EGFR as a Therapeutic Target in Cancer: From Monoclonal Antibodies to Small Molecule Inhibitors

The role of the epidermal growth factor receptor (EGFR) in cancer has been extensively studied over the years, and it has been found to be a crucial transmembrane tyrosine kinase receptor involved in cancer cell proliferation and survival. EGFR was the first molecular target against which monoclonal antibodies (mAbs) were developed for cancer therapy. Cetuximab and Panitumumab are two mAbs which have been introduced into clinical practice for the treatment of metastatic colorectal and head and neck cancer by targeting the external part of the EGFR. These mAbs also have an effect on the immune system in determining the overall anti-tumor response. While mAbs have been developed as a targeted therapy against EGFR, small molecule EGFR tyrosine kinase inhibitors (TKIs) have also been explored as a potential treatment option in recent years. Unlike mAbs, small molecule inhibitors are synthetic and can be orally administered, making them more convenient for patients. These inhibitors have been categorized into first, second, third, and fourth-generation and have exhibited clinical efficacy in relevant models. However, resistance to the current inhibitors remains a concern, and ongoing studies are focused on finding more efficient EGFR inhibitors. In this article, we will highlight the importance of targeting the EGFR signaling pathway in cancer therapy and the related epigenetic mutations. We will also consider recent advances in the discovery and development of different EGFR inhibitors, as well as the use of various therapeutic strategies such as multi-targeting agents and combination therapies.

Monoclonal antibodies against EGFR target the extracellular domain of the receptor and block ligand-induced EGFR activation by competing for receptor binding with the ligand. Small-molecule EGFR tyrosine kinase inhibitors bind reversibly to the intracellular catalytic domain of the receptor and inhibit EGFR auto-phosphorylation and downstream signaling. In addition, some small-molecule inhibitors can also block other growth factor receptor tyrosine kinases, including members of the EGFR family and the vascular endothelial growth factor receptor.

While monoclonal antibodies are highly selective to EGFR, intrinsic or acquired resistance to EGFR inhibitors limits their effectiveness in cancer therapy. This resistance can be due to constitutive activation of downstream mediators, over-expression of other tyrosine kinase receptors, or up-regulation of the vascular endothelial growth factor, which promotes angiogenesis and resistance to EGFR inhibition. For example, the persistent activation of signaling pathways such as MAPK and PI3K/Akt can promote cell proliferation, survival, differentiation, and motility, leading to a resistant phenotype that is unaffected by treatment with anti-EGFR monoclonal antibodies such as Cetuximab.

Cetuximab (also known as C225 or Erbitux™) is a chimeric monoclonal antibody that binds to EGFR. It is composed of a human immunoglobulin (Ig) G1 and a murine monoclonal antibody M225. Compared to the natural ligands TGF-α and EGF, cetuximab binds to EGFR with a much higher affinity (2-log). Upon binding, cetuximab promotes the internalization and degradation of the EGFR without activating it, leading to a reduction in the amount of available EGFR on the cell surface and blocking the downstream signaling pathways. Additionally, cetuximab can also bind to the mutant receptor EGFRvIII, leading to the internalization of 50% of the antibody-receptor complexes within 3 hours and an 80% reduction in phosphorylated EGFRvIII.

Cetuximab binding to EGFR induces cell cycle arrest at the G0/G1 boundary and increases the expression of p27KIP1, a cell cycle regulator. It also induces apoptosis by increasing the expression of pro-apoptotic proteins such as Bax and caspase-3, -8, and -9, and by inactivating anti-apoptotic proteins such as Bcl-2, leading to decreased expression or phosphorylation. Furthermore, cetuximab has been shown to inhibit the production of pro-angiogenic factors like vascular endothelial growth factor, interleukin-8, and the basic fibroblast growth factor, resulting in a decrease in new blood vessel formation and the development of distant metastases in orthotopic cancer models.

Panitumumab, marketed as Vectibix by Amgen in Thousand Oaks, CA, is a monoclonal antibody that targets the extracellular domains of EGFR, which is fully humanized with no murine proteins due to its development by Abgenix’s XenoMouse technology. Panitumumab demonstrates high affinity with EGFR, with a dissociation constant of 5 × 10−11 M, and minimal allergic reactions or anaphylaxis. The drug is well-tolerated, and its primary toxic effect is dermatological, with no reports of grade 4 toxic effects in clinical trials. As a fully human antibody, panitumumab produces minimal infusion-related reactions, with only one of 148 patients experiencing a grade 3 reaction and one of 463 patients discontinuing treatment due to a grade 2 hypersensitivity reaction. Moreover, the drug can be administered at various intervals, such as weekly, fortnightly or every 3 weeks, without significant changes in its pharmacokinetic parameters.

Anti-EGFR monoclonal antibodies have been widely used in cancer therapy, but their clinical effectiveness has been limited, with only 15% of patients showing responses. The modest overall survival benefit, coupled with the economic crisis, has led to a progressive reduction in their use in clinical practice. Therefore, identifying biomarkers to predict and target eligible patients for anti-EGFR antibody treatment is a major challenge for the future. Efforts are needed to consider not only the mechanistic effect of blocking EGFR but also the further exploiting of mAb effects on the immune system. Although there is now an impressive amount of data in the field, a thorough understanding of the scenario is still lacking due to its complexity. Nevertheless, the goal of reaching the final destination will be worth all the efforts made.

Following the discovery of anti-EGFR therapies for cancer treatment, numerous small molecules have been synthesized and evaluated as EGFR TKIs, classified into first to third generations of EGFR TKIs. The EGFR gatekeeper T790M mutation, which is the primary mechanism of resistance against the first- and second-generation EGFR TK inhibitors, has led to the discovery and development of third-generation TK inhibitors. However, the application of these inhibitors is limited due to rapid resistance through the EGFR C797S mutation. To address this, fourth-generation EGFR TK inhibitors have been introduced for clinical evaluation, targeting EGFR tertiary mutation (C797S). Specific inhibitors targeting the mutant EGFR have also been designed and discovered. Small molecule EGFR degraders have been evaluated to address epigenetic mutations in the tyrosine kinase domain of EGFR. In addition, multi-target agents and combination therapy have been examined to overcome epigenetic mutations and enhance the effectiveness of EGFR TK inhibitors.

Numerous small molecules have been synthesized and assessed for their efficacy as EGFR TKIs, with favorable outcomes in clinical trials when compared to traditional treatments like platinum-based chemotherapy. The majority of these compounds act as reversible competitors to ATP for binding to the tyrosine kinase’s intracellular catalytic domain. The first-generation EGFR-TKIs, such as gefitinib and erlotinib, which are quinazoline-based derivatives, were approved by the US Food and Drug Administration (FDA) for treating patients with non-small cell lung cancer in 2003 and 2004, respectively.

The second generation of EGFR TKIs, including afatinib, dacomitinib, neratinib, canertinib, and pelitinib, were initially developed to overcome T790M mutation. These inhibitors contain an electrophilic acrylamide group that covalently interacts with Cys797 at the ATP binding cleft of EGFR. Additionally, they contain an aniline moiety that could potentially clash with the gatekeeper Met790 residue. However, most of the second-generation inhibitors suffer from low maximum tolerated dose and dose-limiting toxicity such as skin rash and gastrointestinal issues, which greatly limit their clinical use. The dose-limiting toxicity may result from targeting both mutant T790M and wild-type EGFR, as well as targeting other members of the HER family, such as HER2. These limitations have hindered further clinical development of these agents.

The third-generation EGFR TKIs are also known as mutant-selective EGFR TKIs and have demonstrated promising efficacy in NSCLC patients who are resistant to the first- and second-generation EGFR TKIs. These inhibitors, which typically have an aminopyrimidine scaffold, covalently bind to the active thiols of Cys797 through their electrophilic acrylamide Michael-acceptors. They selectively and irreversibly target the mutant EGFRT790M over wild-type EGFR. The X-ray technique has shown that this class of inhibitor adopts a U-shaped conformation in the mutant EGFRT790M active site to selectively form the covalent complex. Several third-generation EGFR TKIs have been synthesized and developed for clinical practice.

Osimertinib (AZD9291) and olmutinib (HM61713) are third-generation EGFR TKIs that are effective in treating NSCLC patients with the T790M mutation. Osimertinib has been approved for clinical use due to its high efficacy and low adverse effects. Olmutinib is approved for clinical use in Korea for the treatment of patients with locally advanced or metastatic NSCLC who have the T790M mutation. However, despite their effectiveness, the development of epigenetic mutations and acquired resistance have limited the clinical use of third-generation EGFR TKIs. Tertiary C797S point mutation is reported to be the main mechanism of acquired resistance to these inhibitors.

It is clear that combination therapy and multitarget agents are important approaches to overcome the issue of drug resistance in NSCLC patients. Combination therapy involves using more than one medication to treat a single disease, and combining EGFR TK inhibitors with other agents has been shown to improve antitumor activity and overcome drug resistance. Blocking the EGFR signaling pathway in combination with conventional cytotoxic drugs or radiotherapy has been found to enhance cancer cell damage and programmed cell death, leading to better anticancer activity. In addition, combining drugs that target different signaling pathways in cancer growth has been shown to enhance antitumor activity.

Dual inhibition of both the extracellular and intracellular domains of EGFR receptors is another useful strategy. Combination therapy of anti-EGFR monoclonal antibody with different EGFR TKIs is an example of dual inhibition of both the extracellular and intracellular domains of EGFR receptor. The tolerable toxicity profile of third-generation EGFR TKIs, such as osimertinib, makes them good candidates for the development of more efficacious combination regimens. Several clinical trials in combination therapy have been investigated for osimertinib targeting different pathways such as TORC1/2, Bcl2, VEGFR2, MEK, MET, Abl1/Src, JAK-1, FGFR, and AKT pathways, exhibiting promising results which will be released in the near future. Therefore, the development of new therapeutic regimens and multiple trials of such combination therapy are needed to overcome the issue of drug resistance in NSCLC patients.

The development of fourth-generation EGFR inhibitors is focused on overcoming the EGFR C797S mutation, which is the leading mechanism of resistance to third-generation EGFR inhibitors. The rational design of specific small molecule inhibitors involves various scientific disciplines, such as medicinal chemistry, preparative organic chemistry, structural biology, crystallography, and computational methods. One such fourth-generation EGFR inhibitor is the thiazole amide derivative 57 (EAI001), which was obtained through a high-throughput screening strategy from a library of 2.5 million compounds at 1 µM ATP concentration. EAI001 is a novel allosteric EGFR inhibitor with potent activity against mutant EGFRL858R/T790M and selectivity over wild-type EGFR. The X-ray crystal structure between 57 and mutant EGFRT790M revealed that EAI001 bound to the allosteric site near the classic ATP binding pocket, forming the C-helix in the inactive conformation. The direct contact between the aminothiazole group of EAI001 and the gatekeeper Met790 residue justified the specificity of mutant EGFRT790M compared to EGFRWT.

Indeed, the development of next generation small molecule EGFR TKIs with different scaffolds and binding interactions is crucial for overcoming resistance caused by T790M and C797S mutations. In addition, the use of small molecule degraders targeting EGFR through the allosteric site has emerged as a promising strategy to overcome resistance problems. These degraders utilize the cell’s own protein degradation machinery to eliminate EGFR from cancer cells, which can be particularly effective against tumors with acquired resistance to traditional TKI therapy. The design and optimization of these small molecule degraders require a multidisciplinary approach that involves chemistry, biology, and pharmacology. Nevertheless, this approach holds great potential for improving the long-term efficacy of EGFR targeted therapies in cancer treatment.

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