Development of monoclonal antibodies for the treatment of solid tumors resistant to the current pharmacological treatments and overexpressing Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1)

Background: The role of Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) as a recently characterized immune-checkpoint inhibitor as well as marker for tumor progression and metastasis development of several solid tumors, makes it an attractive target for cancer immunotherapy. Herein, we describe the development and characterization of DIA 12.3, a new fully human IgG1 antibody against CEACAM1, which derives from the previously developed anti-CEACAM1 single chain variable fragment (scFv) antibody, termed DIATHIS1. Methods: Bicistronic vectors were designed for IgG1 and IgG4 antibody expression and stable cell lines were developed for their production. In vitro assays were performed to evaluate the physico-chemical properties of the antibodies and their biological activity. In addition, the mutated mtN297A IgG1 was developed in order to reduce the effector functions of the antibody Fc region and, thus, to prevent excessive cytotoxicity. After antibody DOTAylation and radiolabeling with 64Cu, PET experiments with NSG and hCEACAM1 and hCEA double transgenic mice were performed to evaluate the in vivo tumor targeting and distribution of 64Cu-DOTAylated-DIA 12.3 antibody. Results: The current project is focused on the IgG1 antibody, since it showed better structural and functional performances compared to IgG4. More in detail, we found that DIA 12.3 purified by Protein A Affinity Chromatography has a high-purity grade monomeric formulation, with structural as well as biological stability eight months after the purification and of storage at 4°C. ELISA assays revealed that DIA 12.3 is able to bind hCEACAM1 antigen with a linear activity and high affinity. Flow cytometry analyses demonstrated high antibody immunoreactivity with hCEACAM1 expressed on the cell surface membrane of human metastatic melanoma, bladder and colon cancer cell lines. Furthermore, cytotoxic assays displayed the capability of DIA 12.3 to enhance the NK-92 cell-mediated cytotoxicity against metastatic melanoma, bladder, colon and head&neck cancer cells. ELISA and flow cytometry assays showed that the N297A mutation does not affect the antibody binding ability, but it results in a reduced stimulation of NK cell-mediated tumor killing, as expected. DIA 12.3 and mtN297A DIA 12.3 antibodies also show binding activity to CEACAM1 antigen on neutrophil cell surface, without inducing toxicity or triggering unnecessary activation of neutrophils. Biodistribution analyses revealed specific localization of radiolabeled DIA 12.3 antibody to hCEACAM1-positive xenograft in NSG mice. Conclusion: These findings highlight the potential of DIA 12.3 antibody as a candidate in immunotherapy treatments of CEACAM1-expressing solid tumors and indicate the need for further in vitro and in vivo evaluation of DIA 12.3 pharmacokinetic and pharmacodynamic profile.