Reviews

Antitumour immunity regulated

by aberrant ERBB family signalling
Shogo Kumagai1,2,3, Shohei Koyama2,3 and Hiroyoshi Nishikawa 1,2,3 ✉

Abstract | Aberrant signalling of ERBB family members plays an important role in tumorigenesis
and in the escape from antitumour immunity in multiple malignancies. Molecular-targeted
agents against these signalling pathways exhibit robust clinical efficacy, but patients inevitably
experience acquired resistance to these molecular-targeted therapies. Although cancer
immunotherapies, including immune checkpoint inhibitors (ICIs), have shown durable antitumour
response in a subset of the treated patients in multiple cancer types, clinical efficacy is limited in
cancers harbouring activating gene alterations of ERBB family members. In particular, ICI
treatment of patients with non-small cell lung cancers with epidermal growth factor receptor
(EGFR) alterations and breast cancers with HER2 alterations failed to show clinical benefits,
suggesting that EGFR and HER2 signalling may have an essential role in inhibiting antitumour
immune responses. Here, we discuss the mechanisms by which the signalling of ERBB family
members affects not only autonomous cancer hallmarks, such as uncontrolled cell proliferation,
but also antitumour immune responses in the tumour microenvironment and the potential
application of immune-genome precision medicine into immunotherapy and molecular-targeted
therapy focusing on the signalling of ERBB family members.

Neoantigens
Antigens derived from gene
alterations in tumour cells.
As they are recognized as
foreign bodies by the immune
system, strong immune
responses are generally
induced.
1
Department of Immunology,
Nagoya University Graduate
School of Medicine,
Nagoya, Japan.
2
Division of Cancer
Immunology, Research
Institute, National Cancer
Center, Tokyo, Japan.
3
Division of Cancer
Immunology, Exploratory
Oncology Research & Clinical
Trial Center (EPOC), National
Cancer Center, Chiba, Japan.
✉e-mail: hnishika@ncc.go.jp
https://doi.org/10.1038/
s41568-020-00322-0
Members of the ERBB tyrosine kinase family present
some of the most commonly altered proteins in cancer,
and aberrant tyrosine kinase activation through gene
alterations can drive tumorigenesis, tumour growth
and progression. Oncogenic alterations of genes encod-
ing members of the ERBB family, leading to aberrant
ERBB signalling and driving tumour growth, have
been reported in various types of cancer including
breast, lung, head and neck, brain and gastrointestinal
cancers1,2. Thus, molecular-targeted therapies, including
tyrosine kinase inhibitors (TKIs), monoclonal antibodies
(mAbs) and antibody–drug conjugates (ADCs), target-
ing oncogenic epidermal growth factor receptor (EGFR)
or human epidermal growth factor receptor 2 (HER2)
signalling have been developed and successfully used in
the clinic, resulting in improved survival of patients with
cancers harbouring these gene alterations3–5. Yet, it is dif-
ficult to gain a long-lasting clinical benefit with these
molecular-targeted therapies, indicating the importance
of developing more effective cancer therapies6.
Based on the cancer immuno-editing hypothesis,
the immune system eradicates developing cancer cells
(elimination). In the initial phase of tumour develop-
ment, some cancer cells reduce their immunogenicity by
decreasing the expression of abnormal proteins that can
otherwise be readily recognized by the immune system
as neoantigens. A previous study showed that tumours
with sparse infiltration of T cells exhibit a waning of
neoantigen expression or copy-number loss of clonal
neoantigens during tumour development, suggesting
previous immuno-editing7. In addition, cancer cells
develop certain assets, such as inactivating alterations in
genes encoding interferon-γ (IFNγ) receptor and major
histocompatibility complex (MHC) molecules, which
can hamper and gradually decay antitumour immune
responses (equilibrium). Cancer cells acquire various
immune escape mechanisms, including the recruitment
of immunosuppressive cells and elevated expression of
various immunosuppressive molecules, such as immune
checkpoint molecules (escape). Consequently, these cells
form clinically apparent cancers8. Thus, the expression of
immune checkpoint molecules by cancer cells is one
of the essential requirements for cancer development9,
indicating the possibility that full engagement of anti­
tumour immune responses may regain control of tumour
growth and progression. Indeed, immune checkpoint
inhibitors (ICIs) have been proven to be clinically effi-
cient and have become a new standard treatment in
various types of cancer. Although patients with cancer
with a high tumour mutation burden (TMB) are thought
to respond to ICIs10–13, not only the absolute number of
non-synonymous mutations but also mutational signa-
tures, such as smoking-related or APOBEC signatures,
have been demonstrated to correlate with the sensitivity

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Reviews
Antibody-dependent
cellular cytotoxicity
(ADCC). When antibodies bind
to the target cells, especially
cancer cells, immune cells such
as macrophages and natural
killer cells are attracted. These
attracted immune cells possess
activating Fc receptors that
recognize the Fc region of the
antibody, and kill the targeted
cells to which the antibody
binds through releasing
cytotoxic molecules.
182 | March 2021 | volume 21
to ICIs in several cancer types10,11,14,15. In addition, the
gene expression profile related to T cell accumulation
and activation and specific somatic mutations in cancers
could have a positive or negative impact on the ther-
apeutic outcome of ICIs independent of the TMB16–18.
Moreover, cancer cells with oncogenic driver mutations
often harbour low numbers of non-synonymous muta-
tions, and some oncogenic alterations (for example,
alterations in STK11, PTEN, RHOA and so on) are asso-
ciated with the resistance of ICIs19–25. By contrast, certain
inactivating genomic alterations (for example, ARID1A
and so on) correlate with a favourable response to ICIs26.
Therefore, clarifying how specific oncogene alterations
and the resulting intercellular and intracellular signalling
immunologically modulate the tumour microenviron-
ment (TME) becomes more important with the expan-
sion of cancer immunotherapies to a broad spectrum of
cancer. Recent studies have revealed important roles
of the ERBB family in evading antitumour immunity
in many cancer types, including breast, lung, head and
neck, brain and colon cancers3,27.
Despite the striking antitumour effects of ICIs28,29,
their clinical efficacy against cancers harbouring gene
alterations of ERBB family members is limited, indicat-
ing that ERBB family members may play an important
role in evading the antitumour immune response20,30,31.
Here, we summarize how the aberrant signalling of
ERBB family members impairs antitumour immune
responses by modulating the immunological landscape
of the TME and how therapeutic targeting helps to over-
come the resistance of tumours with alterations in ERBB
family genes to ICIs.
ERBB family signalling
ERBB family members
ERBB family members (ERBB1–ERBB4; also known
as EGFR, HER2, HER3 and HER4) are receptor tyro­
sine kinases (RTKs), each comprising an extracellular
ligand-binding domain, a single hydrophobic transmem-
brane region and an intracellular segment with a tyrosine
kinase domain32. The ligands for EGFR, HER3 and HER4
are well studied33 and downstream signalling pathways
activated by ERBB family members are interconnected
and overlapping1,2,34,35 (Fig. 1). Two major representa-
tive signalling pathways are the phosphatidylinositol-3
kinase (PI3K)–AKT–mammalian target of rapamycin
(mTOR) and mitogen-activated protein kinase (MAPK)
pathways1,2,36,37, in addition to the phospholipase Cγ
(PLCγ)–protein kinase C (PKC)38,39 and Janus kinase 2
(JAK2)–signal transducer and activator of transcription
3 (STAT3) pathways40,41. From an immunological view-
point, EGFR or HER2 signalling inhibits IFNγ responses
and suppresses expression of interferon regulatory fac-
tors (IRFs) and inflammatory chemokine production via
PI3K–AKT pathways20,42–45 (Fig. 2).
ERBB gene alterations in tumours
Gene alterations of the ERBB family cause tumorigen­
esis, tumour growth and progression in multiple solid
cancers1,3,6,27,46–50. Aberrant activation of EGFR and HER2
in cancer cells can be induced by various mechanisms,
including gene amplification, point mutations, deletions
and autocrine ligand-receptor stimulation35,51–54 (Table 1).
These gene alterations abnormally activate EGFR and
HER2 signalling, independent of ligand-receptor stim-
ulation, resulting in tumorigenesis, tumour growth
and progression. The oncogenic functions of HER3
are largely mediated via its overexpression and interac-
tion with EGFR or HER2 because of its limited kinase
activity55–58. The role of HER4 is inconsistent in tumour
development as it has several isoforms, oncogenic iso-
forms and tumour-suppressive isoforms, harbouring
different activities59–61.
Targeting aberrant ERBB signalling
Based on significant roles of ERBB signalling in tumor-
igenesis, tumour growth and progression, specific types
of agents targeting aberrantly activated ERBB signalling,
such as TKIs, mAbs and ADCs, have been developed
and established as standard treatment strategies3–5,27.
Several TKIs targeting EGFR inhibit kinase domain
binding to endogenous ATP1, suppress the tyrosine-
phosphorylating activities of EGFR and prevent its
downstream signalling. Subgroups of patients with
non-small cell lung cancer (NSCLC) harbour hyperac-
tivated EGFR based on the L858R mutation in exon 21
or small deletions in exon 19 (ref.62) — these subgroups
of patients experience tumour regression in response
to first-generation EGFR-TKIs6,49. However, acquired
resistance to EGFR-TKIs develops after 9–14 months
and about half of the cases with acquired resistance
harbour the T790M mutation in EGFR63. Lapatinib,
one of the first-generation EGFR–HER2-TKIs, revers-
ibly binds to the ATP-binding pocket and suppresses
the PI3K–AKT and MAPK pathways in HER2+ solid
cancers64–66. HER2 reactivation through acquisition
of the HER2 L755S mutation has been reported as a
mechanism of acquired resistance to lapatinib in HER2+
breast cancer67.
Compared with TKIs, mAbs inhibit ERBB family
members (EGFR, HER2 and HER4) in different ways.
EGFR-targeted mAbs prevent ligands from binding
to EGFR and exhibit clinical efficacy through attenu-
ating downstream ERK signalling in cancers harbour-
ing wild type (WT) EGFR activated by ligand-receptor
stimulation68–72. In inflammatory breast cancer, in vitro
experiments with human cancer cell lines showed that
EGFR signalling promotes inflammation and cancer
stem cell-like activity73. The phase II study evaluating the
safety and efficacy of panitumumab plus neoadjuvant
chemotherapy in patients with HER2– inflammatory
breast cancer showed fairly good efficacy (pathological
complete response rate: 28%)72. The result of an ongoing
randomized study should be awaited.
mAbs targeting HER2 are often used in the treatment
of HER2+ solid cancers4,74–80 and inhibit dimerization of
HER2 and/or other ERBB family members, which is
independent of ligand-receptor stimulation, leading to
inhibition of downstream signalling and induction of
antibody-dependent cellular cytotoxicity (ADCC)81–86.
Antitumour activities of several mAbs against HER3
have also been investigated. mAbs against HER3 pre-
vent phosphorylation induced by ligand-receptor bind-
ing, resulting in reduced HER3 expression on the cell
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 Reviews

Multiple ligands: EGF, Impaired Multiple ligands: surface87,88. However, sufficient clinical efficacy of these
HB-EGF, BTC, TGFα, ligand-binding NRG1–NRG4 , BTC, mAbs has not been shown89.
AREG, EPGN or EREG domain NRG1–NRG4 HB-EGF or EREG
In addition to these reagents, one promising strategy
is an ADC that can specifically deliver its payload, a cyto-
EGFR HER2 HER3 HER4 toxic agent, to the targeted cancer cells by binding to tar-
get molecules, resulting in the internalization and release
Impaired of the cytotoxic agent in the cancer cells90. In terms of
Tyrosine
tyrosine ERBB family members, ADCs are being investigated
kinase with mAbs against EGFR, HER2 and HER3. Of these,
kinase domain
domain several ADCs derived from anti-HER2 mAbs have been
investigated91 and have exhibited encouraging clinical
efficacy and been approved for patients with solid cancers
with HER2 amplification5,92–96. Clinical efficacy of a novel
RAS NCK1 PLCγ PI3K JAK
HER3-targeting ADC, U3-1402, is under investigation97.
Mechanisms of resistance to ERBB-targeted therapies
RAF PAK PKC AKT STAT3 have been investigated over the last decades. The com-
mon resistance mechanisms of TKIs, mAbs and ADCs
MEK JNK TSC2 include upregulation of downstream signalling pathways,
signalling through alternate RTK pathways, upregulation
of anti-apoptotic pathways and histological transforma-
ERK JUN AP1 FOS RHEB tions. One of the TKI resistance mechanisms is based
on acquisition of additional mutations in the tyrosine
mTOR kinase domain. Secondary gene alterations in EGFR or
HER2 include second-site mutations that weaken the
efficacy of TKIs and, less often, amplifications or loss of
S6K 4E-BP1 the targeted genes. There are several forms of second-site
mutations with structural and functional character-
S6 istics: gatekeeper mutations (such as EGFR T790M),
covalent binding site mutations (such as EGFR C797S,
HER2 C805S), solvent-front mutations (such as EGFR
Nucleus Gene transcription Translation G796S/R) and so on98–101. Defects in binding to the tar-
get cause resistance to anti-EGFR mAb or anti-HER2
mAb. The EGFR S492R mutation contributes to acquired
DNA resistance to cetuximab but keeps tumours sensitive
Cell proliferation and survival Tumour cell
to panitumumab102. HER2 truncations (p95) are not
recognized by trastuzumab, pertuzumab or T-DM1
(ref.103). The mechanisms of resistance to ERBB-targeted

Fig. 1 | Signalling pathways caused by ERBB family members. ERBB family
members (ERBB1–ERBB4; also known as epidermal growth factor receptor (EGFR),
human epidermal growth factor receptor 2 (HER2), HER3 and HER4) are receptor
tyrosine kinases (RTKs) harbouring an analogous structure, which comprise an
extracellular ligand binding domain, a single hydrophobic transmembrane region
and an intracellular tyrosine kinase domain (except for HER3). Among the EGFR
ligands, EGF, transforming growth factor-α (TGFα), amphiregulin (AREG) and epigen
(EPGN) interact solely with EGFR, whereas epiregulin (EREG), heparin-binding
EGF-like growth factor (HB-EGF) and betacellulin (BTC) also bind to and activate
HER4. A family of EGF-related ligands, neuregulins (NRGs; composed of NRG1–NRG4)
bind to HER3 and HER4. HER2 directly binds to none of these EGF-related ligands.
HER3 is considered to have an impaired tyrosine kinase domain and to provide little
kinase activity. Therefore, in order to activate and provide signalling of HER2 and
HER3, their heterodimerization with other ERBB family members is needed32,320–323.
Downstream signalling pathways activated by ERBB family members overlap and
influence each other. In phosphatidylinositol-3 kinase (PI3K)–AKT–mammalian
target of rapamycin (mTOR) pathways, AKT phosphorylates and inhibits the tumour
suppressor TSC2, thereby activating RHEB, which positively regulates mTOR.
mTOR upregulates the canonical mRNA translation through activation of ribosomal
protein S6 kinase (S6K) and suppression of eukaryotic translation initiation factor
4E-BP1, and is strongly associated with cell proliferation. RAS–RAF–MEK–ERK
contributes to cell survival, proliferation and growth. NCK adaptor protein 1
(NCK1)–p21-activated kinase (PAK)–JNK and phospholipase Cγ (PLCγ)–protein
kinase C (PKC) lead to activation of the dimeric activator protein 1 (AP1)
transcription factor that promotes tumorigenesis. The Janus kinase 2 (JAK2)–signal
transducer and activator of transcription 3 (STAT3) pathway contributes to cell
proliferation.
therapies are summarized in Table 2.
Immunology of EGFR-mutant tumours
Tumour-infiltrating immune cells
Tumour mutation burden and CD8+ T cell infiltration.
Cancer cells harbour somatic mutations that generate
non-self proteins, peptides of which can be presented
to the immune system as tumour-specific antigens
such as neoantigens, and/or epigenetic alterations that
lead to high or aberrant expression of normal proteins,
which are referred to as tumour-associated antigens in
the process of tumour development13,104. It is thought
that tumours harbouring a high level of neoantigens
exhibit robust inflammation, with high levels of effec-
tor T cells that are specific for these antigens. The TMB
is the total amount of gene alterations (substitutions,
insertions and deletions) and is thought to correlate
with higher levels of neoantigens. The TMB is there-
fore considered to be a biomarker of the clinical efficacy
of ICIs105. There is intertumoural diversity of the lev-
els of TMB and the potential neoantigenicity of each
mutation is variable depending on the diversity of the
patients, such as MHC12. Consequently, the TME exhib-
its different immune landscapes, referred to as inflamed
or non-inflamed (indicative of the high or low levels of

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effector T cell infiltration observed in these tumours,
respectively). Tumours with alterations in ERBB family
genes, particularly EGFR alterations, frequently develop
non-inflamed TMEs 20,30,31,106,107. The non-inflamed
TMEs of tumours with EGFR alterations are thought to
be reflective of and caused by their low TMB20,30,31,106,108.
A previous clinical study suggests that the TMB of
EGFR-mutated NSCLCs is significantly lower than that
of EGFR-WT NSCLCs, resulting in impaired clinical
responses to ICIs108. The relatively low TMB in patients
with EGFR-mutated NSCLCs could be explained by
the high prevalence of non-smokers among patients
with EGFR-mutated NSCLCs and sufficient strength
of oncogenic EGFR signalling for carcinogenesis109.
Additionally, patients with EGFR-mutated NSCLCs
reportedly show lower APOBEC signatures than
non-smoking patients with EGFR-WT NSCLCs,
which potentially leads to the relatively low TMB of
EGFR-mutated NSCLCs110.
Regulatory T cell infiltration. Regulatory T cells
(Treg cells) are immunosuppressive CD4+ T cells express-
ing the master transcription factor, forkhead box pro-
tein P3 (FOXP3), and play essential roles in maintaining
self-tolerance111. Tumour-infiltrating Treg cells also sup-
press antitumour immunity112, and predict unfavourable
outcomes of PD1 blockade therapy, particularly when
harbouring high PD1 expression113,114. As Treg cell migra-
tion is mostly induced by chemokine networks in the
TME, Treg cells are often accompanied by effector T cells
in the inflamed TME112,115,116. However, EGFR-mutated
NSCLCs with a non-inflamed TME contain abundant

EGFR

Tumour Tyrosine kinase domain Activating gene alteration IFNγ

cell

IFNγ
NCK1 PLCγ RAS JAK2 PI3K RAS JAK2 PI3K
receptor

NF-κB IRF1
IL-6 NF-κB
CCL22 • IL-6 CXCL10
• IL-8
• IL-10 Myeloid cell
• VEGF Pro-tumoral
↑ NKG2D inflammation
ligand • Immuno-
suppressive
cytokines
• Pro-tumoral
cytokines
NKG2D EGFR ligand • EGFR ligands

NK PI3K
cell

↑ Treg M1 ↑ M2 ↓ CD8+ GSK3β

↓ DC
cell TAM TAM T cell
FOXP3 degradation
EGFR EGFR EGFR EGFR
signalling signalling signalling signalling Treg cell
inhibition inhibition inhibition inhibition

↓ Treg ↑ M1 ↓ M2 ↑ CD8+ ↑ Adenosine AMP ATP

cell ↑ DC T cell
TAM TAM
CD73 CD39

Tumour cell

Fig. 2 | The effects of EGFR signalling on immune cells in the TME.
Cancers with activating alterations in the epidermal growth factor
receptor (EGFR) gene commonly establish the non-inflamed tumour
microenvironment (TME). EGFR signalling downregulates CXC-chemokine
ligand 10 (CXCL10), concurrent with interferon regulatory factor 1 (IRF1), a
positive regulator of CXCL10, via phosphatidylinositol-3 kinase (PI3K)–AKT
pathways, resulting in the reduction of effector CD8+ T cells. Regulatory
T cells (Treg cells) are recruited in the non-inflamed TME of EGFR-mutated
cancers by upregulating CC-chemokine ligand 22 (CCL22) expression
through JNK–JUN activation. RAS–RAF–MEK–ERK, Janus kinase 2 (JAK2)–
signal transducer and activator of transcription 3 (STAT3) and PI3K–AKT–
nuclear factor-κB (NF-κB) pathways produce pro-tumoural cytokines and
EGFR ligands. In addition, EGFR signalling in myeloid cells induces
pro-tumoural inflammation and leads to increased expression of EGFR
ligands, such as HB-EGF. EGFR signalling in Treg cells prevents glycogen
synthase kinase 3β (GSK3β) from degrading forkhead box protein P3
(FOXP3), enhancing the immunosuppressive function of Treg cells.
Consequently, EGFR-tyrosine kinase inhibitor or anti-EGFR treatment
(summarized as EGFR signalling inhibition in the figure) increases
the numbers of CD8 + T cells, dendritic cells (DCs) and M1-like
tumour-associated macrophages (TAMs) and decreases Treg cell infiltration,
resulting in the inhibition of conversion of M1-like TAMs to M2-like TAMs.
IFNγ, interferon-γ; NCK1, NCK adaptor protein 1; NK cell, natural killer cell;
PLCγ, phospholipase Cγ.

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Table 1 | Alterations of genes encoding ERBB family members and their ligands
Gene Alteration Type of cancer
EGFR Mutation NSCLC and glioma
Amplification Head and neck cancer, oesophageal cancer, NSCLC,
colorectal cancer and glioma
HER2 Mutation Breast cancer, NSCLC and gastric, bladder and endometrial
cancer
Amplification Breast, gastric and oesophageal cancer
HER3 Mutation Breast and gastric cancer
HER4 Mutation NSCLC, melanoma and medulloblastoma
TGFA Overexpression Lung, pancreas, prostate, ovary, colon and head and
neck cancer
NREG Overexpression Colorectal and head and neck cancer
NSCLC, non-small cell lung cancer.
levels of Treg cells and macrophages, the latter of which
can produce chemokines recruiting more Treg cells in the
inflamed TME (Fig. 2). A preclinical lung cancer mouse
model with activating EGFR mutations also demon-
strated the upregulation of Treg cell-associated genes in
the lung early after induction of aberrantly upregulated
EGFR signalling117. We have recently shown that, in
addition to the low TMB, EGFR signalling downreg-
ulates CXC-chemokine ligand 10 (CXCL10), which is
known to recruit effector CD8+ T cells44,45, concurrent
with IRF1, a positive regulator of CXCL10, via PI3K–
AKT pathways20,42,43. Moreover, Treg cells are recruited to
the non-inflamed TMEs of tumours with EGFR muta-
tions by upregulating CCL22 expression via the JNK–
cJun pathway, which has been shown using a murine
model with cell lines transduced with EGFR 19del20,116.
Consequently, EGFR-TKI treatment can promote the
recruitment of CD8+ T cells and inhibit Treg cell infiltra-
tion in the TME of EGFR-mutated tumours in vivo20.
Thus, the mechanism behind the non-inflamed pheno­
type seems to be that EGFR signalling not only pre-
vents the recruitment of effector CD8+ T cells but also
promotes Treg cell infiltration. Furthermore, it has been
shown that EGFR blockade by antibodies or kinase
inhibitors promotes the secretion of chemokines (CCL2,
CCL5 and CXCL10) in head and neck squamous cell
carcinoma (HNSCC) cells and keratinocytes upon
IFNγ and tumour necrosis factor (TNF) stimulation
in vitro118,119. Together, EGFR signalling negatively affects
T cell accumulation in the TME through modulating the
chemokine milieu.
Innate immune cells and stromal cells. In addition to
modulating acquired immunity such as T cell balance
through chemokines, EGFR signalling influences the
characteristics of innate immune cells and stromal cells
in the TME. EGF secreted by colon cancer cells pro-
motes M2 polarization of tumour-associated macro­
phages (TAMs), which are associated with suppression
of cytotoxic T cell function120. In some cases, podopla-
nin (PDPN)+ cancer-associated fibroblasts infiltrate into
the TME of EGFR-mutated lung adenocarcinomas and
induce primary resistance to EGFR-TKIs in patients121.
PDPN + cancer-associated fibroblasts regulate the
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motility and localization of tumour-specific cytotoxic
T cells through nitric oxide synthase (iNOS)122 and
induce M2 TAMs123, resulting in impaired antitumour
immunity. By contrast, a study has revealed that EGFR
expression positively correlates with the expression of
ligands for natural killer cell activating receptor NKG2D
in cancer cells, which is reduced by EGFR-TKIs124. It is
therefore important to test in clinical studies whether
patients with EGFR-overexpressing or EGFR-mutated
tumours express NKG2D ligand at higher levels than
patients with EGFR-WT tumours or whether natu-
ral killer cells accumulate and activate in the TME of
EGFR-overexpressing or EGFR-mutated tumours.
EGFR inhibition interacts with immune cell infiltration.
EGFR-mutated cancer cells also overproduce negative
immune modulators, such as TGFβ, IL-10, VEGF, IDO,
ARG1 and adenosine, which can directly suppress nat-
ural killer cell killing, dendritic cell maturation and
cytotoxic T cell function and proliferation, although
the detailed mechanism(s) remains to be determined125.
Therefore, EGFR-TKI treatment potentially breaks this
negative spiral of immune suppression in the TME of
EGFR-overexpressing or EGFR-mutated tumours. As
such, short-term EGFR-TKI treatment of EGFR-mutated
lung tumours in mouse models increased the numbers
of CD8+ T cells, dendritic cells and M1-like TAMs and
decreased Treg cell infiltration, resulting in the inhibition
of the conversion of M1-like TAMs to M2-like TAMs126.
The percentage of mononuclear myeloid-derived sup-
pressor cells (MDSCs), which are immunosuppressive,
in lung tumours was elevated throughout the treat-
ment period. After prolonged EGFR-TKI treatment,
no significant change or even a decrease in antitumour
effector cells and increasing secretion of IL-10 and
CCL2 in serum are observed as compared with the
untreated group126.
In clinical studies, CD8 + or FOXP3 + tumour-
infiltrating lymphocyte (TIL) densities were investi-
gated in paired clinical EGFR-mutated NSCLC tis-
sue samples obtained from patients before and after
acquired resistance to EGFR-TKI treatment127. The
densities of both CD8+ and FOXP3+ TILs were signifi-
cantly decreased after acquired resistance to EGFR-TKI
treatment. In this study, however, 48% of patients
received cytotoxic chemotherapy between the termi-
nation of EGFR-TKI treatment and re-biopsy of tis-
sue specimens, thus it is difficult to conclude that this
immunological phenotype derived only from acquired
resistance to EGFR-TKI. Single-cell RNA sequencing of
EGFR-mutated NSCLC biopsies from patients showed
that whereas EGFR-TKIs were effective, more CD8+
T cell infiltration and less macrophage infiltration were
observed than before EGFR-TKI treatment. On the
other hand, when tumours were resistant to EGFR-TKI
treatment, the infiltration of fewer CD8+ T cells and
of more macrophages in the TME were found than
when EGFR-TKIs were effective128. However, epithelial–
mesenchymal transition is one of the major mechanisms
of acquired resistance to EGFR-TKI treatment129–131. TGFβ
is often upregulated in cancer cells with mesenchymal
phenotypes132, resulting in inhibition of effector T cell
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Table 2 | Mechanisms of resistance to ERBB-targeted therapies

Mechanism Therapy Molecular mechanism Refs
Mutation of the tyrosine kinase EGFR-TKI EGFR T790M, exon 20 insertions, C797S, 98–101

domain G796S/R
HER2-TKI HER2 T798I, C805S, exon 20 insertions 266,267

EGFR mAb EGFR S492R 102

HER2 mAb/HER2-targeted ADC p95HER2 (lacking extracellular domains) 103

Mucin 4 (masking the epitope) 268

CD44–hyaluronan polymer complex 269,270

(masking the epitope)

Upregulation of downstream EGFR-TKI, EGFR mAb RAS–RAF pathway upregulation 271–277

signalling pathways
HER2 and EGFR-TKI, HER2 mAb, PI3K–AKT pathway upregulation 129,278–286

EGFR-TKI, HER2-targeted ADC

EGFR-TKI JAK–STAT pathway upregulation 287–290

EGFR-TKI SRC activation 291,292

Signalling through alternate EGFR-TKI, HER2 and EGFR-TKI, Other ERBB members upregulation 293–296

RTK pathways HER2 mAb (including their ligands)

EGR-TKI IGF1R upregulation 297,298

EGFR-TKI, HER2 mAb MET upregulation 129,299–302

EGFR-TKI AXL upregulation 303,304

Upregulation of anti-apoptotic HER2 and EGFR-TKI, EGFR-TKI Low expression of BIM 305–307

pathways
EGFR-TKI High expression of YAP1 308

HER2 mAb Downregulation of CDK inhibitors 85,309

Histological transformation EGFR-TKI Small cell transformation 129,310

EGFR-TKI Epithelial–mesenchymal transformation 129–131

Defects in ADCC HER2 mAb FcγR III polymorphism 311

Drug efflux pump HER2-targeted ADC MRP1/MDR1/MDR4 induction 286,312

Trafficking protein modulation HER2-targeted ADC CAV1 upregulation (co-localizing in 313,314

caveolae and not lysosomes)

Lysosomal defects HER2-targeted ADC SLC46A3 (lysosomal transporter) 286,315

downregulation
ADC, antibody–drug conjugate; ADCC, antibody-dependent cellular cytotoxicity; EGFR, epidermal growth factor receptor;
HER2, human epidermal growth factor receptor 2; JAK, Janus kinase; mAb, monoclonal antibody; mTOR, mammalian target
of rapamycin; PI3K, phosphatidylinositol-3 kinase; RTK, receptor tyrosine kinase; STAT, signal transducer and activator of
transcription; TKI, tyrosine kinase inhibitor.

function, downregulation of MHC class I in cancer cells
and accumulation of immunosuppressive cells such as
Treg cells and macrophages133–137 and cancer-associated
fibroblasts138. Therefore, larger clinical studies addressing
these issues are warranted.
MHC expression
MHC is important for the presentation of tumour anti-
gens to T cells. MHC class I molecules bind antigen
peptides of 8–10 amino acids and are expressed on the
surface of almost all nucleated cells, and MHC class I–
peptide complexes present antigenic information to
CD8+ T cells. CD8+ T cells recognize cognate antigens
and are thus involved in immune surveillance against
non-self components such as malignancies and infec-
tions. MHC class II molecules bind antigen peptides
of 10–20 amino acids and are constitutively expressed
on the surface of antigen-presenting cells, including
B cells, macrophages and dendritic cells139, and MHC
class II–peptide complexes present antigenic informa-
tion to CD4+ T cells. Activated CD4+ T cells help various
immune responses against infection and cancer140,141.
Two different mechanisms regulating MHC expres-
sion in human malignancies have been noted142. One
mechanism is the transcriptional regulation of MHC
expression under physiologic conditions143. Constitutive
MHC expression is regulated by distinct regions within
MHC promoters, to which transcription factors such as
nuclear factor-κB (NF-κB), IRF1 and cAMP-responsive
element-binding protein (CREB) for MHC class I and
class II major histocompatibility complex transactiva-
tor (CIITA) for MHC class II can bind. Another mech-
anism is the induced regulation of MHC expression in
response to certain cytokines: type I interferons, IFNγ
and TNF for MHC class I; and IFNγ for MHC class II.
MHC molecules are upregulated via changes in tran-
scription factors and/or co-activators caused by these
cytokines144. In cancer settings, MHC molecules play an
important role in presenting tumour antigens to T cells
to induce antitumour immune responses145,146. However,
tumours are equipped with several immunosuppressive
mechanisms to escape antitumour immune responses147.

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 Reviews

One typical example is the downregulation of the surface
expression of MHC molecules. This downregulation is
caused by various mechanisms, including the genetic
loss of the MHC locus and epigenetic silencing145,148–151.
Some studies, which focus on the interaction between
EGFR signalling and MHC molecules, have shown that
EGFR ligands, EGF and TGFα, reduce the expression
of both MHC class I and MHC class II molecules152–155
(Fig. 3). PI3K–AKT pathways, which are downstream of
EGFR signalling, also suppress MHC class I and MHC
class II expression156–158. Accordingly, EGFR inhibition
in human cell lines in vitro increased the expression of
both MHC class I and MHC class II along with other
important components of antigen presentation, includ-
ing transporters associated with antigen processing 1
(TAP1), TAP2 and β2-microglobulin (β2M)106. In EGFR-
mutated human lung cancer cell lines, MHC class I
expression is significantly downregulated compared
with that in EGFR-WT cell lines in response to IFNγ
in vitro159. As discussed above20, the downregulation
of IRF1 via PI3K–AKT pathways in lung cancer cells
with EGFR alterations supports the notion that mutated
EGFR signalling negatively regulates IFNγ signalling,
which is essential for MHC class I expression. In addi-
tion to the PI3K–AKT pathways, EGFR-downstream
MAPK pathways, especially phospho-ERK, are negative
regulators of MHC class I expression160. Indeed, MHC
class I and MHC class II expression is elevated when
MAPK pathways in cancer cells are suppressed by several
inhibitors, such as BRAF or MEK inhibitors160–164. TKIs
(such as PD168393 and AG1478) as well as cetuximab,
an anti-EGFR mAb, could upregulate MHC class I and
MHC class II expression induced by IFNγ in primary
human keratinocytes and a malignant keratinocyte
cell line165. In head and neck cancer cell lines, erlotinib,
dacomitinib (a pan-ERBB inhibitor) and cetuximab
promote the induction of MHC class II expression by
IFNγ166,167. In studies using NSCLC cell lines with EGFR
mutations, gefitinib increased MHC class I expression
in cancer cells168. Additionally, the tumour-derived
exosomes containing activated EGFR signalling play
a role in the suppression of interferon signalling by
triggering the activation of MEK kinase 2, leading to a
decrease in MHC expression169.
Immunosuppressive molecule expression
The expression of PDL1, which is one of the most
important immune checkpoint molecules, is induced
by two different mechanisms: genetic alterations (that
is, amplification, fusion and 3ʹ untranslated region dis-
ruption, leading to intrinsic expression) and inflam-
mation (that is, IFNγ stimulation, leading to acquired
expression) in cancer cells170–172. PDL1 expression is
reportedly affected by EGFR signalling via two oppo-
site mechanisms in NSCLCs20,117,173 (Fig. 3). Intrinsically,
PDL1 expression is upregulated by activation of the
PI3K–AKT, RAS–RAF–MEK–ERK or JAK–STAT3
pathway by EGFR signalling117,173–176. Accordingly, a
sublethal dose of in vitro EGFR-TKI treatment reduces
PDL1 expression in cell lines through inhibiting EGFR
signalling117. On the other hand, PDL1 expression is also
regulated by IFNγ via IRF1 signalling20. In our study,

EGFR ERBB HER2

Tyrosine kinase Activating Tumour

domain gene alteration cell IFNγ
IFNγ
receptor
RAS JAK2 PI3K RAS PI3K
↓ IRF1 ↓ IRF3
AKT Endosome
↓ Type I/II interferon ↓ Type I/II interferon TBK1

EGFR HER2
heterodimer dsDNA
signalling cGAS or
signalling STING
inhibition cGAMP
inhibition

↑ Type I/II interferon ↑ Type I/II interferon

↓ MHC PDL1
class I or II HER2
heterodimer
EGFR signalling signalling
inhibition inhibition
↑ MHC class I or II

Fig. 3 | Reduced antigenicity in EGFR-mutated or HER2-amplified
cancer. In epidermal growth factor receptor (EGFR)-mutated cancers,
phosphatidylinositol-3 kinase (PI3K)–AKT pathways downregulate interferon
signalling, via interferon regulatory factor 1 (IRF1; one of the transcriptional
factors of major histocompatibility complex (MHC)), and RAS–RAF–MEK–
ERK pathways also negatively regulate MHC expression. PDL1 expression by
EGFR-mutated cancers is affected by EGFR signalling via two opposite
mechanisms. In cancer cells, PDL1 expression is upregulated by the
activation of PIK3–AKT, RAS–RAF–MEK–ERK and Janus kinase 2 (JAK2)–signal
transducer and activator of transcription 3 (STAT3) pathways. On the other
hand, PDL1 expression is also upregulated by interferon-γ (IFNγ) via IRF1
signalling. In cancers with human epidermal growth factor receptor 2 (HER2)
amplification, HER2 induces loss of phosphorylation of TANK-binding kinase 1
(TBK1) and attenuates stimulator of interferon genes (STING) signalling,
resulting in the impairment of interferon signalling, especially IRF3, one of
the transcriptional factors of MHC. Molecular-targeted therapies that inhibit
EGFR or HER2 heterodimer signalling increase the expression of MHC class I
and MHC class II in EGFR-mutated and HER2+ cancers.

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Reviews
NKT cells
A type of T cells that also
possess the characteristics of
natural killer cells. The T cell
receptors of natural killer
T (NKT) cells recognize
glycolipids presented on
CD1d molecules as antigens.
188 | March 2021 | volume 21
EGFR signalling inhibited IRF1 expression, thereby
inhibiting acquired PDL1 expression by IFNγ in in vitro
experiments using human cell lines20. Therefore, how
EGFR signalling affects PDL1 expression is still under
debate. Nevertheless, in clinical samples, many studies
have shown that PDL1 expression assessed by immuno­
histochemistry is significantly higher in EGFR-WT
NSCLCs than EGFR-mutated NSCLCs108,177–181 and that
PDL1 expression is elevated following EGFR-TKI treat-
ment in EGFR-mutated NSCLCs30,127,182. Furthermore, in
one of these studies, two of the five patients who showed
an increase in PDL1 expression in cancer cells tended
to exhibit a durable clinical response to subsequent ICI
treatment, suggesting that EGFR-TKI treatment may
favourably affect ICI efficacy127. In addition to PDL1,
the expression of PDL2, another ligand for PD1, in
in vitro experiments with human cancer cell lines could
also be regulated by EGFR signalling in NSCLCs183. For
another B7 family molecule, the expression of HERV-H
LTR-associating 2 (HHLA2), which could inhibit T cell
function as an immune checkpoint184, is upregulated in
clinical specimens from patients with EGFR-mutated
NSCLC as compared with patients with EGFR-WT
NSCLC185. Further investigation of the mechanism(s)
regulating HHLA2 expression in EGFR-mutated cancers
is warranted.
CD73 is one of the negative immunomodulatory
molecules expressed by multiple cancer types and
immune cells that promotes production of adenosine186.
Adenosine functions as an immunosuppressive media-
tor in the TME. When adenosine stimulates adenosine
receptors, including A2AR, on each immune cell, both
infiltration and immunosuppressive activity of Treg cells
and MDSCs are enhanced187–189 and activity of dendritic
cells, effector T cells, natural killer cells and NKT cells is
inhibited190–197. Supporting the notion that CD73 pro-
motes immunosuppression via adenosine and allows
tumour growth, high expression of CD73 is associated
with poor prognosis in patients with NSCLCs, although
these data are preliminary198. In addition, higher levels
of baseline tumour adenosine signalling are associated
with resistance to ICIs in various types of cancer199. In
preliminary analysis of clinical samples, higher expres-
sion of CD73 is related to EGFR-mutated NSCLCs than
to EGFR-WT NSCLCs198,200. CD73 expression is induced
by EGF and is inhibited by EGFR-TKIs in in vitro
experiments200. These findings suggest that aberrant
EGFR signalling increases CD73 expression and that the
CD73–adenosine axis is another possible immunosup-
pressive mechanism in EGFR-mutated tumours, which
is potentially targeted by EGFR-TKI.
PD1–PDL1 blockade
All of the immunosuppressive mechanisms in EGFR-
mutated cancer discussed above establish a non-inflamed
TME, resulting in resistance to cancer immunotherapies,
including PD1–PDL1 blockade. Several meta-analyses
on the clinical efficacy of PD1 blockade versus docetaxel
in patients with pretreated advanced NSCLCs have
recently reported that ICIs significantly prolonged
overall survival in the EGFR-WT subgroup but not
in the EGFR-mutated subgroup201–204. In one study on
meta-analyses about the efficacy of PD1 blockade, the
authors analysed the data of not only NSCLCs but also
other types of cancer and demonstrated that signifi-
cantly prolonged progression-free survival and over-
all survival were observed in patients with EGFR-WT
cancers receiving PD1 blockade but not in those with
EGFR-mutated cancers205. Therefore, EGFR-mutated
cancer is generally resistant to ICIs mainly due to its
non-inflamed TME.
However, the level of ICI efficacy in EGFR-mutated
NSCLCs varies depending on the types of EGFR
alterations206,207. As such, EGFR exon 19-deleted cancers
exhibit worse response rates than EGFR L858R-mutated
cancers206. This may be partially due to the substantially
lower TMB of EGFR exon 19-deleted cancers compared
with EGFR L858R-mutated cancers207. The TMB is
known to increase significantly with age208. The lower
TMB of EGFR exon 19-deleted cancers than EGFR
L858R-mutated cancers may be caused by older age at
diagnosis for EGFR L858R-mutated cancers209,210. TP53
mutations are reported to be associated with higher
TMB among EGFR-mutated cancers, which may reflect
genetically unstable states caused by co-occurring
mutations207. Recently, several groups have addressed the
relationship between EGFR T790M mutation, one of
the major causes of acquired resistance to EGFR-TKI,
and immunological phenotypes. Lower PDL1 expres-
sion and higher levels of FOXP3+ T cell infiltration were
observed in EGFR T790M-positive tumours compared
with EGFR T790M-negative tumours, although the
under­lying mechanism(s) remains to be elucidated211,212.
Moreover, EGFR T790M-mutated tumours show a
significantly worse response rate to ICIs than EGFR
T790M-WT tumours211,213.
EGFR alterations other than L858R mutation and
exon 19 deletion are known as uncommon mutations,
such as L861Q and G719X, and are observed in 10–20%
of patients with NSCLC harbouring activating EGFR
alterations214,215. Uncommon EGFR alterations in patients
with NSCLCs are associated with heavier smoking
history216 and preliminary data show that these types of
patients harbour a higher TMB compared with patients
with NSCLCs harbouring common EGFR alterations217.
The higher TMB may lead to higher PDL1 expression
by tumours and higher frequency of CD8+ T cell infil-
tration in the TME of NSCLCs harbouring uncommon
EGFR alterations218. Patients with NSCLCs harbouring
uncommon EGFR alterations reportedly show signifi-
cantly better response to ICIs than those with NSCLCs
harbouring common EGFR alterations213. Therefore,
ICI treatment may be more favourable for patients with
uncommon EGFR alterations than those with common
EGFR alterations. To understand the molecular mech-
anism of improved sensitivity to ICIs in uncommon
EGFR alteration, immunological analysis comparing
common and uncommon EGFR-mutated NSCLCs will
be necessary in addition to evaluating the TMB.
In EGFR-altered tumours, to variable degrees
depending on the type of EGFR alterations, EGFR sig-
nalling establishes an immunosuppressive environment,
leading to resistance to ICI treatment. Therefore, in
order to improve the efficacy of ICIs in EGFR-altered
www.nature.com/nrc

tumours, it is necessary to target the immunosuppressive
factors caused by aberrant EGFR signalling.
Immunity affected by other ERBB signals
Amplification is the most common alteration in HER2,
which is found in patients with breast, gastric and
oesophageal cancers in contrast to other gene altera-
tions, such as EGFR alterations. HER2 amplification
seems to result in a non-inflamed TME31. In breast
cancers, triple-negative breast cancers harbour the
most abundant TILs, whereas relatively low infiltra-
tion of TILs is observed in breast cancers with HER2
amplification219–221. Additionally, a subset of breast can-
cers with HER2 amplification exhibits an inflammatory
phenotype through activation of the PI3K–AKT–mTOR
pathway, resulting in IL-8 secretion in the TME222,223.
IL-8 not only promotes malignant phenotypes in can-
cer cells, including invasion and metastasis222, but also
recruits neutrophils into the TME, which contributes to
resistance to PD1–PDL1 blockade treatment in several
types of cancer224. HER2 amplification also induces loss
of phosphorylation of TANK-binding kinase 1 (TBK1)
and attenuates stimulator of interferon genes (STING)
signalling, resulting in impairment of the interferon
response and antitumour immune responses225 (Fig. 3).
Associations between MHC expression and HER2
have been investigated for a long time. In studies using
HER2-overexpressing cell lines, the expression of
HER2 inversely correlates with MHC class I expres-
sion and other components of the antigen processing
machinery226. Consistent with these results, knockdown
of HER2 or anti-HER2 mAbs leads to an increase in
MHC class I expression165,227,228.
For PDL1 expression, post-translational modifica-
tions, which include phosphorylation, glycosylation
and ubiquitylation, have been reported to regulate the
protein stability of PDL1 and the interaction between
PD1 and PDL1 (refs229–232). EGF signalling in breast
cancer cells stabilizes PDL1 expression through glyco-
sylation by glycogen synthase kinase 3β (GSK3β)230 and
promotes direct interaction between PDL1 and PD1
through β-1,3-N-acetylglucosaminyl transferase 232.
Recently, the clinical efficacy of PD1 blockade therapies
in tumours with HER2 amplification has been evaluated.
PD1 blockade therapies have shown limited anti­tumour
efficacy in breast cancers and gastric cancers with HER2
amplification233,234, although numerous clinical tri-
als are still ongoing (NCT03032107, NCT03747120,
NCT02605915, NCT03199885, NCT03125928,
NCT03726879 and NCT03417544). Further clinical tri-
als testing the clinical efficacy of combinatory immuno­
therapies with HER2-targeted therapies, including
mAbs or ADCs, are ongoing with much expectation
(see the section Targeted therapies and combinations).
As analyses of the TME of cancers with HER3 or HER4
alterations are limited, further studies are warranted.
The roles of EGFR in immune cells
ERBB family members are potentially expressed in
various haematopoietic cells and play important func-
tions in the TME of cancers without alterations of
ERBB family members. The function of EGFR among
Nature Reviews | CAncER
Reviews
ERBB family members has been investigated in immune
cells, particularly macrophages and T cells, in the TME
(Fig. 2). EGFR is expressed by macrophages in the TME of
both human and mouse cancers, including hepato­
cellular carcinomas and colorectal cancers (CRCs)235,236.
Mice lacking Egfr in myeloid cells showed decreased
carcinogenesis due to the reduction in IL-6 production
via STAT3, suggesting a tumour-promoting function by
myeloid cell-intrinsic EGFR signalling235,236. In addition,
macrophages produce HB-EGF, leading to cell growth
and invasion of myxoid liposarcoma through EGFR
signalling237. The expression of EGFR is also detected
in CD4+ T cells, especially FOXP3+ Treg cells, but not in
CD8+ T cells238. Compared with the other EGFR ligands,
the binding of AREG to EGFR on tumour-infiltrating
Treg cells induces the activation of MAPK pathways,
enhancing the suppressive function of Treg cells 238.
Regarding EGFR ligands, Treg cells themselves pro-
duce AREG, which can promote tumour cell prolifer-
ation via EGFR signalling, in addition to inhibiting the
antitumour immune response239. However, our study
detected little expression of EGFR on tumour-infiltrating
Treg cells20. Therefore, the function of EGFR in Treg cells
remains controversial and may be dependent on the
pathological condition, such as in the context of cancers
and inflammation.
Targeted therapies and combinations
ERBB family signalling, especially EGFR and HER2
signalling, plays a key role in not only promoting
tumour cell proliferation but also inducing a more or
less immunosuppressive TME through multiple mecha­
nisms as described in the above sections. Because the
inhibition of ERBB family signalling potentially switches
non-inflamed and even immunosuppressive profiles
of tumours with ERBB alterations into inflamed pro-
files, several combinations of immunotherapy and
molecular-targeted therapy for ERBB family signal-
ling have been under investigation ( Fig. 4; Table 3;
Supplementary Table 1). Combination immunother-
apies including ICIs and CD73–adenosine axis inhib-
itors with ERBB targeting agents provide synergistic
antitumour immune responses compared with mono­
therapy of either agent alone. TKIs or mAbs against
EGFR alterations prevent the infiltration of immuno-
suppressive cells and potentially strengthen antitumour
responses, especially in NSCLCs and CRCs20, sug-
gesting that combination treatment of EGFR-targeted
therapies with ICIs could be a reasonable option as a
combination immunotherapy. Whereas the clinical
trial of the combination therapy of the third-generation
EGFR-TKI osimertinib with or without durvalumab,
an anti-PDL1 antibody, was closed prematurely due
to the increased incidence of interstitial lung disease240,
the combination of nivolumab, an anti-PD1 antibody,
with the second-generation EGFR-TKI erlotinib was
well tolerated and showed clinical efficacy in patients
with erlotinib-resistant advanced EGFR-mutated
NSCLCs241. Interestingly, adenocarcinoma with EGFR
exon 21 mutations demonstrated favourable clin-
ical outcomes from ICI treatment compared with
EGFR exon 19 deletions due to relatively higher TMB
volume 21 | March 2021 | 189
Reviews

in EGFR exon 21 mutations206,207. Accordingly, fur-
ther investigations are necessary to evaluate whether
there is an increased risk of developing adverse events
when combining PD1–PDL1 blockade with osimerti-
nib compared with the other EGFR-TKIs and which
subset of EGFR-mutated cancers is the optimal target
for combination immunotherapies. Recently, Gong et al.
have shown that EGFR-TKIs upregulate interferons
not only in EGFR-mutated human cancer cell lines
but also in EGFR-WT human cancer cell lines 242.

a EGFR EGFR inhibitor b EGFR inhibitor

Anti-PD1
↑ CD8+ ↑ CD8+
Tyrosine kinase Activating T cell T cell
domain gene alteration PD1

Tumour dsDNA Cytotoxic

↓ Type I/II chemotherapy
cell
interferon ↑ CXCL10

↓ Suppressive
↑ MHC cytokines
class I or II PDL1

↓ EGFR
ligands VEGF

Anti-PDL1 ↑ DC ↑ DC
↓ CCL22 Anti-VEGF

↓ Treg ↑ M1 ↓ M2 ↓ Treg ↑ M1 ↓ M2
cell TAM TAM cell TAM TAM

HER2 Anti-HER2
c d
EGFR, HER3 or HER4 CD16

↑ NK ADCC
cell

dsDNA
↑ Type I/II ↑ CD8+
↑ CD8+ interferon T cell
T cell

↑ MHC Cytotoxic
class I or II ↑ PDL1 chemotherapy
CD39 CD73 ↓ Adenosine
ATP AMP
Anti-CD73 ↑ DC
A2AR ↑ DC
A2AR
antagonist

↓ Treg ↑ M1 ↓ M2 ↓ Treg ↑ M1 ↓ M2 DC maturation

cell TAM TAM cell TAM TAM

Fig. 4 | Targeting molecular pathways of ERBB family signalling for
combination immunotherapies. The concepts of combinations of
immunotherapy and molecular-targeted therapy for molecular pathways
of ERBB family signalling are shown. a | Tyrosine kinase inhibitors (TKIs)
(or monoclonal antibodies (mAbs)) inhibiting epidermal growth factor
receptor (EGFR) signalling prevent the infiltration of immunosuppressive
cells and potentially strengthen antitumour responses. EGFR-TKIs
promote the production of interferons and effector T cell-recruiting
chemokines such as CXC-chemokine ligand 10 (CXCL10) by cancer cells
and decrease the production of immunosuppressive cytokines and
chemokines, resulting in improvement of the efficacy of PD1/PDL1
blockade. b | VEGF inhibitors are one of the options for combination
treatment with immune checkpoint inhibitors (ICIs). The improved efficacy
of ICIs in combination with anti-VEGF mAb may be attributed to the
reduction in immunosuppressive cells, especially regulatory T cells
(Treg cells). This combination treatment strategy may be beneficial to the
treatment of EGFR-TKI-resistant tumours with EGFR mutations. c | CD73
mAbs or A2AR antagonists dampen Treg cells and myeloid-derived
suppressor cells (MDSCs) and enhance the activity of dendritic cells (DCs)
and effector T cells. d | Anti-human epidermal growth factor receptor 2
(anti-HER2) mAbs induce antibody-dependent cellular cytotoxicity
(ADCC), leading to upregulation of PD1 expression by T cells and of PDL1
expression by tumour cells. Antibody–drug conjugates (ADCs) retain the
function of anti-HER2 mAbs and induce immunogenic cell death and
major histocompatibility complex (MHC) class I expression in cancer cells,
resulting in DC maturation, differentiation of tumour-associated
macrophages (TAMs) into the pro-inflammatory M1 type and increased
T cell infiltration.

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 Reviews

Table 3 | Combination treatments including immunotherapies in cancers with aberrant ERBB family signalling
Target Evaluation criteria Type of cancer Treatment Clinical trials Refs
EGFR Mutations NSCLC TKI + anti-PD(L)1 mAb NCT01454102, NCT02013219, NCT02040064, 20,240,241,

(or anti-CTLA4 mAb) NCT02088112, NCT02143466, NCT02364609, 316–318

NCT02039674, NCT02574078, NCT02454933,

NCT03083041
Anti-PD(L)1 mAb + anti-VEGF mAb NCT04147351, NCT04426825 –
Chemotherapy + anti-PD(L)1 mAb NCT04405674, NCT03515837 –
Chemotherapy + anti-PD(L)1 mAb + NCT03647956, NCT03786692, NCT02366143, 246,247

anti-VEGF mAb NCT03802240

CD73–adenosine axis blockade + NCT03454451, NCT02503774, NCT03835949, –
anti-PD(L)1 mAb NCT04262375, NCT04262388, NCT03549000,
NCT02754141, NCT03819465, NCT03833440,
NCT03822351, NCT02740985, NCT03207867,
NCT02403193, NCT04262856, NCT03629756,
NCT03846310, NCT03337698
CD73–adenosine axis blockade + NCT03381274
EGFR-TKI
Overexpression HNSCC Anti-EGFR mAb + anti-PD(L)1 mAb NCT01935921, NCT02764593, NCT04429542, 254

(or anti-CTLA4 mAb) NCT02947386, NCT03051906, NCT04305795,

NCT03082534, NCT04297995
HER2 Overexpression Solid tumours Anti-HER2 mAb + anti-PD(L)1 mAb NCT02605915, NCT02649686, NCT04040699, 257,258

including (and anti-CTLA4 mAb) NCT04162327, NCT02689284, NCT03409848,

BC and GC
Chemotherapy + anti-HER2 mAb +
anti-PD(L)1 mAb
Chemotherapy + anti-HER2 mAb +
HER2-targeted ADC + anti-PDL1 mAb
HER2-targeted ADC + anti-PD(L)1 mAb
NCT03820141, NCT03988036, NCT04082364,
NCT03219268
NCT02605915, NCT02901301, NCT03125928, –
NCT03409848, NCT03414658, NCT04034823,
NCT04082364, NCT03615326
NCT02605915, NCT03894007, NCT03726879 –
NCT03032107, NCT02605915, NCT03523572, 261,262,

(or anti-CTLA4 mAb) NCT04042701, NCT04278144, NCT04280341, 319,320

NCT02924883
HER3 Overexpression BC and GC HER3-targeted ADC + anti-PD(L)1 mAb NA 263

See Supplementary Table 1 for details. ADC, antibody–drug conjugate; BC, breast cancer; EGFR, epidermal growth factor receptor; GC, gastric cancer;
HER, human epidermal growth factor receptor; HNSCC, head and neck squamous cell cancer; mAb, monoclonal antibody; NA, not applicable; NSCLC, non-small
cell lung cancer; TKI, tyrosine kinase inhibitor.

Inhibition of type I interferon signalling enhances
EGFR-TKI sensitivity in EGFR-mutated NSCLCs and
makes EGFR-WT/KRAS-mutated NSCLCs sensitive
to inhibition of EGFR signalling in immunocompetent
animals. Perhaps, a combination of EGFR-TKIs and
interferon-neutralizing antibodies could be efficient in
patients with NSCLCs, regardless of EGFR gene status.
VEGF inhibitors are another option for combi-
nation treatment with ICIs (Fig. 4b). VEGF-targeted
therapies have been demonstrated to improve the
efficiency of EGFR-TKI in EGFR-mutated NSCLCs243
and reduce the suppressive immune cells, includ-
ing Treg cells and MDSCs, in clinical trials244,245. Thus,
the inhibition of VEGF signalling can be a reason-
able option to improve the efficacy of ICIs to treat
EGFR-mutated tumours. A clinical trial has revealed
that EGFR-mutated NSCLCs show a clinical benefit
from the combination of atezolizumab, an anti-PDL1
mAb, with standard first-line platinum-based chemo-
therapy and bevacizumab, an anti-VEGF mAb246. This
combination treatment strategy may be beneficial to the
treatment of EGFR-TKI-resistant tumours with EGFR
mutations. Importantly, the clinical benefit of anti-PDL1
mAb was only confirmed when it was administered
with the anti-VEGF mAb in EGFR-mutated NSCLCs247.
The improved efficacy of ICIs in combination with
anti-VEGF mAb may be attributed to the reduction in
immunosuppressive cells, especially Treg cells20,244, yet fur-
ther studies are needed to elucidate how VEGF blockade
sensitizes EGFR-mutated cancers to ICIs.
Blockade of the CD73–adenosine axis, such as anti-
CD73 mAbs or A2AR antagonists, combined with ICIs
induces T cell activation including cytokine production
(IFNγ and granzyme B) and exhibits antitumour effects
in murine tumours without ERBB alterations248,249 (Fig. 4c).
Some clinical trials evaluating the efficacy of combination
therapies of A2AR inhibitors or anti-CD73 mAbs with
ICIs or of anti-CD73 mAbs and the EGFR-TKI osimer­
tinib, especially in EGFR-mutated NSCLCs, are under
investigation (Table 3).
The anti-EGFR mAb cetuximab is approved for locally
advanced and recurrent and/or metastatic HNSCCs and
metastatic CRCs. Although similar clinical efficacy was
observed in clinical trials of CRCs between cetuximab

Nature Reviews | CAncER volume 21 | March 2021 | 191

Reviews
and the anti-EGFR mAb panitumumab, suggesting that
preventing ligand binding and promoting the internal-
ization of EGFR250 would be the major mechanism of
action in these drugs, several other studies have demon-
strated that cetuximab potentially induces ADCC251,252
and triggers immunogenic cell death253, which is con-
sidered to be an important mechanism of action, when
combined with ICIs. Cetuximab treatment upregulates
PD1 expression in natural killer cells, and PD1 blockade
increases cetuximab-mediated ADCC against PDL1hi
HNSCC cells, where the majority of tumours express
EGFR without EGFR amplification254, indicating that the
combination of anti-EGFR mAb and ICIs could augment
both innate and acquired antitumour immune responses
to HNSCC expressing EGFR. In recurrent and/or meta-
static HNSCC, anti-PD(L)1 mAbs in combination with
cetuximab are being evaluated in phase II clinical trials
(NCT03082534 and NCT03082534).
Similar to anti-EGFR mAbs, anti-HER2 mAbs could
be a rational therapeutic partner of ICIs against can-
cers with HER2 amplification (Fig. 4d). The anti-HER2
mAb trastuzumab prevents HER2 shedding, inhibits
downstream PI3K–AKT signalling and induces ADCC,
leading to upregulation of PD1 expression by T cells and
of PDL1 expression by tumour cells in breast cancers
with HER2 amplification255,256. Indeed, recent phase Ib
and II clinical trials have achieved promising outcomes
in combination therapies of anti-HER2 mAbs with
ICIs in breast cancer and gastric cancer with HER2
amplification257,258, although the results of a randomized
phase III cohort study are still awaited.
ADCs are another attractive treatment modality for
cancers with HER2 amplification when combined with
ICIs. ADCs retain the function of anti-HER2 mAbs259,
and their conjugated cytotoxic reagents exert cytotox-
icity against cancer cells with HER2 amplification as
well as neighbouring cells (called bystander effects)260.
Cancers with HER2 amplification frequently have
intratumoural heterogeneity of HER2 expression. In
these cancers, bystander effects are therefore required
to increase antitumour efficacy by attacking HER2-low
or HER2-negative cancer cells surrounding cancer cells
with HER2 amplification in the TME, suggesting that
ADCs access and kill target cancer cells independent
of either the TMB or TILs. The HER2-targeted ADCs
T-DM1 and DS-8201 or the HER3-targeting ADC
U3-1402 reportedly augment the antitumour effects of
PD1 blockade in preclinical models by inducing immuno­
genic cell death and MHC class I expression in cancer
cells, resulting in dendritic cell maturation, differentia-
tion of TAMs into the pro-inflammatory M1 type and
increased T cell infiltration261–263. T-DM1 combined with
anti-PDL1 mAb atezolizumab versus T-DM1 alone in
previously treated advanced breast cancer with HER2
amplification is being assessed in a randomized phase II
clinical trial (NCT02924883).
Numerous studies have addressed antitumour effi-
cacy of combinations of ICIs and ERBB-targeted ther-
apies. Although anti-VEGF mAbs reportedly improve
the efficacy of ICIs in EGFR-mutated NSCLCs, the
detailed mechanisms remain to be determined espe-
cially in clinical settings. Importantly, anti-VEGF mAbs
192 | March 2021 | volume 21
could improve the efficacy of ICIs even in low TMB
EGFR-mutated tumours, which often exhibit resis­
tance to PD1–PDL1 blockade monotherapy. Recently,
promising outcomes were reported in clinical trials in
which combinatory treatment of multikinase inhibi-
tors and ICIs was evaluated in gastric cancer and renal
cell carcinoma264,265. These results may provide a clue
to understanding underlying mechanisms in which
TKIs can improve the antitumour efficacy of ICIs
even in low TMB tumours, for example, by modulat-
ing immunosuppressive cells. Although the results
in preclinical models clearly show that EGFR-TKI
treatment improves antitumour response in the TME
and strengthens the efficacy of PD1 blockade treat-
ment, long term follow-up studies of combinations of
EGFR-TKI and ICIs or sequential ICI treatment after
EGFR-TKI will be necessary to determine significant
clinical benefits in addition to safety issues. Considering
the mechanisms of action, the combination of ERBB
family-targeted mAbs or ADCs with ICIs could
increase innate and acquired immune activation even
in non-inflamed tumours to potentially switch the TME
towards inflamed tumours. In addition, the combina-
tion of ERBB family-targeted therapies and genetically
engineered immune cell therapies (for example, chi-
meric antigen receptor T cell therapies and TCR T cell
therapies) may be examined as a novel treatment strat-
egy. However, the evidence of clinical benefit for these
combination therapies is still limited. Further studies
determining the cancer types that respond to combi-
nation immunotherapies and elucidating the mecha-
nisms of additional or synergistic effect are warranted.
Altogether, combined comprehensive analyses of the
TME to elucidate both immunological and genomic
landscapes are crucial for achieving immune-genome
precision medicine.
Conclusions
Aberrant signalling of ERBB family members in cancer
cells contributes to tumour development via the follow-
ing two aspects: augmented cell growth and/or survival;
and the establishment of an immunosuppressive TME
via direct regulation of the immune system, enabling
tumours to escape from antitumour immune responses.
Thus, targeting aberrant ERBB family signalling could
be essential for improving the sensitivity to ICIs, possibly
through multiple mechanisms including upregulating
the tumour antigen presentation, disturbing and inac-
tivating immunosuppressive cells, such as Treg cells and
MDSCs, and downregulating immunosuppressive mol-
ecules. To optimize cancer immunotherapies in patients
with gene alterations leading to aberrantly increased
ERBB family signalling, it is necessary to develop novel
therapeutic strategies targeting the immunosuppressive
pathways caused by aberrant signalling of ERBB family
members. Targeting signalling with molecular-targeted
therapies in combination with cancer immunotherapies
could increase antitumour efficacy, leading to the devel-
opment of optimal cancer therapy: immune-genome
precision medicine.
Published online 18 January 2021
www.nature.com/nrc

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Acknowledgements
This study was supported by Grants-in-Aid for Scientific
Research grant no. 17H06162 (to H.N.) from the Ministry of
Education, Culture, Sports, Science and Technology of Japan,
the Projects for Cancer Research by Therapeutic Evolution
(P-CREATE; no. 16cm0106301h0001 to H.N. and no.
19cm0106335h0002 and 19cm0106310h0004 to S.Ko.),
the Development of Technology for Patient Stratification
Biomarker Discovery grant (no. 19ae0101074s0401 to H.N.)
from the Japan Agency for Medical Research and
Development (AMED), and the National Cancer Center
Research and Development Fund (no. 28-A-7 and 31-A-7
to H.N.).
Author contributions
All authors contributed to each stage of the preparation of
this manuscript.
Competing interests
S.Ko. received research funding from Ono Pharmaceutical
and Bristol-Myers Squibb outside this study. H.N. received
honoraria and research funding from Ono Pharmaceutical,
Chugai Pharmaceutical, MSD and Bristol-Myers Squibb, and
research funding from Taiho Pharmaceutical, Daiichi-Sankyo,
Kyowa Kirin, Zenyaku Kogyo, Oncolys BioPharma,
Debiopharma, Asahi-Kasei, Sysmex, Fujifilm, SRL, Astellas
Pharmaceutical, Sumitomo Dainippon Pharma and BD Japan
outside this study. All other authors declare no competing
interests.
Peer review information
Nature Reviews Cancer thanks B. Pollack, F. Skoulidis and
N. Ueno for their contribution to the peer review of this work.
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Supplementary information
Supplementary information is available for this paper at
https://doi.org/10.1038/s41568-020-00322-0.
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