Establishment of a Novel Cancer-Specific Anti-HER2 Monoclonal Antibody H2Mab-250/H2CasMab-2 for Breast Cancers

1. Introduction

Human epidermal growth factor receptor 2 (HER2) is included in the receptor tyrosine kinase family of human epidermal growth factor receptors. To activate the downstream signaling, HER2 must either form heterodimers with other HER members and their specific ligands or self-assemble into ligand-independent homodimers when overexpressed [1]. The HER2 overexpression is observed in approximately 20% of breast cancers [2] and 20% of gastric cancers [3], which are associated with higher rates of recurrence, poor prognosis, and shorter overall survival. A monoclonal antibody (mAb) against HER2, trastuzumab, exhibited an anti-proliferating effect in vitro and a potent antitumor effect in vivo [4,5]. The addition of trastuzumab to chemotherapy improves objective response rates, progression-free survival, and overall survival in HER2-positive breast cancer patients with metastasis [6]. Trastuzumab has become the standard treatment for HER2-positive breast cancers [7] and HER2-positive gastric cancers [8]. Trastuzumab has been the most effective therapy for HER2-positive breast cancer for more than 20 years [9].

The major adverse effect associated with anti-HER2 therapeutic mAbs is cardiotoxicity, thereby necessitating routine cardiac monitoring in clinics [10]. Furthermore, mice lacking ErbB2 (ortholog of HER2) displayed embryonic lethal due to the dysfunctions associated with a lack of cardiac trabeculae [11]. Ventricular-restricted ErbB2-deficient mice showed the features of dilated cardiomyopathy [12]. These results indicate that HER2 is vital for normal heart development and homeostasis. Therefore, more selective anti-HER2 mAbs against tumors, which can reduce heart failures are required.

We previously established several anti-HER2 mAbs, such as H₂Mab-19 (IgG_2b, kappa) [13], H₂Mab-41 (IgG_2b, kappa) [14], H₂Mab-77 (IgG₁, kappa) [15], H₂Mab-119 (IgG₁, kappa) [16], H₂Mab-139 (IgG₁, kappa) [17], and H₂Mab-181 (IgG₁, kappa) [18] by the immunization of HER2 ectodomain (HER2ec). We further engineered the mAbs into the mouse IgG_2a type (H₂Mab-77-mG_2a, H₂Mab-119-mG_2a, and H₂Mab-139-mG_2a, respectively), and produced the core fucose-deficient types (H₂Mab-77-mG_2a-f, H₂Mab-119-mG_2a-f, and H₂Mab-139-mG_2a-f, respectively) to potentiate the antibody-dependent cellular cytotoxicity (ADCC) and antitumor effect in vivo [19,20,21]. In this study, we developed and characterized a novel HER2 mAb, named H₂Mab-250/H₂CasMab-2.

2. Materials and Methods

2.1. Cell culture

Chinese hamster ovary (CHO)-K1, BT-474, SK-BR-3, MDA-MB-468, MCF 10A, hTERT TIGKs, HBEC3-KT, hTERT-HME1, RPTEC/TERT1, and P3X63Ag8U.1 (P3U1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Human keratinocyte HaCaT was purchased from Cell Lines Service GmbH (Eppelheim, Germany). hTCEpi, hTEC/SVTERT24-B, and HCEC-1CT were purchased from EVERCYTE (Vienna, Austria).

The cDNA of HER2 (wild type; WT) and deletion mutants (dN218, dN342, and dN511) were cloned into the pCAG-nPA16 vector. A HER2 point mutant (W614A) and HER2 WT were cloned into the pCAG-nPA-cRAPMAP vector. CHO-K1 cells were transfected with the above-mentioned vectors using a Neon transfection system (Thermo Fisher Scientific Inc., Waltham, MA). A few days after transfection, PA tag-positive cells were sorted by the cell sorter (SH800; Sony Corp., Tokyo, Japan) using NZ-1, which was originally developed as an anti-human PDPN mAb [22]. Finally, CHO/HER2 and CHO/HER2 (dN218, dN342, dN511, and W614A) cell lines were established.

CHO-K1, CHO/HER2 (WT, deletion, and point mutants), and P3U1 were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), and BT-474, SK-BR-3, MDA-MB-468, HEK293T, and HaCaT were cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium (Nacalai Tesque, Inc.), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific Inc.), 100 units/mL of penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc.). Mammary epithelial cell line, MCF 10A was cultured in Mammary Epithelial Cell Basal Medium BulletKit^TM (Lonza, Basel, Switzerland) supplemented with 100 ng/mL cholera toxin (Sigma-Aldrich Corp., St. Louis, MO).

Immortalized normal epithelial cell lines were maintained, as follows; hTERT TIGKs, Dermal Cell Basal Medium and Keratinocyte Growth Kit (ATCC); HBEC3-KT, Airway Epithelial Cell Basal Medium and Bronchial Epithelial Cell Growth Kit (ATCC); hTERT-HME1, Mammary Epithelial Cell Basal Medium BulletKit^TM without GA-1000 (Lonza); hTCEpi, KGMTM-2 BulletKit^TM (Lonza); hTEC/SVTERT24-B, OptiPROTM SFM and GlutaMAX^TM-I (Thermo Fisher Scientific Inc.); RPTEC/TERT1, DMEM/F-12 and hTERT Immortalized RPTEC Growth Kit with supplement A and B (ATCC); HCEC-1CT, DMEM / M199 (4:1, Thermo Fisher Scientific Inc.), 2 % Cosmic Calf Serum (Cytiva, Marlborough, MA), 20 ng/mL hEGF (Sigma-Aldrich Corp.), 10 μg/mL insulin (Sigma-Aldrich Corp.), 2 μg/mL apo-transferrin (Sigma-Aldrich Corp.), 5 nM sodium-selenite (Sigma-Aldrich Corp.), 1 μg/mL hydrocortisone (Sigma-Aldrich Corp.).

All cell lines were cultured at 37°C in a humidified atmosphere with 5% CO₂ and 95% air.

2.2. Development of hybridomas

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).

Anti-HER2 mAbs, such as H₂Mab-19 (IgG_2b, kappa) [13], H₂Mab-41 (IgG_2b, kappa) [14], H₂Mab-77 (IgG₁, kappa) [15], H₂Mab-119 (IgG₁, kappa) [16], H₂Mab-139 (IgG₁, kappa) [17], and H₂Mab-181 (IgG₁, kappa) [18] were established previously. In this study, H₂Mab-193 (IgG₁, kappa), H₂Mab-215 (IgG₁, kappa), and H₂Mab-250 (IgG₁, kappa) were established by the same strategy. Briefly, BALB/c mice were immunized with recombinant HER2ec produced by LN229 cells together with Imject Alum (Thermo Fisher Scientific, Inc.). After several additional immunizations, spleen cells were fused with P3U1 cells. The culture supernatants of hybridomas were screened using enzyme-linked immunosorbent assay (ELISA) with recombinant HER2ec and flow cytometry with cell lines.

2.3. Production of recombinant mAbs

To generate recombinant H₂Mab-250 and H₂Mab-119, their V_H cDNAs and the C_H cDNA of mouse IgG₁ were cloned into the pCAG-Neo vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The V_L cDNAs and C_L cDNA of the mouse kappa light chain were also cloned into the pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation). The vectors were transfected into ExpiCHO-S cells using the ExpiCHO Expression System (Thermo Fisher Scientific, Inc.), and Ab-Capcher (ProteNova, Kagawa, Japan) was used to purify the recombinant H₂Mab-250 and H₂Mab-119.

To generate mouse IgG_2a-type H₂Mab-250 (H₂Mab-250-mG_2a), we cloned the V_H cDNA of H₂Mab-250 and the C_H of mouse IgG_2a into the pCAG-Neo vector. The mouse kappa light chain vector of H₂Mab-250 was described above. To produce the defucosylated form (H₂Mab-250-mG_2a-f), the vectors were transfected into BINDS-09 (fucosyltransferase 8-knockout ExpiCHO-S) cells using the ExpiCHO Expression System. H₂Mab-250-mG_2a-f was purified using Ab-Capcher.

2.4. Flow cytometry

Cells were collected using 0.25% trypsin and 1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.). The cells (1 × 10⁵ cells/sample) were treated with mouse anti-HER2 mAbs (10 μg/mL), trastuzumab (10 μg/mL), NZ-1 (10 μg/mL), or blocking buffer [control; 0.1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS)] for 30 min at 4˚C. Next, the cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG (1:1,000; Cell Signaling Technology, Danvers, MA) for mouse anti-HER2 mAbs, Alexa Fluor 488-conjugated anti-human IgG (1:1,000; Sigma-Aldrich, Corp.) for trastuzumab, or Alexa Fluor 488-conjugated anti-rat IgG (1:1,000; Cell Signaling Technology) for NZ-1 for 30 min at 4˚C. The fluorescence data was collected using EC800 or SA3800 Cell Analyzer (Sony Corp), and the data were analyzed using FlowJo (BD Biosciences, Franklin Lakes, NJ).

2.5. ADCC reporter bioassay

The ADCC reporter bioassay was performed using an ADCC Reporter Bioassay kit (Promega Corporation, Madison, WI), according to the manufacturer’s instructions. Target cells (BT-474 and HaCaT, 12,500 cells per well) were cultured in a 96-well white solid plate. H₂Mab-250-mG_2a-f and trastuzumab (Herceptin; Chugai Pharmaceutical Co., Ltd, Tokyo, Japan) were serially diluted and added to the target cells. Jurkat cells stably expressing the human FcγRIIIa receptor and a nuclear factor of activated T-cells (NFAT) response element driving firefly luciferase, were used as effector cells. The engineered Jurkat cells (75,000 cells in 25 μl) were then added and co-cultured with antibody-treated target cells at 37˚C for 6 h. Luminescence using the Bio-Glo Luciferase Assay System was measured using a GloMax luminometer (Promega Corporation).

2.6. Immunohistochemical analysis

Formalin-fixed paraffin-embedded (FFPE) tissue of HER2-positive breast cancer was obtained from Sendai Medical Center [15]. Informed consent for sample procurement and subsequent data analyses was obtained from the patient or the patient’s guardian at Sendai Medical Center. Normal tissues were purchased from BioChain Institute Inc. (Eureka Drive Newark, CA) or Cybrdi Inc. (Frederick, MD). The tissue sections were autoclaved in citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20 min. The blocking was performed using SuperBlock T20 (Thermo Fisher Scientific Inc.). The sections were incubated with H₂Mab-250 (1, 0.5, or 0.1 μg/mL) and H₂Mab-119 (0.5 or 0.1 μg/mL), and then treated with the EnVision+ Kit for mouse (Agilent Technologies, Inc., Santa Clara, CA). The chromogenic reaction was performed using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies, Inc.). Counterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation) and Leica DMD108 (Leica Microsystems GmbH, Wetzlar, Germany) was used to obtain images and examine the sections.

2.7. ELISA

Synthesized peptides covering the HER2 extracellular domain IV and point mutant peptides were synthesized by Sigma-Aldrich Corp. The peptides (10 µg/mL) and HER2ec (1 µg/mL) were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc.). Plate washing was performed with PBS containing 0.05% (v/v) Tween 20 (PBST; Nacalai Tesque, Inc.). After blocking with 1% (w/v) BSA in PBST, H₂Mab-250 (10 µg/mL) was added to each well. Then, the wells were further incubated with peroxidase-conjugated anti-mouse immunoglobulins (1:2000 dilution; Agilent Technologies, Inc). Enzymatic reactions were conducted using One-Step Ultra TMB. The optical density at 655 nm was measured using an iMark microplate reader.

2.8. Determination of K_D via surface plasmon resonance (SPR)

Measurement of K_D between H₂Mab-250 and the HER2 peptides was performed using SPR. H₂Mab-250 was immobilized on the sensor chip CM5 in accordance with the manufacturer’s protocol by Cytiva. Immobilization of H₂Mab-250 (10 μg/mL in acetate buffer (pH 4.0); Cytiva) was carried out using an amine coupling reaction. The surface of the flow cell 2 of the sensor chip CM5 was treated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide (NHS), followed by the injection of H₂Mab-250. The K_D between H₂Mab-250 and the peptides was determined using Biacore X100 (Cytiva). The binding signals were measured using a single-cycle kinetics method. The data were analyzed by 1:1 binding kinetics using Biacore X100 evaluation software (Cytiva) to determine the association rate constant (ka) and dissociation rate constant (kd) and K_D. The affinity constant (K_A) at equilibrium was calculated as 1/K_D.

3. Results

3.1. Establishment of H₂Mab-250

We immunized mice with HER2ec produced by LN229 cells. The culture supernatants of hybridomas were screened using ELISA with HER2ec. We further screened the reactivity to HER2-positive breast cancers (BT-474 and SK-BR-3) and non-transformed normal epithelial cells including HaCaT (keratinocyte) and MCF 10A (mammary gland) using flow cytometry. One of the established hybridomas, H₂Mab-250 reacted with CHO/HER2, HER2-positive BT-474, and SK-BR-3 cells, but not with triple-negative MDA-MB-468 cells. H₂Mab-250 did not react with HaCaT and MCF 10A cells (Figure 1A). In contrast, H₂Mab-119 showed similar reactivity to both cancer and normal epithelial cells (Figure 1A).

We next investigated the difference in the reactivity to immortalized normal epithelial cells, including hTERT TIGKs (gingiva), HBEC3-KT (lung bronchus), hTERT-HME1 (mammary gland), hTCEpi (corneal), hTEC/SVTERT24-B (thymus), RPTEC/TERT1 (kidney proximal tubule), and HCEC-1CT (colon). H₂Mab-250 did not react with those normal cells, while H₂Mab-119 was reactive with all immortalized normal epithelial cells (Figure 1B), indicating that H₂Mab-250 possesses cancer-specific reactivity against HER2.

We further compared the reactivity of H₂Mab-250 with our established anti-HER2 mAbs and trastuzumab. As shown in Supplementary Figures 1 and 2, anti-HER2 domain I mAbs (H₂Mab-77 [15] and H₂Mab-139 [17]), anti-HER2 domain II mAbs (H₂Mab-193 and H₂Mab-215), anti-HER2 domain III mAbs (H₂Mab-19 [13] and H₂Mab-181 [18]), and anti-HER2 domain IV mAbs (H₂Mab-41 [14] and trastuzumab) reacted with HER2-positive breast cancers, non-transformed normal epithelial cells, and immortalized normal epithelial cells [16]. These results indicated that H₂Mab-250 exhibits an exceptional reactivity compared to other anti-HER2 mAbs.

3.2. The ability of effector cell activation by H₂Mab-250 and trastuzumab

The ADCC reporter bioassay is a bioluminescent reporter gene assay to quantify the biological activity of the antibody via FcγRIIIa-mediated pathway activation in an ADCC mechanism of action [23]. We next produced H₂Mab-250-mG_2a-f, the core-fucose deleted IgG_2a version of H₂Mab-250 using fucosyltransferase 8-deficient ExpiCHO-S (BINDS-09) cells and examined whether H₂Mab-250-mG_2a-f could activate ADCC program in the presence of BT-474 and HaCaT cells. To compare the ADCC pathway activation by H₂Mab-250-mG_2a-f and trastuzumab, we treated BT-474 and HaCaT cells with serially diluted mAbs, and then incubated with effector Jurkat cells, which express the human FcγRIIIa receptor and an NFAT response element driving firefly luciferase. As shown in Figure 2A, H₂Mab-250-mG_2a-f could activate the effector (EC₅₀: 9.9 µg/mL), but it was less effective than trastuzumab (EC₅₀: 0.053 µg/mL). Importantly, H₂Mab-250-mG_2a-f did not activate the effector in the presence of HaCaT cells. In contrast, trastuzumab activated the effector with similar EC₅₀ (0.036 µg/mL) to BT-474 cells (Figure 2B). These results indicated that H₂Mab-250-mG_2a-f selectively activates the effector cells against breast cancer cells.

3.3. Immunohistochemical analysis of H₂Mab-250 in breast cancer and normal epithelium

Immunohistochemical analysis was performed to examine the reactivity of H₂Mab-250 with normal and tumor tissue sections. In contrast to flow cytometry, H₂Mab-250 exhibited more potent reactivity to the HER2-positive breast cancer section than H₂Mab-119 (Figure 3A). Since all anti-HER2 therapeutic mAbs are associated with cardiotoxicity, a major adverse effect [10], the reactivity of H₂Mab-250 to a normal heart was further investigated. Even with higher concentrations of H₂Mab-250 (1 µg/mL), no reactivity with the normal heart could be detected (Figure 3B). Finally, the reactivity of H₂Mab-250 to other normal tissues was investigated. As shown in Figure 3C, no reactivity of H₂Mab-250 with any normal tissues, including breast, stomach, lung, colon, kidney, and esophagus could be observed.

3.4. Epitope identification for H₂Mab-250

To determine the epitope for H₂Mab-250, we examined the reactivity to CHO/HER2 (WT) and the N-terminal HER2 deletion mutants (dN218, dN342, and dN511)-expressed CHO-K1 cells (Figure 4A, left). H₂Mab-250 reacted with dN218, dN342, dN511, and HER2 (WT). In contrast, H₂Mab-119 reacted with only WT, but not with dN218, dN342, and dN511. Since HER2 (WT) and the deletion mutants possess PA16 tag at the N-terminus, all expression on the cell surface could be confirmed by anti-PA16 tag mAb, NZ-1 (Figure 4A, right). These results suggest that H₂Mab-250 and H₂Mab-119 recognize the domain IV and domain I, respectively.

For further assessment of the H₂Mab-250 epitope, ELISA was performed using synthetic peptides that cover the HER2 domain IV. As shown in Figure 4B, H₂Mab-250 reacted with HER2 domain IV peptide, amino acids 603–622, 613–632, but not with 593–612, 623–642, and 633–652, indicating that H₂Mab-250 recognizes the 613–622 of HER2 domain IV. We further used alanine-substituted peptides of the 603–622 in HER2 domain IV. A potent reduction of the reactivity was observed in the W614A peptide (Figure 4C). We confirmed that the reactivity of H₂Mab-250 completely disappeared in CHO/HER2 W614A cells in flow cytometry (Figure 4D).

The K_D of H₂Mab-250 with the alanine-substituted peptides of HER2 domain IV (603–622) was measured using Biacore X100 (Table 1). The affinity constant (K_A) at equilibrium was calculated as 1/K_D (Figure 4E). Compared to the K_A of the 603–622 (WT) peptide, decreased K_A values were observed from the 613–617 region, suggesting that the 613–617 region is involved in the binding to H₂Mab-250. A remarkable reduction was measured in the W614A peptide, indicating that Trp614 is mainly involved in the recognition by H₂Mab-250.

4. Discussion

In this study, we developed a cancer-specific mAb targeting HER2. H₂Mab-250 can recognize breast cancer cells, but not normal cells in flow cytometry (Figure 1) and immunohistochemistry (Figure 3). H₂Mab-250-mG_2a-f could activate ADCC against breast cancer cells, but not against normal epithelial cells (Figure 2). We also identified the H₂Mab-250 epitope sequence (_613-IWKFP_-617) by SPR analysis (Figure 4). The _613-IWKFP_-617 sequence is partially included with the wider binding epitope of trastuzumab (residues 579-625) [24]. Furthermore, no reaction was observed between H₂Mab-250 and CHO/HER2 W614A in flow cytometry (Figure 4), indicating that Trp614 plays a central role in recognition by H₂Mab-250. Although H₂Mab-250 possesses a high affinity to epitope-containing peptide (603–622) in SPR analysis, the recognition in flow cytometry using cell lines was lower than that of H₂Mab-119 (Figure 1). In contrast, H₂Mab-250 exhibited a higher reactivity than H₂Mab-119 in the immunohistochemical analysis using breast cancer tissues (Figure 3). This discrepancy might be induced by the possibility that the epitope sequence is partially exposed in cancer cells, but not in normal cells in clinical cancer tissues. The mechanism of recognition by H₂Mab-250 should be further investigated in future studies.

For the clinical treatment of metastatic breast cancer, trastuzumab is administered in patients with HER2-overexpressing tumors, which are defined by strong and complete IHC membranous staining of more than 10% of cells (IHC 3+) and/or in situ hybridization (ISH)-amplified. Furthermore, trastuzumab-based antibody-drug conjugates (ADCs) such as trastuzumab-deruxtecan (T-DXd) have been evaluated in various clinical trials. Based on the studies, T-DXd has been approved in not only HER2-positive breast cancers [25,26], but also HER2-mutant lung cancer [27] and HER2-low (IHC 1+ or IHC 2+ / ISH-non-amplified) advanced breast cancer [28]. A significant number of patients can benefit from T-DXd therapy since approximately half of all breast cancers are classifiable as HER2-low [29]. Meanwhile, cardiotoxicity is the most significant toxicity associated with T-DXd [30]. Further studies are essential to evaluate in vivo toxicities of H₂Mab-250.

H₂Mab-250-mG_2a-f could trigger the ADCC activity to BT-474 selectively (Figure 2). Although the effect of H₂Mab-250-mG_2a-f is lower than that of trastuzumab, we should consider that effector Jurkat cells express human FcγRIIIa receptor. In contrast, H₂Mab-250 exhibited a superior reactivity to HER2-positive breast cancer tissue sections in immunohistochemistry (Figure 3). Chimeric antigen receptor (CAR)-T cell therapy against HER2 has been evaluated in clinical studies [29]. It would be worthwhile to investigate the cancer specificity of H₂Mab-250 scFv and the efficacy of CAR-T against HER2-positive tumors in future studies. In the future, H₂Mab-250 could contribute to the development of CAR-T or ADCs without adverse effects for breast cancer therapy.