Development of a novel antibody-drug conjugate targeting both CEACAM5 and CEACAM6

Background: Carcinoembryonic antigen cell adhesion molecule (CEACAM) family members 5 and 6 are tumor-associated antigens frequently upregulated in epithelial cancers, including pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC), where they contribute to invasion, metastasis, survival, resistance to chemotherapy, and immune escape. CT109 is a novel antibody with dual specificity for both CEACAM5 and 6, mediated by binding to a shared glycoepitope. CT109 exhibits a high affinity (in the single nM range) to the target. Recently CEACAM6 was identified as a novel immune checkpoint. To further develop CT109, in vitro and in vivo characterization of the antibody-drug conjugate (ADC), CT109-SN-38, was performed. Materials and Methods: Immunoblots were performed against recombinant CEACAM5/6 and lysates of PDAC and CRC cell lines to assess the specificity of CT109. Immunohistochemistry (IHC) was also performed to establish the expression prevalence in epithelial cancers and confirm specificity. Internalization kinetics were assessed by pHrodo conjugation and flow cytometry in two PDAC cell lines. ADC IC50 values were determined in the antigen-expressing PDAC line, BxPC-3, and non-expressing line, PANC-1, using CellTiter Glo. Effects of CT109-SN-38 in vivo were performed in a heterotopic xenograft model in nude mice that received triweekly injections of CT109-SN-38 for three weeks. Results: CT109 binds to both cell surface and recombinant CEACAM5 and CEACAM6 in immunoblots. CT109 exhibits remarkable specificity in IHC with high expression prevalence in CRC and PDAC cores (3/3 cores). CT109 is internalized in antigen-expressing, but not in non-expressing, PDAC cells with a half-maximal localization to low pH compartments of 24 hours. In vitro, CT109-SN-38 and auristatin conjugates exhibit robust killing of antigen-expressing cells. in vivo, CT109-SN-38 mediates specific tumor-killing, resulting in reduced tumor volumes (through day 50, p < 0.001) and reduced tumor burden below baseline in a subset of mice (2/10 mice). Conclusions: CT109, as a novel anti-CEACAM5/6 antibody, binds to a high proportion of CRC and PDAC lines and cores. However, binding to CEACAMs 5 and 6 does not compromise specificity in IHC, with no observed staining of normal tissues. While GPI-linked proteins are canonically viewed as slowly or non-internalizing antigens, CT109 is nonetheless internalized. It mediates a dose-dependent cytotoxic effect in vitro as an SN-38, monomethyl auristatin (MMA) F, and MMAE conjugate. CT109-SN-38 similarly exhibits a dose-dependent effect in reducing tumor growth in a heterotopic PDAC tumor xenograft model, with 2/10 mice exhibiting tumor regression throughout the study. Further preclinical and clinical development of CT109-SN-38 is warranted. Conflict of interest: Ownership: Dr. Cohava Gelber has partial ownership and is a shareholder of Stromatis Pharma. Board of Directors: Dr. Cohava Gelber is a co-chairperson and founder of Stromatis Pharma.