International Immunology, Vol. 28, No. 7, pp.

365–370 Published by Oxford University Press on behalf of

Targeting neoantigens for cancer immunotherapy

Correspondence to: P. F. Robbins; E-mail: [email protected]

Review
Abstract
Studies first carried out in the 1980s have demonstrated murine T cells can recognize mutated gene
products, known as neoantigens, and that these T cells are capable of mediating tumor rejection.
The first human tumor antigens isolated in the early 1990s were the products of non-mutated genes
expressed in a tissue-specific manner; subsequent studies have indicated that tumor-infiltrating
lymphocytes that are cultured in vitro frequently recognize mutated gene products. In addition,
correlative studies indicate that clinical responses to therapies involving the use of antibodies
directed against checkpoint inhibitors such as CTLA-4 and PD-1 may be associated with mutational
burden, providing indirect evidence that these responses may primarily be mediated by neoantigen-
reactive T cells. The importance of neoantigen-reactive T cells may be elucidated by the results of
ongoing and future studies aimed at leveraging information gained from mutational profiling to
enhance the potency of immunotherapies.

Keywords:  cancer immunotherapy, mutation, neoantigen, tumor immunology

Introduction
Studies carried out over the last 25 years have provided impor- culture with tumor-infiltrating lymphocytes (TILs) or by in vitro
tant insights into the nature of antigens recognized by human sensitization of PBMCs against autologous tumor cells or
tumor-reactive T cells. These antigens can be grouped into autologous normal cells that were either pulsed with candi-
five general categories based upon their patterns of expres- date T cell epitopes or transfected with genetic constructs
sion in both normal and tumor tissues of origin, as this pro- encoding candidate antigens. The antigens identified using
vides a framework for evaluating therapeutic targets. these approaches can be grouped into five general catego-
The discussion below first centers on the characteristics ries: antigens derived from gene products that are widely
of such antigens identified as targets of tumor-reactive T expressed in normal tissues at relatively low levels in compar-
cells, and the implications of these findings for clinical immu- ison with malignant cells; differentiation antigens expressed
notherapy trials. A  discussion of the results of clinical trials at relatively high levels in a single tissue; antigens that are
that deliberately targeted individual antigens is followed by limited in their expression in adults to germ cells that lack
a discussion of correlative studies analyzing the antigens MHC expression [cancer germline (CG) antigens]; viral anti-
that appear to represent the predominant targets of clini- gens; and mutated antigens (Table 1). Although expression
cally effective bulk populations of adoptively transferred T of the first two categories of antigens in normal cells may trig-
cells. The discussion then centers on analysis of the results ger central and peripheral tolerance mechanisms that lead
of studies in patients receiving antibodies directed against to the selection of low avidity T cells, treatment of patients
checkpoint inhibitors that, while still preliminary, have begun with a high avidity TCR that recognized the melanocyte dif-
to provide clues as to the nature of antigens associated with ferentiation antigen MART-1 resulted in severe skin, eye and
tumor regression. The final section of the review contains a ear toxicity, and resulted in durable responses in only a small
discussion of the implications of these findings for the devel- percentage of patients (1).
opment of future therapies. CG antigens represent gene products whose expression in
the adult is generally limited to germ cells that lack expression
of MHC molecules and thus are not subject to attack by HLA
Tumor antigens: broad categories defined by
class I-restricted or class II-restricted T cells, potentially allowing
expression in tumors and normal tissues
treatment with high avidity T cells while resulting in little or no
Initial studies used to identify tumor antigens were primarily on-target recognition of normal tissues. In clinical trials involving
carried out by screening expression libraries using in vitro adoptive transfer of T cells targeting the CG antigen NY-ESO-1,
366  Neoantigens in cancer immunotherapy
Table 1.  Categories of tumor antigens

Category Normal tissue expression Expression levels in Examples Advantages of Disadvantages of

Over-expressed gene Broadly distributed Low PRAME Expressed in a wide Potential for
products variety of cancer types autoimmunity due to
broad normal-tissue
expression
Tissue-specific Narrow: single tissues Generally high MART-1, CEA Many products Autoimmunity can limit
differentiation expressed at high the ability to develop
antigens levels in one or limited potent therapies
number of tissues
Cancer germline Germ cells Generally high MAGE-A family, Generally not Some family members
antigens NY-ESO-1 expressed in normal are expressed in adult
adult tissues with the normal tissues, leading
exception of germ cells to autoimmunity
Viral antigens None None HPV E6, HPV E7 Not expressed in any The number of
normal tissues therapeutic targets
may be limited by
mechanisms of viral
immune evasion
Mutated antigens None None KRASG12D, KRASG12V Not expressed in any Many are limited to
normal tissues. Some one, or a small number
hotspot mutations are of, tumors
present at relatively
high frequencies in
particular tumor types.
Targeting hotspot
mutations may help to
obviate antigen loss

objective responses were observed in 61% of patients with syno- recognized in all cancers, with the possible exception of some
vial cell sarcoma, 55% of patients with melanoma (2) and 80% of hematological malignancies that possess fewer than 10 non-
patients with myeloma (3). High level expression of NY-ESO-1 has synonymous somatic mutations (10). Recent studies indicating
only been observed in a relatively small percentage of tumors, that that recognition of mutated tumor antigens (neoantigens)
with the exception of synovial cell sarcoma, limiting treatments is associated with clinical responses in a variety of therapeutic
targeting this antigen to relatively small numbers of patients (4). settings have provided an impetus to develop novel therapies
Members of the MAGE-A CG gene family have been targeted based upon analysis of the mutational landscape of tumors.
in multiple trials; however, severe toxicities including four deaths The promises and difficulties with targeting neoantigens are
that were attributed to the transferred T cells were seen in two discussed in the remaining sections of this review.
trials targeting MAGE-A3 epitopes that were attributed to expres-
sion of the related MAGEA12 gene in rare cells present in normal
Correlative studies of neoantigen recognition and
brain tissue (5) or cross-reactivity of an affinity-enhanced TCR
immunotherapy responses
with a protein expressed in cardiomyocytes (6).
These observations led to studies aimed at targeting gene Antigen cloning studies carried out over the past 25 years have
products that are not present in the normal human genome. led to the demonstration that T cells from patients with a variety of
Vaccination of patients against viral antigens such as the cancer types recognize mutated gene products (11); however,
human papillomavirus (HPV) E6 and E7 oncogenes, gene the conventional expression cloning methods used in these
products that are highly expressed in a limited number of tumor studies were time consuming and labor intensive, limiting the
types such as cervical and head and neck cancers, appears to applicability of this approach for identifying targets for therapy.
be effective at preventing disease progression in patients with The advent of relatively inexpensive high throughput sequenc-
premalignant disease (7) but not in patients with invasive cer- ing methods in the last few years, including whole genome
vical cancer (8). In a recent trial, durable complete responses sequencing and whole exome sequencing (WES) analysis of
were seen in two of nine metastatic cervical cancer patients tumor and matched normal DNA and RNA-seq whole transcrip-
receiving autologous TIL cultures that were selected on the tome (RNA-seq) analysis, have provided an opportunity to uti-
basis of several characteristics that included responsiveness lize a variety of reverse immunology approaches (i.e. predicting
to libraries of overlapping peptides that encompassed the E6 and identifying epitopes from nucleotide sequences) to identify
and E7 proteins (9). The degree of reactivity against the E6 and neoepitopes recognized by tumor-reactive T cells.
E7 peptides appeared to be associated with clinical response In a recent study, WES of three metastatic melanomas,
to therapy, a potentially significant finding, particularly if it were combined with expression analysis and use of the MHC
to be replicated in a larger patient population. class I-restricted binding peptide algorithm NetMHCpan, led
Mutated genes encode targets that obviate issues arising to the identification of a total of seven neoepitopes recog-
from expression in normal tissue and that can potentially be nized by three populations of human melanoma TILs (12).
 Neoantigens in cancer immunotherapy  367
Two neoepitopes were also identified as T cell targets in a their ability to recognize panels of 31 amino acid synthetic pep-
patient with metastatic melanoma using an approach where tides encompassing individual mutations (20). Approximately
WES and RNA-seq analysis were used in combination with 4% of the CD4+ T cells present within autologous TILs that were
the NetMHC class I peptide-binding algorithm to generate a administered to one of the patients in this study, who was a
library of MHC–peptide tetramers generated by an ultravio- partial responder to autologous TIL therapy, recognized a sin-
let light-induced peptide exchange approach (13) that were gle neoepitope. In addition, a total of 13% of the CD4+ T cells
screened for binding to the patient TIL sample (14). that were administered to a patient who exhibited a complete
Use of a screening approach based upon transient trans- response to autologous T cells generated by in vitro stimulation
fection of COS-7 or HEK293 cell lines with tandem minigene with autologous tumor cells recognized three neoepitopes.
constructs (TMGs) consisting of 12 minigenes encoding Studies in murine tumor model systems have also pro-
mutated residues plus the 12 flanking normal amino acids vided evidence that neoepitopes can serve as potent tumor
plus constructs encoding autologous HLA gene products rejection antigens. A study carried out using tumors derived
led to the identification of neoepitopes recognized by two from immunodeficient Rag2-knockout mice indicated that
polyclonal populations of melanoma TILs (15). Combining a mutated spectrin-β2 neoepitope represented the domi-
WES with the identification of peptides eluted from cell sur- nant tumor rejection antigen for the murine methylcholan-
face MHC molecules by mass spectrometry represents an threne-induced sarcoma d42m1 (21). In another study, 11
example of another approach that has been used to identify of 50 mutated 27-mer peptides identified by WES of the
neoepitopes recognized by T cells (16). B16F10 murine melanoma were found to induce immune
One question raised by these finding was whether or not this responses preferentially recognizing the mutated epitopes
or a similar approach could be used to identify neoepitopes (22). Immunization of tumor-bearing mice with an immuno-
recognized by T cells in additional cancer types. Using a TMG dominant peptide identified using this approach significantly
screening method, combined with the pulsing of autologous slowed tumor growth and enhanced survival. Additional
antigen-presenting cells with relatively long peptides of 25 murine studies carried out by vaccination with mutated pep-
amino acids, one to three neoepitope targets were identified for tides (16) or synthetic RNA constructs encoding tandem
9 of the 10 gastrointestinal TILs that were evaluated in a recent arrays of epitopes, which appeared to predominantly induce
report (17). All of the T cells evaluated in this study recognized MHC class II restricted responses in immunized mice, con-
neoepitopes that were unique to an individual tumor, with the ferred disease control and survival benefit (23).
exception of T cells from two patients that recognized an iden- In a human vaccine study carried out in three patients with
tical epitope containing a substitution of aspartic acid for gly- metastatic melanoma, vaccination with autologous dendritic
cine at position 12 of the KRAS oncogene (KRASG12D) in the cells that were pulsed with neoepitope peptides identified
context of HLA-C*08:02. Substitution of aspartic acid, valine using WES and RNA-seq in combination with peptide–MHC
and cysteine at this position in the KRAS protein, termed driver binding algorithms led to in vivo expansion of peptide-reac-
mutations, have been shown to stimulate cell growth through tive, and putative tumor-reactive, T cells (24). Although immu-
constitutive activation of the RAS/MAPK signaling pathway, nization did not appear to have a clinical impact on disease
and are commonly found in multiple tumor types including progression in these patients, these results demonstrated the
pancreatic, colon and lung cancers. These results provide feasibility of using this approach to identify neoepitope tar-
support for the application of these methods to the identifica- gets that could potentially be used for combination therapies
tion of neoepitopes recognized in additional cancer types. that involve use of vaccines to boost responses to immune
The ability to readily identify neoepitopes recognized by checkpoint inhibitors or adoptive immunotherapy.
patient T cells has also provided the opportunity to evalu- Analysis of data from clinical trials involving treatments
ate the hypothesis that recognition of mutated antigens may with antibodies directed against inhibitory molecules such as
play an important role in patient responses to cancer immu- CTLA-4 and PD-1, termed checkpoint blockade therapies,
notherapies. In support of this hypothesis, durable tumor has provided further evidence that neoepitope reactivity may
regressions have been observed in melanoma patients who play an important role in mediating responses to immunother-
received autologous TILs that appeared to predominantly apy. Clinical benefit in metastatic melanoma patients treated
recognize neoepitopes expressed by the patients’ tumors with ipilimumab or tremelimumab, antibodies directed against
(12, 18). Treatment of a patient with metastatic cholangiocar- the inhibitory ligand CTLA-4, was associated with mutational
cinoma with an autologous TIL culture, over 95% of which load and the presence of a tetrapeptide signature present on
consisted of CD4+ T cells that recognized a single HLA predicted neoepitopes in patients with long-term benefit but
class II-restricted neoepitope derived from the ERBB2IP pro- not present on predicted neoepitopes in patients with minimal
tein, led to dramatic tumor regression (19). or no clinical benefit (25); however, the lack of an independent
In addition, adoptive transfer of a melanoma TIL population, validation set used to identify the tetrapeptide signature in this
50% of which recognized a mutated HLA class  I  restricted study has called into question the validity of this result (26, 27).
PPP1R3B epitope, but that did not appear to recognize Results of a study involving treatment of metastatic mela-
shared non-mutated antigens, was associated with a com- noma patients with ipilimumab provided further evidence for
plete regression of all metastatic lesions that is ongoing an association between clinical benefit and overall mutational
beyond 10 years (18). load or the overall load of predicted HLA class  I-restricted
In another study, neoantigen-reactive CD4+ T cells were neoepitopes, which, in contrast to results presented in the
identified within populations of tumor-reactive T cells from four previous report, did not appear to contain a tetrapeptide sig-
of five metastatic melanoma patients that were screened for nature (28). The results of a study evaluating the response of
368  Neoantigens in cancer immunotherapy
melanoma patients to treatment with either pembrolizumab or T cells may play important role in mediating clinical responses
nivolumab, two antibodies directed against the immune check- to these therapies, as approximately one quarter of melanoma
point inhibitor PD-1, indicated that neither total mutational load patients receiving adoptive immunotherapy have exhibited
nor predicted HLA class  I  or class  II neoepitope load was long-term durable complete responses (36) and long-term
associated with response to therapy (29). Notably, however, follow up has indicated that approximately one third of mela-
patients in this study whose tumors were within the top third noma patients receiving nivolumab were alive after 5  years
of the distribution of total non-synonymous somatic mutations (37). Treatments combining anti-PD-1 and anti-CTLA-4 immune
survived longer than those whose tumors were within the bot- checkpoint inhibitors aimed at increasing response rates
tom third of the distribution, indicating that multiple factors may appeared in initial studies to result in higher response rates than
influence response to anti-PD-1 therapy. treatment with either agent alone (38), but led to higher levels
Mutational load was, however, associated with response of severe autoimmunity than were seen in patients treated with
to immune checkpoint blockade in a clinical trial evaluating either inhibitor alone. In addition, long-term follow-up of patients
responses to pembrolizumab in patients whose tumors were receiving combinations of immune checkpoint inhibitors have
either mismatch repair-deficient or -proficient (30). In this trial, not been reported, but adjustments of the dosage and timing
patients with mismatch repair-deficient carcinomas derived of infusions of the individual inhibitors are being evaluated to
from either colon or additional tumor types that possessed determine whether or not schedules that limit normal tissue tox-
relatively high mutational burdens exhibited objective clinical icities while maximizing tumor regression can be identified.
response rates of 40% and 71%, respectively, whereas none Further progress in developing novel approaches to the
of the 18 patients bearing mismatch repair-proficient colorec- treatment of patients for whom current immunotherapies
tal cancers responded to therapy. are of limited or no clinical benefit may depend upon gain-
Additional studies have also provided ana indication that ing a better understanding of the biological basis of these
mutational burden may be related to response to immune responses. At the present time it is not possible to determine
checkpoint blockade. Neoantigen burden was positively cor- the percentage of expressed mutated gene products that
related with the clinical benefit and progression-free survival are capable of giving rise to naturally processed and pre-
in patients with non-small cell lung cancer (NSCLC) receiving sented neoepitopes; however, results of screening candidate
the anti-PD-1 antibody pembrolizumab (31). In a recent study epitopes identified using MHC class I peptide-binding algo-
examined the impact of intratumor heterogeneity and pre- rithms indicate that 10% or less of the mutations in a given
dicted neoantigen burden on response to immune checkpoint individual are likely to give rise to an immunogenic epitope. If
blockade in patients with metastatic melanoma or NSCLC (32), true, it would be unlikely that T cells reactive with more than one
a high neoantigen burden was associated with longer overall or two neoepitopes will be present in patients whose tumors
survival in a predominantly early-stage cohort of patients with contain fewer than 100 non-synonymous somatic mutations,
lung adenocarcinoma but not in early-stage patients with squa- many of which will not be expressed or will be expressed at
mous cell carcinoma. A high predicted neoantigen burden was levels that are unlikely to elicit immune responses.
associated with an inflamed tumor signature, as defined by Tumor immunoediting may lead to the elimination of tumors
expression of CD8A, CD8B, and genes associated with anti- that express particularly potent neoepitopes (39); however, the
gen presentation and effector cell function, whereas increased effectiveness of adoptive immunotherapy and immune check-
intratumoral heterogeneity, as determined by evaluating the point inhibitors at mediating regression of multiple cancer types,
clonality of putative neoepitopes expressed by patient tumors, combined with the observation that many of the immunogenic
appeared to have a relatively small but significant negative neoepitopes identified to date appear to be recognized by high
influence on overall survival. Intratumoral heterogeneity also avidity T cells, indicates that this may not play a major role in
appeared to have a negative impact on progression free sur- shaping the neoepitope landscape in most tumors. Variations
vival in patients with advanced NSCLC treated with pembroli- in the levels of inhibitory factors in the tumor microenvironment
zumab and on overall survival in metastatic melanoma patients that may be influenced by intrinsic features of individual tumors
treated with ipilimumab or tremelimumab. may play a more important role in limiting responses to these
Taken together, these results provide support for the hypoth- therapies than the numbers or potency of tumor-reactive T cells
esis that tumor regression in response to adoptive immuno- present within some, and perhaps most, tumors.
therapy and to immune checkpoint inhibitors may primarily be Intratumoral and intertumoral heterogeneity of neoantigen
mediated by T cells that recognize neoepitopes expressed expression of neoantigens targeted by immunotherapy may
by patients’ tumors (reviewed in (33)). Renal cell carcinomas also significantly impact on the effectiveness of treatments
respond to immune checkpoint blockade but possess rela- targeting neoantigens. The ability of adoptive immunotherapy
tively low mutation loads (34), however, indicating that addi- to mediate durable tumor regressions in some patients may
tional factors, such as the expression levels of PD-1 ligand on depend in part upon the fact that many TIL populations con-
tumors or normal tumor-infiltrating cells (reviewed in (35)), are tain T cells that recognize multiple neoantigens. Targeting
likely to play important roles in modulating clinical responses single neoantigens derived from trunk mutations (40) or driver
to immune checkpoint blockade. mutations that are essential for carcinogenesis or metastasis
may represent the most effective strategy, although defining
driver mutations for some tumors remains a challenge.
Summary and future perspectives
Common hotspot driver mutations, such as the KRASG12D
Overall, the results of adoptive immunotherapy and check- mutation that is expressed by approximately 45% of pan-
point blockade studies suggest that neoantigen-reactive creatic adenocarcinoma (41) and 13% of colorectal
 Neoantigens in cancer immunotherapy  369
adenocarcinomas (42) and for which HLA-C*08:02-restricted 11 van der Bruggen, P., Stroobant, V., Vigneron, N. and Van den
T cells have been identified represent attractive targets for Eynde, B. 2013. Peptide database: T cell-defined tumor antigens.
Cancer Immun. Available at: http://www.cancerimmunity.org/
the development of TCR-based therapies. The HLA-C*08:02 peptide/
allele is expressed by nearly 10% of patients bearing tumors 12 Robbins, P. F., Lu, Y. C., El-Gamil, M. et  al. 2013. Mining

commonly containing KRASG12D mutations, which would allow exomic sequencing data to identify mutated antigens recog-
treatment of a relatively small percentage of patients with nized by adoptively transferred tumor-reactive T cells. Nat.
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13 Toebes, M., Coccoris, M., Bins, A. et al. 2006. Design and use of
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of KRASG12V and KRASG12D epitopes in the context of HLA- 14 van Rooij, N., van Buuren, M. M., Philips, D. et  al. 2013. Tumor
A*11:01 (43), an HLA class  I  allele expressed by approxi- exome analysis reveals neoantigen-specific T-cell reactivity in an
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15 Lu, Y. C., Yao, X., Crystal, J. S. et  al. 2014. Efficient identi-
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