Cancer immunotherapy: Co-stimulatory agonists and co-inhibitory antagonists

Article · August 2009with51 Reads
DOI: 10.1111/j.1365-2249.2009.03912.x · Source: PubMed
Abstract
The generation and maintenance of immune responses are controlled by both co-stimulatory and co-inhibitory signalling through T cell co-receptors, many of which belong to the immunoglobulin-like superfamily or the tumour necrosis factor receptor superfamily. Agonistic or antagonistic monoclonal antibodies targeting these co-receptors have the potential to enhance immunity. Furthermore, their activity on the immunosuppressive regulatory T cell populations which are prevalent within many tumours provides an additional rationale for their use as anti-cancer therapies. This review summarizes the interactions between cancer and the immune system, highlighting the ways in which these new classes of immunostimulatory antibodies might enhance anti-tumour immunity and summarizing early clinical experience with their use.
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Cancer immunotherapy: co-stimulatory agonists and
co-inhibitory antagonistscei_3912 9..19
K. S. Peggs,* S. A. Quezada†‡ and
J. P. Allison†‡
*Department of Haematology, UCL Cancer
Institute, Paul O’Gorman Building, University
College London, 72 Huntley Street, London, UK,
and Ludwig Center for Cancer Immunotherapy,
Howard Hughes Medical Institute, and
Department of Immunology, Memorial
Sloan-Kettering Cancer Center, 1275 York
Avenue,NewYork,NewYork,USA
Summary
The generation and maintenance of immune responses are controlled
by both co-stimulatory and co-inhibitory signalling through T cell co-
receptors, many of which belong to the immunoglobulin-like superfamily or
the tumour necrosis factor receptor superfamily. Agonistic or antagonistic
monoclonal antibodies targeting these co-receptors have the potential to
enhance immunity. Furthermore, their activity on the immunosuppressive
regulatory T cell populations which are prevalent within many tumours pro-
vides an additional rationale for their use as anti-cancer therapies. This review
summarizes the interactions between cancer and the immune system, high-
lighting the ways in which these new classes of immunostimulatory anti-
bodies might enhance anti-tumour immunity and summarizing early clinical
experience with their use.
Keywords: cancer immunotherapy,co-inhibition,co-stimulation,CTLA-4,
regulatory T cells
Accepted for publication 6 February 2009
Correspondence: K. S. Peggs, Department of
Haematology, UCL Cancer Institute, Paul
O’Gorman Building, University College
London, 72 Huntley Street, London WC1E 6BT,
UK.
E-mail: k.peggs@cancer.ucl.ac.uk
Cancer and the immune system
The immune surveillance hypothesis posits that the immune
system plays a key role in suppressing tumour growth and
that the incidence of cancer would be much greater were it
not for the ability of the immune system to identify and
eliminate nascent tumour cells. Widespread acceptance of
this concept was hampered for many years by a lack of firm
supportive experimental evidence until the turn of this
century, when a series of influential papers demonstrated
that lymphocytes and interferon-gco-operate to inhibit the
development of spontaneous and carcinogen-induced
tumours in mice engineered genetically to lack a functional
immune system [1–4]. While the immune system appears
capable of eliminating or containing early tumour growth,
some tumour cells escape detection and eventually cause
cancer. It is hypothesized that selective pressure exerted by
the immune system drives the cellular composition of these
tumours to become serially less immunogenic (immuno-
editing), as demonstrated by the finding that tumour cells
from immunodeficient mice are more immunogenic than
those from immunocompetent mice [1].
Immunoediting may be considered to consist of three pro-
cesses occurring either independently or sequentially [5].
First, ‘elimination, in which immunity functions as an
extrinsic tumour suppressor; secondly, ‘equilibrium’, in
which cancerous cells survive but are held in check by the
immune system [6]; and thirdly, ‘escape’, in which tumour
cell variants with either reduced immunogenicity or the
capacity to attenuate or subvert immune responses grow into
clinically apparent cancers [7]. The changes occurring in the
escape phase may be considered broadly as those intrinsic to
the tumour cells themselves, including enhanced resistance
to apoptosis and down-regulation of co-stimulatory ligands,
and those involving the local tumour microenvironment.
These mechanisms are neither mutually exclusive nor
entirely separable. Anti-tumour responses may be frustrated
by regulatory mechanisms which normally act to limit
T cell responses following chronic exposure to antigen [e.g.
up-regulation of cytotoxic T lymphocyte-associated-antigen
4 (CTLA-4) or programmed cell death-1 (PD-1) receptors],
or by tumour-induced subversion of other regulatory path-
ways [e.g. expression of T cell inhibitory molecules such as
PD-ligand 1 (PD-L1), B7-H3 or B7x, or accumulation of
immunosuppressive T cell or antigen-presenting cell (APC)
populations]. Further proposed mediators of local immune
suppression include soluble suppressive factors elaborated
by the tumour or parenchyma such as interleukin (IL)-10
or transforming growth factor (TGF)-b, and indoleamine
2,3-dioxygenase (IDO) expressed by tumour cells or IDO-
competent APCs, which may cause both direct suppression
of T cells and enhancement of local regulatory T cell-
Clinical and Experimental Immunology REVIEW ARTICLE doi:10.1111/j.1365-2249.2009.03912.x
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mediated suppression [8]. The presence of an array of other
cell types capable of actively suppressing immune reactions,
such as CD4+CD25+FoxP3+regulatory T cells (Treg), IL-10-
secreting regulatory T cells, CD1d-restricted natural killer
(NK) T cells, immature and plasmacytoid dencritic cells
(DCs) (iDCs and pDCs) and myeloid-derived suppressor
cells within the tumour or tumour-draining lymph nodes is
clearly critical to induction and/or maintenance of local
immune privilege in a number of systems [9]. Such cells may
be recruited preferentially to these sites, or expanded or
induced therein.
The apparent confirmation of the validity of the immune
surveillance hypothesis led to great enthusiasm for the devel-
opment of immune-based anti-cancer therapies. On the
basis of growing evidence that tumours express antigens that
can be presented by professional APCs to induce the genera-
tion of tumour-specific cytotoxic T lymphocytes (CTLs),
tumour immunotherapists aimed to parallel the successes
achieved in developing vaccines for infectious diseases. Strat-
egies included vaccination with peptide, DNA or antigen-
pulsed DCs, either alone or coupled with approaches based
on directly enhancing effector number or function by adop-
tive transfer of tumour-reactive T cells.However,attempts to
target human cancers have been significantly less successful
than was initially envisaged possible. While resulting in some
impressive responses it is perhaps, in hindsight,unsurprising
that, given the multitude of locally immunosuppressive
mechanisms engaged within an actively growing tumour,
attainment of clinically significant responses are rare even
with therapies that succeed in inducing systemic immunity.
The presence of large numbers of T cells capable of recog-
nizing tumours is not singularly sufficient to mediate
tumour regression, as evidenced by unrestricted tumour
growth in T cell receptor (TCR) transgenic mice in which all
the T cells are capable of recognizing the tumour antigen
[10]. Clinical studies of active immunization have shown
that despite expansion of tumour-reactive T cells to levels of
up to 40% of the circulating CD8+T cell repertoire, tumour
growth may continue apparently unimpeded [11]. There is
now ample experimental evidence that functional systemic
anti-tumour activity may not translate into tumour rejec-
tion, either because of lack of infiltration of T cells into the
tumour [12] or because of local suppression of function
within the tumour microenvironment. Although tumour-
specific immunity is compromised in tumour-bearing mice,
there is often no generalized immune deficiency [13], indi-
cating that tumours can suppress specifically the induction
of effective anti-tumour immunity. This concept is perhaps
highlighted most clearly by concomitant immunity, wherein
a mouse injected with a tumour will reject a subsequent
challenge with the same tumour at a distant site, despite
continued growth at the site of initial challenge [14]. Because
tumours share many similarities with chronic pathogens
such as Mycobacterium tuberculosis, the challenges of deliv-
ering effective vaccines or immunotherapies are aligned
much more closely with those associated with therapy of
these established chronic infections than with acute infec-
tious pathogens, in which the majority of the successes have
come with prophylactic vaccination strategies. Addressing
the factors conferring local immune privilege will probably
prove critical to improving outcomes following anti-cancer
immunotherapies.
Monoclonal antibodies in cancer immunotherapy
Monoclonal antibodies have revolutionized the treatment of
a number of malignancies. Their mechanisms of action vary,
but can be categorized broadly as direct or indirect. The
former include blockade of function (e.g. hindering ligand
binding, increasing internalization of receptors) and stimu-
lation of function (e.g. inducing apoptosis) on the tumour
cells. In addition, monoclonal antibodies can be used to
target local delivery of conjugated therapeutics such as
toxins or radioisotopes. The indirect mode of action is
mediated by the immune system and includes the acti-
vation of complement-dependent cytoxicity and both
complement-dependent and antibody-dependent cellular
cytotoxicity involving macrophages and NK cells,effectors of
the innate immune system. The development of antibodies
that can interfere with the function of co-stimulatory and
co-inhibitory pathways on effector T cells has provided a
novel mechanism for indirect anti-cancer activity. The
primary targets for such interventions were envisaged ini-
tially to be the effector T cells, but it is becoming clear that
regulatory T cell populations might also be important targets
for their overall activity. A number of regulatory CD4+Tcell
subtypes are recognized, including those which are produced
by the thymus, express CD4, CD25, 4-1BB, OX40,
glucocorticoid-induced tumour necrosis factor receptor
(TNFR) family-related gene (GITR) and CTLA-4, and
appear crucially dependent upon the expression of the fork-
head box P3 transcription factor (FoxP3) for their develop-
ment (so-called ‘naturally occurring’ Treg,nT
reg); and those
which arise from naive CD4+T cells as a result of ‘tolero-
genic’ encounters in the periphery. The latter ‘inducible’ Treg
include IL-10-producing, FoxP3-negative Tr1 cells, TGF-b-
producing Th3 cells and extra-thymically generated
CD4+CD25+FoxP3+iTreg cells. The acquisition of regulatory
phenotype and suppressive functions by conventional non-
regulatory CD4+T cells following exposure to antigens under
certain conditions is now recognized as a major contributor
to the maintenance of T cell homeostasis and control of
inflammation. Characterization of the conditions that drive
such peripheral conversion is ongoing, but factors such as
suboptimal antigen stimulation in combination with TGF-b
appear to be important, both of which are likely to be rel-
evant within the tumour microenvironment [15]. IDO pro-
duced by either tumour cells or parenchyma (eg. pDCs) also
favours conversion [16]. The dominant inhibitory potential
of Treg cell populations in murine models of malignancy is
K. S. Peggs et al.
10 © 2009 British Society for Immunology, Clinical and Experimental Immunology,157: 9–19
well established [17], and more recently their potential role
in human malignancies has been demonstrated [18]. Their
relative abundance predicts for tumour outcomes in murine
models [19], and correlates inversely with outcomes in
several epithelial carcinomas [20]. Intriguingly, in haemato-
logical malignancies this association is reversed and high
levels of Treg appear to confer improved prognoses [21,22].
Because the targets of many cancer vaccination strategies are
self-antigens, it is perhaps no surprise that ‘therapeutic’
cancer vaccines can induce amplification of tumour-specific
Treg [23].
Promoting T cell function via modulation of
co-stimulation or co-inhibition
Immune activation is regulated by two major families of
co-receptors expressed by T cells: the immunoglobulin-like
(Ig) superfamily and the TNFR superfamily [24,25] (Fig. 1).
Co-stimulatory members of the former include CD28 and
inducible T cell co-stimulator (ICOS), while OX40, CD27,
4-1BB, CD30, GITR and herpes-virus entry mediator
(HVEM) are members of the latter. CTLA-4 and PD-1 are
the most well-established inhibitory members of the Ig
superfamily. The B and T lymphocyte attenuator (BTLA) is
the most recently described inhibitory member of this family
[26]. The identities of the receptors for the newer B7 ligands
(B7-H3 and B7x/B7-H4) remain elusive, but these receptors
may also mediate significant inhibitory activity, perhaps
more so in the periphery, given the tissue distribution of the
ligands. Stimulatory or blocking monoclonal antibodies are
being investigated extensively for their abilities to enhance T
cell numbers, function and maintenance of immunological
memory [27,28].
Stimulatory antibodies to 4-1BB (CD137), OX40
(CD134) and GITR
The TNFR family members are appealing candidates for the
development of targeted therapeutics. The greatest attention
has been focused upon 4-1BB and OX40. 4-1BB is expressed
on activated T cells (including Treg and NK T cells), activated
NK and dendritic cells, eosinophils, mast cells and endothe-
lial cells in some metastatic tumours [25]. Its ligand 4-1BBL
is expressed on activated DCs, B cells and macrophages.
Ligation on T cells results in up-regulation of anti-apoptotic
genes and protection from activation-induced cell death
(AICD), enhancing establishment of durable memory CTLs
[29]. The up-regulation of 4-1BB on antigen-experienced T
cells suggests that anti-4-1BB may target these primed T cells
differentially, influencing those T cells preferentially with
highest avidity receptors and explaining partially why 4-1BB
co-stimulation may be superior to CD28 co-stimulation for
the generation of antigen-specific cells for adoptive therapies
[30]. While it is assumed that co-stimulation of CD8+Tcells
is the principal mechanism of action of anti-4-1BB, various
other immunomodulatory activities may contribute. In this
respect, a common theme developing in our understanding
of the function of immunostimulatory antibodies is their
possible multiplicity of function, reflecting the cellular dis-
tribution of the receptors. Thus, (i) activation of APCs; (ii)
reductioninT
reg suppressive capacity or enhancement of
effector resistance to suppression; and (iii) co-stimulation of
CD4+and CD8+T cells are all supported by experimental
data [31,32]. Furthermore, activated NK cells and NK T cells
may be relevant targets for anti-tumour activity [33,34].
Reverse signalling into cells expressing the ligands for a
number of Ig or TNFR superfamily members is another
recurring theme of recent investigations into immune
modulating functions of these molecules. In the case of
4-1BBL, this may result in enhanced production of inflam-
matory mediators or enhanced cell adhesion, facilitating
egress of immune effectors into sites of inflammation
[35,36]. There are conflicting data as to whether 4-1BB liga-
tion on Treg results in enhanced or reduced suppressive
capacity [31,37,38], and synergy of anti-tumour activity
with approaches that are thought to target Treg number or
function has been taken as evidence that any 4-1BB-
mediated inhibitory effects on Treg function may be relatively
modest [39]. Forced expression of 4-1BBL in murine
tumours enhances immunogenicity, reducing engraft-
ment rates in immune-competent recipients, although
growth of untransfected cells is affected only modestly in
relatively poorly immunogenic tumours [40]. Agonistic
anti-4-1BB monoclonal antibodies enhance anti-tumour
Fig. 1. Potential immunomodulatory targets for monoclonal antibody
therapy. In the case of effector T cells, agonistic antibodies directed
towards co-stimulatory pathways and antagonistic or blocking
antibodies directed towards co-inhibitory pathways can directly
enhance effector numbers, functions or persistence. The situation with
respect to regulatory T cell populations remains less clear, but it
appears likely from the current literature that agonists of classic
co-stimulatory molecules may abrogate function, whereas blockade of
cytotoxic T lymphocyte-associated-antigen 4 may also reduce
suppressor function.
Immunostimulatory antibodies in cancer immunotherapy
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© 2009 British Society for Immunology, Clinical and Experimental Immunology,157: 9–19
CTL responses, enabling rejection of established syngeneic
tumour cell lines [41–43]. Activity appears critically depen-
dent upon CD8+T cells and (in most studies) NK cells,
with the role of CD4+T cells varying in different tumour
models [34,42–44]. As with other immunostimulatory anti-
bodies, combination with vaccination strategies enhances
activity in poorly immunogenic tumour models [45,46].
Co-administration with transgenic tumour-specific CTL
enhances anti-tumour activity, apparently via a reduction in
AICD rather than enhanced proliferation [47]. Early experi-
ence with a humanized clinical grade antibody (BMS-
663513) in patients with melanoma and renal cell carcinoma
suggests that the antibody is well tolerated with some
evidence of activity (6% partial responses in melanoma
patients), demonstrating the probable need to evaluate
combinatorial approaches [39,48–50]. Two particularly
intriguing and seemingly paradoxical features of anti-4-1BB
monoclonal antibodies are their ability to ameliorate
autoimmunity and to suppress humoral immunity in mice
[51–53]. While this suppressive activity has been proposed to
be advantageous in terms of limiting possible antibody-
mediated toxicities, it is recognized that it might also be
deleterious to the development of optimal anti-cancer
immunity.
OX40 is expressed transiently on activated CD4+and
CD8+T cells, functioning as a late co-stimulatory receptor
[25,54]. It is also expressed by NK T cells and Treg. Its ligand
OX40L is expressed in a similar distribution to 4-1BBL, on
activated DCs, B cells and macrophages, as well as activated
T cells and endothelial cells [25]. OX40 ligation regulates
CD4+and CD8+T cell survival and memory generation,
preventing T cell tolerance [55–58]. It also impairs the sup-
pressor functions of Treg [59,60], allowing tumour-resident
DCs to traffic to draining lymph nodes to prime tumour-
specific CD8+CTL [61]. Furthermore, OX40 triggering
appears to be antagonistic for FoxP3 induction in antigen-
responding naive CD4+T cells, effectively suppressing the
generation of iTreg [62,63], and blocks the generation of
IL-10-producing Tr1 cells [64], suggesting that OX40 may
antagonize the generation of a number of different inducible
Treg populations. 4-1BB and OX40 act independently to
facilitate robust CD8 and CD4 recall responses, overlapping
in their intracellular signalling pathways, yet neither 4-1BB
nor GITR signalling seem to block the generation of Tr1
[64], and there are currently no reports illustrating whether
they influence FoxP3+iTreg induction.As with 4-1BBL, forced
expression of OX40L by tumour cells increases immunoge-
nicity, with tumour rejection dependent upon both CD4+
and CD8+T cells [65]. Furthermore, intratumoural injection
of DCs modified to have enhanced expression of OX40L can
effect tumour rejection in murine models that are dependent
upon CD8+CTL responses, themselves dependent upon
CD4+T cells and NK T cells [66]. Agonistic anti-OX40 anti-
bodies increase anti-tumour activity in a number of trans-
plantable tumour models [67]. Concomitant activity on
both effector and regulatory compartments may be a pre-
requisite of effective rejection of established tumours [61]. In
preclinical models, OX40 ligation enhances several other
immunostimulatory approaches [68–71].
The GITR is also expressed transiently on activated T cells
[72,73], and expressed constitutively at high levels on Treg
with further induction following activation [74,75]. Its
ligand GITRL is expressed at low levels on B cells, macro-
phages and some DCs, increasing transiently following
activation. GITR ligation stimulates both proliferation and
function of CD4+and CD8+T cells [76]. Its activity on Treg
has remained more contentious [77–79]. Anti-GITR anti-
bodies reduce suppression in co-cultures of CD4+CD25
effectors and CD4+CD25+Treg, but whether this relates to
reduced Treg suppressor function, increased resistance of
effectors to the preserved suppressor function of Treg,ora
combination of both, has yet to be demonstrated definitively.
Experiments using mixtures of GITR+/+and GITR–/– effector
and regulatory cells suggest that ligation of GITR on the
effector population rather than the regulatory population is
critical for abrogating suppression [79], suggesting that
enhanced effector resistance to suppression may be key in in
vitro assays. Injection of adenovirus expressing recombinant
GITRL into B16 melanoma promotes T cell infiltration and
reduced tumour volumes [80], while agonistic anti-GITR
antibodies have been shown to enhance both rejection of
established methylcholanthrene-induced fibrosarcomas, and
to enhance systemic anti-tumour responses and concomi-
tant immunity when given following B16 melanoma chal-
lenge [81,82]. Furthermore, the same antibody also enhances
the impact of DNA-vaccination in terms of generation of
systemic immunity and enhancing resistance to challenge
with murine melanoma [83].
Stimulation through checkpoint blockade: CTLA-4
(CD152), PD-1 (CD279) and PD-L1 (CD274)
In contrast to the TNFR superfamily, the existence of
co-inhibitory receptors mediating direct down-regulation of
lymphocyte activation and/or effector function has been a
recognized feature of the Ig superfamily for some time.
Indeed, the co-inhibitory receptor-ligand members outnum-
ber the co-stimulatory members within this superfamily,
engendering the idea of regulatory or inhibitory checkpoint
blockade as a therapeutic anti-cancer strategy [84]. Blockade
of inhibitory immune checkpoints for therapeutic benefit
offers considerable promise, particularly as combination
with other treatment modalities that promote cross-priming
of anti-tumour immunity may yield additive or synergistic
activity. The strategy that is the most advanced in clinical
development involves antibodies which block CTLA-4 [28].
The CTLA-4 is expressed by activated CD4+and CD8+T
cells, although its surface expression is regulated tightly with
a short half-life. It influences some of the earliest events in T
cell activation, being mobilized rapidly from intracellular
K. S. Peggs et al.
12 © 2009 British Society for Immunology, Clinical and Experimental Immunology,157: 9–19
vesicles to the immune synapse after TCR engagement,but is
endocytosed promptly in the unphosphorylated state. It is
expressed constitutively by nTreg and iTreg, although the
majority is found intracellularly, even following activation.
CTLA-4 shares the B7-1 (CD80) and B7-2 (CD86) ligands
with CD28, a critical co-stimulatory molecule. Ligation of
CD28 in concert with TCR stimulation enhances T cell pro-
liferation by inducing production of IL-2 and anti-apoptotic
factors, decreasing the number of ligated TCR that are
required for a given biological response. CTLA-4 engage-
ment blocks augmentation of gene regulations by CD28-
mediated co-stimulation, and its function as a negative
regulator of CD28-dependent T cell responses is demon-
strated strikingly by the phenotype of CTLA-4 knock-out
mice, which succumb to a rapidly lethal polyclonal CD4-
dependent lymphoproliferation within 3–4 weeks of birth
[85,86]. CTLA-4 has significantly higher affinities for both
B7 ligands than does CD28. Accumulation of both receptors
at the synapse is influenced by ligand binding. CD28 is
recruited in the absence of B7-1 and B7-2 binding but is not
effectively stabilized there, and its localization can be dis-
rupted by CTLA-4. The latter is dependent more critically
upon ligand binding for concentration at the synapse.
CTLA-4 may, therefore, both out-compete CD28 for ligand,
particularly when ligand densities are limiting, and be able to
exclude CD28 from the immunological synapse by virtue of
the generation of extended high affinity lattices of alternat-
ing CTLA-4 and B7-1 homodimers [87]. For this reason the
tight spatial and temporal regulation of CTLA-4 expression
is likely to be critical for determining the outcome of CD28-
mediated signalling. Furthermore, CTLA-4 ligation induces
decreased production of cytokines (particularly IL-2, and its
receptor) and cell cycle arrest in G1, suggesting that ligation-
dependent mechanisms also contribute to its negative regu-
latory function. Finally, CTLA-4 has an important role in
Treg-mediated suppression, as evidenced by the recent dem-
onstration that Treg-specific CTLA-4 deficiency in condi-
tional knock-out (CKO) mice is associated with a profound
reduction in their suppressive capacity [88]. CKO mice
developed a lethal autoimmune lymphoproliferative syn-
drome with a slightly slower tempo than CTLA-4–/– mice.
The mechanism(s) by which CTLA-4 mediates these Treg-
associated effects remain(s) unclear, but may be dependent
upon reverse signalling into B7-expressing cells [89,90]. Fur-
thermore, Treg-mediated suppression during in vitro suppres-
sor assays is associated with reduced activation of APCs
(evidenced by reduced surface expression of B7 molecules
[88]).
Antibody-mediated blockade of CTLA-4 is particularly
effective at enhancing secondary immune responses, more
markedly in CD4+T cells. While often having only modest
effects as a monotherapy in preclinical tumour models of
poorly immunogenic tumours, anti-CTLA-4 synergizes with
a number of other anti-tumour immunotherapies [24,28].
Furthermore, early clinical studies have shown that CTLA-4
blockade has activity as a monotherapy (5–15% objective
response rates in melanoma and renal carcinoma) and, in
keeping with murine models, enhanced activity in combina-
tion with a number of other therapies in the treatment of
human malignancies including melanoma, renal, ovarian
and prostatic carcinomas [24,91–95]. More than 4000
patients have been treated to date with anti-CTLA-4 (ipili-
mumab or tremelimumab). Adverse immunological events
have been a feature of some of the early studies, often asso-
ciating with clinical responses, although they have generally
proved manageable and the majority reversible, allaying
some of the concerns that the use of therapeutics designed to
enhance immune reactivity non-specifically and to interfere
with tumour-induced tolerance might uncouple mecha-
nisms of self-tolerance systemically, resulting in uncon-
trolled autoimmunity. This is a theoretical concern for
agents inducing immunostimulation either by agonism of
co-stimulatory pathways, antagonism of co-inhibitory path-
ways or subversion of Treg-mediated suppression. The severe
toxicity experienced by normal volunteers receiving a ‘super-
agonistic’ co-stimulatory antibody directed towards CD28
(TGN1412) highlights the need for careful evaluation of
these new therapeutics [96], although other targets which do
not obviate the requirement for TCR signalling in inducing
T cell activation (a feature of super-agonists) will probably
have more favourable toxicity profiles, as is the case with
CTLA-4 blockade. The association between adverse immune
events and responses with anti-CTLA-4 is apparent across
tumour types. For example, in patients with enterocolitis,
response rates of 45% and 46% have been reported for meta-
static melanoma and renal cell cancer respectively [97].What
remains less clear is whether this association is an inevitable
outcome of the mechanism of action of this new class of
immunotherapeutics, or whether a narrow therapeutic
window exists in which beneficial anti-tumour activity can
be dissociated from adverse immune events, as has been
hinted in some studies.
The PD-1 is expressed by activated CD4+and CD8+T cells,
as well as B cells, monocytes and at lower levels on NK T
cells. It binds to two separate ligands, PD-L1 and PD-L2,
which exhibit distinct expression profiles [98]. PD-L1 is
expressed broadly, and can be detected on resting and acti-
vated T cells (including nTreg), B cells, macrophages, DCs and
mast cells. In addition, its expression on non-haematopoietic
cells may have physiological relevance, suggesting that inhi-
bition through PD-L1/PD-1 may not be restricted solely to
the interaction of T cells and professional APCs, but that it
may also occur during the effector phase of the immune
response in peripheral tissues, perhaps helping to prevent
immune-mediated tissue damage directly at the tissue
interface. By comparison, PD-L2 has a much more limited
expression profile. It is not expressed on naive or activated T
cells, but is instead restricted to activated macrophages,
myeloid dendritic cells and mast cells, suggesting that it
fulfils a role that differs from that of PD-L1. PD-1–/– mice
Immunostimulatory antibodies in cancer immunotherapy
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develop an array of autoimmune pathologies characterized
by high titres of autoantibodies, in keeping with a negative
regulatory effect on T and/or B cells [99,100]. PD-L1 and
PD-L2 may also regulate T cell responses through reverse
signalling. Cross-linking antibodies against PD-L2 directly
induce dendritic cells to produce immunomodulatory
cytokines such as IL-6 and tumour necrosis factor-a, at the
same time as protecting them from cell death [101,102].
Furthermore, PD-1-Ig inhibits DC activation and increases
IL-10 production independently of any influence on IDO.
PD-L1 binds B7-1 with an affinity intermediate between
those of CTLA-4 and CD28 for B7-1, allowing suppression of
T cell proliferation and cytokine production through either
B7-1 or PD-L1 signalling [103]. Thus, blockade of both
CTLA-4 and PD-L1 might eliminate simultaneously cell
intrinsic negative signalling through CTLA-4, B7-1, PD-L1
and PD-1, while favouring positive signalling through CD28.
Recent data highlight the relevance of this pathway to
chronic T cell responses to pathogens, wherein ‘exhausted’ T
cells demonstrate a selective up-regulation of PD-1 and
administration of anti-PD-L1 antibodies in vivo restores
their activity [104,105]. Together, these data suggest that
blockade of PD-1 and/or PD-L1 can restore functionality of
the T cell compartment and might be applied to enhance T
cell activity towards other chronic pathologies such as
cancer.
The PD-L1 is expressed by a variety of human and murine
tumours, and PD-1 is expressed by tumour-infiltrating lym-
phocytes, leading to the hypothesis that they may be impor-
tant in restricting intratumour effector T cell responses
[106]. In humans, myeloid DCs isolated from tumour or
lymph nodes from ovarian carcinoma patients express high
levels of PD-L1, and are capable of enhancing T cell activity
only following PD-L1 blockade [107]. Similarly, plasmacy-
toid DCs in tumour-draining lymph nodes produce high
levels of IDO which results in Treg activation, up-regulation
of PD-L1 on the DCs and negative regulation of T cell
responses [108]. PD-L1 expression has been correlated
directly with poor prognosis in a number of human cancers
[109–111]. Forced expression of PD-L1 on murine tumour
lines diminished T cell activation and tumour killing in vitro,
and enhanced tumour growth markedly in vivo, while anti-
PD-L1 antibodies blocked these effects [112,113]. In the 4T1
mammary carcinoma model PD-L1 is up-regulated in vivo
by the tumour, making it refractory to immunotherapy with
the anti-4-1BB antibody. Co-administration with anti-
PD-L1 resulted in dramatic tumour rejection [114]. Simi-
larly, anti-PD-L1 antibody delayed in vivo growth of PD-L1-
expressing murine myeloma cell lines. PD-L1 blockade has
also been shown to synergize with adoptive cellular therapy
to induce rejection of squamous cell carcinoma [113]. Fur-
thermore, adoptive transfer of PD1–/– tumour-reactive CD8+
T cells caused rejection of B16 melanoma, while neither
wild-type nor CTLA-4–/– tumour-reactive CD8+Tcellswere
capable of inducing rejection [115].
Very few studies have examined the ability of PD-1 block-
ade to directly promote anti-tumour T cell responses in vivo.
Anti-PD-1 antibodies reduce dissemination of B16 mela-
noma and CT26 colon carcinoma [116], and induce a small
but significant decrease in growth of murine pancreatic car-
cinoma [117]. A fully human anti-PD-1 monoclonal anti-
body has been developed recently and its ability to enhance
the function of human tumour-specific T cells has been
tested in vitro [118]. Vaccine-induced melanoma-reactive
CD8+T cells showed an increase in number and function
upon restimulation in vitro with the blocking anti-PD-1
antibody. There were no apparent effects on cell death, sug-
gesting that the augmented in vitro responses were due
mainly to increased proliferation of tumour-reactive T cells
rather than decreased cell death. While these results are
promising, a better understanding of the mechanisms
underpinning bidirectional regulation through PD-L1 and
B7-1 may help to inform future trials and perhaps to help
decide which molecule (PD-L1 or PD-1) makes the optimal
target for cancer immunotherapy.
Other inhibitory members of the Ig superfamily offer pos-
sible targets for co-inhibitory blockade, although the impact
such interventions would have on anti-tumour activity
remains more speculative at present. Thus, the as-yet uni-
dentified receptor for the B7x (B7-H4) ligand offers one such
possibility. The current literature on B7-H3 and anti-tumour
responses remains somewhat contradictory [24]. The BTLA
(CD272) is also a potential target, offering the unique
example of an Ig superfamily member whose ligand is a
member of the TNFR family (HVEM).
Redefining response criteria
The mechanisms underlying the anti-tumour activity of
immunostimulatory therapies are indirect, relying upon the
activation of tumour-reactive immune effector cells, and
contrasting with the direct activity of most conventional
chemotherapeutics. The kinetics of clinical responses may
therefore differ significantly, potentially taking longer to
become manifest. This issue is well illustrated by early expe-
rience with CTLA-4 blockade. While the development and
application of response evaluation criteria in solid tumours
(RECIST) has greatly enhanced objectivity in clinical trials
reporting, it is now clear that the tempo of responses with
anti-CTLA-4 is such that disease stabilization over several
months, or possibly even progression, may occur before the
development of a clinical response [119–121]. RECIST cri-
teria were not designed to allow for this pattern of response.
In a report summarizing the pooled experience from six of
the larger ipilimumab studies in stage III–IV melanoma
(including monotherapy and combination with peptide
vaccine, dacarbazine or IL-2), 46 of 356 (12·9%) patients
receiving 0·1–20 mg/kg in single or multiple doses achieved a
response [11 complete (CR) and 35 partial (PR) responses]
[119]. Many responses occurred later than is typical with
K. S. Peggs et al.
14 © 2009 British Society for Immunology, Clinical and Experimental Immunology,157: 9–19
cytotoxic agents. In 28 of 46 patients CR/PR was docu-
mented at time-points beyond 12 weeks from initiation of
treatment. In four of 46 patients (or 1·1% of the total
number treated) responses occurred following initial
progression. Delayed response onset occurred irrespective of
dose, regimen and therapeutic partner. The results suggest
that continued treatment/observation may be beneficial
despite initial progressive or stable disease. In addition, this
pattern does not appear to be confined to patients with mela-
noma, as similarly delayed responses have been reported
with renal cancer [120]. It is therefore important to accept
this new concept of delayed response assessment so that
clinical activity is not rejected prematurely and falsely.
Combinatorial immunotherapeutics
It is clear from preclinical models and early clinical experi-
ence that multi-modal approaches may be required to eradi-
cate poorly immunogenic tumours. The recent literature
demonstrates the potential for many combinations to give
synergistic or additive effects. Attempting to choose ratio-
nally which are likely to be the best approaches remains
challenging, but considering approaches under a number of
basic headings allows the identification of potentially attrac-
tive combinations. Thus, modern immunotherapeutic strat-
egies can be divided according to those which: (i) improve
antigen presentation or immunogenicity (e.g. vaccines, CpG
oligodeoxynucleotides); (ii) improve T effector function,
numbers or persistence directly (e.g. agonistic anti-TNFR
antibodies, cytokines); (iii) remove or disable immunologi-
cal checkpoints, either cell intrinsic or cell extrinsic (e.g.
CTLA-4 or PD-1 blockade, Treg depletion and possibly ago-
nistic anti-TNFR antibodies); (iv) ‘reset’ the system taking
advantage of proliferative advantages in a lymphopenic envi-
ronment (e.g. adoptive cellular therapy); and (v) improve
antigen specificity or TCR avidity for tumour antigens (e.g.
TCR gene therapies). It has also become apparent that some
agents bridge these categories, so the duality of enhancing
effector function and reducing suppression afforded by, e.g.
CTLA-4 blockade, or OX40 stimulation, may be achieved
with one agent. Because recent data highlight the ability of
regulatory checkpoints to limit the efficacy of any directly
stimulatory strategy, the inclusion of at least one therapy
aimed at disabling immune checkpoints is theoretically
attractive. So, for example, the combination of anti-CTLA-4
with vaccines, CpG oligodeoxynucleotides, regulatory T cell
depletion or anti-4-1BB enhances activity markedly [122–
125]. Similarly diverse synergy is seen when combining anti-
4-1BB antibodies with other modalities [48,49,68,126]. One
potential advantage of approaches relying on the synergy of
multiple components is that they might reduce the toxicity
induced by higher doses of each agent administered as
monotherapy (e.g. immune responses may be constrained
towards tumour-related antigens if combined with antigen-
specific vaccination or adoptive cellular therpaies rather than
ubiquitous self-antigens). Appropriate timing of sequential
therapies is likely to become an important factor in such
combinatorial approaches.
Conclusions
Major challenges remain, including identification of the best
combinatorial strategies. These will continue to be informed
by careful mechanistic studies in mouse models, while rec-
ognizing potential differences compared with humans. The
identification of robust predictors of response may parallel
attempts to tailor chemotherapeutics according to the
genetic profile of the tumour or of tumour infiltrates. Ulti-
mately, attempts to manipulate the host in order to achieve
such favourable immunological profiles will need to demon-
strate improved clinical outcomes in comparative studies,
and in this regard the application of novel classes of immu-
nostimulatory antibodies offers great promise.
Acknowledgements
Karl S. Peggs is currently an investigator at the Department
of Haematology, UCL Cancer Institute, University College
London, UK, and receives funding from the Leukaemia
Research Fund, UK. Sergio A. Quezada is a Research Fellow
funded by the Irvington Institute Fellowship Program of the
Cancer Research Institute USA, and a junior member of the
Millennium Nucleus on Immunology and Immunotherapy,
Pontifícia Universidad Católica de Chile. James P. Allison is
an investigator of the Howard Hughes Medical Institute and
holds the David H. Koch Chair in Immunological Studies at
the Memorial Sloan-Kettering Cancer Center.
Disclosure
James P. Allison is co-inventor of intellectual property con-
cerning CTLA-4 that is held by University of California,
Berkeley and is a consultant for Medarex and Bristol Meyers
Squibb, who are involved in the clinical development of
anti-CTLA-4.
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Immunostimulatory antibodies in cancer immunotherapy
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    • Successful clinical trials resulted in FDA approval of both antiCTLA4 and antiPD1 [135,136]. T cells also express stimulatory receptors from the TNFR superfamily including OX40 and 4-1BB, whose ligation by agonistic antibodies has a stimulatory effect on T cell survival and on memory T cells [137]. Finally, DCs and other APCs can be activated by agonistic antibodies against the stimulatory molecule CD40, resulting in DC maturation which is crucial for the induction of a potent immune response [138,139] (Figure 5).
    Full-text · Thesis · Jul 2015 · British Journal of Cancer
    • Apart from targeted therapies, agents modulating immunological checkpoints have shown great promise in the clinical management of patients with metastatic melanoma. Cytotoxic Tlymphocyte-associated antigen 4 (CTLA-4) is an immune checkpoint molecule that downregulates T-cell activation, and its blockade by agonistic antibodies enhances antitumor immu- nity [14]. Ipilimumab, a fully human monoclonal antibody against CTLA-4, has shown an overall survival benefit in previously treated and treatment-naïve patients with metastatic melanoma in two randomized phase III trials [15,16] .
    [Show abstract] [Hide abstract] ABSTRACT: Up to 50% of patients with uveal melanoma (UM) develop metastatic disease with limited treatment options. The immunomodulating agent ipilimumab has shown an overall survival (OS) benefit in patients with cutaneous metastatic melanoma in two phase III trials. As patients with UM were excluded in these studies, the Dermatologic Cooperative Oncology Group (DeCOG) conducted a phase II to assess the efficacy and safety of ipilimumab in patients with metastatic UM. We undertook a multicenter phase II study in patients with different subtypes of metastatic melanoma. Here we present data on patients with metastatic UM (pretreated and treatment-naïve) who received up to four cycles of ipilimumab administered at a dose of 3 mg/kg in 3 week intervals. Tumor assessments were conducted at baseline, weeks 12, 24, 36 and 48 according to RECIST 1.1 criteria. Adverse events (AEs), including immune-related AEs were graded according to National Cancer Institute Common Toxicity Criteria (CTC) v.4.0. Primary endpoint was the OS rate at 12 months. Forty five pretreated (85%) and eight treatment-naïve (15%) patients received at least one dose of ipilimumab. 1-year and 2-year OS rates were 22% and 7%, respectively. Median OS was 6.8 months (95% CI 3.7-8.1), median progression-free survival 2.8 months (95% CI 2.5-2.9). The disease control rate at weeks 12 and 24 was 47% and 21%, respectively. Sixteen patients had stable disease (47%), none experienced partial or complete response. Treatment-related AEs were observed in 35 patients (66%), including 19 grade 3-4 events (36%). One drug-related death due to pancytopenia was observed. Ipilimumab has very limited clinical activity in patients with metastatic UM. Toxicity was manageable when treated as per protocol-specific guidelines. ClinicalTrials.gov NCT01355120.
    Full-text · Article · Mar 2015
    • T cells hamper tumor development [9,10] but unfortunately tumors can also prevent themselves from sustained T cell responses via so called immune checkpoints like CTLA-4, PD-1 and PD-L1 etc. [11]. Studies in mouse models have revealed that manipulation of inhibitory immune checkpoints could reduce T cell responses against tumors [12]. In additon, we know that NSCLC induces pro-tumorigenic immunosuppressive changes to evade the immune system, and these changes can be elicited by the inhibitors [13].
    [Show abstract] [Hide abstract] ABSTRACT: Immunotherapy has become a crucial modality for non-small-cell lung cancer treatment. Recently, two immune checkpoints, PD-1 and PD-L1, have emerged as important targets for immunotherapy. Their antitumor efficacy has been confirmed by in vitro and in vivo studies. But the correlation between PD-1/PD-L1 expression and EGFR expression was controversial and needs more evidences to support the combination of PD-1/PD-L1 inhibitors and tyrosine kinase inhibitors.
    Full-text · Article · Jan 2015
    • In the last few years, the antitumoral potential of modulating the immune response via costimulatory and coinhibitory receptors of the CD28 family and tumor necrosis factor receptor (TNFR) superfamily has attracted great attention, notably for cancer therapy [60]. Promising results in preclinical studies have led to the development of an increasing number of corresponding agonistic and antagonistic antibodies, many of them being already evaluated in clinical trials [61][62][63]. However, it is also becoming clear that systemic application of such antibodies can potentially lead to severe adverse events and autoimmunity [64, 65].
    [Show abstract] [Hide abstract] ABSTRACT: Treatment with cytokines holds great potential for cancer immunotherapy, but is generally restricted by systemic toxicity. Tumor-directed targeting in the form of antibody fusion proteins appears to be an attractive strategy to overcome this problem. In the last twenty years, continuous efforts in developing appropriate molecules have retrieved a variety of antibody fusion proteins that reveal promising therapeutic effects in preclinical studies. Currently, several candidates are in clinical evaluation. Here, recent developments exploring diverse antibody formats, tumor targets and cytokines of different families as well as strategies addressing cytokine modification or presentation are discussed and clinical trials summarized at a glance. Thus, antibody-cytokine fusion proteins are becoming progressively improving immunologic reagents that raise expectations mainly for combinatorial cancer therapies.
    Article · Sep 2013
    • It is increasingly appreciated that cancers are recognized by the immune system, and under certain circumstances, the immune system may control or even eliminate tumors [5] . Studies in mouse models of transplantable tumors have demonstrated that manipulation of costimulatory or coinhibitory signals can amplify T cell responses against tumors [6]. This may be accomplished by blockade of coinhibitory molecules, such as CTLA-4, PD-1, and LAG-3, or by enhanced signaling of costimulatory molecules, such as GITR, OX40, and 4-1BB78910111213141516171819.
    [Show abstract] [Hide abstract] ABSTRACT: It is increasingly appreciated that cancers are recognized by the immune system, and under some circumstances, the immune system may control or even eliminate tumors. The modulation of signaling via coinhibitory or costimulatory receptors expressed on T cells has proven to be a potent way to amplify antitumor immune responses. This approach has been exploited successfully for the generation of a new class of anticancer therapies, "checkpoint-blocking" antibodies, exemplified by the recently FDA-approved agent, ipilimumab, an antibody that blocks the coinhibitory receptor CTLA-4. Capitalizing on the success of ipilimumab, agents that target a second coinhibitory receptor, PD-1, or its ligand, PD-L1, are in clinical development. Lessons learned from treating patients with CTLA-4 and PD-1 pathway-blocking antibodies will be reviewed, with a focus on concepts likely to inform the clinical development and application of agents in earlier stages of development. See related review At the bench: Preclinical rationale for CTLA-4 and PD-1 blockade as cancer immunotherapy.
    Article · May 2013
    • Early evidence for its potential as a target for enhancing antitumour immunity came from murine models (Leach et al, 1996), followed shortly thereafter by clinical evaluation of fully human anti-CTLA-4 antibodies. Objective responses as defined by Response Evaluation Criteria in Solid Tumours (RECIST) were documented in 10–15% of patients, though immune-related adverse events (IRAEs) involving a variety of tissues, including the gastrointestinal tract, were documented in 25–30% of cases treated at the higher doses (reviewed in Peggs et al, 2009a), highlighting the relatively narrow therapeutic index. Clinical responses appeared to correlate with the development of IRAEs, though this correlation was not absolute in either direction (Downey et al, 2007).
    [Show abstract] [Hide abstract] ABSTRACT: The past few years have witnessed something of a renaissance in the field of cancer immunotherapy, relating largely to the clinical advances that have been associated with the development of monoclonal antibodies targeting the immune inhibitory co-receptors CTLA-4 and PD-1 and to the pursuit of genetically modified antigen-redirected adoptive T-cell therapies. These advances are based on a more substantial understanding of the factors restricting effective immune therapies that has been derived from the study of pre-clinical models of tumour growth in immune competent mice. Just as the recognition of the importance of positive co-stimulatory signaling has been instrumental to recent advances in the development of genetically modified antigen-specific adoptive cellular therapies, an increasing awareness of the ability of tumours to subvert multiple immune inhibitory pathways, effectively blunting the development or expansion of any anti-tumour immunity, is fostering the development of novel therapies that appear active as monotherapies but may achieve their greatest impact in combinatorial regimens. This mini-review will focus on attempts to target co-inhibitory members of the immunoglobulin superfamily.British Journal of Cancer advance online publication, 19 March 2013; doi:10.1038/bjc.2013.117 www.bjcancer.com.
    Full-text · Article · Mar 2013
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