In Vitro Characterization of 177Lu-DOTA-M5A Anti-Carcinoembryonic Antigen Humanized Antibody and HSP90 Inhibition for Potentiated Radioimmunotherapy of Colorectal Cancer

Tabassom Mohajershojai; Preeti Jha; Anna Boström; Fredrik Y Frejd; Paul J Yazaki; Marika Nestor

doi:10.3389/fonc.2022.849338

In Vitro Characterization of ¹⁷⁷Lu-DOTA-M5A Anti-Carcinoembryonic Antigen Humanized Antibody and HSP90 Inhibition for Potentiated Radioimmunotherapy of Colorectal Cancer

Front Oncol. 2022 Mar 31:12:849338. doi: 10.3389/fonc.2022.849338. eCollection 2022.

Authors

Tabassom Mohajershojai¹, Preeti Jha^{1

2}, Anna Boström³, Fredrik Y Frejd¹, Paul J Yazaki⁴, Marika Nestor¹

Affiliations

¹ Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
² Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden.
³ Ridgeview Instruments AB, Uppsala, Sweden.
⁴ Department of Immunology and Theranostics, Beckman Research Institute, City of Hope, Duarte, CA, United States.

Abstract

Carcinoembryonic antigen (CEA) is an antigen that is highly expressed in colorectal cancers and widely used as a tumor marker. ¹³¹I and ⁹⁰Y-radiolabeled anti-CEA monoclonal antibodies (mAbs) have previously been assessed for radioimmunotherapy in early clinical trials with promising results. Moreover, the heat shock protein 90 inhibitor onalespib has previously demonstrated radiotherapy potentiation effects in vivo. In the present study, a ¹⁷⁷Lu-radiolabeled anti-CEA hT84.66-M5A mAb (M5A) conjugate was developed and the potential therapeutic effects of ¹⁷⁷Lu-DOTA-M5A and/or onalespib were investigated. The ¹⁷⁷Lu radiolabeling of M5A was first optimized and characterized. Binding specificity and affinity of the conjugate were then evaluated in a panel of gastrointestinal cancer cell lines. The effects on spheroid growth and cell viability, as well as molecular effects from treatments, were then assessed in several three-dimensional (3D) multicellular colorectal cancer spheroid models. Stable and reproducible radiolabeling was obtained, with labeling yields above 92%, and stability was retained at least 48 h post-radiolabeling. Antigen-specific binding of the radiolabeled conjugate was demonstrated on all CEA-positive cell lines. Dose-dependent therapeutic effects of both ¹⁷⁷Lu-DOTA-M5A and onalespib were demonstrated in the spheroid models. Moreover, effects were potentiated in several dose combinations, where spheroid sizes and viabilities were significantly decreased compared to the corresponding monotherapies. For example, the combination treatment with 350 nM onalespib and 20 kBq ¹⁷⁷Lu-DOTA-M5A resulted in 2.5 and 2.3 times smaller spheroids at the experimental endpoint than the corresponding monotreatments in the SNU1544 spheroid model. Synergistic effects were demonstrated in several of the more effective combinations. Molecular assessments validated the therapy results and displayed increased apoptosis in several combination treatments. In conclusion, the combination therapy of anti-CEA ¹⁷⁷Lu-DOTA-M5A and onalespib showed enhanced therapeutic effects over the individual monotherapies for the potential treatment of colorectal cancer. Further in vitro and in vivo studies are warranted to confirm the current study findings.

Keywords: 177Lu-DOTA-M5A mAb; HSP90 (heat shock protein 90); carcinoembryonic antigen (CEA); colorectal cancer; combination (combined) therapy; molecular radiotherapy; multicellular 3D spheroids; onalespib (AT13387).