Introduction
Metastatic lesions of renal cell
carcinoma (RCC) occasionally regress spontaneously following removal of the
primary disease, though this is exceptionally rare. This is thought to
represent an immune response, and RCC, like melanoma, has thus been considered
to be immunogenic. Although very few urologists have been fortunate enough to
experience such a rare event, these anecdotes suggest that RCC may be
immunogenic in nature. Before the era of molecular targeting drugs, cytokine
therapy with interferon (IFN) or interleukin-2 (IL-2) was widely used, and
partial response (PR) and complete response (CR) were achieved in 15.9% and
1.8%, respectively, of patients with advanced RCC treated with IFN (1). Despite
the low response rate, it is noteworthy that cytokine therapy can achieve a CR
or cure in patients with advanced metastatic RCC.
Furthermore, even though prostate
cancer, unlike RCC, is not generally described as immunogenic, we experienced a
patient with castration-resistant prostate cancer (CRPC) with bone metastases
who maintained CR for a long period after hormonal manipulation and
chemotherapy with docetaxel. In another case of CRPC, the tumor was reported to
vanish spontaneously during therapy (2). These outcomes could be attributed to
an immune response. These rare but important clinical observations suggest that
specific cancer immunity can be triggered by certain conditions, and that
cancer immunity has the potential to achieve a cure in some cases. Immunotherapy
has thus been a focus of research for advanced RCC, even in the era of
molecular targeting drugs.
History of therapies for RCC
The history of therapies for RCC is
summarized in Table 1 (3). IFNα and
IL-2 were approved in Japan in 1987 and 1999, respectively, and
reduced-intensity stem cell transplantation (RIST) was reported by Child et al.
(4) in 2000. This ‘mini-transplantation’, aimed to damage tumor cells via a
graft versus host reaction, was expected to demonstrate good clinical efficacy;
however, the results in Japan have been disappointing (5).
Identification of the von Hippel-Lindau
(VHL) tumor suppressor gene in 1993 (6) and subsequent discovery of a high rate
of VHL gene mutations in patients with sporadic RCC (7) formed the theoretical
basis for molecular targeting drugs for advanced RCC, such as tyrosine kinase
and mammalian target of rapamycin inhibitors. The VHL gene product functions as
an ubiquitin ligase targeting hypoxia-inducible factor-1α (HIF-1α). HIF-1α
levels are regulated by its degradation in the ubiquitin/proteasome system.
Excess HIF-1α caused by malfunctioning of the VHL protein results in
up-regulation of vascular endothelial growth factor (VEGF) and glucose
transporter-1, which are responsible for the characteristic RCC phenotypes of
hypervascularity and clear cytoplasm on hematoxylin and eosin staining,
respectively.
Molecular targeting drugs target the
cascade mediated by VHL protein, and several such agents, including sorafenib,
sunitinib, axitinib, temsirolimus, and everolimus, have been approved in Japan
since 2008.
Therapeutic approaches for RCC: immunotherapy and
molecular targeting drugs
The extremely rare spontaneous
regression of metastatic lesions following surgical removal of the primary
lesion, e.g., by nephrectomy, is thought to be caused by an immunologic
response. This characteristic of RCC and the consequent notion that RCC is
immunogenic, form the basis for non-specific immunotherapy with cytokines,
which has been employed for some time. However, molecular targeting drugs
currently form the mainstream of treatment for metastatic RCC. The use of IFNα
is currently limited to lung metastases. In this review, we consider recent
progress in immunotherapy for RCC and discuss future prospects with regard to
the role of immunotherapy as a mainstream therapy for RCC.
Overview of cytokine therapy for advanced,
metastatic RCC
Urologists have continued to treat
patients with advanced metastatic RCC using IFNα for the past 20 years.
Although a previous study reported response rates to IFNα as low as 15.9% and
1.8% for PR and CR, respectively (1), it is noteworthy that cytokine therapy
resulted in some instances of CR, which were not achieved with molecular
targeting drugs. Furthermore, a Japanese multicenter study reported a survival
benefit of cytokine therapies in RCC patients with lung metastases (8).
Cytokine therapy may thus be generally beneficial in many patients with RCC.
The combination of IFN and IL-2 resulted in response rates of 35.7% and 4.8%
for CR+PR and CR, respectively, indicating further possibilities of cytokine
therapy (9).
The anti-tumor mechanisms of IFN
involve activation of macrophages and monocytes, enhancement of natural killer
(NK) cell activities, induction of antigen presentation on the cell surface,
and enhancement of cytotoxic T lymphocyte (CTL) activities (10). Although IL-2
enhances the activities of NK cells, B cells and T cells, including CTLs, it
also temporarily activates regulatory T cells (Tregs) via the IL-2 receptor
α-chain on Tregs, and may thus suppress CTL activities with possible
unfavorable effects.
Peptide-based vaccines
Peptide-based vaccines represent a
rational approach to inducing cancer-specific immunity against cancer antigens.
Tumor-associated antigens (TAA) are incorporated into antigen-presenting cells
(APCs), broken down into pieces, processed, and presented on HLA class I (MHC
class I) in the form of peptides. The presented peptide antigens stimulate CD8+
effector cells (CTLs) that specifically recognize the antigen and attack cancer
cells bearing the TAA. The amino acid sequences of the peptides within TAA
sequences have been investigated with the aim of producing peptide-based
vaccines. Candidate peptides capable of binding HLA class I are synthesized and
used to stimulate peripheral blood mononuclear cells in vitro to induce
antigen-specific CTLs. Peptides capable of inducing CTLs with high, specific
cytotoxic activity toward cancer cells are then selected for use as vaccines.
These peptides, mixed with adjuvant, are presented on HLA class I of APCs
following injection, resulting in lymphocyte stimulation and induction of tumor
antigen-specific CTLs.
Regarding the clinical efficacy of
peptide-based vaccines for RCC, Uemura et al. (11) reported three PR and six
stable diseases (SD) among 23 patients with metastatic disease in a phase I
trial of carbonic anhydrase 9-derived peptides. Limited efficacy (SD) was
achieved following immunotherapy using Wilms’ tumor 1 peptide in two of three
RCC patients enrolled in one study (12). Hypoxia-inducible factor prolyl
hydroxylase 3 (HIFPH3) was shown to be over-expressed in primary RCC tissues
and many RCC cell lines, and an HIFPH3-derived peptide induced CTLs in three of
six RCC patients, though no clinical trials have been reported (13).
A phase II randomized, clinical trial
of IMA901, a mixture of multiple TAA-derived peptides, showed that Tregs were
reduced by a single dose of cyclophosphamide, and patients responded
immunologically to IMA901 had longer overall survival (14). A phase I clinical
trial of human VEGF receptor 1-derived peptide vaccines in patients with
metastatic RCC demonstrated PR in two and SD for more than 5 months in eight of
18 patients (15).
Peptide-based vaccine therapy emulates
naturally occurring antigen presentation, and targets specific cellular
immunity against cancer cells using TAA-derived peptides. Peptide vaccine
therapy is thus rational with the potential for high efficacy. However, it has
so far demonstrated limited clinical effects in patients with metastatic RCC,
probably because of the lack of defined, appropriate antigens.
Innate and acquired immune systems in cancer
immunotherapy
The innate immune system seems to play
an important role in the initial stages of anti-cancer immunity. Concerning the
implications of innate immunity for immunotherapy of RCC, several reports have
focused on NK cells. Combination therapy with IFNα and IL-2 has been reported
to enhance the cytotoxic activities of NK cells in patients with advanced RCC
(16), and NK cells have been shown to be necessary for anti-tumor activities in
RCC patients treated with cytokines (17). Furthermore, low numbers of
peripheral NK cells (NK-Kir+) correlated with shorter disease-free survival in
patients with RCC (18).
NKT cells also contribute to
anti-cancer immunity, and a lack of these cells is associated with impaired
anti-cancer immunity (19). The exogenous NKT cell ligand α-galactosylceramide
was tested in a clinical trial for lung cancer and head and neck cancer (20).
NKT cells are activated as follows: Toll-like receptors activate dendritic
cells to produce IL-2. Stimulation by IL-2 and recognition of a self-ligand
presented on CD1d of APCs then cause activation of NKT cells, which in turn
activate specific helper T cells, together with CD8+ CTLs. The NKT cell system
thus acts as a functional bridge between the innate and acquired immune systems
in the development of specific immunity. The innate immune system, represented
by the NK and NKT systems, thus provides an initial step in anti-cancer
immunity eventually leading to acquired and cancer-specific immunities.
It is likely that the development of
specific and effective immunity against RCC could achieve a cure, though no effective
means of achieving this goal have yet been identified.
Galectin 9 and PINCH: isolation and evaluation of
new tumor antigens for peptide-based vaccine therapies
Despite a low response rate, cytokine
therapy can offer an effective therapeutic option for advanced RCC. Survival
benefit of cytokine therapy has been reported in patients with metastatic RCC
(8), and the observation that cytokine therapy can induce a CR in a very
limited number of patients with metastatic RCC indicates the curative potential
of immunotherapy. The key to understanding the successful induction of
anti-cancer immunity involves knowing which antigens are important and how
specific immunity is acquired in those few patients who respond well to
cytokine therapy. It is also important to establish if the mechanisms of
specific immunity in those successful cases are applicable to RCC patients in
general. If so, general methods for inducing cancer immunity based on these
specific cases may provide effective and potentially curative therapies for
patients with advanced RCC. This could reduce the need for expensive, molecular
targeting drugs, thus helping to limit the expanding medical expenditure in
Japan.
We investigated ‘true cancer antigens’
by screening an RCC expression library using sera from patients with metastatic
disease who responded well to cytokine therapy as probes, under the assumption
that these responders had acquired specific antibodies against RCC together
with specific cellular immunity. We identified two novel genes, galectin 9 and
PINCH, as RCC-specific antigens that were specifically highly expressed in all
the tested clear cell carcinoma samples, compared with normal renal tissues
(21). Using peptides derived from these two antigens, we induced
HLA-A*2402-restricted CTL clones and HLA-A*0201-restricted CTLs with high
antigen-specific killing activities toward RCC cells (21) (Figure 1). These antigens seemed to be closely related to an
immune escape mechanism, cancer cell survival, and metastases of RCC.
Figure 1.
Antigen-specific, HLA-A*2402-restricted cytotoxicities of CTLs induced with
galectin 9-derived (A) and PINCH-derived peptides (B). A, B. Peripheral blood
mononuclear cells from healthy volunteers (HLA-A*2402) were stimulated with
galectin 9-derived and PINCH-derived peptides, respectively, to induce CTLs.
The cytotoxicity of the CTLs toward RCC cells was assessed by a 51Cr-release
assay (21).CTLs induced by both peptides showed high cytotoxic activity toward
TUHR-10TKB RCC cells (HLA-A*2402+, galectin 9+, PINCH+) and SKGIIIa uterine
cancer cells (HLA-A*2402+, galectin 9+, PINCH+). CTLs were not active toward
cells with different HLA-A or negative antigens. Bars represent means +/- standard
errors of assays performed in triplicate. A. CTLs induced by the galectin
9-derived peptide exhibited cytotoxicity toward OCUU-1 RCC cells (HLA-A*0206+,
galectin 9+), implying less stringency in terms of HLA-A for the CTLs used.
Galectin 9 modulates cellular immunity
by suppressing excess immune reactions such as allergies. Its receptor is T
cell immunoglobulin and mucin domain 3 (TIM-3), located on the surface of T
cells. Galectin 9 causes apoptosis of activated T cells through TIM-3 (22, 23),
which is an immune checkpoint molecule, as are programmed cell death protein-1
(PD-1) and CTL-associated protein 4 (CTLA-4) (24). These immune checkpoint
molecules mediate immunosuppressive signals causing inactivation of T cells.
Anti-cancer drugs targeting these immune checkpoints are currently under
development, and have attracted considerable attention and expectations (25).
PINCH contributes to apoptosis
resistance in cancer cells (26) and promotes epithelial-mesenchymal transition
in renal tubular cells (27), and thus plays an important role in cancer
survival and metastases.
Galectin 9 and PINCH produce favorable
environments for RCC cells and therefore provide promising targets for
immunotherapy. Our method of identifying potentially useful cancer antigens was
unique in that we screened for antigens with central roles in developing
specific anti-cancer immunity by reacting them with sera from responders to
cytokine therapy. The abilities of the identified antigens to induce CTLs with
specific, high cytotoxicity was then evaluated. This method involved basic
molecular cloning techniques using clinical samples, and was developed by a
urologist with extensive experience, including many clinical cases of RCC (21).
Peptide-based vaccines have the problem
that various peptide sequences of the same antigen need to be prepared in
accordance with the HLA types of the patients. TAAs are presented on HLA class
I in a form of peptides that comprise part of the whole antigen: different
peptides derived from the same whole antigen associate with HLA class I of
different HLA types. The binding motif of the antigen for each HLA type should
be predicted in silico, and the ability of each predicted peptide to induce
CTLs by stimulating lymphocytes of the same HLA type needs to be tested.
We previously identified galectin 9-
and PINCH-derived peptides that induced HLA-A*02-restricted CTLs,
HLA-A*24-restricted CTLs, and HLA-A*33-restricted CTLs (28), respectively, all
of which exhibited specific and highly cytotoxic activities toward RCC cells.
The frequency of HLA-A*02 is high in Europe and North America, while HLA-A*24
is a major HLA type, and HLA-A*33 is also common in Japan. These peptides thus
represent important candidates for future peptide-based vaccine therapies.
New anti-cancer drugs targeting immune checkpoint
molecules
Cancer cells have recently been shown
to exploit immune checkpoint molecules that mediate the suppression of immune
signals, thus facilitating escape from the immune surveillance system. These
checkpoint molecules, including CTLA-4, PD-1, programmed death ligand 1
(PD-L1), and TIM-3, mediate T cell suppression. Drugs (antibodies) targeting
these checkpoint molecules, referred to as checkpoint inhibitors, including
antibodies against CTLA-4, PD-1, and PD-L1, have been developed (25) and in
some cases approved (Table 2).
Clinical studies of an anti-PD-1
antibody (nivolumab; BMS-936558) (29, 30) and a humanized monoclonal antibody
against PD-L1 (MPDL-3280A) (30) have been reported in patients with RCC. A
response rate (PR + CR) of 27% was achieved with nivolumab in patients with RCC
(29).
Increasing attention has been paid to
TIM-3 as a target (24). TIM-3 binds to its ligand galectin 9, causing
suppression of activated T cells (22, 23). Combined therapy with anti-TIM-3,
anti-PD-1, and anti-CTLA-4 antibodies demonstrated an additive anti-tumor
effect in experimental animal models (31). Our study suggested that galectin
9-derived peptides would induce CTLs that attack RCC, and also remove the
immunosuppressive environment caused by activation of the immune checkpoint
TIM-3.
Summary
Progress in molecular biology following
the discovery of the VHL gene has resulted in the development of various
molecular targeting drugs and advancements in therapies for advanced RCC. On
the other hand, following on from the long-standing use of cytokines such as
IFN and IL-2, immunotherapy is about to enter a new era represented by immune
checkpoint inhibitors. However, the costs associated with the clinical study,
development, and approval of these immune checkpoint inhibitors, marketed as
humanized antibodies, are likely to be enormous.
Peptide-based vaccine therapy aims to
induce specific immunity, and appears to offer potential survival benefits and
cure; however, their efficacy is currently poor. Innate immunity provides an
initial step leading to specific anti-cancer immunity, and non-specific immune
reactions initiated by cytokines may also cause specific anti-tumor immunity.
The combination of ‘real’ cancer antigens and new, immune-activating agents
targeting molecules such as immune checkpoints may thus induce strong
cancer-specific immunity.
Cytokine therapy is highly effective in
a very limited number of patients, indicating the high potential of
immunotherapy. However, the mechanisms responsible for the development of
immunity in these patients with RCC remain unclear. We identified novel RCC
antigens that reacted with sera from cytokine-therapy responders. We
successfully induced antigen-specific, HLA-restricted CTLs with high activities
by stimulating lymphocytes with peptides derived from those antigens. However,
the mechanisms responsible for the effects of the cytokines in particular cases
are unknown. Further, detailed investigations of successfully treated cases and
analysis of the general mechanisms are likely to lead to promising new
therapies for RCC.
Conflict of interest
The authors declare that they have no
competing interests.
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