Abstract 2763: Characterization of a novel immunohistochemistry (IHC) assay for CEACAM5 using a commercial antibody
Authors: Cheng Chin Leng , Chen Hongli , Kirchner Karen , Cesarone Gregory 
Published: April 2023

Abstract
Introduction: Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is expressed on the surface of some tumor epithelial cells and is a potential therapeutic target for antibody-drug conjugates such as tusamitamab ravtansine in development. An IHC assay has been fit-for-purpose validated in non-small cell lung cancer (NSCLC) for evaluation of clinical samples using a proprietary CEACAM5-specific murine antibody (Sanofi clone 769) and the Dako/Agilent Autostainer Link 48 platform. Here we compare 5 commercial antibody clones to the validated IHC assay for CEACAM5.
Methods: We tested 5 commercial clones (CI-P83-1 [Santa Cruz Biotechnology], 487609 [R&D Systems], OTI1D4 [Thermo Fisher], EPCEAR7 [Abcam], and 327 [Sino Biological]) to evaluate CEACAM5 detection in formalin-fixed, paraffin-embedded (FFPE) human cancer tissues from Discovery Life Sciences using the Leica BOND III platform. The staining pattern and intensity of CEACAM5 reactivity were compared for clone 769 and the commercial clones in the same FFPE samples. Initial IHC testing used gastric cancer tissues with continued assay development in colorectal cancer tissues. Commercially available (HT-29, MKN-45, BxPC-3, and PC-3) cell lines and CHO cells overexpressing other CEACAM targets (Sanofi) were used to test for cross reactivity and specificity. IHC staining in cell line controls and human cancer tissues (small cell lung cancer, NSCLC adenocarcinoma, squamous cell NSCLC, and pancreatic cancer), was performed to further evaluate staining accuracy to CEACAM5 and to better compare clones.
Results: Of the 5 antibodies tested, 3 were deemed unsatisfactory due to diffuse cytoplasmic staining or overstaining and were not evaluated further. Clones CI-P83-1 and 487609 best matched the pattern and intensity of clone 769 staining and were evaluated at various dilutions in gastric and colorectal cancer samples to determine optimal antibody concentration and to demonstrate range and linearity of the IHC assays. Clones CI-P83-1 and 487609 accurately and specifically detected CEACAM5 without cross reactivity to similar targets in cell lines. In the human cancer tissues, clone CI-P83-1 tended to show greater parity to clone 769 than clone 487609, which sometimes showed slightly higher CEACAM5 signal than clone 769. Clone CI-P83-1 was appropriately reactive in CEACAM5 expression control cells, highly reactive in CHO cells overexpressing CEACAM5, and lacked cross reactivity in CHO cells overexpressing CEACAM types 1, 6, 7, or 8. A fit-for-purpose validation of clone CI-P83-1 in non-squamous NSCLC with clone 769 showed comparable staining in this indication.
Conclusions: Among the 5 tested clones, there were differences in staining pattern, intensity, and cross reactivity. CI-P83-1 was the closest to 769 among the 5 clones. Further testing and validation are required to understand the finer differences between 769 and CI-P83-1.
Citation Format: Chin Leng Cheng, Hongli Chen, Karen Kirchner, Gregory Cesarone. Characterization of a novel immunohistochemistry (IHC) assay for CEACAM5 using a commercial antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2763.