT), and five T cell engagers (TCEs) (Figure 4A).
<table><caption>Table 1. MSLN-targeted vaccines in clinical trials</caption><thead><tr><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td>NCT05014776</td><td>II</td><td>estimated 2026-04-03</td><td>17</td><td>Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins</td><td>evaluation of safety and efficacy of CRS-207 in combination with pembrolizumab, ipilimumab, and tadalafil despite the use of CRS-207, this clinical trial is not focused on MSLN</td><td>PDAC</td><td>posology: CRS-207 1 × 10<sup>9</sup> CFU i.v. on day 2 of cycles 1–6 (3-week cycles) + pembrolizumab 200 mg i.v. on day 1 of cycles 1–6 + ipilimumab 50 mg i.v. on day 1 of cycles 1, 3, and 5 + tadalafil 20 mg po. on days 3–21 of cycles 1–6<br>safety: N/A<br>efficacy: N/A</td><td>Gross et al.<sup>113</sup></td></tr><tr><td></td><td>NCT03006302</td><td>II</td><td>2024-08-05</td><td>41</td><td>Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins</td><td>evaluation of safety, tolerability, and efficacy of CRS-207 in combination with epacadostat and pembrolizumab with or without CY/GVAX despite the use of CRS-207, this clinical trial is not focused on MSLN</td><td>pancreatic</td><td>posology: CRS-207 1 × 10<sup>9</sup> CFU i.v. on day 2 of cycles 3–6 (3-week cycles). (For epacadostat, pembrolizumab, cyclophosphamide, and GVAX pancreas vaccine details: NCT03006302)<br>safety (n = 40): 40% of patients experienced SAEs<br>efficacy (n = 26): OS: 46% of patients survived more than 6 months</td><td>-</td></tr><tr><td>CRS-207</td><td>NCT03190265</td><td>II</td><td>2022-08-23</td><td>61</td><td>Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins</td><td>evaluation of safety and efficacy of nivolumab and ipilimumab in combination with either sequential administration of CY/GVAX followed by CRS-207 or with administration of CRS-207 alone despite the use of CRS-207, this clinical trial is not focused on MSLN</td><td>pancreatic</td><td>posology: CRS-207 1 × 10<sup>9</sup> CFU i.v. on day 2 of cycles 3–6 (3-week cycles). (For nivolumab, ipilimumab, cyclophosphamide, and GVAX pancreas vaccine details: NCT03190265)<br>safety: without CY/GVAX, 78% of patients experienced SAEs and 37% experienced grade ≥3 TRAEs. With CY/GVAX, 80% of patients experienced SAEs and 33% experienced grade ≥3 TRAEs<br>efficacy nivolumab + ipilimumab + CRS-207 (n = 30): OR: CR + PR 7%<br>efficacy nivolumab + ipilimumab + CY/GVAX + CRS-207 (n = 31): OR: CR + PR 0%</td><td>-</td></tr><tr><td></td><td>NCT03122548*</td><td>II</td><td>2018-01-31 (terminated due to low enrollment and lack of clinical activity in other CRS-207 studies)</td><td>5</td><td>Aduro Biotech</td><td>evaluation of safety and efficacy of CRS-207 in combination with pembrolizumab</td><td>gastric, GEJ, and esophageal</td><td>posology: CRS-207 1 × 10<sup>9</sup> CFU i.v. on day 2 of cycle 1 (3-week cycles), day 1 of cycles 2–4 and then on day 1 every 6 weeks + pembrolizumab 200 mg i.v. on day 1 of each 3-week cycle<br>safety (n = 5): 80% of patients experienced SAEs<br>efficacy (n = 5): OS (n = 5): 2.5 months. PFS (n = 5): 1.2 months. OR (n = 3): CR 0%, PR 0%, SD 0%, and PD 100%</td><td>Kelly et al.<sup>114</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 1. Continued**</caption><thead><tr><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td>NCT03175172*</td><td>II</td><td>2018-01-31 (terminated due to low enrollment and lack of clinical activity)</td><td>10</td><td>Aduro Biotech</td><td>evaluation of safety and efficacy of CRS-207 in combination with pembrolizumab despite the use of CRS-207, this clinical trial is not focused on MSLN</td><td>MPM</td><td>posology: CRS-207 1 × 10<sup>9</sup> CFU i.v. on day 2 (3-week cycles) + pembrolizumab 200 mg i.v. on day 1. If tolerated, pembrolizumab and CRS-207 were administered on the same day for subsequent cycles. After 4 cycles, pembrolizumab was administered on day 1 every 3 weeks and CRS-207 once every 6 weeks<br>safety (n = 10): 40% of patients experienced SAEs<br>efficacy (n = 10): OS (n = 10): 2.8 months. PFS (n = 10): 1.4 months. OR (n = 9): CR 0%, PR 10%, SD 11%, and PD 89%</td><td>Alley et al.<sup>115</sup></td></tr><tr><td>CRS-207</td><td>NCT02243371</td><td>II</td><td>2017-07-21</td><td>93</td><td>Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins</td><td>evaluation of safety and efficacy of CY/GVAX followed by CRS-207 with or without nivolumab despite the use of CRS-207, this clinical trial is not focused on MSLN</td><td>pancreatic</td><td>posology: CRS-207 1 × 10<sup>9</sup> CFU i.v. on day 2 of cycles 3–6 (3-week cycles) + cyclophosphamide 200 mg/m<sup>2</sup> i.v. on day 1 of cycles 1 and 2 + GVAX 5 × 10<sup>8</sup> cells in 6 i.d. injections on day 2 of cycles 1 and 2, w/ or w/o nivolumab 3 mg/kg i.v. on day 1 of cycles 1–6<br>safety (n = 93): 12% of patients treated with CY/GVAX + CRS-207 experienced grade ≥3 TRAEs (n = 42). 35% of patients treated with CY/GVAX + CRS-207 + nivolumab experienced grade ≥3 TRAEs (n = 51)<br>efficacy CY/GVAX + CRS-207 (n = 42): OS: 6.1 months. PFS: 2.2 months. OR: CR 0%, PR 2%, SD 7%, and PD 67%<br>efficacy CY/GVAX + CRS-207 + nivolumab (n = 51): OS: 5.9 months. PFS: 2.2 months. OR: CR 0%, PR 2%, SD 12%, and PD 69%</td><td>Tsujikawa et al.<sup>118</sup></td></tr><tr><td></td><td>NCT01417000</td><td>II</td><td>2017-02-10</td><td>93</td><td>Aduro Biotech</td><td>evaluation of safety and efficacy of CY/GVAX in combination with CRS-207 and CY/GVAX alone</td><td>PDAC</td><td>posology: in CY/GVAX alone condition, the treatment was on weeks 1, 4, 7, 10, 13, and 16. In combination condition, CY 200 mg/m<sup>2</sup> i.v. on day 1 of weeks 1 and 4 + GVAX 1 × 10<sup>8</sup> cells i.d. on day 2 of weeks 1 and 4 + CRS-207 1 × 10<sup>9</sup> CFU i.v. on day 1 of weeks 7, 10, 13, and 16<br>safety: in CY/GVAX alone condition, 4 grade ≥3 AEs</td><td>Le et al.<sup>192</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 1. Continued**</caption><thead><tr><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>occurred (n = 29). In combination condition, 29 grade ≥3 AEs occurred (n = 61)<br><b>efficacy CY/GVAX alone (n = 29):</b><br>OS: 3.9 months. OR: SD 24%<br><b>efficacy CY/GVAX + CRS-207 (n = 61):</b> OS: 6.1 months. OR: SD 31%</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td><b>posology:</b> CRS-207 1 × 10<sup>9</sup> CFU i.v. on day 1 of weeks 7, 10, 13, and 16. (For cyclophosphamide, GVAX pancreas vaccine, gemcitabine, capecitabine, fluorouracil, leucovorin, irinotecan, and erlotinib details: NCT02004262)<br><b>safety (n = 169):</b> in the below order of treatment conditions, 47%, 37%, and 28% of patients experienced SAEs. Most frequently reported AEs in all treatment groups were chills, pyrexia, fatigue, and nausea. No treatment-related deaths occurred</td><td>Le et al.<sup>193</sup></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td><b>efficacy single-agent chemotherapy (n = 43, primary cohort):</b> OS: 4.7 months. PFS: 2.1 months<br><b>efficacy CRS-207 alone (n = 58, primary cohort):</b> OS: 5.2 months. PFS: 2.1 months<br><b>efficacy CRS-207 + CY/GVAX (n = 68, primary cohort):</b> OS: 4.2 months. PFS: 2.3 months</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td><b>posology:</b> CRS-207 1 × 10<sup>9</sup> CFU i.v. Q3W for 6 cycles, then Q6W (For epacadostat, and pembrolizumab details: NCT02575807)<br><b>safety (n = 32):</b> In combination with CRS-207, epacadostat RP2D was determined as 300 mg po. All patients included, 44% experienced SAEs<br><b>efficacy epacadostat 300 mg (n = 28):</b> OS (n = 28): 2.1 (CRS-207 + epacadostat + pembrolizumab, n = 1) to 11.3 months (CRS-207 alone, n = 8). PFS (n = 28): 1.2 (CRS-207 + epacadostat + pembrolizumab, n = 1) to 1.8</td><td></td></tr><tr><td>CRS-207</td><td>NCT02575807*</td><td>I/II</td><td>2018-05-08 (terminated due to low enrollment and lack of clinical activity)</td><td>35</td><td>Aduro Biotech</td><td>evaluation of safety and efficacy of CRS-207 alone and in combination with epacadostat and/or pembrolizumab despite the use of CRS-207 this clinical trial is not focused on MSLN</td><td>ovarian, fallopian and peritoneal</td><td></td><td></td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 1. Continued**</caption><thead><tr><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>months (CRS-207 alone and CRS-207 + epacadostat, n = 8 and n = 16). OR (n = 24): All patients combined the best response was SD (SD 31% with CRS-207 + epacadostat, n = 13)</td><td></td></tr><tr><td>NCT01675765</td><td>I</td><td>2019-08-19</td><td>60</td><td>Aduro Biotech</td><td>evaluation of safety and efficacy of CRS-207 in combination with pemetrexed and cisplatin w/ or w/o cyclophosphamide</td><td>MPM</td><td></td><td>**posology:** CRS-207 1 × 10<sup>9</sup> CFU on weeks 1, 3, 23, 26 and every 8 weeks from week 34 + pemetrexed 500 mg/m<sup>2</sup> and cisplatin 75 mg/m<sup>2</sup> on weeks 5, 8, 11, 14, 17, and 20 (up to six 3-week cycles), w/ or w/o cyclophosphamide 200 mg/m<sup>2</sup> on weeks 1, 3, 23, and 26<br>**safety (n = 60):** 39% of patients treated with CRS-207 + pemetrexed + cisplatin experienced SAEs (n = 38). 50% of patients treated with CRS-207 + pemetrexed + cisplatin + cyclophosphamide experienced SAEs (n = 22)<br>**efficacy CRS-207 + pemetrexed + cisplatin (n = 21):** OR: CR 3%, PR 53%, SD 14%, PD 3% and NE 3%<br>**efficacy CRS-207 + pemetrexed + cisplatin + cyclophosphamide (n = 36):** OR: CR 0%, PR 52%, SD 38%, and PD 10%</td><td>Hassan et al.<sup>194</sup></td></tr><tr><td>NCT00585845</td><td>I</td><td>2009-02-01</td><td>17</td><td>Anza Therapeutics</td><td>evaluation of safety, tolerability, and pharmacokinetics of CRS-207</td><td>solid</td><td></td><td>**posology:** CRS-207 1 × 10<sup>8</sup> to 1 × 10<sup>10</sup> CFU i.v. up to 4 administrations every 3 weeks<br>**safety (n = 17):** MTD was determined to be 1 × 10<sup>9</sup> CFU. At the MTD, 100% of patients experienced grade 4 lymphopenia, 33% experienced grade 3 hypophosphatemia and 17% experienced grade 3 elevated gamma-glutamyl transferase (n = 6)<br>**efficacy CRS-207 1 × 10<sup>9</sup> CFU (n = 6):** OS: 33% of patients survived more than 15 months. OR: CR 0%, PR 0%, SD 33%, and PD 67%</td><td>Le et al.<sup>116</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 1. Continued**</caption><thead><tr><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>JNJ-757 (JNJ-64041757)</td><td>NCT03371381*</td><td>I/II</td><td>2018-10-09 (terminated due to lack of clinical benefit)</td><td>12</td><td>Janssen-Johnson &amp; Johnson</td><td>evaluation of safety, pharmacokinetics, and efficacy of JNJ-757 in combination with nivolumab</td><td>NSCLC</td><td>posology: JNJ-757 1 × 10<sup>9</sup> CFU i.v. Q4W + nivolumab 240 mg i.v. Q2W<br/>safety (n = 12): 50% of patients experienced grade ≥3 AEs, including two cases of treatment-related fatal pneumonitis<br/>efficacy (n = 11): OR: CR 0%, PR 0%, SD 36%, PD 45%, and NE 18%</td><td>Brahmer et al.<sup>119</sup></td></tr><tr><td>Neoantigen DNA vaccine</td><td>NCT03122106*</td><td>I</td><td>2022-08-13 (terminated due to loss of funding)</td><td>15</td><td>Washington University School of Medicine</td><td>evaluation of safety and efficacy of neoantigen DNA vaccine</td><td>pancreatic</td><td>posology: 2 injections (1 into each deltoid or lateralis) on days 1, 5, 9, 13, 17, and 21<br/>safety (n = 11): no patients experienced SAE. No grade ≥3 AEs were reported<br/>efficacy (n = 9): 100% of patients analyzed by ELISPOT experienced a neoantigen-specific immune response</td><td>Panni et al.<sup>120</sup></td></tr><tr><td>Neoantigen peptide vaccine</td><td>NCT03956056*</td><td>I</td><td>2023-07-06 (terminated due to insufficient funding/staff)</td><td>12</td><td>Washington University School of Medicine</td><td>evaluation of safety and efficacy of neoantigen peptide vaccine in combination with poly-ICLC</td><td>pancreatic</td><td>posology: neoantigen peptide vaccine + poly-ICLC on days 1, 4, 8, 15, 22, 50, and 78 sc<br/>safety (n = 10): 10% of patients experienced SAEs<br/>efficacy (n = 9): 100% of patients analyzed by ELISPOT experienced a neoantigen-specific immune responses</td><td>Panni et al.<sup>120</sup></td></tr></tbody></table>

Discontinued clinical trials are shown with the symbol *. AE, adverse event; CFU, colony-forming units; CR, complete response; CY/GVAX, cyclophosphamide + GVAX pancreas vaccine; GEJ, gastroesophageal junction; i.d., intradermal; i.v., intravenous; MPM, malignant pleural mesothelioma; MTD, maximum tolerated dose; NE, not evaluated; NSCLC, non-small-cell lung cancer; OR, overall response; OS, overall survival; PD, progressive disease; PDAC, pancreatic ductal adenocarcinoma; PFS, progression-free survival; po., per os; PR, partial response; Q2W, once every 2 weeks; Q3W, once every 3 weeks; Q4W, once every 4 weeks; Q6W, once every 6 weeks; RP2D, recommended phase 2 dose; SAE, serious adverse event; sc., subcutaneous; SD, stable disease; TRAE, treatment-related adverse event.
A

<figure><img src="image_7.png" /></figure>

B

<figure><img src="image_8.png" /></figure>

The development of anti-MSLN treatment was initiated in 1998 under the direction of Ira Pastan. Ira Pastan's team used a mesothelin expression plasmid to immunize mice. This approach led to the isolation of an anti-mesothelin single-chain variable fragment (scFv) known as SS scFv.<sup>121</sup> Mutations were introduced in the complementarity determining region 3 (CDR3) of SS V<sub>L</sub> to enhance its binding affinity, resulting in the variant named SS1.<sup>122</sup> Notably, SS1 competes with MUC16 for MSLN binding.<sup>25</sup> SS1 was regularly used to develop anti-MSLN drugs including SS1P, LMB-100, amatuximab, and even CAR-T cells (Figure 4B).<sup>122,123</sup>

<caption>Figure 4. Schematic representation of antibody-based immunotherapies targeting MSLN that have advanced to the clinical trial phase, along with the duration of their clinical study</caption>

(A) The molecules were separated based on the targeting epitope on MSLN. Regarding molecules that compete with MUC16, based on SS1 three molecules were engineered: SS1P (immunotoxin, dsFv conjugated to PE38), LMB-100 (immunotoxin, Fab conjugated to PE24), and amatuximab (ADCC and ADCP mediator, chimeric human antibody). Regarding molecules that do not compete with MUC16, based on BAY 86-1903, two molecules were engineered: BAY 94-9343 (ADC, payload: DM4) and BAY 2287 (TRT, radionuclide: <sup>227</sup>Th). Based on h7D9.v3, DMOT4039A was engineered (ADC, payload: MMAE). Concerning SGN-MesoC2 (ADC, payload: SGD-12280) and NI-1801 (ADCC and ADCP mediator, bispecific), it is likely that the MSLN arm targets a membrane-proximal epitope on MSLN. JNJ-79032421 (TCE, bispecific) and CT-95 (TCE, bispecific) were engineered to target a membrane-proximal epitope on MSLN. For seven molecules no information was found about their competition with MUC16: BMS-986148 (ADC, payload: tubulysin), RC88 (ADC, payload: MMAE), ABBV-428 (immunostimulator, bispecific), AMG 994 (immunostimulator, bispecific), HPN536 (TCE, trispecific), AMG 305 (TCE, trispecific), and ZW171 (TCE, trispecific). (B) Representation of the clinical trial periods for the 17 anti-MSLN antibody-based immunotherapies. ADC, antibody-drug conjugate; ADCC, antibody-dependent-cell-mediated cytotoxicity; ADCP, antibody-dependent-cell-mediated phagocytosis; DM4, ravtansine; dsFv, disulfide-stabilized Fv; has, human serum albumin; ICE, immune cell-engager; MMAE, monomethyl auristatin E; MSLN, mesothelin; MUC16, mucin 16; PE, pseudomonas Exotoxin A; SGD-12280, novel topoisomerase 1 inhibitor; TCE, T cell engager; TRT, targeted radionuclide therapy.

Immunotoxins: SS1P and LMB-100

A recent review by Hagerty et al. provides a comprehensive summary of both pre-clinical and clinical data regarding MSLN-targeted immunotoxin (iTOX).<sup>124</sup> The two iTOXs that advanced to the clinical trial stage are derived from the scFv SS1 and conjugated to truncated forms of *Pseudomonas* exotoxin A (PE) (Figure 4A). The mechanism of action of these iTOXs involves the impairment of cellular functions, inducing cell death through apoptosis.<sup>125</sup> Following MSLN binding, the iTOX is internalized through receptor-mediated endocytosis. During intracellular trafficking, the cleaved toxin part induces the irreversible ADP ribosylation of elongation factor-2 (EF-2), which is crucial for protein elongation during translation.

SS1P (SS1(dsFv)PE38 or CAT-5001), developed by the National Cancer Institute (NCI), is the disulfide-stabilized SS1 fused to the
truncated PE form known as PE38 (38 kDa).¹²²,¹²⁶ SS1P has been evaluated in clinical trials both as monotherapy and in combination with bevacizumab, cisplatin, carboplatin, cyclophosphamide, paclitaxel, pemetrexed, and pentostatin, mainly in patients with MPM or ovarian and pancreatic cancers (Table 2). Despite some evidence of anti-tumor effectiveness, reported clinical activity has been modest, and its development has faced critical challenges due to immunogenicity with the development of neutralizing anti-drug antibodies (ADA) against PE and toxicity.¹²⁹

To overcome the immunogenicity issues, the research shifted to a second generation of iTOX with LMB-100. LMB-100 is a humanized anti-MSLN Fab fused to the deimmunized domain III of *Pseudomonas* exotoxin A (24 kDa) to reduce immunogenicity.¹²⁶ Nine clinical trials with LMB-100 alone or in combination with chemotherapy or ICIs have been reported, most of them in MPM (Table 2). LMB-100 alone was found to be well tolerated with manageable side effects.¹³¹,¹³² However, despite a lower immunogenicity than SS1P, it still faces the formation of ADA, limiting its long-term use in patients.¹³³ According to the latest update from AdisInsight, its development has been discontinued for solid tumors.

Interestingly, although most epithelioid MPMs express MUC16, the impact of its presence on the therapeutic efficiency of iTOX that compete with MUC16 has not been thoroughly investigated.

As of March 2025, there is no evidence of ongoing clinical trials with SS1P or LMB-100 (Figure 4B).

**ADCs: Anetumab ravtansine, DMOT4039A, BMS-986148, RC88, and SGN-MesoC2**

Antibody-drug conjugates (ADCs) combine the specificity of monoclonal antibodies with the potency of cytotoxic payloads.¹³⁴

Anetumab ravtansine (BAY 94-9343 or AR) is an ADC developed by Bayer. It is a human anti-mesothelin antibody that does not compete with MUC16. The antibody is conjugated to the maytansinoid tubulin inhibitor DM4 via a non-cleavable disulfide-containing linker (Figure 4A).¹³⁵ To date, 15 clinical trials have been conducted with AR, the first of which was initiated in 2011 (Table 2).¹³⁶ AR has been evaluated both as monotherapy and in combination with immune checkpoint inhibitors (ICIs) (atezolizumab, bevacizumab, ipilimumab, nivolumab, and pembrolizumab), chemotherapy (cisplatin, gemcitabine, pegylated liposomal doxorubicin [PLD], and pemetrexed), or fungicide (itraconazole). Regarding safety, clinical studies have demonstrated that AR exhibits a satisfactory level of tolerability and favorable pharmacokinetics across multiple studies. Concerning its efficacy, there is evidence supporting the clinical potential of AR in some MSLN-expressing tumors, with cases of complete responses (CRs) and partial responses (PRs) reported.¹³⁶,¹³⁷ However, a direct comparison with other treatments, such as vinorelbine, has not yet demonstrated the superiority of AR in terms of progression-free survival (PFS) or overall survival (OS).¹³⁶,¹³⁷ While it has shown promise as part of combination therapies in certain con-

texts, particularly in high mesothelin-expressing tumors, its clinical efficacy remains inconsistent across indications.¹³⁸⁻¹⁴⁰ AR is still in an active phase 2 clinical trial, notably in combination with ICI.¹³⁸

DMOT4039A is an ADC developed by Genentech, a subsidiary of Roche. The structure of DMOT4039A consists of a humanized anti-MSLN monoclonal antibody (mAb) (h7D9.v3 or MMOT0530A), which does not compete with MUC16 binding conjugated to the tubulin inhibitor monomethyl auristatin E (MMAE) via a cathepsin-cleavable linker (Figure 4A).¹⁴¹,¹⁴² To date, two related phase 1 clinical trials have been conducted with DMOT4039A as monotherapy, focusing on pancreatic and ovarian cancers (Table 2). The first study, initiated in 2011, demonstrated acceptable tolerability but yielded disappointing results regarding clinical activity, particularly in patients with pancreatic cancer.¹⁴³ A confirmed partial response was observed in only 8% of patients, compared to 30% in ovarian cancer patients.¹⁴³ The second one, a multicenter imaging study (NCT01832116) performed in parallel using the same antibody conjugated to zirconium-89 showed a lower uptake in the pancreatic tumors than in ovarian tumors, which may be due to the high level of fibrosis in the pancreatic microenvironment.¹⁴⁴ Since 2015, the development status of DMOT4039A remains unknown.

The ADC BMS-986148, a product of Bristol-Myers Squibb, is a fully human anti-MSLN IgG1 conjugated to a cytotoxic peptide, tubulysin, a tubulin polymerization inhibitor via a cleavable linker (Figure 4A).¹⁴⁵ There is no information on the epitope targeted by BMS-986148. BMS-986148 has been evaluated both in monotherapy and in combination with nivolumab in different indications (Table 2). The phase 1 assay as monotherapy in advanced and/or metastatic solid tumors was completed in 2017, and the second trial was prematurely terminated. In both trials, BMS-986148 demonstrated a manageable safety profile in patients with advanced tumors and a durable antitumor activity was observed in some patients. Notably, treatment discontinuations were observed in both assays, primarily due to treatment-related adverse events (TRAEs).¹⁴⁵ The status of any ongoing research by Bristol-Myers Squibb on this compound remains uncertain.

RC88, developed by RemeGen, consists of an anti-mesothelin antibody linked to MMAE via a cleavable linker (Figure 4A).¹⁴⁶ No information regarding the targeted epitope on MSLN has been disclosed. At present, five clinical trials are underway with RC88, either as monotherapy or in combination with monoclonal antibodies (mAbs), including RC48 (anti-HER2), bevacizumab (anti-VEGF), and sintilimab (anti-PD1) (Table 2). To date, there have been no published clinical trial data, apart from a poster presented at the 2024 ASCO meeting that reported the tolerability and clinical efficacy of RC88 in patients with mesothelin-expressing solid tumors.¹⁴⁷ Following the FDA's approval of the fast track designation for RC88 in December 2023, a phase 2 clinical trial has been initiated to assess the efficacy of RC88 in patients diagnosed with platinum-resistant recurrent epithelial ovarian, fallopian tube, and primary peritoneal cancer.
<table><caption>Table 2. MSLN-targeted antibody-based therapies in clinical trials</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="2">Immunotoxin</td><td rowspan="2">SS1P (SS1(dsFv) PE38 or CAT-5001)</td><td>NCT01362790</td><td>I/II</td><td>2017-08-07</td><td>55</td><td>National Cancer Institute</td><td>evaluation of safety, tolerability, and efficacy of SS1P in combination with pentostatin and cyclophosphamide</td><td>mesothelioma, pancreatic and lung adenocarcinoma</td><td>posology: SS1P 35 µg/kg i.v. on days 10, 12, and 14 for 1 cycle then on days 2, 4, and 6 for a maximum of 6 cycles + pentostatin + cyclophosphamide (regimen A). SS1P 35 µg/kg i.v. on days 18, 20, and 22 for 1 cycle then on days 6, 8, and 10 for a maximum of 6 cycles + pentostatin + cyclophosphamide (regimen B). (For pentostatin and cyclophosphamide details: NCT01362790)<br/>safety depending on the regimen (n = 53): at 35 µg/kg, 25%-100% patients experienced SAEs (n = 45). No SS1P-related grade 4 AEs reported. MTD was determined to be 25 µg/kg (n = 8)<br/>efficacy depending on the regimen (n = 45): OS: 4.2-29.3 months. PFS: 3.9-11.8 months. OR: CR 0%, PR 5%, SD 5%, PD 7% and NE 82%</td><td>Hassan et al.<sup>127</sup></td></tr><tr><td>NCT01445392</td><td>I</td><td>2016-10-03</td><td>24</td><td>National Cancer Institute</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of SS1P in combination with pemetrexed and cisplatin</td><td>MPM</td><td>posology: SS1P 25-55 µg/kg i.v. on days 1, 3, and 5 for 2 cycles (3-week cycle) + pemetrexed 500 mg/m² i.v. on day 1 for up to 6 cycles + cisplatin 75 mg/m² i.v. on day 1 for up to 6 cycles<br/>safety: MTD was determined to be 45 µg/kg. 100% of patients experienced AEs. No SS1P-related grade 4 AEs<br/>efficacy (n = 24): OS: Hassan et al.<sup>128</sup></td><td>Hassan et al.<sup>128</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>Table 2. Continued</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td>NCT01051934</td><td>I</td><td>2011-09-28</td><td>2</td><td>National Cancer Institute</td><td>evaluation of safety, tolerability, and pharmacokinetics of SS1P in combination with paclitaxel, carboplatin and bevacizumab</td><td>NSCLC</td><td>13.6 months. PFS: 6.0 months. OR: CR 0%, PR 50%, SD 13%, PD 21% and NE 17%<br>efficacy SS1P 45 µg/ mg (n = 13): OR: CR 0%, PR 77%, SD 8% and PD 15%</td><td></td></tr><tr><td></td><td></td><td>NCT00006981</td><td>I</td><td>N/A (publication 2009)</td><td>24</td><td>National Cancer Institute</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of SS1P</td><td>mesothelioma, ovarian and pancreatic</td><td>posology: 10 injections of SS1P 4 to 25 µg/kg/day i.v.<br>safety (n = 24): MTD was determined to be 25 µg/kg/day. All patients experienced at least 1 AE. 13% of patients experienced grade ≥3 AEs<br>efficacy (n = 24): OR: CR 0%, PR 4%, SD 50%, and PD 46%</td><td>Kreitman et al.<sup>129</sup></td></tr><tr><td></td><td></td><td>NCT00066651</td><td>I</td><td>N/A (publication 2007)</td><td>34</td><td>National Cancer Institute</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of SS1P</td><td>mesothelioma, ovarian and pancreatic</td><td>posology: with 6 injections QOD of SS1P: 8 to 25 µg/kg i.v. With 3 injections QOD of SS1P: 25 to 60 µg/kg i.v.<br>safety (n = 34): MTD with 6 injections was determined to be 18 µg/kg/dose (n = 17) and with 3 injections to be 45 µg/kg/dose (n = 17). SS1P is well-tolerated with pleuritis as the DLT at the highest dose level<br>efficacy (n = 34): OR: CR 0%, PR 12%, SD 56%, PD 29% and NE 3%</td><td>Hassan et al.<sup>130</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 2. Continued**</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="2">Immunotoxin</td><td rowspan="2">SS1P<br>(SS1(dsFv)PE38 or<br>CAT-5001)</td><td>NCT00024674*</td><td>I</td><td>2001-02-01<br>(withdrawn,<br>continued by NCI<br>NCT00006981)</td><td>0</td><td>INSYS<br>Therapeutics</td><td>evaluation of safety<br>and efficacy of SS1P</td><td>N/A</td><td>posology: 10-day<br>continuous i.v.<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>NCT00024687*</td><td>I</td><td>2000-11-01<br>(withdrawn,<br>continued by NCI<br>NCT00006981)</td><td>0</td><td>INSYS<br>Therapeutics</td><td>evaluation of safety,<br>tolerability,<br>pharmacokinetics and<br>efficacy of SS1P</td><td>N/A</td><td>posology: 6 injections<br>i.v. QOD<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td rowspan="3">Immunotoxin</td><td rowspan="3">LMB-100 (RG7787<br>or RO6927005)</td><td>NCT04027946*</td><td>II</td><td>2023-12-22<br>(terminated due to<br>slow accrual)</td><td>6</td><td>National Cancer<br>Institute</td><td>evaluation of efficacy<br>of LMB-100 followed<br>by pembrolizumab</td><td>NSCLC</td><td>posology: LMB-100<br>140 µg/kg on days 1, 3,<br>and 5 of 3-week cycles<br>(up to 2) + 200 mg of<br>pembrolizumab on<br>day 1 of each<br>subsequent 3-week<br>cycles<br>safety (n = 3): 0% of<br>patients experienced<br>grade ≥3 TRAEs.<br>100% of patients<br>analyzed experienced<br>SAEs.<br>efficacy (n = 3): OS:<br>22.9 months. PFS: 3<br>months. OR: CR 0%,<br>PR 0%, SD 67%, and<br>PD 33%</td><td>Hassan<br>et al.<sup>131</sup></td></tr><tr><td>NCT03644550*</td><td>II</td><td>2020-11-02<br>(terminated due to<br>principal investigator<br>decision after FDA's<br>approval of<br>nivolumab +<br>ipilimumab as first<br>line treatment for<br>mesothelioma)</td><td>18</td><td>National Cancer<br>Institute</td><td>evaluation of safety<br>and efficacy of LMB-<br>100 followed by<br>pembrolizumab</td><td>Mesothelioma</td><td>posology: LMB-100<br>140 µg/kg i.v. on days<br>1, 3, and 5 of two 3-<br>week cycles +<br>pembrolizumab 200<br>mg i.v. Q3W from<br>cycle 3<br>safety (n = 18): 78% of<br>patients experienced<br>SAEs<br>efficacy (n = 18): OS<br>(n = 18): 17.1 months.<br>PFS (n = 18): 7.1<br>months. OR (n = 17):<br>PR 18%</td><td>Hassan<br>et al.<sup>131</sup></td></tr><tr><td>NCT02810418</td><td>I/II</td><td>2021-06-22</td><td>40</td><td>National Cancer<br>Institute</td><td>evaluation of safety,<br>tolerability, and<br>efficacy of LMB-100<br>alone and in<br>combination with<br>nab-paclitaxel</td><td>PDAC</td><td>posology: LMB-100<br>65 or 100 µg/kg i.v. on<br>days 1, 3, and 5 of 3-<br>week cycles for 1-3<br>cycles w/ or w/o nab-<br>paclitaxel 125 mg/m<sup>2</sup><br>i.v. on days 1 and 8</td><td>Alewine<br>et al.<sup>132</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 2. Continued**</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td><strong>safety (n = 20)</strong>: with nab-paclitaxel, LMB-100 MTD was determined to be 65 µg/kg. Most common AEs were hypoalbuminemia (100%), edema (65%), fatigue (50%), hyponatremia (50%), and myalgia (45%)<br/><strong>efficacy LMB-100 65 µg/kg + nab-paclitaxel 125 mg/m² (n = 14)</strong>: OS: 5.3 months. OR: PR 7%. Although clinical activity was observed, the combination was not well tolerated</td><td></td></tr><tr><td></td><td></td><td>NCT05375825*</td><td>I</td><td>2024-01-31 (withdrawn)</td><td>0</td><td>National Cancer Institute</td><td>evaluation of safety, tolerability, and pharmacokinetics of LMB-100</td><td>MPM</td><td><strong>posology</strong>: LMB-100 1 mg/mL intrathoracic perfusion using a closed circuit and roller pump with a heat exchanger following maximal cytoreductive surgery<br/><strong>safety</strong>: N/A<br/><strong>efficacy</strong>: N/A</td><td>-</td></tr><tr><td></td><td></td><td>NCT02798536</td><td>I</td><td>2022-04-21</td><td>21</td><td>National Cancer Institute</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of LMB-100 alone and in combination with nab-paclitaxel</td><td>Mesothelioma</td><td><strong>posology</strong>: LMB-100 100 to 170 µg i.v. on days 1, 3, and 5 of each 3-week cycle (up to 2-4), w/ or w/o nab-paclitaxel 125 mg/m² i.v. on days 1 and 8 of each 3-week cycle (up to 6)<br/><strong>safety (n = 21)</strong>: LMB-100 alone RP2D was determined to be 140 µg (n = 10). In combination with nab-paclitaxel, LMB-100 RP2D was determined to be 100 µg (n = 11). 0% of patients treated with LMB-100 at 140 µg</td><td>Alewine et al.<sup>132</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 2. Continued**</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>experienced SAEs (n = 7). 33% of patients treated with LMB-100 at 170 µg experienced SAEs (n = 3). 36% of patients treated with LMB-100 at 100 µg + nab-paclitaxel experienced SAEs (n = 11)<br><b>efficacy LMB-100 140 µg (n = 7):</b> OS: 16.9 months. PFS: 2.7 months. OR: CR 0% and PR 0%<br><b>efficacy LMB-100 170 µg (n = 3):</b> OS: 33.5 months. PFS: 2.7 months<br><b>efficacy LMB-100 100 µg + nab-paclitaxel (n = 11):</b> OS: 10.1 months. PFS: 2.7 months. OR: CR 0% and PR 0%</td><td></td></tr><tr><td rowspan="2">Immunotoxin</td><td rowspan="2">LMB-100 (RG7787 or RO6927005)</td><td>NCT04840615*</td><td>I</td><td>2022-01-12 (terminated due to slow accrual)</td><td>2</td><td>National Cancer Institute</td><td>evaluation of feasibility and safety of LMB-100 intratumoral injections in combination with ipilimumab</td><td>mesothelioma</td><td><b>posology:</b> LMB-100 400 µg itu. + ipilimumab itu. on days 1 and 4 each 3-week cycle (up to 4)<br><b>safety (n = 2):</b> 100% of patients experienced SAEs<br><b>efficacy (n = 2):</b> OS: 3.4 months. PFS: 1.4 months. OR: CR 0% and PR 0%</td><td>-</td></tr><tr><td>NCT04034238</td><td>I</td><td>2021-11-19</td><td>19</td><td>National Cancer Institute</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of LMB-100 in combination with tofacitinib</td><td>mesothelioma, CHO, and PDAC</td><td><b>posology:</b> LMB-100 65 to 140 µg/kg i.v. on days 4, 6, and 8 each 3-week cycles + tofacitinib 10 mg po. twice daily on days 1-10<br><b>safety (n = 10):</b> with tofacitinib, LMB-100 MTD was determined to be 100 µg/kg on days 4, 6, and 8. At the MTD, 14% of patients</td><td>Pegna et al.<sup>195</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>Table 2. Continued</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>experienced SAEs that may be related to LMB-100 (n = 7)<br><b>efficacy:</b> N/A</td><td></td></tr><tr><td></td><td></td><td>NCT03436732*</td><td>I</td><td>2019-04-30<br>(terminated due to fatal occurrence attributed to one of the study drugs)</td><td>5</td><td>National Cancer Institute</td><td>evaluation of safety and tolerability of LMB-100 in combination with SEL-110</td><td>Mesothelioma</td><td><b>posology:</b> LMB-100 100 or 140 µg/kg on days 1, 3, and 5 each 3-week cycles (up to 4) + SEL-110 on day 1 of each cycle at escalating doses<br><b>safety (n = 5):</b> the study was closed due to a fatal occurrence of pneumonitis attributed to one of the drugs<br><b>efficacy (n = 5):</b> OR: CR 0% and PR 0%</td><td>-</td></tr><tr><td></td><td></td><td>NCT02317419*</td><td>I</td><td>2015-08-01<br>(terminated due to the emerging benefit:risk ratio that did not justify continuing dosing patients)</td><td>15</td><td>Roche</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of LMB-100 alone and in combination with gemcitabine and nab-paclitaxel</td><td>Solid</td><td><b>posology:</b> LMB-100 i.v. on days 1, 3, and 5 of each 3-4-week cycles, w/ or w/o gemcitabine + nab-paclitaxel on days 1, 8, and 15 of each 4-week cycles<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td>Alewine et al.<sup>132</sup></td></tr><tr><td>Antibody-drug conjugate</td><td>Anetumab ravtansine (BAY 94-9343)</td><td>NCT03587311</td><td>II</td><td>estimated 2025-10-21</td><td>96</td><td>National Cancer Institute</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of AR in combination with bevacizumab vs. paclitaxel in combination bevacizumab</td><td>HGOC</td><td><b>Data cutoff date:</b> 2024-11-27<br><b>posology:</b> AR 2.2 mg/kg i.v. QW + bevacizumab 10 mg/kg i.v. Q2W. Paclitaxel 80 mg/m² i.v. QW + bevacizumab 10 mg/kg i.v. Q2W<br><b>safety (n = 57):</b> with bevacizumab, AR RP2D was determined to be 2.2 mg/kg. 26 grade ≥3 TRAEs were reported with paclitaxel + bevacizumab (n = 29). 29 grade ≥3 TRAEs were reported with AR + bevacizumab (n = 28)</td><td>Alqaisi et al.<sup>138</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 2. Continued**</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td><strong>efficacy paclitaxel + bevacizumab (n = 29):</strong> PFS: 12.7 months. OR: CR 0%, PR 66%, SD 20%, and PD 14%<br><strong>efficacy AR + bevacizumab (n = 28):</strong> PFS: 5.3 months. OR: CR 4%, PR 18%, SD 50%, and PD 28%</td><td></td></tr><tr><td></td><td></td><td>NCT03926143*</td><td>II</td><td>2022-05-18 (terminated due to strategic company decisions)</td><td>9</td><td>Bayer</td><td>evaluation of long-term safety and efficacy of AR alone and in combination with gemcitabine</td><td>Solid</td><td><strong>posology: N/Asafety (n = 9):</strong> 100% of patients experienced TEAEs. 89% of patients experienced TRAEs. 22% of patients experienced TESAEs. 0% of patients experienced TRSAEs<br><strong>efficacy (n = 9):</strong> OS: 34.1 months</td><td>-</td></tr><tr><td></td><td></td><td>NCT03023722</td><td>II</td><td>2019-12-11</td><td>18</td><td>Yale University</td><td>evaluation of safety and efficacy of AR</td><td>pancreatic</td><td><strong>posology: AR 6.5 mg/kg i.v. Q3W.</strong><br><strong>safety (n = 18):</strong> 61% of patients experienced SAEs<br><strong>efficacy (n = 14):</strong> TTP: 2.1 months. OR: CR 0%, PR 0%, SD 14%, and PD 86%</td><td>Chokshi and Hochster<sup>196</sup></td></tr><tr><td></td><td></td><td>NCT02610140</td><td>II</td><td>2019-09-06</td><td>248</td><td>Bayer</td><td>evaluation of safety, pharmacokinetics, and efficacy of AR vs. vinorelbine</td><td>MPM</td><td><strong>posology: AR 6.5 mg/kg Q3W. Vinorelbine 30 mg/m<sup>2</sup> Q3W</strong><br><strong>safety (n = 235):</strong> 74% of patients treated with vinorelbine experienced grade ≥3 AEs and 15% experienced TESAEs (n = 72). 48% of patients treated with AR experienced grade ≥3 AEs and 7% experienced TESAEs (n = 163)<br><strong>efficacy vinorelbine (n = 82):</strong> OS (n = 82): 11.6 months. PFS (n = 82): 4.5 months. OR (n</td><td>Kindler et al.<sup>137</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>Table 2. Continued</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>= 65): CR 0%, PR 9%, SD 77%, and PD 14%<br>efficacy AR (n = 166): OS (n = 166): 9.5 months. PFS (n = 166): 4.3 months. OR (n = 146): CR 0%, PR 11%, SD 73%, and PD 16%<br>posology: AR i.v. Q3W<br>safety (n = 2): 50% of patients experienced an SAE (respiratory failure)<br>efficacy (n = 2): OS: 1.7 months. PFS: 1.3 months. OR: CR 0% and PR 0%</td><td></td></tr><tr><td></td><td></td><td>NCT02839681*</td><td>II</td><td>2018-07-25 (terminated due to slow/insufficient accrual)</td><td>2</td><td>National Cancer Institute</td><td>evaluation of safety, tolerability, and efficacy of AR</td><td>Lung</td><td></td><td></td></tr><tr><td></td><td></td><td>NCT03126630</td><td>I/II</td><td>estimated 2026-09-18</td><td>49</td><td>National Cancer Institute</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of AR in combination with pembrolizumab</td><td>MPM</td><td>Data cutoff date: 2024-08-08<br>posology: pembrolizumab 200 mg i.v. w/ or w/o AR 6.5 mg/kg i.v. Q3W. (up to 1-2 years)<br>safety (n = 35): 53% of patients treated with pembrolizumab alone experienced grade ≥3 AEs (n = 17) and 61% of patients treated with AR + pembrolizumab experienced grade ≥3 AEs (n = 18)<br>efficacy pembrolizumab alone (n = 17): PFS: 3.9 months. OR: CR 0%, PR 6%, SD 29%, PD 53% and NE 12%<br>efficacy AR + pembrolizumab (n = 18): PFS: 12.2 months. OR: CR 0%, PR 17%, SD 50%, PD 20% and NE 13%.</td><td>Mansfield et al.<sup>139</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 2. Continued**</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="2">Antibody-drug conjugate</td><td rowspan="2">Anetumab ravtansine (BAY 94-9343)</td><td rowspan="2">NCT03816358</td><td rowspan="2">I/II</td><td rowspan="2">2020-01-07 (terminated due to slow accrual)</td><td rowspan="2">1</td><td rowspan="2">Mayo Clinic</td><td rowspan="2">evaluation of safety, tolerability, and efficacy of AR in combination with atezolizumab</td><td rowspan="2">NSCLC</td><td><strong>Study outcomes:</strong><br/><strong>posology:</strong> AR i.v. + atezolizumab i.v. Q3W<br/><strong>safety:</strong> N/A<br/><strong>efficacy:</strong> N/A</td><td rowspan="2">Hassan et al.<sup>136</sup></td></tr><tr><td><strong>Data cutoff date:</strong><br/><strong>2022-01-22</strong><br/><strong>posology:</strong> AR 5.5 or 6.5 mg/kg i.v. on day 1 (3- to 4-week cycles) + nivolumab i.v. on day 8 of cycle 1 and on day 1 of subsequent cycles (up to 17), w/ or w/o ipilimumab i.v. on day 8 of cycle 1 and on day 1 of cycles 2-4 or gemcitabine on days 1 and 8<br/><strong>safety (n = 28):</strong> 2 DLTs were reported for AR 6.5 mg/kg + nivolumab + ipilimumab. RP2D was determined to be 6.5 mg/kg. At 6.5 mg/kg, 5, 17 and 20% of patients experienced TRAEs, for AR + nivolumab, AR + nivolumab + ipilimumab, AR + nivolumab + gemcitabine, respectively<br/><strong>efficacy (n = 33):</strong> following data correspond to AR + nivolumab (n = 11), AR + nivolumab + ipilimumab (n = 13), AR + nivolumab + gemcitabine (n = 9). OR: CR 0%, PR 0%, SD 18, 15, 89%, PD 64, 46, 0% and NE 18, 38, 11%</td><td></td></tr><tr><td></td><td>NCT03102320</td><td>I</td><td>2021-07-26</td><td>173</td><td>Bayer</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of AR</td><td>gastric, NSCLC, TNBC, thymic, and PDAC</td><td><strong>Study outcomes:</strong><br/><strong>posology:</strong> AR 6.5 mg/kg i.v. on day 1 (3-week cycle), w/ or w/o cisplatin 25 mg/m<sup>2</sup> i.v.</td><td>Adjei et al.<sup>198</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>Table 2. Continued</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>alone and in combination with cisplatin or gemcitabine</td><td></td><td>or gemcitabine 1000 mg/m² on day 1 and 8 (up to 6 cycles)<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td></td></tr><tr><td></td><td></td><td>NCT02751918</td><td>I</td><td>2019-10-31</td><td>65</td><td>Bayer</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of AR in combination with PLD</td><td>ovarian, fallopian, and peritoneal</td><td><b>posology:</b> AR 5.5 or 6.5 mg/kg i.v. + PLD 30 mg/m² i.v. Q3W<br><b>safety (n = 65):</b> with PLD, AR MTD was determined to be 6.5 mg/kg (n = 9). No DLTs reported. More than 10% of patients experienced grade ≥3 AEs, fatigue (12%), neutrophil count decrease (11%) and neutropenia (11%) (n = 65)<br><b>efficacy (n = 65):</b> PFS: 5.0 months. OR: CR 2%, PR 26%, SD 43%, PD 17% and NE 12%. High MSLN expression increases treatment efficacy</td><td>Santin et al.<sup>140</sup></td></tr><tr><td></td><td></td><td>NCT02639091</td><td>I</td><td>2019-10-17</td><td>36</td><td>Bayer</td><td>evaluation of safety, tolerability, and pharmacokinetics of AR in combination with pemetrexed and cisplatin</td><td>mesothelioma and NSCLC</td><td><b>Data cutoff date:</b><br><b>2017-06-12</b><br><b>posology:</b> AR 5.5 or 6.5 mg/kg i.v. + pemetrexed 500 mg/m² i.v. + cisplatin 75 mg/m² i.v. Q3W.<br><b>safety (n = 25):</b> with pemetrexed + cisplatin, AR MTD was determined to be 6.5 mg/kg, 2 grade 3 AEs occurred (corneal microcystic and hypertension)<br><b>efficacy (n = 17):</b> OR (n = 17): PR 47% and NE 6%. OR with AR 6.5 mg/kg (n = 13): PR 46%</td><td>Hassan et al.<sup>199</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 2. Continued**</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="2"></td><td rowspan="2">Anetumab ravtansine (BAY 94-9343)</td><td>NCT02696642</td><td>I</td><td>2019-08-19</td><td>54</td><td>Bayer</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of AR</td><td>solid</td><td>posology: AR 6.5 mg/kg i.v. Q3W<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>NCT02824042</td><td>I</td><td>2019-08-05</td><td>63</td><td>Bayer</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of AR alone and in combination with itraconazole</td><td>solid</td><td>posology: AR i.v. + atezolizumab i.v. on day 1 (3-week cycles), w/ or w/o itraconazole 200 mg po. twice daily on day 18, once daily on days 19-21, and once daily on days 22-30<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td rowspan="2"></td><td rowspan="2"></td><td>NCT01439152</td><td>I</td><td>2019-07-30</td><td>148</td><td>Bayer</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of AR</td><td>solid</td><td>posology: AR 0.15 to 7.5 mg/kg Q3W. or AR 1.8 or 2.2 mg/kg QW<br>safety (n = 148): Q3W MTD was determined to be 6.5 mg/kg (n = 45), 39% of patients experienced grade ≥3 TESAEs (n = 38). QW MTD was determined to be 2.2 mg/kg (n = 71), 42% of patients experienced grade ≥3 TESAEs (n = 36)<br>efficacy (n = 148): PFS (n = 103): 2.8 months. PFS AR 6.5 mg/kg Q3W (mesothelioma-ovarian, n = 12-20): 14.1-4.1 months. PFS AR 2.2 mg/kg QW (mesothelioma-ovarian, n = 15-21): 2.6-2.2 months. OR (n = 148): CR 1%, PR 7%, SD 45%, PD 41% and NE 7%</td><td>Hassan et al.<sup>136</sup></td></tr><tr><td>NCT02485119</td><td>I</td><td>2017-07-04</td><td>12</td><td>Bayer</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of AR</td><td>solid</td><td>posology: AR 4.5 or 6.5 mg/kg i.v. Q3W<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr></tbody></table>

(Continued on next page)
<table><caption>Table 2. Continued</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="2"></td><td rowspan="2">DMOT4039A</td><td rowspan="2">NCT01469793</td><td rowspan="2">I</td><td rowspan="2">2015-12-01</td><td rowspan="2">71</td><td rowspan="2">Genentech - Roche</td><td rowspan="2">evaluation of safety, tolerability, pharmacokinetics, and efficacy of DMOT4039A</td><td rowspan="2">pancreatic and ovarian</td><td>posology: DMOT4039A 0.2 to 2.8 mg/kg i.v. Q3W, or 0.8 to 1.2 mg/kg i.v. QW</td><td rowspan="2">Weekes et al.<sup>143</sup></td></tr><tr><td>safety: Most frequent TRAEs occurred in 10% of patients on Q3W and QW schedules. RP2D was determined to be 2.4 mg/kg for Q3W regimen and 1 mg/kg for QW regimen</td></tr><tr><td></td><td></td><td>NCT01832116</td><td>I</td><td>2014-05-01</td><td>11</td><td>University Medical Center Groningen</td><td>evaluation of pharmacokinetics of 89Zr-MMOT0530A before the clinical trial NCT01469793</td><td>pancreatic and ovarian</td><td>efficacy pancreatic cancer DMOT4039A 2.4 mg/kg Q3W. (n = 26): PFS: 1.7 months. OR: CR 0%, PR 8%, SD 35%, and PD 57% efficacy ovarian cancer DMOT4039A 2.4 mg/kg Q3W. (n = 10): PFS: 4.9 months. OR: CR 0% and PR 30%</td><td>ter Weele et al.<sup>200</sup></td></tr><tr><td></td><td>BMS-986148</td><td>NCT02341625*</td><td>I/II</td><td>2020-05-07 (terminated due to business reasons)</td><td>126</td><td>Bristol-Myers Squibb</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of BMS-986148 alone and in combination with nivolumab</td><td>solid</td><td>posology: monotherapy, BMS-986148 0.1 to 1.6 mg/kg i.v. Q3W, or BMS-986148 0.4 to 0.6 mg/kg i.v. QW. In combination, BMS-986148 0.8 mg/kg i.v. Q3W + nivolumab 360 mg i.v. Q3W safety (n = 126): BMS-986148 MTD and RP2D was determined to be 1.2 mg/kg Q3W (n = 84). The dose level of 0.6 mg/kg QW was considered above the MTD (n = 12). 49% of</td><td>Rottey et al.<sup>145</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 2. Continued**</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>patients treated with BMS-986148 monotherapy Q3W experienced grade ≥3 TRAEs (n = 84). 25% of patients treated with BMS-986148 monotherapy QW experienced grade ≥3 TRAEs (n = 12). 33% of patients treated with BMS-986148 + nivolumab Q3W experienced grade ≥3 TRAEs (n = 30) efficacy BMS-986148 alone 1.2 mg/kg Q3W. (n = 51): PFS (n = 51): 2.8 months. PFS (mesothelioma, n = 25): 3.8 months. OR (n = 44): CR 0%, PR 7%, SD 57%, and PD 36% efficacy BMS-986148 0.8 mg/kg + nivolumab 360 mg Q3W. (n = 30): PFS (n = 30): N/A. PFS (mesothelioma, n = 13): 6.5 months. OR (n = 26): CR 0%, PR 23%, SD 54%, and PD 23%</td><td></td></tr><tr><td></td><td></td><td>NCT02884726</td><td>I</td><td>2017-09-06</td><td>8</td><td>Bristol-Myers Squibb</td><td>evaluation of safety and tolerability of BMS-986148</td><td>N/A</td><td>posology: BMS-986148 0.8 mg/kg i.v. Q3W safety (n = 7): 29% of patients experienced grade 3 AEs. No grade 4 AEs or SAEs were reported. One DLT was reported efficacy: N/A</td><td>Rottey et al.<sup>145</sup></td></tr><tr><td rowspan="2">Antibody-drug conjugate</td><td rowspan="2">RC88</td><td>NCT06173037</td><td>II</td><td>estimated 2026-12-31</td><td>88</td><td>RemeGen</td><td>evaluation of safety, pharmacokinetics, and efficacy of RC88</td><td>EOC, fallopian, and peritoneal</td><td>posology: RC88 2 mg/kg i.v. Q3W safety: N/A efficacy: N/A</td><td>-</td></tr><tr><td>NCT06016062</td><td>I/II</td><td>estimated 2025-12-31</td><td>221</td><td>RemeGen</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of RC148</td><td>solid</td><td>posology: RC88 2 mg/kg i.v. Q3W + RC148 20 mg/kg i.v. Q3W, w/or w/o bevacizumab</td><td>-</td></tr></tbody></table>

(Continued on next page)
<table><caption>Table 2. Continued</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>alone and in combination with docetaxel, RC48, RC88 or RC88 with bevacizumab</td><td></td><td>15 mg/kg i.v. Q3W. (For docetaxel and RC48 details: NCT06016062)<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td></td></tr><tr><td></td><td></td><td>NCT05804526</td><td>I/II</td><td>estimated 2025-12-01</td><td>82</td><td>RemeGen</td><td>evaluation of safety, pharmacokinetics, and efficacy of RC88 in combination with sintilimab</td><td>solid</td><td><b>posology:</b> RC88 1.5 to 2.5 mg/kg i.v. Q3W + sintilimab 200 mg i.v. Q3W<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td>-</td></tr><tr><td rowspan="2">RC88</td><td></td><td>NCT04175847</td><td>I/II</td><td>estimated 2025-12-01</td><td>200</td><td>RemeGen</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of RC88</td><td>solid</td><td><b>Data cutoff date:</b> 2023-12-19<br><b>posology:</b> RC88 0.1-2.5 mg/kg i.v. Q3W<br><b>safety (n = 164):</b> 40% of patients experienced grade ≥3 TEAEs. 14% of patients experienced TRSAEs. RP2D was determined to be 2 mg/kg Q3W<br><b>efficacy (n = 6-43):</b> OR: CR 0%-7% and PR 22%-46%</td><td>Liu et al.<sup>147</sup></td></tr><tr><td>NCT05508334</td><td>I</td><td>estimated 2025-12-30</td><td>81</td><td>RemeGen</td><td>evaluation of safety, tolerability, and efficacy of RC88</td><td></td><td>solid</td><td><b>posology:</b> RC88 dose escalation i.v. Q2W<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td>Li et al.<sup>146</sup></td></tr><tr><td></td><td>SGN-MesoC2 (PF-08052666 or HBM9033)</td><td>NCT06466187</td><td>I</td><td>estimated 2029-02-16</td><td>365</td><td>Seagen - Pfizer</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of SGN-MesoC2</td><td></td><td><b>posology:</b> SGN-MesoC2 dose escalation i.v.<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td>Cornelison et al.<sup>148</sup></td></tr><tr><td>Radionuclide therapy</td><td>BAY 2287411</td><td>NCT03507452</td><td>I</td><td>2022-03-29</td><td>36</td><td>Bayer</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of BAY2287411</td><td>mesothelioma and ovarian</td><td><b>posology:</b> Thorium-227 starts at 1.5 MBq and increased in steps of 1.0-1.5 MBq, with antibody doses of 10-400 mg i.v. Q6W<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td>Roy et al.<sup>201</sup></td></tr><tr><td>ADCC and ADCP mediator</td><td>Amatuximab (MORAb-009)</td><td>NCT02357147*</td><td>II</td><td>2018-11-30 (terminated due to business reason)</td><td>124</td><td>Morphotek</td><td>evaluation of safety and efficacy of amatuximab in combination with</td><td>MPM</td><td><b>posology:</b> amatuximab 5 mg/kg i.v. + pemetrexed 500 mg/m² i.v. + cisplatin</td><td>Hassan et al.<sup>202</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>Table 2. Continued</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>pemetrexed and cisplatin</td><td></td><td>75 mg/m² i.v. Q3W for 6 cycles<br><b>safety (n = 106)</b>: 20% of patients treated with placebo + pemetrexed + cisplatin experienced SAEs (n = 54). 29% of patients treated with amatuximab + pemetrexed + cisplatin experienced SAEs (n = 52)<br><b>efficacy</b>: N/A</td><td></td></tr><tr><td></td><td></td><td>NCT00738582</td><td>II</td><td>2014-01-10</td><td>89</td><td>Morphotek</td><td>evaluation of safety, tolerability, and efficacy of amatuximab in combination with pemetrexed and cisplatin</td><td>MPM</td><td><b>posology:</b><br>amatuximab 5 mg/kg i.v. on days 1 and 8 + pemetrexed 500 mg/m² i.v. on day 1 + cisplatin 75 mg/m² i.v. on day 1 of each 3-week cycle for 5 cycles<br><b>safety (n = 89)</b>: 100% of patients experienced TEAEs. 43% of patients experienced SAEs<br><b>efficacy (n = 89)</b>: OS (n = 89): 14.8 months. PFS (n = 84): 6.1 months. OR (n = 83): CR 0%, PR 40%, SD 51%, and PD 10%</td><td>Hassan et al.<sup>153</sup></td></tr><tr><td></td><td></td><td>NCT00570713</td><td>II</td><td>2009-12-01</td><td>155</td><td>Morphotek</td><td>evaluation of safety and efficacy of amatuximab in combination with gemcitabine</td><td>pancreatic</td><td><b>posology:</b><br>amatuximab 5 mg/kg i.v. + gemcitabine 200 mg/m² i.v. QW<br><b>safety:</b> 72% of patients treated with placebo + gemcitabine experienced SAEs (n = 75). 67% of patients treated with amatuximab + gemcitabine experienced SAEs (n = 73)<br><b>efficacy placebo + gemcitabine (n = 77)</b>: OS: 6.9 months. PFS: 3.5 months. OR: CR</td><td>Hassan et al.<sup>152</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>Table 2. Continued</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0%, PR 8%, SD 56%, PD 23%, and NE 13% efficacy amatuximab + gemcitabine (n = 78): OS: 6.5 months. PFS: 3.4 months. OR: CR 0%, PR 6%, SD 47%, PD 19%, and NE 27%</td><td></td></tr><tr><td></td><td></td><td>NCT01413451</td><td>I</td><td>2013-11-15</td><td>7</td><td>National Cancer Institute</td><td>evaluation of safety, tolerability, and pharmacokinetics of <sup>111</sup>Indium-radiolabeled amatuximab</td><td>mesothelioma and PDAC</td><td>posology: single-dose injection<br>safety (n = 6): radiotracer dose of 5 mCi was well tolerated efficacy (n = 6): tumor and metastatic sites imaged. Higher uptake in mesothelioma than pancreatic cancer</td><td>Lindenberg et al.<sup>156</sup></td></tr><tr><td></td><td></td><td>NCT01521325</td><td>I</td><td>2013-03-01</td><td>6</td><td>Morphotek</td><td>evaluation of safety, tolerability, and pharmacokinetics of <sup>111</sup>Indium-radiolabeled amatuximab</td><td>mesothelioma and PDAC</td><td>posology: single-dose injection.<br>safety (n = 6): radiotracer dose of 5 mCi was well tolerated. efficacy (n = 6): tumor and metastatic sites imaged. Higher uptake in mesothelioma than pancreatic cancer</td><td>Lindenberg et al.<sup>156</sup></td></tr><tr><td></td><td></td><td>NCT01018784</td><td>I</td><td>2013-02-01</td><td>17</td><td>Eisai</td><td>evaluation of safety and tolerability of amatuximab</td><td>solid</td><td>posology: amatuximab 50 to 200 mg/m<sup>2</sup> i.v. QW<br>safety (n = 17): MTD was determined to be 200 mg/m<sup>2</sup>. 77% of patients experienced AEs<br>efficacy (n = 17): OR: CR 0%, PR 0%, SD 18%, and PD 82%</td><td>Fujisaka et al.<sup>151</sup></td></tr><tr><td></td><td></td><td>NCT00325494</td><td>I</td><td>2008-09-01</td><td>24</td><td>Morphotek</td><td>evaluation of safety, tolerability, and pharmacokinetics of amatuximab</td><td>mesothelioma, pancreatic and ovarian</td><td>posology: amatuximab 12.5 to 400 mg/m<sup>2</sup> i.v. on days 1, 8, 15, and 22.<br>safety (n = 24): MTD was determined to be</td><td>Hassan et al.<sup>152</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 2. Continued**</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>200 mg/m².41% patients experienced SAEs, among them 2 patients treated at 400 mg/m² experienced DLT<br><b>efficacy (n = 24)</b>: OS: 6.5 months. PFS: 3.4 months. OR: CR 0%, PR 0%, SD 46%, PD 38%, and NE 17%.</td><td></td></tr><tr><td></td><td>NI-1801</td><td>NCT05403554</td><td>I</td><td>estimated 2026-09-30</td><td>70</td><td>Light Chain Bioscience - NovImmune</td><td>evaluation of safety, tolerability, pharmacokinetics and efficacy of NI-1801 alone and in combination with pembrolizumab or paclitaxel</td><td>ovarian, TNBC, NSCLC, and PDAC</td><td><b>Data cutoff date: 2025-08-01</b><br><b>posology:</b> NI-1801 Q2W or Q3W, w/ or w/o pembrolizumab 400 mg Q6W (up to 4 cycles) or paclitaxel 80 mg/m² QW (up to 4 cycles)<br><b>safety (n = 42)</b>: well tolerated both as monotherapy and in combination with pembrolizumab<br><b>efficacy NI-1801 alone (n = 21)</b>: OS (1 year): 75%<br><b>efficacy NI-1801 + pembrolizumab (n = 21)</b>: OS (1 year): 35%. PFS (9 months): 33%</td><td>-</td></tr><tr><td rowspan="2">Immunostimulator</td><td>ABBV-428</td><td>NCT02955251</td><td>I</td><td>2019-10-29</td><td>61</td><td>AbbVie</td><td>evaluation of safety, tolerability, pharmacokinetics and efficacy of ABBV-428</td><td>Solid</td><td><b>posology:</b> ABBV-428 0.01 to 3.6 mg/kg i.v. Q2W<br><b>safety (n = 59)</b>: 3 grade ≥3 AEs were reported. RP2D was determined to be 3.6 mg/kg<br><b>efficacy at 3.6 mg/kg (n = 25)</b>: OR: CR 0%, PR 0%, SD 36%, and PD 64%</td><td>Luke et al.<sup>159</sup></td></tr><tr><td>AMG 994 (Inezetamab)</td><td>NCT04727554</td><td>I</td><td>2023-06-05</td><td>11</td><td>Amgen</td><td>evaluation of safety, tolerability, pharmacokinetics and efficacy of AMG 994 alone and in combination with</td><td>Solid</td><td><b>posology:</b> AMG 994 (low or high dose) i.v. QW + AMG 404 i.v. Q4W. for 12 cycles (4-week cycles)<br><b>safety (n = 11)</b>: with</td><td>-</td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 2. Continued**</caption><thead><tr><th>Drug type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>AMG 404 (anti-PD-1 IgG1)</td><td></td><td>low-dose regimen, 0% of patients experienced DLTs and 100% experienced SAEs (n = 3). With high-dose regimen, 38% of patients experienced DLTs and 75% experienced SAEs (n = 8). 100% of patients experienced TEAEs (n = 11) efficacy low dose (n = 3): OS: 6.0 months. PFS: 2.6 months. OR: CR 0% and PR 0% efficacy high dose (n = 8): OS: 12.5 months. PFS: 1.6 months. OR: CR 0% and PR 25%</td><td></td></tr><tr><td rowspan="4">T cell engager</td><td>HPN536</td><td>NCT03872206</td><td>I</td><td>2023-01-04</td><td>95</td><td>Harpoon Therapeutics - Merck</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of HPN536</td><td>N/A</td><td>posology: HPN536 multiple injection doses of 6-14,400 ng/kg i.v.<br>safety: N/A<br>efficacy: N/A</td><td>Molloy et al.<sup>160</sup></td></tr><tr><td>AMG 305</td><td>NCT05800964</td><td>I</td><td>estimated 2028-08-12</td><td>220</td><td>Amgen</td><td>evaluation of safety and tolerability of AMG 305</td><td>Solid</td><td>posology: i.v.<br>safety: N/A<br>efficacy: N/A</td><td>Pham et al.<sup>162</sup></td></tr><tr><td>JNJ-79032421</td><td>NCT06255665</td><td>I</td><td>estimated 2028-03-07</td><td>120</td><td>Janssen - Johnson &amp; Johnson</td><td>evaluation of safety and tolerability of JNJ-79032421</td><td>Solid</td><td>posology: N/A<br>safety: N/A<br>efficacy: N/A</td><td>Smans et al.<sup>163</sup></td></tr><tr><td>ZW171</td><td>NCT06523803*</td><td>I</td><td>2025-10-01 (terminated due to sponsor decision)</td><td>160</td><td>Zymeworks</td><td>evaluation of safety, tolerability, and efficacy of ZW171</td><td>N/A</td><td>posology: N/A<br>safety: N/A<br>efficacy: N/A</td><td>–</td></tr><tr><td></td><td>CT-95</td><td>NCT06756035</td><td>I</td><td>estimated 2028-12-01</td><td>50</td><td>Context Therapeutics</td><td>evaluation of safety, tolerability, and efficacy of CT-95</td><td>solid</td><td>posology: CT-95 dose escalation i.v. QW.<br>safety: N/A<br>efficacy: N/A</td><td>–</td></tr></tbody></table>

Discontinued clinical trials are shown with the symbol *. ADCC, antibody-dependent-cell-mediated cytotoxicity; ADCP, antibody-dependent-cell-mediated phagocytosis; AE, adverse event; AR, anetumab ravansine; CHO, cholangiocarcinoma; CR, complete response; DLT, dose-limiting toxicity; EOC, epithelial ovarian cancer; FDA, Food and Drug Administration; HGOC, high-grade ovarian cancer; itu., intratumoral; i.v., intravenous; MPM, malignant pleural mesothelioma; MTD, maximum tolerated dose; NCI, National Cancer Institute; NE, not evaluated; NSCLC, non-small-cell lung cancer; OR, overall response; OS, overall survival; PD, progressive disease; PDAC, pancreatic ductal adenocarcinoma; PFS, progression-free survival; PLD, pegylated liposomal doxorubicin; po., per os; PR, partial response; QW, once weekly; Q2W, once every 2 weeks; Q3W, once every 3 weeks; Q6W, once every 6 weeks; QOD, every other day; RP2D, recommended phase 2 dose; SAE, serious adverse event; SD, stable disease; TEAE, treatment emergent adverse event; TESAE, treatment emergent serious adverse event; TNBC, triple negative breast cancer; TRAE, treatment-related adverse event; TRSAE, treatment-related serious adverse event; TTP, time to progression.
SGN-MesoC2 (or PF-08052666) is an ADC initially developed by Harbour BioMed under the name HBM9033 but now licensed by Pfizer. SGN-MesoC2, described as binding to membrane-bound MSLN, comprises a camptothecin-based topoisomerase 1 inhibitor payload (SGD-12280) coupled to a human immunoglobulin G1 (IgG1) mAb via a protease-cleavable linker (Figure 4A).¹⁴⁸ SGN-MesoC2 is the only anti-MSLN ADC in clinical trials that does not use a microtubule inhibitor as payload.¹³⁴ The first clinical trial started in 2024.

**TRT: BAY 2287411**

BAY 2287411 is a mesothelin-targeted thorium-227 conjugate developed by Bayer. The human anti-mesothelin antibody (BAY 86-1903) is conjugated to the 3,2-HOPO chelator and radiolabeled with the alpha emitter thorium-227 (Figure 4A).¹⁴⁹ To date, only one clinical trial has been conducted with BAY 2287411: a phase 1 monotherapy study that began in 2018 and ended in 2022 (Table 2). The results of this study are not available. Preclinical studies have demonstrated that BAY 2287411 is well tolerated in mice and in monkeys, induces a specific and significant antitumor activity, and activates several immunostimulatory pathways while modulating the surface expression of various immunomodulatory markers.¹⁴⁹ Furthermore, the combination of BAY 2287411 with either ICIs or DNA damage response inhibitors led to a significant potentiation of the antitumor activity of the compound.¹⁴⁹,¹⁵⁰

**ADCC and ADCP mediators: Amatuximab and NI-1801**

Amatuximab (or Morab-009), developed by Morphotek, is a humanized chimeric IgG1 derived from SS1, therefore competing with MUC16 (Figure 4A). Its mechanism of action includes inhibition of the MSLN/MUC16 axis and ADCC/ADCP. Six clinical trials have been conducted with amatuximab alone or in combination with pemetrexed and cisplatin (Table 2), in MPM and PDAC. However, despite a good safety profile, clinical studies concluded that amatuximab cannot be regarded as an effective treatment.¹⁵¹,¹⁵² Interestingly, based on the clinical trial NCT00738582, which did not achieve its targeted PFS primary endpoint, Nicolaides et al. investigate the correlation between MUC16 expression levels and the efficacy of amatuximab-based treatments.¹⁵³,¹⁵⁴ The authors demonstrated that amatuximab treatment induced a reversible increase in serum CA-125 levels due to the blockade of MUC16-MSLN interactions. Furthermore, soluble CA-125 was shown to impair amatuximab-mediated immune effector functions by binding to its (Fab')₂ domain, thereby disrupting its interactions with CD16a (ADCC) and CD32a (ADCP) receptors.¹⁵⁴,¹⁵⁵ No major studies have been published since the discontinued trial NCT02357147.

Amatuximab has also been investigated as an imaging agent. Two correlated phase 1 clinical trials were conducted with ¹¹¹Indium-radiolabeled amatuximab in mesothelioma and PDAC patients.¹⁵⁶ The efficacy of the agent in imaging tumor and metastatic sites was demonstrated, thus supporting its potential application as a diagnostic tool.

NI-1801, developed by Light Chain Bioscience-NovImmune, is a bispecific human IgG1 κλ-body targeting mesothelin and CD47 on tumors cells (Figure 4A). As described by the company, NI-1801 is made of a common human IgG1 heavy chain and two light chains (one kappa and one lambda) with distinct specificities. NI-1801 relies on ADCC and ADCP mechanisms, the ADCP process being enhanced by the blocking of the CD47 “don’t eat me signal.” Based on the publication of Hatterer et al., it is likely that the MSLN arm targets a membrane-proximal epitope on MSLN, thus not competing with MUC16.¹⁵⁷ A phase 1 study has started in 2022 with solid tumors.

**Immunostimulators: ABBV-428 and AMG 994**

ABBV-428 and AMG 994, developed by AbbVie and Amgen, respectively, are bispecific antibodies that exhibit bivalency toward mesothelin and CD40. ABBV-428 is an scFv-based antibody composed of a silent human IgG1 Fc fused to anti-CD40 and anti-MSLN scFv at the N- and C-terminus, respectively (Figure 4A).¹⁵⁸ As described in the Amgen protocol 20190136, AMG 994, also known as inezetamab, is an effector-silenced anti-CD40 IgG1 fused to an anti-MSLN scFv at the C-terminus. There is no information about the targeted epitope on MSLN. The mechanism of action of both molecules relies on the MSLN-dependent activation of CD40-expressing immune cells, leading to an enhanced priming and activation of cytotoxic T cells, activation of B lymphocytes, and reprogramming of type 2 macrophages into type 1.¹⁵⁸,¹⁵⁹ Phase 1 clinical trials have been conducted to assess the efficacy of both molecules in MSLN-positive solid tumors (Table 2). ABBV-428 evaluated as monotherapy demonstrated acceptable tolerability, but only minimal clinical activity. No further development of this molecule has been reported. Preliminary results suggest that AMG 994 demonstrates higher efficacy, yet it concomitantly induces toxicity. However, the development of AMG 994 was discontinued following a business decision.

**T cell engagers: HPN536, AMG 305, JNJ-79032421, ZW171, and CT-95**

HPN536, AMG 305, JNJ-79032421, ZW171, and CT-95 are all anti-MSLN/anti-CD3 TCEs able to recruit and activate T cells against MSLN⁺ tumor cells (Figure 4A).

HPN536 is a trispecific T-cell-activating construct (TriTAC) developed by Harpoon Therapeutics, a subsidiary of Merck.¹⁶⁰ It consists of a fusion of a humanized anti-MSLN llama single-domain antibody (sdAb), an anti-human serum albumin (HSA) sdAb for half-life extension through the FcRn loop, and a humanized scFv anti-CD3ε.¹⁶¹ The building blocks are connected by G₄SG₃S linkers.¹⁶⁰

AMG 305, a trispecific antibody-like TCE directed against MSLN (monovalent), p-cadherin (CDH3) (monovalent), and CD3 (bivalent), was developed by Amgen. This agent is an scFv-based construct that requires binding to both CDH3 and MSLN for potent cytotoxic activity, CDH3 and MSLN being co-expressed in multiple solid tumor types.¹⁶²
JNJ-79032421 is a membrane-restricted mesothelin targeting Fab fused to an anti-CD3 scFv developed by Janssen, a Johnson & Johnson subsidiary.¹⁶³ The precise structure of the compound remains undisclosed.

ZW171 is a 2 + 1 scFv-Fab-based anti-MSLN (bivalent) and anti-CD3 (monovalent) TCE, developed by Zymeworks.¹⁶⁴ The only clinical trial (NCT06523803) conducted with this therapeutic agent was discontinued, likely due to on-target/off-tumor toxicity.

CT-95 is a 2 + 2 bispecific scFv-Fab-based TCE anti-MSLN (bivalent) and anti-CD3 (bivalent) developed by Context Therapeutics. As described by the company, it has been engineered to target a membrane proximal epitope on MSLN.

In cynomolgus monkeys, HPN536 demonstrated both good tolerability and MSLN-dependent pharmacologic activity.¹⁶⁰ In cynomolgus monkeys, AMG 305 and ZW171 also demonstrated good tolerance.¹⁶² In a CDH3⁺ MSLN⁺ xenograft tumor model, AMG 305 has been shown to demonstrate *in vivo* antitumor activity that is dose dependent.¹⁶²

To date, HPN536, AMG 305, JNJ-79032421, ZW171, and CT-95 are all involved in only one monotherapy phase 1 study. The trials were initiated in 2019, 2023, 2024, 2024, and 2025, respectively. The results of these studies are not yet available (Table 2).

## Adoptive cell therapies targeting mesothelin

Mesothelin-targeted cell therapies have made notable progress in recent years, with several approaches progressing toward early-phase clinical trials. While the majority of adoptive cell therapies targeting mesothelin are based on conventional αβ T cell engineering, in particular chimeric antigen receptor (CAR)-T cells, the field is actively exploring other immune cell types (such as NK cells, γδ T cells, or macrophages) for CAR-based therapies, especially in the context of solid tumors.

## MSLN-targeting T cells

The clinical research on anti-MSLN CAR-T cells was initiated in 2011 with the mRNA-transfected SS1/CD3ζ/4-1BB CAR-T cells developed by the University of Pennsylvania.¹⁶⁵ Fifteen years later, various strategies employing CAR-T cells have advanced to the clinical stage with currently 63 clinical trials registered (Table 3).

Different strategies have been developed to improve MSLN CAR-T cells efficiency and address the immunosuppressive TME and CAR-T cell exhaustion: (1) CAR-T cells secreting ICI, cytokine, or chemokine; (2) multispecific CAR-T cells; (3) logic-gated CAR-T cells; (4) allogenic CAR-T cells; and (5) PD-1 KO CAR-T cells. Different routes of administration are also being evaluated to improve tumor targeting and reduce systemic toxicity, notably regional delivery such as intrapleural or intraperitoneal administration. In some cases, CAR-T cells have undergone modifications during their development.

For instance, M28z cells have been updated to M28z1XXPD1DNR, featuring a modified CD3ζ and a PD-1 dominant negative receptor. Similarly, αPD-1-mesoCAR-T cells, initially developed to secrete anti-PD-1 antibody, have been re-engineered to secrete anti-PD-1 sdAb.¹⁶⁷⁻¹⁶⁹

Anti-mesothelin CAR-T trials are mainly in the early stages (phase I/II), to assess safety, tolerability, and early signs of efficacy. Data on the design and available results are limited. However, the results published to date show acceptable tolerability and clinical responses in certain patients, notably in malignant pleural mesothelioma using regional intrapleural delivery, particularly when combined with pembrolizumab.¹⁶⁷

Beyond the traditional CAR-T cell approach, alternative strategies for engineering T cells have also been explored. These include (1) T-cell-receptor-engineered T cells (TCR-T cells), in which patient TCR is genetically modified to recognize a mesothelin-specific peptide presented by a specific human leukocyte antigen (HLA) molecule, typically HLA-A*02:01, as exemplified by the FH-TCR-T<sub>MSLN</sub> cells developed at the Fred Hutchinson Cancer Center, and (2) T cell receptor fusion construct (TRuC) T cells, which involve the fusion of an anti-mesothelin sdAb directly to the CD3ε subunit of the native CD3/TCR complex.¹⁷⁰,¹⁷¹ This platform has been tested both without (NCT03907852) and with (NCT05451849) the co-expression of a chimeric switch receptor designed to convert inhibitory signals from the TME, such as PD-L1, into stimulatory signals for the T cell.¹⁷¹

To date, a single clinical study has been conducted on CAR-γδT cells, and no data are yet available.

## Other immune cell types: CAR-PBMCs, CAR-NK cells, and CAR-macrophages

Since 2018, there has been an expansion of research interest on immune cell populations other than T cells, which has led to the development of novel cell therapy approaches (Table 3).

A single clinical study has been conducted on CAR-PBMCs targeting MSLN. However, this study was prematurely stopped due to a change in the sponsor's strategic priorities. Despite an acceptable tolerability, the available data fail to provide compelling evidence of efficacy.¹⁷²

Two exploratory clinical studies have been conducted with autologous CAR-NK cells for evaluation of safety issues and preliminary efficacy mainly in gynecological cancers. No results have yet been published.

In addition, two clinical studies have been conducted on CAR-macrophages, with results only available for SY001 cells in combination with tislelizumab (anti-PD-1). The study's findings revealed that 100% of patients experienced grade ≥3 adverse events (AEs), and no responses to the treatment were observed.¹⁷³
<table><caption>Table 3. MSLN-targeted cell therapies in clinical trials</caption><thead><tr><th>Cell type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td>anti-MSLN CAR-T cells (mRNA transfected SS1/CD3ζ/ 4-1BB CAR-T cells)</td><td>NCT01897415</td><td>I</td><td>2017-03-01</td><td>16</td><td>University of Pennsylvania</td><td>evaluation of feasibility, safety, and efficacy of anti-MSLN CAR-T cells</td><td>PDAC</td><td>posology: no LD. 1 to 3 × 10<sup>8</sup> cells/m<sup>2</sup> i.v. 3 times weekly for 3 weeks.<br/>safety (n = 6): no DLT reported. No CRS reported. 3 grade 3 AEs were reported. 2 grade 4 TRAEs were reported.<br/>efficacy (n = 6): PFS: 4.6 months. OR: CR 0%, PR 0%, SD 33%, and PD 67%.</td><td>Beatty et al.<sup>166</sup></td></tr><tr><td></td><td>CAR-T cells</td><td>NCT01355965</td><td>I</td><td>2015-10-01</td><td>18</td><td>University of Pennsylvania</td><td>evaluation of feasibility, safety, pharmacokinetics, and efficacy of anti-MSLN CAR-T cells</td><td>MPM and PDAC</td><td>Data cutoff date: 2014-02-01<br/>efficacy: no LD. Different protocols were used with multiple injections at 1 × 10<sup>8</sup> cells i.v., 1 × 10<sup>9</sup> cells i.v., 3 × 10<sup>8</sup> cells/m<sup>2</sup> i.v. and/or 2 × 10<sup>8</sup> cells i.tu.<br/>safety (n = 2): treatment not tolerated, the first patient died because of the treatment and the second experienced 2 grade ≥3 AEs<br/>efficacy (n = 2): results suggest a role of anti-MSLN CAR-T cells in inducing an antitumor effect</td><td>Beatty et al.<sup>165</sup></td></tr><tr><td></td><td>anti-MSLN CAR-T cells (retroviral transduced CAR-T cells)</td><td>NCT01583686*</td><td>I/II</td><td>2018-12-17 (terminated due to slow/insufficient accrual)</td><td>15</td><td>National Cancer Institute</td><td>evaluation of safety, tolerability, and efficacy of anti-MSLN CAR-T cells in combination with aldesleukin</td><td>solid</td><td>posology: LD. Single-dose injection of 1 × 10<sup>6</sup> to 1 × 10<sup>8</sup> cells i.v. + aldesleukin 72,000 IU/kg i.v. (up to 15 dose injections)<br/>safety (n = 15): 45% of patients experienced SAEs. The only group without SAEs is the one with 1 × 10<sup>7</sup> cells<br/>efficacy (n = 15): OR: CR 0%, PR 0%, SD 7%, and PD 93%. The only</td><td>—</td></tr></tbody></table>

(Continued on next page)
<table><caption>Table 3. Continued</caption><thead><tr><th>Cell type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>patient with an SD was injected with 3 × 10<sup>6</sup> CAR-PBL</td><td></td></tr><tr><td></td><td></td><td>NCT05057715</td><td>I</td><td>estimated 2038-09-01</td><td>13</td><td>University of Pennsylvania</td><td>evaluation of feasibility and safety of huCART-meso cells in combination with VCN-01</td><td>pancreatic and ovarian</td><td>posology: No LD. VCN-01 3.3 × 10<sup>12</sup> vp i.v. on day 0 + huCART-meso 5 × 10<sup>7</sup> cells i.v. on day 14 (regimen A). VCN-01 1 × 10<sup>13</sup> vp i.v. on day 0 + huCART-meso 5 × 10<sup>7</sup> cells i.v. on day 14 (regimen B). huCART-meso cells i.v. on day 0 + VCN-01 3.3 × 10<sup>12</sup> vp i.v. on day 14 (regimen C)</td><td></td></tr><tr><td rowspan="3">huCART-meso cells(lentiviral transduced SS1/4-1BB/CD3ζ CAR-T cells)</td><td>NCT05623488</td><td>I</td><td>estimated 2038-02-01</td><td>12</td><td>University of Pennsylvania</td><td>evaluation of feasibility and safety of intratumoral huCART-meso cells injection</td><td>TNBC</td><td></td><td>safety: N/A<br>efficacy: N/A</td><td></td></tr><tr><td>NCT03323944</td><td>I</td><td>estimated 2025-09-01</td><td>18</td><td>University of Pennsylvania</td><td>evaluation of feasibility and safety of i.v. injection and local delivery of huCART-meso cells</td><td>pancreatic</td><td></td><td>safety: no LD. Single-dose injection of 1 × 10<sup>6</sup> to 3 × 10<sup>7</sup> cells/m<sup>2</sup> i.v., i.p., or intrahepatic.<br>safety: N/A<br>efficacy: N/A</td><td>Haas et al.<sup>123</sup></td></tr><tr><td>NCT03054298</td><td>I</td><td>estimated 2024-07-30</td><td>54</td><td>University of Pennsylvania</td><td>evaluation of feasibility and safety of i.v. injection and local delivery of huCART-meso cells</td><td>mesothelioma, ovarian, and pancreatic</td><td></td><td>Data cutoff date: 2019-07-17<br>posology: w/ or w/o LD. Single-dose injection of 1–3 × 10<sup>7</sup> cells/m<sup>2</sup> i.v. or 1 to 3 × 10<sup>8</sup> cells/m<sup>2</sup> i.v.<br>safety (n = 15): 7 grade ≥3 AEs were reported<br>efficacy (n = 15): PFS: 2.1 months. OR: CR 0%, PR 0%, SD 73%, and PD 27%</td><td>Haas et al.<sup>123</sup></td></tr><tr><td>CCAR-T cells</td><td>huCART-meso cells (lentiviral transduced SS1/4-1BB/CD3ζ CAR-T cells)</td><td>NCT02465983*</td><td>I</td><td>2017-11-01 (teminated due to lack of efficacy and funding)</td><td>4</td><td>University of Pennsylvania</td><td>evaluation of feasibility, safety, pharmacokinetics, and efficacy of huCART-meso cells</td><td>PDAC</td><td>posology: LD. huCART-meso cells 3 × 10<sup>7</sup> cells/m<sup>2</sup> i.v. + anti-CD19 CAR-T</td><td>Ko et al.<sup>203</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 3. Continued**</caption><thead><tr><th>Cell type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>meso cells in combination with anti-CD19 CAR-T cells</td><td></td><td>cells 3 × 10<sup>7</sup> cells/m<sup>2</sup> i.v. (two separate injections)<br><b>safety (n = 3)</b>: no DLT reported<br><b>efficacy (n = 3)</b>: OR: CR 0%, PR 0%, SD 33%, PD 67%. CAR-T-19 cells deplete normal B cells but at the dose tested no improvement of huCART-meso cells persistence</td><td></td></tr><tr><td></td><td></td><td>NCT02159716</td><td>I</td><td>2015-11-01</td><td>19</td><td>University of Pennsylvania</td><td>evaluation of feasibility, safety, pharmacokinetics, and efficacy of huCART-meso cells</td><td>PDAC, EOC, and MPM</td><td><b>posology</b>: w/ or w/o LD. Single-dose injection of 1–3 × 10<sup>7</sup> cells/m<sup>2</sup> i.v. or 1 to 3 × 10<sup>8</sup> cells/m<sup>2</sup> i.v.<br><b>safety (n = 15)</b>: without LD, at 1 to 3 × 10<sup>7</sup> cells/m<sup>2</sup> a DLT occurred<br><b>efficacy (n = 15)</b>: PFS: 2.1 months. OR: CR 0%, PR 0%, SD 73%, and PD 27%</td><td>Haas et al.<sup>123</sup></td></tr><tr><td></td><td></td><td>NCT02388828</td><td>N/A</td><td>2019-07-01</td><td>10</td><td>University of Pennsylvania</td><td>evaluation of long-term safety of huCART-meso cells</td><td>N/A</td><td><b>posology</b>: no LD<br><b>safety</b>: N/A<br><b>efficacy</b>: N/A</td><td>–</td></tr><tr><td>M28z cells (retroviral transduced anti-MSLN M912 scFv/CD28/CD3ζ CAR-T cells)</td><td>NCT02414269</td><td>I/II</td><td>estimated 2026-04-30</td><td>113</td><td>Memorial Sloan Kettering Cancer Center</td><td>evaluation of safety and efficacy of M28z cells alone and in combination with pembrolizumab</td><td>solid</td><td><b>Data cutoff date:</b> 2019-04-01<br><b>posology</b>: w/ or w/o LD. Single-dose injection of 1.3 × 10<sup>5</sup> to 8.6 × 10<sup>7</sup> cells/kg ip. (at PD stage 15% of patients received a second injection) + pembrolizumab 200 mg i.v. 3 times and 4 weeks after completing CAR-T cells injection<br><b>safety (n = 27)</b>: no DLTs reported. 22% of patients experienced grade 3 AEs. Grade 4 AEs were reversible<br><b>efficacy M28z + pembrolizumab (n = 16)</b>: OS: 23.9 months.</td><td>Adusumilli et al.<sup>167</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>Table 3. Continued</caption><thead><tr><th>Cell type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td>anti-MSLN CAR-T cells (retroviral transduced anti-MSLN/4-1BB/CD3ζ CAR-T cells)</td><td>NCT02580747**</td><td>I</td><td></td><td>estimated 2018-11-01 (unknown status)</td><td>20</td><td>Chinese PLA General Hospital</td><td>evaluation of feasibility, safety, and efficacy of anti-MSLN CAR-T cells</td><td>solid</td><td>OR: CR 0%, PR 13%, SD 56%, and PD 31%<br>posology: no LD. Injection on days 0, 1, and 2<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>TAI-meso-CART cells</td><td>NCT02706782**</td><td>I</td><td></td><td>estimated 2018-09-01 (unknown status)</td><td>30</td><td>Shanghai GeneChem</td><td>evaluation of safety and efficacy of TAI-meso-CART cells</td><td>pancreatic</td><td>posology: LD. Single-dose injection of 1 × 10<sup>6</sup> to 10 × 10<sup>6</sup> cells/kg i.v.<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>anti-MSLN CAR-T cells (transduced anti-MSLN CAR-T cells)</td><td>NCT02792114</td><td>I</td><td></td><td>estimated 2026-06-01</td><td>186</td><td>Memorial Sloan Kettering Cancer Center</td><td>evaluation of safety and tolerability of anti-MSLN CAR-T cells</td><td>breast</td><td>posology: LD. Single-dose injection i.v.<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>anti-MSLN CAR-T cells (lentiviral transduced CAR-T cells)</td><td>NCT02959151**</td><td>I/II</td><td></td><td>estimated 2018-07-01 (unknown status)</td><td>20</td><td>Shanghai GeneChem</td><td>evaluation of safety and efficacy of anti-MSLN CAR-T cells in combination with interventional therapy</td><td>pancreatic</td><td>posology: no LD. Single-dose injection of 1.25-4 × 10<sup>7</sup> cells/cm<sup>3</sup> of tumor bulk i.v. or itu<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>anti-MSLN CAR-T cells</td><td>NCT02930993**</td><td>I</td><td></td><td>estimated 2019-08-01 (unknown status)</td><td>20</td><td>China Meitan General Hospital</td><td>evaluation of safety and efficacy of anti-MSLN CAR-T cells</td><td>solid</td><td>posology: LD. 3-day split-dose of 5 × 10<sup>4</sup> to 1 × 10<sup>7</sup> cells/kg i.v. (day 0, 10%; day 1, 30%; day 2, 60%).<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>αPD-1-mesoCAR-T cells (DNA transfected anti-MSLN CAR-T cells secreting PD-1 antibody)</td><td>NCT03615313**</td><td>I/II</td><td></td><td>estimated 2020-12-03 (unknown status)</td><td>50</td><td>Shanghai Cell Therapy Research Institute</td><td>evaluation of safety and efficacy of αPD-1-mesoCAR-T cells</td><td>solid</td><td>Data cutoff date:<br>2020-05-25<br>posology: LD. 5 × 10<sup>6</sup> cells/kg, 5 × 10<sup>7</sup> cells/kg or 1 × 10<sup>8</sup> cells/kg i.v. from day 1 to day 3<br>safety (n = 10): most common AEs were mild fatigue and fever<br>efficacy (n = 10): PFS: 3.2 months. OR: CR 0%, PR 20%, SD 40%, and PD 40%<br>posology: no LD. Single-dose injection i.v.</td><td>Fang et al.<sup>169</sup></td></tr><tr><td>CAR-T cells</td><td>αPD-1-mesoCAR-T cells (DNA transfected anti-MSLN CAR-T cells)</td><td>NCT03030001**</td><td>I/II</td><td></td><td>estimated 2019-02-01 (unknown status)</td><td>40</td><td>Ningbo Cancer Hospital</td><td>evaluation of safety and efficacy of anti-MSLN CAR-T cells</td><td>solid</td><td></td><td>-</td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 3. Continued**</caption><thead><tr><th>Cell type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td>cells secreting PD-1 antibody)</td><td></td><td></td><td></td><td></td><td></td><td>expressing PD-1 antibody</td><td></td><td>safety: N/A<br>efficacy: N/A</td><td></td></tr><tr><td></td><td>anti-MSLN CAR-T cells secreting CTLA-4/PD-1 antibodies</td><td>NCT03182803**</td><td>I/II</td><td>estimated 2019-04-20 (unknown status)</td><td>40</td><td>Shanghai Cell Therapy Research Institute</td><td>evaluation of safety, pharmacokinetics, and efficacy of anti-MSLN CAR-T cells secreting CTLA-4/PD-1 antibodies</td><td>solid</td><td>posology: LD. 2 to 5 × 10<sup>7</sup> cells/kg i.v. from day 18 to day 19 (±2 days), 2 cycles<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td></td><td>anti-MSLN CAR-T cells</td><td>NCT03267173**</td><td>I</td><td>estimated 2019-06-01 (unknown status)</td><td>10</td><td>First Affiliated Hospital of Harbin Medical University</td><td>evaluation of safety and efficacy of anti-MSLN CAR-T cells</td><td>pancreatic</td><td>posology: no LD.<br>Single-dose injection<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td></td><td>anti-MSLN CAR-T cells (anti-MSLN CD8+ CAR-T cells + anti-TGF-β CD4+ CAR-T cells secreting IL-7/CCL19 and/or anti-PD-1/CTLA-4/ TIGIT scFvs)</td><td>NCT04203459**</td><td>N/A</td><td>estimated 2022-12-31 (unknown status)</td><td>80</td><td>First Affiliated Hospital of Harbin Medical University</td><td>evaluation of the effects and mechanism of gut microbiota on anti-MSLN CAR-T cells</td><td>pancreatic</td><td>posology: N/A<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td></td><td>anti-MSLN-7 × 19 CAR-T cells (lentiviral transduced anti-MSLN scFv/TLR2/ CD3ζ CAR-T cells secreting human IL-7 and CCL19)</td><td>NCT03198052</td><td>I</td><td>estimated 2036-08-01</td><td>30</td><td>Second Affiliated Hospital of Guangzhou Medical University</td><td>evaluation of safety, tolerability, and efficacy of anti-MSLN CD8+ CAR-T cells + anti-TGF-β CD4+ CAR-T cells secreting IL-7/CCL19 and/or anti-PD-1/CTLA-4/ TIGIT scFvs</td><td>lung</td><td>posology: no LD. 1 to 10 × 10<sup>6</sup> cells/kg i.v. or regional injections for 3 or more cycles<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td></td><td>anti-MSLN CAR-T cells(lentiviral transduced anti-MSLN scFv/4-1BB/ CD3ζ CAR-T cells)</td><td>NCT03497819**</td><td>I</td><td>estimated 2020-10-31 (unknown status)</td><td>10</td><td>First Affiliated Hospital of Wenzhou Medical University</td><td>evaluation of safety and efficacy of anti-MSLN-7 × 19 CAR-T cells</td><td>pancreatic and ovarian</td><td>Data cutoff date: 2021-07-17<br>posology: no LD.<br>Injections every 1 to 2 months i.v. or local<br>safety (n = 6): injection every 1 to 2 months. No grade ≥2 AEs reported<br>efficacy (n = 2): OR: CR 50%, PR 0%, SD 0%, and PD 50%</td><td>Pang et al.<sup>204</sup></td></tr><tr><td></td><td>anti-MSLN CAR-T cells (GIMI-IRB cells)</td><td>NCT03356808**</td><td>I/II</td><td>estimated 2020-12-31 (unknown status)</td><td>20</td><td>Shenzhen Geno-Immune Medical Institute</td><td>evaluation of safety and efficacy of anti-MSLN CAR-T cells in combination with anti-CD19 CAR-T cells</td><td>pancreatic</td><td>posology: LD. i.v. or artery injection<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr></tbody></table>

(Continued on next page)
<table><caption>Table 3. Continued</caption><thead><tr><th>Cell type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="10">CAR-T cells</td><td rowspan="3">anti-MSLN CAR-T cells (GIMI-IRB cells)</td><td rowspan="3">NCT03356795**</td><td rowspan="3">I/II</td><td rowspan="3">estimated 2020-12-01 (unknown status)</td><td rowspan="3">20</td><td rowspan="3">Shenzhen Geno-Immune Medical Institute</td><td rowspan="3">evaluation of feasibility, safety, pharmacokinetics, and efficacy of anti-MSLN CAR-T cells</td><td rowspan="3">cervical</td><td><strong>posology:</strong> no LD.<br>Single-dose injection of 1 × 10<sup>6</sup> to 1 × 10<sup>7</sup> cells/kg i.v.</td><td rowspan="3">-</td></tr><tr><td><strong>safety:</strong> N/A</td></tr><tr><td><strong>efficacy:</strong> N/A</td></tr><tr><td rowspan="2">anti-MSLN CAR-T cells</td><td rowspan="2">NCT03638206**</td><td rowspan="2">I/II</td><td rowspan="2">estimated 2023-03-01 (unknown status)</td><td rowspan="2">73</td><td rowspan="2">Shenzhen BinDeBio</td><td rowspan="2">evaluation of safety and efficacy of anti-MSLN CAR-T cells</td><td rowspan="2">gastric, pancreatic, and mesothelioma</td><td><strong>posology:</strong> LD. Single-dose injection</td><td rowspan="2">-</td></tr><tr><td><strong>safety:</strong> N/A</td></tr><tr><td rowspan="2">anti-MSLN CAR-T cells</td><td rowspan="2">NCT03941626**</td><td rowspan="2">I/II</td><td rowspan="2">estimated 2021-12-01 (unknown status)</td><td rowspan="2">50</td><td rowspan="2">Shenzhen BinDeBio</td><td rowspan="2">evaluation of safety and efficacy of anti-MSLN CAR-T cells</td><td rowspan="2">gastric</td><td><strong>posology:</strong> no LD</td><td rowspan="2">-</td></tr><tr><td><strong>safety:</strong> N/A</td></tr><tr><td rowspan="2">MPTK-CAR-T cells (PD-1 and TCR deficient lentiviral transduced P4 scFv/CD28/CD3ζ CAR-T cells)</td><td rowspan="2">NCT03545815**</td><td rowspan="2">I</td><td rowspan="2">estimated 2020-12-30 (unknown status)</td><td rowspan="2">15</td><td rowspan="2">Chinese PLA General Hospital</td><td rowspan="2">evaluation of feasibility, safety, pharmacokinetics, and efficacy of MPTK-CAR-T cells</td><td rowspan="2">solid</td><td><strong>posology:</strong> no LD.<br>Single-dose injection of 0.1–9 × 10<sup>6</sup> cells/kg i.v. If undetectable circulating MPTK-CAR-T cells, possibility of a second or a third infusion (8/15)</td><td rowspan="2">Wang et al.<sup>205</sup></td></tr><tr><td><strong>safety (n = 15):</strong> 5 grade ≥3 AEs were reported</td></tr><tr><td rowspan="2">GC008t cells (PD-1 deficient lentiviral transduced P4 scFv/CD28/CD3ζ CAR-T cells))</td><td rowspan="2">NCT03747965**</td><td rowspan="2">I</td><td rowspan="2">estimated 2020-05-01 (unknown status)</td><td rowspan="2">10</td><td rowspan="2">Chinese PLA General Hospital</td><td rowspan="2">evaluate the feasibility, safety, and efficacy of GC008t cells</td><td rowspan="2">pancreatic, ovarian, and colorectal</td><td><strong>posology:</strong> w/ or w/o LD. 1 × 10<sup>6</sup> to 5 × 10<sup>7</sup> cells/kg. 4/9 patients received repeat injections of GC008t</td><td rowspan="2">Wang et al.<sup>206</sup></td></tr><tr><td><strong>safety (n = 9):</strong> CRS noticed only at the highest dose</td></tr><tr><td>anti-MSLN CAR-T cells (lentiviral)</td><td>NCT03638193**</td><td>N/A</td><td></td><td></td><td>10</td><td>Shenzhen BinDeBio</td><td></td><td>pancreatic</td><td><strong>posology:</strong> LD. Single-dose injection of 1 ×</td><td>-</td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 3. Continued**</caption><thead><tr><th>Cell type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td>transduced SS1/TCRζ/ 4-1BB CAR-T cells)</td><td></td><td></td><td>estimated 2022-02-01 (unknown status)</td><td></td><td></td><td>evaluation of safety and efficacy of CART-meso cells</td><td></td><td>10<sup>7</sup> to 3 × 10<sup>8</sup> cells/ m<sup>2</sup> i.v.<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td></td></tr><tr><td></td><td>anti-MSLN CAR-T cells (retroviral transduced CAR-T cells)</td><td>NCT03799913**</td><td>I</td><td>estimated 2022-04-01 (unknown status)</td><td>20</td><td>Zhejiang University</td><td>evaluation of feasibility, safety, pharmacokinetics, and efficacy of anti-MSLN CAR-T cells</td><td>ovarian</td><td><b>posology:</b> LD.<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td>–</td></tr><tr><td></td><td>gavo-cel (gavocabtagene autoleucel or TC-210 or lentiviral transduced anti-MSLN sdAb (MH1)/ CD3ζ CAR-T cells)</td><td>NCT03907852</td><td>I/II</td><td>estimated 2028-11-02</td><td>36</td><td>TCR2 Therapeutics</td><td>evaluation of safety and efficacy of gavo-cel</td><td>MPM, ovarian, and CHO</td><td><b>Data cutoff date:</b><br>2023-01-17<br><b>posology:</b> w/ or w/o LD. Single-dose injection of 5 × 10<sup>7</sup> to 5 × 10<sup>8</sup> cells/m<sup>2</sup> i.v.<br><b>safety (n = 32):</b> 97% of patients experienced AEs. 78% of patients experienced CRS. The RP2D was determined to be 1 × 10<sup>8</sup> cells/m<sup>2</sup>, after LD. At the RP2D, no cases of pneumonitis were reported and severe CRS events (2/13, 15%) were rapidly reversible<br><b>efficacy in MPM (n = 23):</b> OS: 11.1 months. PFS: 5.6 months<br><b>efficacy in ovarian cancer (n = 8):</b> OS: 9.4 months. PFS: 5.8 months</td><td>Hassan et al.<sup>174</sup></td></tr><tr><td></td><td>anti-MSLN CAR-T cells (retroviral transduced CAR-T cells)</td><td>NCT03916679**</td><td>I/II</td><td>estimated 2023-04-20 (unknown status)</td><td>20</td><td>Second Affiliated Hospital, School of Medicine, Zhejiang University</td><td>evaluation of feasibility, safety, pharmacokinetics, and efficacy of anti-MSLN CAR-T cells</td><td>EOC</td><td><b>posology:</b> LD.<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td>–</td></tr><tr><td rowspan="2">CAR-T cells</td><td>anti-MSLN CAR-T cells (transduced anti-MSLN CAR-T cells)</td><td>NCT03814447**</td><td>I</td><td>estimated 2023-01-01 (unknown status)</td><td>10</td><td>Shanghai 6th People's Hospital</td><td>evaluation of feasibility, safety, pharmacokinetics, and efficacy of anti-MSLN CAR-T cells</td><td>Ovarian</td><td><b>posology:</b> LD. Single-dose injection of 5 × 10<sup>6</sup> cells/kg<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td>–</td></tr><tr><td>αPD1-MSLN-CAR T cells (anti-MSLN CAR-T cells secreting anti-PD-1 sdAb)</td><td>NCT05089266</td><td>I</td><td>estimated 2025-01-30</td><td>30</td><td>Shanghai Cell Therapy Group</td><td>evaluation of safety and efficacy of αPD1-MSLN-CAR T cells</td><td>Colorectal</td><td><b>posology:</b> No LD.<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td>–</td></tr></tbody></table>

(Continued on next page)
<table><caption>Table 3. Continued</caption><thead><tr><th>Cell type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="5">M28z1XXPD1DNR cells (M28z equipped with a modified CD3ζ (1XX), and a PD-1 dominant negative receptor (PD1DNR))</td><td rowspan="2">NCT04489862**</td><td rowspan="2">I</td><td>estimated 2023-08-01 (unknown status)</td><td rowspan="2">10</td><td rowspan="2">Shanghai Mengchao Cancer Hospital</td><td>evaluation of safety, tolerability, pharmacokinetics and efficacy of αPD1-MSLN-CAR T cells</td><td rowspan="2">NSCLC and mesothelioma</td><td>posology: no LD.<br>Single-dose injection of 1 × 10<sup>5</sup> to 3 × 10<sup>6</sup> cells/kg<br>safety: N/A<br>efficacy: N/A</td><td rowspan="2">-</td></tr><tr><td>estimated 2022-12-01 (unknown status)</td><td>evaluation of safety, tolerability, pharmacokinetics and efficacy of αPD1-MSLN-CAR T cells</td><td>posology: LD. Single-dose injection of 1 × 10<sup>5</sup> to 3 × 10<sup>6</sup> cells/kg i.v.<br>safety: N/A<br>efficacy: N/A</td></tr><tr><td rowspan="2">NCT04503980**</td><td rowspan="2">I</td><td>estimated 2022-06-01 (unknown status)</td><td rowspan="2">10</td><td rowspan="2">Shanghai Cell Therapy Group</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of αPD1-MSLN-CAR T cells</td><td rowspan="2">colorectal and ovarian</td><td>safety: LD. 1 × 10<sup>5</sup> to 3 × 10<sup>6</sup> cells/kg i.v.<br>safety: N/A<br>efficacy: N/A</td><td rowspan="2">-</td></tr><tr><td>estimated 2028-09-30</td><td>evaluation of safety and tolerability of M28z1XXPD1DNR cells</td><td>posology: no LD.<br>Single-dose injection i.p.<br>safety: N/A<br>efficacy: N/A</td></tr><tr><td>NCT06623396</td><td>I</td><td>estimated 2028-09-30</td><td>18</td><td>Memorial Sloan Kettering Cancer Center</td><td>evaluation of safety and tolerability of M28z1XXPD1DNR cells</td><td>esophagogastric</td><td>posology: no LD.<br>Single-dose injection i.p.<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>NCT04577326</td><td>I</td><td>estimated 2026-09-30</td><td>14</td><td>Memorial Sloan Kettering Cancer Center</td><td>evaluation of safety and pharmacokinetics of M28z1XXPD1DNR cells</td><td>MPM</td><td>Data cutoff date: 2021-12-01<br>posology: LD. Single-dose injection of 1–3 × 10<sup>6</sup> cells/kg ip.<br>safety (n = 4): No grade ≥3 TRAEs reported.<br>efficacy: N/A</td><td>Adusumilli et al.<sup>168</sup></td></tr><tr><td>LCAR-M23 cells</td><td>NCT04562298*</td><td>I</td><td>2022-06-07 (terminated due to sponsors and collaborator decision)</td><td>15</td><td>Shanghai East Hospital</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of LCAR-M23 cells</td><td>EOC</td><td>posology: LD. Single-dose injection<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>PD-1 KO anti-MSLN CAR-TILs secreting anti-PD-1/CTLA-4 scFvs</td><td>NCT04842812</td><td>I</td><td>estimated 2035-01-01</td><td>40</td><td>Second Affiliated Hospital of Guangzhou Medical University</td><td>evaluation of safety, tolerability, and efficacy of PD-1 KO anti-MSLN CAR-TILs secreting anti-PD-1/CTLA-4 scFvs</td><td>Solid</td><td>posology: No LD. 1 to 10 × 10<sup>7</sup> cells/kg for each treatment, 3 or more cycles<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>anti-MSLN CAR-T cells</td><td>NCT05531708*</td><td>I</td><td>2021-04-02 (withdrawn)</td><td>0</td><td>Shanghai Pudong Hospital</td><td>evaluation of safety and efficacy of anti-MSLN CAR-T cells</td><td>Solid</td><td>posology: LD. Single-dose injection i.v.<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>anti-MSLN CAR-T cells (mRNA</td><td>NCT04981691**</td><td>I</td><td>estimated 2022-07-09 (unknown status)</td><td>12</td><td>Ruijin Hospital</td><td>evaluation of safety, pharmacokinetics, and</td><td>Solid</td><td>posology: LD. First week, 1 to 3 × 10<sup>9</sup> cells i.v. divided into 3</td><td>-</td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 3. Continued**</caption><thead><tr><th>Cell type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td>transfected CAR-T cells)</td><td></td><td></td><td></td><td></td><td></td><td>efficacy of anti-MSLN CAR-T cells</td><td></td><td>injections. Second week, 1 to 3 × 10<sup>9</sup> cells i.v. for each injection (3 injections)<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td></td></tr><tr><td></td><td>FH-TCR-TMSLN cells</td><td>NCT04809766</td><td>I</td><td>2025-01-15</td><td>9</td><td>Fred Hutchinson Cancer Center</td><td>evaluation of safety, pharmacokinetics, and efficacy of FH-TCR-TMSLN</td><td>PDAC</td><td>Data cutoff date: 2023-07-01<br><b>posology:</b> LD. 3 injections of 1 × 10<sup>9</sup> or 3.3 × 10<sup>9</sup> cells Q3W<br><b>safety (n = 6):</b> no DLTs occurred. 17% of patients experienced SAEs<br><b>efficacy:</b> N/A</td><td>Chiorean et al.<sup>170</sup></td></tr><tr><td></td><td>RD133 cells</td><td>NCT05166070</td><td>I</td><td>estimated 2037-01-01</td><td>24</td><td>The First Affiliated Hospital with Nanjing Medical University</td><td>evaluation of safety, tolerability, and efficacy of RD133 cells</td><td>solid</td><td>posology: LD. Single-dose injection of 1–6 × 10<sup>6</sup> cells/kg i.v.<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td>–</td></tr><tr><td></td><td>LD013 cells</td><td>NCT05372692</td><td>N/A</td><td>2023-02-01</td><td>3</td><td>Weijia Fang, MD</td><td>evaluation of safety of LD013 cells</td><td>ovarian</td><td>posology: no LD<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td>–</td></tr><tr><td rowspan="4">CCAR-T cells</td><td>TC-510 cells (lentiviral transduced anti-MSLN sdAb (MH1)/CD3ζ TCR-T cells with PD-1;CD28 switch receptor)</td><td>NCT05451849</td><td>I/II</td><td>estimated 2028-10-30</td><td>6</td><td>TCR2 Therapeutics</td><td>evaluation of safety and efficacy of TC-510</td><td>solid</td><td>posology: LD<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td>McCarthy et al.<sup>171</sup></td></tr><tr><td>BZE2209 cells (anti-MSLN CAR-T cells secreting anti-PD-1/CTLA-4 sdAbs)</td><td>NCT06248697</td><td>I</td><td>estimated 2026-12-31</td><td>16</td><td>Shanghai Cell Therapy Group</td><td>evaluation of safety and tolerability of BZE2209 cells</td><td>solid</td><td>posology: no LD. i.v.<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td>–</td></tr><tr><td></td><td>NCT05783089</td><td>I</td><td>estimated 2027-04-01</td><td>87</td><td>Cancer Institute and Hospital, Chinese Academy of Medical Sciences</td><td>evaluation of safety, tolerability, and efficacy of anti-MSLN CAR-T cells</td><td>Solid</td><td>posology: LD. Single-dose injection of 0.1–2 × 10<sup>6</sup> cells/kg i.v. or local injection, w/ or w/o checkpoint inhibitors<br><b>safety:</b> N/A<br><b>efficacy:</b> N/A</td><td>–</td></tr><tr><td></td><td>NCT05848999**</td><td>I</td><td>estimated 2025-07-01 (unknown status)</td><td>87</td><td>Zhejiang University</td><td>evaluation of safety, tolerability, and efficacy of UCLM802</td><td>Solid</td><td>posology: LD. Single-dose injection of 0.1–2 × 10<sup>6</sup> cells/kg i.v.</td><td>–</td></tr></tbody></table>

(Continued on next page)
<table><caption>Table 3. Continued</caption><thead><tr><th>Cell type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="10">CAR-T cells</td><td>NCT05775666</td><td>I</td><td>estimated 2025-01-01</td><td>87</td><td>Peking University</td><td>evaluation of safety, tolerability, and efficacy of UCLM802 cells</td><td>Solid</td><td>posology: LD<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>NCT05779917</td><td>I</td><td>estimated 2036-03-10</td><td>30</td><td>Second Affiliated Hospital of Guangzhou Medical University</td><td>evaluation of safety, tolerability, and efficacy of anti-MSLN CAR-T cells secreting IL-21 and anti-PD-1 scFv</td><td>pancreatic</td><td>posology: no LD<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>NCT05568680</td><td>I</td><td>estimated 2027-12-01</td><td>42</td><td>Verismo Therapeutics</td><td>evaluation of feasibility, safety, tolerability, and efficacy of SynKIR-110 cells</td><td>ovarian, CHO, and mesothelioma</td><td>posology: no LD.<br>Single-dose injection i.v.<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>NCT06701201</td><td>N/A</td><td>estimated 2042-02-01</td><td>60</td><td>Verismo Therapeutics</td><td>evaluation of long-term safety and efficacy of SynKIR-110 cells</td><td>N/A</td><td>posology: N/A<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>NCT06377202*</td><td>N/A</td><td>2023-11-15 (withdrawn and replaced by NCT06701201)</td><td>0</td><td>Verismo Therapeutics</td><td>evaluation of long-term safety of SynKIR-110 cells</td><td>Solid</td><td>posology: no LD.<br>Single-dose injection<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>NCT06249256</td><td>I</td><td>estimated 2026-12-31</td><td>12</td><td>Shanghai Cell Therapy Group</td><td>evaluation of safety, tolerability, and efficacy of BZT2312 cells</td><td>Solid</td><td>posology: LD. Single-dose injection of 5 × 10<sup>5</sup> to 5 × 10<sup>6</sup> cells/kg i.v.<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>NCT05963100</td><td>I/II</td><td>estimated 2026-05-16</td><td>10</td><td>Zhongda Hospital</td><td>evaluation of safety and efficacy of anti-MSLN TCR-like CAR-T cells</td><td>Ovarian</td><td>safety: No LD. 1 × 10<sup>6</sup> to 2 × 10<sup>7</sup> cells/kg<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>NCT06054308*</td><td>N/A</td><td>2024-02-02 (withdrawn)</td><td>0</td><td>Chinese University of Hong Kong</td><td>evaluation of feasibility of endoscopic ultrasound-guided injection of anti-MSLN CAR-T cells</td><td>PDAC</td><td>posology: no LD.<br>Endoscopic ultrasound-guided injection<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr><tr><td>NCT06256055</td><td>I</td><td>estimated 2025-04-01</td><td>24</td><td>UTC Therapeutics</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of UCMYM802 cells</td><td>solid</td><td>posology: 1 × 10<sup>8</sup> to 2 × 10<sup>9</sup> cells i.v. QW (4 injections)<br>safety: N/A<br>efficacy: N/A</td><td>-</td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 3. Continued**</caption><thead><tr><th>Cell type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td>BZE2203 cells (anti-MSLN CAR-T cells secreting anti-PD-1 sdAb)</td><td>NCT06327997</td><td>I</td><td>estimated 2027-12-31</td><td>20</td><td>Shanghai Cell Therapy Group</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of BZE2203 cells</td><td>Solid</td><td>posology: no LD.<br>Single-dose injection of 5 × 10<sup>5</sup> to 5 × 10<sup>6</sup> cells/kg i.v.<br>safety: N/A<br>efficacy: N/A</td><td>—</td></tr><tr><td></td><td>A2B694 cells (logic-gated Tmod™ CAR-T cells with anti-MSLN activating receptor and HLA-A*02 blocking receptor)</td><td>NCT06051695</td><td>I/II</td><td>estimated 2029-06-01</td><td>230</td><td>A2 Biotherapeutics</td><td>evaluation of safety, tolerability, and efficacy of A2B694 cells</td><td>solid</td><td>posology: LD. Single-dose injection i.v.<br>safety: N/A<br>efficacy: N/A</td><td>Punekar et al.<sup>207</sup></td></tr><tr><td></td><td>CHT102 cells (anti-MSLN UCAR-T cells)</td><td>NCT06760364</td><td>I</td><td>estimated 2039-12-24</td><td>21</td><td>Tianjin Medical University Cancer Institute and Hospital</td><td>evaluation of safety, pharmacokinetics, and efficacy of CHT102 cells</td><td>Pancreatic</td><td>posology: No LD.<br>Arterial injection.<br>safety: N/A<br>efficacy: N/A</td><td>—</td></tr><tr><td></td><td></td><td>NCT06717022</td><td>N/A</td><td>estimated 2039-05-01</td><td>24</td><td>Tianjin Medical University Cancer Institute and Hospital</td><td>evaluation of safety and efficacy of CHT102 cells</td><td>N/A</td><td>posology: no LD.<br>2.5 × 10<sup>6</sup> to 3 × 10<sup>7</sup> cells/kg i.v. on days 1, 5, and 9<br>safety: N/A<br>efficacy: N/A</td><td>—</td></tr><tr><td>CAR-T cells</td><td>MSLN-CAR-γδT cells</td><td>NCT06196294</td><td>I</td><td>estimated 2036-12-30</td><td>30</td><td>Second Affiliated Hospital of Guangzhou Medical University</td><td>evaluation of safety, tolerability, and efficacy of MSLN-CAR-γδT cells</td><td>pancreatic, lung, liver, and mesothelioma</td><td>posology: no LD<br>safety: N/A<br>efficacy: N/A</td><td>—</td></tr><tr><td>CAR-PBMCs</td><td>MCY-M11 cells (mRNA transfected CAR-PBMCs)</td><td>NCT03608618*</td><td>I</td><td>2021-08-24 (terminated due to shift of sponsor's focus)</td><td>14</td><td>MaxCyte</td><td>evaluation of feasibility, safety, and tolerability of MCY-M11 cells administered intraperitoneally</td><td>solid</td><td>Data cutoff date: 2020-01-01<br>posology: no LD.<br>Injections of 1 × 10<sup>7</sup> to 5 × 10<sup>8</sup> cells i.p. QW for 3 weeks<br>safety (n = 11): no TRAEs reported. No DLTs reported<br>efficacy at 5 × 10<sup>7</sup> (n = 3): PFS: 4 months. OR: CR 0%, PR 0%, SD 100%, and PD 0%.</td><td>Annunziata et al.<sup>172</sup></td></tr><tr><td rowspan="2">CAR-NK cells</td><td>anti-MSLN CAR-NK cells</td><td>NCT03692637**</td><td>I</td><td>estimated 2021-11-01 (unknown status)</td><td>30</td><td>Allife Medical Science and Technology</td><td>evaluation of safety, tolerability, and efficacy of anti-MSLN CAR-NK cells</td><td>EOC</td><td>posology: no LD.<br>Single-dose injection of 5 × 10<sup>5</sup> to 3 × 10<sup>6</sup> cells/kg i.v.<br>safety: N/A<br>efficacy: N/A</td><td>—</td></tr><tr><td>anti-MSLN CAR-NK cells; anti-MSLN CAR-NK cells</td><td>NCT05410717</td><td>I</td><td>estimated 2036-05-31</td><td>200</td><td>Second Affiliated Hospital of</td><td>evaluation of safety and efficacy of anti-MSLN CAR-NK cells; ovarian, testis, and endometrial</td><td></td><td>posology: no LD<br>safety: N/A<br>efficacy: N/A</td><td>Li et al.<sup>208</sup></td></tr></tbody></table>

(Continued on next page)
<table><caption>Table 3. Continued</caption><thead><tr><th>Cell type</th><th>Drug name</th><th>NCT number</th><th>Phase</th><th>Completion date</th><th>Enrollment</th><th>Sponsor</th><th>Study</th><th>Tumor type</th><th>Study outcomes</th><th>Reference</th></tr></thead><tbody><tr><td></td><td>secreting IL-7/CCL19; anti-MSLN CAR-NK secreting anti- PD-1/ PD-L1/CTLA-4 scFvs</td><td></td><td></td><td></td><td></td><td>Guangzhou Medical University</td><td>anti-MSLN CAR-NK cells secreting IL-7/ CCL19; anti-MSLN CAR-NK secreting anti- PD-1/PD-L1/ CTLA-4 scFvs alone and in combination with cannabidiol or nicotinamide adenine dinucleotide</td><td></td><td>posology: no LD.<br>Single-dose injection of 1 × 10<sup>6</sup> to 5 × 10<sup>8</sup> cells i.v.<br>safety: N/A<br>efficacy: N/A</td><td></td></tr><tr><td></td><td>TAK-103 cells</td><td>NCT05164666</td><td>I</td><td>estimated 2027-10-31</td><td>2</td><td>Takeda</td><td>evaluation of safety, pharmacokinetics, and efficacy of TAK-103 cells</td><td>solid</td><td></td><td></td></tr><tr><td>CAR-macrophages</td><td>SY001 cells(adenoviral transduced anti-MSLN scFv/4-1BB/CD3ζ CAR-macrophages)</td><td>NCT06562647</td><td>N/A</td><td>estimated 2025-04-01</td><td>2</td><td>CellOrigin Biotech</td><td>evaluation of safety, tolerability, pharmacokinetics, and efficacy of SY001 cells in combination with tislelizumab</td><td>ovarian</td><td>posology: no LD.<br>SY001 cells injections i.v. on days 1, 3, and/or 5 + tislelizumab i.v. on day 1<br>safety (n = 2): 100% of patients experienced transient cytokine fluctuations and reductions in lymphocytes or neutrophils post-treatment but no other grade ≥3 AEs or CRS were observed<br>efficacy (n = 2): OR: SD 100% (28-day follow-up period)</td><td>Li et al.<sup>173</sup></td></tr></tbody></table>

Discontinued clinical trials are shown with the symbol *. Clinical trials with an unknown status are shown with the symbol **. AE, adverse event; CHO, cholangiocarcinoma; CR, complete response; CRS, cytokine release syndrome; DLT, dose-limiting toxicity; EOC, epithelial ovarian cancer; i.p., intrapleural; itu., intratumoral; i.v., intravenous; LD, lymphodepletion; MPM, malignant pleural mesothelioma; NE, not evaluated; NSCLC, non-small-cell lung cancer; OR, overall response; OS, overall survival; PD, progressive disease; PDAC, pancreatic ductal adenocarcinoma; PFS, progression-free survival; PR, partial response; Q3W, once every 3 weeks; QW, once weekly; RP2D, recommended phase 2 dose; SAE, serious adverse event; SD, stable disease; TNBC, triple-negative breast cancer; TRAE, treatment-related adverse event.
CONCLUSION

Mesothelin has been established as a compelling target due to its high expression in several aggressive tumors and limited expression in healthy tissues. Over recent years, a broad range of mesothelin-targeted therapies have progressed into clinical trials, with satisfactory safety profiles and encouraging early signs of efficacy, notably in mesothelioma, with consistent objective radiologic responses following administration of anti-mesothelin CAR-T cells.¹⁷⁴

However, despite substantial supporting evidence for MSLN-targeted therapies (Tables 1, 2, and 3), several factors may collectively contribute to their modest clinical outcomes.

The “antigen sink” phenomenon, driven by the presence of soluble mesothelin in both tumor tissue and circulation, is frequently cited as a contributing factor to the heterogeneous outcomes observed across clinical trials. However, its impact remains poorly documented due to the inconsistent measurement, reporting, and consideration of circulating mesothelin levels in clinical studies. To mitigate the decoy effect of soluble mesothelin, strategies targeting membrane proximal epitopes have been developed, including the engineering of bispecific antibodies and CAR-T cells designed to preferentially engage membrane-bound targets despite the presence of soluble antigen.¹⁷⁵

Patient selection in clinical trials should take into account prior exposure to chemotherapy. Several studies have shown that chemotherapy can stimulate tumor cells to activate proteases such as ADAM17, which is implicated in mesothelin shedding.¹⁷⁶,¹⁷⁷ This increase in protease activity may raise sMSLN levels. Therefore, it would be valuable to stratify patients based on their chemotherapy history and measure baseline sMSLN. Such stratification could help interpret therapeutic responses and guide dose-exposure analyses more accurately.

It should be noted that the occurrence of soluble forms of membrane receptors in patient serum is not exclusive to mesothelin. Soluble form of many TAAs have been detected, some of them (EGFR, CEA, and HER2) at concentrations comparable to those of mesothelin.¹⁷⁸⁻¹⁸⁰ Immune checkpoint receptors, including PD-1, PD-L1, and CTLA-4, are also present as circulating molecules.¹⁸¹,¹⁸² These soluble factors can either hinder or enhance antitumor therapy efficacy depending on their concentrations, context, and interactions within the TME.

Although mesothelin targeting is primarily considered in its membrane-associated form, the soluble fraction may also represent a therapeutically relevant target. From a therapeutic standpoint, engaging soluble MSLN could facilitate passive diffusion within the tumor and help mitigate classical limitations such as the binding site barrier, thereby improving tissue penetration.¹⁸³,¹⁸⁴ In addition, certain therapeutic modalities, particularly nuclear medicine approaches such as beta-emitting radiotherapy, do not necessarily require direct membrane engagement to achieve cytotoxic effects, which further sup-

ports the potential interest of targeting non-membrane-associated MSLN.¹⁸⁵

Another important but often insufficiently documented aspect in clinical trial reports is the MUC16 status of enrolled patients. Beyond its role in contributing to local immunosuppression and affecting therapeutic efficacy, MUC16 may modulate the retention of circulating mesothelin in tumors by facilitating mesothelin rebinding to the surface of MUC16-expressing tumor cells, resulting in localized mesothelin enrichment.³⁴,¹⁸⁶ However, both membrane-bound and soluble forms of MUC16/CA125 can interfere with the antibody recognition of mesothelin, particularly for antibodies targeting epitopes overlapping with the MUC16-binding site, thereby impacting patient stratification.

Additional factors may also be considered, including the heterogeneity of MSLN expression, which likely contributes to therapeutic resistance, as well as the association of high MSLN expression with a cold and immunosuppressive TME, characterized by limited infiltration of cytotoxic T cells and upregulation of immune checkpoint molecules such as PD-L1.

In parallel, several therapeutic failures have stemmed from molecule-intrinsic limitations rather than from the mesothelin target itself. Immunotoxins were restricted by immunogenicity and systemic toxicity,¹²⁹,¹³³ while amatuximab, though well tolerated, exhibited reduced effector functions due to MUC16-MSLN and MUC16-Fab interactions.¹⁵⁴,¹⁵⁵ In addition, the stroma of solid tumors may limit drug penetration and therapeutic efficacy. Conversely, early-phase clinical trials of mesothelin-directed CAR-T cells have reported encouraging outcomes though larger studies remain necessary to confirm their long-term safety and effectiveness.

Interestingly, these limitations are largely representative of the obstacles faced by tumor-antigen-targeted therapies in the context of solid tumors.

A better understanding of the biological and clinical implications of mesothelin, combined with the systematic integration of circulating mesothelin measurement and MUC16 status in clinical trials, could enhance the interpretation of results and enable more appropriate patient selection.

Several emerging research directions are being pursued to develop novel mesothelin-targeting therapies addressing current limitations and aiming to enhance efficacy, safety, and durability.

Innovative approaches such as conditional mesothelin binding via dual or multi-targeting and affinity tuning are currently evaluated.¹⁸⁷⁻¹⁹⁰ Moreover, although amatuximab alone is not effective, the “theranostic strategy,” combining ⁸⁹Zr- and ¹⁷⁷Lu-labeled amatuximab, has shown promise with *in vivo*-specific tumor accumulation in PANC-1 MSLN⁺ models and therapeutic efficacy with minimal side effects.¹⁹¹
In summary, mesothelin remains an attractive therapeutic target, offering considerable hope for the treatment of aggressive cancers. Nonetheless, it continues to hold many unresolved questions, making it a particularly stimulating field for future research.

## ACKNOWLEDGMENTS

R.B. was supported by Aix Marseille University. P.C. and B.K. were supported by CNRS. This work was supported by institutional grants from CNRS and Inserm.

## AUTHOR CONTRIBUTIONS

R.B., conceptualization, literature search and data curation, formal analysis, visualization, writing—original draft, and writing—review & editing. P.C., validation. B.K., conceptualization, formal analysis, writing—review & editing, supervision, and validation. All authors reviewed and approved the final version of the manuscript.

## DECLARATION OF INTERESTS

The authors have declared that no competing interest exists.

## REFERENCES

1. Chang, K., Pastan, I., and Willingham, M.C. (1992). Isolation and characterization of a monoclonal antibody, K1, reactive with ovarian cancers and normal mesothelium. Int. J. Cancer 50, 373-381. https://doi.org/10.1002/ijc.2910500308.

2. Chang, K., and Pastan, I. (1996). Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers. Proc. Natl. Acad. Sci. USA 93, 136-140. https://doi.org/10.1073/pnas.93.1.136.

3. Weidemann, S., Gagelmann, P., Gorbokon, N., Lennartz, M., Menz, A., Luebke, A.M., Kluth, M., Hube-Magg, C., Blessin, N.C., Fraune, C., et al. (2021). Mesothelin Expression in Human Tumors: A Tissue Microarray Study on 12,679 Tumors. Biomedicines 9, 397. https://doi.org/10.3390/biomedicines9040397.

4. Bera, T.K., and Pastan, I. (2000). Mesothelin is not required for normal mouse development or reproduction. Mol. Cell Biol. 20, 2902-2906. https://doi.org/10.1128/MCB.20.8.2902-2906.2000.

5. Sathyanarayana, B.K., Hahn, Y., Patankar, M.S., Pastan, I., and Lee, B. (2009). Mesothelin, Stereocilin, and Otoancorin are predicted to have superhelical structures with ARM-type repeats. BMC Struct. Biol. 9, 1. https://doi.org/10.1186/1472-6807-9-1.

6. Zhang, X.-Y., Hong, L.-L., and Ling, Z.-Q. (2025). MUC16/CA125 in cancer: new advances. Clin. Chim. Acta 565, 119981. https://doi.org/10.1016/j.cca.2024.119981.

7. Rupert, P.B., Buerger, M., Friend, D.J., and Strong, R.K. (2024). Structural elucidation of the mesothelin-mucin-16/CA125 interaction. Structure 32, 1049-1054.e2. https://doi.org/10.1016/j.str.2024.04.011.

8. Aithal, A., Rauth, S., Kshirsagar, P., Shah, A., Lakshmanan, I., Junker, W.M., Jain, M., Ponnusamy, M.P., and Batra, S.K. (2018). MUC16 as a novel target for cancer therapy. Expert Opin. Ther. Targets 22, 675-686. https://doi.org/10.1080/14728222.2018.1498845.

9. Rihs, H.-P., Casjens, S., Raiko, I., Kollmeier, J., Lehnert, M., Nöfer, K., May-Taube, K., Kaiser, N., Taeger, D., Behrens, T., et al. (2022). Mesothelin Gene Variants Affect Soluble Mesothelin-Related Protein Levels in the Plasma of Exposed Males and Mesothelioma Patients from Germany. Biology 11, 1826. https://doi.org/10.3390/biology11121826.

10. Muminova, Z.E., Strong, T.V., and Shaw, D.R. (2004). Characterization of human mesothelin transcripts in ovarian and pancreatic cancer. BMC Cancer 4, 19. https://doi.org/10.1186/1471-2407-4-19.

11. Chang, K., and Pastan, I. (1994). Molecular cloning and expression of a cDNA encoding a protein detected by the K1 antibody from an ovarian carcinoma (OVCAR-3) cell line. Int. J. Cancer 57, 90-97. https://doi.org/10.1002/ijc.2910570117.

12. Hassan, R., and Ho, M. (2008). Mesothelin targeted cancer immunotherapy. Eur. J. Cancer 44, 46-53. https://doi.org/10.1016/j.ejca.2007.08.028.

13. Joseph, S., Zhang, X., Smith, L.K., and Alewine, C. (2021). Furin is not required for processing of mesothelin precursor protein. Biochim. Biophys. Acta Mol. Cell Res. 1868, 118967. https://doi.org/10.1016/j.bbamcr.2021.118967.

14. Sharon, E., Zhang, J., Hollevoet, K., Steinberg, S.M., Pastan, I., Onda, M., Gaedcke, J., Ghadimi, B.M., Ried, T., and Hassan, R. (2012). Serum mesothelin and megakaryocyte potentiating factor in pancreatic and biliary cancers. Clin. Chem. Lab. Med. 50, 721-725. https://doi.org/10.1515/CCLM.2011.816.

15. Liu, X., Chan, A., Tai, C.-H., Andresson, T., and Pastan, I. (2020). Multiple proteases are involved in mesothelin shedding by cancer cells. Commun. Biol. 3, 728. https://doi.org/10.1038/s42003-020-01464-5.

16. Zhan, J., Lin, D., Watson, N., Esser, L., Tang, W.K., Zhang, A., Liu, X., Hassan, R., Gleinich, A., Shajahan, A., et al. (2023). Structures of Cancer Antigen Mesothelin and Its Complexes with Therapeutic Antibodies. Cancer Res. Commun. 3, 175-191. https://doi.org/10.1158/2767-9764.CRC-22-0306.

17. Lin, I., Rupert, P.B., Pilat, K., Ruff, R.O., Friend, D.J., Chan, M.K., Clarke, M., Hoffstrom, B.G., Carter, J., Meshinchi, S., et al. (2023). Novel mesothelin antibodies enable crystallography of the intact mesothelin ectodomain and engineering of potent, T cell-engaging bispecific therapeutics. Front. Drug Discov. 3, 1216516. https://doi.org/10.3389/fddsv.2023.1216516.

18. Inaguma, S., Wang, Z., Lasota, J., Onda, M., Czapiewski, P., Langfort, R., Rys, J., Szpor, J., Waloszczyk, P., Okoń, K., et al. (2017). Comprehensive immunohistochemical study of mesothelin (MSLN) using different monoclonal antibodies 5B2 and MN-1 in 1562 tumors with evaluation of its prognostic value in malignant pleural mesothelioma. Oncotarget 8, 26744-26754. https://doi.org/10.18632/oncotarget.15814.

19. Einama, T., Kamachi, H., Nishihara, H., Homma, S., Kanno, H., Ishikawa, M., Kawamata, F., Konishi, Y., Sato, M., Tahara, M., et al. (2015). Importance of luminal membrane mesothelin expression in intraductal papillary mucinous neoplasms. Oncol. Lett. 9, 1583-1589. https://doi.org/10.3892/ol.2015.2969.

20. Lamberts, L.E., de Groot, D.J.A., Bense, R.D., de Vries, E.G.E., and Fehrmann, R.S.N. (2015). Functional genomic mRNA profiling of a large cancer data base demonstrates mesothelin overexpression in a broad range of tumor types. Oncotarget 6, 28164-28172. https://doi.org/10.18632/oncotarget.4461.

21. Kaeding, A.J., Barwe, S.P., Gopalakrishnapallai, A., Ries, R.E., Alonzo, T.A., Gerbing, R.B., Correnti, C., Loken, M.R., Broderson, L.E., Pardo, L., et al. (2021). Mesothelin is a novel cell surface disease marker and potential therapeutic target in acute myeloid leukemia. Blood Adv. 5, 2350-2361. https://doi.org/10.1182/bloodadvances.2021004424.

22. Hucl, T., Brody, J.R., Gallmeier, E., Iacobuzio-Donahue, C.A., Farrance, I.K., and Kern, S.E. (2007). High Cancer-Specific Expression of Mesothelin (MSLN) Is Attributable to an Upstream Enhancer Containing a Transcription Enhancer Factor-Dependent MCAT Motif. Cancer Res. 67, 9055-9065. https://doi.org/10.1158/0008-5472.CAN-07-0474.

23. Sato, N., Maitra, A., Fukushima, N., van Heek, N.T., Matsubayashi, H., Iacobuzio-Donahue, C.A., Rosty, C., and Goggins, M. (2003). Frequent Hypomethylation of Multiple Genes Overexpressed in Pancreatic Ductal Adenocarcinoma. Cancer Res. 63, 4158-4166.

24. Park, G.B., and Kim, D. (2017). TLR5/7-mediated PI3K activation triggers epithelial-mesenchymal transition of ovarian cancer cells through WAVE3-dependent mesothelin or OCT4/SOX2 expression. Oncol. Rep. 38, 3167-3176. https://doi.org/10.3892/or.2017.5941.

25. Kaneko, O., Gong, L., Zhang, J., Hansen, J.K., Hassan, R., Lee, B., and Ho, M. (2009). A Binding Domain on Mesothelin for CA125/MUC16. J. Biol. Chem. 284, 3739-3749. https://doi.org/10.1074/jbc.M806776200.

26. Gubbels, J.A.A., Belisle, J., Onda, M., Rancourt, C., Migneault, M., Ho, M., Bera, T.K., Connor, J., Sathyanarayana, B.K., Lee, B., et al. (2006). Mesothelin-MUC16 binding is a high affinity, N-glycan dependent interaction that facilitates peritoneal metastasis of ovarian tumors. Mol. Cancer 5, 50. https://doi.org/10.1186/1476-4598-5-50.

27. Koyama, Y., Wang, P., Liang, S., Iwaisako, K., Liu, X., Xu, J., Zhang, M., Sun, M., Cong, M., Karin, D., et al. (2017). Mesothelin/mucin 16 signaling in activated portal fibroblasts regulates cholestatic liver fibrosis. J. Clin. Investig. 127, 1254-1270. https://doi.org/10.1172/JCI88845.

28. Nishio, T., Koyama, Y., Liu, X., Rosenthal, S.B., Yamamoto, G., Fuji, H., Baglieri, J., Li, N., Brenner, L.N., Iwaisako, K., et al. (2021). Immunotherapy-based targeting of MSLN + activated portal fibroblasts is a strategy for treatment of cholestatic liver fibrosis.
fibrosis. Proc. Natl. Acad. Sci. USA 118, e2101270118. https://doi.org/10.1073/pnas.2101270118.

29. Fuji, H., Miller, G., Nishio, T., Koyama, Y., Lam, K., Zhang, V., Loomba, R., Brenner, D., and Kisseleva, T. (2021). The role of Mesothelin signaling in Portal Fibroblasts in the pathogenesis of cholestatic liver fibrosis. Front. Mol. Biosci. 8, 790032. https://doi.org/10.3389/fmolb.2021.790032.

30. Lin, Y., and Lubman, D.M. (2024). The role of N-glycosylation in cancer. Acta Pharm. Sin. B 14, 1098-1110. https://doi.org/10.1016/j.apsb.2023.10.014.

31. Thomas, D., Rathinavel, A.K., and Radhakrishnan, P. (2021). Altered glycosylation in cancer: A promising target for biomarkers and therapeutics. Biochim. Biophys. Acta Rev. Cancer 1875, 188464. https://doi.org/10.1016/j.jbcan.2020.188464.

32. Duran, A., Guerrero, P.E., Ortiz, M.R., Pérez Del Campo, D., Castro, E., Garcia-Velasco, A., Fort, E., de Llorens, R., Saldova, R., Llop, E., and Peracaula, R. (2022). Characterization of Mesothelin Glycosylation in Pancreatic Cancer: Decreased Core Fucosylated Glycoforms in Pancreatic Cancer Patients' Sera. Biomedicines 10, 1942. https://doi.org/10.3390/biomedicines10081942.

33. Fujihira, H., Takakura, D., Matsuda, A., Abe, M., Miyazaki, M., Nakagawa, T., Kajino, K., Denda-Nagai, K., Noji, M., Hino, O., and Irimura, T. (2021). Bisecting-GlcNAc on Asn388 is characteristic to ERC/mesothelin expressed on epithelioid mesothelioma cells. J. Biochem. 170, 317-326. https://doi.org/10.1093/jb/mvab044.

34. Zhang, X., Yu, Y., Peer, C.J., Landsman, R., Skorupan, N., Cao, L., and Alewine, C. (2022). Low serum mesothelin in pancreatic cancer patients results from retention of shed mesothelin in the tumor microenvironment. Transl. Oncol. 21, 101440. https://doi.org/10.1016/j.tranon.2022.101440.

35. Hagerty, B.L., and Takabe, K. (2023). Biology of Mesothelin and Clinical Implications: A Review of Existing Literature. World J. Oncol. 14, 340-349. https://doi.org/10.14740/wjon1655.

36. Shimizu, A., Hirono, S., Tani, M., Kawai, M., Okada, K.I., Miyazawa, M., Kitahata, Y., Nakamura, Y., Noda, T., Yokoyama, S., and Yamaue, H. (2012). Coexpression of MUC16 and mesothelin is related to the invasion process in pancreatic ductal adenocarcinoma. Cancer Sci. 103, 739-746. https://doi.org/10.1111/j.1349-7006.2012.02214.x.

37. Chen, S.-H., Hung, W.-C., Wang, P., Paul, C., and Konstantopoulos, K. (2013). Mesothelin binding to CA125/MUC16 promotes pancreatic cancer cell motility and invasion via MMP-7 activation. Sci. Rep. 3, 1870. https://doi.org/10.1038/srep01870.

38. Einama, T., Kamachi, H., Nishihara, H., Homma, S., Kanno, H., Takahashi, K., Sasaki, A., Tahara, M., Okada, K., Muraoka, S., et al. (2011). Co-expression of mesothelin and CA125 correlates with unfavorable patient outcome in pancreatic ductal adenocarcinoma. Pancreas 40, 1276-1282. https://doi.org/10.1097/MPA.0b013e318221bed8.

39. Kakimoto, S., Miyamoto, M., Einama, T., Takihata, Y., Matsuura, H., Iwahashi, H., Ishibashi, H., Sakamoto, T., Hada, T., Suminokura, J., et al. (2021). Significance of mesothelin and CA125 expression in endometrial carcinoma: a retrospective analysis. Diagn. Pathol. 16, 28. https://doi.org/10.1186/s13000-021-01093-4.

40. Kakimoto, S., Miyamoto, M., Einama, T., Matsuura, H., Iwahashi, H., Ishibashi, H., Sakamoto, T., Hada, T., and Takano, M. (2020). Co-Expression of Mesothelin and CA125 Is Associated with the Poor Prognosis of Endometrial Serous Carcinoma and Mixed Carcinomas Including Serous Carcinoma. Pathol. Oncol. Res. 26, 2299-2306. https://doi.org/10.1007/s12253-020-00823-1.

41. Takihata, Y., Einama, T., Kobayashi, K., Suzuki, T., Yonamine, N., Fujinuma, I., Tsunenari, T., Yamagishi, Y., Iwasaki, T., Miyata, Y., et al. (2021). Different role of MSLN and CA125 co-expression as a prognostic predictor between perihilar and distal bile duct carcinoma. Oncol. Lett. 21, 414. https://doi.org/10.3892/ol.2021.12675.

42. Einama, T., Yamagishi, Y., Takihata, Y., Suzuki, T., Yamasaki, T., Hirose, Y., Kobayashi, K., Yonamine, N., Fujinuma, I., Tsunenari, T., et al. (2021). Co-expression of mesothelin and CA125/MUC16 is a prognostic factor for breast cancer, especially in luminal-type breast cancer patients. Biomark. Res. 9, 78. https://doi.org/10.1186/s40364-021-00335-3.

43. Shen, J., Sun, X., and Zhou, J. (2020). Insights Into the Role of Mesothelin as a Diagnostic and Therapeutic Target in Ovarian Carcinoma. Front. Oncol. 10, 1263.

44. Montemagno, C., Cassim, S., Trichanh, D., Savary, C., Pouyssegur, J., Pagès, G., Fagret, D., Broisat, A., and Ghezzi, C. (2019). 99mTc-A1 as a Novel Imaging Agent Targeting Mesothelin-Expressing Pancreatic Ductal Adenocarcinoma. Cancers (Basel) 11, 1531. https://doi.org/10.3390/cancers11101531.

45. Winter, J.M., Tang, L.H., Klimstra, D.S., Brennan, M.F., Brody, J.R., Rocha, F.G., Jia, X., Qin, L.-X., D'Angelica, M.I., DeMatteo, R.P., et al. (2012). A novel survival-based tissue microarray of pancreatic cancer validates MUC1 and mesothelin as biomarkers. PLoS One 7, e40157. https://doi.org/10.1371/journal.pone.0040157.

46. Weidemann, S., Perez, D., Izbicki, J.R., Neipp, M., Mofid, H., Daniels, T., Nahrstedt, U., Jacobsen, F., Bernreuther, C., Simon, R., et al. (2021). Mesothelin is Commonly Expressed in Pancreatic Adenocarcinoma but Unrelated to Cancer Aggressiveness. Cancer Invest. 39, 711-720. https://doi.org/10.1080/07357907.2021.1943747.

47. Einama, T., Kawamata, F., Kamachi, H., Nishihara, H., Homma, S., Matsuzawa, F., Mizukami, T., Konishi, Y., Tahara, M., Kamiyama, T., et al. (2016). Clinical impacts of mesothelin expression in gastrointestinal carcinomas. World J. Gastrointest. Pathophysiol. 7, 218-222. https://doi.org/10.4291/wjgp.v7.i2.218.

48. Nagata, K., Shinto, E., Shiraishi, T., Yamadera, M., Kajiwara, Y., Mochizuki, S., Okamoto, K., Einama, T., Kishi, Y., and Ueno, H. (2021). Mesothelin Expression is Correlated with Chemoresistance in Stage IV Colorectal Cancer. Ann. Surg Oncol. 28, 8579-8586. https://doi.org/10.1245/s10434-021-10507-y.

49. Yoon, D.H., Ibrahim, A., Tatishchev, S., Duldulao, M.P.N., Lee, S.W., and Shin, J. (2021). Prevalence of mesothelin expression in peritoneal disease from colorectal and appendiceal cancers. J. Surg. Oncol. 124, 1091-1097. https://doi.org/10.1002/jso.26610.

50. Malla, M., Deshmukh, S.K., Wu, S., Xiu, J., Farrell, A.P., Lenz, H.-J., Lou, E., Goel, S., Spetzler, D., Parikh, A.R., et al. (2023). Comprehensive characterization of mesothelin expression in colorectal cancer. J. Clin. Oncol. 41, 245. https://doi.org/10.1200/JCO.2023.41.4_suppl.245.

51. Inoue, S., Tsunoda, T., Riku, M., Ito, H., Inoko, A., Murakami, H., Ebi, M., Ogasawara, N., Pastan, I., Kasugai, K., et al. (2020). Diffuse mesothelin expression leads to worse prognosis through enhanced cellular proliferation in colorectal cancer. Oncol. Lett. 19, 1741-1750. https://doi.org/10.3892/ol.2020.11290.

52. Shiraishi, T., Shinto, E., Mochizuki, S., Tsuda, H., Kajiwara, Y., Okamoto, K., Einama, T., Hase, K., and Ueno, H. (2019). Mesothelin expression has prognostic value in stage II/III colorectal cancer. Virchows Arch. 474, 297-307. https://doi.org/10.1007/s00428-018-02514-4.

53. Tozbikian, G., Brogi, E., Kadota, K., Catalano, J., Akram, M., Patil, S., Ho, A.Y., Reis-Filho, J.S., Weigelt, B., Norton, L., et al. (2014). Mesothelin expression in triple negative breast carcinomas correlates significantly with basal-like phenotype, distant metastases and decreased survival. PLoS One 9, e114900. https://doi.org/10.1371/journal.pone.0114900.

54. Suzuki, T., Yamagishi, Y., Einama, T., Koiwai, T., Yamasaki, T., Fukumura-koga, M., Ishibashi, Y., Takihata, Y., Shiraishi, T., Miyata, Y., et al. (2020). Membrane mesothelin expression positivity is associated with poor clinical outcome of luminal-type breast cancer. Oncol. Lett. 20, 193. https://doi.org/10.3892/ol.2020.12055.

55. Li, Y.R., Xian, R.R., Ziober, A., Conejo-Garcia, J., Perales-Puchalt, A., June, C.H., Zhang, P.J., and Tchou, J. (2014). Mesothelin expression is associated with poor outcomes in breast cancer. Breast Cancer Res. Treat. 147, 675-684. https://doi.org/10.1007/s10549-014-3077-5.

56. Wang, L., Niu, Z., Zhang, L., Liu, X., Wang, X., Li, F., and Wang, Y. (2012). Clinicopathological significance of mesothelin expression in invasive breast cancer. J. Int. Med. Res. 40, 909-916. https://doi.org/10.1177/147323001204000309.

57. Bayoglu, I.V., Kucukzeybek, B.B., Kucukzeybek, Y., Varol, U., Yildiz, I., Alacacioglu, A., Akyol, M., Demir, L., Dirican, A., Yildiz, Y., et al. (2015). Prognostic value of mesothelin expression in patients with triple negative and HER2-positive breast cancers. Biomed. Pharmacother. 70, 190-195. https://doi.org/10.1016/j.biopharma.2015.01.019.

58. Parinyanitkul, N., Blumenschein, G.R., Wu, Y., Lei, X., Chavez-Macgregor, M., Smart, M., and Gonzalez-Angulo, A.M. (2013). Mesothelin expression and survival outcomes in triple receptor negative breast cancer. Clin. Breast Cancer 13, 378-384. https://doi.org/10.1016/j.clbc.2013.05.001.
59. Cheng, W.F., Huang, C.Y., Chang, M.C., Hu, Y.H., Chiang, Y.C., Chen, Y.L., Hsieh, C.Y., and Chen, C.A. (2009). High mesothelin correlates with chemoresistance and poor survival in epithelial ovarian carcinoma. Br. J. Cancer **100**, 1144-1153. https://doi.org/10.1038/sj.bjc.6604964.

60. Hanaoka, T., Hasegawa, K., Kato, T., Sato, S., Kurosaki, A., Miyara, A., Nagao, S., Seki, H., Yasuda, M., and Fujiwara, K. (2017). Correlation Between Tumor Mesothelin Expression and Serum Mesothelin in Patients with Epithelial Ovarian Carcinoma: A Potential Noninvasive Biomarker for Mesothelin-targeted Therapy. Mol. Diagn. Ther. **21**, 187-198. https://doi.org/10.1007/s40291-017-0255-2.

61. Li, Y., Tian, W., Zhang, H., Zhang, Z., Zhao, Q., Chang, L., Lei, N., and Zhang, W. (2022). MSLN Correlates With Immune Infiltration and Chemoresistance as a Prognostic Biomarker in Ovarian Cancer. Front. Oncol. **12**, 830570. https://doi.org/10.3389/fonc.2022.830570.

62. Yen, M.J., Hsu, C.-Y., Mao, T.-L., Wu, T.C., Roden, R., Wang, T.-L., and Shih, I.-M. (2006). Diffuse mesothelin expression correlates with prolonged patient survival in ovarian serous carcinoma. Clin. Cancer Res. **12**, 827-831. https://doi.org/10.1158/1078-0432.CCR-05-1397.

63. Yildiz, Y., Kabadayi, G., Yigit, S., Kucukzeybek, Y., Alacacioglu, A., Varol, U., Taskaynatan, H., Salman, T., Oflazoglu, U., Akyol, M., and Tarhan, M.O. (2019). High expression of mesothelin in advanced serous ovarian cancer is associated with poor prognosis. J. BUON. **24**, 1549-1554.

64. Baba, K., Ishigami, S., Arigami, T., Uenosono, Y., Okumura, H., Matsumoto, M., Kurahara, H., Uchikado, Y., Kita, Y., Kijima, Y., et al. (2012). Mesothelin expression correlates with prolonged patient survival in gastric cancer. J. Surg. Oncol. **105**, 195-199. https://doi.org/10.1002/jso.22024.

65. Saha, S., Mukherjee, C., Basak, D., Panja, P., Mondal, P.K., Ghosh, R., Halder, A., Chowdhury, A., Dhali, G.K., Chattopadhyay, B.K., et al. (2023). High expression of mesothelin in plasma and tissue is associated with poor prognosis and promotes invasion and metastasis in gastric cancer. Adv. Cancer Biol. Metastasis **7**, 100098. https://doi.org/10.1016/j.adcanc.2023.100098.

66. Han, S.-H., Joo, M., Kim, H., and Chang, S. (2017). Mesothelin Expression in Gastric Adenocarcinoma and Its Relation to Clinical Outcomes. J. Pathol. Transl. Med. **51**, 122-128. https://doi.org/10.4132/jptm.2016.11.18.

67. Einama, T., Homma, S., Kamachi, H., Kawamata, F., Takahashi, K., Takahashi, N., Taniguchi, M., Kamiyama, T., Furukawa, H., Matsuno, Y., et al. (2012). Luminal membrane expression of mesothelin is a prominent poor prognostic factor for gastric cancer. Br. J. Cancer **107**, 137-142. https://doi.org/10.1038/bjc.2012.235.

68. Feng, F., Zhang, H., Zhang, Y., and Wang, H. (2020). Level of mesothelin expression can indicate the prognosis of malignant pleural mesothelioma. Transl. Cancer Res. **9**, 7479-7485. https://doi.org/10.21037/tcr-19-2027.

69. Chu, G.J., Linton, A., Kao, S., Klebe, S., Adelstein, S., Yeo, D., Rasko, J.E.J., and Cooper, W.A. (2023). High mesothelin expression by immunohistochemistry predicts improved survival in pleural mesothelioma. Histopathology **83**, 202-210. https://doi.org/10.1111/his.14916.

70. Forest, F., Patoir, A., Dal Col, P., Sulaiman, A., Camy, F., Laville, D., Bayle-Bleuez, S., Fournel, P., and Habougit, C. (2018). Nuclear grading, BAP1, mesothelin and PD-L1 expression in malignant pleural mesothelioma: prognostic implications. Pathology **50**, 635-641. https://doi.org/10.1016/j.pathol.2018.05.002.

71. Roe, O.D., Creaney, J., Lundgren, S., Larsson, E., Sandeck, H., Boffetta, P., Nilsen, T.I., Robinson, B., and Kjaerheim, K. (2008). Mesothelin-related predictive and prognostic factors in malignant mesothelioma: a nested case-control study. Lung Cancer **61**, 235-243. https://doi.org/10.1016/j.lungcan.2007.12.025.

72. Schneider, J., Hoffmann, H., Dienemann, H., Herth, F.J.F., Meister, M., and Muley, T. (2008). Diagnostic and prognostic value of soluble mesothelin-related proteins in patients with malignant pleural mesothelioma in comparison with benign asbestosis and lung cancer. J. Thorac. Oncol. **3**, 1317-1324. https://doi.org/10.1097/JTO.0b013e318187491c.

73. Nichetti, F., Marra, A., Corti, F., Guidi, A., Raimondi, A., Prinzi, N., de Braud, F., and Pusceddu, S. (2018). The Role of Mesothelin as a Diagnostic and Therapeutic Target in Pancreatic Ductal Adenocarcinoma: A Comprehensive Review. Target. Oncol. **13**, 333-351. https://doi.org/10.1007/s11523-018-0567-0.

74. Arnold, D.T., De Fonseka, D., Hamilton, F.W., Rahman, N.M., and Maskell, N.A. (2017). Prognostication and monitoring of mesothelioma using biomarkers: a systematic review. Br. J. Cancer **116**, 731-741. https://doi.org/10.1038/bjc.2017.22.

75. Castelletti, L., Yeo, D., van Zandwijk, N., and Rasko, J.E.J. (2021). Anti-Mesothelin CAR T cell therapy for malignant mesothelioma. Biomark. Res. **9**, 11. https://doi.org/10.1186/s40364-021-00264-1.

76. Poteet, E., Liu, D., Liang, Z., Van Buren, G., Chen, C., and Yao, Q. (2019). Mesothelin and TGF-α predict pancreatic cancer cell sensitivity to EGFR inhibitors and effective combination treatment with trametinib. PLoS One **14**, e0213294. https://doi.org/10.1371/journal.pone.0213294.

77. Hussein, N.H., Amin, N.S., and El Tayebi, H.M. (2020). GPI-AP: Unraveling a New Class of Malignancy Mediators and Potential Immunotherapy Targets. Front. Oncol. **10**, 537311. https://doi.org/10.3389/fonc.2020.537311.

78. Chang, M.-C., Chen, C.-A., Hsieh, C.-Y., Lee, C.-N., Su, Y.-N., Hu, Y.-H., and Cheng, W.-F. (2009). Mesothelin inhibits paclitaxel-induced apoptosis through the PI3K pathway. Biochem. J. **424**, 449-458. https://doi.org/10.1042/BJ20082196.

79. Lurie, E., Liu, D., LaPlante, E.L., Thistlethwaite, L.R., Yao, Q., and Milosavljevic, A. (2020). Histoepigenetic analysis of the mesothelin network within pancreatic ductal adenocarcinoma cells reveals regulation of retinoic acid receptor gamma and AKT by mesothelin. Oncogenesis **9**, 62. https://doi.org/10.1038/s41389-020-00245-3.

80. Brunet, A., Bonni, A., Zigmond, M.J., Lin, M.Z., Juo, P., Hu, L.S., Anderson, M.J., Arden, K.C., Blenis, J., and Greenberg, M.E. (1999). Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell **96**, 857-868. https://doi.org/10.1016/s0092-8674(00)80595-4.

81. Huo, Q., Xu, C., Shao, Y., Yu, Q., Huang, L., Liu, Y., and Bao, H. (2021). Free CA125 promotes ovarian cancer cell migration and tumor metastasis by binding Mesothelin to reduce DKK1 expression and activate the SGK3/FOXO3 pathway. Int. J. Biol. Sci. **17**, 574-588. https://doi.org/10.7150/ijbs.52097.

82. Liu, Y., Ao, X., Ding, W., Ponnusamy, M., Wu, W., Hao, X., Yu, W., Wang, Y., Li, P., and Wang, J. (2018). Critical role of FOXO3a in carcinogenesis. Mol. Cancer **17**, 104. https://doi.org/10.1186/s12943-018-0856-3.

83. Montero, J., and Letai, A. (2018). Why do BCL-2 inhibitors work and where should we use them in the clinic? Cell Death Differ. **25**, 56-64. https://doi.org/10.1038/cdd.2017.183.

84. Hu, J., Wang, J., Guo, X., Fan, Q., Li, X., Li, K., Wang, Z., Liang, S., Amin, B., Zhang, N., et al. (2024). MSLN induced EMT, cancer stem cell traits and chemotherapy resistance of pancreatic cancer cells. Heliyon **10**, e29210. https://doi.org/10.1016/j.heliyon.2024.e29210.

85. Yang, X., Huang, M., Zhang, Q., Chen, J., Li, J., Han, Q., Zhang, L., Li, J., Liu, S., Ma, Y., et al. (2021). Metformin Antagonizes Ovarian Cancer Cells Malignancy Through MSLN Mediated IL-6/STAT3 Signaling. Cell Transplant. **30**, 9636897211027819. https://doi.org/10.1177/09636897211027819.

86. Fukuda, A., Wang, S.C., Morris, J.P., Folias, A.E., Liou, A., Kim, G.E., Akira, S., Boucher, K.M., Firpo, M.A., Mulvihill, S.J., and Hebrok, M. (2011). Stat3 and MMP7 contribute to pancreatic ductal adenocarcinoma initiation and progression. Cancer Cell **19**, 441-455. https://doi.org/10.1016/j.ccr.2011.03.002.

87. Bharadwaj, U., Marin-Muller, C., Li, M., Chen, C., and Yao, Q. (2011). Mesothelin overexpression promotes autocrine IL-6/sIL-6R trans-signaling to stimulate pancreatic cancer cell proliferation. Carcinogenesis **32**, 1013-1024. https://doi.org/10.1093/carcin/bgr075.

88. Cruceriu, D., Baldasici, O., Balacescu, O., and Berindan-Neagoe, I. (2020). The dual role of tumor necrosis factor-alpha (TNF-α) in breast cancer: molecular insights and therapeutic approaches. Cell. Oncol. **43**, 1-18. https://doi.org/10.1007/s13402-019-00489-1.

89. Bharadwaj, U., Marin-Muller, C., Li, M., Chen, C., and Yao, Q. (2011). Mesothelin confers pancreatic cancer cell resistance to TNF-α-induced apoptosis through Akt/PI3K/NF-κB activation and IL-6/Mcl-1 overexpression. Mol. Cancer **10**, 106. https://doi.org/10.1186/1476-4598-10-106.

90. Chonov, D.C., Ignatova, M.M.K., Ananiev, J.R., and Gulubova, M.V. (2019). IL-6 Activities in the Tumour Microenvironment. Part I. J. Med. Sci. **7**, 2391-2398. https://doi.org/10.3889/oamjms.2019.589.

91. Bromberg, J., and Wang, T.C. (2009). Inflammation and cancer: IL-6 and STAT3 complete the link. Cancer Cell **15**, 79-80. https://doi.org/10.1016/j.ccr.2009.01.009.
92. Kumari, N., Dwarakanath, B.S., Das, A., and Bhatt, A.N. (2016). Role of interleukin-6 in cancer progression and therapeutic resistance. Tumour Biol. 37, 11553-11572. https://doi.org/10.1007/s13277-016-5098-7.

93. Bharadwaj, U., Li, M., Chen, C., and Yao, Q. (2008). Mesothelin-Induced Pancreatic Cancer Cell Proliferation Involves Alteration of Cyclin E via Activation of signal transducer and activator of transcription protein 3. Mol. Cancer Res. 6, 1755-1765. https://doi.org/10.1158/1541-7786.MCR-08-0095.

94. Du, W.W., Yang, W., Liu, E., Yang, Z., Dhaliwal, P., and Yang, B.B. (2016). Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2. Nucleic Acids Res. 44, 2846-2858. https://doi.org/10.1093/nar/gkw027.

95. Servais, E.L., Colovos, C., Rodriguez, L., Bograd, A.J., Nitadori, J.i., Sima, C., Rusch, V.W., Sadelain, M., and Adusumilli, P.S. (2012). Mesothelin overexpression promotes mesothelioma cell invasion and MMP-9 secretion in an orthotopic mouse model and in epithelioid pleural mesothelioma patients. Clin. Cancer Res. 18, 2478-2489. https://doi.org/10.1158/1078-0432.CCR-11-2614.

96. Wang, Y., Wang, L., Li, D., Wang, H.B., and Chen, Q.F. (2012). Mesothelin promotes invasion and metastasis in breast cancer cells. J. Int. Med. Res. 40, 2109-2116. https://doi.org/10.1177/03006051204000608.

97. Ueda, T., Bruchovsky, N., and Sadar, M.D. (2002). Activation of the androgen receptor N-terminal domain by interleukin-6 via MAPK and STAT3 signal transduction pathways. J. Biol. Chem. 277, 7076-7085. https://doi.org/10.1074/jbc.M108255200.

98. Liao, H.-Y., Da, C.-M., Liao, B., and Zhang, H.-H. (2021). Roles of matrix metalloproteinase-7 (MMP-7) in cancer. Clin. Biochem. 92, 9-18. https://doi.org/10.1016/j.clinbiochem.2021.03.003.

99. Avula, L.R., Rudloff, M., El-Behaedi, S., Arons, D., Albalawy, R., Chen, X., Zhang, X., and Alewine, C. (2020). Mesothelin Enhances Tumor Vascularity in Newly Forming Pancreatic Peritoneal Metastases. Mol. Cancer Res. 18, 229-239. https://doi.org/10.1158/1541-7786.MCR-19-0688.

100. Gopinathan, G., Milagre, C., Pearce, O.M.T., Reynolds, L.E., Hodivala-Dilke, K., Leinster, D.A., Zhong, H., Hollingsworth, R.E., Thompson, R., Whiteford, J.R., and Balkwill, F. (2015). Interleukin-6 Stimulates Defective Angiogenesis. Cancer Res. 75, 3098-3107. https://doi.org/10.1158/0008-5472.CAN-15-1227.

101. Patel, S.A., Nilsson, M.B., Le, X., Cascone, T., Jain, R.K., and Heymach, J.V. (2023). Molecular Mechanisms and Future Implications of VEGF/VEGFR in Cancer Therapy. Clin. Cancer Res. 29, 30-39. https://doi.org/10.1158/1078-0432.CCR-22-1366.

102. He, X., Wang, L., Riedel, H., Wang, K., Yang, Y., Dinu, C.Z., and Rojanasakul, Y. (2017). Mesothelin promotes epithelial-to-mesenchymal transition and tumorigenicity of human lung cancer and mesothelioma cells. Mol. Cancer 16, 63. https://doi.org/10.1186/s12943-017-0633-8.

103. Kang, E., Seo, J., Yoon, H., and Cho, S. (2021). The Post-Translational Regulation of Epithelial-Mesenchymal Transition-Inducing Transcription Factors in Cancer Metastasis. Int. J. Mol. Sci. 22, 3591. https://doi.org/10.3390/ijms22073591.

104. Uehara, N., Matsuoka, Y., and Tsubura, A. (2008). Mesothelin Promotes Anchorage-Independent Growth and Prevents Anoikis via Extracellular Signal-Regulated Kinase Signaling Pathway in Human Breast Cancer Cells. Mol. Cancer Res. 6, 186-193. https://doi.org/10.1158/1541-7786.MCR-07-0254.

105. Xia, S., Duan, W., Xu, M., Li, M., Tang, M., Wei, S., Lin, M., Li, E., Liu, W., and Wang, Q. (2024). Mesothelin promotes brain metastasis of non-small cell lung cancer by activating MET. J. Exp. Clin. Cancer Res. 43, 103. https://doi.org/10.1186/s13046-024-03015-w.

106. Silvestri, R., Colucci, E., Piccardi, M., Landi, S., and Gemignani, F. (2025). Decoding the role of mesothelin in tumor dynamics and targeted treatment innovations. Mol. Biomed. 6, 131. https://doi.org/10.1186/s43556-025-00379-z.

107. Malla, M., Deshmukh, S.K., Wu, S., Samec, T., Olevian, D.C., El Naili, R., El-Rayes, B., Xiu, J., Farrell, A., Lenz, H.-J., et al. (2024). Mesothelin expression correlates with elevated inhibitory immune activity in patients with colorectal cancer. Cancer Gene Ther. 31, 1547-1558. https://doi.org/10.1038/s41417-024-00816-1.

108. Liang, O., White, A.L., Fielder, T., Shin, J.-S., Sagnella, S.M., Schmitz, U., and Yeo, D. (2025). High mesothelin expression is associated with low cytotoxic T cell infiltration in pancreatic cancer. Front. Immunol. 16, 1651687. https://doi.org/10.3389/fimmu.2025.1651687.

109. Luckett, T., Abudula, M., Ireland, L., Glenn, M., Bellomo, G., Stafferton, R., Halloran, C., Ghaneh, P., Jones, R., Schmid, M.C., and Mielgo, A. (2024). Mesothelin Secretion by Pancreatic Cancer Cells Co-opts Macrophages and Promotes Metastasis. Cancer Res. 84, 527-544. https://doi.org/10.1158/0008-5472.CAN-23-1542.

110. Qualiotto, A.N., Baldavira, C.M., Balancin, M., Ab'Saber, A., Takagaki, T., and Capelozzi, V.L. (2023). Mesothelin expression remodeled the immune-matrix tumor microenvironment predicting the risk of death in patients with malignant pleural mesothelioma. Front. Immunol. 14, 1268927. https://doi.org/10.3389/fimmu.2023.1268927.

111. Brockstedt, D.G., Giedlin, M.A., Leong, M.L., Bahjat, K.S., Gao, Y., Luckett, W., Liu, W., Cook, D.N., Portnoy, D.A., and Dubensky, T.W. (2004). Listeria-based cancer vaccines that segregate immunogenicity from toxicity. Proc. Natl. Acad. Sci. USA 101, 13832-13837. https://doi.org/10.1073/pnas.0406035101.

112. Kelly, R.J., Sharon, E., Pastan, I., and Hassan, R. (2012). Mesothelin-targeted agents in clinical trials and in preclinical development. Mol. Cancer Ther. 11, 517-525. https://doi.org/10.1158/1535-7163.MCT-11-0454.

113. Gross, N.E., Zhang, Z., Mitchell, J.T., Charmsaz, S., Hernandez, A.G., Coyne, E.M., Shin, S.M., Vargas Carvajal, D.C., Sidiropoulos, D.N., Cho, Y., et al. (2024). Phosphodiesterase-5 inhibition collaborates with vaccine-based immunotherapy to reprogram myeloid cells in pancreatic ductal adenocarcinoma. JCI Insight 9, e179292. https://doi.org/10.1172/jci.insight.179292.

114. Kelly, R.J., Abrams, T.A., Catenacci, D.V.T., Wainberg, Z.A., Lin, B.S.-L., Enstrom, A., Whiting, C.C., Wason, P., Murphy, A., Brockstedt, D.G., et al. (2018). A phase 2 trial of CRS-207 and pembrolizumab in adults with recurrence of metastatic gastric, gastroesophageal junction (GEJ), or esophageal adenocarcinomas. J. Clin. Oncol. 36, TP500. https://doi.org/10.1200/JCO.2018.36.4_suppl.TP500.

115. Alley, E.W., Tanvetyanon, T., Jahan, T.M., Gandhi, L., Peikert, T., Stevenson, J., Schlienger, K., Liu, W., Nair, N., Honarmand, S., and Kindler, H.L. (2021). A phase II single-arm study of CRS-207 with pembrolizumab (pembro) in previously treated malignant pleural mesothelioma (MPM). J. Clin. Oncol. 37, 29. https://doi.org/10.1200/JCO.2019.37.8_suppl.29.

116. Le, D.T., Brockstedt, D.G., Nir-Paz, R., Hampl, J., Mathur, S., Nemunaitis, J., Sterman, D.H., Hassan, R., Lutz, E., Moyer, B., et al. (2012). A live-attenuated Listeria vaccine (ANZ-100) and a live-attenuated Listeria vaccine expressing mesothelin (CRS-207) for advanced cancers: phase I studies of safety and immune induction. Clin. Cancer Res. 18, 858-868. https://doi.org/10.1158/1078-0432.CCR-11-2121.

117. Hassan, R., Alley, E., Kindler, H., Antonia, S., Jahan, T., Honarmand, S., Nair, N., Whiting, C.C., Enstrom, A., Lemmens, E., et al. (2019). Clinical Response of Live-Attenuated, Listeria monocytogenes Expressing Mesothelin (CRS-207) with Chemotherapy in Patients with Malignant Pleural Mesothelioma. Clin. Cancer Res. 25, 5787-5798. https://doi.org/10.1158/1078-0432.CCR-19-0070.

118. Tsujikawa, T., Crocenzi, T., Durham, J.N., Sugar, E.A., Wu, A.A., Onners, B., Nauroth, J.M., Anders, R.A., Fertig, E.J., Laheru, D.A., et al. (2020). Evaluation of Cyclophosphamide/GVAX Pancreas Followed by Listeria-Mesothelin (CRS-207) with or without Nivolumab in Patients with Pancreatic Cancer. Clin. Cancer Res. 26, 3578-3588. https://doi.org/10.1158/1078-0432.CCR-19-3978.

119. Brahmer, J.R., Johnson, M.L., Cobo, M., Viteri, S., Sarto, J.C., Sukari, A., Awad, M.M., Salgia, R., Papadimitrakopoulou, V.A., Rajan, A., et al. (2021). JNJ-64041757 (JNJ-757), a Live, Attenuated, Double-Deleted Listeria monocytogenes-Based Immunotherapy in Patients With NSCLC: Results From Two Phase 1 Studies. JTO Clin. Res. Rep. 2, 100103. https://doi.org/10.1016/j.jtocrr.2020.100103.

120. Panni, U.Y., Chen, M.Y., Zhang, F., Cullinan, D.R., Li, L., James, C.A., Zhang, X., Rogers, S., Alarcon, A., Baer, J.M., et al. (2023). Induction of cancer neoantigens facilitates development of clinically relevant models for the study of pancreatic cancer immunobiology. Cancer Immunol. Immunother. 72, 2813-2827. https://doi.org/10.1007/s00262-023-03463-x.

121. Chowdhury, P.S., Viner, J.L., Beers, R., and Pastan, I. (1998). Isolation of a high-affinity stable single-chain Fv specific for mesothelin from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with anti-tumor activity. Proc. Natl. Acad. Sci. USA 95, 669-674. https://doi.org/10.1073/pnas.95.2.669.
122. Hassan, R., Lerner, M.R., Benbrook, D., Lightfoot, S.A., Brackett, D.J., Wang, Q.-C., and Pastan, I. (2002). Antitumor activity of SS(dsFv)PE38 and SS1(dsFv)PE38, recombinant antimesothelin immunotoxins against human gynecologic cancers grown in organotypic culture in vitro. *Clin. Cancer Res.* 8, 3520-3526.

123. Haas, A.R., Tanyi, J.L., O'Hara, M.H., Gladney, W.L., Lacey, S.F., Torigian, D.A., Soulen, M.C., Tian, L., McGarvey, M., Nelson, A.M., et al. (2019). Phase I Study of Lentiviral-Transduced Chimeric Antigen Receptor-Modified T Cells Recognizing Mesothelin in Advanced Solid Cancers. Mol. Ther. 27, 1919-1929. https://doi.org/10.1016/j.ymthe.2019.07.015.

124. Hagerty, B.L., Pegna, G.J., Xu, J., Tai, C.-H., and Alewine, C. (2020). Mesothelin-Targeted Recombinant Immunotoxins for Solid Tumors. *Biomolecules* 10, 973. https://doi.org/10.3390/biom10070973.

125. Havaei, S.M., Aucoin, M.G., and Jahanian-Najafabadi, A. (2021). Pseudomonas Exotoxin-Based Immunotoxins: Over Three Decades of Efforts on Targeting Cancer Cells With the Toxin. *Front. Oncol.* 11, 781800. https://doi.org/10.3389/fonc.2021.781800.

126. Bauss, F., Lechmann, M., Krippendorff, B.-F., Staack, R., Herting, F., Festag, M., Imhof-Jung, S., Hesse, F., Pompiati, M., Kollmorgen, G., et al. (2016). Characterization of a re-engineered, mesothelin-targeted Pseudomonas exotoxin fusion protein for lung cancer therapy. Mol. Oncol. 10, 1317-1329. https://doi.org/10.1016/j.molonc.2016.07.003.

127. Hassan, R., Miller, A.C., Sharon, E., Thomas, A., Reynolds, J.C., Ling, A., Kreitman, R.J., Miettinen, M.M., Steinberg, S.M., Fowler, D.H., and Pastan, I. (2013). Major cancer regressions in mesothelioma after treatment with an anti-mesothelin immunotoxin and immune suppression. *Sci. Transl. Med.* 5, 208ra147. https://doi.org/10.1126/scitranslmed.3006941.

128. Hassan, R., Sharon, E., Thomas, A., Zhang, J., Ling, A., Miettinen, M., Kreitman, R.J., Steinberg, S.M., Hollevoet, K., and Pastan, I. (2014). Phase I study of the anti-mesothelin immunotoxin SS1P in combination with pemetrexed and cisplatin for front-line therapy of pleural mesothelioma and correlation of tumor response with serum mesothelin, megakaryocyte potentiating factor, and cancer antigen. Cancer 120, 3311-3319. https://doi.org/10.1002/cncr.28875.

129. Kreitman, R.J., Hassan, R., Fitzgerald, D.J., and Pastan, I. (2009). Phase I trial of continuous infusion anti-mesothelin recombinant immunotoxin SS1P. *Clin. Cancer Res.* 15, 5274-5279. https://doi.org/10.1158/1078-0432.CCR-09-0062.

130. Hassan, R., Bullock, S., Premkumar, A., Kreitman, R.J., Kindler, H., Willingham, M.C., and Pastan, I. (2007). Phase I study of SS1P, a recombinant anti-mesothelin immunotoxin given as a bolus I.V. infusion to patients with mesothelin-expressing mesothelioma, ovarian, and pancreatic cancers. *Clin. Cancer Res.* 13, 5144-5149. https://doi.org/10.1158/1078-0432.CCR-07-0869.

131. Hassan, R., Alewine, C., Mian, I., Spreafico, A., Siu, L.L., Gomez-Roca, C., Delord, J.-P., Italiano, A., Lassen, U., Soria, J.-C., et al. (2020). Phase I study of the immunotoxin LMB-100 in patients with mesothelioma and other solid tumors expressing mesothelin. *Cancer* 126, 4936-4947. https://doi.org/10.1002/cncr.33145.

132. Alewine, C., Ahmad, M., Peer, C.J., Hu, Z.I., Lee, M.-J., Yuno, A., Kindrick, J.D., Thomas, A., Steinberg, S.M., Trepel, J.B., et al. (2020). Phase I/II Study of the Mesothelin-targeted Immunotoxin LMB-100 with Nab-Paclitaxel for Patients with Advanced Pancreatic Adenocarcinoma. *Clin. Cancer Res.* 26, 828-836. https://doi.org/10.1158/1078-0432.CCR-19-2586.

133. Skorupan, N., Peer, C.J., Zhang, X., Choo-Wosoba, H., Ahmad, M.I., Lee, M.-J., Rastogi, S., Sato, N., Yu, Y., Pegna, G.J., et al. (2024). Tofacitinib to prevent anti-drug antibody formation against LMB-100 immunotoxin in patients with advanced mesothelin-expressing cancers. *Front. Oncol.* 14, 1386190. https://doi.org/10.3389/fonc.2024.1386190.

134. Shen, L., Sun, X., Chen, Z., Guo, Y., Shen, Z., Song, Y., Xin, W., Ding, H., Ma, X., Xu, W., et al. (2024). ADCdb: the database of antibody-drug conjugates. Nucleic Acids Res. 52, D1097-D1109. https://doi.org/10.1093/nar/gkad831.

135. Mauricio, D., Harold, J., Tymon-Rosario, J.R., Zeybek, B., and Santin, A.D. (2021). Novel mesothelin antibody-drug conjugates: current evidence and future role in the treatment of ovarian cancer. Expert Opin. Biol. Ther. 21, 1087-1096. https://doi.org/10.1080/14712598.2021.1869210.

136. Hassan, R., Blumenschein, G.R., Moore, K.N., Santin, A.D., Kindler, H.L., Nemunaitis, J.J., Seward, S.M., Thomas, A., Kim, S.K., Rajagopalan, P., et al. (2020). First-in-Human, Multicenter, Phase I Dose-Escalation and Expansion

Study of Anti-Mesothelin Antibody-Drug Conjugate Anetumab Ravtansine in Advanced or Metastatic Solid Tumors. *J. Clin. Oncol.* 38, 1824-1835. https://doi.org/10.1200/JCO.19.02085.

137. Kindler, H.L., Novello, S., Bearz, A., Ceresoli, G.L., Aerts, J.G.J.V., Spicer, J., Taylor, P., Nackaerts, K., Greystoke, A., Jennens, R., et al. (2022). Anetumab ravtansine versus vinorelbine in patients with relapsed, mesothelin-positive malignant pleural mesothelioma (ARCS-M): a randomised, open-label phase 2 trial. Lancet Oncol. 23, 540-552. https://doi.org/10.1016/S1470-2045(22)00061-4.

138. Alqaisi, H.A., Cohn, D.E., Chern, J.-Y., Duska, L.R., Jewell, A., Corr, B.R., Winer, I.S., Girda, E., Crispens, M.A., Dhani, N.C., et al. (2025). Randomized phase II study of bevacizumab with weekly anetumab ravtansine or weekly paclitaxel in platinum-resistant/refractory high grade ovarian cancer (NCI trial). *Clin. Cancer Res.* 31, 993-1001. https://doi.org/10.1158/1078-0432.CCR-24-3128.

139. Mansfield, A.S., Vivien Yin, J., Bradbury, P., Kwiatkowski, D.J., Patel, S., Bazhenova, L.A., Forde, P., Lou, Y., Dizona, P., Villaruz, L.C., et al. (2024). Randomized trial of anetumab ravtansine and pembrolizumab compared to pembrolizumab for mesothelioma. *Lung Cancer* 195, 107928. https://doi.org/10.1016/j.lungcan.2024.107928.

140. Santin, A.D., Vergote, I., González-Martín, A., Moore, K., Oaknin, A., Romero, I., Diab, S., Copeland, L.J., Monk, B.J., Coleman, R.L., et al. (2023). Safety and activity of anti-mesothelin antibody-drug conjugate anetumab ravtansine in combination with pegylated-liposomal doxorubicin in platinum-resistant ovarian cancer: multicenter, phase Ib dose escalation and expansion study. *Int. J. Gynecol. Cancer* 33, 562-570. https://doi.org/10.1136/ijgc-2022-003927.

141. Dennis, M., Scales, S.J., Spencer, S.D., and Zhang, Y. (2012). *Anti-Mesothelin Antibodies and Immunconjugates*. patent 91. WO2012087962A2.

142. Best, R.L., LaPointe, N.E., Azarenko, O., Miller, H., Genualdi, C., Chih, S., Shen, B.-Q., Jordan, M.A., Wilson, L., Feinstein, S.C., and Stagg, N.J. (2021). Microtubule and tubulin binding and regulation of microtubule dynamics by the antibody drug conjugate (ADC) payload, monomethyl auristatin E (MMAE): Mechanistic insights into MMAE ADC peripheral neuropathy. *Toxicol. Appl. Pharmacol.* 421, 115534. https://doi.org/10.1016/j.taap.2021.115534.

143. Weekes, C.D., Lamberts, L.E., Borad, M.J., Voortman, J., McWilliams, R.R., Diamond, J.R., de Vries, E.G.E., Verheul, H.M., Lieu, C.H., Kim, G.P., et al. (2016). Phase I Study of DMOT4039A, an Antibody-Drug Conjugate Targeting Mesothelin, in Patients with Unresectable Pancreatic or Platinum-Resistant Ovarian Cancer. Mol. Cancer Ther. 15, 439-447. https://doi.org/10.1158/1535-7163.MCT-15-0693.

144. Lamberts, L.E., Menke-van der Houven van Oordt, C.W., ter Weele, E.J., Bensch, F., Smeenk, M.M., Voortman, J., Hoekstra, O.S., Williams, S.P., Fine, B.M., Maslyar, D., et al. (2016). ImmunoPET with Anti-Mesothelin Antibody in Patients with Pancreatic and Ovarian Cancer before Anti-Mesothelin Antibody-Drug Conjugate Treatment. *Clin. Cancer Res.* 22, 1642-1652. https://doi.org/10.1158/1078-0432.CCR-15-1272.

145. Rottey, S., Clarke, J., Aung, K., Machiels, J.-P., Markman, B., Heinhuis, K.M., Millward, M., Lolkema, M., Patel, S.P., de Souza, P., et al. (2022). Phase I/II Trial of BMS-986148, an Anti-mesothelin Antibody-drug Conjugate, Alone or in Combination with Nivolumab in Patients with Advanced Solid Tumors. *Clin. Cancer Res.* 28, 95-105. https://doi.org/10.1158/1078-0432.CCR-21-1181.

146. Li, Q., Wang, L., Zhang, J., Zhao, G., Liu, Z., Ma, X., and Jiang, J. (2023). Translation of the efficacy of antibody-drug conjugates from preclinical to clinical using a semi-mechanistic PK/PD model: A case study with RC88. *Clin. Transl. Sci.* 16, 1232-1242. https://doi.org/10.1111/cts.13526.

147. Liu, Y., Li, G., Yang, R., Huang, Y., Luo, S., Dang, Q., Li, Q., Huang, D., Huang, Y., Tang, D., et al. (2024). The efficacy and safety of RC88 in patients with ovarian cancer, non-squamous-non-small-cell lung-carcinoma and cervical cancer: Results from a first-in-human phase 1/2 study. *J. Clin. Oncol.* 42, 5551. https://doi.org/10.1200/JCO.2024.42.16_suppl5551.

148. Cornelison, G.L., Zhang, M., Nie, C., and Zhu, G. (2025). Abstract 324: PF-08052666 (SGN-MesoC2; HBM9033), a first-in-class topoisomerase 1 inhibitor-based ADC targeting MSLN, demonstrates potent antitumor activity in preclinical models of ovarian, lung, and colorectal cancers. *Cancer Res.* 85, 324. https://doi.org/10.1158/1538-7445.AM2025-324.