Claudin6 (CLDN6): A Emerging Target For Solid Tumor

In June 2023, BioNTech announced the first human trial data of BNT211 at the American Society of Clinical Oncology (ASCO). TORL BioTherapeutics also disclosed the first clinical data of its CLDN6 ADC drug TORL-1-23. These early clinical results provide strong support for Claudin-6 (CLDN6) as a potential target in solid tumors, especially ovarian cancers and testicular cancers. CLDN6, as a member of the Claudins family, will be a emerging target for solid tumor therapy after Claudin 18.2 (ClDN18.2).

BNT211 is a combination therapy of CLDN6-targeting CAR-T and mRNA vaccine, including autologous CAR-T targeting CLDN6 and CLDN6-encoded nanoparticle mRNA vaccine CARVac. The mRNA vaccine enhances its efficacy by stimulating further expansion of CAR-T cells in vivo by expressing CLDN6 on the surface of antigen-presenting cells. Combined with the newly published study data, the researchers said that CLDN6 CAR-T infusion alone or in combination with CARVac was safe and showed efficacy in patients with CLDN6-positive cancers, and the results were superior to data from other solid tumor CAR T trials.

1. Introduction to CLDN6

The Claudins (CLDNs) protein family was first cloned and named from chicken liver by Furuse Mikio et al. in 1998, and is an important part of tight junctions (TJs). Tight junctions are mainly composed of occludin, CLDNs, and tight junction adhesion molecules. At present, 27 members of the Claudin family have been discovered, and with the deepening of research, members of the Claudins family will continue to grow, 24 of which are expressed in mammals. A large number of studies have confirmed that the Claudins family is an essential skeleton protein of TJs, mainly expressed in a tissue-specific manner in endothelial cells or epithelial cells.

According to the level of sequence homology of different claudins, these members can be divided into two types, namely classic claudins and non-classical claudins. CLDN6 belongs to the classical claudins and is homologous to CLDN1, CLDN2, CLDN3, CLDN4, CLDN5, CLDN7, CLDN8, CLDN9, CLDN10, CLDN14, CLDN15, CLDN17 and CLDN19. 2001, Turksen K. Identification of CLDN6 from differential display analysis of differentiated embryoid bodies (EBs). However, Turksen K. was not the first to isolate CLDN6. CLDN6 cDNA was first isolated from an ectoderm-specific library by Harrison S.M.'s team in 1995.

Claudins members are widely distributed in different tissues and organs, and their expression has diversified characteristics. In recent years, a large number of research data have revealed that abnormal expression of Claudins is associated with a variety of tumors, and more potential targets of the Claudins family have emerged for clinical diagnosis and treatment of tumors (Figure 1). It plays a role not only in cancer, but also in other diseases.

For example, aberrant expression of CLDN1 can lead to sclerosing cholangitis and ichthyosis. Increased CLDN3 is associated with decreased alveolar barrier function. In the field of cancer immunotherapy, CLDN18.2, an isomer of Claudins family member CLDN18, has become a hot target for competition by pharmaceutical companies. Now, CLDN6 is a potential target of the Claudins family, and the development of tumor-targeted drugs is also in full swing.

Clinical applications of claudin-targeting agents

Figure 1 Clinical applications of claudin-targeting agents in the diagnosis, prognosis and treatment of cancer.
(Source: References [1])

2. Structure And Function of CLDN6

As one of the 27 members of the CLDN family, CLDN6 is an important molecule that makes up the tight junction between cells. The CLDN6 gene, located on chromosome 16p13.3, is composed of 220 amino acids with a molecular weight of 23kDa and contains three exons. Claudins members have similar structures. As shown in Figure 2, CLDN6 has four transmembrane domains, a short cytoplasmic N-terminal, a C-terminal cytoplasmic domain, two extracellular domains (a larger ECL1 and a smaller ECL2), and a short intracellular ring, consistent with other CLDNNS. CLDN6 has a C-terminal PDZ binding site, which enables CLDN6 to interact with some proteins in the cell, and plays an important role in cell junctions and epithelial tissue permeability.

The PDZ binding domain of ECL1 and cytoplasmic C-terminal is a typical domain of CLDN. ECL1 crosses between cells to form ion-selective pores. The position and charge of amino acids in the ECL1 region of different claudins are very different, and the difference of C-terminal sequence is another difference point that distinguishes the members of claudin. The PDZ binding sequence at the C-terminal and some potential phosphorylation sites enable claudin to directly act on its specific structural domain with ZO-1, ZO-2, ZO-3, MUPP1, etc. which contain PDZ structure in the cytoplasm, and ZO-1 and ZO-2 can directly connect with actin filament. Thus TJ protein can form a stable system with the intracellular skeleton structure.

Structure diagram of CLDN6

Figure 2. Structure diagram of CLDN6
(Source: References [2])

As a member of the claudin protein family, CLDN6 also has the functions of tight junction permeability regulation and barrier formation. Regarding the permeability regulation function, CLDN6 is critical for the regulation of chloride ion permeability in neonatal renal proximal tubules and the regulation of ion transport in the lymphatic sac epithelium of the inner ear. On the barrier side, CLDN6 is important for maintaining the lung epithelial barrier. In addition, CLDN6 is mainly involved in the formation of the epidermal permeability barrier (EPB) during preterm birth. Unlike other claudin members, CLDN6 is the only CLDN with potential specificity because it activates cell adhesion signals and regulates the activity of nuclear receptors. CLDN6 is regulated by stimulation and transcription factors, DNA methylation, phosphorylation, and palmitoylation.

3. Research Projects of CLDN6

CLDN6 studies emerging immunotherapies for the treatment of cancer, including monoclonal antibodies, bispecific antibodies (BsAbs), antibody drug conjugates (ADCs) and CAR-T-cells therapy. CLDN6 is applied in monoclonal antibodies for testicular germ cell tumor, BsAbs for ovarian carcinoma, ADCs for hepatocellular carcinoma, and CAR-T-cells therapy for ovarian carcinoma. At present, there are a total of 7 clinical projects, including 1 monoclonal antibodies project, 2 bispecific antibodies projects, 2 ADC drug projects, and 2 CAR-T therapy projects.

Clinical application of CLDN6 immunotherapy

Figure 3. Clinical application of CLDN6 immunotherapy
(Source: References [2])

3.1 BNT211 & BNT142

BioNTech announced several updates to its CLDN portfolio at the 2023 ASCO Annual Meeting, including BNT211 and BNT142. Among them, the most striking is the latest research results of BNT211 for the treatment of solid tumors.

BNT211 is a CLDN6-targeted CAR T cell therapy developed by BioNetech. The therapy is designed to induce robust immune responses against various CLDN6-positive solid tumors, such as ovarian, sarcoma, testicular, endometrial and gastric cancers. BNT211 is currently in a phase 1/2 trial with or without CARVac in patients with CLDN6-positive relapsed/refractory advanced solid tumors. The latest results show that in patients with CLDN6-positive relapsed/refractory advanced solid tumors, CARVac, with or without CLDN6-encoding mRNA vaccine, exhibited positive activity and manageable safety data. As of March 2023, among the 17 evaluable patients who received BNT211 with or without CARVac, the overall objective response rate (ORR) was 41%, and the disease control rate (DCR) reached 65%. One of the patients with testicular cancer who received automated BNT211 treatment achieved surgical complete response (sCR). Five additional patients achieved a partial response (PR) and three patients achieved stable disease (SD). During the automated manufacturing process, BNT211 maintained its efficacy, which supports the scale-up of the production process.

BNT142 is an mRNA vaccine encoding a CD3xCLDN6 bispecific. After intravenous injection, BNT142 is directed to the liver, where the two RNAs are translated, assembled and secreted into the circulation as antibodies. Since mRNA vaccine toxicity tends to accumulate in the liver, the report of hepatotoxicity is of great significance. BNT142 is currently undergoing a Phase I dose escalation clinical trial.

3.2 TORL-1-23

TORL-1-23 is an ADC drug developed by TORL BioTherapeutics by conjugating anti-CLDN6 monoclonal antibody and tubulin inhibitor MMAE via a cleavable linker. TORL BioTherapeutics presented the first clinical data of its Claudin6 ADC drug TORL-1-23 at ASCO 2023. Phase I clinical trials have shown positive efficacy, among 25 refractory cancer patients who received an average of 5 previous treatments, 28% of patients achieved confirmed remission. After establishing the recommended phase II dose (RP2D), TORL plans to conduct an expansion cohort study in CLDN6-positive ovarian cancer and non-small cell lung cancer (NSCLC), as well as other CLDN6-positive cancers.

3.3 DS-9606

DS-9606 is an ADC drug targeting CLDN6 developed by DaiichiSankyo. The results of preclinical research data show that the targeted Claudin6 ADC constructed on this technology platform exhibits significant anti-tumor activity. DS-9606 is currently undergoing a Phase 1 clinical study, which aims to evaluate the safety and tolerability of DS9606a in patients with advanced solid tumors. Secondary objectives of the study will evaluate the pharmacokinetic properties of DS-9606a, investigate the duration of response and progression-free survival of DS-9606a, and assess the immunogenicity of DS-9606a.

3.4 AMG 794

AMG794 is a bispecific antibody with extended half-life developed based on the bispecific T cellengager (BITE) technology platform, which can bind to human and cynomolgus monkey CLDN6 and CD3. AMG794 can reprogram T cells to kill CLDN6-expressing tumor cells and is currently enrolling patients in a Phase I clinical trial.

3.5 TJ-C64B

TJ-C64B is a bispecific antibody that simultaneously targets the tumor antigen claudin6 and the T cell costimulatory molecule 4-1BB for the treatment of Claudin6-positive tumors. TJ-C64B can conditionally activate T cells by binding to CLDN6, thereby providing more precise and efficient activation of the immune system while reducing the risk of systemic toxicity. Currently, TJ-C64B is in the preclinical research stage.

4. CLDN6 In tumors And Other Diseases

CLDN6 and other family proteins play an important role in the formation of tight junctions between epithelial cells and endothelial cells. The CLDN family maintains the balance of the intracellular environment through cell barriers, paracellular transport and signal transduction. Abnormal expression of family proteins such as CLDN6 can impair the function of TJs and reduce the barrier function, leading to increased tissue permeability, and ultimately leading to the occurrence of various diseases including genetics, allergic diseases, infectious diseases of various systems, and even tumors . At present, the research on CLDN6 mainly focuses on tumors, especially ovarian cancer, testicular cancer, breast cancer, cervical cancer and endometrial cancer.

4.1 CLDN6 and tumors

Tumor cells often exhibit loss of intercellular tight junctions, which may also be an important step in the development of tumor metastasis. Altered tight junction protein permeability will result in more nutrients and other factors infiltrated for tumor growth. As the main structure of tight junctions, abnormal expression of Claudins can lead to structural damage and functional impairment of epithelial cells and endothelial cells, and it is abnormally expressed in tumors of various epithelial origins. For example, the expression of Cliaudin2 in prostate cancer and breast cancer, Claudin3 in kidney cancer and bladder cancer, and Claudin4 in kidney cancer and liver cancer are down-regulated. Unlike other CLDNNS, CLDN6 is a tumor-specific protein that is present in a variety of solid tumors, including the ovaries, endometrium, lungs, stomach, and testes, but not in healthy adult tissue. Based on this, more and more studies on the role of CLDN6 in cancer have been conducted in recent years. With the continuous disclosure of research results, CLDN6 has gradually become an ideal target for tumor therapy.

4.2 CLDN6 and other diseases such as polycystic ovary syndrome

Studies have confirmed that CLDN6 is highly expressed in polycystic ovary syndrome (PCOS) and Granular Cells (GC), weakening the expression of CLDN6 can inhibit the proliferation of ovarian GC in PCOS rats and induce GC apoptosis. PCOS is characterized by oligomenorrhea, hyperandrogenism, and polycystic ovarian changes, affecting 5% to 20% of women of reproductive age worldwide. In women with PCOS, GCs proliferate abnormally and are associated with overproduction of androgens in the ovaries. Another study reported that inhibiting the expression of CLDN6 can inhibit the proliferation, migration and invasion of endometriosis cells. Endometriosis is a common clinical gynecological disease, and the incidence rate is relatively high in women of childbearing age. Patients often have symptoms such as excessive menstrual flow and dysmenorrhea. The above studies imply that CLDN6 may serve as a potential target for the treatment of PCOS or endometriosis.

5. Conclusion

There are a variety of drug therapies targeting Claudin6, including monoclonal antibodies, ADCs, bispecific antibodies, and CAR-T cells. With more and more studies on the tight junction protein family, the molecular mechanism and drug research related to tumors of the CLDNs family is becoming a hot focus. Following CLDN18.2, CLDN6 is also a key member of tight junction proteins. Abnormal expression of CLDN6 plays an important role in ovarian cancer and other malignant tumors, especially gynecological tumors. At present, some positive early clinical results have been announced by ASCO in 2023, which is expected to set off a new upsurge in solid tumor research and development in the future, which deserves continuous attention.

As a professional PEG supplier, Biopharma PEG supplies a variety of PEG linkers with various reactive groups in GMP or non-GMP grades to continuously assist your ADC & PROTAC research and development.

References:
[1] Li, Jian. "Targeting claudins in cancer: diagnosis, prognosis and therapy." American journal of cancer research 11.7 (2021): 3406.
[2] Huinan Qu, Qiu Jin and Chengshi Quan“CLDN6: From Traditional Barrier Function to Emerging Roles in Cancers”Int. J. Mol. Sci. 2021, 22(24)
[3] Turksen K., Troy T.C. Claudin-6: A novel tight junction molecule is developmentally regulated in mouse embryonic epithelium. Dev. Dyn. Off. Publ. Am. Assoc. Anat. 2001;222:292–300.
[4]. Timothy A. Yap, et al. A phase I/II dose escalation trial with expansion cohorts to evaluate safety and preliminary efficacy of BNT142 in patients with prospectively confirmed claudin 6-positive solid tumors. 2023 ASCO, Abs TPS2669.
[5]. Gottfried E. Konecny, et al. Initial results of dose finding in a first-in-human phase 1 study of a novel Claudin 6 (CLDN6) targeted antibody drug conjugate (ADC) TORL-1-23 in patients with advanced solid tumors. 2023 ASCO, Abs 3082.
[6] Du H, Yang X, Fan J, et al. Claudin 6: Therapeutic prospects for tumours, and mechanisms of expression and regulation[J]. Molecular Medicine Reports, 2021, 24(3): 1-9.
[7] ASCO 2023 – Claudin 6 Makes A Splash In Ovarian Cancer，https://www.benzinga.com/general/biotech/23/06/32780740/asco-2023-claudin-6-makes-a-splash-in-ovarian-cancer
[8]. John B. A. G. Haanen,et al. CLDN6 CAR-T cell therapy of relapsed/refractory solid tumors ± a CLDN6-encoding mRNA vaccine: Dose escalation data from the BNT211-01 phase 1 trial using an automated product. 2023 ASCO, Abs 2518.