Overcoming claudin family homology: discovery of ARC101, a highly potent CLDN6-specific T-cell engager with a novel CD3 binder for ovarian adenocarcinoma

Background Claudin 6 (CLDN6) is a tight junction membrane protein whose expression in normal tissue is confined to embryonic cells, but is aberrantly expressed in various human cancers, such as ovarian cancer (OC) and testicular cancer (TC). A monoclonal antibody against CLDN6, IMAB027, has shown promising antitumor activity in preclinical human CLDN6-positive (CLDN6+) cancer models. In this series of nonclinical studies, we investigated CLDN6 expression in normal and cancer tissues, as well as the localization and possible function of CLDN6 in cancer cells. Methods Expression of CLDN6 was assessed in a wide range of human tissues (eg, lung, colon, skin, ovary) and cultured cells by quantitative RT-PCR, immunohistochemistry (IHC), flow cytometry, and western blotting. To investigate the effect of dedifferentiation on CLDN6 expression, human-induced pluripotent cells were generated by transfecting foreskin fibroblasts with a reprogramming cocktail, and then CLDN6 expression was evaluated. To characterize CLDN6 as a potential novel marker to identify cancer stem cells (CSCs) in OC, coexpression of CLDN6 with known CSC surface markers were analyzed by flow cytometry, and CLDN6+ and CLDN6-negative cells were tested in colony formation and sphere formation assay. Human OC cell lines were transplanted intraperitoneally into nude mice and assessed for metastasis to investigate tumorigenecity of CLDN6+ cells. Results Except for low mRNA levels measured in placenta, testis, umbilical cord, cerebellum, and lung samples, no CLDN6 (mRNA or protein) was detected in the vast majority of normal tissues. Additionally, there was also a lack of CLDN6 protein expression in tissue zones where stem cells for tissue homeostasis would normally be found as determined by IHC with an anti-CLDN6 antibody. CLDN6 was expressed on the cell surface of several solid tumors, including ovarian, testicular, uterine, and lung cancer tissues; OC and TC samples had high level expression. CLDN6 expression was strongly activated in human-induced pluripotent stem cells generated from fibroblasts. CLDN6 showed selective coexpression with known CSC markers such as CD44, CD24, and CD90 in OC and TC cell lines. In addition, some CLDN6+ OC cells exhibited CSC-like behavior in vitro: CLDN6+ populations were clonogenic and formed well-defined spheres in low attachment conditions; these spheres had the ability to self-renew into secondary spheres. Analysis of OC metastases in mouse xenografts showed when xenografts were generated by OC cells that had <10% of CLDN6+ cells, the metastases were enriched in CLDN6+ cells, suggesting CLDN6+ cells had selective growth advantage. Conclusions CLDN6 is a cancer cell-specific surface molecule aberrantly expressed in several cancers, and its expression may be an identifier for cells with CSC-like traits. These characteristics make CLDN6 an attractive target candidate for tumor-specific therapeutic antibodies. Citation Format: Özlem Türeci, Meike Wagner, Claudia Paret, Maria M. Kreuzberg, Stefan Wöll, Korden Walter, Sabine C. Häcker, Ikumi Nakajo, Tomohiro Yamada, Ugur Sahin. Claudin 6 is a carcinoembryonic antigen with cancer stem cell marker features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1907.

Claudin 6 (CLDN6) is a tight junction molecule that is involved in cell to cell adhesion of epithelial and endothelial cell sheets. CLDN6 is considered an oncofetal protein which is not expressed in normal human tissue but is expressed in some cancers such as endometrial, ovarian and testis cancer. Expression of CLDN6 in endometrial and urothelial cancer leads to a poor prognosis. The problem of developing antibodies against CLDN6 is that the family member claudin 9 (CLDN9) is highly homologous, only varying by 2 amino acids in the extracellular domain. To address this need, Integral Molecular has developed the MPS Discovery Engine® to enable the isolation, characterization, and engineering of monoclonal antibodies for tight junction proteins, GPCRs, ion channels, and transporters. MPS utilizes a collection of technologies to address each of the barriers to monoclonal antibody development against the native extracellular epitopes of multispan membrane proteins. These include, antigen engineering to attain high levels of surface expression, DNA and Lipoparticle immunization to present native epitopes to the immune system, diverse immunization host species to deal with highly conserved proteins, Lipoparticles (high concentration native membrane proteins) to enable phage display, microfludic B-cell isolation to isolate rare MAbs, and shotgun mutagenesis (comprehensive alanine scanning) for epitope mapping. Using the MPS Discovery Engine® we were able to successfully screen a large panel of clones for claudin 6 specificity. From these clones there were 72 potential antibodies that reacted with either claudin 6 or 6/9. A subset of these antibodies reacted only to claudin 6 and not to claudin 9 which has led to our lead drug candidate. With our MPS Discovery Engine® platform, we have the ability to target intact, conformationally specific, and functional antibodies to multipass membrane proteins. Citation Format: Lewis J. Stafford, Brad Screnci, Chidananda Sulli, Erin Rosenberg, Nicholas Molino, David Tucker, Jonathan Sullivan, Trevor Barnes, Jennifer Pfaff, Tanmayee Hazarika, Thomas Charpentier, Samantha Gilman, Rebecca Rimkunas, Rona Wilf, Sharon Willis, Benjamin Doranz, Joseph Rucker, Ross Chambers. Discovery of a novel claudin 6 (CLDN6) specific monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5759.

Claudin 6 (CLDN6), a member of the claudin family of tight junction proteins, is highly expressed in multiple solid tumors such as ovarian and endometrial cancers with limited expression in normal tissues. Here we developed an anti-CLDN6 ADC C6P comprising a Fc-silenced anti-CLDN6 monoclonal antibody, a cleavable linker and a modified PBD (compound A). C6P selectively bound to CLDN6 without cross-reactivity to other claudin family members such as CLDN 3/4/5/8/9. Additionally, it also bound to I143V-mutated CLDN6, a major single nucleotide polymorphism (SNP) in human population. The antibody demonstrated strong binding affinity to several ovarian cancer cell lines with different CLDN6 expression levels and showed good endocytosis. Compound A was an optimized mono-imine PBD dimer with less cytotoxicity than conventional bis-imine PBD dimers. High systemic clearance of this payload was also designed to minimize toxicities. To achieve homogeneous ADC, a glycan mediated site-specific conjugation was used to generate C6P. C6P showed CLDN6-dependent growth inhibition against various cell lines, without effect on CLDN6 negative cells. In PA-1 and OVCAR3 xenograft mouse models, C6P showed a dose-dependent anti-tumor effect and led to robust and sustained tumor regression. C6P demonstrated good stability and safety profiles, likely due to site-specific conjugation and the higher systemic clearance of compound A. Less than 1% payload release was observed in human plasma after a 21-days incubation. C6P was well-tolerated at a single dose of 0.5 mg/kg in cynomolgus monkeys and did not show hematology-related toxicity. The intact ADC/total antibody AUC ratio was above 95%, and the payload was undetectable in the plasma of cynomolgus monkeys after injection with C6P. Overall, the highly specific CLDN6-targeting antibody, the optimized PBD payload, site-specific conjugation and well-controlled DAR value collectively contributed to the potent anti-tumor efficacy, and good safety profile of C6P, supporting its future clinical research. Citation Format: Xiaolan Yu, Shuting Wang, Lingfeng You, Zhibin Xu, Zhendong Xue, Yuchang Mao, Shuyu Cai, Jun Feng, Ting Zhang, Xin Ye, Min Hu, Feng He. C6P, a claudin 6 (CLDN6) directed ADC containing pyrolobenzodiazepine (PBD) for the treatment of advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5460.

Background: Claudin 6 (CLDN6) is a clinically validated target for many solid tumors notably ovarian, endometrial, and testicular cancer. Its expression is restricted to tumor cells and fetal tissues with little or no detectable in normal tissues. As such, CLDN6 represents an attractive therapeutic target for the development of an antibody drug conjugate (ADC). Here, we describe the development and preclinical characterization of a novel ADC called PLB-002, which consists of a highly selective CLDN6 targeting antibody, PLB-002-ab7, conjugated to a FDA-approved microtubule inhibitor, eribulin, via a Primelink Biotherapeutics proprietary enzyme-cleavable linker with an optimized average drug-to-antibody ratio (DAR) of 4.0. PLB-002-ab7 is a novel humanized anti-CLDN6 single-domain antibody (Mw. 78.8 kDa) with highly binding affinity and selectivity to CLDN6 with barely binding to related CLDN family members (CLDN9, CLDN3, and CLDN4) in protein and cell based binding assays. Methods: The in vitro binding affinity of the PLB-002 was determined by flow cytometry on OVCAR3, PA-1, OVCA429, OV90, and NEC-8 cells. Cell internalization effect was evaluated by an in-direct flow cytometry assay on OVCAR3, PA-1, and OV90 cells. In vitro cytotoxicity was measured using Cell-Titer Glo assay on OVCAR3, OV90, and NEC-8 cells. In vivo anti-tumor efficacy was investigated using several cancer cell derived xenograft (CDX) models of OVCAR3, OV90, and NEC-8 cells, as well as patient derived xenograft (PDX) models of ovarian cancer with CLDN6 high, moderate, low and negative expression. A dose-range finding (DRF) safety study of PLB-002 was performed in cynomolgus monkeys. Pharmacokinetics (PK) and pharmacodynamics (PD) data of PLB-002 in CDX mice were determined by LC-MS/MS method. Results: PLB-002 exhibited strong binding, rapid internalization effect, and potent antitumor activity in vitro on OV90 (CLDN6 low expression) and OVCAR3 (CLDN6 high expression) cells with nano-molar range of IC50. When evaluated in OVCAR3 and OV90 CDXs of ovarian cancer, NEC-8 CDX of testicular germ cell tumor, and OV PDXs, PLB-002 showed strong tumor regression in a dose-dependent manner, which is consistent with the PK analyses showing high exposure, slow clearance and long half-life for both total antibody and conjugated drug. In a non-human primate study using cynomolgus monkey, PLB-002 showed favorable safety profile with no clinical symptoms of toxicities up to the highest tested dose. Conclusions: These results show that PLB-002 has remarkable antitumor activity and support the clinical development as a therapeutic ADC for the treatment of ovarian cancer and other CLDN6 expressing solid cancer. Citation Format: Haitao Pan, Qing Zhang, Ganyuan Xiao, Guangchao Zhang, Jiaxin Li, Ling Xin, Kia J. Puan, Ling Xu, Yingdong Lu, Mao Yin. PLB-002 is a novel Claudin 6 antibody-drug conjugate for ovarian cancer and testicular germ cell cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2945.

<div>Abstract<p>Purpose: Claudin-6 (CLDN6) is expressed at elevated levels in multiple human cancers including ovarian and endometrial malignancies, with little or no detectable expression in normal adult tissue. This expression profile makes CLDN6 an ideal target for development of a potential therapeutic antibody-drug-conjugate (ADC). This study describes the generation and preclinical characterization of CLDN6-23-ADC, an ADC consisting of a humanized anti-CLDN6 monoclonal antibody coupled to MMAE via a cleavable linker. Experimental Design: A fully humanized anti-CLDN6 antibody was conjugated to MMAE resulting in the potential therapeutic ADC, CLDN6-23-ADC. The anti-tumor efficacy of CLDN6-23-ADC was assessed for anti-tumor efficacy in CLDN6-positive (CLDN6+) and negative (CLDN6-) xenografts and patient-derived xenograft (PDX) models of human cancers. Results: CLDN6-23-ADC selectively binds to CLDN6, versus other CLDN family members, inhibits the proliferation of CLDN6+ cancer cells in vitro and is rapidly internalized in CLDN6+ cells. Robust tumor regressions were observed in multiple CLDN6+ xenograft models and tumor inhibition lead to markedly enhanced survival of CLDN6+ PDX tumors following treatment with CLDN6-23-ADC. IHC assessment of ovarian cancer tissue microarrays demonstrate elevated levels of CLDN6 in 29% of ovarian epithelial carcinomas. Approximately 45% of high-grade serous ovarian carcinomas and 11% of endometrial carcinomas are positive for the target. Conclusions: We report the development of a novel antibody-drug conjugate, CLDN6-23-ADC, that selectively targets CLDN6, a potential onco-fetal-antigen which is highly expressed in ovarian and endometrial cancers. CLDN6-23-ADC exhibits robust tumor regressions in mouse models of human ovarian and endometrial cancers and is currently undergoing Phase I study.</p></div>

<div>AbstractPurpose:<p>Claudin-6 (CLDN6) is expressed at elevated levels in multiple human cancers including ovarian and endometrial malignancies, with little or no detectable expression in normal adult tissue. This expression profile makes CLDN6 an ideal target for development of a potential therapeutic antibody–drug conjugate (ADC). This study describes the generation and preclinical characterization of CLDN6–23-ADC, an ADC consisting of a humanized anti-CLDN6 monoclonal antibody coupled to monomethyl auristatin E (MMAE) via a cleavable linker.</p>Experimental Design:<p>A fully humanized anti-CLDN6 antibody was conjugated to MMAE resulting in the potential therapeutic ADC, CLDN6–23-ADC. The antitumor efficacy of CLDN6–23-ADC was assessed for antitumor efficacy in CLDN6-positive (CLDN6+) and -negative (CLDN6−) xenografts and patient-derived xenograft (PDX) models of human cancers.</p>Results:<p>CLDN6–23-ADC selectively binds to CLDN6, versus other CLDN family members, inhibits the proliferation of CLDN6+ cancer cells <i>in vitro</i>, and is rapidly internalized in CLDN6+ cells. Robust tumor regressions were observed in multiple CLDN6+ xenograft models and tumor inhibition led to markedly enhanced survival of CLDN6+ PDX tumors following treatment with CLDN6–23-ADC. IHC assessment of cancer tissue microarrays demonstrate elevated levels of CLDN6 in 29% of ovarian epithelial carcinomas. Approximately 45% of high-grade serous ovarian carcinomas and 11% of endometrial carcinomas are positive for the target.</p>Conclusions:<p>We report the development of a novel ADC, CLDN6–23-ADC, that selectively targets CLDN6, a potential onco-fetal-antigen which is highly expressed in ovarian and endometrial cancers. CLDN6–23-ADC exhibits robust tumor regressions in mouse models of human ovarian and endometrial cancers and is currently undergoing phase I study.</p></div>

<div>AbstractPurpose:<p>Claudin-6 (CLDN6) is expressed at elevated levels in multiple human cancers including ovarian and endometrial malignancies, with little or no detectable expression in normal adult tissue. This expression profile makes CLDN6 an ideal target for development of a potential therapeutic antibody–drug conjugate (ADC). This study describes the generation and preclinical characterization of CLDN6–23-ADC, an ADC consisting of a humanized anti-CLDN6 monoclonal antibody coupled to monomethyl auristatin E (MMAE) via a cleavable linker.</p>Experimental Design:<p>A fully humanized anti-CLDN6 antibody was conjugated to MMAE resulting in the potential therapeutic ADC, CLDN6–23-ADC. The antitumor efficacy of CLDN6–23-ADC was assessed for antitumor efficacy in CLDN6-positive (CLDN6+) and -negative (CLDN6−) xenografts and patient-derived xenograft (PDX) models of human cancers.</p>Results:<p>CLDN6–23-ADC selectively binds to CLDN6, versus other CLDN family members, inhibits the proliferation of CLDN6+ cancer cells <i>in vitro</i>, and is rapidly internalized in CLDN6+ cells. Robust tumor regressions were observed in multiple CLDN6+ xenograft models and tumor inhibition led to markedly enhanced survival of CLDN6+ PDX tumors following treatment with CLDN6–23-ADC. IHC assessment of cancer tissue microarrays demonstrate elevated levels of CLDN6 in 29% of ovarian epithelial carcinomas. Approximately 45% of high-grade serous ovarian carcinomas and 11% of endometrial carcinomas are positive for the target.</p>Conclusions:<p>We report the development of a novel ADC, CLDN6–23-ADC, that selectively targets CLDN6, a potential onco-fetal-antigen which is highly expressed in ovarian and endometrial cancers. CLDN6–23-ADC exhibits robust tumor regressions in mouse models of human ovarian and endometrial cancers and is currently undergoing phase I study.</p></div>

Purpose:Claudin-6 (CLDN6) is expressed at elevated levels in multiple human cancers including ovarian and endometrial malignancies, with little or no detectable expression in normal adult tissue. This expression profile makes CLDN6 an ideal target for development of a potential therapeutic antibody–drug conjugate (ADC). This study describes the generation and preclinical characterization of CLDN6–23-ADC, an ADC consisting of a humanized anti-CLDN6 monoclonal antibody coupled to monomethyl auristatin E (MMAE) via a cleavable linker.Experimental Design:A fully humanized anti-CLDN6 antibody was conjugated to MMAE resulting in the potential therapeutic ADC, CLDN6–23-ADC. The antitumor efficacy of CLDN6–23-ADC was assessed for antitumor efficacy in CLDN6-positive (CLDN6+) and -negative (CLDN6−) xenografts and patient-derived xenograft (PDX) models of human cancers.Results:CLDN6–23-ADC selectively binds to CLDN6, versus other CLDN family members, inhibits the proliferation of CLDN6+ cancer cells in vitro, and is rapidly internalized in CLDN6+ cells. Robust tumor regressions were observed in multiple CLDN6+ xenograft models and tumor inhibition led to markedly enhanced survival of CLDN6+ PDX tumors following treatment with CLDN6–23-ADC. IHC assessment of cancer tissue microarrays demonstrate elevated levels of CLDN6 in 29% of ovarian epithelial carcinomas. Approximately 45% of high-grade serous ovarian carcinomas and 11% of endometrial carcinomas are positive for the target.Conclusions:We report the development of a novel ADC, CLDN6–23-ADC, that selectively targets CLDN6, a potential onco-fetal-antigen which is highly expressed in ovarian and endometrial cancers. CLDN6–23-ADC exhibits robust tumor regressions in mouse models of human ovarian and endometrial cancers and is currently undergoing phase I study.

The principal site of hepatitis C virus (HCV) replication is the liver. HCV pseudoparticles infect human liver derived cell lines and this suggests that liver-specific receptors contribute to defining HCV hepatotropism. At least three host cell molecules have been reported to be important for HCV entry: the tetraspanin CD81, scavenger receptor class B member I (SR-BI), and the tight junction (TJ) protein Claudin 1 (CLDN1). Hepatocytes in liver tissue coexpress CD81, SR-BI, and CLDN1, consistent with their ability to support HCV entry. CLDN1 localized at the apical-canalicular TJ region and at basolateral-sinusoidal hepatocyte surfaces in normal tissue and colocalized with CD81 at both sites. In contrast, CLDN1 appeared to colocalize with SR-BI at the basolateral-sinusoidal surface. CLDN1 expression was increased on basolateral hepatocyte membranes in HCV-infected and other chronically inflamed liver tissue compared with normal liver. In contrast, CLDN4 hepatocellular staining was comparable in normal and diseased liver tissue. HCV infection of Huh-7.5 hepatoma cells in vitro significantly increased CLDN1 expression levels, consistent with a direct modulation of CLDN1 by virus infection. In HCV infected livers, immunohistochemical studies revealed focal patterns of CLDN1 staining, suggesting localized areas of increased CLDN1 expression in vivo which may potentiate local viral spread within the liver.

Background/Objectives: The oncofetal membrane protein Claudin 6 (CLDN6) is an attractive target for T cell-based therapies. There is a lack of detailed analyses on the age-dependent expression of CLDN6 in normal tissues is lacking, which limits the expansion of CLDN6 CAR-T cell clinical trials to pediatric populations. Methods: We analyzed CLDN6 expression in extracranial solid tumors and normal tissues of children using RNA-sequencing data from over 500 pediatric solid tumor samples, qRT-PCR and immunohistochemistry (IHC) in more than 100 fresh-frozen tumor samples and, approximately, 250 formalin-fixed paraffin-embedded (FFPE) samples. We examined normal tissue expression via qRT-PCR in 32 different infant tissues and via IHC in roughly 290 tissues from donors across four age groups, as well as in fetal autopsy samples. Results: In fetal tissues, we detected CLDN6 expression primarily in the epithelial cells of several organs, including the skin, lungs, kidneys, intestinal tract, and pancreas, but not in undifferentiated blastemal cells. Postnatally, we found CLDN6-positive epithelial progenitors only during the first few weeks of life. In older-age groups, isolated clusters of CLDN6-positive progenitors were present, but in scarce quantities. In tumor tissues, we found strong and homogeneous CLDN6 expression in desmoplastic small round cell tumors and germ cell tumors. Wilms tumors demonstrated heterogeneous CLDN6 expression, notably absent in the blastemal component. Conclusions: These findings highlight an organ-specific presence of CLDN6-positive epithelial precursors that largely disappear in terminally differentiated epithelia within weeks after birth. Therefore, our data support CLDN6 as a viable therapeutic target in pediatric patients and justify their inclusion in basket studies for anti-CLDN6-based therapies.

Claudin 6 (CLDN6) is an oncofetal protein involved in tight junction formation and is responsible for regulating cell polarity, adhesion and permeability. CLDN6 has recently emerged as an ideal target for novel oncology therapeutics, given the expression in various solid tumors, with limited normal tissue expression. High-levels of CLDN6 cell-surface protein expression are commonly detected in testicular cancer and epithelial ovarian cancer (EOC), with lower frequency expression in additional solid tumors such as gastric-, cervical-, endometrial-, and lung cancer. Using normal human tissue microarrays (TMA) and a highly selective commercially available CLDN6 antibody, minimal staining of CLDN6 was confirmed across a large panel of normal tissues by IHC, where sparse and limited apical staining of CLND6 was detected in pancreas, stomach, salivary gland, kidney and uterine tissues. High CLDN6 expression in ovarian tumors was demonstrated with the same selective antibody, confirming CLDN6 as a unique solid tumor target with limited normal tissue expression. ARC101 is a bispecific antibody that targets CLDN6 on tumor cells and CD3 on T cells, leading to T-cell-mediated tumor cell lysis. Generation of CLDN6-specific therapeutic antibodies has been challenging given the high degree of homology that exists between CLDN6 and CLN9, and to a lesser extent CLDN4. ARC101 was selected as a therapeutic candidate, due to the high degree of specificity obtained with this molecule. ARC101 bound to and induced cytotoxicity of a CLDN6 overexpressing SKOV3 ovarian cell line. No significant binding or cytotoxicity was observed for CLDN9 or CLDN4 overexpressing SKOV3 cell lines, underscoring the high specificity of ARC101 for CLDN6 over other highly homologous claudins. ARC101 bound to CLDN6-positive human ovarian cancer cell lines OVCAR3, PA1, and OV90, while binding to OVCAR3 cells was completely abolished when CLDN6 expression was deleted via knock out. ARC101 induced potent T cell activation and cytotoxicity at low effector to target (E:T) cell ratios across the panel of CLDN6-positive cells (2D and 3D models), with no activity observed on the CLDN6-negative OVCAR3 knock out cell line. In vivo, ARC101 demonstrated potent and dose-dependent anti-tumor activity in an ovarian tumor xenograft model. Overall, ARC101 is a highly specific CLDN6-directed T-cell engager with significant therapeutic potential for CLDN6-positive solid tumors. A Phase 1 first-in-human (FIH) clinical trial of ARC101 (NCT06672185) is planned to commence in 2025. Citation Format: Sanjaya Singh, Danlin Yang, Joseph Erhardt, Theresa McDevitt, Scott R. Brodeur, Bridget Larkin, Cassandra Lowenstein, Desmond Lewis, Tabitha Soto, Brinda Shah. Pharmacologic characterization of a highly specific and potent CLDN6xCD3 T cell engager (TCE), ARC101, for the treatment of CLDN6+ cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7325.

There is a large unmet need for targeted therapies to treat ovarian cancer and other solid tumors. A promising strategy is the use of T cell engaging bispecific antibodies that recruit T cells to kill cancer cells by simultaneously binding to tumor-associated antigens (TAA) on cancer cells and CD3 on T cells. Effective and safe use of these therapeutics depends on selective targeting of cancer cells over normal tissue, feasible when a TAA is more highly expressed on cancer cells versus normal tissues. Selectivity can also be improved by using a mixed valency “2 + 1” bispecific format, coupled with target affinity tuning, to achieve avid binding to the TAA. Claudin-6 (CLDN6) is a tetraspan membrane protein, a member of the claudin family of tight junction proteins, and has been identified as a promising TAA for ovarian cancer due to its high expression on ovarian and other cancer tissues and low expression on normal tissue. However, a complicating factor is that many proteins in the claudin family have high sequence identity. Most similar to CLDN6 is CLDN9, and the two proteins differ at only 3/76 residues in their extracellular loops, creating a challenge for the development of highly selective CLDN6 antibodies. We analyzed CLDN6 and CLDN9 expression using a combination of normal tissue gene expression data (GTEx), cancer cell genomics data (TCGA), and immunohistochemistry (IHC) on a panel of cancer and normal tissues. Data confirmed the tumor-specific expression pattern of CLDN6 but indicated high CLDN9 expression in normal tissues, suggesting that strong antibody selectivity for CLDN6 versus CLDN9 is critical. We created CLDN6-selective T cell engaging bispecific antibodies by starting with a highly selective monoclonal antibody humanized from a mouse hybridoma. Selectivity was further improved by engineering the CDR regions. Variant antibodies were screened for binding to transiently transfected HEK293E cells that highly expressed either CLDN6, CLDN9, or other homologous claudin family members. Selective variants were converted into the XmAb® 2 + 1 bispecific antibody format and tested in T cell-dependent cellular cytotoxicity (TDCC) assays using cancer cell lines expressing CLDN6, or HEK293E cells transfected with claudin homologs. A set of lead CLDN6 x CD3 bispecifics displaying a range of potencies on CLDN6+ cancer cell lines and high selectivity for CLDN6 over CLDN9 and other homologous claudins was identified. Tolerability and pharmacokinetics of these molecules are currently being assessed in non-human primates, and activity will be evaluated in humanized mouse models of ovarian cancer. Citation Format: Matthew S. Faber, Sung-Hyung Lee, Yoon Kyung Kim, Jing Qi, Kendra N. Avery, Duc-Hanh T. Nguyen, Rumana Rashid, Araz Eivazi, Seung Y. Chu, Juan E. Diaz, Connie Ardila, Ruschelle Love, Alex Nisthal, Norman J. Barlow, Christine Bonzon, Umesh S. Muchhal, Matthew J. Bernett, John R. Desjarlais. Bispecific claudin-6 x CD3 antibodies in a 2 + 1 format demonstrate selectivity and activity on human ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1860.

<div>Abstract<p>Purpose: Claudin-6 (CLDN6) is expressed at elevated levels in multiple human cancers including ovarian and endometrial malignancies, with little or no detectable expression in normal adult tissue. This expression profile makes CLDN6 an ideal target for development of a potential therapeutic antibody-drug-conjugate (ADC). This study describes the generation and preclinical characterization of CLDN6-23-ADC, an ADC consisting of a humanized anti-CLDN6 monoclonal antibody coupled to MMAE via a cleavable linker. Experimental Design: A fully humanized anti-CLDN6 antibody was conjugated to MMAE resulting in the potential therapeutic ADC, CLDN6-23-ADC. The anti-tumor efficacy of CLDN6-23-ADC was assessed for anti-tumor efficacy in CLDN6-positive (CLDN6+) and negative (CLDN6-) xenografts and patient-derived xenograft (PDX) models of human cancers. Results: CLDN6-23-ADC selectively binds to CLDN6, versus other CLDN family members, inhibits the proliferation of CLDN6+ cancer cells in vitro and is rapidly internalized in CLDN6+ cells. Robust tumor regressions were observed in multiple CLDN6+ xenograft models and tumor inhibition lead to markedly enhanced survival of CLDN6+ PDX tumors following treatment with CLDN6-23-ADC. IHC assessment of ovarian cancer tissue microarrays demonstrate elevated levels of CLDN6 in 29% of ovarian epithelial carcinomas. Approximately 45% of high-grade serous ovarian carcinomas and 11% of endometrial carcinomas are positive for the target. Conclusions: We report the development of a novel antibody-drug conjugate, CLDN6-23-ADC, that selectively targets CLDN6, a potential onco-fetal-antigen which is highly expressed in ovarian and endometrial cancers. CLDN6-23-ADC exhibits robust tumor regressions in mouse models of human ovarian and endometrial cancers and is currently undergoing Phase I study.</p></div>

AMG 794 is a half-life extended BiTE® immune therapy targeting the oncofetal antigen Claudin 6 (CLDN6). AMG 794 redirects T cells to kill CLDN6-expressing tumor cells and is being developed for the treatment of non-small cell lung cancer (NSCLC) and epithelial ovarian cancer (EOC). CLDN6 is a compelling tumor antigen that is expressed during embryonic and fetal development, transcriptionally silenced in adult tissues, and re-expressed on the surface of NSCLC and EOC cells. By immunohistochemistry, CLDN6 staining of the cell membrane was observed in 27% of non-squamous NSCLC (n = 63) and 69% of EOC (n = 92) samples, the majority of which were of the high-grade serous ovarian cancer subtype. Expression of CLDN6 protein was not detected in most normal adult tissues, with rare CLDN6 immunostaining limited to individual cells in the pituitary, pancreas, small intestine, kidney, and female reproductive organs. AMG 794 is a fully human BiTE® molecule that binds both human and cynomolgus monkey CLDN6 and CD3. AMG 794 binds human CLDN6 and CD3 with equilibrium dissociation constant (KD) of 13 nM and 36 nM, respectively. In vitro, AMG 794 redirects human T cells to kill CLDN6-expressing cancer cells with a half-maximal lysis concentration (EC50) of 2.6 ± 1.1 pM to 127.4 ± 53.4 pM. Consistent with the mechanism of action of BiTE® immune therapy, AMG 794 induces T cell activation and transient production of cytokines in co-cultures of T cells and CLDN6-expressing tumor cells. Remarkably, AMG 794 binding and cytotoxic activity is selective for CLDN6 over other claudin family proteins, despite high homology in the extracellular loops with CLDN9. Weekly dosing of AMG 794 significantly inhibited the growth of established lung and ovarian xenograft tumors in immunocompromised mice injected with human T cells. Anti-tumor activity was associated with an increase in tumor-infiltrating T cells. AMG 794 was well tolerated in a one-month repeat-dose toxicology study in cynomolgus monkey, with evidence for target engagement. The potent, selective activity of AMG 794 for CLDN6-expressing NSCLC and EOC cells, together with an acceptable nonclinical safety profile, supported the advancement of AMG 794 into clinical development. A first-in-human study to explore the safety, tolerability, pharmacokinetics, and anti-tumor activity of AMG 794 in patients with CLDN6-positive advanced/metastatic non-squamous NSCLC or EOC will be enrolling patients in March 2022. Citation Format: Elizabeth Pham, Anja Henn, Beate Sable, Joachim Wahl, Kip Conner, Katja Matthes, Shivani Gupta, Rodolfo Yabut, Famke Aeffner, Kristin Lewis Wilson, Jonas Anlahr, Christoph Dahlhoff, Vijay Kale, Matthias Friedrich, Tobias Raum, Peter Kufer, Angela Coxon, Sabine Stienen, Julie M. Bailis. AMG 794, a Claudin 6-targeted half-life extended (HLE) bispecific T cell engager (BITE®) molecule for non-small cell lung cancer and epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5202.

Background Claudin 6 (CLDN6) is a tight junction membrane protein whose expression in normal tissue is confined to embryonic cells, but is aberrantly expressed in various human cancers. The anti-CLDN6 monoclonal antibody (mAb), IMAB027, has shown promising antitumor activity in preclinical human CLDN6-positive (CLDN6+) cancer models. Conjugation of IMAB027 with monomethyl auristatin E (MMAE) may utilize the precision tumor-targeting of the mAb to deliver a highly effective cytotoxic drug to the tumor. In this report we present the preclinical characterization of this antibody–drug conjugate, IMAB027–vcMMAE. Methods Internalization of IMAB027 in various CLDN6+ human ovarian (OC) and testicular cancer (TC) cell lines was assessed by immunofluorescence, flow cytometry, and Fab-ZAP internalization assay. Binding characteristics of IMAB027–vcMMAE were examined by flow cytometry. Cell viability and IMAB027–vcMMAE-mediated cytotoxic effects (direct and indirect [bystander]) were assessed in cell cultures by the XTT metabolic assay. Apoptosis was evaluated by caspase 3/7, annexin V, and TUNEL assays. Xenograft mouse tumors were generated by injecting human OC cells into nude mice to assess the safety and antitumor activity of IMAB027–vcMMAE. Results IMAB027–vcMMAE binds robustly to, and is internalized by, cell lines expressing CLDN6. IMAB027–vcMMAE reduced the viability of CLDN6+ OC and TC cells by up to 100% with EC50 values in the ng/mL order. IMAB027–vcMMAE induced apoptosis in CLDN6+ cells in a dose-dependent manner. Additionally, after conjugation, IMAB027–vcMMAE retained IMAB027's ability to induce CLDN6+ cell death via antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Cell lines that did not express CLDN6 were unaffected by IMAB027–vcMMAE in monocultures; however, in cocultures of CLDN6+ and CLDN6-negative cells, IMAB027–vcMMAE exerted bystander effect, resulting in the death of cocultured CLDN6-negative cells in addition to the target-bearing CLDN6+ cells. In vivo, significant antitumor effects were observed after a single intravenous administration of 16 mg/kg IMAB027–vcMMAE in mouse OC xenografts. Further, xenograft tumors with low and/or heterogeneous CLDN6 expression treated with IMAB027–vcMMAE showed efficient tumor size reduction. Repeated dosing of IMAB027–vcMMAE was well tolerated in mice, with no physical abnormalities, changes in behavior, or alterations in appearance observed. Conclusions IMAB027–vcMMAE is a specific antibody–drug conjugate against CLDN6 that induces potent antitumor activity in CLND6+ tumor cells in vitro and in vivo. Furthermore, IMAB027–vcMMAE was able to induce antitumor effects in tumors with low and/or heterogeneous target expression, which may be driven by bystander activity. Citation Format: Özlem Türeci, Maria Kreuzberg, Korden Walter, Stefan Wöll, Ramona Schmitt, Tomohiro Yamada, Ikumi Nakajo, Ugur Sahin. Preclinical characterization of the safety and antitumor activity of IMAB027-vcMMAE, an anticlaudin 6 antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1778.