Novel Claudin18.2-Targeted Therapies in Gastrointestinal Tumors

Abstract

Gastrointestinal (GI) tumors are a type of malignant tumor characterized by their increasing incidence and lethality. Thus, there is an urgent need to explore more peculiar treatment targets. Recently, the adhesion protein claudin18.2 (CLDN18.2) has emerged as a promising target for expanding the scope of targeted therapy for GI tumors because it facilitates the proliferation and evasion of tumor cells through mediation of PI3K-AKT and RAF-MAPK signaling pathways and tumor microenvironment. Therapies targeting CLDN18.2 have advanced considerably, prompting research into new combination regimens and modified CLDN18.2 targeted drug using nanoparticles and CAR-T-modified drugs. There is a compelling rationale to prioritize the investigation of the significance of CLDN18.2 in GI tumors. The unique expression of CLDN18.2 renders it as a promising biomarker for developing precision-guided therapeutic interventions for GI tumors.

Introduction

Gastrointestinal (GI) tumors are associated with a high morbidity and mortality rate and account for about 25% of the incidence and 35% of the mortality of all cancers worldwide. , GI tumors consist of a variety of cancers, including but not limited to pancreatic cancer (PC), gastric cancer (GC), colorectal cancer (CRC), and esophageal cancer (ESCA). Conventional clinical treatment encompasses a range of therapeutic modalities, including surgical interventions, chemotherapy, radiotherapy, and targeted therapy. The advent of molecularly targeted therapeutic agents that target human epidermal growth factor receptor 2 (ERBB2 or HER2) and epidermal growth factor receptor (EGFR) has signified a paradigm shift in the realm of developing effective treatments for digestive tract tumors. Although targeted drugs have significantly progressed in treating certain cancers, they are not the first choice in curing digestive tract tumors. Therefore, it is imperative to develop more effective and novel treatment approaches. Claudin18.2 (CLDN18.2) is an essential protein in the tight junctions (TJs) of normal tissues and is highly expressed in digestive tract tumors, particularly in GC and PC. − The expression level of CLDN18.2 is lower in CRC than in other digestive tract tumors. In recent years, Zolbetuximab targeting CLDN18.2 has been approved by the Food and Drug Administration (FDA) for the treatment of patients with gastric/gastroesophageal junction (G/GEJ) adenocarcinoma who have a positive diagnosis for CLDN18.2. Consequently, CLDN18.2 has also gradually garnered attention in the domain of other GI tumors. Other therapies, such as chimeric antigen receptor T (CAR-T), bispecific antibodies (BiTE), and antibody-drug conjugates (ADC), have achieved remarkable progress, especially in the treatment of GCs, ESCAs, and PCs. In addition, these novel therapies are efficacious in a broader range of tumor types, even in patients with low levels of CLDN18.2 expression. Most reviews have focused more on the advancement of CLDN18.2 in GCs and PCs. , This review provides a synopsis of recent advancements in CLDN18.2 inhibitors in GI tumors, and updates in combination therapy and the presence of potential biomarkers.

The CLDN Protein Family

The CLDNs represent a family of integral membrane proteins that comprise TJs, which are major intercellular junctions that control the flow of molecules in the intercellular space among epithelial cells. , The structure of CLDNs is comprised of four transmembrane domains (TM1–4), an intracellular N-terminal and C-terminal region, and two extracellular loops (ECL1 and ECL2). , The ECL1 domain comprises four β-strands and an extracellular helix (ECH), while the ECL2 domain contains a single β-strand and a cell-surface-exposed portion of the transmembrane domain. , The CLDN family comprises 34 genes, including CLDN1–12, 14–20, 22–25, and 34. , Specifically, CLDN1, 2, 4, 6,7, and 18.2 have been identified as therapeutic targets for the treatment methods of GI cancers. CLDN1 is upregulated in head and neck squamous cell carcinoma, breast, colorectal, gastric, ovarian, pancreatic and thyroid cancer. CLDN4 demonstrated a significant increase in solid tumors, such as triple-negative breast cancer, colorectal and GC, non-small cell lung cancer (NSCLC), pancreatic and prostate cancer, and urothelial carcinoma. , CLDN6 is overexpressed in ovarian, endometrial and GC, and NSCLC. , CLDN18 exhibited upregulation of gastric, esophageal and ovarian adenocarcinoma, pancreatic ductal adenocarcinoma (PDAC), and NSCLC. ,

The Identification and Development of Therapies for CLDN18.2

The CLDN18 belongs to a TJs family and was first identified in 1998 by Tsukita S. The CLDN18 is selectively spliced to form two distinct variants (CLDN18.1 and CLDN18.2) with highly homologous amino acid sequences. Subsequent investigations revealed that CLDN18.2 is exclusively expressed in differentiated gastric mucosa epithelial cells in normal tissues and various tumor tissues. , CLDN18.2 has shown itself as a potential therapeutic target for cancers.

A series of phase III clinical trials were conducted to formally assess the efficacy of Zolbetuximab to target CLDN18.2 in advanced GCs, including the FAST, SPOTLIGHT, and GLOW studies. , In 2023, the results of phase III clinical trials demonstrated the potential of Zolbetuximab as a first-line treatment option for G/GEJ adenocarcinoma patients with locally advanced, unresectable, or metastatic HER2- and CLDN18.2⁺. , Furthermore, the VENTANA CLDN18 (43–14A) RxDx assay is the companion diagnostic tool for this purpose. The development of CLDN18.2 targeted therapies is underway to assess their efficacy in numerous clinical trials. The field is also witnessing the emergence of CLDN18.2-targeted therapy in combination with other therapeutic regimens.

The Oncogenic Mechanisms of CLDN18.2 in GI Cancers

CLDN18.2 interferes with the immune system in several ways (Figure ). It has been demonstrated that CLDN18.2 facilitates the adhesion between GC cells and cancer-associated fibroblasts (CAFs), contributing to the clustering of cancer cells and CAFs into emboli and thereby promoting the metastatic progression of GC (Figure ). In an immunohistochemistry (IHC) analysis of tumor tissues, paraneoplastic, and lymph nodes, CLDN18.2 was associated with higher CD4⁺ T cells, CD8⁺ T cell infiltration, and the low-level expression of programmed death-ligand 1 (PD-L1). , Tumor cells expressing CLDN18.2 are often found within a highly immunosuppressive tumor microenvironment. However, another study further elucidates the role of CLDN18 in fostering robust immunological synapses (ISs) between cytotoxic T lymphocytes (CTLs) and CLDN18 positive cancer cells. This is achieved by promoting the adhesion protein-activated leukocyte cell adhesion molecule (ALCAM) to interact with the lipid rafts of the tumor cell membrane, thereby enhancing T-cell activation (Figure ). The absence of CLDN18 contributes cancer cells to evade immune recognition. The clustering of T cell activation is associated with heightened responsiveness to immunotherapy. This finding provides a novel direction for CLDN18.2-targeted therapies combined with immunotherapy.

1.

High expression of CLDN18.2 regulates the malignant behaviors of tumors: 1) CLDN18.2 plays a pivotal role in the adhesion between cancer cells and CAFs and contributes to the clustering of cancer cells. 2) CLDN18 engages in the establishment of robust ISs between CTLs and CLDN18-positive cancer cells by secreting ALCAM. 3) CLDN18.2-ARHGAP promotes the activation of RhoA and FAK, thereby boosting the PI3K-AKT and RAF-MAPK signaling pathways. 4) CLDN18.2 activates the PI3K-AKT signaling pathway, and promotes the aggregation of Treg cells, which is conducive to immunosuppression. 5) The RAF-MAPK signaling pathway regulates the expression of CLDN18.2. 6) MicroRNA N-72 impedes the transcription of CLDN18.2, reduces the ability of intercellular junctions, and favors the angiogenesis of tumors.

Besides, CLDN18.2 promotes tumor cell proliferation and survival by regulating downstream signaling pathways, such as phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) and rapidly accelerated fibrosarcoma (RAF)-mitogen-activated protein kinase (MAPK) signaling pathways (Figure ). The CLDN18-Rho GTPase-activating protein (ARHGAP) fusion is specific type in early onset GC, and is associated with more advanced disease and metastases, and poor response to chemotherapy , (Figure ). Moreover, CLDN18-ARHGAP fusions share a common Rho GTPase-activating protein domain after gene translocation, further activate the focal adhesion kinase (FAK) and the Yes-associated protein (YAP) pathway , (Figure ). Ultimately, the process directly or indirectly promotes the activation of PI3K-AKT and RAF-MAPK signaling pathways. As a novel fusion gene, CLDN18-ARHGAP induces a regulatory T (Treg) cell-enriched microenvironment and achieves immune suppression through activation of PI3K-AKT signaling (Figure ). The study elucidates the role of epidermal growth factor (EGF) and rat sarcoma (RAS) in inducing CLDN18 expression via the activation of MAPK, contributing to the malignant potential of cancer. Angiogenesis plays an essential role in the growth and metastasis of CRC. MicroRNA N-72 is secreted into endothelial cells via exosomes and binds to the 3′ untranslated region of CLDN18’ mRNA, decreasing the expression of CLDN18.2, blocking cell junctions, thereby promoting angiogenesis (Figure ). However, CLDN18.1 impedes insulin-like growth factor-1 receptor (IGF1R) and the phosphorylation of AKT. It reduces the expression of transcriptional coactivators of postsynaptic density protein 95/discs large/zonula occludens-1 (PDZ)-binding motifs and YAP. These multifaceted mechanisms contribute to the suppression of lung cancer.

CLDN18.2 and Targeted Therapies

CLDN18.2 has emerged as a promising target for developing novel therapeutic agents in the area of PCs and GCs, the success observed offers novel avenues for treating other GI cancers. To advance the field of CLDN18.2 targeted therapies, researchers have developed various therapeutic agents, including monoclonal antibodies (Zolbetuximab), bispecific antibodies (BsAbs: IBI389 and ABL111), Antibody-Drug Conjugates (ADCs include microtubule inhibitors (LM-302, SYSA1801, and ATN-022) and Topoisomerase inhibitors (XNW27011 and SHR-A1904)), and CAR-T (CT041) (Figure ).

2.

Categorization of CLDN18.2 targeted therapies.

Monoclonal Antibodies

Monoclonal antibodies specifically recognize and bind to specific antigens on the surface of tumor cells, and directly destroy cellular membranes, block immune checkpoints, and suppress new blood vessel formation.

Zolbetuximab

As a structurally chimeric IgG1 monoclonal antibody, Zolbetuximab specifically binds to CLDN18.2 on the surface of tumor cells, thereby triggering antibody-dependent cell-mediated toxicity (ADCC), complement-dependent cell-mediated toxicity (CDC), apoptosis, and inhibition of cell proliferation. Furthermore, Zolbetuximab potentiated the antimouse programmed cell death-1 (PD-1) antibody, augmenting its capacity to impede tumor growth. In a murine model, the combination of Zolbetuximab and chemotherapy was associated with an increased frequency of tumor-infiltrating CD8⁺ T cells compared to mice treated with a control.

In patients with advanced G/GEJ adenocarcinoma exhibiting high CLDN18.2 expression, the FAST, GLOW, or SPOTLIGHT studies (NCT01630083, NCT03653507 and NCT03504397) demonstrated that adding Zolbetuximab to chemotherapy (EOX/CAPOX) significantly extended both progression-free survival (PFS) and overall survival (OS), respectively ,, (Table S1). A meta-analysis of Zolbetuximab plus chemotherapy incorporating of the above studies, Zolbetuximab plus chemotherapy prolonged the median PFS (HR 0.64; 95% CI 0.49–0.84; p < 0.01; I² = 59%) and OS (HR 0.72; 95% CI 0.62–0.83; p < 0.01; I² = 31%) compared to receiving single chemotherapy. It is worth noting that the adverse effects of Zolbetuximab mainly include nausea, anemia, neutrophil count decreased and vomiting etc., mostly belonging to grade 1–2 (mild to moderate) (Table S1). But the side effects are manageable and interventional.

However, Zolbetuximab has demonstrated clinical efficacy primarily in patients with high CLDN18.2 expression. Consequently, increasing research efforts are focused on developing monoclonal antibodies targeting tumors with low CLDN18.2 levels. In contrast to Zolbetuximab, ZL-1211 targets GC cells across both high and low CLDN18.2 expression levels, broadening its therapeutic scope. In preclinical studies, ZL-1211 suppressed tumor growth through multiple mechanisms, including ADCC, CDC, proinflammatory cytokine secretion, and natural killer (NK) cell activation. In summary, ZL-1211 has the potential to benefit a broader spectrum of patients with CLDN18.2 positive gastric cancer.

Bispecific Antibodies

BsAbs are recombinant molecules containing two distinct binding domains, each recognizing a different antigen or epitope, which have demonstrated clinical activity in a variety of tumor types. BiTE is a specialized form of BsAb. BiTE’s mechanism of action involves its attachment to proteins on the surface of T cells, which are concurrently binding to specific proteins on tumor cells. , This process induces T-cell activation, promotes the production of cytolytic proteins, the release of inflammatory cytokines, and the further proliferation of T-cells, thereby controlling tumor growth. , Although the antitumor activity of BsAbs is more potent, the risk of adverse effects - particularly cytokine release syndrome (CRS) - is significantly higher than that of monoclonal antibodies (Table S1).

IBI389

IBI389, a type of BiTE targeting CLDN18.2 and CD3⁺, is being evaluated in clinic trials (Table S1). In an open, multicenter phase Ia/Ib study, the results showed that in the patients with GC treated with ≥ 10 μg/kg of IBI389, the objective remission rate (ORR) and disease control rate (DCR) were 30.8% and 73.1%, respectively, while in pancreatic cancer patients treated with 600 μg/kg IBI389, the ORR was 30.3% and DCR was 69.6% (Table S1). An early phase trial of IBI389 has been conducted in CLDN18.2 positive solid tumors.

ABL111

As a novel BiTE designed to target CLDN18.2 and 4–1BB, ABL111 demonstrates the capacity to restrict the activation of 4–1BB⁺ T cells within the GC microenvironment. By activating the 4–1BB signaling pathway, ABL111 enhances the proliferation of T cells and the expansion of memory T cells while avoiding the hepatotoxicity caused by widespread 4–1BB expression. ABL111 and CLDN18.2 had a high affinity, including tumor cells that express a low level of CLDN18.2. Targeting CLDN18.2 by BiTE shows great antitumor activity in treating solid tumors, particularly in combination with immune checkpoint inhibitors or other therapeutic modalities. A phase I clinical trial assessed the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity of a ABL111 in patients with CLDN18.2 positive solid tumors (≥1% tumor cells showing ≥ 1+ membrane staining intensity). Among GC patients with CLDN18.2 expression, the ORR reached 16% at doses ≥ 5 mg/kg.

Antibody-Drug Conjugates

ADC is characterized by the high targeting properties of monoclonal antibodies and the potent killing power of small-molecule cytotoxic drugs. Targeting CLDN18.2 by ADC drugs is a promising area of research, as evidenced by the findings presented in Table S1. ADCs comprise target-specific monoclonal antibodies (mAbs), ultrapotent cytotoxic agents, and cleavable/noncleavable linkers, to balance systemic stability and release intracellular drug. The structural architecture of the ADC contributes to a distinct adverse effect profile, characterized by gastrointestinal toxicity and hematologic disorders (Table S1). The management of adverse reactions has emerged as a key factor in successful drug launches.

LM-302

LM-302 is a novel ADC that is directed against CLDN18.2. It consists of a recombinant humanized anti-CLDN18.2 IgG1 mAb conjugated with the cytotoxic payload monomethyl auristatin E (MMAE) through a type of cleavable linker. In a phase I/II study, 36 patients with CLDN18.2 positive (≥50% of tumor cell staining intensity ≥ 2+) refractory advanced G/GEJ cancer treated with LM-302 have an ORR of 30.6%, median PFS of 7.16 months (95% CI, 2.72-NA) and DCR of 75.0% (NCT05161390). Besides, the manageable safety of LM-302 was monitored. In another randomized phase Ib/II clinical trial recruiting 18 patients with CLDN18.2⁺ biliary tract cancer (BTC) that failed chemotherapy, the result displays manageable safety (NCT05994001). Of the six efficacy-evaluable patients, three patients with CLDN18.2 expression in 30%, 40%, and 80% achieved partial response (PR), resulting in the best of response (BOR) of 50% in Phase Ib (Table S1).

SYSA1801

SYSA1801 is a recombinant antihuman CLDN18.2 monoclonal antibody-MMAE conjugate drug, consisting of one fully human mAb against CLDN18.2 conjugated with MMAE derivative (LND002) through a cleavable linker. In a phase I trial, SYSA1801 was administered to 26 patients diagnosed with drug-resistant GCs and PCs (NCT05009966). Among 21 patients evaluable for efficacy, the ORR was 38.1% (95% CI, 18.1–61.6%), and DCR was 57.1% (95% CI, 34.0–78.2%). Besides, among the 17 evaluable patients diagnosed with GC, the ORR was 47.1% (95% CI, 23.0–72.2%), and DCR was 64.7% (95% CI, 38.3–85.8%) (Table S1).

ATN-022

ATN-022 comprises a humanized monoclonal antibody conjugated to vc-MMAE (a protease-cleavable linker-payload), with the antibody exhibiting subnanomolar affinity for CLDN18.2. ATN-022 is observed to be effective in low expression of CLDN18.2. In the dose escalation study, an ORR was 47.1%, and a DCR was 100%, including 9 PR and 11 disease stable (SD) patients among 21 GC patients with at least one tumor assessment (NCT05718895). One CR and one PR were observed in three patients with GC who received 2.4 mg/kg of the treatment with low expression of CLDN18.2 expression (5% of tumor cell staining intensity <2+) (Table S1). This can bring a breakthrough to part of the population.

XNW27011

XNW27011 is an ADC directed against CLDN18.2, delivering a proprietary topoisomerase I inhibitor (Topo1i) as its cytotoxic payload. A multicenter, open-label, dose-escalation phase of phase I/II study recruits G/GEJ cancer, pancreatic, and ovarian tumors with CLDN18.2 positive (≥5% of tumor cell staining intensity ≥ 2+) (CTR20231735). Among 75 G/GEJ cancer patients with evaluable efficacy, the BOR was 46.7%, and the DCR was 88%, with a favorable safety profile with a large therapeutic window (Table S1).

SHR-A1904

SHR-A1904 is a novel an ADC composed of CLDN18.2-targeting monoclonal antibody, a DNA Topo1i payload and a cleavable peptide-based linker. In phase I trials to value SHR-A1904 in advanced solid tumor patients who received systemic therapy, 17.9% of patients had low expression of CLDN18.2 (1+ membrane staining intensity in >1% but <50% of tumor cells, or 2+/3+ intensity in <50% of tumor cells), and 82.1% of patients had moderately high expression of CLDN18.2 (2+/3+ membrane staining intensity in≥ 50% of tumor cells). Of the 74 evaluable moderately high expression patients, 1 patient achieved complete remission and 25 patients achieved PR, an objective remission rate ORR of 35.1% (95% CI, 24.4–47.1). This preliminarily proves the effective response of SHR-A1904 to patients with CLDN18.2 positive expression (Table S1).

Chimeric Antigen Receptor T Cells

CAR-T, a revolutionary treatment involving the genetic engineering of T cells to target and destroy tumor cells, has demonstrated remarkable potential in managing cancers. CAR-T therapy offers another creative approach to the development of CLDN18.2-targeted drugs. In clinical reports, two patients with metastatic PC failing standard therapy achieved tumor control after treatment with CLDN18.2 CAR-T. In a preclinical model of pancreatic tumors, the dual-targeted fibroblast activation protein (FAP) and CLDN18.2 CAR-T inhibitor enhanced the recruitment of myeloid-derived suppressor cells (MDSCs), inhibited T-cell exhaustion and ameliorated the tumor microenvironment (TME). The CT041-CG4006 study (NCT04581473) to assess the efficacy of CT041 (Satri-cel), which enrolled 98 patients with GI cancers for CLDN18.2 positive (≥40% of tumor cells with membrane staining intensity ≥ 2+), including 73 cases of G/GEJ cancer, 10 instances of PCs, 8 cases of bowel cancers (colon and small bowel), 4 cases of biliary system tumors and 3 cases of other tumors, showed an ORR of 38.8%, a DCR of 91.8%, a median PFS of 4.4 months (95% CI, 3.7–6.6, and an OS of 8.8 months (95% CI, 7.1–10.2) (Table S1). Besides, treatment-related adverse events (TRAEs) and CRS are common toxicities of CLDN18.2 CAR-T therapy (Table S1).

Resistance Mechanisms to CLDN18.2 Targeted Therapy

Despite significant advances in CLDN18.2-targeted therapy for GI tumors, drug resistance remains a major challenge. Under selective pressure from therapy or through clonal evolution, tumor cells can modify surface antigen expression to evade immune-mediated destruction, manifested as loss of CLDN18.2 expression or its downregulation. The most widely validated strategies to overcome this type of resistance involve combination regimens, particularly combinations incorporating immunotherapy with CLDN18.2-targeted agents. Furthermore, BsAbs targeting multiple antigens help counteract escape mechanisms.

Immune-suppressive cells in the TME, along with the heterogeneous expression of target molecules, are great challenge for resistance to CAR-T targeting CLDN18.2. , For instance, hypoxia, elevated levels of transforming growth factor-beta (TGF-β), and Tregs are conducive to immune effector functions. CAR-T integrated with interleukin-15 (IL-15) promotes the survival and function of T cell in suppressive TME. Enhancing stromal modulation and immune infiltration within the TME improve penetration and function of CLDN18.2-targeted therapies, such as pegvorhyaluronidase alfa and oncolytic viruses.

Furthermore, compensatory signaling pathways that bypass the CLDN18.2 target also drive resistance to CLDN18.2-targeted therapies. In such circumstances, CLDN18.2-targeted therapies in combination with pathway inhibitors represent promising agents for inhibiting downstream signaling cascades. Developing dual-targeted CAR-T therapies against CLDN18.2 and other antigens represents a promising therapeutic strategy.

The Promising Prospects of CLDN18.2 in Cancers

CLDN18.2-targeted therapy is generally more appropriate for patients with advanced-stage tumors or those experiencing significant adverse effects from standard treatments. However, the heterogeneous expression levels of the target antigen limit the applicability of single-agent CLDN18.2-targeted therapy across all tumor populations. This underscores the need for a more comprehensive therapeutic approach and drug modification (Figure ). Determining the appropriate patient populations and optimal scheduling for individualized precision therapy represents another highly promising approach (Figure ).

3.

Categorization of modification of CLDN18.2 inhibitors and CLDN18.2 as biomarkers. *: the type of drug or assay is in preclinical trial; #: the type of drug or assay is in clinical trial.

Modification of the CLDN18.2 Therapy

CLDN18.2 is generally highly expressed in PCs and GCs, but its significance in tumors with low expression, such as GC, is restricted. Issues with drug safety - such as linker instability and excessive payload toxicity - limit the broader clinical application of ADCs. Failure to meticulously screen high CLDN18.2 expression and to determine the optimal therapeutic window has been shown to contribute to clinical trial collapse. Additionally, extended administration of targeted therapeutics substantially contributes to the emergence of drug resistance in clinical practice. Improving precision therapy with CLDN18.2 - targeted therapy is a future direction, including novel modifications of drugs, combination regimens, and precision of assays.

Nanoparticle-Modified Drugs

Nanoparticles constitute a promising approach to enhance the targeting of tumors exhibiting low expression of CLDN18.2 while concomitantly reducing damage to normal cells through surface modification. This characterization holds significant promise for patients with low expression of CLDN18.2, offering a potential avenue for targeted therapeutic interventions. BNT141 is a nucleoside-modified RNA-based therapeutic encoding a sequence of Zolbetuximab, delivered to the tumor site via lipid nanoparticles (LNPs) to achieve high local expression. Preclinical studies of surface B significantly inhibited the growth of ovarian cancer cells (Table ). Besides, researchers have genetically engineered cell membranes to obtain bispecific A single-chain variable fragments (SCVFs) consisting of anti-CD40 SCVFs and anti-CLDN18.2 SCVFs. These have then been covered in PLGA nanocores. The presence of anti-CLDN18.2 SCVFs has enabled macrophages to specifically recognize CLDN18.2 positive tumor cells to achieve better immune response. In a murine model, the type of SCVFs was observed to have a good tumor suppression effect and promote the killing ability of T cells.

1. Summary of Preclinical CLDN18.2-Targeted Therapies.

Cancer model	Posology	Treatment route	Biological effects in cancers	Side Effects	PMID
Panc4.14-luc orthotopic model (NSG or c57BL/6)	0.1 mg/kg AMG 910 or 5 mg/kg anti-PD-1 antibody or the combination of both,TVI	Tumor volume was measured using an IVIS imaging system at different time point.	Reduced tumor volume, combined anti-PD-1 therapy further prolong survival.	NA	37507075
BGC823 CLDN18.2 mice	7.4 MBq of Zirconium-89 labled-TST001, TVI	PET scans were acquired at each time point (2,24,48, and 72 h postinjection).	Higher SUVmax and T/NT ratio	No weight loss, no acute toxicity, no significant immune response	37181294
PDAC PDX model with various level of TGFβ	1e6 AZD6422, TVI	Tumor volumes and body weights were measured biweekly	Greater antitumor activity	Better safety range	39321207
PDX gastric model	3, 10, and 30 μg BNT141 RNA-LNP and 800 μg IMAB362, TVI	Measure the maximum and minimum tumor diameters with a caliper every 2 to 4 days	Inhibiting tumor growth	No signs of toxicity	37860278
BGC823 CLDN18.2/AGSCLDN18.2 mouse models	5.55 MBq and 11.10 MBq [177Lu]Lu-TST001	Evaluated cell uptake, imaging and biodistribution experiments.	Inhibiting tumor growth	No significant short-term toxicity	38062170
KPC tumor mice model (C57BL/6), Panc0218.2/Panc0218.2-shIDO1 tumor-bearing mice (NCG mice)	50 mg/kg/day IDO1 inhibitor or DMSO, 5 × 106 cells per mouse CAR-T, TVI	Measure the body weight and tumor volume twice a week	Inhibiting tumor growth	NA	40045363

In another study, researchers designed a monoclonal antibody (1D5) and developed an antibody-dependent fluorescence-magnetic nanoparticle for specific detection and magnetic hyperthermia (MHT). The MHT has been shown to overcome an immune-depressive TME by recruiting CD11c⁺ dendritic cells, compensating PD-1 in CD8⁺ T cells, and enhancing CD86⁺ macrophage polarization. Thus, the MHT combined PD-1 inhibitor is a treatment for enhancing the therapeutic effect for CLDN18.2 positive individuals.

CAR-T Modified Drugs

The CAR-T therapy has several serious side effects, including cytokine release syndrome, neurotoxicity, infections, hematogenic and allergic reactions. Researchers have combined the targeting domain against CLDN18.2 with endogenous TCR complexes to develop a T cell antigen coupler (TAC-T). In vitro and in vivo models, the TAC receptor showed potent antitumor activity against CLDN18.2 positive tumors and no evidence of serious side effects associated with CAR-T therapy. These novel modified CLDN18.2 therapies have been demonstrated to target tumor cells with high precision, offering a potentially significant benefit for patients’ low expression of CLDN18.2. Investigators are currently conducting clinical trials of TAC-T, aiming to expand the scope of its practical applications (NCT05862324).

The challenge of CAR-T therapy for solid tumors is the immunosuppressive TME, usually characterized by immune and stromal cells secreting high levels of TGFβ. AZD6422Z, a novel CAR-T, integrates dominant negative TGFβ receptor II (dnTGFβR II) and CAR in T cells. This can resist TGFβ-mediated immunosuppression, thus enhancing its antitumor activity. AZD6422 demonstrated notable antitumor activity and tolerability in various patient-derived tumor xenograft models with multiple CLDN18.2 and TGFβ (Table ).

Biomarker

The low expression of CLDN18.2 in normal tissues and its specific expression in various malignant tumors, including GC and PC, contributes to CLDN18.2 as a promising biomarker.

Positron Emission Tomography (PET)

A growing body of studies has focused on developing novel approaches for detecting CLDN18.2. The utilization of [⁶⁸Ga] Ga-PMD22 as a positron emission tomography (PET) visualizer, with a targeted focus on the CLDN18.2, has been demonstrated in clinical studies. The extant research has corroborated the efficacy of [⁶⁸Ga] Ga-PMD22 in facilitating precise tumor imaging, a process that is characterized by its high specificity. [^{99 m}Tc] Tc-PHG102 is a nanoantibody-based radioactive probe that employs a combination of PHG102 and CLDN18.2 to achieve single-photon emission computed tomography (SPECT) imaging of tumors by utilizing the ^{99 m} Tc’s radioactive signal. In the future, the stability of the targeted probes will be further updated and combined with other traditional assays to achieve better precision diagnosis of cancers with CLDN18.2 expression.

Single Cell Sequencing Technology

CT041 CAR-T treatment performs well in open-label, single-arm phase 1 study, but not all CLDN18.2 positive patients benefit from CT041 CAR-T treatment. A single-cell transcriptome sequencing technology analyzed the cell types and proportions of blood and the therapeutic effect before and after treatment. As a result, the high-level expression of CLDN18 expression in ascitic-fluid epithelial cells (ECs) was a good forecast indicator of CAR-T therapy. In contrast, a high myelocytomatosis viral oncogene homologue (MYC) expression in ascitic ECs and a strong interaction between tumor cells and T cells were unfavorable prognostic factors. The advent of single cell sequencing technology has empowered clinicians to identify the beneficiary population of CT041 accurately.

Combination Therapies

The CLDN18.2 combinations, including monoclonal antibody combined chemotherapy (FG-M108 plus CAPOX), monoclonal antibody combined immunotherapy and chemotherapy (ASKB589 plus CAPOX and PD-1 inhibitor, TST001 plus CAPOX and PD-1 inhibitor), CLDN8.2 CAR-T combined immunotherapy (CAR-T plus IDO1), Bispecific antibody combined immunotherapy (AMG910 plus PD-1 inhibitor), combined radiotherapy ([177Lu] Lu-labeled anti-CLDN18.2 antibody and 131I-HLX58-Der), exert tumor-suppressing effects in a coordinated and overlapping manner, thereby reducing the toxic side effects of single agents (Figure ).

4.

Categorization of combination of CLDN18.2 targeted therapies.

Monoclonal Antibody Combined Chemotherapy

Combination chemotherapies and CLDN18.2 targeted therapies coordinate the tumor-suppressing effects. In previous studies, combined chemotherapy has produced good results in several clinical trials, demonstrating the feasibility of combination treatments. FG-M108 is a monoclonal antibody targeting CLDN18.2 characterized by enhanced ADCC activity. Some studies that evaluated FG-M108 combined CAPOX for advanced G/GEJ adenocarcinoma or PC display satisfactory results , (Table ).

2. Comparison of Single and Combinational Therapies for CLDN18.2 Monoclonal Antibodies .

Drugs	Tumors	Dosages	Phase	ORR	DCR	Subgroup ORR	DOI	NCT number
ASKB589	Advanced solid tumors	≥6 mg/kg; Q3W	I/II	22%	89%		10.1200/JCO.2023.41.4_suppl.397	NCT04632108
ASKB589 plus chemotherapy	Advanced solid tumors	6 mg/kg; Q3W	I/II	34.2%	71.1%		10.1200/JCO.2025.43.16_suppl.4044	NCT04632108
ASKB589+CAPOX/PD-1 inhibitor	Advanced G/GEJ adenocarcinoma	6 mg/kg; Q3W	Ib/II	73.5%	100%	H:77.8%; M:61.5% PD-L1 CPS<1:76.2%;PD-L1 CPS 1–5:70.0%;PD-L1 CPS ≥ 5:72.2%	10.1200/JCO.2025.43.4_suppl.454	NCT05632939
TST001 plus CAPOX and Nivolumab	Advanced G/GEJ adenocarcinoma	3 mg/kg or 6 mg/kg Q3W	I/IIa	55.7%	NA		10.1200/JCO.2025.43.16_suppl.4032	NCT04495296
FG-M108 plus AG	Locally advanced unresectable or metastatic PC	300 mg/m2; Q3W	Ib	32.4%	100%		10.1200/JCO.2025.43.4_suppl.729	NCT04894825
FG-M108 plus CAPOX	Locally advanced unresectable or metastatic G/GEJ adenocarcinoma	300 mg/m2; Q3W	I/IIa	77.8%			10.1200/JCO.2024.42.16_suppl.4049	NCT06177041
FG-M108 plus AG	Locally advanced unresectable or metastatic G/GEJ adenocarcinoma	600 mg/m2; Q3W	I/II	30%	90%		10.1200/JCO.2025.43.4_suppl.729	NCT04894825

^aH/M: high/median expression of CLDN18.2; L: low expression of CLDN18.2;G/GEJ: gastric/gastroesophageal junction; PC: pancreatic cancer; PD-1: programmed cell death protein 1; nivolumab: a PD-1 inhibitor; CAPOX: a chemotherapy including capecitabine and oxaliplatin; AG: a chemotherapy including albumin-bound paclitaxel and gemcitabine; ORR: objective response rate; DCR: Disease Control Rate; CPS: combined positive score; *: dosage of CLDN18.2 inhibitors.

A phase I/IIa trial for FG-M108 and CAPOX recruited the HER2^– and CLDN18.2⁺ population locally advanced unresectable or metastatic G/GEJ adenocarcinoma (NCT06177041). As a result, in a low CLDN18.2 expressions (IHC 1+/2+/3+≥10% and 2+/3+ < 40%), the ORR and DCR were 46.7% and 100.0%, and ORR was 81% and DCR was 97% in medium or high expressions of CLDN18.2 (IHC 2+/3+≥40%) (Table ). The treatment of FG-M108 plus CAPOX prolonged the population of high expression of CLDN18.2 but not low-expressing populations. Thus, it is a big challenge for low-expressing populations.

Monoclonal Antibody Combined Immunotherapy and Chemotherapy

The expression of PD-L1 displays a low level in the populations of moderate/high CLDN18.2 expression. However, targeted CLDN18.2 treatment increased PD-L1 expression on the surface of tumor cells, thereby enhancing the efficacy of immunotherapy. In another retrospective analysis, 98 patients with CLDN18.2 positive demonstrated a 74.2% positivity expression level of PD-L1. Regardless of PD-L1 expression levels, the combination of Zolbetuximab and CAPOX can increase the PFS of CLDN18.2 positive patients from 6.8 months to 8.21 months. These data confirm the feasibility of combining CLDN18.2 targeted therapies with immunotherapy in tumor-bearing patients. Thus, a range of data, from single agents to combination treatment to triple drug combination, demonstrate the feasibility of triple therapy. ASKB589 displays promising antitumor activity and acceptable safety in patients with CLDN18.2 positive G/GEJ adenocarcinoma. In an I/II clinical trial of ASKB589 for advanced tumors, the ORR and DCR were 22% and 89% (NCT04632108). Moreover, the results obtained better ORR combined with ASKB589 and CAPOX. ASKB589 combined with CAPOX and PD-1 inhibitors against G/GEJ adenocarcinoma with moderate or high expression level of CLDN18.2 (≥2+ membrane staining intensity in ≥ 40% of tumor cells) showed PFS rate at 9 months was 58.1% (95% CI, 42.2–71.0) and the OS rate at 12 months was 77.1% (95% CI, 61.2–87.2) (NCT05632939) (Table ).

A TranStar102 is a phase I/IIa study focusing on advanced G/GEJ cancer NCT04495296). Among 45 efficacy-evaluable patients, 27 achieved PR following treatment with TST001 plus CAPOX. TST001 combined with nivolumab (a PD-1 inhibitor) and CAPOX showed significantly better therapeutic efficacy than Zolbetuximab combined with CAPOX. , In another cohort of TranStar102, the group of high expressions of PD-L1 combined positive score (CPS)<5 and CLDN18.2 can also benefit from triple therapy. , There is a synergistic mechanism between CLDN18.2 targeted therapies and checkpoint inhibitors.

CLDN18.2 CAR-T Combined Immunotherapy/Chemotherapy

Indoleamine 2,3-Dioxygenase 1 (IDO1), a pivotal rate-limiting enzyme, facilitates tryptophan metabolism, leading to subsequent accumulation of immunosuppressive tryptophan catabolic metabolites and kynurenine. This process impedes T cell proliferation, regulatory T cell activation, and the suppression of antitumor immune responses. In addition, IDO1 leads to the accumulation of kynurenine. It increases thymocyte selection-associated high mobility group box protein (TOX), which regulates the expression of multiple genes and drives CAR-T cells into a state of exhaustion. In a vivo study, the combined IDO1 inhibitors and CAR-T effectively suppressed the growth of tumors in models of GC and PC (Table ). Therefore, targeted IDO1 is considered a promising option. Further, preconditioning chemotherapy drugs such as fludarabine and cyclophosphamide have been shown to overcome the metabolic inhibition of the TME by inhibiting IDO1. These underscore the potential of targeted IDO1 therapy in enhancing the efficacy of CLDN18.2 CAR-T and the significance of preemptive chemotherapeutic agents in CAR-T.

Bispecific Antibody Combined Immunotherapy with/without Chemotherapy

AMG910 treatment has been found to show antitumor activity in preclinical models, including GC and PDAC, to shift Treg function from suppressing the immune system to enhancing the immune system, resulting in tumor shrink. In GSU and SNU-620 GC xenograft models, AMG910 significantly inhibits tumor growth by binding CD3⁺ T cells. The combination of AMG910 and PD-1 inhibitor displays great therapeutic promise (Table ). The clinical trials of AMG910 are still in phase I, testing the safety and tolerability of the drug, and the clinical results are expected to be optimiztic. Another clinical trial assesses the safety and efficacy, PK, and pharmacodynamics of ABL111 combined with nivolumab (a PD-1 inhibitor) and mFOLFOX standard chemotherapy in CLDN18.2 positive (1% tumor cells with 1+ membrane staining intensity). Notably, the ORR was 80% within the group of CLDN18.2 expression <75%, while this reached 100% in the recommended dose group. The low expression group benefits from the ABL111 combined therapy. It suggests enhanced efficacy of the combination therapy in low CLDN18.2-expressing tumors.

Combined Radiotherapy

Radiotherapy activates the systemic immune system, and radiotherapy combined with CLDN18.2 targeted therapies is a selective treatment. In a murine PC model to evaluate the combination, the results demonstrated that the radiotherapy group and the CAR-T targeted CLDN18.2 inhibited tumor growth. The combination significantly delayed tumor growth in both local and distant untreated lesions.

[177Lu] Lu-Labeled Anticlaudin-18.2 Antibody

Tumor radiation therapy technology is a combination of molecular imaging and radionuclide-targeted therapy (TRT) that both monitor the target at the diseased site and kill tumor cells at the diseased site. Monoclonal antibodies are desirable for targeted tumor therapy and have a high binding affinity for targeted ligands. Combining TRT and monoclonal antibodies is a potential treatment option for solid tumors. Researchers have combined the TST001 with DOTA (a type of chelator) and radiolabeled it with the radionuclide 177 Lu to develop a [177Lu] Lu-labeled anti-CLDN18.2 antibody. In a GC murine model, [177Lu] Lu-labeled anti-CLDN18.2 antibody effectively inhibited tumor growth.

¹³¹I-HLX58-Der

Other researchers struggle to combine radiation therapy with ADC drugs to develop radio-antibody-drug conjugates (RADCs). 131I-HLX58-Der, the first RADCs, is connected by CLDN18.2 specific antibody HLX58 and Deruxtecan (DXd) and was labeled with the internal radiation therapy of 131I. In a mouse model, the half inhibitory concentration (IC50) of 131I-HLX58-Der was 11.28 ng/mL, showing potent cytotoxicity and significantly reducing the tumor size. Moreover, the biodistribution of 131I-HLX58-Der by microSPECT/CT imaging revealed that 131I-HLX58-De was specifically distributed in CLDN18.2 positive tumors. However, proving its value will require more advanced modeling, clinical trials, etc.

In summary, the high-expression population of CLDN18.2 demonstrated a substantial response to combination therapy. However, further trials need to validate the efficacy of combination therapy in the low-expression population. CLDN18.2, in combination with immunotherapy and chemotherapy, contributes to expanding the population, regardless of PD-L1 expression. The accumulation of clinical trials leads CLDN18.2 to be an essential target in GI tumors and even change the therapeutic landscape of some cancer types.

Future Prospects

The efficacy of targeted therapy has not demonstrated more significant potential in GI tumors. Consequently, elevated morbidity and mortality rates have precipitated the development of additional therapeutic modalities for GI tumors. Monoclonal antibody inhibitors targeting CLDN18.2 have demonstrated encouraging results in treating G/GEJ cancer and PC, and the approval of more targeted drugs by the FDA further validates the efficacy of this approach. The subsequent development of various medications targeting CLDN18.2, such as BiTE, ADC, and CAR-T, gives a new opportunity for CLDN18.2 positive patients, especially those with high expression.

The success of these drugs in G/GEJ cancer and PC underscores the potential for targeted therapeutic interventions to address other GI tumors. Consequently, it is imperative to investigate the expression pattern and prognostic value of CLDN18.2 in other GI tumors to identify potential benefits of CLDN18.2. More researchers expand the value of CLDN18.2 targeted therapies, particularly in tumor populations where CLDN18.2 is expressed low. The development of supplementary clinical research protocols, such as a combination of monoclonal/bispecific antibody and immunotherapy and/or chemotherapy, is necessary to broaden the benefits to a more substantial number of GI tumor patients. Exploiting more assays to detect CLDN18.2 contributes to identifying the population that will benefit from CLDN18.2 targeted therapies.

Conclusions

CLDN18.2 targeted therapies have demonstrated noteworthy efficacy in clinical trials, thus prompting heightened scrutiny regarding the target of CLDN18.2. CLDN18.2 exerts its regulatory influence over various downstream pathways and modulates the tumor microenvironment, thereby contributing to immune evasion and tumor proliferation. Nevertheless, the single CLDN18.2 targeted therapy frequently encounters limitations, such as therapeutic effects influenced by the expression level. Recent clinical trials are increasingly focusing on the combination of CLDN18.2 and modification of the CLDN18.2 targeted therapies. This emerging oncology trend can potentially transform the realm of tumor therapy. Improving the sensitivity of the CLDN18.2 assay also enables further screening of the beneficiary population and optimizing therapeutic strategies.

All data generated or analyzed during this study are included in this published article.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c06506.

• Summary of CLDN18.2 inhibitors (PDF)

W.B.O. designed the study, W.Y., M.X., and Y.G. performed the experiments and acquired the data. W.Y. and W.B.O. analyzed and interpreted the acquired data. W.Y. and W.B.O. participated in the scientific discussion and drafting of the manuscript. All authors reviewed the manuscript.

The authors declare no competing financial interest.