Targeting neoantigens to augment antitumour immunity
Mark Yarchoan
*
, Burles A. Johnson III
*
, Eric R. Lutz, Daniel A. Laheru, and Elizabeth M.
Jaffee
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland
21231, USA
Abstract
The past decade of cancer research has been marked by a growing appreciation of the role of
immunity in cancer. Mutations in the tumour genome can cause tumours to express mutant
proteins that are tumour specific and not expressed on normal cells (neoantigens). These
neoantigens are an attractive immune target because their selective expression on tumours may
minimize immune tolerance as well as the risk of autoimmunity. In this Review we discuss the
emerging evidence that neoantigens are recognized by the immune system and can be targeted to
increase antitumour immunity. We also provide a framework for personalized cancer
immunotherapy through the identification and selective targeting of individual tumour
neoantigens, and present the potential benefits and obstacles to this approach of targeted
immunotherapy.
Recent advances in genome sequencing have revealed that during the process of initiation
and progression, cancers acquire tens to thousands of different somatic mutations. Most of
these mutations confer no intrinsic growth advantage (passenger mutations) and are often the
result of genomic instability within the tumour. A smaller number of cancer mutations
interfere with normal cell regulation and help to drive cancer growth and resistance to
targeted therapies (driver mutations)
1
. To date, approximately 140 genes have been
identified that can drive tumorigenesis
2
. However, both driver mutations and passenger
mutations can alter amino acid coding sequences, collectively known as nonsynonymous
mutations, causing tumours to express mutant proteins that are not expressed by normal
cells
3,4
. These abnormal protein sequences are processed into short peptides (epitopes) and
presented on the cell surface in the context of major histocompatibility complex (MHC; also
known as human leukocyte antigen (HLA) in humans), thereby becoming recognizable to T
cells as foreign antigens (FIG. 1).
Cancers with a single dominant mutation can often be treated effectively by targeting the
dominant driver mutation
5
. Former American Society for Clinical Oncology (ASCO)
president George Sledge has termed such cancers “stupid cancers” for their sensitivity to
targeted therapy
6
. By contrast, “smart cancers” have a much higher number of mutations,
Correspondence to E.M.J. ejaffee@jhmi.edu.
*
These authors contributed equally to this work.
Competing interests statement
The authors declare competing interests: see Web version for details.
HHS Public Access
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Nat Rev Cancer
. Author manuscript; available in PMC 2018 April 01.
Published in final edited form as:
Nat Rev Cancer
. 2017 April ; 17(4): 209–222. doi:10.1038/nrc.2016.154.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
multiple simultaneous driver mutations and are less amenable to treatment with traditional
targeted therapies
6
. However, these smart cancers with high mutational burdens often have
high expression of aberrant proteins
6–8
. In the current immunotherapy era, these aberrant
proteins are increasingly recognized as opportunities for the immune system to recognize
and control tumour growth.
Owing to their selective expression in tumours, the tumour-specific antigens (TSAs) that
arise from non-synonymous mutations and other genetic alterations are called neoantigens
9
(TABLE 1). In the subset of human tumours with a viral aetiology, as with Merkel cell
carcinoma (MCC) associated with the Merkel cell polyomavirus (MCPyV) and cancers of
the cervix, oropharynx and other sites associated with the human papillomavirus (HPV), the
proteins encoded by viral open reading frames are another type of neoantigen
10–12
. In
addition to TSAs, there are two other broad categories of tumour antigen. Tumour-associated
antigens (TAAs) are over-expressed in malignant cells but are also present in normal cells at
low levels of expression
9
. Cancer/testis antigens (CTAs) are expressed by various tumour
types and by reproductive tissues (for example, testes, fetal ovaries and trophoblasts) but
have limited expression on other normal tissues in the adult and are generally not presented
on normal reproductive cells, because these tissues do not express MHC class I
molecules
13,14
.
Initial attempts to target TSAs, including efforts targeting virus-associated antigens
15
,
mutated growth factor receptors
16,17
or mutated KRAS
18,19
, have provided an early
validation of TSAs as tumour antigen targets. However, most neoantigens arise from unique
mutations, and technical challenges related to the identification and targeting of neoantigens
specific to individual tumours have prevented widespread adoption of an individualized
approach. This Review discusses how such limitations are beginning to be overcome through
major advancements in genomics and bioinformatics, including massively parallel
sequencing (MPS) and epitope prediction algorithms. It is now widely recognized that the
development of immune checkpoint inhibitors is transforming cancer care by providing
unprecedented clinical benefit for some tumour types
20,21
. However, novel approaches to
immunotherapy are now required to improve on current objective response rates (ORRs) and
to extend benefit to all tumour types. In this Review we describe the relationship between
neoantigens and sensitivity to current immunotherapies, and also provide a framework for
the targeting of individual tumour neoantigens, with the potential to make antitumour
immunity more specific, more effective and less toxic.
TSAs, TAAs and CTAs as targets
TSAs, TAAs and CTAs have all been considered as targets for immunotherapy (TABLE 1).
Until now, most cancer vaccines have focused on commonly overexpressed TAAs because of
the potential to treat many patients with the same therapeutic vaccine. Although CTA targets
are also shared between tumours, they are known to be expressed in only a limited number
of tumour types, potentially reducing the applicability of CTA-targeted immunotherapy.
Unfortunately, most attempts to target TAAs with vaccines have met with limited success
22
,
potentially because TAAs are normal host proteins and are therefore subject to both central
and peripheral tolerance mechanisms
13
. Although some TAA-specific T cells do avoid
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negative selection, high-affinity T cell receptors (TCRs) for TAAs are preferentially depleted
and the range of affinities of the remaining TCRs for TAAs is lower than TCR affinities for
typical TSAs and other foreign antigens
23,24
. T cell cytotoxicity and activation are correlated
with TCR binding affinity
25
, which supports the idea that immune responses against TAAs
are less vigorous than those against TSAs.
A separate concern with TAAs and CTAs is the potential for autoimmune toxicities related
to immune activation in non-target tissues, because these targets are expressed on normal
host cells. Concerns about collateral damage to non-target tissues have received increased
attention as modern immunotherapies have become potent enough to induce lethal
autoimmunity
26
. In some cases the immune destruction of normal organs can be managed.
For example, the destruction of all CD19-positive B lineage cells (both healthy and
malignant) with anti-CD19 chimeric antigen receptor (CAR) T cells in patients with acute
lymphoblastic leukaemia (ALL)
27,28
can be managed with immunoglobulin replacement, or
through reconstitution of B lineage cells with allogeneic stem-cell transplantation. However,
in other cases, toxicity from off-target immune activation has led to serious complications.
For example, attempts to target the TAA carbonic anhydrase 9 in renal cell carcinoma (RCC)
led to severe liver toxicity owing to expression of this antigen in bile duct epithelial cells
29
,
and likewise, an attempt to target ERBB2 (also known as HER2 or NEU) in a patient with a
metastatic colon cancer that overexpressed ERBB2 led to rapid respiratory failure and death,
probably due to low levels of ERBB2 expression on lung epithelial cells
30
. Similarly,
attempts to use adoptive cell therapy (ACT) to target the CTA melanoma-associated antigen
3 (MAGE-A3) resulted in severe neurological toxicity and death, which was probably
related to previously unrecognized expression of MAGE-A family members in the brain
31
.
This case challenges the concept of high CTA specificity. Although CAR T cells have shown
considerable promise against some haematological cancers
27,28
, the ability to treat solid
cancers with CAR T cells and other targeted immunotherapies has thus far been severely
limited by the lack of suitable TAA and CTA targets.
TSAs are theoretically a more attractive immunotherapy target because they may be
recognized as non-self by the host immune system, and are therefore less likely to establish
complex immune tolerance mechanisms. Additionally, immunotherapies targeting TSAs
should theoretically be less likely to induce autoimmunity, because the target is not
expressed on normal cells. Despite these theoretical advantages, T cell responses against
TSAs do not ensure clinically detectable anti-tumour activity, as evidenced by the inability
of a host to eradicate melanoma despite the presence of high frequencies of cytotoxic T
lymphocytes (CTLs) before vaccination
32
. Furthermore, neoantigen-specific approaches to
immunotherapy cannot completely eliminate the risk of autoimmunity, because neoantigen-
specific T cells can be cross-reactive with the non-mutated version of the antigen. This is
exemplified by cancer-induced auto-immune disease. A recent study identified eight patients
with cancer who concurrently developed scleroderma, an autoimmune disease, with
detectable autoantibodies against RNA polymerase III subunit RPC1. Although these
patients had various malignancies (breast, ovarian and colon), it was demonstrated that most
tumours from these patients (six of eight) had specific genetic alterations in
POLR3A
, the
gene that encodes RPC1. Therefore, the neoantigen-specific T cells against mutated
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POLR3A
in the context of a malignancy seem to be driving the autoimmune disease
process
33
.
Neoantigens and the immune response
Evidence for immune system recognition of tumour neoantigens
The immune system has an extraordinary ability to distinguish self from non-self. This
inherent property of the immune system gives it the ability to recognize and target non-self
antigens on cancer cells to control cancer. In 1943, Gross and colleagues
34
were among the
first to report that the immune system can recognize and destroy cancer cells. They showed
that mice with resected tumours were protected against subsequent re-exposure from the
same tumour cells, and that similar protection against tumour cells could be induced by first
exposing mice to lethally irradiated tumour cells. However, at the time, the nature of the
antigens that enabled the immune system to reject cancers was not clear. In the late 1980s
the Boon group
35,36
made the important initial observation in a mouse model that
antitumour T cells can recognize aberrant peptides derived from tumour-specific mutations.
Several years later, somatic mutations were also shown by several separate groups to be a
source of neo-antigens recognized by T cells in human tumours
37–40
. Another important
advance in our understanding of TSAs occurred in 2005, when Wölfel and colleagues
41
analysed the naturally occurring antitumour T cell response against melanoma in a single
patient. They found that the T cells of the patient were reactive against five mutated epitopes
resulting from nonsynonymous mutations and that this immunoreactivity against melanoma
neoantigens predominated over the response to TAAs. Similarly, the Rosenberg group
42
showed that the adoptive transfer of
ex vivo
-expanded tumour-reactive tumour-infiltrating
lymphocytes (TILs) into a patient with melanoma could cause complete tumour regression,
and that these T cells reacted to mutated forms of two neoantigens on the melanoma cells.
These neoantigen-specific T cells persisted at high levels in the tumour 1 month after
transfer. Together, these studies provided support for the role of neoantigens in the naturally
occurring antitumour T cell response.
More recent support for neoantigens as important tumour antigens for the human immune
system has emerged from studies of novel immune checkpoint inhibitors targeting cytotoxic
T lymphocyte-associated protein 4 (CTLA4) and programmed cell death protein 1 (PD1),
which are expressed by activated T cells
43,44
. The remarkable clinical activity of immune
checkpoint inhibitors against a wide variety of human cancers has provided indisputable
evidence that the immune system can recognize and destroy established cancers
20,21
. To
destroy established cancers in the presence of immune checkpoint inhibitors, T cells must
recognize antigens displayed by MHCs on tumour cells (FIG. 1). Theoretically, any class of
tumour antigen could be recognized by T cells; however, it is increasingly clear that TSAs
serve as the most important tumour antigens in certain cancers where immune checkpoint
inhibitors have shown clinical efficacy. For example, TSAs have been demonstrated to be T
cell targets of sarcoma-bearing mice treated with immune checkpoint inhibitors, and
administration of peptide vaccines incorporating these antigens into mice could achieve
similar outcomes to immune checkpoint blockade
45
. More recently, PD1-expressing
neoantigen-specific T cells have been identified in the peripheral blood of patients with
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melanoma, correlating with the recently documented activity of PD1 inhibitors in this
population
46
.
Tumour somatic mutation frequency and sensitivity to immune checkpoint blockade
Although the full therapeutic potential of immune checkpoint inhibitors remains undefined,
it is evident from the available human clinical data that the frequency of somatic mutations
within a tumour type, and by extension, the potential for neoantigens within a tumour type,
is largely correlated with sensitivity to immune checkpoint inhibitors (FIG. 2). Two tumour
types with notably high ORRs to immune checkpoint blockade are melanoma
47–51
and non-
small-cell lung cancer (NSCLC)
52–54
. Both cancers are usually formed in the presence of
mutagens (ultraviolet light in the case of melanoma and tobacco smoke in the case of
NSCLC
55
), and as a result they have amongst the highest somatic mutation burdens of any
cancer type. By comparison, immune checkpoint inhibitors thus far have shown little or no
activity in the subset of cancers with lower mutation burdens, such as Ewing sarcoma
56
and
prostate cancer
26,57
. The clinical observation that somatic mutations and potential for
neoantigens in a tumour type are correlated with objective clinical responses to immune
checkpoint inhibitors strongly supports the idea that neoantigens are important
immunotherapy tumour antigens.
Somatic mutation burden seems to be an important marker of immune response even within
a particular tumour type. This was recently illustrated in a series of clinical trials in
colorectal cancer. Although most colorectal cancer has DNA mismatch repair proficiency
(MMR-P), a small subset of colorectal cancer (<5%) have DNA mismatch repair deficiency
(MMR-D), also referred to as microsatellite instability (MSI). As mismatch repair proteins
correct errors in DNA replication, colorectal cancers with MMR-D are marked by genomic
instability, a remarkably high mutation burden and the potential for high numbers of
neoantigens. In clinical trials of PD1 inhibitors in unselected populations of patients with
colorectal cancer, little to no activity was observed
26,58
. However, a clinical trial of the PD1
inhibitor pembrolizumab in a selected population of patients with MMR-D colorectal cancer
demonstrated high response rates
58
. Therefore, within colon cancer, MMR-D predicts
responses to immune checkpoint inhibition. A similar correlation between intratumour
mutation burden and clinical benefit from immune checkpoint inhibition has been
demonstrated in melanomas
43
and lung cancers
44
, providing additional support for the role
of tumour mutations and associated neoantigens in the antitumour immune response.
Although tumour somatic mutation burden is largely correlated with clinical response to
currently available immunotherapy agents, the neoantigens resulting from somatic mutations
are only one component dictating the rate and quality of immune response to immune
checkpoint inhibitors. As a result, the association between potential neoantigens and
response to immune checkpoint blockade is not linear (FIG. 2) and does not preclude
multiple additional factors influencing responses to immune checkpoint inhibitors. For
example, in the study discussed above that correlated intratumour mutation burden in
melanoma with response to immune checkpoint blockade, there were multiple patients with
high mutational burden who did not benefit from CTLA4 blockade, indicating that mutation
burden alone is not sufficient to predict benefit
43
. One possible explanation for this varied
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