A Cancer-Specific Monoclonal Antibody against HER2 Exerts Antitumor Activities in Human Breast Cancer Xenograft Models

Monoclonal antibody (mAb)-based and/or cell-based immunotherapies provide innovative approaches to cancer treatments. However, safety concerns over targeting normal cells expressing reactive antigens still exist. Therefore, the development of cancer-specific mAbs (CasMabs) that recognize cancer-specific antigens with in vivo antitumor efficacy is required to minimize the adverse effects. We previously screened anti-human epidermal growth factor receptor 2 (HER2) mAbs and successfully established a cancer-specific anti-HER2 mAb, H2Mab-250/H2CasMab-2 (IgG1, kappa). In this study, we showed that H2Mab-250 reacted with HER2-positive breast cancer cells but did not show reactivity to normal epithelial cells in flow cytometry. In contrast, a clinically approved anti-HER2 mAb, trastuzumab, recognized both breast cancer and normal epithelial cells. We further compared the affinity, effector activation, and antitumor effect of H2Mab-250 with trastuzumab. The results showed that H2Mab-250 exerted a comparable antitumor effect with trastuzumab in the mouse xenograft models of BT-474 and SK-BR-3, although H2Mab-250 possessed a lower affinity and effector activation than trastuzumab in vitro. H2Mab-250 could contribute to the development of chimeric antigen receptor-T or antibody–drug conjugates without adverse effects for breast cancer therapy.


Introduction
The overexpression of human epidermal growth factor receptor 2 (HER2) is observed in approximately 20% of breast cancers [1] and 20% of gastric cancers [2], which are associated with higher rates of recurrence and shorter overall survival.HER2 forms heterodimers with other HER members and the ligands or ligand-independent homodimers when overexpressed [3].The formation of hetero-or homodimers leads to the activation of downstream signaling such as RAS-ERK and PI3K-AKT pathways, which promote cancer cell proliferation, survival, and invasiveness [3].A clinically approved anti-HER2 monoclonal antibody (mAb), trastuzumab, showed an anti-proliferative effect in vitro and a potent antitumor efficacy in vivo [4,5].In the treatment of breast cancer patients with metastasis, trastuzumab is administered in patients with HER2-overexpressed tumors, which are defined by strong and complete membranous staining of more than 10% of cells in immunohistochemistry (IHC 3+) and/or in situ hybridization (ISH)-amplified [6].The combination therapy of chemotherapeutic agents with trastuzumab improves objective response rates, progression-free survival, and overall survival in HER2-positive breast cancer patients with metastasis [7].Therefore, trastuzumab has become the most effective therapy for HER2-positive breast cancers [8] and HER2-positive gastric cancers [9].
The trastuzumab-based antibody-drug conjugates (ADCs) such as trastuzumabderuxtecan (T-DXd) have been evaluated in various clinical trials [10].Based on the studies, T-DXd has been approved in not only HER2-positive breast cancer [11,12] but also HER2-mutant lung cancer [13] and HER2-low (IHC 1+ or IHC 2+/ISH-non-amplified) advanced breast cancer [14].Since approximately half of all breast cancers are classifiable as HER2-low, a significant number of patients can benefit from T-DXd therapy [15].
Anti-HER2 therapeutic mAbs and the ADCs have common adverse effects such as cardiotoxicity [16].Patients must receive routine cardiac monitoring [17].Moreover, ErbB2 (ortholog of HER2)-knockout mice showed embryonic lethal phenotype because of the lack of cardiac trabeculae [18].The ErbB2-conditional knockout mice in the ventricular area displayed the features of dilated cardiomyopathy [19].These results indicate that HER2 is essential for normal heart development and homeostasis.Therefore, more selective anti-HER2 mAbs against cancers are necessary to reduce heart failures.
Trastuzumab is a humanized IgG 1 mAb that binds to Fcγ receptors (FcγRs) on various immune cells [23].The FcγR binding activates macrophages, dendritic cells, and neutrophils, which change adaptive immune responses by antigen presentation, cytokine production, and chemotaxis [4].Moreover, the FcγR engagement activates natural killer (NK) cells and macrophages, which can result in the target cell lysis, termed antibodydependent cellular cytotoxicity (ADCC) [4].In mouse mAbs, IgG 2a or IgG 2b can bind to FcγR with high affinity [24].Furthermore, a core fucose deficiency on the Fc N-glycan has been shown to enhance the binding of IgG to FcγR on effector cells [25] and exert potent antitumor effects [26].The defucosylated recombinant mAbs can be produced using fucosyltransferase 8-knockout Chinese hamster ovary (CHO) cells [27].
In this study, we produced a mouse IgG 2a -type mAb and a human IgG 1 -type mAb from H 2 Mab-250.We then compared the affinity, effector activation, and antitumor effect of H 2 Mab-250 with trastuzumab.

The Binding Affinity of H 2 Mab-250 and Trastuzumab
H 2 Mab-250 recognized HER2 expressed in breast cancers (BT-474 and SK-BR-3), but it did not recognize HER2 in normal epithelial cells, although trastuzumab recognized both types of HER2 [22].Since H 2 Mab-250 and trastuzumab are mouse IgG 1 and human IgG 1 , respectively, we generated the same isotype of recombinant mAbs (mouse IgG 2a or human IgG 1 ), which possess ADCC, to compare the antitumor activities between H 2 Mab-250 and trastuzumab.H 2 Mab-250-mG 2a and tras-mG 2a are mouse IgG 2a -type mAbs derived from H 2 Mab-250 and trastuzumab, respectively.H 2 Mab-250-hG 1 and trastuzumab are human IgG 1 -type mAbs.

The Ability of Effector Cell Activation by the Derivatives of H 2 Mab-250 and Trastuzumab
The mAb-FcγRIIIa binding-mediated ADCC pathway activation in effector cells can be quantified by a bioluminescent reporter gene assay, called the ADCC reporter bioassay [28].We previously showed that H 2 Mab-250-mG 2a selectively activates the effector cells against breast cancer cells, but not against normal cells.In contrast, trastuzumab activated the effector with a similar 50% effective concentration (EC 50 ) against breast cancer and normal cells [22].We next examined whether the derivatives of H 2 Mab-250 and trastuzumab could activate the ADCC pathway in the presence of BT-474 and SK-BR-3 cells.To compare the ADCC pathway activation by the derivatives of H 2 Mab-250 and trastuzumab, we treated BT-474 and SK-BR-3 cells with serially diluted mAbs, and then we incubated them with effector Jurkat cells, which express the human FcγRIIIa, and a firefly luciferase reporter gene driven by a nuclear factor of activated T cell (NFAT)-responsive element.As shown in Figure 3A, H 2 Mab-250-mG 2a could activate the effector (EC 50 : 1.6 × 10 −5 g/mL), but it was less effective than tras-mG 2a (EC 50 : 3.7 × 10 −8 g/mL) in the presence of BT-474 cells.H 2 Mab-250-mG 2a and tras-mG 2a also exhibited a similar relationship to EC 50 (3.9× 10 −6 g/mL and 9.0 × 10 −9 g/mL, respectively) in SK-BR-3 cells (Figure 3B).Furthermore, H 2 Mab-250-hG 1 could activate the effector (EC 50 : 1.4 × 10 −6 g/mL), but it was less effective than trastuzumab (EC 50 : 1.5 × 10 −8 g/mL) in BT-474 cells (Figure 3A).H 2 Mab-250-hG 1 and trastuzumab also exhibited a similar relationship to EC 50 (2.2× 10 −7 g/mL and 1.5 × 10 −9 g/mL, respectively) in SK-BR-3 cells (Figure 3B).These results indicated that H 2 Mab-250-mG 2a and H 2 Mab-250-hG 1 possess a lower ability of effector cell activation than tras-mG 2a and trastuzumab, respectively.The EC 50 was calculated using GraphPad PRISM 6.

Immunohistochemical Analysis by H 2 Mab-250 and Trastuzumab in Breast Cancer Tissue
We previously showed that H 2 Mab-250 could stain the HER2-positive breast cancer tissue, but not normal tissues, including the heart, breast, stomach, lung, colon, kidney, and esophagus in IHC [22].We next compared the reactivity of H 2 Mab-250 with tras-mG 2a using a formalin-fixed paraffin-embedded (FFPE) tissue of HER2-positive breast cancer.In contrast to the binding affinity, H 2 Mab-250 exhibited superior reactivity to the breast cancer cells over tras-mG 2a (Figure 4).H 2 Mab-250 showed clear tumor staining even at 1/20 concentration of tras-mG 2a (0.5 µg/mL) (Figure 4A).We also confirmed that H 2 Mab-250 did not stain normal breast and heart tissues (Figure 4B).
The body weight loss was slightly observed on days 6 and 13 in H 2 Mab-250-mG 2atreated BT-474 xenograft-bearing mice (Figure 5G), but there was no significant difference in SK-BR-3 xenograft-bearing mice (Figure 5H).
The body weight loss was slightly observed on days 6 and 13 in H2Mab-250-mG2atreated BT-474 xenograft-bearing mice (Figure 5G), but there was no significant difference in SK-BR-3 xenograft-bearing mice (Figure 5H).

Antitumor Activities by H 2 Mab-250-hG 1 and Trastuzumab
We next evaluated the antitumor activity of H 2 Mab-250-hG 1 and trastuzumab in the BT-474 and SK-BR-3 xenograft models.We injected H 2 Mab-250-hG 1 and trastuzumab, as well as control human IgG, intraperitoneally on days 7, 14, and 21 after BT-474 and SK-BR-3 inoculation.Furthermore, human NK cells were injected around the tumors on the same days of the mAb injection.We measured the tumor volume on days 7, 14, 21, and 28 following the inoculation.The H 2 Mab-250-hG 1 administration led to a significant
The benefit of low-affinity mAbs for therapeutic applications has been discussed.A low-affinity anti-EGFR mAb (K D : 3.4 × 10 −7 M) was efficiently taken up by cancer cells but not normal cells, which resulted in sufficient efficacy against tumor cells, but low toxicity against normal keratinocytes [33].An anti-HER3 mAb, Ab562, possesses ≈10-fold lower affinity (K D : 2~3 × 10 −8 M) than patritumab.The ADC AMT-562 exhibited sufficient antitumor effects with minimizing potential toxicity [34].Since H 2 Mab-250 exhibited no reactivity to normal epithelial cells in flow cytometry (Figure 1) and IHC [22], H 2 Mab-250 or H 2 Mab-250-ADC could exhibit antitumor efficacy with lower side effects.
H 2 Mab-250-mG 2a and H 2 Mab-250-hG 1 could trigger the ADCC activity to BT-474 and SK-BR-3 cells; however, the effects of H 2 Mab-250-mG 2a and H 2 Mab-250-hG 1 were also lower than that of tras-mG 2a and trastuzumab, respectively (Figure 3).In contrast, H 2 Mab-250 exhibited a superior reactivity to HER2-positive breast cancer tissue in immunohistochemistry (Figure 4).H 2 Mab-250 also recognized the HER2-positive breast cancer tissue in the absence of antigen retrieval (Figure S1).We previously identified the H 2 Mab-250 epitope as 613-IWKFP -617 in the HER2 domain IV.The epitope sequence is partially included with the wider binding epitope of trastuzumab (residues 579-625) [35].Furthermore, we identified Trp614 as a central amino acid in the recognition by H 2 Mab-250 [22].Since the similar in vivo antitumor efficacy of H 2 Mab-250-mG 2a and H 2 Mab-250-hG 1 was shown compared with tras-mG 2a and trastuzumab, respectively (Figures 5 and 6), the 613-IWKFP -617 sequence may be highly accessible by H 2 Mab-250 in vivo due to the unknown mechanism.Compared to the in vitro cell culture condition, in vivo tumor cells received various stresses such as hypoxia [36], nutrient deprivation [37], and abnormal redox state [38].Further studies are required for the influence of those stresses on the recognition by H 2 Mab-250 in cancer cells.
Chimeric antigen receptor (CAR)-T cell therapy against HER2 has been evaluated in clinical trials [15].CAR-T cells against CD19 have improved outcomes for patients with B-cell lymphoma.However, disease relapse commonly occurs in many patients [39,40].Although different mechanisms of immune escape have been demonstrated [41], the tumor cells from many relapsed patients did not exhibit the features of immune escape.Recently, additional mechanisms, such as trogocytosis, have been proposed [42].When CAR-T cells, possessing the high-affinity anti-CD19 FMC63-based CAR, are co-cultured with CD19positive lymphoma cells, the CAR-T cells strip CD19 from lymphoma cells and incorporate it into their plasma membrane [42].This is called "trogocytosis", which results in the emergence of antigen-negative target cells.Furthermore, the CAR-T cells that acquired CD19 by trogocytosis can be killed by the other CD19 CAR-T cells [42].A promising approach to limit CAR-T cell-mediated trogocytosis is the reduction of CAR affinity [43].CD19-targeting CAR with ≈40-fold lower affinity (K D : 1.4 × 10 −8 M) than the clinically approved FMC63-based CAR (K D : 3.3 × 10 −10 M) was developed [44].The reduced affinity CAR-T cells exhibited higher efficacy and persistence than FMC63-based CAR-T cells in a mouse model [44], as well as robust antitumor efficacy and persistence in two clinical trials [44,45].These data show that it is possible to significantly limit trogocytosis by reducing CAR affinity while maintaining antitumor activity as well as clinical efficacy.The property of H 2 Mab-250 could contribute to the development of HER2-targeting CAR-T cells (now in a clinical phase I study in the US) by limiting trogocytosis and maintaining cancer specificity.
All cell lines were cultured at 37 • C in a humidified atmosphere with 5% CO 2 and 95% air.

Production of Recombinant mAbs
To generate recombinant H 2 Mab-250, the V H cDNAs and the C H cDNA of mouse IgG 1 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 Co., Ltd., Kagawa, Japan) was used to purify the recombinant H 2 Mab-250.
To generate mouse IgG 2a -type H 2 Mab-250 (H 2 Mab-250-mG 2a ), we cloned the V H cDNA of H 2 Mab-250 and C H of mouse IgG 2a into the pCAG-Ble vector.The mouse kappa light chain vector of H 2 Mab-250 was described above.To generate a mouse IgG 2a type of trastuzumab (tras-mG 2a ), the V H cDNA of trastuzumab and the C H cDNA of mouse IgG 2a were cloned into the pCAG-Neo vector, and the V L cDNA of trastuzumab and the C L cDNA of mouse kappa light chain were cloned into the pCAG-Ble vector.
Normal human IgG was purchased from Sigma-Aldrich Corp.

ADCC Reporter Bioassay
The ADCC reporter bioassay was performed using an ADCC Reporter Bioassay kit (Promega Corporation, Madison, WI, USA), according to the manufacturer's instructions.Target cells (BT-474 and SK-BR-3, 12,500 cells per well) were cultured in a 96-well white solid plate.H 2 Mab-250-mG 2a , H 2 Mab-250-hG 1 , tras-mG 2a , and trastuzumab were serially diluted and added to the target cells.Jurkat cells stably expressing the human FcγRIIIa receptor and a 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).

Immunohistochemical Analysis
FFPE tissue of HER2-positive breast cancer was obtained from the Sendai Medical Center [20].Informed consent for sample procurement and subsequent data analyses was obtained from the patient or the patient's guardian at the Sendai Medical Center.A normal tissue array (B901064) was purchased from BioChain Institute Inc. (Eureka Drive, Newark, CA, USA).The antigen retrieval was performed by autoclave 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 H 2 Mab-250 (10, 1, 0.5 or 0.1 µg/mL) and tras-mG 2a (10 µg/mL) and then treated with the EnVision+ Kit for mouse (Agilent Technologies, Inc., Santa Clara, CA, USA).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.To examine the antitumor effect of H 2 Mab-250-mG 2a tras-mG 2a , H 2 Mab-250-hG 1 , and trastuzumab, animal experiments were approved by the Institutional Committee for Experiments of the Institute of Microbial Chemistry (approval no.2023-060 or 2023-066).During the experimental period, we monitored mice maintained in a pathogen-free environment on an 11 h light/13 h dark cycle with food and water supplied ad libitum.Mice were monitored for health and weight every one or five days.We identified body weight loss exceeding 25% and maximum tumor size exceeding 3000 mm 3 as humane endpoints and terminated the experiments.
The tumor volume was calculated using the following formula: volume = W 2 × L/2, where W is the short diameter and L is the long diameter.All mice were euthanized by cervical dislocation.

Conclusions
A cancer-specific anti-HER2 mAb, H 2 Mab-250, exhibited antitumor efficacy in vivo.In the future, H 2 Mab-250 could contribute to the development of CAR-T or ADCs without adverse effects for breast cancer therapy.
Institutional Review Board Statement: The animal experiment to examine the antitumor effect was approved by the Institutional Committee for Experiments of the Institute of Microbial Chemistry (approval nos.2023-060 and 2023-066).
Informed Consent Statement: Informed consent for sample procurement and subsequent data analyses was obtained from the patient or the patient's guardian at the Sendai Medical Center.

Figure 3 .
Figure 3.The ADCC reporter assay by H 2 Mab-250-mG 2a , H 2 Mab-250-hG 1 , tras-mG 2a , and trastuzumab in the presence of BT-474 and SK-BR-3 cells.Target HER2-positive breast cancer cells such as BT-474 (A) or SK-BR-3 (B) were cultured in a 96-well white solid plate.H 2 Mab-250-mG 2a , H 2 Mab-250-hG 1 , tras-mG 2a , and trastuzumab were serially diluted and added to the target cells (n = 3).The engineered Jurkat cells were then added and co-cultured with antibody-treated target cells.Luminescence using the Bio-Glo Luciferase Assay System was measured using a GloMax luminometer (Promega Corporation, Madison, WI, USA).Values are presented as the mean ± SD.The EC 50 was calculated using GraphPad PRISM 6.