**Nanodelivery Systems Targeting Epidermal Growth Factor Receptors for Glioma Management**

**Sathishbabu Paranthaman <sup>1</sup> , Meghana Goravinahalli Shivananjegowda <sup>1</sup> , Manohar Mahadev <sup>1</sup> , Afrasim Moin <sup>2</sup> , Shivakumar Hagalavadi Nanjappa <sup>3</sup> , Nandakumar Dalavaikodihalli Nanjaiyah <sup>4</sup> , Saravana Babu Chidambaram <sup>5</sup> and Devegowda Vishakante Gowda 1,\***


Received: 30 September 2020; Accepted: 18 October 2020; Published: 10 December 2020

**Abstract:** A paradigm shift in treating the most aggressive and malignant form of glioma is continuously evolving; however, these strategies do not provide a better life and survival index. Currently, neurosurgical debulking, radiotherapy, and chemotherapy are the treatment options available for glioma, but these are non-specific in action. Patients invariably develop resistance to these therapies, leading to recurrence and death. Receptor Tyrosine Kinases (RTKs) are among the most common cell surface proteins in glioma and play a significant role in malignant progression; thus, these are currently being explored as therapeutic targets. RTKs belong to the family of cell surface receptors that are activated by ligands which in turn activates two major downstream signaling pathways via Rapidly Accelerating Sarcoma/mitogen activated protein kinase/extracellular-signal-regulated kinase (Ras/MAPK/ERK) and phosphatidylinositol 3-kinase/a serine/threonine protein kinase/mammalian target of rapamycin (PI3K/AKT/mTOR). These pathways are critically involved in regulating cell proliferation, invasion, metabolism, autophagy, and apoptosis. Dysregulation in these pathways results in uncontrolled glioma cell proliferation, invasion, angiogenesis, and cancer progression. Thus, RTK pathways are considered a potential target in glioma management. This review summarizes the possible risk factors involved in the growth of glioblastoma (GBM). The role of RTKs inhibitors (TKIs) and the intracellular signaling pathways involved, small molecules under clinical trials, and the updates were discussed. We have also compiled information on the outcomes from the various endothelial growth factor receptor (EGFR)–TKIs-based nanoformulations from the preclinical and clinical points of view. Aided by an extensive literature search, we propose the challenges and potential opportunities for future research on EGFR–TKIs-based nanodelivery systems.

**Keywords:** glioblastoma; receptor tyrosine kinases; epidermal growth factor receptor; small molecule inhibitors; nanoformulations

#### **1. Introduction**

Gliomas are the most common and lethal solid brain tumors and are known to affect about 0.02% of the worldwide population [1]. The occurrence of malignant gliomas and the frequency of cancer deaths have increased at an amplified rate across the world [2]. More than 330,000 new Central Nervous system (CNS) tumor cases and 227,000 brain cancer-related deaths were documented globally in the Global Burden of Disease (GBD) 2016 tumor database [3]. Despite the increase in cancer awareness programs, advancement in diagnostic tools, and treatment strategies in the United States, the prevalence of gliomas has been unstoppable [4–6].

Based on the molecular characteristics and origin of apparent cell types, the CNS tumors were classified using I to IV grade criteria by the World Health Organization (WHO) in 2007 and 2016 [7]. Accordingly, Grade, I, II, III, and IV are pilocytic astrocytomas, gliomas including diffuse astrocytomas, anaplastic astrocytomas, and glioblastoma multiforme (GBM), respectively [8,9]. The current standard treatment approaches across the world are dependent on surgery, radiotherapy, and chemotherapeutic drugs, i.e., temozolomide (TMZ), which resulted in the average survival rate of about 14 months [10–12]. Therefore, there is a definite need for understanding the molecular pathways and mechanisms involved in GBM pathology and thereby determine better management [13].

Generally, Receptor Tyrosine Kinase (RTKs) are commonly identified cell surface receptors that are considered to be pivotal regulators of critical cellular processes (epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR)). EGFR is a transmembrane receptor tyrosine kinase that controls cancer cell proliferation, migration, differentiation, and homeostasis [14]. Nearly 50–60% of GBMs have EGFR genetic variants, with mutations, readjustments, selective linking, and amplification [15].

Over the past decades, many investigators have hundreds of designs and synthesized small molecular drugs as RTK inhibitors with extensive research. The Food and Drug Administration (FDA) has approved a few medications as first-line therapy for various forms of cancer (Table 1) [16]. However, the significant development of anti-cancer components has developed new problems [17]. For example, clinical studies that were conducted for the first and second generation of anti-EGFR drugs on the inhibition of cell growth, angiogenesis, and proliferation were found to be of no therapeutic benefit in GBM treatment. Many researchers also reported significant limitations such as low solubility, poor oral bioavailability, and severe adverse effects in the existing EGFR–TKIs drugs. In addition, the gradual rise of drug resistance during therapy instantly needs to be addressed [18]. The third generation of EGFR–TKIs drug (AZD9291) was developed recently and confirmed to have an effective preclinical investigation in GBM the in vitro and in vivo models' above-listed drawbacks [15]. The advantages of nanotechnology offer a potential drug delivery approach with apparent benefits of nanoformulations such as lesser particle size, bulky surface area, excellent surface reactivity, active sites, and appropriate adsorption ability. Nano particles (NPs) applied as drug transporters have the potential to increase drug absorption and bioavailability, enrich effective targeting delivery, prolong the circulation time, and limit the dangerous side effects on healthy tissues [19].

In the present review, the authors have summarized epidemiology and risk factors associated with GBM, RTKs, and their inhibitors of intracellular signaling pathways in glioma, the clinical profile of small molecule inhibitors (EGFR–TKIs) drugs, and the associated multiple failures/resistance. In addition, the current progressive research of various nanopreparations for EGFR–TKIs and the combination of chemotherapeutic drugs to target GBM have been discussed. Aided by an extensive literature search and review, the authors have also proposed the possibilities and challenges for upcoming research on EGFR-TKIs and other chemotherapeutic agents.


**Table 1.** Approved small molecular tyrosine kinase inhibitors for cancer therapy [16,19].

#### **2. Molecular Pathology of Glioma**

Different genetic investigations determine various noteworthy biomarkers. Many of these were utilized in neuro-oncology to identify glioma patients, specifically the combined losses of the chromosome arms 1p and 19q in oligodendroglial cancer. Then, the methylation status of O-6 methylguanine–DNA methyltransferase gene promoter and modifications in the EGFR pathway in GBM, isocitrate dehydrogenase 1 (IDH1) and IDH2 gene mutations in diffuse gliomas, as well as B-Raf status in pilocytic astrocytomas. These groups are associated with different prognosis, germline variants, and the median age at diagnosis, highlighting different pathogenic mechanisms [20]. Although most GBM patients receive standard treatments, significant variations in clinical outcomes are often seen due to the heterogeneity of the tumors [21,22]. Hence, it is essential to determine more significant and practical biomarkers for analyzing the prognosis in GBM patients. Inflammation and immunity are critically involved in glioma initiation and progression [23,24], and various study reports suggested that inflammatory response cells such as neutrophils [25], lymphocytes [26], and platelets [27] are associated with the prognosis of cancer patients. In recent years, the prognostic value of preoperative hematological markers, such as albumin-to-globulin ratio (AGR), monocyte-to-lymphocyte ratio (MLR), median platelet volume (MPV), neutrophil-to-lymphocyte ratio (NLR), platelet distribution width (PDW), and platelet-to-lymphocyte ratio (PLR), have been investigated in several cancers, including gliomas [28–32]. However, there were no scientific investigations to the prognostic value of hematological biomarkers in a cohort of gliomas, mainly in relation to the various molecular classes. Therefore, a study examined the predictive value of preoperative hematological biomarkers (AGR, MLR, MPV, NLR, PDW, and PLR) alone and in combination with the five glioma molecular groups on the clinical trial results of a comparatively great cohort (n = 592) of Grade II–IV GBM patients. Based on these results, we suggest an analytical model for Grade II–IV GBM based on molecular pathology and NLR, and identify for lower-grade gliomas (LGG) four risk groups with distinct overall survival [33].

#### **3. Epidemiology and Risk Factors of Glioma**

In the past few decades, the investigation of adult glioma was prioritized because of a lesser global incidence of GBM, i.e., 10 per 100,000 people. Due to the lack of new and efficient diagnostic strategies, the survival rate (SR) of 15 months after diagnosis creates a critical public health problem [2,34,35]. GBM accounts for 50% of all gliomas in various age groups [36]. Although the peak incidence is between 55 and 60 years of age, GBM could occur at any age, with a mortality rate of 2.5% of the worldwide cancer death toll. GBM accounts for the third foremost cause of deaths due to cancer in patients from 15 to 34 years [37,38]. The GBM incidence ratio was more in men when compared to females [39]. The Western world reported a higher incidence of gliomas than less developed countries, which could be recognized as due to under-recording glioma cases, narrow contact to health care, and alterations in diagnostic practices [40–42]. A few studies showed that blacks were less prone to GBM. Further, the incidence of GBM was reported to be higher in Asians, Latinos, and Whites [43].

The current global standard for the catalog and identification of gliomas is as per WHO classification. WHO categorizes gliomas as Grade I to IV based on malignancy level, which is committed by the histopathological measures. Class I to III gliomas relay to abrasions with less proliferative potential and can be managed surgically with chemo and/or radiotherapy. In contrast, Grade IV gliomas are highly malignant and invasive. GBM is the utmost aggressive, offensive, and identical type of cancer and was labeled as Grade IV [44].

A positive family history, absence of atrophic conditions, longer length of leukocyte telomere, and risk alleles at more than twenty genetic loci are a few of the endogenous factors that enhance glioma risk. A high dose of ionizing radiation is also one of the environmental factors attributed to a higher risk of glioma [45]. Identifying modifiable factors that would enable primary prevention approaches remain the quintessential goal of glioma epidemiologic research.

Several studies on the allergic and nutritional epidemiology of glioma showed an inverse association between allergy and gliomas but did not provide any causal relationship between them [46]. The nutritional epidemiology studies suggested that various food groups and nutrients were associated with glioma risk; the results were inconclusive and not replicable in subsequent research [24,47]. The results of epidemiological analysis also suggested the presence of an inverse association between cancer and certain neurological conditions, mainly age-related neurodegenerative diseases [48]. In recent research, a potent negative association was observed between the expression levels of microRNAs in GBM than Alzheimer's disease (AD), suggesting that although the molecular pathways behind the development of these two pathologies are the same, they appear to be inversely controlled by microRNAs [49]. Another epidemiological study indicated that the patients suffering from AD have a lower risk of developing lung cancer (LC) and suggest a higher risk of developing GBM [50].

#### **4. Receptors Tyrosine Kinase (RTK) and Their Inhibitors of Intracellular Signaling Pathways in Glioma**

RTKs belong to the family of cell surface receptors and are receptors for hormones, growth factors, neurotrophic factors, cytokines, and extracellular signaling molecules. The tyrosine kinase (TK) comprises the intracellular TK domain, extracellular ligand-binding domain, and a hydrophobic transmembrane domain. The domains as mentioned above get activated upon binding of the ligand, leading to the TK domain's autophosphorylation and dimerization. This receptor, when activated by ligands in turn, activates two downstream signaling pathways, Rapidly Accelerating Sarcoma/mitogen activated protein kinase/extracellular-signal-regulated kinase (Ras/MAPK/ERK) and phosphatidylinositol 3-kinase/a serine/threonine protein kinase/mammalian target of rapamycin (PI3K/AKT/mTOR) [51] (Figure 1), which play a prominent role in cell differentiation, survival, proliferation, and angiogenesis. Thus, RTKs and their ligands were proven to be promising targets in the treatment of GBM. Among the several receptors belonging to the RTK group in human glioma, the signaling pathways such as EGFR and VEGF receptor mutations have played a significant role in GBM described below in detail [52].

GBM described below in detail [52].

the treatment of GBM. Among the several receptors belonging to the RTK group in human glioma,

**Figure 1.** Schematic representation of Receptor Tyrosine Kinases (RTK) activation and the downstream signaling. RTKs, particularly epidermal growth factor receptors (EGFR), are amplified in glioblastoma, which significantly alters the nutrient uptake and utilization. The Rapidly Accelerating Sarcoma/mitogen activated protein kinase/extracellular-signal-regulated kinase (Ras/MAPK/ERK) and phosphatidylinositol 3-kinase/a serine/threonine protein kinase/mammalian target of rapamycin (PI3K/AKT/mTOR) pathways get activated through the stimulation by growth factor receptor (GFR). Physiologically, these two pathways orchestrate to execute cell proliferation, survival, motility, adhesion, and angiogenesis. Any deregulation in these pathways leads to an activation of oncogenic signaling cascades causing glioma. **Figure 1.** Schematic representation of Receptor Tyrosine Kinases (RTK) activation and the downstream signaling. RTKs, particularly epidermal growth factor receptors (EGFR), are amplified in glioblastoma, which significantly alters the nutrient uptake and utilization. The Rapidly Accelerating Sarcoma/mitogen activated protein kinase/extracellular-signal-regulated kinase (Ras/MAPK/ERK) and phosphatidylinositol 3-kinase/a serine/threonine protein kinase/mammalian target of rapamycin (PI3K/AKT/mTOR) pathways get activated through the stimulation by growth factor receptor (GFR). Physiologically, these two pathways orchestrate to execute cell proliferation, survival, motility, adhesion, and angiogenesis. Any deregulation in these pathways leads to an activation of oncogenic signaling cascades causing glioma.

#### 4.1. EGFR Family and Its Mutations *4.1. EGFR Family and Its Mutations*

RTKs that generally control the proliferation, migration, and differentiation of neural progenitors via signaling EGFR and its downstream MAPK and PI3K/AKT/mTOR pathways (Figure 1) [17]. EGFR, a member of the ErbB family, is commonly expressed in neural progenitors during brain growth and initiated stem cell astrocytes and transit-amplifying cells in the adult rodent subventricular zone (SVZ) [53,54]. Among 45–57% of GBM patients, the mutation and amplification in EGFR ErbB1 (EGFR, HER1) were detected, which indicated its major role in the pathogenesis of GBM [55,56]. In addition, about 8–41% of GBM patients showed mutations in erythroblastic oncogenic B/human epidermal growth factor receptor (ErbB2/HER-2) [55,57]. Its expression reduces significantly in the adult human cortex (Cx) and white matter (WM) under non-reactive conditions but is retained within the adult human SVZ astrocyte ribbon. The mechanisms maintaining more EGFR expression in human neural growth and its silencing upon difference are not well understood and not have been investigated before at the epigenetic level [58]. Excitingly, the most diffuse gliomas of LGG and high-grade glioma (HGG) have shown the pathological expression of EGFR. Generally, EGFR overexpression in gliomas has been mainly recognized to gene amplification, the activating mutation EGFRvIII, and gene fusion events, which overall comprise approximately half of GBM and are rarely observed in LGG [59,60]. EGFRvIII, a truncated species, is often expressed in GBM and independently activated by a ligand, resulting in cell survival and proliferation. Despite the growthenhancing properties of the EGFRvIII, its expression has been linked to the increasing overall survival of patients. Furthermore, EGFRvIII, being a neoantigen, equally elicits an immune response [60,61]. RTKs that generally control the proliferation, migration, and differentiation of neural progenitors via signaling EGFR and its downstream MAPK and PI3K/AKT/mTOR pathways (Figure 1) [17]. EGFR, a member of the ErbB family, is commonly expressed in neural progenitors during brain growth and initiated stem cell astrocytes and transit-amplifying cells in the adult rodent subventricular zone (SVZ) [53,54]. Among 45–57% of GBM patients, the mutation and amplification in EGFR ErbB1 (EGFR, HER1) were detected, which indicated its major role in the pathogenesis of GBM [55,56]. In addition, about 8–41% of GBM patients showed mutations in erythroblastic oncogenic B/human epidermal growth factor receptor (ErbB2/HER-2) [55,57]. Its expression reduces significantly in the adult human cortex (Cx) and white matter (WM) under non-reactive conditions but is retained within the adult human SVZ astrocyte ribbon. The mechanisms maintaining more EGFR expression in human neural growth and its silencing upon difference are not well understood and not have been investigated before at the epigenetic level [58]. Excitingly, the most diffuse gliomas of LGG and high-grade glioma (HGG) have shown the pathological expression of EGFR. Generally, EGFR overexpression in gliomas has been mainly recognized to gene amplification, the activating mutation EGFRvIII, and gene fusion events, which overall comprise approximately half of GBM and are rarely observed in LGG [59,60]. EGFRvIII, a truncated species, is often expressed in GBM and independently activated by a ligand, resulting in cell survival and proliferation. Despite the growth-enhancing properties of the EGFRvIII, its expression has been linked to the increasing overall survival of patients. Furthermore, EGFRvIII, being a neoantigen, equally elicits an immune response [60,61]. Recent investigations have started to explore EGFR overexpression mechanisms in gliomas outside of genetic alterations, including the role of epigenetics. Still, there is no study that has analyzed the EGFR promoter in human glioma samples.

#### *4.2. VEGF Family and Its Mutations* EGFR promoter in human glioma samples.

VEGF, a potent angiogenic protein, is known to enhance vascular permeability. Although VEGF has a role in normal tissues, malignant transformation has been shown to induce VEGF expression—especially under hypoxic conditions inducing the transcription factors (HIF1α and HIF1β) to translocate to the nucleus, thereby activating the VEGF gene [61] (Figure 1). Upon activation of the VEGF gene, angiogenesis is enhanced to neutralize the hypoxia. GBM tumors enhanced the expression of VEGF and hypoxia, which in turn caused irregular vasculature [62]. The enhanced expression of VEGF in GBM tissues was due to the up-regulation of the VEGF receptor, VEGFR2, which acted by RAS (Rapidly Accelerating Sarcoma) or PI3K (phosphatidylinositol 3-kinase) or the PLCγ–PKC–MAPK pathway in contrast to RTKs [63]. VEGFR3 operates similarly to TK activities. The PKC and RAS pathway is known to be stimulated by lymphangiogenesis in VEGFR-3. VEGF was also shown to play a vital role in vascularization and endothelial cells' neoplastic growth [64]. 4.2. VEGF Family and Its Mutations VEGF, a potent angiogenic protein, is known to enhance vascular permeability. Although VEGF has a role in normal tissues, malignant transformation has been shown to induce VEGF expression especially under hypoxic conditions inducing the transcription factors (HIF1α and HIF1β) to translocate to the nucleus, thereby activating the VEGF gene [61] (Figure 1). Upon activation of the VEGF gene, angiogenesis is enhanced to neutralize the hypoxia. GBM tumors enhanced the expression of VEGF and hypoxia, which in turn caused irregular vasculature [62]. The enhanced expression of VEGF in GBM tissues was due to the up-regulation of the VEGF receptor, VEGFR2, which acted by RAS (Rapidly Accelerating Sarcoma) or PI3K (phosphatidylinositol 3-kinase) or the PLCγ–PKC–MAPK pathway in contrast to RTKs [63]. VEGFR3 operates similarly to TK activities. The PKC and RAS pathway is known to be stimulated by lymphangiogenesis in VEGFR-3. VEGF was also shown to play a vital role in vascularization and endothelial cells' neoplastic growth [64].

*Pharmaceutics* **2020**, *12*, x 6 of 28

of genetic alterations, including the role of epigenetics. Still, there is no study that has analyzed the

#### **5. Molecular Drug Therapy Targets and Its Clinical Profile of EGFR Family in Glioma 5. Molecular Drug Therapy Targets and Its Clinical Profile of EGFR Family in Glioma**

#### *5.1. Small-Molecule Kinase Inhibitors 5.1. Small-Molecule Kinase Inhibitors*

Various active components prevent the EGFR activity, and its ligands have been under progress since the starting of this era. Small molecular EGFR protein tyrosine kinase inhibitors (EGFR–TKIs) have become the most innovative active component in anti-cancer management [52]. EGFR–TKIs are a 4-anilinoquinazoline structure that could covalently link with the ATP binding site of the RTK to procedure the dynamic conformation. The initiation loop was phosphorylated and consequently inhibited the phosphorylation of TK (Figure 2) [65]. Various active components prevent the EGFR activity, and its ligands have been under progress since the starting of this era. Small molecular EGFR protein tyrosine kinase inhibitors (EGFR–TKIs) have become the most innovative active component in anti-cancer management [52]. EGFR–TKIs are a 4-anilinoquinazoline structure that could covalently link with the ATP binding site of the RTK to procedure the dynamic conformation. The initiation loop was phosphorylated and consequently inhibited the phosphorylation of TK (Figure 2) [65].

**Figure 2.** RTKs (EGFR) signal transduction and are a target site for small molecule and monoclonal antibody in glioma treatment. Small-molecule tyrosine kinase inhibitors block the downstream signaling by competing for ATP at the catalytic site of the kinase domain whilst the monoclonal antibodies (mAbs), which have an outstanding degree of specificity, block downstream signaling by binding to the leucine-rich and cysteine-rich ectodomains. Compounds that inhibit mammalian target of rapamycin (mTOR), a downstream signal in the EGFR pathway, facilitate the autophagic clearance of cancerous cells. **Figure 2.** RTKs (EGFR) signal transduction and are a target site for small molecule and monoclonal antibody in glioma treatment. Small-molecule tyrosine kinase inhibitors block the downstream signaling by competing for ATP at the catalytic site of the kinase domain whilst the monoclonal antibodies (mAbs), which have an outstanding degree of specificity, block downstream signaling by binding to the leucine-rich and cysteine-rich ectodomains. Compounds that inhibit mammalian target of rapamycin (mTOR), a downstream signal in the EGFR pathway, facilitate the autophagic clearance of cancerous cells.

Erlotinib, an EGFR-TKI drug, prevents the phosphorylation of the TK intracellular domain of EGFR [66]. Several phase II studies for GBM were not efficient in recurrent GBM [67] patients. In contrast, Erlotinib's combination therapy with temozolamide was well tolerated and enhanced the survival rate in the newly diagnosed GBM patients [68,69]. Gefitinib (ZD1839/Iressa®), another EGFR–TKI, radio sensitized U251 GBM cells in vitro [70]. Still, there was no improvement in the survival rate shown in the phase II clinical trial with newly diagnosed GBM patients [71]. AEE788 and Vandetanib inhibited both EGFR and VEGFR TK (Table 2), but when tested on GBM, patients showed lesser efficacy or enhanced toxicity. AEE788, in a phase I clinical trial, exhibited less efficacy and higher toxicity in treating recurrent GBM patients [72]. Although AEE788 showed very little efficacy in an in vitro GBM cell line, it decreased cell proliferation in vitro when administered in combination with histone deacetylase inhibitors (HDACIs) [73]. In a phase II trial, when incorporated into the standard regimen (surgery + chemotherapy + radiotherapy), AEE788 showed no/little effects, due to which the study was terminated [74].

Lapatinib, an inhibitor of both EGFR and HER2 TKs, showed little effect in a phase I/II clinical trial [75], but in combination therapy with CUDC-101, an HDAC inhibitor, it enhanced the radiosensitivity of the GBM cell line in vitro [76]. Few VEGFR–TKIs such as vatalanib (PTK787), sorafenib, and tivozanib showed lesser efficacy when individually administered (Table 2). Vatalanib and tivozanib did not affect the tumor volume; however, they were well tolerated in a phase II trial [77,78]. Sorafenib's combination therapy with the standard regimen had little effect on recurrent GBM patients in a phase II trial [79]. A phase III trial of Cediranib (AZD2171), another VEGFR–TKI, failed to improve the progression-free survival, both in monotherapy and lomustine recurrent GBM patients [80].

#### *5.2. Targeting Extracellular Domain of RTKs through Antibody Therapies*

Among the various therapies targeted toward the kinase domains of RTK, the extracellular domain also served as a probable target for antibody therapy. The antibodies antagonized the ligand-binding site of RTKs, preventing the ligand binding and thereby activating the kinase domains [81]. An EGFR targeting the antibody cetuximab showed antagonistic activity by inhibiting the activation of RTKs, which in turn inhibited the tumor malignancy [82]. The antibody was used as rescue therapy in patients who have not responded to standard treatment. In addition, cetuximab monotherapy was well tolerated, and minimal recurrence of GBM was reported by a phase II clinical trial [83]. Another monoclonal antibody (mAb), ornartuzumab, targeting the hepatocyte growth factor receptor/tyrosine-protein kinase Met (HGFR/c-MET) receptor's extracellular domain was reported to prevent the cancer growth in orthotopic U87 GBM xenograft. MK-0646 (H7C10/F50035/dalotuzumab), a humanized monoclonal insulin-like growth factor receptor type 1 (IGF-1R) antibody, was shown to be an antagonist that decreased cell proliferation and induced apoptosis [84].

The antibody therapies are still in the preliminary stages of investigation and are promising therapeutic targets for GBM compared to the small molecule kinase inhibitors [85,86]. In addition, the primary constraint faced in the antibody therapy was the Blood Brain Barrier (BBB) penetrability and the large size of molecules, which could be overcome by engineered antibodies capable of penetrating the BBB [87]. Antibodies binding with the transferrin receptors were used to cross the BBB in both murine and primate models. On the other hand, using Ommaya reservoirs or during surgery, the antibodies could directly be delivered to the brain, bypassing the BBB [88].


**Table 2.** Ongoing trials targeting the EGFR in glioblastoma (GBM).


**Table 2.** *Cont.*

R—Recruiting, C—Completed, A—Active, NR—Not Recruiting, T—Terminated, W—Withdrawn, and #—Study has passed its completion date and status has not been verified in more than two years.

#### *5.3. Therapies Directed at RTK Ligands*

Antibodies not only bind to the extracellular domains but also are capable of trapping the ligands that activate the RTK signaling pathways [89]. Targeting the ligands might serve as an attractive means for GBM therapy. However, the usage of antibody was reduced due to various factors such as mutations in EGFRvIII and the inability to cross the BBB, which limited the tumor penetration and efficacy of the therapy. Bevacizumab, a humanized murine mAb, was reported to bind to VEGF and prevent it from binding to the receptor [90]. Bevacizumab was granted accelerated approval by the FDA in 2009; however, the drug demonstrated reduced efficacy against the newly diagnosed GBM and had no benefit on the patient's overall survival [91]. Aflibercept, another trap for VEGF, prevented its binding to the receptor, and recurrent GBM patients were proven to have only 7.7% of participants resulting in progression-free survival rates after six months in a phase II trial. Rilotumumab (AMG102), an anti-HGF mAb, was shown to bind to HGF, thus preventing the binding to the HGFR/c-MET and thereby activating downstream targets. In combination with temozolomide in vitro, rilotumumab

was shown to inhibit the growth of U87MG glioblastoma cells. The combination showed only minimal effects on GBM in the phase II clinical trial [18].
