Establishment of a novel anti-TROP2 monoclonal antibody TrMab-29 for immunohistochemical analysis

TROP2 is a type I transmembrane glycoprotein originally identified in human trophoblast cells that is overexpressed in several types of cancer. To better understand the role of TROP2 in cancer, we herein aimed to develop a sensitive and specific anti-TROP2 monoclonal antibody (mAb) for use in flow cytometry, Western blot, and immunohistochemistry using a Cell-Based Immunization and Screening (CBIS) method. Two mice were immunized with N-terminal PA-tagged and C-terminal RAP/MAP-tagged TROP2-overexpressed Chinese hamster ovary (CHO)–K1 cells (CHO/PA-TROP2-RAP-MAP), and hybridomas showing strong signals from PA-tagged TROP2-overexpressed CHO–K1 cells (CHO/TROP2-PA) and weak-to-no signals from CHO–K1 cells were selected using flow cytometry. We demonstrated using flow cytometry that the established anti-TROP2 mAb, TrMab-29 (mouse IgG1 kappa), detected TROP2 in MCF7 breast cancer cell line as well as CHO/TROP2-PA cells. Western blot analysis showed a 40 kDa band in lysates prepared from both CHO/TROP2-PA and MCF7 cells. Furthermore, TROP2 was strongly detected by immunohistochemical analysis using TrMab-29, indicating that TrMab-29 may be a valuable tool for the detection of TROP2 in cancer.


Introduction
The trophoblast cell-surface antigen (TROP2) is a type I transmembrane glycoprotein that was originally identified in human trophoblast cells [1][2][3]. TROP2 is highly expressed in many cancers and may play a critical role in tumor progression [4,5]. Because increased TROP2 expression has been reported in more than 85% of all solid cancers, TROP2 may be a useful marker for cancer diagnosis and immunotherapy [6][7][8]. It has also been identified in stem cells of various tissues, including basal cells, all of which are capable of self-renewal, regeneration, and differentiation [6,9,10]. Further, several monoclonal antibodies (mAbs) against TROP2 are currently evaluated in clinical cancer trials, including DS-1062a [4,11], PF-06664178 [4,12], and IMMU-132 [4,13,14].

Hybridoma production
Female BALB/c mice (6 weeks old) were purchased from CLEA Japan (Tokyo, Japan) and kept under specific pathogen-free conditions. All animal experiments were conducted in accordance with relevant guidelines and regulations to minimize animal suffering and distress in the laboratory. The Animal Care and Use Committee of Tohoku University approved all animal experiments performed in this study (Permit number: 2019NiA-001). Mice were euthanized by cervical dislocation under anesthesia, and death was verified by respiratory and cardiac arrest. We employed CBIS to develop new mAbs against TROP2. Two mice were immunized with CHO/PA-TROP2-RAP-MAP cells (1 × 10 8 cells/mouse) via intraperitoneal (i.p.) injection with Imject Alum (Thermo Fisher Scientific, Inc.). After several additional immunizations, a booster immunization was administered by i. p. injection 2 days before spleen cells were collected. The collected spleen cells were fused with P3U1 cells using polyethylene glycol 1500 (Roche Diagnostics, Indianapolis, IN, USA), and the resulting hybridomas were selected in RPMI 1640 medium containing hypoxanthine, aminopterin, and thymidine (Thermo Fisher Scientific, Inc.). Culture supernatants were screened by flow cytometry. The isotype of mAbs were determined using isotype-specific secondary antibodies (SouthernBiothech, Birmingham, AL).

Flow cytometry
Cells were collected following a brief exposure to 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). The cells were then washed with 0.1% bovine serum albumin (BSA) in phosphatebuffered saline (PBS) and treated with primary mAbs or hybridoma culture supernatants for 30 min at 4 • C. After incubation, cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA). Fluorescence data were collected using SA3800 Cell Analyzer (Sony Corp.) and analyzed using FlowJo (BD Biosciences, Franklin Lakes, NJ, USA).

Immunohistochemical analysis
Paraffin-embedded tissue sections of a breast cancer tissue array (Cat#T8235721-5, Lot#B104066; BioChain, San Francisco, CA, USA) were autoclaved in EnVision FLEX Target Retrieval Solution High pH (Agilent Technologies, Inc.) for 20 min. After blocking with SuperBlock T20 (Thermo Fisher Scientific, Inc.), tissue sections were incubated with TrMab-29 (5 μg/mL) for 1 h at room temperature and then treated with the EnVision + Kit for mouse (Agilent Technologies Inc.) for 30 min. Color was developed using 3,3 ′ -diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) for 2 min. Counterstaining was performed with hematoxylin (FUJIFILM Wako Pure Chemical Corporation). Hematoxylin & eosin (HE) staining (FUJIFILM Wako Pure Chemical Corporation) was performed using consecutive tissue sections. Leica DMD108 (Leica Microsystems GmbH, Wetzlar, Germany) was used to examine the sections and obtain images.

Establishment of anti-TROP2 mAbs
Two mice were immunized with CHO/PA-TROP2-RAP-MAP cells to develop anti-TROP2 mAbs. Hybridomas showing strong signals from CHO/TROP2-PA cells and weak-to-no signals from CHO-K1 cells were selected using flow cytometry. After additional Western blot and immunohistochemical analyses, we established a novel anti-TROP2 mAb, TrMab-29 (mouse IgG 1 kappa). Hybridoma cells of TrMab-29 could stably grow and secret mAbs using serum-free medium.
Further, we evaluated the usefulness of TrMab-29 for Western blot analysis. We found a TrMab-29-positive 40 kDa band in lysates from both CHO/TROP2-PA and MCF7 cells (Fig. 1B). In contrast, no band was observed in lysates from CHO-K1 and TROP2-knockout MCF7 (BINDS- Finally, we investigated whether TrMab-29 is suitable for immunohistochemical analysis. As shown in Fig. 2, we found a TrMab-29positive membrane-staining pattern in invasive ductal carcinoma ( Fig. 2A and B) and adenocarcinoma ( Fig. 2E and F) tissue sections from patients with breast cancer. Hematoxylin and eosin (H&E) staining of invasive ductal carcinoma sections (Fig. 2C and D) and adenocarcinoma sections ( Fig. 2G and H) are also presented for reference. These findings demonstrate that TrMab-29 is well suited for immunohistochemical analysis using paraffin-embedded tissue sections.

Discussion
Using the CBIS method, in which antigen-expressing cell lines are used for both immunization and screening [15], we have developed many useful mAbs against membrane proteins, including CD19 [16], CD20 [17], CD44 [18], CD133 [15], and PD-L1 [19], for research applications. Of these mAbs, we recently developed clone C 20 Mab-11 to reliably detect CD20 by flow cytometry, Western blot, and immunohistochemical analysis [17]. CD20 has four membrane-spanning domains and only two small extracellular domains that include amino acids 72-80 and 142-182 [31,32]. Although there are several commercially available mAbs that interact with amino acids 142-182 of CD20 and are specifically useful in flow cytometry, there are no available anti-CD20 mAbs that are effective not only in flow cytometry but also in Western blot and immunohistochemical analyses [17], indicating that establishing multipurpose mAbs remains difficult. In this study, we aimed to establish a multipurpose anti-TROP2 mAb using the CBIS method. To that end, we successfully developed a sensitive and specific novel anti-TROP2 mAb (TrMab-29), which can be used in different research applications, such as flow cytometry (Fig. 1A), Western blot (Fig. 1B), and immunohistochemical analyses (Fig. 2).
As a next step, it would be of interest to determine whether TrMab-29 is suitable for use in targeted molecular therapy against TROP2expressing cancers. Among mouse immunoglobulins, IgG 2a and IgG 2b can induce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) [33,34]. As TrMab-29 was determined to belong to the mouse IgG 1 subclass, which does not exert ADCC/CDC, it should be changed to the IgG 2a or IgG 2b subclass. Recently, we have focused on various modalities that can be used to promote antibody therapy, including antibody-drug conjugates (ADCs), radioimmunotherapy (RIT), photoimmunotherapy (PIT), and chimeric antigen receptor T-cell (CAR-T) therapy. ADCs include a mAb, a linker, and a "payload," providing a means in which a cytotoxic drug can be delivered directly to cancer tissues via antibody targeting [35]. RIT is a modality permitting selective internal radiation therapy using radiolabeled mAbs against cancer-associated antigens [36], whereas near-infrared PIT (NIR-PIT) is a novel cancer therapy that combines the specificity of mAbs with directed toxicity induced by a photoabsorber following exposure to NIR light [37]. Alternatively, CAR-T therapy enables T-cells to detect predefined surface antigens on cancer cells independent of major histocompatibility restriction [38]. Following the establishment and characterization of our novel anti-TROP2 mAb described in the current study, we will investigate different treatment modalities, including ADC, RIT, PIT, and CAR-T-cell therapy, to explore TrMab-29-mediated antitumor activities in TROP2-expressing cancers.