New anti-mesothelin single-domain antibodies and cell models for developing targeted breast cancer therapy

About authors

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia

Correspondence should be addressed: Stepan P. Chumakov
Miklouho-Maclaya, 16/10, Moscow, 117997; moc.liamg@lukhtah

About paper

Funding: the study was supported by the Russian Ministry of Education and Science (Project ID RFMEFI60418X0205).

Compliance with ethical standards: the study was conducted in compliance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International and the Directive 86/609/EEC dated November 24, 1986.

Author contribution: Kravchenko JE — cell culture, real-time PCR and ELISA; Chumakov SP — preparation of immune libraries, protein purification, flow cytometry, manuscript preparation; Frolova EI — study design, immunization and blood collection, primary cultures, manuscript preparation.

Received: 2020-09-28 Accepted: 2020-10-20 Published online: 2020-10-31

Most triple negative breast cancers (TNBC) are characterized by elevated expression of mesothelin (MSLN), a cell surface antigen and one of the preferred targets for the therapy of solid tumors. Most continuous TNBC cell lines are MSLN-negative, which obstructs the development of MSLN-targeted therapy for TNBC. The aim of this study was to identify TNBC cell lines with MSLN hyperexpression and to obtain single-domain antibodies (nanobodies) capable of recognizing MSLN in TNBC cells. Mesothelin expression levels were measured in the panel of TNBC cell lines by real-time reverse-transcription PCR. PCR results were verified by measuring concentrations of the megakaryocyte potentiating factor (the secreted fragment of the mesothelin precursor) using sandwich ELISA. Immune phage-display VHH fragment libraries were prepared from mononuclear cells of Vicugna pacos using a modified library enrichment protocol. Two nanobody variants with high specificity for the target and Kd of about 140 and 95 nmol, respectively were obtained. Two MSLN+ and three MSLN– cell lines were identified in the TNBC cell lines panel. The nanobodies demonstrated the ability to recognize the target antigen in MSLN+ cells and had the low ability to bind to MSLN– cells. Thus, we found a convenient MSLN+ TNBC cell model for MSLN-targeted therapy testing. The new single-domain antibodies can be used as targeting components of chimeric antigen receptors.