TARGETING CEACAM5/6 WITH A NOVEL DUAL SPECIFIC ANTIBODY-DRUG CONJUGATE AS A THERAPEUTIC STRATEGY IN GASTROINTESTINAL CANCERS

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), where they contribute to invasion, metastasis, survival, resistance to chemotherapy, and immune escape. CT109 is a novel humanized antibody with dual specificity for both CEACAM5 and 6, mediated by binding to a shared glycoepitope. CT109 exhibits a high affinity to both CEACAMs. To further develop CT109, in vitro and in vivo characterization of antibody-drug conjugates (ADCs) was performed. Methods: Immunoblots were performed against recombinant CEACAM5 and 6 and lysates of PDAC and CRC cell lines to assess the specificity of CT109. For CT109 cognate epitope characterization, PNGase F treatment, mutation of N-linked and glycosylation sites (Asn>Gln) was performed on recombinant CEACAM6-Fc expressed in HEK293T. Immunohistochemistry (IHC) was also performed to establish expression prevalence in epithelial cancers and confirm target expression specificity in tissues. Internalization kinetics were assessed by pHrodo conjugation and flow cytometry in two PDAC cell lines. For CT109 antibody-drug conjugates (ADCs), IC50 values were determined in the antigen-expressing PDAC line, BxPC-3, and non-expressing line, PANC-1. Effects of CT109-SN-38 and CT109-MMAF in vivo were evaluated in a heterotopic BxPC-3 xenograft model. Results: CT109 bound to both cell surface and recombinant CEACAM5 and CEACAM6 on immunoblots. CT109 exhibited remarkably high tissue specificity in IHC and high expression prevalence in PDAC and colorectal cancer (CRC). CT109 was internalized in antigen-expressing, but not in non-expressing, PDAC cells with a half-maximal localization to low pH compartments after 2.3-9.6 hours. In vitro, the CT109-SN-38 and auristatin ADCs exhibited robust dose and antigen-dependent cell killing. In vivo, CT109-SN-38 mediated specific tumor-killing, resulting in reduced tumor growth rates and volumes (through day 50, p = 0.02) and reduced tumor burden below baseline in a subset of mice (3/10 mice in the high dose group). Conclusions: CT109, an anti-CEACAM5/6 antibody with a unique glycoepitope, bound to a high proportion of CRC and PDAC lines and tumors. However, binding to CEACAMs 5 and 6 does not compromise specificity in IHC, with a vast majority of normal tissues exhibiting no expression. In a small subset of epithelial tissues, expression was barely detectable and relegated to the epithelial lumen in normal tissues. While GPI-linked proteins such as CEACAM 5 and 6 are canonically viewed as slow or non-internalizing antigens, CT109 was nonetheless internalized. As SN-38, monomethyl auristatin (MMA) F, and MMAE conjugates, CT109 ADCs mediated a dose and antigen-dependent cytotoxic effect in vitro. CT109-SN-38 similarly exhibited a dose-dependent effect in reducing tumor growth in a heterotopic PDAC tumor xenograft model, with regression observed in the subset of mice receiving the ADC. Further preclinical and clinical development of CT109-SN-38 is warranted.

Author

• Arias Cardenas, Kelly Concepcion

• ADC
• CEACAM5
• CEACAM6
• PANCREATIC ADENOCARCINOMA
• SN-38
• XENOGRAFT MODEL

Identifier

hdl:1920/13681

OAI identifier oai:identifier

oai:MARS:1920/13681