A Review of Margetuximab-Based Therapies in Patients with HER2-Positive Metastatic Breast Cancer

Simple Summary HER2+ metastatic breast cancer (MBC) is a highly prevalent type of breast cancer owing to its resistance to conventional anti-HER2 drugs. Therefore, novel agents that can arrest tumor progression and enhance the overall survival rates of HER2+ breast cancer patients, which would represent a major advancement toward the treatment of HER2 + MBC, need to be developed. This review provides insights into the clinical implication and utility of margetuximab, an anti-HER2 drug, in HER2 + MBC treatment, focusing on studies on the efficacy of margetuximab. Margetuximab is indeed an excellent addition to the arsenal of anti-HER2 mAb drugs that can be used for treating HER2 + MBC. Abstract Breast cancer (BC) is the most commonly diagnosed cancer globally, with high mortality rates. Targeted drug therapies have revolutionized cancer treatment. For example, treatment with human epidermal receptor 2 (HER2) antagonists has markedly improved the prognosis of patients with HER2-positive BC (HER2 + BC). However, HER2+ metastatic BC (MBC) remains prevalent owing to its resistance to conventional anti-HER2 drugs. Therefore, novel agents are needed to overcome the limitations of existing cancer treatments and to enhance the progression-free and overall survival rates. Progress has been made by optimizing the fragment crystallizable (Fc) domain of trastuzumab, an IgG1 monoclonal, chimeric anti-HER2 antibody, to develop margetuximab. The modified Fc domain of margetuximab enhances its binding affinity to CD16A and decreases its binding affinity to CD32B, thereby promoting its antitumor activity. This review summarizes studies on the efficacy of margetuximab, discusses its utility as an anti-HER2 monoclonal antibody drug for the treatment of HER2 + BC, and presents the latest advances in the treatment of BC. This review provides insights into the clinical implication of margetuximab in HER2 + MBC treatment.

HER2 (also known as ErbB2, C-erbB2, or Her2/neu) is a tyrosine kinase protein belonging to the epidermal growth factor receptor (EGFR) family [6]. HER2 expression (iii) cytoplasmic carboxyl-terminal tail with multiple phosphorylation sites, is essential for the activation of downstream signaling pathways [18]. The tyrosine kinase domain is essential for the dimerization of HER receptors. Ligand binding by the receptors is essential for the activation of the tyrosine kinase domains, which facilitates their dimerization. Dimerization can take place between molecules of the same receptor (homodimerization) or between two different HER receptors (heterodimerization) [19][20][21][22].
When inactive, i.e., in the absence of a ligand, these receptors exist as monomers in a tethered conformation due to the intramolecular interactions between domains II and IV. In this tethered conformation the "dimerization arm" (β-hairpin/loop) that exists in cysteinerich domain II, is completely buried in the tethering arm of domain IV. This restricts the movement of receptor ECD arms and stabilizes the tethered receptor conformation, which is responsible for the auto-inhibition of ligand binding and dimerization [20,23]. However, ligand binding to domains I and III induces a conformational change that destabilizes the intramolecular tether and prevents the autoinhibitory effect. This exposes the dimerization arm and consequently allows dimerization.
Ligand-induced dimerization activates intracellular tyrosine kinase and enables it to assume an asymmetric active structure to create pTyr docking sites via kinase trans-tyrosine phosphorylation. These docking sites lodge the Src homology 2 (SH2) and phosphotyrosine binding (PTB) motifs of phosphotyrosine-binding proteins (e.g., Grb2, Shc, and PLCγ) [23]. The specific interaction of the pTyr-binding motifs with signaling proteins activates downstream signaling pathways, including the Ras-mitogen activated protein kinase (MAPK), Ras/Raf/MEK/MAPK, phosphoinositide-3-kinase (PI3K)-Akt, phospholipase C-gamma (PLC-γ), Src signaling, and signal transducer and activator of transcription (STAT) pathways [20]. These signaling pathways regulate essential cellular processes, including cell proliferation, migration, motility, differentiation, and apoptosis [24]. However, the cellular effects mainly depend on the intermediate pathway that is activated, and the magnitude and duration of ligand binding, which is further diversified by various ligands and the dimer in question [21].
Except for HER2, all ErbB family receptors directly bind to ligands. This is due to HER2's ECD lacking a known ligand-binding domain [18,19]. It is therefore thought that HER2 functions as a co-receptor with, or dimerization partner of, other ErbB receptors. Similarly, all ErbB receptors exhibit tyrosine kinase activity, except for HER3, which has no or minimal catalytic kinase activity [22]. Interestingly, heterodimerization between HER2 and HER3 exhibits the most potent pro-tumorigenic and mitogenic signaling activity [23].

Unique Characteristics of HER2 Promote Tumor Progression in BC
The HER2 protein has several fates ( Figure 1). For example, as a monomer, as a homodimer, and as a HER2/HER3 or HER2/HER4 heterodimer [19]. The TK domain of HER2 is activated upon homo/hetero dimerization, evoking a signaling cascade that activates the receptive gene, leading to cell proliferation, migration, invasion, or cell survival, which are the hallmarks of cancer. The activation of the signaling cascades contributes to the heterogeneity of different cancer types (e.g., ovarian cancer, BC, and non-small cell lung cancer).
The overexpression/amplification of ErbB receptors shows a significant correlation with poor prognosis, cancer metastasis, decreased survival rates, and enhanced drug resistance [25]. The activation of HER2 signaling pathways is mainly responsible for cellular proliferation and cell survival phenomena, which are regarded as dominant drivers that cause tumor development and progression in nearly 85% of BC cases [26]. The amplification/overexpression of the HER-neu proto-oncogene and the HER2/HER3 heterodimer that activates oncoproteins have promiscuous roles in the pathogenesis of solid tumors [27]. Moreover, a truncated HER2 protein that lacks ECD-p95 (p95HER2) and an active Cterminal fragment, is detected in nearly 40% of HER2 + BC cases [28]. Therefore, p95HER2 is used as a predictive biomarker for cancer prognosis. Moreover, it is used to evaluate the efficacy of, or resistance to, existing treatments for BC [29,30]. terminal fragment, is detected in nearly 40% of HER2+BC cases [28]. Therefore, p95HER2 is used as a predictive biomarker for cancer prognosis. Moreover, it is used to evaluate the efficacy of, or resistance to, existing treatments for BC [29,30].
The HER family of tyrosine kinases (TK) receptors have various ligand-binding capabilities that help orchestrate several biological processes in normal cells. The HER family of receptors comprises HER1, HER2, HER3, and HER4, depicted in blue, magenta, golden, and green, respectively. All members of the HER receptor family are structurally similar, containing an extracellular domain with a ligand-binding site (except HER2), a lipophilic transmembrane domain, and a TK intracellular domain (except for HER3). The TK domain of HER1, HER2, and HER4 is activated following homo-or heterodimerization and evokes a signaling cascade that activates a receptive gene, leading to cell proliferation, migration, invasion, or cell survival.

Current Insights on Margetuximab, a Novel Anti-HER2 Drug for Treatment of Positive Metastatic Breast Cancer
Research to develop anti-HER2-personalised therapeutic agents is being conducted at a rapid pace, which is evident based on the number of molecules being introduced into clinical trials. Rituximab initiated an era of immunotherapeutics, with mAbs extensively employed afterward to target tumors [47][48][49][50]. The therapeutic functionality of mAbs depends on the interactions of two regions thereof with components of the host immune system: the fragment antigen binding (Fab) region that binds to the antigen and the fragment crystallizable (Fc) region that interacts with the FcγRs and C1 complex (C1q) components of the immune system [51][52][53][54]. The mAbs exert their cytotoxic actions by promoting the interaction between Fc and FcγRs to activate the innate immune response by engaging complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent cellular cytotoxicity (ADCC) [51,[55][56][57].
Antibody-dependent cellular cytotoxicity involves a cascade of mechanisms that target the FcRIIIa (CD16A) receptor on the cell surface and the Fc domains of immunoglobulins. It acts by enhancing natural killer (NK) cells, monocytes or macrophages (CD16+ subsets), and NKT cells or γδ T cells to enhance cytolysis and exert antitumor effects [58][59][60][61][62][63]. The FcγR family in humans comprises activating receptors and inhibitory receptors. Activating receptors include FcγRI (CD64, a high-affinity receptor), FcγRIIa (CD32a), FcγRIIIa (CD16a), and GPI-linked FcγRIIIb (low affinity); inhibitory receptors include FcγRIIb. The CD16A, CD32A, and CD32B receptors are expressed on effector cells and regulate immune activation processes. Human CD16a is a transmembrane low-affinity IgG Fc receptor [64] that triggers immune NK cells for their ADCC effects via the lone FcγR present in the NK cells [65,66]. Mutations in the FcγRIIIa gene generate two FcγRIIIa polymorphs with valine (V) and phenylalanine (F) at amino acid position 158 (FcγRIIIA-V158F). This polymorphism is considered crucial as it influences the rate of tumor cell lysis via ADCC, with the high-affinity valine v/v allele being responsible for more lysis than the V/F or FF alleles [51,52].
HER2 + MBC was successfully treated using the humanized IgG1 antibody, trastuzumab [53]. This was the first anti-HER2 mAb approved by the US FDA. It hinders HER1 activity by modulating extracellular HER2-neu [54]. Specifically, trastuzumab binds to the ECD of the HER2 receptor to inhibit its homodimerization and destabilize its heterodimers. Furthermore, it inhibits the formation of p95HER2, implicated in tumor progression. Moreover, trastuzumab can mediate ADCC via the activation of NK cells, made possible by the detection of the Fc portion thereof, ultimately leading to the death of cancer cells. Trastuzumab triggers HER2 internalization followed by lysosomal degradation and activates the c-Cbl-ubiquitin ligase-mediated ubiquitination and degradation of HER2. Trastuzumab also prevents matrix metalloproteinase (MMP)-mediated HER2 shedding. All of these effects ultimately lead to the inhibition of downstream signaling cascades [55,56]. The pathways inhibited by trastuzumab include the PI3K pathway, where decreasing phosphatase and tensin Homologue (PTEN) phosphorylation and AKT dephosphorylation increases cell death. Trastuzumab also inhibits the MAPK pathway, which activates the cyclin-dependent kinase inhibitor p27 KIP1 and promotes cell-cycle arrest and apoptosis. These actions show that trastuzumab exhibits anti-proliferative and anti-angiogenic effects, ultimately leading to the death of cancer cells. However, a major setback of this drug is the occurrence of drug resistance [54].
Novel immunotherapeutic agents are being developed to overcome resistance and enhance the overall survival of HER2 + BC patients. One such drug, margetuximab (MGAH22), was authorized by the US FDA on 16 December 2020 [57,58].

Pharmacology of Margetuximab
Fc-engineering strategies have been used over the years to customize mAbs to enhance their cytotoxic and antitumor potencies, margetixumab is the consequence of that effort. This drug, developed by MacroGenics, is a novel IgG1 monoclonal human/mouse chimeric antibody engineered in its Fc-domain to target HER2 ( Figure 2) and is derived from 4D5, a precursor to trastuzumab [59]. The Fc-engineered domain of margetuximab exhibits mutations of five amino acid components (L235V, F243L, R292P, Y300L, and P396L) [67][68][69].
setback of this drug is the occurrence of drug resistance [54].
Novel immunotherapeutic agents are being developed to overcome resistance enhance the overall survival of HER2+BC patients. One such drug, margetux (MGAH22), was authorized by the US FDA on 16 December 2020 [57,58].

Pharmacology of Margetuximab
Fc-engineering strategies have been used over the years to customize mAbs to hance their cytotoxic and antitumor potencies, margetixumab is the consequence of effort. This drug, developed by MacroGenics, is a novel IgG1 monoclonal human/m chimeric antibody engineered in its Fc-domain to target HER2 ( Figure 2) and is der from 4D5, a precursor to trastuzumab [59]. The Fc-engineered domain of margetux exhibits mutations of five amino acid components (L235V, F243L, R292P, Y300L, P396L) [67][68][69].

Mechanism of Action
Similar to Trastuzumab, Margetixumab works by binding to the Fab epitope o HER2 receptor with comparable specificity and affinity (Figure 3) [68] and exhibit independent antiproliferative effects. It shows elevated relative affinity towards bot lelic variants of CD16A. The low-affinity allelic variant (FcγRIIIA-V158F) was found associated with a decreased clinical response to trastuzumab. The Fc-engineered dom

Mechanism of Action
Similar to Trastuzumab, Margetixumab works by binding to the Fab epitope of the HER2 receptor with comparable specificity and affinity ( Figure 3) [68] and exhibits Fcindependent antiproliferative effects. It shows elevated relative affinity towards both allelic variants of CD16A. The low-affinity allelic variant (FcγRIIIA-V158F) was found to be associated with a decreased clinical response to trastuzumab. The Fc-engineered domain of margetuximab is specifically optimized to increase its binding to all FcγRIIIA-V158F allelic variants, compared to the wild-type IgG1 [60,70]. This increased binding to FcγRIIIA is associated with the enhanced ADCC activity of human NK cells, which leads to the suppression of cell proliferation [71]. Additionally, the Fc-engineered domain of margetuximab lowers its affinity or decreases its binding to the inhibitory receptor CD32B (FcγRIIB) [53,72]. These altered binding capabilities of margetuximab, particularly in cells with lower levels of HER2, in cells resistant to trastuzumab, and in patients bearing FcγRIIIA-V158F, result in increased ADCC and enhanced anti-tumor effects [73].
FcγRIIIA is associated with the enhanced ADCC activity of human NK cells, which lea to the suppression of cell proliferation [71]. Additionally, the Fc-engineered domain margetuximab lowers its affinity or decreases its binding to the inhibitory receptor CD3 (FcγRIIB) [53,72]. These altered binding capabilities of margetuximab, particularly in ce with lower levels of HER2, in cells resistant to trastuzumab, and in patients beari FcγRIIIA-V158F, result in increased ADCC and enhanced anti-tumor effects [73].

Pharmacokinetic Properties
The molecular formula of margetuximab is C6484H10010N1726O2024S42 [74]. Its pharmac kinetics have been thoroughly investigated in Phase 1 clinical trials using pharmacokine two-compartmental models with parallel linear and Michaelis-Menten elimination. T approved dose used for the pharmacokinetic studies was 15 mg/kg Q3W or 6.0 mg/kg Q [73]. Bang et al. (2017) reported that the pharmacokinetic parameters of clearance (C central volume (V1), inter-compartment clearance (Q), and peripheral volume (V2) we 0.299, 3.73, 0.885, and 3.73 L/day, respectively. Moreover, the authors reported the dist bution (t.5 dist) and half-life (t.5) at 1.12 and 15.5 days, respectively [73]. The Cmax was 4 µ g/mL (20%), whereas the AUC0-21d was 4,120 µ g/day/mL (21%) following administ tion to patients with advanced HER2+BC and relapsed or refractory status, with a volum of distribution of 5.47 L (22%). Margetuximab is metabolized through several catabo pathways into smaller peptides via proteases in a pattern similar to that of the other mA The terminal half-life of Margetuximab was 19.2 days (28%), with a clearance rate of 0. L/day (24%). An approximately 3% concentration decrease in the serum levels of M getuximab was observed four months post-discontinuation [61].

Pharmacokinetic Properties
The molecular formula of margetuximab is C 6484 H 10010 N 1726 O 2024 S 42 [74]. Its pharmacokinetics have been thoroughly investigated in Phase 1 clinical trials using pharmacokinetic two-compartmental models with parallel linear and Michaelis-Menten elimination. The approved dose used for the pharmacokinetic studies was 15 mg/kg Q3W or 6.0 mg/kg QW [73]. Bang et al. (2017) reported that the pharmacokinetic parameters of clearance (CL), central volume (V1), inter-compartment clearance (Q), and peripheral volume (V2) were 0.299, 3.73, 0.885, and 3.73 L/day, respectively. Moreover, the authors reported the distribution (t .5 dist) and half-life (t .5 ) at 1.12 and 15.5 days, respectively [73]. The C max was 466 µg/mL (20%), whereas the AUC0-21d was 4,120 µg/day/mL (21%) following administration to patients with advanced HER2 + BC and relapsed or refractory status, with a volume of distribution of 5.47 L (22%). Margetuximab is metabolized through several catabolic pathways into smaller peptides via proteases in a pattern similar to that of the other mAbs. The terminal half-life of Margetuximab was 19.2 days (28%), with a clearance rate of 0.22 L/day (24%). An approximately 3% concentration decrease in the serum levels of Margetuximab was observed four months post-discontinuation [61].

Indications/Therapeutic Use
Margetuximab, used synergistically with chemotherapy, is prescribed to adult patients with BC previously treated with two or more anti-HER2 agents, of which at least one was administered to treat metastatic diseases [61].

Tolerability and Toxicity
Treatment with margetuximab is well accepted and tolerated [61,73]. The SOPHIA clinical trial showed the safety profile of margetuximab. In this clinical trial, fatal adverse reactions were reported in 1.1% of margetuximab-receiving patients, with viral and aspiration pneumonia at 0.8% and 0.4%, respectively. Serious adverse reactions occurred in 16% of margetuximab-treated patients. The most prominent of these were left ventricular (LV) dysfunction and infusion-related reactions (IRRs) [61]. Although LV cardiac dysfunction was observed in 1.9% of margetuximab-treated patients, a lack of studies on margetuximab-treated patients with a LV ejection fraction of less than 50% or a history of the cardiac disease led to the addition to the warning and precautions section in FDA drug label [61]. The SOPHIA clinical trial showed that IRRs occurred in 13% of margetuximabreceiving patients. The majority of IRRs were reported during cycle 1 of therapy and resolved within 24 h. These infusion-related reactions were associated with symptoms such as nausea, fever, hypotension, anemia, diarrhea, headache, vomiting, fatigue, tachycardia, and certain cutaneous manifestations such as urticaria or rash. However, therapy for 9% of margetuximab-receiving patients was interrupted due to IRRS, and IRRS was discontinued in margetuximab therapy in 0.4% of treated patients. Patients experiencing severe infusion-related reactions must be carefully screened. Several toxicity parameters, including risk assessment during pregnancy and lactation, were not evaluated. Data on margetuximab-associated toxicity remains limited [61].

Dosage and Administration
A dosage of 15 mg/kg has been approved for administration via intravenous infusion. The initial dose is administered over 120 min, followed by another for approximately 30 min every three weeks until disease progression or unacceptable toxicity is observed. For therapy using a combined regimen, Margetuximab should be administered immediately following the completion of chemotherapy [61,75].

Clinical Trials of Margetuximab in BC
Clinical trials of Margetuximab began in 2010 and were conducted by MacroGenics [60,73,76]. A phase 1 trial (NCT01148849) was developed to assess the safety, efficacy, pharmacokinetic properties, pharmacodynamics, and antitumor activities of Margetuximab [73]. A total of 66 patients were included, all of whom presented with HER2overexpressing breast and/or gastric carcinomas. Patients were divided into two groups, whereby 34 patients were treated with regimen A (intravenous infusion at a dose range of 0.1-6.0 mg/kg weekly for 3 weeks), while 32 were treated with regimen B (intravenous infusion at a dose range of 10-18 mg/kg once every 3 weeks). Margetuximab was well tolerated at the given doses without any attainable maximum tolerated dose for either regimen. The trial results indicated that, of the 60 response-evaluable patients, 12% showed partial responses, while 52% reached stable disease. Moreover, a reduction in tumor size was observed in 78% of patients. Adverse effects were mild, with constitutional symptoms such as nausea and pyrexia. The authors concluded that Margetuximab exhibited promising activity and could be used alone to treat patients with HER2+ tumors. This trial facilitated further research on the potential clinical application of Margetuximab as a single agent or in combination therapy [73,77]. Nordstrom et al. (2011) reported that margetuximab exhibited a promising safety profile, with 0.1 mg/kg as a minimum human equivalent dose in trials on cynomolgus monkeys receiving 150 mg/kg [60]. 'No observed adverse effect level' (NOAEL) was observed; the dose was derived from the minimum effective dose of 1 mg/kg used in transgenic mice bearing human CD16A-V158F (xenograft models). Furthermore, margetuximab treatment was considered acceptable at a dose range of 15-150 mg/kg [60]. This study was followed by a phase II clinical trial (NCT01828021) that employed a Simon 2-stage design in 41 patients to investigate the efficacy and activity of margetuximab in patients with advanced BC, in either relapsed or refractory status, and low HER2 expression, as evidenced using a fluorescence ISH test [78]. A series of trials involving Chinese patients has also been conducted (NCT04398108 and NCT04262804). The most prominent clinical trial was a phase III trial (SOPHIA, NCT02492711). This randomized, parallel assignment, open-label trial design comprised 536 patients aged ≥ 18 years with confirmed HER2 + MBC or unresectable BC previously treated with at least two HER2-directed therapies in the metastatic setting [76]. A 1:1 randomization was performed based on the chemotherapy treatment (capecitabine, gemcitabine, eribulin, or vinorelbine) administered along with margetuximab ( Although this trial exhibited a comparable safety profile between margetuximab and trastuzumab-treated groups, IRRs were commonly seen in the margetuximab-treated group (13.6%) compared to the trastuzumab group [79]. Margetuximab was approved following a series of clinical trials (Table 2) to treat HER2 + MBC in December 2020. Margetuximab is currently undergoing several other clinical trials to further investigate its usage, safety, and efficacy in other cancer types, such as gastric and gastroesophageal junction cancer [80,81]. The trials currently registered with Clinicaltrials.gov are listed in Table 3.

Conclusions
ErbB2 receptors are capable of enhancing the malignancy of solid tumors, including breast and gastric cancers, and sustaining cancer types resistant to conventional therapies. The scientific community deemed HER2 an effective target for cancer treatment, prevention, and diagnosis. The development of novel cancer-targeted therapies is a major advancement toward the treatment of HER2 + MBC. A step towards this was achieved by optimizing the Fc domain of trastuzumab to create the novel mAb margetuximab. Clinical trials with margetuximab have demonstrated its efficacy in treating HER2 + MBC. Importantly, compared with the standard trastuzumab combined with chemotherapy, the combination of margetuximab with chemotherapy showed favorable results in terms of overall response rate (ORR) and PFS. However, further evidence is required to determine its optimal use in a variety of clinical settings. Studies on treatment resistance to this drug and toxicity profiling are also warranted.