**Targeting the Tetraspanins with Monoclonal Antibodies in Oncology: Focus on Tspan8/Co-029**

#### **Mathilde Bonnet 1,†, Aurélie Maisonial-Besset 2,†, Yingying Zhu <sup>3</sup> , Tiffany Witkowski <sup>2</sup> , Gwenaëlle Roche <sup>1</sup> , Claude Boucheix <sup>3</sup> , Céline Greco 3,4 and Françoise Degoul 2,\***


Received: 19 December 2018; Accepted: 30 January 2019; Published: 4 February 2019

**Abstract:** Tetraspanins are exposed at the surface of cellular membranes, which allows for the fixation of cognate antibodies. Developing specific antibodies in conjunction with genetic data would largely contribute to deciphering their biological behavior. In this short review, we summarize the main functions of Tspan8/Co-029 and its role in the biology of tumor cells. Based on data collected from recently reported studies, the possibilities of using antibodies to target Tspan8 in immunotherapy or radioimmunotherapy approaches are also discussed.

**Keywords:** tetraspanins; cancer; Tspan8; immunotherapy; radioimmunotherapy

#### **1. Introduction**

Tspan8 belongs to the tetraspanin molecular family of surface glycoproteins containing 33 members in humans, which are now referred to as Tspan1–33 (Figure 1). Tetraspanins are small membrane proteins (200–350 amino acids), which interact laterally with multiple partner proteins and with each other to form the so-called TEMs (tetraspanin-enriched microdomains). To qualify for membership in the tetraspanin family, a protein must have four transmembrane domains and several conserved amino acids, including an absolutely conserved CCG motif and two other cysteine residues that contribute to two crucial disulfide bonds within the second extracellular loop (EC2) (Figure 2). One or two additional disulfide bonds may also be found in EC2. The biological importance of tetraspanins is supported by functional consequences of genetic modifications that occur either spontaneously in humans or experimentally in mice. For instance, gene inactivation may affect fertility (CD9, CD81), and visual (RDS), kidney (CD151) or immunological functions (CD81, CD37) [1–4]. Since tetraspanins are not adhesion or signaling molecules, receptors or enzymes, the properties of these molecules are highly dependent on their ability to form TEMs with a hierarchical organization. Indeed, each tetraspanin has specific partners, including integrins, ADAM metalloproteases, growth factor receptors and histocompatibility antigens, that they are directly associated with through protein–protein interactions and form primary complexes with [5,6]. Coupled together, the latter can form second-order complexes through tetraspanin–tetraspanin interactions that may involve cholesterol and palmitoylation. In some cases, the function of these associated molecules has not yet been elucidated for the CD9P-1 and EWI2 (official protein names of PTGFRN

and IGSF8) [7,8]. Some molecular experiments showed that tetraspanins may interfere with the properties (affinity of the integrin α6β1 for laminin-1 is modulated by the tetraspanin CD151) [9], the trafficking (CD81 controls the expression of CD19 at the B-lymphoid cell surface) [10] or membrane compartmentalization of associated molecules (such as ADAM10 by TspanC8) [11]. Detailed proteomic analysis of membrane molecules that are able to associate with CD9 and Tspan8 has been reported recently in colon carcinoma cell lines. Among other membrane proteins, E-cadherin and EGFR are associated with TM4 complexes. More specifically, the presence of Tspan8 in the membrane drives EGFR to tetraspanin complexes, which results in changes in motility behavior (see next paragraph) [12].

**Figure 1.** Homology tree between human tetraspanins. Protein sequences have been aligned to generate this distance tree. Bolded names correspond to commonly used ones. The number of cysteines in the extracellular loop EC2 is given when they are different from six cysteines.

**Figure 2.** Schematic representation of Tspan8 in membrane. The transmembrane (TM) domains inserted in the phospholipid bilayer are palmitoylated with a cholesterol molecule in between these domains (by analogy with CD81 structure [13]). Among the two extracellular (EC) regions, the larger region contains six cysteines involved in disulfide bonds. EC1 and EC2 = extracellular domains 1 and 2; Ch = cholesterol; and PL = phospholipid.

Some tetraspanins are widely expressed (for example, CD81 and CD151) while others are more restricted (for example, CD37 and CD53 on lymphoid cells or RDS in retina). Several clinical observations and experimental data have correlated the expression of some tetraspanins with tumor metastatic potential. CD82 or CD9 expression is generally associated with favorable prognosis in different cancers [3]. On the contrary, CD151 and Tspan8 expression in tumor cells has been frequently associated with increased migration, proliferation and angiogenesis induction (Table 1). Mechanisms that could potentially explain the role of tetraspanins in tumors have been investigated experimentally using cellular models of overexpression in addition to knockdown or knockout animal models (reviewed in references [3,4,14–16]). The modulation of cellular properties, such as proliferation, cell migration and apoptosis, has been previously reported. Among other mechanisms, the knockout of CD37 leads to the occurrence of lymphomas that appear to be linked to the constitutive activation of the IL6 signaling pathway [17]. At a tissue level, an effect of Tspan8 on angiogenesis could be partially mediated by exosomes. More generally, the role of tetraspanins in tumor cell communication with their microenvironment through an effect on exosomal biogenesis is considered to be an important function of these molecules [18]. The changes in cell properties induced by the (over)expression of Tspan8 have been investigated in preclinical models. For example, the Isreco1 cell line derived from primary colorectal cancer (CRC) that does not express Tspan8 was compared with Is1-Co029, which was obtained by transduction to express Tspan8 at the same level as the two cell lines derived from metastases of the same patient. There was no difference in the motility of single cells plated on collagen, but RNAi targeting various surface molecules, such as E-cadherin, p120-catenin or EGFR, increased the motility of Is1-Co029, whereas no effect was observed on Isreco1 [12,19]. A possible link between Tspan8, E-cadherin and motility could be the signaling molecule p120-catenin, which is retained at the cell membrane through its affinity for E-cadherin. Furthermore, it has been reported to regulate Rho and Rac functions in cell adhesion and motility (reviewed in reference [19]). However, models using long-term established tumor cell lines in 2D settings could be misleading and it would be important to conduct experiments on cells derived from fresh tumors and cultured in 3D conditions as organoids that would better reflect the in-vivo conditions.

**Table 1.**Summary of studies dealing with biological consequences of Tspan8 targeting using specific antibodies.


i.v.: intravenous, i.p.: intraperitoneal, i.f.p: intrafootpad and s.c.: subcutaneous.

Experimental results and clinical trials using monoclonal antibodies (mAbs) suggest that some tetraspanins may be targeted in hematological malignancies and carcinomas [26]. In a similar way, Tspan8 appears to be an interesting candidate for the development of therapeutic antibodies, and different methods are discussed in the last part of this review.

#### **2. Mechanisms and Requirements for the Use of Therapeutic Anti-Tetraspanin Antibodies**

#### *2.1. Molecular Mechanisms*

Since antibodies are large proteins, their ability to reach their target may be limited by their diffusion inside different tissues and their components due to certain barriers, such as the hemato-encephalic barrier. However, since the permeability of tumor vessels is usually abnormal, diffusion appears to be increased in such tumor tissues [27,28]. Once located close to their biological target, the antibodies can enact different mechanisms of action.

#### 2.1.1. Blocking Antibodies

According to current knowledge, the action of tetraspanins relies on their ability to regulate the function of their partner molecules. Even if the detailed molecular basis remains mostly unknown, the binding of mAbs on Tspan8 may result in the inhibition of cell migration, invasion, proliferation and angiogenesis in organized tissues (Table 1).

#### 2.1.2. Cytotoxic Antibodies

Unconjugated antibodies mediate ADCC (antibody-dependent cell cytotoxicity) via the activation of accessory cytotoxic cells for killing target cells. Upon fixation on the cell surface, they recruit macrophages or NK cells through their Fc fragment, which can further destroy the target cell. The subclass and glycosylation of the Fc region are important parameters that determine the efficacy of ADCC. Furthermore, the ability of antibodies to mediate ADCC may be optimized through genetic modification of their Fc region.

On the contrary, radionuclide- or drug-conjugated antibodies have directly toxic effects in vivo.

Depending on the radionuclide used (nature of emissions, energy and so on), radionuclide-conjugated antibodies can efficiently treat the target cells and the neighboring cells, which can be of high value in the case of heterogeneous cell expression of the target molecule. In addition, radionuclide-conjugated antibodies can also allow for a combination of imaging and radiotoxicity. This will be detailed in the radionuclide-conjugated Tspan8 antibodies section.

When antibodies are conjugated with cytotoxic drugs, an internalization of the antigen/antibody complex is often necessary to allow drug delivery [29]. However, if the targeted protein is widely distributed, the use of these two main categories of antibody–drug conjugates may have deleterious effects on normal tissues. As a consequence, the careful evaluation of the pattern of expression of the target is a critical prerequisite for the development of a therapeutic antibody. However, recent progress in antibody-conjugated drugs has allowed one to target more specific released molecules in tumors. For instance, cleavage in acidic zones that occur in the highly proliferative zone or proteolytic cleavage by tumor enzymes, such as MMPs, increase specificity. Another stage was added with a pro-body approach, which consists of modifying the antibody with a small peptide that needs to be cleaved by a specific protease for antigen recognition [30].

#### *2.2. Pattern of Expression*

An ideal antigenic target should be expressed at a high intensity on the surface of tumor cells, especially tumor stem cells and not on normal cells. None of the molecules that are currently targeted by mAbs are able to fulfil these criteria and not surprisingly, tetraspanins do not escape this rule.

As mentioned above, the tissue distribution of tetraspanins is highly variable. A very restricted distribution of some tetraspanins has already been observed for peripherin/RDS found in the photoreceptors, UPk1a found on the urinary bladder epithelium or CD37 expressed mainly on B lymphoid cells. In contrast, other tetraspanins, such as CD9, CD63, CD81 or CD151, have a very large distribution and may be difficult to target in vivo. Tspan8 is expressed in a limited number of tissues and is mainly found on epithelial cells of the digestive tract. However, it is also located on the epithelial cells of the kidney, prostate and trachea [23]. The second aspect relates to the intensity ratio of Tspan8 in tumor compared to normal tissues, which is another crucial issue that needs to be addressed in order to avoid side effects. Increased Tspan8 expression in tumor tissues (colorectal and ovarian cancer, melanoma, hepatocellular and pancreatic carcinoma) is generally related to worse prognosis [19,25,31–33]. Therefore, Tspan8 appears to be an interesting target candidate for cancer treatment with mAbs.

#### *2.3. Dodging Immune Neutralization*

This important point has been widely studied and previously reviewed. Thus, this will not be detailed in this article. This is mainly realized through the humanization of the molecule but other methods have been previously reported [34].

#### **3. Therapeutic Antibodies Directed toward the Tetraspanins CD9, CD151 and CD37 in Cancer**

There are a few proofs of principle that have shown that targeting tetraspanins with antibodies might inhibit tumor growth or even induce partial or complete remission [35].

The effects of the anti-CD9 mAb ALB6 (IgG1) injected intravenously were investigated in a model of human gastric cancer (MKN-28) implanted subcutaneously in nude mice. A reduction of 60–70% in the size of the tumor in the treated group was observed compared to the control IgG-treated mice. At the same time, a significant reduction in cell proliferation and angiogenesis and an increase in apoptotic signals were observed [36]. Since this mAb is directed towards human CD9, damages to normal tissues were not evaluated. As CD9 is strongly expressed on many cellular types and particularly on platelets in humans, the use of this anti-CD9 antibody could lead to a loss of treatment efficiency and may trigger platelet activation or lysis depending on the nature of the Fc fragment [37]. However, Fc-mediated side effects could be avoided by genetic modifications of the antibody.

There have only been a few studies targeting tetraspanins in humans for therapeutic purposes. A cocktail of antibodies Ba1/2/3 directed respectively toward CD9, CD24 and CD10 to deplete the bone marrow of acute lymphoblastic leukemia patients for autologous bone marrow transplantation was investigated. Even if the specific role of each individual antibody was difficult to evaluate, an absence of toxicity against hematological stem cells was observed in these antibodies [38].

Due to its restricted specificity for differentiated cells of the B-cell lineage, CD37 was considered to be a potential target for the treatment for B-cell lymphoma using an anti-CD37 antibody radiolabeled with iodine-131 (β <sup>−</sup>; T1/2 = 8.02 d; 606 keV). Very encouraging results were obtained in comparison with an anti-CD20 antibody labelled with the same radionuclide [39]. However, since the use of unconjugated humanized anti-CD20 mAbs for B-cell lymphoma has been a very straightforward method for the improvement of treatment protocols, the use of anti-CD37 mAbs for the treatment of lymphoma and chronic lymphocytic leukemia was abandoned and only recently reintroduced. Several forms of therapeutic anti-CD37 antibodies, whether they are human or humanized, used in combination or alone (unconjugated antibodies (Bi 836826 and otlertuzumab) and drug conjugates (monomethyl auristatin E: AGS67E, maytansine: IMGN529)), or radiolabeled antibodies (177Lu: belatutin) [40], have been developed and are currently undergoing clinical tests. Promising results have been obtained in the resistant forms of NHL (Non-Hodgkin Lymphomas) and CLL (Chronic Lymphocytic Leukemias).

Anti-CD151 antibodies inhibited metastasis spread and primary tumor growth in human tumor mouse models [41,42]. Despite their variable ability to disrupt the complex between CD151 and α3β1 integrin, different anti-CD151 antibodies were reported to prevent metastasis formation in clinical models [35].

#### **4. Treatment with Unconjugated Anti-Tspan8 Antibodies**

Unconjugated mAbs may act through two different mechanisms, which are namely the mediation of ADCC or interference with molecular functions that are required for malignant cells to express their tumorigenicity. The functional activity of the anti-Tspan8 mAbs can be assessed to some extent in vitro but the link between tumor growth inhibition and either ADCC or functional inhibition could be difficult to determine in vivo.

Several studies were performed by Zöller's group with the rat pancreatic adenocarcinoma cells (AS-Tspan8 compared to AS). The anti-rat Tspan8 mAb D6.1 was found to inhibit cell proliferation in vitro [20]. Furthermore, in rat mesentery fragments cultured with tumor cells or their exosomes, increased endothelial cell branching linked to Tspan8 expression was blocked by mAb D6.1 [21]. These studies suggest that Tspan8 overexpression in rats promotes angiogenic activity and supports tumor growth while anti-rat Tspan8 mAbs can efficiently inhibit this process. In addition, increased vessel density observed by intravital microscopy was abolished after treatment with D6.1 in an in-vivo model of peritoneal carcinosis [21].

Anti-human Tspan8 mAbs (Ts29.1: IgG1 and Ts29.2: IgG2b) were produced by Boucheix's team. These antibodies had no effect on cell proliferation, migration or apoptosis for colorectal cancer (CRC) cell lines Isreco-1/Is1-Co029, SW480 (presenting spontaneously weak Tspan8 expression) and SW480-Co029 (overexpressing Tspan8 after gene transduction) in vitro. However, the 2D motility of Is1-Co029 was increased by RNAi targeting E-cadherin and p120-catenin while it only decreased after specific co-treatment with anti-Tspan8 mAbs [12,19]. EGFR blocking (mAb cetuximab or AG1478, a chemical EGFR inhibitor) in Is1-Co029 cells also induced an increase in cell motility, which was further blocked by treatment with anti-Tspan8 mAbs [12]. The observations in relation to EGFR inhibition were unexpected since Isreco-1 cell lines have a KRAS mutation, which should be associated with an inhibition of the EGFR function. Thus, this suggested that EGFR signaling may still be influenced when Tspan8 is expressed.

In a mouse model of CRC (SW480 vs. SW480-Co029), the growth of SW480-Co029 tumors was inhibited by up to 70% when treated in the early stages with the IgG2b anti-human Tspan8 mAbTs29.2 in vivo (initially 2 mg intraperitoneally, followed by 1 mg twice a week for 4 weeks). The same results were also observed in another CRC mouse model, which expressed spontaneously high levels of Tspan8 (HT29). The inhibition of the cell proliferation in vivo was demonstrated by a reduction of the mitotic index in HT29 tumor cells in Ts29.2-treated mice. These in-vivo data underlined the crucial role of Tspan8 in tumor growth and the therapeutic potential of anti-Tspan8 mAbs as a CRC treatment. The discrepancy between the in-vitro and in-vivo data on cell proliferation suggested that the binding of Ts29.2 to tumor cells may modify their response to signaling from the microenvironment. No significant differences between the treated and control mice were found when assessing the inflammatory infiltrate, angiogenesis (CD34) and apoptotic signal (Casp3). These findings did not support the hypothesis of ADCC.

In another approach targeting Tspan8, Park et al. [25] used phage display technology to produce a fully human mAb directed against Tspan8 LEL (large extra loop = EC2). For an in-vivo experiment, the mice that were intraperitoneally injected with SK-OV3-luc human ovarian cell line intravenously received either IgG or Tspan8–LEL IgG (10 mg/kg) twice a week until day 42 post inoculation. In the control IgG-treated group, a detectable luminescence signal in removed organs (ovary, pancreas, colon, heart, liver, spleen and kidney) was observed in 24 of 31 control mice whereas the incidence fell to 50% (15 of 30 mice) in the Tspan8–LEL IgG-treated group. This reduction of 35% was considered to be significant and the mice did not show any signs of severe toxicity.

#### **5. Treatment with Radionuclide-Conjugated Anti-Tspan8 Antibodies**

#### *5.1. Radiolabeling of Antibodies*

For radiotherapeutic purposes, antibodies are usually modified with grafted chelating moieties to allow radiolabeling with β <sup>−</sup>-emitter radionuclides like yttrium-90 (β <sup>−</sup>; T1/2 = 2.67 d; 2280 keV) or lutetium-177 (β <sup>−</sup>; T1/2 = 6.65 d; 498 keV) [43]. Advantageously, lutetium-177 can be used for both imaging and therapeutic purposes as β <sup>−</sup> and γ radiations are generated during its decay. To date, the only radiolabeled mAb authorized for human treatment is the anti-CD20 [ <sup>90</sup>Y]ibritumomab tiuxetan (Zevalin®), which is administered as a second-line treatment to patients with non-Hodgkin lymphomas (NHLs) that are resistant to chemotherapy [44]. Although they were created several decades ago, radioimmunotherapy (RIT)sing β <sup>−</sup>-emitter—conjugated mAbs remains underused due to inherent issues concerning hematotoxicity, which is induced by the long biological half-life of mAbs in blood, and the low penetration of antibodies in solid tumors [43,45]. Thus, different strategies have been proposed to enhance RIT efficiency, including the use of α-particle emitters, pretargeting protocols and reduction/modification of the antibody size (nanobodies, affibodies and so on).

The use of α-particle emitters is of great interest for delivering high linear energy transfer (LET) in very small volumes (cell diameters of 50–100 µm) without affecting neighboring tissues. Among the increasing number of clinical studies using mAbs/peptides/ligands/radionuclides delivering α-particles [46], two mAbs radiolabeled with bismuth-213 (α; T1/2 = 45.6 min; 5869 keV) ( <sup>213</sup>Bi-cDTPA-9.2.27 targeting MSCP in melanoma and <sup>213</sup>Bi-Hum195 mAb targeting CD33 in acute myeloid leukemia) have had positive results in terms of prognosis [47,48]. Moreover, challenging approaches using DOTA-mAbs radiolabeled with actinium-225 (α; T1/2 = 10.0 d; 5580–5830 keV) are currently under investigation as this radionuclide decay generates francium-221 (α; T1/2 = 4.79 min, 6300 keV), which has an interesting secondary radiotoxic effect [49]. This strategy should be applied for internalizing antibodies to concentrate α-particles in tumor cells.

To decrease hematotoxicity, different pretargeting strategies were developed to dissociate the mAb antibody and radionuclide injections [50]. One of the most interesting approaches involves the use of bio-orthogonal chemistry. In such strategies, the mAb and the delayed injected radioactive molecule are grafted with chemical entities that are highly reactive to each other but inert to chemical functions usually found on in-vivo molecules, such as proteins. This fast and specific reaction can advantageously take place in aqueous media, which is compatible with in-vivo applications. Another way of reducing side effects of RIT is to inject the radioactive molecule in a specific organ to avoid systemic irradiation. For example, this might involve intrahepatic metastases using arterial infusions [51].

Finally, different small protein forms (affibodies, nanobodies and so on) ranging from a few to 30 kDa with a shorter biological half-life in the blood circulation have been tested in preclinical RIT protocols [52] but mostly in nonradioactive applications. In clinical trials, one affibody is currently being investigated in Her2 breast cancer imaging [53].

#### *5.2. Targeting Tspan8 with Radiolabeled Antibodies*

Our team investigated the biodistribution of two monoclonal antibodies targeting human Tspan8, which were namely Ts29.1 and Ts29.2. These were grafted with DOTA and radiolabeled with indium-111 (γ; T1/2 = 2.80 d; 171 keV; 245 keV). The measurement of the immunoreactive fraction revealed that the addition of DOTA-chelating moieties and radiolabeling did not modify the affinity of Ts29.2 for its target. The uptake of [111In]DOTA-Ts29.2 in HT29 tumors was higher than that of [111In]DOTA-Ts29.1 (Figure 3A,B). After this, further experiments were conducted on different models of xenografts [23]. Biodistribution studies on mice with both SW480-Co29/SW480 tumors demonstrated high specificity of [111In]DOTA-Ts29.2 for Tspan8-expressing tumors. The same results were obtained using the Isreco-1 and Is1-Co029 models (Figure 4A,B). Further RIT experiments using this antibody were supported by the promising biodistribution and dosimetry results collected. During therapeutic studies, we observed that

[ <sup>177</sup>Lu]DOTA-Ts29.2 induced a significant reduction in HT29 xenograft growth, with molecular events sustaining the effects of the radiation in this model.

**Figure 3.** In-vivo selection of [111In]DOTA-Ts29 antibodies for imaging and therapy in mice with HT29 colon carcinoma xenografts. Nude NMRI mice with HT-29 tumors were injected (i.v.) with 3.7 MBq of [ <sup>111</sup>In]DOTA-Ts29.1 (upper images) or [111In]DOTA-Ts29.2 (lower images), which were imaged with a planar γ-camera at 4 h, 24 h, 72 h, 120 h and 168 h post injection (**A**). Biodistribution was performed after euthanasia and expressed as the percentage of activity injected per gram of tissue (%AI/g) of [111In]DOTA-Ts29.1 (orange) or [111In]DOTA-Ts29.2 (purple) (**B**). Radioactivity was measured using a γ-counter. Results are presented as the average percentage of injected dose/gram of tissue of three animals for each time point. The error bars represent the standard deviation. Biodistribution difference between the two mAbs: \* *p* < 0.05 Fisher test.

**Figure 4.** In-vivo specificity of [111In]DOTA-Ts29.2 for Tspan8-expressing tumors. Nude NMRI mice with Isreco-1 (left shoulder) and Is1-Co29 (right shoulder) were injected (i.v.) with 3.7 MBq of [ <sup>111</sup>In]DOTA-Ts29.2 and imaged with a planar γ-camera at 4 h, 24 h, 72 h and 120 h post injection (**A**). Ex-vivo biodistribution study (%AI/g) of [111In]DOTA-Ts29.2 (**B**) was determined on the same mice with the same protocol as Figure 3B. Biodistribution difference between the two tumors: \* *p* < 0.05. Fisher test.

To initiate the pretargeting strategies, Ts29.2 was also modified by the addition of a transcyclooctene (TCO) to the lysine residues, which was evaluated in studies conducted in vitro and in vivo using a fluorescent tetrazine. We evaluated the best link size between TCO and TS29.2 and observed a higher fluorescent signal with Ts29.2-TCO without a PEG spacer, which can be explained by a higher isomerization rate of TCO to the inactive CCO form [54]. As tetrazin can be conjugated to a DOTA group, RIT with β <sup>−</sup>-emitters or α-particles will be considered. A recent preclinical study using such an approach had significant effects on mice xenografted with ovarian tumors and treated with an anti-CEA-TCO for 72 h before radionuclide injection [55].

#### *5.3. Pros and Cons of RIT for Human Cancers: Focus on Targeting Tspan8*

Stoichiometrically compared to its corresponding nonradiolabeled antibodies, [ <sup>177</sup>Lu]DOTA-Ts29.2 induced a greater slowing down of tumor growth. The main features in pretargeted radioimmunotherapy PRIT experiments were the reduction of proliferation and increase in apoptosis. As mentioned above, the treatment with nonradioactive antibodies (using 100-times more antibodies than in the [177Lu]DOTA Ts29.2 experiments) also resulted in a slowing down of tumor growth with neither induction of apoptosis nor decrease in angiogenesis. In fact, the nonradioactive antibody should alter the interactions between tumor cells harboring Tspan8 and the microenvironment while its radiolabeled counterpart irradiates all surrounding cells after it attaches to its target antigen. This property should be interesting as it will decrease the number of so-called cancerous stem cells (CSCs) because Tspan8 has been identified on the surface of CSCs in pancreatic tumors [56]. RIT has been proven to be effective in stopping CSCs in melanomas using preclinical models, which utilized an IgM directed toward melanin and radiolabeled with rhenium-188 [57]. Conversely, Tspan8 is exposed on the surface of circulating exosomes [22], leading to potential blood radiotoxicity in RIT experiments. Apart from this potential disadvantage, one can imagine that targeting circulating exosomes will be of interest as these vesicles are implicated in metastatic spread [58]. As mentioned above, the hematotoxicity might be prevented by pretargeting strategies, which will be further reinforced by the use of blood clearing agents such as nonradiolabeled ligands conjugated to albumin [59]. As an example, this might allow their metabolism in the liver.

Tspan8 expression is restricted and this protein has been described as a significant contributor and potential therapeutic target in several cancer types. Even if secondary effects and immune system involvement cannot be evaluated on tumor-grafted mouse models used for these studies, targeting Tspan8 with radiolabeled antibodies seems to be an effective antitumoral therapy.

#### **6. Conclusions**

Tetraspanins may have a broad range of actions in cancers due to their intrinsic membrane localization (cell membrane or exosomes) and high numbers of their interacting molecules [3,26]. The aim of this article was to review recent preclinical attempts at targeting tetraspanins in cancer with a focus on Tspan8. Unconjugated antibodies and radionuclide-conjugated antibodies conceptually represent two different approaches for killing cancer cells through the expression of a surface molecule. Antibodies may have complex effects as they combine cell-mediated cytotoxicity and functional deleterious effects, such as apoptosis induction, or invasive growth and angiogenesis inhibition. This can occur directly or through microenvironment factors. For tetraspanins, it is still unknown how the targeting can alter the function of tumor cells in vivo, but their association with adhesion molecules, growth factor receptors or enzymes inside membrane molecular complexes leads to disturbance of the structure/composition of these complexes, which may result in modulation of migration and abnormal signaling into the cell and finally, inhibition of invasion/metastasis or even apoptosis. A better view and understanding of the behavior of tumor cells in real life would require improved models (such as 3D in-vitro setups with microenvironment reconstitution or syngeneic models in vivo). Although the mechanism of action of radionuclide antibodies is simple and straightforward, their manufacturing requires careful technical management and radioprotection protocols at all stages of their manipulation. However, these offer interesting potential and should be pursued in the future. Innovative techniques have also been developed to reduce harmful effects that are linked to the antibodies binding to normal healthy tissue.

**Funding:** This research has been supported by INSERM-Transfert CoPOC program (MelCoMAb 2013–2016) and is now granted by the Ligue régionale contre le cancer du Puy de Dome (2019–2020).

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

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **The Changing Paradigm for the Treatment of HER2-Positive Breast Cancer**

#### **Aena Patel <sup>1</sup> , Nisha Unni 1,\* and Yan Peng 2,\***


Received: 30 June 2020; Accepted: 22 July 2020; Published: 28 July 2020

**Abstract:** For decades, HER2-positive breast cancer was associated with poor outcomes and higher mortality rates than other breast cancer subtypes. However, the advent of Trastuzumab (Herceptin) has significantly changed the treatment paradigm of patients afflicted with HER2-positive breast cancer. The discovery of newer HER2-targeted therapies, such as Pertuzumab (Perjeta), has further added to the armamentarium of treating HER2-positive breast cancers. This review highlights recent advancements in the treatment of HER2-positive diseases, including the newer HER2-targeted therapies and immunotherapies in clinical trials, which have paved (and will further update) the way for clinical practice, and become part of the standard of care in the neoadjuvant, adjuvant or metastatic setting.

**Keywords:** HER2-positive breast cancer; metastatic disease; neoadjuvant and adjuvant therapy; targeted therapy; immunotherapy

#### **1. Introduction**

Breast cancer is categorized into four different molecular subtypes: Hormone receptor (HR)-positive (+)/Human epidermal growth factor receptor 2 (HER2)-negative (−) (Luminal A); HR+/HER2+ (Luminal B); HR−/HER2+ (HER2-enriched); and HR−/HER2− (triple negative). The survival rate among patients differ based on the molecular subtype and stage. The survival rate at four years among women with HR+/HER2− is estimated to be 92.5%, followed by HR+/HER2+ at 90.3%, HR−/HER2+ at 82.7%, and HR−/HER2− at 77.0% [1]. The HER2 (human epidermal growth factor receptor 2) oncogene is positive in about 20% of primary invasive breast cancers [2]. It is well known that HER2 overexpression is associated with higher rates of disease recurrence and mortality. In addition, HER2+ breast cancers have a higher predilection to metastasize to the brain. However, during the last two decades, the treatments and outcomes for patients with HER2+ disease have shifted dramatically. Trastuzumab (Herceptin) was first approved in 1998 as the first anti-HER2 directed therapy in metastatic HER2+ invasive breast cancer. Prior to trastuzumab, historically, patients with metastatic HER2+ disease were treated with traditional chemotherapy regimens. A pivotal Phase III trial [3] of 469 women showed that adding trastuzumab to standard chemotherapy (paclitaxel or anthracycline/cyclophosphamide) resulted in improved response rates (50% versus 32%), extended time to progression (7.4 months versus 4.6 months), and improvement in median overall survival (25 versus 20 months) [3]. Relative risk of death was also reduced by 20% at a median follow up of 30 months. Since trastuzumab, multiple agents have been developed to treat patients with HER2+ disease.

The HER superfamily consists of four tyrosine kinase receptors: HER1 (epidermal growth factor receptor), HER2 (neu, c-erbB2), HER3 and HER4. When activated, these receptors cause epithelial cell growth and differentiation. The HER2 oncogene encodes for a glycoprotein receptor with intracellular tyrosine kinase activity, and has no known ligand [4]. On the other hand, the other three HER receptors have known ligands, and form homodimers or heterodimers upon ligand binding, with the HER2 receptor being the preferred dimerization partner. The HER2 receptor can heterodimerize with the other receptors, which results in autophosphorylation of the tyrosine residues. This autophosphosphorylation activates the MAPK (mitogen-activated protein kinase) pathway and the PI3K (phosphatidylinositol 3-kinase) pathways. The HER2, or epidermal growth factor receptor 2, is the target for many HER2-directed therapies.

Herceptin binds to subdomain IV of HER2 in order to disrupt HER2 signaling. HER2 testing is done via immunohistochemistry (IHC), which tests for the overexpression of the HER2 gene product, and fluorescence in situ hybridization (FISH) to test for HER2 gene amplification. The tumor is identified as HER2+ if IHC is 3+ (intense staining within >10% of the tumor cells), or if the ratio of HER2 and the chromosome 17 enumeration probe (HER2/CEP17) is ≥2 and the HER2 copy number signals/cell equals is ≥ 4. It is important to note that those with non-HER2-overexpressing breast cancers do not derive benefits from adjuvant trastuzumab. This was studied in a randomized trial of 3270 patients [5] with invasive breast cancer, with IHC scores of 1+ or 2+ and with FISH <2 (or if the ratio was not performed, HER2 gene copy number <4.0). The study found that adding trastuzumab to chemotherapy (either docetaxel plus cyclophosphamide, or doxorubicin and cyclophosphamide, followed by weekly paclitaxel for 12 weeks) did not improve disease-free survival, distant recurrence-free interval, or overall survival.

This is in line with the updated 2018 ASCO/CAP (American Society of Clinical Oncology/College of American Pathologists) guidelines. The 2018 ASCO/CAP guidelines [6] identify a testing algorithm to address the less commonly found clinical scenarios, in order to address the infrequent HER2 results that are of unclear significance. Another major revision in the 2018 guidelines includes the revision of the definition of IHC 2+. IHC2+ is now defined as invasive breast cancer with weak to moderate complete membrane staining observed in >10% of tumor cells.

Here, we will address the key trials that have led to a major change in how we treat HER2+ invasive breast cancer in the neoadjuvant, adjuvant and metastatic settings. In addition, we will also discuss newer therapies, such as bispecific antibodies, and trials that are ongoing.

#### **2. Neoadjuvant Treatment**

Neoadjuvant HER2-based therapy is typically used (as in other subtypes) in locally advanced breast cancer (Stage IIb with T3 disease, or Stage III), or in patients with an earlier stage HER2+ disease who desire breast conserving therapy, have limited axillary nodal involvement (N1) (which could potentially be converted to node-negative disease and therefore result in sentinel lymph node biopsy), or have had surgery postponed (due to a variety of reasons). Given that pathologic complete response is associated with improved event free survival (EFS) (HR (hazard ratio) of EFS 0.37, 95% CI (confidence interval) 0.32–0.43) and overall survival (HR OS 0.34, 95% CI 0.26–0.42) [7], neoadjuvant therapy can help us gauge which patients are at higher risk of relapse, and more aggressive therapy can be offered. HER2-directed therapy is typically added to a chemotherapy backbone.

Several studies have demonstrated the efficacy of trastuzumab in improving event free survival, pathologic complete response (pCR), and overall survival. Based on a 2012 meta-analysis, herceptin increased pathologic complete response rates from 23% to 40% when added to neoadjuvant chemotherapy [8]. In addition, in the Phase III NOAH (Neoadjuvant Herceptin) trial [9], the addition of herceptin increased pCR rates from 19% to 38%, and improved event free survival (EFS) from 43% to 58% (HR 0.64, 95% CI 0.544–0.930). Given the improvement of event free survival at a median follow up 5 years (from trastuzumab-containing neoadjuvant therapy followed by adjuvant trastuzumab, in patients with locally advanced or inflammatory breast cancer), this trial highlights the association between pathologic complete response and long-term outcomes of patients with HER2+ disease.

Despite the improvement in pathologic complete response and event free survival, 15% of patients will relapse after therapy with trastuzumab due to a resistance to herceptin. The proposed mechanisms have included structural defects within the HER2 receptor [10], the constitutive activation of downstream elements [11], the activation of the downstream pathways by other members of the HER family [10,11], or intracellular alterations that affect the PI3K pathway [10]. Therefore, additional therapeutic targets with different mechanisms of action have been extensively studied in combination with trastuzumab, in order to evaluate whether these combination therapies prolong time to resistance and treatment failure [4].

Pertuzumab (Perjeta) is another biological therapy that has been studied in patients with HER2+ breast cancer. Pertuzumab is a monoclonal antibody that binds to subdomain II of the HER2 receptor and thereby blocks the heterodimerization with HER3, subsequently inhibiting downstream signaling. Given that pertuzumab enhances locoregional responses, it was approved in 2013 for patients with locally advanced, inflammatory, or early stage HER2+ invasive breast cancer (with size >2 cm or node-positive disease). The addition of pertuzumab to herceptin in the neoadjuvant setting was assessed in the NEOSPHERE Trial [12]. In the NEOSPHERE trial, the percentage of patients who achieved pCR was significantly higher in the pertuzumab + herceptin + docetaxel arm (46%) versus the herceptin + docetaxel arm (29%). The addition of pertuzumab led to increased diarrhea, however rates of cardiotoxicity were not higher with the combination of herceptin and pertuzumab, as assessed in the TRYPHAENA Trial [13]. Criticisms of the NEOSPHERE trial include the small sample size, the lack of patients from the United States, the lack of a blinded pathology review and the chosen chemotherapy backbone (which is not typically used in the United States).

If comorbidities preclude the addition of chemotherapy to targeted HER2 therapy, the other options include herceptin + pertuzumab, based on one arm of the NEOSPHERE trial where pcR rates were 16.8% (95% CI: 10.3–25.3, *p* = 0.0198) with pertuzumab + herceptin, versus herceptin + docetaxel. Another option includes the addition of lapatinib + herceptin (given for 18 weeks) based on the PAMELA trial [14]. Lapatinib is a small-molecule inhibitor of the tyrosine kinase inhibitors of both HER1 and HER2. In this trial, pathologically complete response rates were seen in 30% of the patients with previously untreated HER2+, or Stage I–IIIA breast cancer. Although this is an option for patients not wanting chemotherapy, this approach has not been adapted into practice.

Several cytotoxic regimens have proved efficacious in the neoadjuvant/adjuvant setting, including TCH +/− P (docetaxel, carboplatin, herceptin, pertuzumab), PCH +/− P (paclitaxel, carboplatin, herceptin, pertuzumab) or an anthracycline-based regimen, such as AC-TH +/− P (doxorubicin, cyclophosphamide followed by a taxane such as paclitaxel or docetaxel with herceptin +/− pertuzumab) or FEC/EC-TH +/− P (fluoruracil, epirubicin, cyclophosphamide, herceptin, pertuzumab). It is important to note that the safety of pertuzumab has not been established when combined with a doxorubicin-containing regimen. In addition, the safety of pertuzumab for more than six cycles in early-stage breast cancer has also not been established.

For those with low-risk disease, or patients with comorbidities, alternatives include weekly paclitaxel with herceptin (+/− pertuzumab) or a combination of docetaxel with cyclophosphamide, in addition to herceptin, every 3 weeks for four cycles [15], based on the results in the adjuvant setting.

#### **3. Adjuvant Treatment**

Adjuvant chemotherapy is given to patients with HER2+ disease that is a node-positive, or a node-negative disease with tumors >1 cm in size. After completion of chemotherapy and herceptin (given concurrently), the standard of care is to continue herceptin for a total of 52 weeks. Studies have found an improvement in overall survival, with a hazard ratio of 0.67 (95% CI 0.57–0.80) [16], when herceptin is administered for 12 months in the adjuvant setting. Extension to 2 years did not improve the 10-year disease-free survival, as studied in the HERA trial [17]. There was no reported difference in 10-year disease-free survival when herceptin was given for 1 year versus 2 years (HR 1.02, 95% CI 0.89–1.17). In addition, the study found that the incidence of cardiotoxicity was higher in the group who received herceptin for 2 years (7.3% versus 4.4%).

In contrast, a duration of less than 1 year of anti-HER2-directed therapy was proven to be more detrimental in the PHARE trial [18], which showed that treating patients with herceptin for 6 months resulted in more deaths, shorter 2-year disease-free survival rates, and more distant recurrences.

However, there is some data to support the shorter duration of HER2-directed therapy if patients cannot tolerate 12 months. In the recently published PERSEPHONE trial [19], patients with early stage HER2+ breast cancer were randomized to receive either 12 months or 6 months of adjuvant herceptin. Patients who received herceptin for 6 months had 4-year DFS rates similar to those who received adjuvant herceptin for 12 months (89.4% versus 89.8%, HR 1.07, 95% CI 0.93–1.24). It is important to note that the absolute difference in the 4-year DFS was only 0.4%. The difference between the discordant results between the PHARE and PERSEPHONE trials, despite a near equivalence of hazard ratios in both studies, has been attributed to the chosen non-inferiority margin of each trial [20]. The PHARE trial had a non-inferiority margin of 1.15. The upper bound of the two-sided 95% confidence interval was less than 1.15 in the PHARE trial. However, in the PERSEPHONE trial, the non-inferiority margin was defined as an absolute decrease in the 4-year DFS rate of 3%, which resulted in a non-inferiority margin of 1.316. In addition, the two-sided confidence interval was 90% in the PERSEPHONE trial, which thereby increasing the chance of concluding non-inferiority. Pondé et al. [20] note that if the non-inferiority margin of 1.15 was used in the PERSEPHONE trial, then the non-inferiority goal would not have been reached. In terms of safety, the PERSEPHONE trial demonstrated that patients who received 6 months of adjuvant herceptin experienced less cardiotoxicity (3% versus 8%, *p* < 0.0001) and fewer severe adverse events (19% in the 6-month group versus 24% in the 12-month group, *p* < 0.0002). However, it is important to note that 90% of the patients in the PERSEPHONE trial received anthracycline-based treatments. In addition, given that more non-anthracycline-based regimens are used in the current climate, the benefit of shortening the duration of trastuzumab to 6 months, in terms of cardiac safety, is not clear.

The KATHERINE trial evaluated the safety and efficacy of kadcyla (ado-trastuzumab emtansine or T-DM1) for 14 cycles vs. herceptin in the adjuvant setting, in patients with residual disease after neoadjuvant HER2-directed therapy [21]. Kadcyla is an antibody–drug conjugate of herceptin linked to an antimicrotubule agent (DM1). Patients with HER2+ early breast cancer with residual disease who received T-DM1 had an improved 3-year DFS, compared to receiving trastuzumab (88% versus 77%; HR 0.50, 95% CI 0.39–0.64). Although the number of serious adverse events was higher in the T-DM1 group (13% versus 8%), switching to T-DM1 in the adjuvant setting is associated with a lower risk of distant recurrence (HR 0.60, 95% CI 0.45–0.79).

The seven-year update of the single-arm, Phase II APT (adjuvant paclitaxel and trastuzumab) trial continued to demonstrate excellent outcomes, with disease-free survival of 93% and a recurrence-free interval (RFI) of 97.5% in small, node-negative HER2+ breast cancers. Based on these encouraging results, herceptin and paclitaxel for 12 weeks, followed by herceptin alone to complete 1 year of treatment, has emerged as a very tolerable and effective treatment option in this subset of patients [22].

Dual anti-HER2-directed therapy is recommended and approved for high-risk disease (node-positive or node-negative, with tumor size >2cm) in the adjuvant setting. In the Phase III APHINITY trial [23], adding pertuzumab to herceptin and chemotherapy led to an improvement in 3-year disease-free survival (94.1 versus 93.2%; HR 0.81, 95% CI 0.66–1.00), with subgroup analysis showing improvement in the patients with node-positive disease (92 vs. 90.2%; HR 0.77, 95% CI 0.62–0.96) but no difference in those with node-negative disease.

Various options exist for the chemotherapy backbone in the adjuvant setting. If an anthracycline-based regimen is used, the recommendation is to administer HER2-directed therapy sequentially, given the increased risk of cardiotoxicity. For example, we administer dose-dense doxorubicin + cyclophosphamide, followed by a taxane + herceptin (+/− pertuzumab). If a nonanthracycline regimen is given, preference is given to chemotherapy and anti-HER2-directed therapy concurrently.

#### **4. Extended Adjuvant Treatment**

Another agent, neratinib (dual kinase inhibitor that irreversibly inhibits the pan-Her receptors), was shown to improve recurrence rates when given after completion of 1 year of herceptin [24]. In the Phase III ExTENET trial, women with early-stage HER2+ disease were randomly assigned to receive neratinib or a placebo after treatment with herceptin. There was an improvement in 5-year invasive disease-free survival (90.2% vs. 87.7%; HR 0.73, 95% CI 0.57–0.92), with a subgroup analysis showing a more pronounced benefit in patients with hormone receptor-positive disease. Diarrheal prophylaxis is recommended, given the high frequency of grade 3–4 diarrhea. The role of neratinib in the post-KATHERINE trial era is unknown.

#### **5. Metastatic Disease**

In patients with HER2+ metastatic breast cancer, four HER2-directed therapies are approved (herceptin, pertuzumab, kadcyla or lapatinib). For patients who want to avoid chemotherapy, single agent herceptin can be used, however with the caveat that with progression, chemotherapy should be considered. For patients with hormone receptor-positive and HER2+ cancers, a combination of endocrine therapy and HER2-directed therapy can be used. However, if the disease is rapidly progressive or if there is visceral involvement, HER2-directed therapy plus chemotherapy is typically recommended.

Patients are typically treated with herceptin and a taxane (docetaxel or paclitaxel) + pertuzumab. Addition of pertuzumab was shown to improve overall response rates (80% versus 69%), progression-free survival (median PFS 19 versus 12 months) and overall survival (medial OS 56.5 versus 40.8 months) in the CLEOPATRA trial [25]. However, the addition of pertuzumab to herceptin and docetaxel led to an increased incidence of diarrhea, neutropenia, rash and serious febrile neutropenia, without increasing the risk of cardiotoxicity.

Cytotoxic chemotherapy is usually discontinued after 6–12 months if patients achieve a response. If patients progress six months after receiving herceptin, triple therapy with herceptin, pertuzumab and a taxane can be re-introduced. However, if patients progress within six months of receiving herceptin, kadcyla (ado-trastuzumab emtansine) is recommended. In the Phase III EMILIA trial [26], patients who were previously treated with herceptin and a taxane were randomized to receive kadcyla or lapatinib + capecitabine. There was an improvement in progression-free survival (10 months vs. 6 months, HR 0.65, 95% CI 0.55–0.77), overall survival (median OS 31 months vs. 25 months; HR 0.68, 95% CI 0.55–0.85) and overall response rate (44% vs. 31%). Serious toxicities included thrombocytopenia (13% versus 0.2%) and a higher incidence of bleeding (30% versus 16% in the lapatinib + capecitabine arm). In the TH3RESA trial [27], for patients with unresectable, locally advanced, metastatic or recurrent HER2+ breast cancer, who had progressed while on two HER2-directed therapies (herceptin and lapatinib), kadcyla resulted in an improvement in progression-free survival (6.2 vs. 3.3 months; HR 0.53, 95% CI 0.42–0.66) and improvement in overall survival (22.7 vs. 15.8 months; HR 0.68, 95% 0.52–0.85). Given the data, kadcyla is usually considered in the second line setting. The Phase III MARIANNE trial [28] studied kadcyla in the first line setting. The study found that there was no significant difference between herceptin with a taxane versus kadcyla with a placebo versus kadcyla with pertuzumab. Upon progression while on kadcyla and trastuzumab-containing regimens, other options, such as lapatinib + capecitabine (given the improvement in time to progression compared to capecitabine alone), or a combination of anti-hormonal therapy along with anti HER2 therapy, can be considered. In the metastatic setting, HER2-directed therapy is often continued even with disease progression.

#### **6. Brain Metastasis in HER2**+ **Disease**

HER2-targeted therapies could be considered instead of locally directed therapies, such as radiation, in patients with brain metastasis from HER2+ breast cancer. After progression while on trastuzumab (with or without pertuzumab) with a taxane, trastuzumab-emtansine is typically utilized, based on the retrospective exploratory analysis from the EMILIA trial [29]. Among patients with CNS (central nervous system) metastasis, there was significant improvement in overall survival in the T-DM1 arm, compared to the lapatinib + capecitabine arm (hazard ratio of 0.38, *p* = 0.008, 26.8 months versus 12.9 months). The efficacy of capecitabine + lapatinib was studied in the LANDSCAPE trial [30]. In this trial, 66% of the patients (29 patients out of 44 patients) were found to have a partial response, with a median time to progression of 5.5 months. The 6-month overall survival was 90%. The combination of lapatinib and capecitabine is typically utilized as a later-line therapy.

The next line of therapy for patients with HER2+ CNS metastasis, after progression while on kadcyla, is typically a combination of tucatinib, capecitabine and trastuzumab [31]. Specifically in relation to brain metastases, 25% of the patients with brain metastases had a one-year PFS when treated with tucatinib with capecitabine and trastuzumab, compared to 0% in the trastuzumab + capecitabine arm (HR 0.48, 95% CI 0.34–0.69). The median PFS in the tucatinib arm was 7.6 months versus 5.4 months in the capecitabine + trastuzumab arm.

Another option for patients with HER2+ metastases is neratinib + capecitabine, based on the Phase II trial [32] that studied 49 patients with HER2+ brain metastasis. Among the lapatinib naïve patients, the objective response rate was 49%, with a median PFS of 5.5 months and overall survival 13.3 months. Among patients who received lapatinib previously, the objective response rate was 33%, with a median PFS of 3.1 months and overall survival of 15.1 months.

Bevacizumab, a monoclonal antibody against vascular endothelial growth factor A, is a systemic therapy option that has been studied in single-arm, Phase II studies [33], with reported response rates of >50% when added to a platinum-based regimen with cisplatin and etoposide.

#### **7. Mechanism of Action of HER2-Directed Therapies and Resistance Mechanisms**

As discussed above, the HER2 receptor is a transmembrane tyrosine kinase receptor that belongs to the human epidermal growth factor receptors (EGFR). It is expressed at a low level on the surface of epithelial cells, and is needed for development in several tissue types, such as the breast, ovary, central nervous system, lung, liver and kidney [10]. It is overexpressed in 25–30% of breast cancer cells. As shown in Figure 1, HER2 forms homodimers (binding of same receptor) or heterodimers (binding of different receptors) with other members of the human epidermal growth factor receptors. The HER2 protein can exist in an inactivated state, and dimerize independent of the binding of a ligand. The binding of a ligand induces phosphorylation of the receptors, which in turn activates the MAPK (mitogen-activated protein kinase) pathway and the PI3K (phosphatidylinositol 3-kinase) pathways.

The development of trastuzumab revolutionized the treatment of HER2+ breast cancer by introducing a monoclonal antibody that specifically targeted breast cancer cells that overexpressed aberrant HER2 receptors. Trastuzumab binds to the domain IV region of the extracellular site of the HER2 protein, thereby preventing dimerization, and subsequently signal transduction and cell survival. Pertuzumab is a monoclonal antibody that binds to domain II of the extracellular component of HER2, thereby preventing dimerization with Her1 and HER3. Ado-trastuzumab-DM1 (T-DM1, Kadcyla) combines the antibody (trastuzumab) with DM1 (anti-microtubule agent derived from maytansine), which then delivers the drug in the intracellular compartment [34]. Trastuzumab, pertuzumab and T-DM1 utilize antibody-dependent cellular cytotoxicity. Lapatinib is a dual tyrosine kinase inhibitor that reversibly binds to the tyrosine kinase receptors (EGFR or Erb1, and HER2 or ErbB2), thereby blocking the phosphorylation and activation of ERK (extracellular signal-regulated kinase) and AKT (protein kinase B). Neratinib, on the other hand, is an irreversible tyrosine kinase inhibitor of HER1, HER2 and HER4, thereby preventing the downstream signaling of the MAP kinase pathway and the AKT signaling pathways.

Although these HER2-targeted therapies have been proven to be efficacious in the various studies previously mentioned, studies have found that less than 35% of patients with HER2+ breast cancer initially respond to trastuzumab [10]. On the other hand, some patients acquire resistance after being on HER2-targeted therapies for several months after initial response. Several mechanisms of resistance to the trastuzumab-based therapy have been proposed [10]. Dr. Vu and colleagues [10] have shown that defects within the HER2 receptor, such as a truncated extracellular domain, prohibits the binding of trastuzumab to the receptor. For example, HER2 can mutate in a fashion that results in a truncated p95HER2 isoform, which inhibits the binding of trastuzumab due to the lack of the extracellular domain [35]. Studies have found that those who acquired the p95HER2 mutation were less likely to respond to trastuzumab as this mutated isoform results in constitutive kinase activity [36]. One way to overcome this form of resistance is to add lapatinib to trastuzumab, given that lapatinib acts intracellularly [37].

Another broad category in the different mechanisms of resistance includes elevations of other tyrosine kinase inhibitors. For example, studies have found that the cross-signaling between Insulin-like Growth factor-1 receptor (IGF-IR) and HER2 induces phosphorylation of HER2, and thereby activates signaling transduction [38]. Overexpression of c-met (tyrosine protein kinase met or hepatocyte growth factor receptor) has been found to confer resistance to trastuzumab [39].

Intracellular alterations can also cause resistance to HER2-targeted therapy. One such alteration involves the hyperactivation of the PI3K pathway by mutations or loss of PTEN (phosphatase and tensin homolog deleted on chromosome 10), which is a tumor suppressor gene that normally inhibits the PI3K pathway [35]. Patients with PTEN-deficient tumors had lower overall response rates to trastuzumab than patients with wild type PTEN [40]. Clinical trials, incorporating different drugs and combination strategies to overcome these mechanisms of resistance, are in progress.

**Figure 1.** HER2 signaling pathway, mechanism of action of targeted therapies, and resistance mechanisms. 1. The truncated P95HER2 isoform results in the loss of the extracellular binding site for trastuzumab. 2.–3. Overexpression of other tyrosine kinase receptors, such as IGF1-R and C-met, can continue to trigger downstream signaling despite blockade by trastuzumab. 4. Mutations or loss of PTEN constitutively activates the PI3K signaling pathway.

Not much is known about the mechanisms of resistance to pertuzumab. However, one theory that has been proposed is the suppression of microRNA-150, which are small, noncoding, single-stranded RNAs, that negatively regulate the PI3K-AKT pathways. Studies in ovarian cancer [41,42] have found that the suppression of miRNA-150 resulted in decreased sensitivity to pertuzumab.

Given that combining trastuzumab with lapatinib targets both the intracellular and extracellular HER2 domains [37], combining these two drugs is an attractive strategy. However, acquired mechanisms of resistance to lapatinib often develop after chronic exposure to lapatinib. One theory is that lapatinib promotes the transcription of estrogen-positive genes, and therefore switches cell survival dependence from HER2 to estrogen receptors [43]. Another proposed mechanism involves the activation of AXL, a membrane-bound tyrosine kinase [44]. AXL has been associated with activation of the AKT/MTOR pathway [37].

The proposed theories concerning resistance to T-DM1 (kadcyla) include low tumor HER2 expression, poor internalization of the HER2-T-DM1 complexes, defective intracellular trafficking of the HER2-T-DM1 complex, and defective lysosomal degradation of T-DM1. These result in inadequate drug concentrations, and therefore cell death is halted [45]. Another proposed mechanism of resistance to T-DM1 is the presence of neuregulin b1 (NRG), which suppresses the cytotoxic activity of T-DM1 by triggering the formation of HER2-HER3 heterodimers. This heteromization activates the PI3K pathway, and thus leads to cell cycle proliferation independently of kadcyla [46,47]. SYD985, another HER2-targeting antibody-drug conjugate [48], is currently being studied in patients who develop resistance to T-DM1.

#### **8. Newer HER2-targeted Therapies**

Although trastuzumab (herceptin), pertuzumab (perjeta), Lapatinib (tykerb) and ado-trastuzumab emtansine (kadcyla) remain the most used HER2-targeted therapies in practice, other new HER2 therapies are being studied in clinical trials.

In the Phase III SOPHIA trial [49], margetuximab was compared to trastuzumab in patients with metastatic HER2+ breast cancer after progression while on the first, second or third line of therapy including kadcyla. In the study, the median age of the patients was 55 years. The backbone chemotherapy was of the investigator's choosing, between four systemic cytotoxic therapies (capecitabine—27% in each arm; eribulin—25% in each arm; gemcitabine—12% in each arm; vinorelbine—33% in each arm). Of note, more than 90% of the patients received kadcyla.

Margetuximab is a novel Fc-engineered monoclonal antibody that targets the HER2 oncoprotein. The Fc portion enhances the immune system in order to provide an added benefit. Of the patients in the study, 85% carried the CD16A 158F allele, which is shown to have a diminished response to trastuzumab. The patients who were homozygous for the CD16A-F allele appeared to attain longer progression-free survival with margetuximab compared to trastuzumab. Patients treated with margetuximab had a higher median PFS and higher overall response rates. The adverse reactions were similar between the two groups.

In the ALTERNATIVE trial [50], the addition of lapatinib to the trastuzumab and endocrine therapy (aromatase inhibitor) was studied in postmenopausal women with HER2+, hormone receptor-positive metastatic breast cancer. The addition of lapatinib to trastuzumab led to higher median progression-free survival (primary end point) and overall survival (see Table 1) rates.


#### **Table 1.**Trials of Newer HER2 Targeted Therapies.

BC, breast cancer; MARG, margetuximab; TRAS, trastuzumab; HR, hormone receptor; MBC, metastatic breast cancer; ET, endocrine therapy; LAP, lapatinib; AI, aromatase inhibitor; ORR, overall response rate; OS, overall survival; PFS, progressive free survival; TUC, tucatinib; CAP, capecitabine; RR, response rates.

The median progression-free survival associated with the combination of lapatinib with trastuzumab was 11 months, versus 5.7 months in the trastuzumab arm (HR 0.62, 95% CI 0.45–0.88). The data for overall survival was immature in the trial. The addition of lapatinib (and removal of pertuzumab) could be considered in patients who progressed after receiving trastuzumab, pertuzumab and an AI, if the disease is not rapidly progressive or if there is not a visceral crisis.

As of May 2020, there are two novel agents approved for the treatment of patients with advanced HER2+ breast cancer, and who have previously been treated.

In December 2019, the FDA approved trastuzumab deruxtecan-nxki (DS-8201a/Enhertu) in patients who have been previously treated with two or more prior anti-HER2 therapies in the metastatic setting. This approval was based on the results of an open-label, single group, phase 2 study with 184 patients who had previously received a median of six lines of therapy [52]. The primary end point of the DESTINY- Breast01trial (Available online: https://clinicaltrials.gov/ct2/show/NCT03248492, identifier NCT03248492, last accessed 7/12/2020) was an overall response rate observed to be 60.9%, with patients achieving a disease control rate of 97.3%, median progression-free survival of 16.4 months and a median duration of response of 14.8 months. The median overall survival has not been reached. DS-8201 is an antibody–drug conjugate like kadcyla, but with a topoisomerase I inhibitor payload linked to a humanized monoclonal antibody, and to HER2, by a cleavable tetrapeptide linker. The most common adverse events noted were neutropenia, interstitial lung disease, anemia, nausea and alopecia. Death due to interstitial lung disease was reported in 2.2% of patients. Currently, ongoing trials are evaluating the effectiveness of DS-8201a against kadcyla in a randomized control trial (DESTINY-Breast03 trial; NCT03529110), as well as the effectiveness of capecitabine and herceptin or lapatinib (DESTINY-Breast02 trial; NCT03523585).

Based on the results of the HER2CLIMB trial [31], another novel HER2-targeted agent, tucatinib, was approved in April 2020 [31]. Tucatinib is an orally bioavailable, highly selective small-molecule HER2 tyrosine kinase inhibitor, which binds to the internal domain of the HER2 protein. Given the small size of the molecule, tucatinib is believed to cross the blood–brain barrier. It causes minimal EGFR inhibition, and hence is relatively well tolerated. Patients with previously treated, unresectable, locally advanced or metastatic HER2+ breast cancer were treated with either tucatinib + trastuzumab + capecitabine or with placebo + trastuzumab + capecitabine. The study allowed patients with untreated but asymptomatic brain metastasis to participate. Tucatinib combination therapy showed statistically significant improvements in progression-free survival at 1 year (primary end point), as well as in the overall survival at 2 years, in the overall population.

Given the high response rate, tucatinib combination is a promising therapy for previously treated HER2+ metastatic breast cancer patients, and could emerge as the go-to option, especially in patients with central nervous system involvement.

Neratinib is a pan-HER inhibitor, which has been recently approved in the metastatic setting, based on the NALA trial [53] and the TBCRC 022 [32]. In the Phase III NALA trial, neratinib plus capecitabine improved progression-free survival compared to lapatinib with capecitabine (12-month PFS was 29%, versus 15%). [53]. However, patients who received neratinib with capecitabine experienced more diarrhea. 25% of the patients in the neratinib plus capecitabine group experienced grade > 3 diarrhea compared to 13% of the patients receiving lapatinib with capecitabine.

Pyrotinib and poziotinib are both irreversible pan-HER inhibitors. Pyrotinib has already received conditional approval in China, based on a Phase II trial which showed improved PFS in combination with capecitabine, with tolerable side effects [54]. A Phase III trial is currently ongoing (NCT03080895).

#### **9. Novel Antibody Drug Conjugates (ADCs)**

Following the success and FDA approval of two Antibody Drug Conjugates (ADCs), there are multiple other agents currently in clinical trials. Like DS-8201, these newer ADCs have a cleavable linker that accounts for what is called the "bystander effect". Bystander effect is responsible for the death of antigen-negative cells (both cancer cells and normal cells), hence it is important in both the efficacy and safety of the drug. Here we discuss a few ADCs with published clinical trial results.

These second-generation ADCs are thought to overcome the resistance of HER2+ cells to T-DM1. One of the ADCs currently in clinical trial, SYD985 (Trastuzumab Duocarmazine), was shown to be effective in T-DM1-resistant patient-derived tumor models [55]. Results of the dose-escalation/dose-expansion study with this agent demonstrated clinical activity in heavily pre-treated HER2+ patients, with a partial response of 33% and a median PFS of 7.6 months [55]. Currently, the Phase III randomized TULIP trial (NCT03262935) is evaluating the drug SYD985 against other standard of care options for previously treated HER2+ breast cancer patients.

Another ADC currently in Phase III trial is BAT8001 (Bio-Thera; NCT04185649), which uses a novel non-cleavable linker between trastuzumab and the maytansine payload. A phase I dose-escalation study (NCT04189211) revealed the drug to be safe, and it also showed efficacy in heavily pre-treated HER2+ patients. A randomized multi-center Phase III trial is ongoing in China [56].

RC48-ADC, or Distamab vedotin (RemeGen), is another antibody–drug conjugate with a cleavable cathepsin linker attached to the monomethyl auristatin E (MMAE) payload. In the Phase Ib/II trial, RC48 demonstrated good tolerability and efficacy. The disease control rate was seen to be 96.7%, with a 46.7% clinical benefit rate (CBR) [57]. A Phase II study is ongoing (NCT03500380).

DHES0815A (Genentech) is an engineered ADC, in which trastuzumab is conjugated with a stable linker to a highly toxic payload, pyrrolobenzodiazepine, and is the subject of an ongoing Phase I study (NCT03451162).

#### **10. Bispecific Antibodies**

These are molecules that recognize two different epitopes of the HER2 protein, and there are several of these currently in trials. In addition to these antibodies blocking tumor signaling pathways, they also engage immune cells and deliver payloads to destroy tumor cells [58].

ZW49 (Zymeworks) is a bispecific, biparatopic antibody, with an auristatin payload of an anti-HER2 biparatopic antibody, ZW25, which binds the same domains as trastuzumab (ECD2) and pertuzumab (ECD4). In pre-clinical breast cancer cell lines and PDX models, ZW49 has demonstrated anti-tumor activity [59]. A Phase I, dose-finding, multicenter, open-label trial is ongoing to assess the safety and tolerability of the drug (NCT03821233).

ZW25 increases tumor cell binding, improves receptor internalization and downregulates HER2 expression. A Phase I study showed a partial response rate of 33%, with a disease control rate of 50% [60].

BTRC4017A (Genentech) is currently being explored in a Phase Ia/b clinical trial (NCT03448042). It has a T-cell-dependent bispecific monoclonal antibody with two antigen recognition sites, one for HER2 and another one for the CD3 complex, which leads to the cross linking of HER2-expressing tumor cells and cytotoxic T lymphocytes.

TrasGex or Timigutuzumab is a glyco-optimized antibody, which has shown efficacy in a dose-escalation Phase I study by enhancing the antibody-dependent cell-mediated cytotoxicity [61]. However, it is unknown if the molecule is still being developed or not.

The experimental drug, NJH395 (Novartis), an immunoconjugate immune stimulator antibody conjugate (ISAC) consisting of a monoclonal anti-ErbB2 antibody conjugated to a TLR7 (Toll-like receptor 7) agonist, is another drug currently being studied in clinical trials (NCT0369771). No pre-clinical data is available yet.

#### **11. Immunotherapy in HER2**+ **Disease**

In addition to newer anti-HER2-targeted therapies, immunotherapy is being extensively studied given the ability of cancers to evade the immune system [62]. Different modalities are being utilized in immunotherapy to boost the patient's immune system so as to attack the cancerous cells. Immune checkpoint inhibitors, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibodies and programmed cell death-1 (PD-1)/programmed cell death-1 ligand (PD-L1) antibodies, are being studied in clinical trials. The expression of PDL-1 has been shown to be associated with unfavorable characteristics, such as HER2+ status in addition to large tumor sizes and high tumor grades [63]. Although PDL-1 therapy is approved for triple negative breast cancers [64], there is no approved PD-1/PDL-1 agent for the treatment of HER2+ breast cancers. In the Javelin Solid Tumor study (Phase Ib) [65], avelumab had an overall response rate of 3% (of the 168 patients in the study; 26 (15.5%) patients were HER2+), but a higher activity was seen if they exhibited PDL-1 expression. Most recently, in the single-arm, multicenter PANACEA trial [66], pembrolizumab was studied in patients with advanced HER2+ breast cancer, who progressed while on the trastuzumab or T-DM1 therapy in this Phase Ib/II trial. While six patients were enrolled in the phase Ib trial (all PDL-1-positive), 52 patients were enrolled in the phase II trial. Patients were tested for PDL-1 expression. Of the 52 patients in the phase II trial, 40 had PDL-1-positive tumors, and the remaining patients had PDL-1-negative tumors. Although no objective responses were seen in the PDL-1-negative patients, the disease control rate was 24% in PD-L1-positive patients, and the overall response rate was 39%. The most common adverse reaction that was noted was fatigue (21%).

#### **12. Future Directions**

Although patients do relatively well on the therapies previously mentioned, a subset of HER2+ breast cancer patients experienced relapse after neoadjuvant and adjuvant therapy, as well as resistance to existing therapies. A dual-targeted HER2 approach is typically used if there are no contraindications. However, what remains to be determined is whether adding a newer HER2-targeted therapy would increase progression-free survival, or whether immunotherapy would help in overcoming the resistance to HER2-targeted therapies. In addition, many trials did not utilize the updated 2018 ASCO (American Society of Clinical Oncology)/CAP (College of American Pathologists) guidelines on HER2 interpretation in breast cancer [6], which generated an increase in negative cases due to the more rigorous algorithm for identifying HER2+ patients. This algorithm helped to reclassify equivocal cases as either HER2+ or HER2−. Treatment of HER2 equivocal cases was not standardized, given the lack of data surrounding the clinical benefits of HER2-targeted therapies in this subset of patients. Since the 2018 guidelines reclassify these patients as either HER2+ or HER2−, the overtreatment of patients can be avoided, and costs can be saved.

There is still an unmet need for treatment of metastatic HER2+ breast cancer. Whether adding or utilizing immunotherapy upfront, like in other subtypes of breast cancer, can improve patient outcomes remains unknown. Different strategies are being employed to improve the efficacy of anti-HER2 therapy by combining existing approved therapies and exploring next-generation sequencing to target potential biomarkers, so as to overcome resistance and reduce side effects. There is much more to be studied in HER2+ breast cancer.

#### **13. Conclusions**

The discovery and implementation of new HER2-directed therapies in clinical practice has significantly changed how patients with HER2+ diseases are being treated. Although major strides have been made in treating patients with HER2+ diseases, studies are ongoing concerning the continued improvement of the outcomes for this subset of patients. Whether it is a combination of multiple HER2-directed therapies in the various settings, or the invention of immunotherapy, the treatment of HER2+ disease has resulted in better outcomes, including in progression-free survival and overall survival, compared to the outcomes of previous decades, and these will continue to evolve.

#### **Funding:** This research received no external funding.

**Conflicts of Interest:** Nisha Unni declares is serving on the advisory board of Eisai Inc. The other authors do not have a conflict of interest.

### **References**


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