Development of Highly Sensitive Anti-Mouse HER2 Monoclonal Antibodies for Flow Cytometry

: Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is an important target of monoclonal antibody (mAb) therapy such as trastuzumab. Due to the development of trastuzumab–deruxtecan, an antibody-drug conjugate, the targetable HER2-positive breast cancer patients have been expanded. To evaluate the developing modalities using anti-HER2 mAbs, reliable preclinical mouse models are required. Therefore, sensitive mAbs against mouse HER2 (mHER2) should be established. This study developed anti-mHER2 mAbs using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mHER2 mAbs, H 2 Mab-300 (rat IgG 2b , kappa) and H 2 Mab-304 (rat IgG 1 , kappa), reacted with mHER2-overexpressed Chinese hamster ovary-K1 (CHO/mHER2) and endogenously mHER2-expressed cell line, NMuMG (a mouse mammary gland epithelial cell) via ﬂow cytometry. Furthermore, these mAbs never recognized mHER2-knockout NMuMG cells. The kinetic analysis using ﬂow cytometry indicated that the dissociation constant ( K D ) values of H 2 Mab-300 and H 2 Mab-304 for CHO/mHER2 were 1.2 × 10 − 9 M and 1.7 × 10 − 9 M, respectively. The K D values of H 2 Mab-300 and H 2 Mab-304 for NMuMG were 4.9 × 10 − 10 M and 9.0 × 10 − 10 M, respectively. These results indicated that H 2 Mab-300 and H 2 Mab-304 could apply to the detection of mHER2 using ﬂow cytometry and may be useful to obtain the proof of concept in preclinical studies.


Introduction
HER2 (human epidermal growth factor receptor 2) also called ERBB2, is a member of the ERBB family tyrosine kinase receptors for the epidermal growth factor (EGF) family. Unlike other HER families, HER2 does not possess ligands and cannot form ligand-induced homodimers. To activate the downstream signaling of HER2, the heterodimer formation with other HER members in the presence of their specific ligands or ligand-independent homodimer formation under the overexpressed condition are required [1]. After the homoand heterodimer formation, HER2 activates the downstream signaling pathways such as the mitogen-activated protein kinases, the phosphoinositide 3-kinase/Akt, and the phospholipase C-γ/protein kinase C pathways [2].
HER2 is overexpressed in~20% of breast cancers and is associated with poor prognosis including a higher recurrence rate and shorter overall survival [3]. Trastuzumab, an anti-HER2 monoclonal antibody (mAb), exhibited a growth inhibitory effect in vitro and a potent antitumor effect in vivo [4]. The combination of trastuzumab with chemotherapy improves the response rates, progression-free survival, and overall survival in HER2overexpressing breast cancer patients with metastasis [5]. Trastuzumab has been a standard therapy for HER2-overexpressing breast cancers for more than 20 years [6]. In the clinic, 311 the HER2-overexpressing tumors are diagnosed through strong and complete immunohistochemistry (IHC) membranous staining of more than 10% of cells (IHC 3+) and/or in situ hybridization (ISH)-amplified.
Trastuzumab-deruxtecan (T-DXd) is a trastuzumab-based antibody-drug conjugate (ADC). The cytotoxicity is achieved through the released topoisomerase I inhibitor, DXd following the endocytosis and enzymatic cleavage of T-DXd [7]. T-DXd first showed beneficial results in metastatic breast cancer patients who had received multiple anti-HER2targeting treatments [8]. Currently, various clinical trials have evaluated the efficacy of T-DXd. Based on the studies, T-DXd has been approved in not only HER2-overexpressing breast cancer [9,10] but also HER2-low (IHC 1+ or IHC 2+/ISH-non-amplified) advanced breast cancer [11].
Since about half of all breast cancers are classifiable as HER2-low [12], a larger number of patients will reap the benefits of T-DXd treatment. The future clinical diagnostics of HER2-low breast cancer is thought to be important to maximize the patient's benefit from treatment. Furthermore, novel modalities against HER2 will be developed and evaluated in preclinical and clinical studies [13]. Therefore, preclinical mouse models are important for the evaluation of novel modalities and the prediction of adverse effects.
Although the development of therapeutic modalities for tumors have been revolutionary, the number of newly approved cancer therapies is limited. Most failure is due to the lack of efficacy [14], suggesting that the current preclinical methods are not sufficient to predict successful outcomes. Preclinical mouse models are essential for the development of cancer therapy. The first models were established through the transplantation of murine tumors into immunocompetent host mice [15]. These models were successful preclinical ones for the evaluation of a chemotherapeutic agent before clinical trials [16]. Furthermore, tumors derived from genetically modified mice can be maintained in fully immunocompetent syngeneic hosts [14]. These syngeneic models were used in preclinical studies to evaluate not only molecular target therapies but also immunotherapies including immune checkpoint inhibitors [14]. However, a preclinical model using anti-mouse HER2 (mHER2) mAb has not been reported.
We have developed the Cell-Based Immunization and Screening (CBIS) method which includes antigen-overexpressing cell immunization and high-throughput screening of hybridoma supernatants using flow cytometry. Using the CBIS method, we have developed mAbs against HER families, including human EGFR [17], HER2 [18], HER3 [19], and mouse EGFR [20]. In this study, we developed novel anti-mHER2 mAbs using the CBIS method and evaluated its application to flow cytometry.
All cells were grown in a humidified incubator at 37 • C, in an atmosphere of 5% CO 2 and 95% air.

Production of Hybridomas
A five-week-old Sprague-Dawley rat was purchased from CLEA Japan (Tokyo, Japan). The animal was housed under specific pathogen-free conditions. All animal experiments were approved by the Animal Care and Use Committee of Tohoku University (Permit number: 2022MdA-001).

Purification of mAbs
The cultured supernatants of H 2 Mab-300 and H 2 Mab-304-producing hybridomas were collected and filtered using Steritop (0.22 µm, Merck KGaA, Darmstadt, Germany). The supernatants were applied to 1 mL of Ab-Capcher (ProteNova, Kagawa, Japan). After washing with phosphate-buffered saline (PBS), the antibodies were eluted with an IgG elution buffer (Thermo Fisher Scientific Inc.). Finally, the eluates were concentrated, and the elution buffer was replaced with PBS using Amicon Ultra (Merck KGaA, Darmstadt, Germany).

Determination of Dissociation Constant (K D ) through Flow Cytometry
CHO/mHER2 and NMuMG cells were suspended in 100 µL serially diluted H 2 Mab-300, H 2 Mab-304, and clone 666521 for 30 min at 4 • C. The cells were treated with 50 µL of Alexa Fluor 488-conjugated anti-rat IgG (1:200). The GeoMean of each histogram including primary mAb + secondary Ab (Alexa Fluor 488-conjugated anti-rat IgG) and only secondary Ab (for background) was determined. We further withdrew the background from each data and determined the apparent K D by the fitting binding isotherms to built-in one-site binding models of GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA).

Development of Anti-mHER2 mAbs using the CBIS Method
To develop anti-mHER2 mAbs, one rat was immunized with LN229/mHER2 cells. ( Figure 1A). The spleen was then excised from the rat, and splenocytes were fused with myeloma P3U1 cells ( Figure 1B). The developed hybridomas were subsequently seeded into ten 96-well plates and cultivated for six days. The positive wells were screened through the selection of mHER2-expressing cell-reactive and CHO-K1-non-reactive supernatants using flow cytometry ( Figure 1C). After the limiting dilution and several additional screenings, anti-mHER2 mAbs, such as H 2 Mab-300 (rat IgG 2b , kappa) and H 2 Mab-304 (rat IgG 1 , kappa), were finally established ( Figure 1D).

Development of Anti-mHER2 mAbs using the CBIS Method
To develop anti-mHER2 mAbs, one rat was immunized with LN229/mHER2 cells. (Figure 1A). The spleen was then excised from the rat, and splenocytes were fused with myeloma P3U1 cells ( Figure 1B). The developed hybridomas were subsequently seeded into ten 96-well plates and cultivated for six days. The positive wells were screened through the selection of mHER2-expressing cell-reactive and CHO-K1-non-reactive supernatants using flow cytometry ( Figure 1C). After the limiting dilution and several additional screenings, anti-mHER2 mAbs, such as H2Mab-300 (rat IgG2b, kappa) and H2Mab-304 (rat IgG1, kappa), were finally established ( Figure 1D).

Discussion
The HER2 extracellular domain is composed of four domains (I-IV) ( Figure 1A). The domain II is essential for the formation of heterodimers with other HER members including EGFR, HER3, and HER4 in the presence of their ligands, such as EGF [22] and neuregulin 1 (NRG1, a HER3 ligand) [23]. The clinically approved anti-HER2 mAbs, trastuzumab and pertuzumab, recognize domains IV and II, respectively [24,25]. Pertuzumab was shown to inhibit the NRG1-induced heterodimerization with HER3 and intracellular signaling [24]. In this study, we developed novel anti-mHER2 mAbs (H 2 Mab-300 and 304) using the CBIS method and showed the application via flow cytometry (Figures 2-4). We should identify these epitopes and determine the property of mAbs including the effect on the homodimer and heterodimer formation of mHER2. We established the RIEDL insertion for epitope mapping (REMAP) method [26][27][28][29] to identify the conformational epitope because most of the mAbs developed using the CBIS method recognized the conformational epitopes. We determined the conformational epitopes of anti-human EGFR mAbs (EMab-51 and EMab-134) [27,29] using the REMAP method.
In a preclinical study, the antitumor activity of anti-HER2 mAbs have been evaluated on the HER2-positive tumor-bearing immunodeficient mice [30] or HER2-overexpressed murine breast cancer-transplanted syngeneic models [31]. Furthermore, mouse mammary tumor virus (MMTV)-driven overexpression of HER2 was generated to investigate the roles of HER2 in mammary adenocarcinoma development and therapeutic effects of anti-HER2 mAb such as 4D5 (murine version of trastuzumab) [32]. The incidence of mammary tumors and tumor growth were significantly reduced in 4D5-treated mice [33].
Currently, targetable HER2-positive tumors are expanded from IHC3+ to HER2-low in breast cancer, suggesting that other tumor types could apply to HER2-targeting therapy. Pancreatic cancer is a difficult-to-treat disease with critical unmet needs. HER2 expression is also observed in pancreatic cancers and is associated with poor prognosis [34]. Recently, new drugs that target KRAS G12D , a common mutation in PDAC, have been developed [35]. One of the drugs, MRTX1133, exhibited specific efficacy in tumor cells harboring KRAS G12D mutations [36,37]. However, MRTX1133 promoted the expression and activation of ERBB receptors including HER2, which is expected as a mechanism of resistance to MRTX1133 [38]. Similar ErbB receptors upregulation was also observed in Kras +/LSL-G12D ; Trp53 +/LSL-R172H ; Pdx1-Cre (KPC) mice, a murine model of pancreatic cancer [39], treated with MEK and AKT inhibitors [40]. Therefore, specific kinase inhibitors or mAbs against ErbB receptors could potentiate the efficacy of the treatment. H 2 Mab-300 and H 2 Mab-304 may be applied to obtain the proof of concept in the above mouse model system. For the preclinical study, the conversion from H 2 Mab-300 and H 2 Mab-304 (rat IgG) to mouse IgG is essential to evaluate the efficacy in mice.
A major adverse effect of anti-HER2 therapeutic mAbs is cardiotoxicity, which is potentiated by co-treatment with chemotherapeutic agents. The cardiotoxic surveillance through routine cardiac monitoring is essential in clinic [41]. Furthermore, ErbB2-deficient mice were embryonic lethal due to cardiac dysfunctions [42]. Mice lacking ErbB2 in ventricular displayed the characteristics of dilated cardiomyopathy [43]. These results indicated that HER2 is essential in normal heart development and homeostasis. However, the detailed mechanism of cardiotoxicity has not been investigated. Therefore, H 2 Mab-300 and H 2 Mab-304 could be used to analyze the cardiotoxicity in various combinations with chemotherapeutic agents in mice. Furthermore, clinical trials combining immunotherapies with HER2-targeted therapies have been conducted [13]. H 2 Mab-300 and H 2 Mab-304 are also important to predict the side effect of the treatment.

Conclusions
H 2 Mab-300 and 304 could be used in preclinical studies to obtain the proof of concept of antitumor effects and predict the side effects of HER2-targeting mAb therapy.
Author Contributions: T.O. and T.T. performed the experiments. M.K.K. and Y.K. designed the experiments. T.O., H.S. and Y.K. analyzed the data. H.S. and Y.K. wrote the manuscript. All authors agreed to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work is appropriately investigated and resolved. All authors have read and agreed to the published version of the manuscript. Data Availability Statement: All related data and methods are presented in this paper. Additional inquiries should be addressed to the corresponding authors.

Conflicts of Interest:
The authors declare no conflict of interest.