Dual Function pH Responsive Bispecific Antibodies for Tumor Targeting and Antigen Depletion in Plasma.

Jan P Bogen,Aileen Ebenig,Julius Grzeschik,Stefan Zielonka,Harald Kolmar,Simon Krah,Steffen C Hinz

doi:10.3389/fimmu.2019.01892

Jan P Bogen, Aileen Ebenig + Show 5 more

Open Access

https://doi.org/10.3389/fimmu.2019.01892

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Abstract

Shedding of membrane-bound cell surface proteins, where the extracellular domain is released and found in the circulation is a common phenomenon. A prominent example is CEACAM5 (CEA, CD66e), where the shed domain plays a pivotal role in tumor progression and metastasis. For treatment of solid tumors, the presence of the tumor-specific antigen in the plasma can be problematic since tumor-specific antibodies might be intercepted by the soluble antigen before invading their desired tumor target area. To overcome this problem, we developed a generic procedure to generate bispecific antibodies, where one arm binds the antigen in a pH-dependent manner thereby enhancing antigen clearance upon endosomal uptake, while the other arm is able to target tumor cells pH-independently. This was achieved by incorporating pH-sensitive binding modalities in the common light chain IGKV3-15*01 of a CEACAM5 binding heavy chain only antibody. Screening of a histidine-doped light chain library using yeast surface display enabled the isolation of pH-dependent binders. When such a light chain was utilized as a common light chain in a bispecific antibody format, only the respective heavy/light chain combination, identified during selections, displayed pH-responsive binding. In addition, we found that the altered common light chain does not negatively impact the affinity of other heavy chain only binders toward their respective antigen. Our strategy may open new avenues for the generation of bispecifics, where one arm efficiently removes a shed antigen from the circulation while the other arm targets a tumor marker in a pH-independent manner.

Highlights

Colorectal cancer (CRC) is the third most diagnosed cancer with approximately 10% of all diagnosed cancers
The vectors used for yeast surface display of the heavy chain and the secretion of the common light chain were based on the pYD1 plasmid backbone (Yeast Display Vector Kit, version D, #V835-01, Thermo Fisher Scientific)
Davis and coworkers recently described a strategy for the generation of bispecific antibodies, where heavy chain heterodimerization is achieved by co-expression of Fcengineered heavy chains that contain mutual exchanges of IgG/IgA segments (SEEDbody technology) [18, 32]

Summary

Introduction

Colorectal cancer (CRC) is the third most diagnosed cancer with approximately 10% of all diagnosed cancers. A specific and sensitive marker for colorectal and gastric carcinomas is the carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5, CEA, CD66e) which is a membrane protein found on the surface of columnar epithelial and goblet cells of the colon [1]. Studies imply that the soluble CEACAM5 is a driving factor for metastatic development in the liver by stimulating kupffer cells to secret proinflammatory cytokines such as IL-10, IL-6, and TNF-α into the hepatic sinusoid [4,5,6]. This in turn leads to the upregulation of cell adhesions proteins, which facilitate the arrest of circulating tumor cells. Liver metastasis is the main reason for CRC-related deaths [7]

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