Defucosylated Monoclonal Antibody (H2Mab-139-mG2a-f) Exerted Antitumor Activities in Mouse Xenograft Models of Breast Cancers against Human Epidermal Growth Factor Receptor 2

The clinically approved human epidermal growth factor receptor 2 (HER2)-targeting monoclonal antibodies (mAbs), trastuzumab, and pertuzumab, target domains IV and II, respectively. Trastuzumab is now the standard treatment for HER2-overexpressed breast and gastric cancers, and trastuzumab in combination with pertuzumab showed clinical benefit. However, there still exist patients who do not respond to the therapy. Furthermore, HER2 mutants that cannot be recognized by pertuzumab were found in tumors. Therefore, novel anti-HER2 mAbs and modalities have been desired. In our previous study, we developed a novel anti-HER2 domain I mAb, H2Mab-139 (mouse IgG1, kappa). We herein produced a defucosylated mouse IgG2a type of mAb against HER2 (H2Mab-139-mG2a-f) to enhance antibody-dependent cellular cytotoxicity (ADCC)-mediated antitumor activity. H2Mab-139-mG2a-f exhibits a high binding affinity in flow cytometry with the dissociation constant (KD) determined to be 3.9 × 10−9 M and 7.7 × 10−9 M against HER2-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/HER2) and HER2-positive BT-474 cells, respectively. Moreover, we showed that H2Mab-139-mG2a-f exerted ADCC and complement-dependent cytotoxicity against CHO/HER2 and BT-474 in vitro and exhibited potent antitumor activities in mouse xenograft models. These results indicated that H2Mab-139-mG2a-f exerts antitumor effects against HER2-positive human breast cancers and is useful as an antibody treatment for HER2-positive human cancers.


Introduction
Human epidermal growth factor receptor 2 (HER2) is included in the receptor tyrosine kinase family of human epidermal growth factor receptor (EGFR).The HER activation is controlled by EGF-family ligands under physiological conditions.The formation of multiple combinations of HER homo-and heterodimers is induced by ligand binding, which triggers the activation of the cytoplasmic tyrosine kinase domain.The activation of several downstream signaling pathways, such as the RAS/RAF/MAPK and PI3K/AKT pathways [1], is induced by the autophosphorylation of specific tyrosine residues.HER2 does not have ligands and cannot form ligand-dependent homodimers, unlike EGFR, HER3, and HER4.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.HER2 possesses four extracellular domains (I-IV) [2].Domain II is known to be essential for the heterodimer formation with other HER members, such as EGFR, HER3, and HER4 in the presence of their ligands, such as EGF [3] and neuregulin 1 (NRG1, a HER3 ligand) [4].
HER2 is overexpressed in approximately 18% of breast cancers and is associated with higher rates of recurrence, poor prognosis, and shorter overall survival [5].HER2 overexpression is also observed in ~20% of gastric cancers [6].A monoclonal antibody (mAb) against domain IV of HER2, trastuzumab, exhibited an anti-proliferating effect in vitro and a potent antitumor effect in vivo [7,8].The addition of trastuzumab to chemotherapy improves objective response rates, progression-free survival, and overall survival in HER2positive breast cancer patients with metastasis [9].Trastuzumab has become the standard treatment for HER2-positive breast cancers [10] and HER2-positive gastric cancers [11].For more than 20 years, trastuzumab has been the most effective therapy for HER2-positive breast cancer [12].
Clinically, the efficacy of trastuzumab involves immunologic engagement [8].The Fc domain of trastuzumab mediates engagement with Fcγ receptors (FcγRs) on various immune cells.The binding of trastuzumab to FcγR facilitates the phagocytosis of antibodybound tumor cells, a process known as antibody-dependent cellular phagocytosis (ADCP).The FcγR engagement also activates macrophages, dendritic cells, and neutrophils, that change adaptive immune responses by antigen presentation, cytokine production, and chemotaxis.Moreover, the FcγR engagement activates natural killer (NK) cells, which can result in the lysis of the target tumor cells, termed antibody-dependent cellular cytotoxicity (ADCC) [13].To improve the FcγRIIIA engagement and ADCC activity, margetuximab was developed by introducing several optimization mutations of trastuzumab [14].Margetuximab was approved by the U.S. Food and Drug Administration (FDA) and showed significant improvement in progression-free survival in heavily pretreated patients [15,16].Moreover, the Fc domain of these mAbs can exert complement-dependent cytotoxicity (CDC) [17,18].
Another clinically approved HER2-targeting mAb, pertuzumab, binds to the domain II and prevents NRG1-induced heterodimerization with HER3 and intracellular signaling [19].The heterodimerization is known to be an important mechanism for resistance to trastuzumab [19].Therefore, pertuzumab is considered to possess a complementary mechanism to trastuzumab [20].The first-line treatment combining trastuzumab, pertuzumab, and chemotherapy has been evaluated and demonstrated clinical benefits [21].The double anti-HER2 blockade has been the standard therapy in the initial management of metastatic HER2-positive breast cancer [9].However, HER2 (S310F/Y) is the most frequent oncogenic missense mutation which cannot be recognized by pertuzumab [22].
In our previous studies, we established anti-HER2 mAbs including H 2 Mab-139 (IgG 1 , kappa) [23] by the immunization of HER2 ectodomain.Those mAbs have been revealed to recognize the domain I of HER2, and are available for flow cytometry, western blotting, and immunohistochemistry (IHC) [23].Mouse IgG 1 cannot bind to mouse FcγRIV which is essential for the activation of effector cells such as macrophage.In contrast, mouse IgG 2a or IgG 2b can bind to it with high affinity [24].Furthermore, a core fucose deficiency on the Fc N-glycan has been shown to enhance the binding to Fc receptors on effector cells [25].Therefore, we have demonstrated that class-switched (from IgG 1 to IgG 2a ) and defucosylated IgG 2a mAbs exhibited a superior ability to activate effector cells and exerted potent antitumor effects in several mouse xenograft models [26].The defucosylated recombinant mAbs can be produced using fucosyltransferase 8 (FUT8)-knockout (KO) Expi-CHO-S cells [27].
In this study, we produced a defucosylated IgG 2a type of anti-HER2 mAb (H 2 Mab-139-mG 2a -f) and evaluated the ability to induce ADCC/CDC in vitro or antitumor efficacy in vivo against HER2-positive and HER2-negative breast cancer cells.

Animal Experiments for ADCC Assay and Mice Xenograft Model
Every animal experiment for ADCC and antitumor activity by H 2 Mab-139-mG 2a -f was approved by the Institutional Committee for Experiments of the Institute of Microbial Chemistry (Numazu, Japan; approval no.2022-056, 2023-001, and 2023-018).Mice were monitored and maintained as described previously [28].

IHC Analysis
A paraffin-embedded breast cancer tissue microarray (T8235721-5, BioChain Institute Inc., Eureka Drive Newark, CA, USA) was autoclaved for 20 min using Envision FLEX TARGET RETRIEVAL SOLUTION High pH.We used SuperBlock T20 (Thermo) for blocking to inhibit the non-specific binding of mAbs to sections.The sections were treated with 10 µg/mL of H 2 Mab-139-mG 2a -f for 1 h at room temperature and then incubated with the EnVision+ Kit for mouse (Agilent) for 30 min.The chromogenic reaction and counterstaining were performed using 3,3 -diaminobenzidine tetrahydrochloride (DAB; Agilent) and hematoxylin (Wako), respectively.
We determined the cytotoxicity (% lysis) as follows: % lysis = (E − S)/(M − S) × 100 "E" indicates the fluorescence in cultures of both effector and target cells, "S" indicates the spontaneous fluorescence of only target cells, "M" indicates the maximum fluorescence following the treatment with a lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM EDTA, and 0.5% Triton X-100].

Statistical Analyses
All data are shown as mean ± standard error of the mean (SEM).Welch's t-test was used for the statistical analyses in ADCC, CDC, and tumor weight.ANOVA with Sidak's post hoc test was used in tumor volume and mouse weight.GraphPad Prism 8 (GraphPad Software, Inc.) was utilized for the calculations.A p < 0.05 was considered to indicate a statistically significant difference.

Detection of HER2 Using H 2 Mab-139-mG 2a -f in Western Blot and IHC Analyses
We next performed western blot analysis using H 2 Mab-139-mG 2a -f.As shown in Figure 2A, H 2 Mab-139-mG 2a -f strongly detected HER2 as more than 180-kDa bands in LN229/HER2 and BT-474 cells.H 2 Mab-139-mG 2a -f faintly detected endogenous HER2 in LN229 cells, but not MDA-MB-468 cells.The expression of IDH1 detected by RcMab-1 was used as an internal control (Figure 2B).These results indicated that H 2 Mab-139-mG 2a -f could detect exogenous and endogenous HER2 in western blot analysis.
could detect exogenous and endogenous HER2 in western blot analysis.
Next, IHC analyses against the formalin-fixed paraffin-embedded (FFPE) sections of breast cancer tissue were performed using H2Mab-139-mG2a-f.As shown in Figure 2C-H H2Mab-139-mG2a-f could distinguish HER2-strong positive (Figure 2C,D), moderate (Figure 2E,F), and negative (Figure 2G,H) breast cancers.The HER2-positive staining was mainly observed on the plasma membrane.We summarized the results of HER2 expression in breast cancer tissue array in Supplementary Table S1.H2Mab-139-mG2a-f stained 10 out of 63 cases (16%) of breast cancers.These results indicated that H2Mab-139-mG2a-f is also available for IHC analysis of FFPE tumor sections.Next, IHC analyses against the formalin-fixed paraffin-embedded (FFPE) sections of breast cancer tissue were performed using H 2 Mab-139-mG 2a -f.As shown in Figure 2C-H S1.H 2 Mab-139-mG 2a -f stained 10 out of 63 cases (16%) of breast cancers.These results indicated that H 2 Mab-139-mG 2a -f is also available for IHC analysis of FFPE tumor sections.
The weight of CHO/HER2 tumors treated with H2Mab-139-mG2a-f was sign lower than that treated with mIgG (66% reduction; p < 0.05; Figure 4B).CHO/HER2 that were resected from mice on day 26 are demonstrated in Figure 4C.
The body weight loss and skin disorders were not detected in CHO/HER2 bearing mice (Figure 4D).The mice on day 26 were shown in Supplementary Figu We then examined whether H 2 Mab-139-mG 2a -f could exhibit CDC against CHO/HER2 cells.As shown in Figure 3C, H 2 Mab-139-mG 2a -f elicited a higher degree of CDC (62.5% cytotoxicity) in CHO/HER2 cells compared with that elicited by control mIgG 2a (15.6% cytotoxicity; p < 0.05).There was no difference between H 2 Mab-139-mG 2a -f and control mIgG 2a in CDC for CHO-K1 (Figure 3D).These results showed that H 2 Mab-139-mG 2a -f exerted significantly high levels of ADCC/CDC against CHO/HER2 cells.
The body weight loss and skin disorders were not detected in CHO/HER2 tumorbearing mice (Figure 4D).The mice on day 26 were shown in Supplementary Figure S1.
The body weight loss was not detected in both BT-474 and MDA-MB-468 xenograftbearing mice (Figure 6G,H).The mice on day 28 about BT-474 and MDA-MB-468 xenograft were demonstrated in Supplementary Figure S2.In the BT-474 xenograft models, we injected H 2 Mab-139-mG 2a -f and control mIgG intraperitoneally on days 7, 14, and 21 after BT-474 inoculation.We measured the tumor volume on days 7, 10, 14, 16, 21, 24, and 28 following the inoculation.The H 2 Mab-139-mG 2af administration led to a significant reduction in BT-474 xenograft on days 21 (p < 0.01), 24 (p < 0.01), and 28 (p < 0.01) compared with that of the control mIgG (Figure 6A).The H 2 Mab-139-mG 2a -f administration resulted in a 36% reduction of tumor volume compared with that of the control mIgG on day 28.
The body weight loss was not detected in both BT-474 and MDA-MB-468 xenograftbearing mice (Figure 6G,H).The mice on day 28 about BT-474 and MDA-MB-468 xenograft were demonstrated in Supplementary Figure S2.

Discussion
Trastuzumab is clinically administered for patients with HER2-overexpressing metastatic breast cancers, 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.Based on clinical studies, the 5th European School of Oncology and the European Society of Medical Oncology guidelines for advanced breast cancer (ABC 5) and the National Comprehensive Cancer Network guidelines consider trastuzumab (anti-HER2 domain IV mAb), pertuzumab (anti-HER2 domain II mAb), and docetaxel as the standard of care for first-line treatment of HER2-positive metastatic breast cancer [29].However, most deaths in the study were due to breast cancer [21].Therefore, better treatments including novel combination therapies and novel modalities are still needed.In this study, we evaluated a novel anti-HER2 domain I mAb, H 2 Mab-139-mG 2a -f, and showed the ADCC activity in vitro (Figures 3 and 5) and antitumor effect in vivo (Figures 4 and 6).Therefore, H 2 Mab-139-mG 2a -f could be an antibody treatment regimen for HER2-positive breast cancer.
The structures of the HER2-HER3-NRG1β complex, revealed by cryo-EM, exhibit a dynamic dimer interface.In the complex, the NRG1β-bound HER3 dimerization arm remains unresolved due to the lack of a ligand-induced conformational change in the apo HER2 monomer, which is essential for the formation of the HER3 dimerization armbinding pocket [22].In contrast, the most frequent oncogenic HER2 mutation (S310F/Y) was found primarily in cancers without HER2 overexpression.The HER2 S310 is localized in the dimerization arm-binding pocket of domain II [30].The structures of HER2 (S310F)-HER3-NRG1β complex exhibited stabilizing interactions with the HER3 dimerization arm and compensate for the inability of HER2 to undergo a needed conformational change [22].Furthermore, HER2-HER3 and HER2 (S310F)-HER3 retain the ability to bind to trastuzumab, but the mutant complex does not bind to pertuzumab [22].These results suggest that pertuzumab is less effective at targeting cancers driven by HER2 (S310F), and different epitope-possessing anti-HER2 mAbs including H 2 Mab-139 could be required for the combination therapy with trastuzumab.
Given that approximately half of all breast cancers are classifiable as HER2-low [36], a greater number of patients may benefit from T-DXd therapy.These results have had a significant impact on the field of breast oncology, particularly in the future clinical diagnostics of HER2-low breast cancer.As a result, future treatment algorithms for both hormone receptor-positive and TNBC are anticipated to change [37].There are several challenges in elucidating the biological roles and pathological significance of HER2-low [38].Since our H 2 Mab-139 is applicable for IHC (Figure 2), it would be valuable to compare its reactivity with approved anti-HER2 diagnostic mAbs such as HercepTest TM and PATHWAY ® .
We achieved increased ADCC activity of H 2 Mab-139-mG 2a -f through class switching and a core fucose deficiency on the N-glycan in the Fc region, which promotes the binding of Fc to FcγRIIIa on effector cells [25].This technique is also applied to mogamulizumab (Poteligeo), a defucosylated humanized mAb targeting CCR4 [39].In contrast, margetuximab is derived from trastuzumab and shares the same epitope with HER2.Five amino acid substitutions in the Fc domain of margetuximab (human IgG 1 ) achieve increased binding to FcγRIIIa and reduced binding to an inhibitory FcγR, FcγRIIb, when compared to trastuzumab [9].We are going to apply the strategy to potentiate the ADCC activity when we generate the humanized H 2 Mab-139 mAb.
We previously produced a bispecific Ab against EGFR and HER2 from our established anti-EGFR mAb (EMab-134) and an anti-HER2 mAb (H 2 Mab-77) [40].The bispecific Ab possesses the tetravalent structure by fusing the single chain Fv of H 2 Mab-77 at the light chains of EMab-134 and showed the antitumor effect in the mouse xenograft model [40].Since we can produce the different types of bispecific Abs and have various clones of anti-HER2 mAbs including H 2 Mab-139 (see Supplementary Materials), we will investigate the activity in future studies.
Previously, we established H 2 Mab-139 using cancer cell-produced HER2 ectodomain as an immunogen.This methodology is essential for the development of cancer-specific mAbs (CasMabs).We have developed CasMabs that target podoplanin (PDPN) [41], which recognize the aberrant glycosylation patterns typical of cancer cells [42].Anti-PDPN-CasMabs are currently applied to CAR-T therapy in preclinical models [43,44].For the development of anti-HER2 CasMab, we need to perform further screening of our already established anti-HER2 mAbs (more than 200 clones), comparing their reactivity against normal cells [45,46].Anti-HER2 CasMabs could be employed in designing modalities including ADCs and CAR-T.

, H 2
Mab-139-mG 2a -f could distinguish HER2-strong positive (Figure 2C,D), moderate (Figure 2E,F), and negative (Figure 2G,H) breast cancers.The HER2-positive staining was mainly observed on the plasma membrane.We summarized the results of HER2 expression in breast cancer tissue array in Supplementary Table