R E V I E W S
`R E V I E W S
`
`The blockade of immune checkpoints
`in cancer immunotherapy
`
`Drew M. Pardoll
`
`Abstract | Among the most promising approaches to activating therapeutic antitumour
`immunity is the blockade of immune checkpoints. Immune checkpoints refer to a plethora of
`inhibitory pathways hardwired into the immune system that are crucial for maintaining
`self-tolerance and modulating the duration and amplitude of physiological immune responses
`in peripheral tissues in order to minimize collateral tissue damage. It is now clear that tumours
`co-opt certain immune-checkpoint pathways as a major mechanism of immune resistance,
`particularly against T cells that are specific for tumour antigens. Because many of the immune
`checkpoints are initiated by ligand–receptor interactions, they can be readily blocked by
`antibodies or modulated by recombinant forms of ligands or receptors. Cytotoxic
`T-lymphocyte-associated antigen 4 (CTLA4) antibodies were the first of this class of
`immunotherapeutics to achieve US Food and Drug Administration (FDA) approval.
`Preliminary clinical findings with blockers of additional immune-checkpoint proteins, such as
`programmed cell death protein 1 (PD1), indicate broad and diverse opportunities to enhance
`antitumour immunity with the potential to produce durable clinical responses.
`
`The myriad of genetic and epigenetic alterations that
`are characteristic of all cancers provide a diverse set of
`antigens that the immune system can use to distinguish
`tumour cells from their normal counterparts. In the
`case of T cells, the ultimate amplitude and quality of
`the response, which is initiated through antigen recogni-
`tion by the T cell receptor (TCR), is regulated by a bal-
`ance between co-stimulatory and inhibitory signals (that
`is, immune checkpoints)1,2 (FIG. 1). Under normal physio-
`logical conditions, immune checkpoints are crucial for
`the maintenance of self-tolerance (that is, the prevention
`of autoimmunity) and also to protect tissues from damage
`when the immune system is responding to pathogenic
`infection. As described in this Review, the expression
`of immune-checkpoint proteins can be dysregulated by
`tumours as an important immune resistance mecha-
`nism. T cells have been the major focus of efforts to
`therapeutically manipulate endogenous anti tumour
`immunity owing to: their capacity for the selective rec-
`ognition of peptides derived from proteins in all cellular
`compartments; their capacity to directly recognize and
`kill antigen-expressing cells (by CD8+ effector T cells; also
`known as cytotoxic T lymphocytes (CTLs)); and their
`ability to orchestrate diverse immune responses (by
`CD4+ helper T cells), which integrates adaptive and innate
`effector mechanisms. Thus, agonists of co-stimulatory
`
`receptors or antagonists of inhibitory signals (the subject
`of this Review), both of which result in the amplifica-
`tion of antigen-specific T cell responses, are the primary
`agents in current clinical testing (TABLE 1). Indeed, the
`blockade of immune checkpoints seems to unleash
`the potential of the antitumour immune response in a
`fashion that is transforming human cancer therapeutics.
`T cell-mediated immunity includes multiple sequen-
`tial steps involving the clonal selection of antigen-
`specific cells, their activation and proliferation in second-
`ary lymphoid tissues, their trafficking to sites of antigen
`and inflammation, the execution of direct effector func-
`tions and the provision of help (through cytokines and
`membrane ligands) for a multitude of effector immune
`cells. Each of these steps is regulated by counterbalanc-
`ing stimulatory and inhibitory signals that fine-tune the
`response. Although virtually all inhibitory signals in
`the immune response ultimately affect intracellular sig-
`nalling pathways, many are initiated through membrane
`receptors, the ligands of which are either membrane-
`bound or soluble (cytokines). As a general rule, co-
`stimulatory and inhibitory receptors and ligands that
`regulate T cell activation are not necessarily over-
`expressed in cancers relative to normal tissues, whereas
`inhibitory ligands and receptors that regulate T cell effec-
`tor functions in tissues are commonly overexpressed on
`
`Amplitude
`In immunology, this refers to
`the level of effector output. For
`T cells, this can be levels of
`cytokine production,
`proliferation or target killing
`potential.
`
`Johns Hopkins University
`School of Medicine, Sidney
`Kimmel Comprehensive
`Cancer Center, CRB1 Room
`444, 1650 Orleans Street,
`Baltimore, Maryland 21287,
`USA
`e-mail: dpardol1@jhmi.edu
`doi:10.1038/nrc3239
`
`252 | APRIL 2012 | VOLUME 12
`
` www.nature.com/reviews/cancer
`
`© 2012 Macmillan Publishers Limited. All rights reserved
`
`Merck Ex. 1029
`Page 1 of 13
`
`

`

` F O C U S O N tUmO U R Im mU N Ol Og y & Im mU N O t hE Ra p y
`R E V I E W S
`
`At a glance
`
`• The huge number of genetic and epigenetic changes that are inherent to most cancer
`cells provide plenty of tumour-associated antigens that the host immune system can
`recognize, thereby requiring tumours to develop specific immune resistance
`mechanisms. An important immune resistance mechanism involves immune-inhibitory
`pathways, termed immune checkpoints, which normally mediate immune tolerance
`and mitigate collateral tissue damage.
`• A particularly important immune-checkpoint receptor is cytotoxic T-lymphocyte-
`associated antigen 4 (CTLA4), which downmodulates the amplitude of T cell
`activation. Antibody blockade of CTLA4 in mouse models of cancer induced
`antitumour immunity.
`• Clinical studies using antagonistic CTLA4 antibodies demonstrated activity in
`melanoma. Despite a high frequency of immune-related toxicity, this therapy
`enhanced survival in two randomized Phase III trials. Anti-CTLA4 therapy was the first
`agent to demonstrate a survival benefit in patients with advanced melanoma and was
`approved by the US Food and Drug Administration (FDA) in 2010.
`• Some immune-checkpoint receptors, such as programmed cell death protein 1 (PD1),
`limit T cell effector functions within tissues. By upregulating ligands for PD1, tumour
`cells block antitumour immune responses in the tumour microenvironment.
`• Early-stage clinical trials suggest that blockade of the PD1 pathway induces sustained
`tumour regression in various tumour types. Responses to PD1 blockade may correlate
`with the expression of PD1 ligands by tumour cells.
`• Multiple additional immune-checkpoint receptors and ligands, some of which are
`selectively upregulated in various types of tumour cells, are prime targets for
`blockade, particularly in combination with approaches that enhance the activation of
`antitumour immune responses, such as vaccines.
`
`tumour cells or on non-transformed cells in the tumour
`microenvironment. It is the soluble and membrane-
`bound receptor–ligand immune checkpoints that are
`the most druggable using agonist antibodies (for co-
`stimulatory pathways) or antagonist antibodies (for
`inhibitory pathways) (TABLE 1). Therefore, in contrast to
`most currently approved antibodies for cancer therapy,
`antibodies that block immune checkpoints do not tar-
`get tumour cells directly, instead they target lymphocyte
`receptors or their ligands in order to enhance endogenous
`antitumour activity.
`Another category of immune-inhibitory molecules
`includes certain metabolic enzymes, such as indoleam-
`ine 2,3-dioxygenase (IDO) — which is expressed by both
`tumour cells and infiltrating myeloid cells — and argi-
`nase, which is produced by myeloid-derived suppres-
`sor cells3–9. These enzymes inhibit immune responses
`through the local depletion of amino acids that are
`essential for anabolic functions in lymphocytes (particu-
`larly T cells) or through the synthesis of specific natural
`ligands for cytosolic receptors that can alter lympho-
`cyte functions. Although this category is not covered in
`this Review, these enzymes can be inhibited to enhance
`intratumoral inflammation by molecular analogues of
`their substrates that act as competitive inhibitors or
`suicide substrates10–12.
`In considering the mechanisms of action of inhibi-
`tors of various immune checkpoints, it is crucial to
`appreciate the diversity of immune functions that they
`regulate. For example, the two immune-checkpoint
`receptors that have been most actively studied in the
`context of clinical cancer immunotherapy, cytotoxic
`T-lymphocyte-associated antigen 4 (CTLA4; also
`known as CD152) and programmed cell death protein 1
`
`Quality
`In immunology, this refers to
`the type of immune response
`generated, which is often
`defined as the pattern of
`cytokine production. This, in
`turn, mediates responses
`against specific types of
`pathogen. For example, CD4+
`T cells can be predominantly:
`TH1 cells (characterized by IFNγ
`production; these cells are
`important for antiviral and
`antitumour responses); TH2
`cells (characterized by IL‑4 and
`IL‑13 production; these cells
`are important for antihelminth
`responses); or TH17 cells
`(characterized by IL‑17 and
`IL‑22 production; these cells
`are important for mucosal
`bacterial and fungal responses).
`
`Autoimmunity
`Immune responses against an
`individual’s normal cells or
`tissues.
`
`CD8+ effector T cells
`T cells that are characterized
`by the expression of CD8. They
`recognize antigenic peptides
`presented by MHC class I
`molecules and are able to
`directly kill target cells that
`express the cognate antigen.
`
`(PD1; also known as CD279) — which are both inhibi-
`tory receptors — regulate immune responses at differ-
`ent levels and by different mechanisms. The clinical
`activity of antibodies that block either of these receptors
`implies that antitumour immunity can be enhanced at
`multiple levels and that combinatorial strategies can be
`intelligently designed, guided by mechanistic consid-
`erations and preclinical models. This Review focuses
`on the CTLA4 and PD1 pathways because these are
`the two immune checkpoints for which clinical infor-
`mation is currently available. However, it is important
`to emphasize that multiple additional immune check-
`points represent promising targets for therapeutic
`blockade based on preclinical experiments, and inhibi-
`tors for many of these are under active development
`(TABLE 1).
`
`CTLA4: the godfather of checkpoints
`The biology of CTLA4. CTLA4, the first immune-
`checkpoint receptor to be clinically targeted, is expressed
`exclusively on T cells where it primarily regulates the
`amplitude of the early stages of T  cell activation.
`Primarily, CTLA4 counteracts the activity of the T cell
`co-stimulatory receptor, CD28 (REFS 13–15). CD28
`does not affect T cell activation unless the TCR is first
`engaged by cognate antigen. Once antigen recognition
`occurs, CD28 signalling strongly amplifies TCR signal-
`ling to activate T cells. CD28 and CTLA4 share identi-
`cal ligands: CD80 (also known as B7.1) and CD86 (also
`known as B7.2)16–20. Although the exact mechanisms
`of CTLA4 action are under considerable debate, because
`CTLA4 has a much higher overall affinity for both
`ligands, it has been proposed that its expression on the
`surface of T cells dampens the activation of T cells by
`outcompeting CD28 in binding CD80 and CD86, as well
`as actively delivering inhibitory signals to the T cell21–26.
`The specific signalling pathways by which CTLA4 blocks
`T cell activation are still under investigation, although a
`number of studies suggest that activation of the protein
`phosphatases, SHP2 (also known as PTPN11) and PP2A,
`are important in counteracting kinase signals that are
`induced by TCR and CD28 (REF. 15). However, CTLA4
`also confers ‘signalling-independent’ T cell inhibition
`through the sequestration of CD80 and CD86 from
`CD28 engagement, as well as active removal of CD80
`and CD86 from the antigen‑presenting cell (APC) sur-
`face27. The central role of CTLA4 for keeping T cell
`activation in check is dramatically demonstrated by the
`lethal systemic immune hyperactivation phenotype of
`Ctla4-knockout mice28,29.
`Even though CTLA4 is expressed by activated CD8+
`effector T cells, the major physiological role of CTLA4
`seems to be through distinct effects on the two major
`subsets of CD4+ T cells: downmodulation of helper
`T cell activity and enhancement of regulatory T (TReg)
`cell immunosuppressive activity14,30,31 (BOX 1). CTLA4
`blockade results in a broad enhancement of immune
`responses that are dependent on helper T cells and,
`conversely, CTLA4 engagement on TReg cells enhances
`their suppressive function. CTLA4 is a target gene of
`the forkhead transcription factor FOXP3 (REFS 32,33),
`
`NATURE REVIEWS | CANCER
`
` VOLUME 12 | APRIL 2012 | 253
`
`© 2012 Macmillan Publishers Limited. All rights reserved
`
`Merck Ex. 1029
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`
`

`

`Figure 1 | Multiple co-stimulatory and inhibitory
`interactions regulate T cell responses. Depicted are
`various ligand–receptor interactions between T cells and
`antigen-presenting cells (APCs) that regulate the T cell
`response to antigen (which is mediated by peptide–
`major histocompatibility complex (MHC) molecule
`complexes that are recognized by the T cell receptor
`(TCR)). These responses can occur at the initiation of
`T cell responses in lymph nodes (where the major APCs
`are dendritic cells) or in peripheral tissues or tumours
`(where effector responses are regulated). In general,
`T cells do not respond to these ligand–receptor
`interactions unless they first recognize their cognate
`antigen through the TCR. Many of the ligands bind to
`multiple receptors, some of which deliver co-stimulatory
`signals and others deliver inhibitory signals. In general,
`pairs of co-stimulatory–inhibitory receptors that bind the
`same ligand or ligands — such as CD28 and cytotoxic
`T-lymphocyte-associated antigen 4 (CTLA4) — display
`distinct kinetics of expression with the co-stimulatory
`receptor expressed on naive and resting T cells, but the
`inhibitory receptor is commonly upregulated after T cell
`activation. One important family of membrane-bound
`ligands that bind both co-stimulatory and inhibitory
`receptors is the B7 family. All of the B7 family members
`and their known ligands belong to the immunoglobulin
`superfamily. Many of the receptors for more recently
`identified B7 family members have not yet been identified.
`Tumour necrosis factor (TNF) family members that bind
`to cognate TNF receptor family molecules represent a
`second family of regulatory ligand–receptor pairs. These
`receptors predominantly deliver co-stimulatory signals
`when engaged by their cognate ligands. Another major
`category of signals that regulate the activation of T cells
`comes from soluble cytokines in the microenviron-
`ment. Communication between T cells and APCs is
`bidirectional. In some cases, this occurs when ligands
`themselves signal to the APC. In other cases, activated
`T cells upregulate ligands, such as CD40L, that engage
`cognate receptors on APCs. A2aR, adenosine A2a
`receptor; B7RP1, B7-related protein 1; BTLA, B and T
`lymphocyte attenuator; GAL9, galectin 9; HVEM,
`herpesvirus entry mediator; ICOS, inducible T cell
`co-stimulator; IL, interleukin; KIR, killer cell immunoglobulin-
`like receptor; LAG3, lymphocyte activation gene 3;
`PD1, programmed cell death protein 1; PDL, PD1 ligand;
`TGFβ, transforming growth factor-β; TIM3, T cell
`membrane protein 3.
`
`+
`
`–
`
`+
`
`–
`
`+
`
`–
`
`–
`
`– –
`
`Signal 1
`
`–
`
`+ + +
`
`– –
`
`R E V I E W S
`R E V I E W S
`
`Antigen-presenting cell
`
`PDL1 or PDL2
`
`T cell
`
`?
`
`PDL1 or PDL2
`
`CD80 or CD86
`
`CD80 or CD86
`
`B7RP1
`
`B7-H3
`
`B7-H4
`
`HVEM
`
`Peptide
`
`MHC class I or II
`
`CD137L
`
`OX40L
`
`CD70
`
`+
`
`CD40
`
`GAL9
`
`Adenosine
`
`Cytokines
`(TGFβ, IL-1,
`IL-6, IL-10,
`IL-12, IL-18)
`
`PD1
`
`CD28
`
`CTLA4
`
`ICOS
`
`?
`
`?
`
`BTLA
`
`KIR
`
`TCR
`
`LAG3
`
`CD137
`
`OX40
`
`CD27
`
`CD40L
`
`TIM3
`
`A2aR
`
`Nature Reviews | Cancer
`the expression of which determines the TReg cell line-
`age34,35, and TReg cells therefore express CTLA4 consti-
`tutively. Although the mechanism by which CTLA4
`enhances the immunosuppressive function of TReg
`cells is not known, TReg cell-specific CTLA4 knockout
`or blockade significantly inhibits their ability to regu-
`late both autoimmunity and antitumour immunity30,31.
`Thus, in considering the mechanism of action for
`CTLA4 blockade, both enhancement of effector CD4+
`T cell activity and inhibition of TReg cell-dependent
`immunosuppression are probably important factors.
`
`Clinical application of CTLA4‑blocking antibodies —
`the long road from mice to FDA approval. Initially,
`the general strategy of blocking CTLA4 was ques-
`tioned because there is no tumour specificity to the
`
`expression of the CTLA4 ligands (other than for some
`myeloid and lymphoid tumours) and because the dra-
`matic lethal autoimmune and hyperimmune pheno-
`type of Ctla4-knockout mice predicted a high degree of
`immune toxicity associated with blockade of this recep-
`tor. However, Allison and colleagues36 used preclinical
`models to demonstrate that a therapeutic window was
`indeed achieved when CTLA4 was partially blocked with
`antibodies. The initial studies demonstrated significant
`antitumour responses without overt immune toxicities
`when mice bearing partially immunogenic tumours were
`treated with CTLA4 antibodies as single agents. Poorly
`immunogenic tumours did not respond to anti-CTLA4 as
`a single agent but did respond when anti-CTLA4 was
`combined with a granulocyte–macrophage colony-
`stimulating factor (GM-CSF)-transduced cellular
`
`CD4+ helper T cells
`T cells that are characterized
`by the expression of CD4. They
`recognize antigenic peptides
`presented by MHC class II
`molecules. This type of T cell
`produces a vast range of
`cytokines that mediate
`inflammatory and effector
`immune responses. They also
`facilitate the activation of CD8+
`T cells and B cells for antibody
`production.
`
`254 | APRIL 2012 | VOLUME 12
`
` www.nature.com/reviews/cancer
`
`© 2012 Macmillan Publishers Limited. All rights reserved
`
`Merck Ex. 1029
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`

`

` F O C U S O N tUmO U R Im mU N Ol Og y & Im mU N O t hE Ra p y
`R E V I E W S
`
`PD1
`
`Inhibitory receptor
`
`PDL1
`
`Ligand for PD1
`
`LAG3
`
`Inhibitory receptor
`
`Table 1 | The clinical development of agents that target immune-checkpoint pathways
`Target
`Biological function Antibody or Ig fusion protein
`State of clinical development*
`CTLA4
`Inhibitory receptor
`Ipilimumab
`FDA approved for melanoma, Phase II and
`Phase III trials ongoing for multiple cancers
`Previously tested in a Phase III trial of patients
`with melanoma; not currently active
`Phase I/II trials in patients with melanoma and
`renal and lung cancers
`Phase I trial in multiple cancers
`Phase I trial in multiple cancers
`Phase I trial in multiple cancers
`Phase I trial in multiple cancers
`Phase I trials planned for 2012
`Phase III trial in breast cancer
`Preclinical development
`Phase I trial in multiple cancers
`Inhibitory ligand
`B7-H3
`Preclinical development
`Inhibitory ligand
`B7-H4
`Preclinical development
`Inhibitory receptor
`TIM3
`CTLA4, cytotoxic T-lymphocyte-associated antigen 4; FDA, US Food and Drug Administration; Ig, immunoglobulin; LAG3, lymphocyte
`activation gene 3; mAbs, monoclonal antibodies; PD1, programmed cell death protein 1; PDL, PD1 ligand; TIM3, T cell membrane protein 3.
`*As of January 2012. ‡PD1 specificity not validated in any published material. §PDL2–Ig fusion protein. ||LAG3–Ig fusion protein.
`
`Tremelimumab
`
`MDX-1106 (also known as
`BMS-936558)
`MK3475
`CT-011‡
`AMP-224§
`MDX-1105
`Multiple mAbs
`IMP321||
`Multiple mAbs
`MGA271
`
`Myeloid cells
`Any white blood cell
`(leukocyte) that is not a
`lymphocyte: macrophages,
`dendritic cells and granulocytic
`cells.
`
`Suicide substrates
`Molecules that inhibit an
`enzyme by mimicking its
`substrate and covalently
`binding to the active site.
`
`Antigen-presenting cell
`(APC). Any cell that displays on
`its surface an MHC molecule
`with a bound peptide antigen
`that a T cell recognizes through
`its TCR. This can be a dendritic
`cell or a macrophage, or any
`cell that expresses antigen and
`would be killed by an activated
`CD8+ effector T cell‑specific
`response (such as a tumour cell
`or virally infected cell).
`
`Regulatory T (TReg) cell
`A type of CD4+ T cell that
`inhibits, rather than promotes,
`immune responses. They are
`characterized by the
`expression of the forkhead
`transcription factor FOXP3, the
`lack of expression of effector
`cytokines such as IFNγ and the
`production of inhibitory
`cytokines such as TGFβ, IL‑10
`and IL‑35.
`
`Immunogenic tumours
`In the case of tumours in mice,
`this refers to a tumour that
`naturally elicits an immune
`response when growing in a
`mouse. With regard to human
`tumours, melanoma is typically
`considered immunogenic
`because patients with
`melanoma often have increased
`numbers of T cells that are
`specific for melanoma antigens.
`
`Objective clinical responses
`A diminution of total
`cross‑sectional area of all
`metastatic tumours — as
`measured by a CT or MRI scan
`— by >30% (corresponding to
`~50% decrease in volume)
`with no growth of any
`metastatic tumours.
`
`Response rate
`The proportion of treated
`patients that achieve an
`objective response.
`
`vaccine37. These findings suggested that, if there is an
`endogenous antitumour immune response in the ani-
`mals after tumour implantation, CTLA4 blockade could
`enhance that endogenous response, which ultimately can
`induce tumour regression. In the case of poorly immuno-
`genic tumours, which do not induce substantial endoge-
`nous immune responses, the combination of a vaccine and
`a CTLA4 antibody could induce a strong enough immune
`response to slow tumour growth and in some cases
`eliminate established tumours.
`These preclinical findings encouraged the produc-
`tion and testing of two fully humanized CTLA4 anti-
`bodies, ipilimumab and tremelimumab, which began
`clinical testing in 2000. As with virtually all anticancer
`agents, initial testing was as a single agent in patients
`with advanced disease that were not responding to con-
`ventional therapy38. Both antibodies produced objective
`clinical responses in ~10% of patients with melanoma,
`but immune-related toxicities involving various tissue
`sites were also observed in 25–30% of patients, with coli-
`tis being a particularly common event39–41 (FIG. 2). The
`first randomized Phase III clinical trial to be completed
`was for tremelimumab in patients with advanced mela-
`noma. In this trial, 15 mg per kg tremelimumab was given
`every three months as a single agent and compared with
`dacarbazine (also known as DTIC), a standard mela-
`noma chemotherapy treatment. The trial showed no
`survival benefit with this dose and schedule relative to
`dacarbazine42.
`However, ipilimumab fared better. Even though the
`intrinsic activity, response rates in Phase II trials and
`immune toxicity profiles were similar for both antibod-
`ies, ipilimumab was more carefully evaluated at different
`doses and schedules. Additionally, more careful definition
`of algorithms for improved clinical management of the
`immune toxicities (using steroids and tumour necrosis
`
`factor (TNF) blockers) mitigated the overall morbidity
`and mortality that were associated with immunological
`toxicities. Interestingly, although there is evidence that
`clinical responses might be associated with immune-
`related adverse events, this correlation is modest43. Finally,
`in a randomized three-arm clinical trial of patients with
`advanced melanoma that received either: a peptide
`vaccine of melanoma-specific gp100 (also known as
`PMEL) alone; the gp100 vaccine plus ipilimumab; or ipil-
`imumab alone, there was a 3.5 month survival benefit for
`patients in both groups receiving ipilimumab (that is, with
`or without the gp100 peptide vaccine) compared
`with the group receiving the gp100 peptide vaccine
`alone44. As ipilimumab was the first therapy to dem-
`onstrate a survival benefit for patients with metastatic
`melanoma, it was approved by the US Food and Drug
`Administration (FDA) for the treatment of advanced
`melanoma in 2010 (dacarbazine was approved on the
`basis of response rate but has not been shown to provide
`a survival benefit in patients with melanoma).
`More impressive than the mean survival benefit was
`the effect of ipilimumab on long-term survival: 18%
`of the ipilimumab-treated patients survived beyond two
`years (compared with 5% of patients receiving the gp100
`peptide vaccine alone)44. In this and other studies, the
`proportion of long-term survivors was higher than
`the proportion of objective responders. The finding of
`ongoing responses and survival long after completion
`of a relatively short course of therapy (four doses of
`10 mg per kg over 3 months) support the concept that
`immune-based therapies might re-educate the immune
`system to keep tumours in check after completion of the
`therapeutic intervention.
`As with all oncology agents that benefit a limited pro-
`portion of treated patients, there has been much effort
`in defining biomarkers that predict clinical responses
`
`NATURE REVIEWS | CANCER
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` VOLUME 12 | APRIL 2012 | 255
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`

`R E V I E W S
`R E V I E W S
`
`Natural killer (NK) cells
`Immune cells that kill cells
`using mechanisms similar to
`CD8+ effector T cells but do
`not use a clonal TCR for
`recognition. Instead, they are
`activated by receptors for
`stress proteins and are
`inhibited through distinct
`receptors, many of which
`recognize MHC molecules
`independently of the bound
`peptide.
`
`Anergy
`A form of T or B cell
`inactivation in which the cell
`remains alive but cannot be
`activated to execute an
`immune response. Anergy is a
`reversible state.
`
`to anti-CTLA4 therapy. To date, no such pretreatment
`biomarker has been validated to the point at which it
`could be applied as part of standard-of-care therapeu-
`tic decision-making, although insights have emerged
`from the identification of certain post-treatment
`immune responses that seem to correlate with clinical
`outcome45–47.
`An important feature of the anti-CTLA4 clinical
`responses that distinguishes them from conventional
`chemotherapeutic agents and oncogene-targeted small
`molecule drugs is their kinetics. Although responses to
`chemotherapies and tyrosine kinase inhibitors (TKIs)
`commonly occur within weeks of initial administration,
`the response to immune-checkpoint blockers is slower
`and, in many patients, delayed (up to 6 months after
`treatment initiation). In some cases, metastatic lesions
`actually increase in size on computed tomography (CT)
`or magnetic resonance imaging (MRI) scans before
`regressing, which seems to occur owing to increased
`immune cell infiltration. These findings demand a re-
`evaluation of response criteria for immunotherapeu-
`tics away from the conventional time-to-progression
`or Response Evaluation Criteria in Solid Tumours
`(RECIST) objective response criteria, which were devel-
`oped on the basis of experiences with chemotherapeutic
`agents and as the primary measure of drug efficacy48.
`
`Blockade of the PD1 pathway
`Another immune-checkpoint receptor, PD1, is emerg-
`ing as a promising target, thus emphasizing the diversity
`of potential molecularly defined immune manipula-
`tions that are capable of inducing antitumour immune
`responses by the patient’s own immune system.
`
`The biology of the PD1 pathway. In contrast to CTLA4,
`the major role of PD1 is to limit the activity of T cells
`in peripheral tissues at the time of an inflammatory
`
`Box 1 | TReg cells in the maintenance of immune tolerance in cancer
`
`Regulatory T (TReg) cells are crucial for the maintenance of self-tolerance. Their unique
`genetic programme is driven by the forkhead transcription factor FOXP3, which is
`encoded on the X chromosome. Foxp3-knockout mice, and humans with homozygous
`mutation of FOXP3 (which causes immunodysregulation, polyendocrinopathy,
`enteropathy and X-linked (IPEX) syndrome) develop autoimmune syndromes involving
`multiple organs30–33. The inhibitory activity of TReg cells on immune responses remains to
`be completely understood, but involves the production of inhibitory cytokines, such as
`transforming growth factor-β (TGFβ), interleukin-10 (IL-10) and IL-35. They are
`subdivided into ‘natural’ TReg (nTReg) cells, which develop in the thymus, and ‘induced’
`TReg (iTReg) cells, which accumulate in many tumours and are thought to represent a
`major immune resistance mechanism. They are therefore viewed as important cellular
`targets for therapy. TReg cells do not express cell surface molecules that are unique to
`either subset, but they do express high levels of multiple immune-checkpoint receptors,
`such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), programmed cell death
`protein 1 (PD1), T cell membrane protein 3 (TIM3), adenosine A2a receptor (A2aR) and
`lymphocyte activation gene 3 (LAG3). Genes encoding some of these immune-
`checkpoint receptors, such as CTLA4, are actually FOXP3 target genes. Paradoxically,
`although inhibiting effector T cells, these receptors seem to enhance TReg cell activity or
`proliferation. Although an antibody that specifically targets TReg cells has not yet been
`produced, many of the immune-checkpoint antibodies in clinical testing probably block
`the immunosuppressive activity of TReg cells as a mechanism of enhancing antitumour
`immunity.
`
`response to infection and to limit autoimmunity49–55
`(FIG. 3). This translates into a major immune resistance
`mechanism within the tumour microenvironment56–58.
`PD1 expression is induced when T cells become acti-
`vated49. When engaged by one of its ligands, PD1 inhib-
`its kinases that are involved in T cell activation through
`the phosphatase SHP250, although additional signalling
`pathways are also probably induced. Also, because PD1
`engagement inhibits the TCR ‘stop signal’, this pathway
`could modify the duration of T cell–APC or T cell–
`target cell contact59. Similarly to CTLA4, PD1 is highly
`expressed on TReg cells, where it may enhance their
`proliferation in the presence of ligand60. Because many
`tumours are highly infiltrated with TReg cells that prob-
`ably further suppress effector immune responses, block-
`ade of the PD1 pathway may also enhance antitumour
`immune responses by diminishing the number and/or
`suppressive activity of intratumoral TReg cells.
`The two ligands for PD1 are PD1 ligand 1 (PDL1; also
`known as B7-H1 and CD274) and PDL2 (also known
`as B7-DC and CD273)50,61–63. These B7 family members
`share 37% sequence homology and arose through gene
`duplication, which has positioned them within 100 kb
`of each other in the genome63. Recently, an unexpected
`molecular interaction between PDL1 and CD80 was dis-
`covered64, whereby CD80 expressed on T cells (and pos-
`sibly APCs) can potentially behave as a receptor rather
`than a ligand by delivering inhibitory signals when
`engaged by PDL1 (REFS 65,66). The relevance of this inter-
`action in tumour immune resistance has not yet been
`determined. Finally, genetic evidence from PD1-deficient
`T cells suggests that both PDL1 and PDL2 may bind to
`a co-stimulatory receptor that is expressed on T cells67.
`These complex binding interactions are reminiscent
`of the CD80 and CD86 ligand pair, each of which binds
`the co-stimulatory receptor CD28 that is expressed on
`resting T cells and the inhibitory receptor CTLA4 that is
`expressed on activated T cells. However, as stated above,
`PD1 predominantly regulates effector T cell activity
`within tissue and tumours, whereas CTLA4 predomi-
`nantly regulates T cell activation (FIG. 3). Understanding
`the role of these various interactions in different can-
`cer settings is highly relevant for the selection of both
`antibodies and recombinant ligands for use in the clinic.
`PD1 is more broadly expressed than CTLA4: it is
`induced on other activated non-T lymphocyte subsets,
`including B cells and natural killer (NK) cells68,69, which
`limits their lytic activity. Therefore, although PD1
`blockade is typically viewed as enhancing the activity
`of effector T cells in tissues and in the tumour micro-
`environment, it also probably enhances NK cell activity
`in tumours and tissues and may also enhance antibody
`production either indirectly or through direct effects on
`PD1+ B cells70.
`In addition, chronic antigen exposure, such as
`occurs with chronic viral infection and cancer, can lead
`to high levels of persistent PD1 expression, which
`induces a state of exhaustion or anergy among cognate
`antigen-specific T cells. This state, which has been
`demonstrated in multiple chronic viral infections in
`mice and humans, seems to be partially reversible by
`
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