The ErbB receptor family belongs to the receptor tyrosine kinases (RTKs) and consists of four members: ErbB1/EGFR/HER1, ErbB2/HER2/Neu, ErbB3/HER3 and ErbB4/HER4¹. Based on the protein structure, the ErbB receptors comprise an extracellular ligand binding domain, a hydrophobic transmembrane domain, an intracellular tyrosine kinase domain and a C-terminal tyrosine-rich tail². The activity of these receptors is tightly controlled and regulated by their ligands under normal conditions. Numerous ligands of ErbB receptors have been described, including epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), amphiregulin (AREG), betacellulin (BTC), heparin binding epidermal growth factor (HB-EGF), epiregulin (EPR), neuregulins (NRGs) and epigen (EPI). EGF, TGF-α, and AREG are specific ligands only for the EGFR. BTC, HB-EGF, and EPR bind to both EGFR and ErbB4. NRG-1 and 2 selectively bind to ErbB3, and BTC, HB-EGF, EPR, NRG1-4 bind to ErbB4^3,4. ErbB2 has no known ligand and can be activated by forming homodimers or heterodimers with other ErbB receptors^1,5,6.

Upon activation, the receptors are induced to form homo- or heterodimers, trigger the phosphorylation of intracellular domains, recruit related adaptor proteins, and subsequently activate downstream signaling⁷. The signaling pathways activated by ErbB receptors mainly comprise the PI3K-Akt-mTOR and RAS-RAF-MEK-ERK pathways, which are involved in several important physiological events including cell proliferation, apoptosis, differentiation, migration, embryonic development, and organogenesis⁸. However, abnormal activation of these pathways is considered as pro-oncogenic and the aberrations of ErbB receptors; including overexpression, amplification and active mutation, often occur in many types of cancers,  such as non-small-cell lung cancer (NSCLC), metastatic colorectal cancer (mCC), glioblastoma (GBM), head and neck cancer, pancreatic, ovarian, bladder and breast cancers^9,10. According to the prominent role of EGFR family signaling in cancer development, EGFR and its family members are very attractive therapeutic targets.

ErbB targeted therapies

Two therapeutic approaches have been employed to target ErbB receptors and inhibit their downstream signaling pathways. One is monoclonal antibodies, which are developed to directly target the extracellular domains of the receptors, block ligand binding or prevent receptor dimerization, then subsequently inhibit phosphorylation of intracellular domain and downstream signaling activation. Currently, cetuximab and panitumumab are two clinical available antibodies targeting EGFR¹¹. Cetuximab is a human/mouse chimeric IgG1 antibody which has been approved for metastatic colorectal cancer (mCRC) and squamous cell carcinoma of the head and neck (SCCHN) with wild type KRAS and NRAS^12,13. Panitumumab is a fully humanized IgG2 antibody and has been approved for the treatment of mCRC¹⁴. In addition, trastuzumab and pertuzumab are approved to treat ErbB2/HER2 positive tumors. Both of these two are humanized antibodies, trastuzumab targets the extracellular domain of ErbB2 to induce the internalization of cell surface ErbB2¹⁵, while pertuzumab targets the dimerization arm of extracellular ErbB2 domain to block its dimerization with other ErbB receptors¹⁶.

The second therapeutic approach is tyrosine kinase inhibitors (TKIs), which are designed to bind the intracellular tyrosine kinase domain (TKD) of the receptors; thereby, blocking the activation of downstream signaling pathways^9,17. The TKIs of ErbB receptor have been developed to the third generation. The first generation, gefitinib, erlotinib and lapatinib, can target the receptor through competitive and reversible binding at the tyrosine kinase domain¹⁸. Unlike the first generation, the second generation TKIs, afatinib, dacomitinib and neratinib, have the advantage of forming covalent, irreversible bonds with the target domain, and this will increase their effectiveness through a prolonged inhibition of ErbB signaling^19-21. The most common mechanism of resistance to first- or second-generation TKIs is the development of a mutation in the EGFR gene called T790M. Therefore, the third generation TKIs, such as osimertinib and olmutinib, are designed to target this specific mutation^22,23. Lapatinib is currently clinically approved for patients with ErbB2/HER2 positive breast cancer²⁴. Gefitinib, erlotinib, afatinib, and osimertinib are currently clinically approved for the treatment of EGFR driven NSCLC⁷, and erlotinib is also approved for use in pancreatic cancer in combination with gemcitabine^25,26. Clinical studies have shown that ErbB-targeted therapies, especially new generation of irreversible EGFR TKIs, could significantly improve the outcome of patients with late-stage ErbB-dependent cancers and had a high response rate. However, almost all patients eventually relapse and develop resistance to treatment; no reports on a cure have yet been seen²⁷. A major challenge for EGFR TKI treatment is developing new inhibitors or combinational therapies that can overcome drug resistance and limit tumor relapses.

In addition to inhibiting tumor growth by inducing tumor cell apoptosis through directly blocking oncogenic signaling, recent studies indicate that the antitumor efficacy of these ErbB targeted therapeutics also relies on the host immune system. Trastuzumab and cetuximab have been reported to mediate tumor killing by engaging several innate immune effectors, including complement-dependent cytotoxicity (CDC), antibody-dependent cell cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) by myeloid cells, and NK cells²⁸. Moreover, both clinical and preclinical studies have observed that the antitumor efficacy of ErbR2/HER2 and EGFR targeted antibodies strongly relay on the host adaptive T cell immunity^29-32. EGFR TKIs have also been reported to influence the host immune system, either by modulating tumor plasticity to enhance immune cell attack³³, or altering tumor microenvironment, including reducing regulatory T cells³⁴ and increasing CD8⁺ T cells infiltration³⁵. Whereas another study reported that EGFR TKIs could inhibit T cell activation through inhibiting the c-Raf/ERK (extracellular signal–regulated kinase) and AKT signaling pathways in vitro³⁶. The mechanisms by which EGFR TKIs influence the host immune system are still not well defined. Interestingly, our recent study observed that manipulating the dosing and timing of EGFR TKIs could enhance the  antitumor efficacy in host T cell immunity dependent fashion. We demonstrated that compared with standard of care hyperfractionated EGFR TKI treatment (low dose with daily treatment, HyperTKI), hypofractionated EGFR TKI treatment (high dose with a low frequency treatment, HypoTKI) not only could significantly reduce tumor burden, but also could enhance tumor specific T cell responses through triggering great innate sensing for type I IFN and CXCL10 production³⁷. Further investigations with syngeneic mouse models need to be done to explore whether EGFR TKIs have other impacts on the host immune system.

Immune checkpoint blockade therapy for ErbB related cancers

Accumulating clinical evidence has proven that cancer immunotherapy is a promising treatment for patients with advanced cancers. Due to the great success at the clinical level, cancer immunotherapy was named as “Breakthrough of the Year” by Science in 2013^38,39.Of even greater concern, the Nobel prize for physiology or medicine in 2018 was awarded to James P. Allison and Tasuku Honjo for their discovery of cytotoxic T-lymphocyte-associated protein (CTLA-4) and programmed cell death protein 1 / programmed cell death protein ligand 1 (PD-1 / PD-L1), the inhibitory antibodies of which have been widely and successfully used in clinic as cancer immunotherapy and have been approved for the treatment of at least ten types of  cancers, such as melanoma, lung cancer, CRC, head and neck squamous cell carcinoma (HNSCC), Hodgkin’s lymphoma, urothelial carcinoma, gastric cancer, and cervical cancer^23,40,41. Immunotherapy, especially PD-L1/PD-1 immune checkpoint blockade (ICB), has shown encouraging antitumor efficacy in lung cancer patients, including both NSCLC and SCLC. However, several clinical trials have shown that PD-L1/PD-1 blockade did not benefit cancer patients with EGFR driver mutation regardless of PD-L1 expression^42,43. The underlining mechanisms are still largely unclear. PD-L1 expression level, tumor mutation burden (TMB) and immunosuppressive tumor microenvironment (TME) are the most potential reasons.

Previous preclinical study has reported that EGFR active mutation could induce PD-L1 expression in lung epithelial cells, and EGFR TKI could reverse this effect⁴⁴. Later, some studies confirmed this correlation with using the tumor samples from clinical patients^45-47. In contrast, there are also some studies demonstrated that PD-L1 expression in tumor tissues was negatively correlated with EGFR mutation status. A pooled analysis found that compared to NSCLC patients with wild type EGFR, patients with EGFR mutations were less likely to be PD-L1-positive⁴⁸. In addition, the analysis of the Cancer Genome Atlas and the Guangdong Lung Cancer Institute (GCLI) database have also confirmed the lower PD-L1 expres­sion in tumors with EGFR mutation^49,50. Therefore, the relationship between EGFR mutation and PD-L1 expression is still conflicting, PD-L1 expression may not be the major reason why NSCLC cancer patients with EGFR mutation show poor response to PD-1/PD-L1 blockade.

Tumor mutation burden (TMB) is becoming a promising biomarker for the responses to immunotherapy in cancer patients^51-54. Low TMB is correlated with low response to immunotherapy, which suggests that EGFR mutant NSCLC tumor may harbor low TMB. Indeed, studies have reported that patients with EGFR mutations showed a significantly decreased TMB ⁴⁹. In addition, the EGFR mutation status is associated with TMB, NSCLC tumors with EGFR^Δ19alterations harbored lower TMB compared with EGFR^L858R tumors^43,55. Consistently, NSCLC patients with EGFR^L858R have better response to PD-L1/PD-1 blockade than patients with EGFR^Δ19alterations⁴³. Therefore, low TMB is one of the reasons for the low response rate to immunotherapy.

Tumor microenvironment (TME) is another key factor that impacts the patients’ response rate to immunotherapy⁵⁶. Based on the presence/absence and the location of tumor infiltrating lymphocytes (TIL), tumors can be classified into three types: immune-desert TME, immune-excluded TME, and the inflamed TME. Immune-desert and immune-excluded TME are also considered to be uninflamed TME, which has been reported to be resistant to ICB, whereas tumors with inflamed TME are sensitive to ICB⁵⁷. Indeed, it has been reported that EGFR active mutations were associated with the uninflamed TME⁴⁹, and EGFR signaling pathway activation in immunosuppressive cells was related to the immunosuppressive TME⁵⁰. Previous studies have revealed that EGFR signaling activation played vital roles in generating regulatory T cells (Tregs) and maintaining their suppressive functions^58,59. In addition, other studies also showed that STAT3 (downstream of EGFR) activation could inhibit the maturation of dendritic cells (DCs), and promote the IDO expression in both DCs and myeloid-derived suppressor cells (MDSCs)^60-62. However, how the activated EGFR signaling can be transferred from tumor cells to immunosuppressive cells is still not well studied. A recent study has observed that EGFR expressed on tumor cells could be transferred to Tregs through exosomes and then promote the generation of tumor specific Tregs⁶³. This also indicated that exosomes could transfer EGFR to DCs and MDSCs for their immunosuppressive function. Taken together, all these studies suggest that tumor cells with activated EGFR signaling can induce the immunosuppressive TME to mediate immune tolerance for tumor development and resistance to immunotherapy.    

Combination of EGFR targeted therapies and immune checkpoint blockade

Reducing tumor burden and increasing immunogenicity are considered as two key factors for effective tumor control⁶⁴. Combinational strategies that achieve these two goals will improve the outcomes of patients clinically⁶⁵.  EGFR targeted therapies, especially EGFR TKIs, have shown promising antitumor efficacy in cancer patients with EGFR active mutations. However, the major problem for EGFR targeted therapies is that they can initially reduce tumor burden and significantly improve the outcomes, but patients eventually encounter tumor relapse and develop drug resistance to EGFR TKIs²⁷. While the strength of emerging immunotherapies, especially ICB, is the long-term response during the treatment, the weakness is low response rate and only a small fraction of patients respond to treatment⁶⁵. According to the strength and weakness of EGFR targeted therapies and ICB, combination of these two therapies may achieve both initial high response rate and later long-term response to treatment, improved initial outcomes and overall survival of cancer patients³⁷. Notably, many clinical trials with the combination of EGFR TKIs and ICB have been conducted. Although combinational treatment showed promising synergistic antitumor effect, several trials have been terminated due to severe toxicities⁶⁶. One study showed that combination of Eroltinib with ICB could benefit patients with tolerable toxicity⁶⁷. Another study showed that sequential PD-L1/PD-1 blockade and Osimertinib could cause severe immune related side effects⁶⁸. Interestingly, our recent study proposed that optimizing the dosing and timing of EGFR TKI and ICB could achieve both efficient tumor control without severe toxicities. We demonstrated that combining HypoTKI and PD-L1 blockade could effectively control advanced large tumors, increase overall survival, and limit tumor relapse. We also observed that compared with HyperTKI plus PD-L1 blockade, HypoTKI plus PD-L1 blockade has fewer side effects³⁷. Therefore, several factors, such as selected EGFR TKIs, manipulating the dosing and timing of EGFR TKI, and the schedule of treatment, need to be considered to optimize the combinational strategies of EGFR targeted therapies and ICB to achieve the maximized antitumor effects without severe side effects.       

Conclusion remarks

ErbB driven cancer patients are resistant to emerging ICB treatment. ErbB targeted therapies, especially EGFR TKIs, are still considered the frontline therapies. According to the strength of EGFR TKI in reducing tumor burden and the long-lasting response of ICB, combination of EGFR TKIs and ICB has been tested in clinical trials; however, severe toxicities resulted in the premature termination of several trials. Although a preclinical study has shown that optimizing the dosing and timing of EGFR TKI and ICB could achieve synergistic antitumor effect without severe side effects in mouse models, whether the proposed strategy can be applied to the clinic still needs further investigations. Moreover, to explore the reasons of toxicities caused by the EGFR TKI and ICB will facilitate the development of potential combination strategies for the clinic.

Acknowledgments

Y.-X.F. holds the Mary Nell and Ralph B. Rogers Professorship in Immunology. This work was supported in part by Texas CPRIT grant RP180725 and RR150072 (CPRIT scholar in Cancer Research) to Y.-X. F.

Competing interests

The authors declare that they have no competing interests.

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