Novel antibodies targeting immune regulatory checkpoints for cancer therapy

Novel antibodies targetingimmune regulatorycheckpoints forcancer therapyChern Siang Lee, Mark Cragg, Martin Glennie & Peter Johnson

Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital,

Southampton SO16 6YD, UK

CorrespondenceProfessor Martin Glennie, Cancer SciencesUnit, Faculty of Medicine, University ofSouthampton, Southampton GeneralHospital, Southampton SO16 6YD, UK.Tel.: +02 3 8079 6593Fax: +02 3 8070 4061E-mail: M.J.Glennie@soton.ac.uk-----------------------------------------------------------------------

Keywordscancer, immunomodulatory antibodies,immunotherapy, monoclonal antibodies-----------------------------------------------------------------------

Received2 October 2012

Accepted17 April 2013

Accepted ArticlePublished Online23 May 2013

Cancers must evade or suppress the immune system in order to develop. Better understanding of the molecular regulation governingtumour detection and effective activation of the immune system (so called immune regulatory checkpoints) has provided new targetsfor cancer immunotherapy. Therapeutic monoclonal antibodies against these targets are currently undergoing clinical evaluation withmore in pre-clinical development; buoyed by the recent licence approval of the anti-CTLA-4 antibody, ipilumumab, for use inmelanoma. This article will review the current status of the various antibodies and target molecules being investigated.

Introduction

The use of monoclonal antibodies (mAbs) as therapeuticshas increased rapidly over the last 30 years. The first gen-eration of therapeutic mAbs were of mouse origin. Thisoften resulted in a rapid induction of human anti-mouseantibody (HAMA) responses when infused into humans;limiting their half-life and therapeutic efficacy [1–4]. Sub-stitution of the murine Fc domain with human sequencesthrough recombinant DNA technology resulted in chi-meric mAbs with improved pharmacokinetic and thera-peutic efficacy [5–7]. Progressive improvements inrecombinant DNA and cloning techniques eventually ledto the development of humanized mAbs carrying mousehypervariable loops in the human V-region framework [8,9]. Fully human mAbs are now available through the use ofphage display libraries or genetically modified mice carry-ing human rather than mouse immunoglobulin loci [10–13]. Current mAb nomenclature attaches suffixes todiscriminate these various mouse, chimeric, humanizedand human mAbs as well as mAb-derivatives such as Fcfusions and bispecific reagents (Table 1).

Alongside small molecule target inhibitors, immunetherapies and therapeutic mAbs, in particular, havebecome an important treatment strategy in oncology with

numerous mAbs approved by the United States Food andDrug Administration (FDA) and the European MedicinesAgency (EMA), and with many more undergoing clinicalevaluation and pre-clinical development. Those such asrituximab, which directly target molecules expressed onthe tumour (i.e. CD20 on lymphoma) can achieve theirtherapeutic effects through Fc-dependent cytotoxiceffects, interacting with Fc receptor-expressing cells ofthe innate immune effector system to trigger antibody-dependent cell mediated cytotoxicity (ADCC) or serumproteins to elicit complement dependent cytotoxicity(CDC) [14, 15]. Others, such as trastuzumab and cetuximab,can also function by blocking key survival signals in thetumour cell provoking cell cycle arrest and cell death inaddition to ADCC [16–19]. A detailed discussion of theactivity and modes of action of these so-called direct tar-geting mAbs is beyond the scope of this article. Table 2provides a brief overview of these agents, their targets andmechanisms of action with Table 3 summarising a selec-tion of promising agents currently undergoing clinicalevaluation. In this review we will examine a different andfast-developing class of mAbs, those that do not targetthe tumour directly but instead target receptors of theimmune system to promote anti-cancer immunity,so-called immunomodulatory mAb.

British Journal of ClinicalPharmacology

DOI:10.1111/bcp.12164

Br J Clin Pharmacol / 76:2 / 233–247 / 233© 2013 The British Pharmacological Society

mailto:[email protected]

Immunomodulatory mAbs forcancer immunotherapy

Immunomodulatory mAbs can provoke immune rejectionof the tumour without having to identify directly tumourantigens that may be unique to an individual cancer. Theadvantage of such an approach, in comparison with con-ventional therapies and even direct targeting mAbs, isthe potential for developing immunological memory tothe tumour, preventing its recurrence, the main issue ofmodern cancer treatment.

Regulation of T-cell responsesThe recognition of antigenic peptides (e.g. tumour-associated antigens) bound to the major histocompatibil-ity complex (MHC) by the T-cell receptor (TCR) alone isinsufficient for T- cell activation to effect immune destruc-tion of the cancer cell. Co-regulatory (inhibitory orstimulatory) signals are also required, transmitted bya series of receptor-ligand pairs (see Figure 1). Theseco-regulatory molecules can be constitutively (e.g. CD28on naive T-cells) or variably expressed on the cell surface atdifferent stages of an immune response (early activatoryphase or late executory phase) and interact in immuno-logical synapses formed between the different cell types ofthe adaptive immune system and tumour cells. Ultimately,they determine the fate of the T-cell response; activationand differentiation into effector T-cells, deletion or anergy.Such regulatory checkpoints are crucial for the mainte-nance of self-tolerance under physiological conditions butthere is an increasing recognition of the important role theimmune system plays in cancer formation [20]. Cancer cellscan co-opt these mechanisms or release immunosuppres-sive cytokines such as transforming growth factor β(TGF-β) to evade and suppress the immune system inorder to develop into a tumour [21]. Tumour infiltratinglymphocytes (TILs) that fail to eliminate the tumour but yet

possess cytolytic functions in vitro suggest an endogenousanti-tumour effect that can be harnessed if immune toler-ance can be broken [22]. Such immune tolerance of thecancer by the host can be disrupted by using mAbs totarget immune checkpoint receptors in order to boostweak, ineffectual endogenous anti-tumour immuneresponses to therapeutic levels. For example, immunecheckpoint receptors that inhibit effector T-cell functionand boost regulatory T-cell functions (regulatory T-cellsplay an immunosuppressive role in the tumourmicroenvironment by inhibiting anti-tumour immuneresponses) are called checkpoint blockers and serve aspromising therapeutic targets.

Immunomodulatory mAbs can be either activatory(agonists) towards a stimulatory receptor by mimickingligand engagement (immunostimulatory mAb) or inhibi-tory (antagonistic) against a checkpoint blocker by block-ing ligand engagement. Figure 2 illustrates the multipletherapeutic targets that may be manipulated with theseimmunomodulatory mAbs. Examples of these mAbs aredetailed below and a selection of agents undergoing clini-cal evaluation are summarized in Table 4.

Antagonists of checkpoint blockersLigation of the T-cell activator CD28 by a member of the B7family of co-stimulatory molecules, B7-1 (also known asCD80) or B7-2 (also known as CD86), greatly enhances TCR-induced survival, proliferation, differentiation and eventualactivation of the naive T-cell. Concurrently as one of theimmunological counterbalances to attenuate unwanted orexcessive T-cell responses, cytotoxic T-lymphocyte associ-ated antigen 4 (CTLA4), is transported from intracellularstores to the cell surface where it serves to down-regulatethe T-cell activation signal by avid competitive binding toB7 molecules. For this reason, CTLA4 has been referred toas a brake on the immune system and was proposed as aviable target for blocking with mAbs to enhance anti-cancer responses.

Ipilimumab is a fully human mAb that blocks CTLA4function. It is the first immunomodulatory mAb to belicensed after demonstrating a statistically and clinicallysignificant survival benefit in a pivotal phase III clinical trial[23]. Patients with metastatic melanoma who had receivedat least one previous line of treatment were randomizedto receive ipilimumab with a glycoprotein 100 (gp100)peptide vaccine, ipilimumab alone or gp100 alone as thecontrol arm. The median overall survival (OS) was 10months [95% confidence interval (CI) 8.5, 11.5] in patientsreceiving both ipilimumab and gp100 vs. 6.4 months (95%CI 5.5, 8.7) in the gp100 alone group [hazard ratio (HR) fordeath 0.68, P < 0.001]. The median OS in the ipilumumabonly group was 10.1 months (95% CI 8.0, 13.8) and thehazard ratio of death was 0.66 (P = 0.003) when comparedwith gp100 alone. There was no difference in OS betweenthe two ipilimumab containing groups. Further clinicaltrials are on-going in multiple tumour types as either

Table 1Antibody nomenclature

Substem-suffix Type of molecule

-a-mab Rat-o-mab Mouse

-e-mab Hamster-i-mab Primate

-xi-mab Chimeric-xizu-mab Chimeric-humanized

-zu-mab Humanized-u-mab Fully human antibody from phage display or transgenic mice

technology

-stim Fc fusion peptide-cept Fc fusion protein

-axo-mab Bispecific mouse-rat hybrid mAb

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Table 2Monoclonal antibodies approved for use in oncology

Approved monoclonal antibodiesAntibody Target Mechanism of action Cancer type EMA approval Trial reference

Trastuzumab HER2 Inhibition of HER2 signalling Early or advanced breast cancerAdvanced gastric or gastro-oesophageal junction

adenocarcinoma

Approved [127, 128]

Cetuximab EGFR Inhibition of EGFR signalling and ADCC Locally advanced or recurrent/metastatic squamouscell head and neck cancer

Wild-type KRAS metastatic colorectal cancer incombination with chemotherapy

Approved [129–131]

Panitumumab EGFR Inhibition of EGFR signalling Wild-type KRAS metastatic colorectal cancer Approved [132]Bevacizumab VEGF Inhibition of VEGF signalling Metastatic colorectal cancer

Metastatic breast cancerMetastatic non-small cell lung cancerMetastatic renal cell cancerRecurrent glioblastoma multiformeAdvanced ovarian cancer

Approved [133–139]

Ipilimumab CTLA4 Inhibition of CTLA4 signalling Second line treatment for unresectable/metastaticmelanoma

Approved [23]

Denosumab RANKL Inhibition of RANKL on osteoclast Prevention of skeletal related events from bonemetastases from solid tumours

Approved [140]

Rituximab CD20 ADCC, induction of apoptosis and CDC NHLRelapsed CLL

Approved [141, 142]

Alemtuzumab CD52 ADCC, induction of apoptosis and CDC CLL for which fludarabine is inappropriate Approved [143]

Ofatumumab CD20 ADCC and CDC Fludarabine and alemtuzumab refractory CLL Approved [144]90Y-Ibritumomab-

tiuxetanCD20 Radioisotope conjugated mAb Follicular lymphoma (as consolidation treatment) or

relapsed diseaseApproved [145]

Brentuximab vedotin CD30 MMAE conjugated mAb Relapsed or refractory CD30+ Hodgkin lymphoma Approved [146]131I-tositumomab CD20 Radioisotope conjugated mAb NHL EMA orphan drug status [147]

Gemtuzumabozogamicin

CD33 Calicheamicin conjugated mAb Acute myeloid leukaemia EMA orphan drug status [148]

EMA, European Medicines Agency; HER2, human epidermal growth factor receptor 2; EGFR, epidermal growth factor receptor; ADCC, antibody-dependent cell-mediatedcytotoxicity; VEGF, vascular endothelial growth factor; CTLA4, cytotoxic T lymphocyte antigen 4; RANKL, receptor activator for nuclear factor kappa B ligand; NHL, Non-Hogkin’slymphoma; CDC, complement dependent cytotoxicity; CLL, chronic lymphocytic leukaemia; MMAE, monomethyl auristatin E.

Table 3A selection of novel direct tumour targeting mAbs undergoing clinical trials

Antibody Target Mechanism of action Cancer typePhase ofclinical trial NCT identifier

Brentuximab vedotin CD30 MMAE conjugated mAb Hodgkin’s lymphoma III NCT01100502Ch14.8 GD2 ADCC and CDC Neuroblastoma III NCT01041638

Ganitumab IGF1R Inhibition of IGF1R Pancreatic cancer III NCT01231347Necitumumab EGFR Inhibition of EGFR Non-small cell lung cancer III NCT00981058

NCT00982111

Ramucirumab VEGFR2 Inhibition of VEGF signalling Gastric cancerGastro-oesophageal junction cancerHepatocellular carcinoma

III NCT00917384NCT01140347

Nimotuzumab EGFR Inhibition of EGFR signalling Nasopharyngeal cancerHead and neck cancerOesophageal cancerSquamous cell carcinoma

III NCT01074021NCT00957086NCT01249352NCT01402180

Siltuximab IL-6 Induction of apoptosis Relapsed multiple myeloma III NCT01266811Racotumumab NGc-containing gangliosides Anti-idiotype mAb vaccine Non-small cell lung cancer III NCT01460472

Elotuzumab CS1 Induction of apoptosis and ADCC Relapsed multiple myeloma III NCT01239797Trastuzumab emtansine HER2 Mertansine conjugated mAb Refractory HER2+ breast cancer III NCT01419197

MMAE, monomethyl auristatin E; NCT, http://clinicaltrials.gov identifier number; GD2, disialoganglioside 2; IGF1R, insulin-like growth factor 1 receptor; VEGFR2, vascular endothelialgrowth factor receptor 2; IL-6, interleukin-6; NGc, N-glycolyl; CS1, CD2 subset 1; HER2, human epidermal growth factor receptor 2.

Novel antibodies targeting immune regulatory checkpoints for cancer therapy

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monotherapy or in combination with other modalities(see Table 4). The question of scheduling ipilimumab withcytotoxic chemotherapy is of particular importancebecause although chemotherapy may increase antigendelivery it may also delete key immune cells. A recentphase II, three-armed, placebo-controlled study (CA184-041) randomized 204 treatment naive patients withadvanced non-small cell lung cancer to chemotherapy

only, chemotherapy and concurrent ipilimumab or twocycles of chemotherapy before ipilimumab was phased in.Non-progressive patients who tolerated treatment thenreceived ipilimumab or placebo maintenance every 12weeks. The study met its primary end-point of immune-related progression free survival (irPFS) for the phasedtreatment group (HR 0.72, P = 0.05) but not for the concur-rent treatment group (HR 0.81, P = 0.13) compared with the

Antigen presenting cellor tumour cell

(depending on context) T-cell

BTLAHVEM

CTLA4B7 1/2

TCR

CD28

PD-1PD-L1

MHC

B7 1/2

T-cellResponse?

Immunoglobulinsuperfamily members

CD40

4-1BB

CD27

OX40

CD40L

4-1BBL

CD70

OX40L

TNFR superfamilymembers

TIM3GAL-9

LAG3MHC2

CD40

Signal 2: Co-stimulatory signals

Signal 2: Co-Inhibitory signals

Outcome

4-1BB 4-1BBL

CD40L

Signal 1:Antigenic

stimulation

Signal 3:Cytokines

AnergyDeletion

Effector T-cell

Activation

Proliferation

Differentiation

Abortiveactivation

VISTAUnidentified

receptor

Figure 1Schematic representation of an immune synapse and downstream events. Signal 1 is generated by T-cell receptor (TCR) recognition of peptide antigenpresented on major histocompatibility complex (MHC) by antigen-presenting cells (APCs, e.g. dentritic cells) or tumour cells. Signal 2 is provided byco-regulatory (co-stimulatory or inhibitory) molecules interacting with their cognate ligands expressed on APCs. These co-regulatory molecules can beexpressed constitutively in naive T-cells (e.g. CD28 and CD27) or induced by TCR engagement leading to expression at different time points of T-cellactivation. Signal 3 is mostly provided by cytokines secreted by APCs or other immune cells including CD4 T-helper cells to help regulate the effectordifferentiation pathway. Successful activation of a naive CD8 T-cell allows survival and proliferation of the reactive clone, allowing their progeny todifferentiate into cytotoxic T lymphocytes that kill tumour cells expressing the tumour associated antigen. Unsuccessful activation leads to anergy ordeletion of the T-cell. Transient, abortive activation of T-cells leading to deletion or anergy may occur in the context of antigen presentation by immature(or insufficiently activated) dendritic cells. CD40/CD40L and 4-1BB/4-1BBL are known as bi-directional signalling molecules and ligand engagementactivates intra-cellular signalling transduction pathways. Furthermore the receptors can also be expressed on the target cell and may play a part in therapywith mAb. LAG3, lymphocyte activation gene 3; TIM3, T-cell immunoglobulin and mucin domain 3; GAL-9, galectin-9; PD-1, programmed death-1; PDL-1,programmed death ligand-1; HVEM, herpes virus entry mediator; BTLA, B and T lymphocyte activator; CTLA4, cytotoxic T-lymphocyte associated antigen 4;VISTA, V-domain Ig suppressor of T-cell activation; TNFR, tumour necrosis factor receptor

C. S. Lee et al.


control group. There was also an improvement in WHO-criteria progression free survival (HR 0.69, P = 0.02) [24]. Aphase III trial (NCT01285609) examining this regime insquamous cell lung cancer is now underway as a subsetanalysis suggested greater efficacy in favour of squamoushistology. The parallel study examining extensive stagesmall cell lung cancer patients (n = 130) also arrived at asimilar conclusion favouring the phased ipilimumabregime for irPFS (HR 0.63, P = 0.03) over the concurrentregime (HR 0.75, P = 0.11) compared with the control group[25]. Although small, these clinical studies highlight theneed for more well-designed phase II trials in other cancersto test the effect of scheduling for these novel mAbs incombination with other modalities.

Another CTLA4 mAb, tremelimumab (previouslyticilimumab; Pfizer-MedImmune), suffered initial disap-

pointment after a phase III clinical trial in metastatic mela-noma was stopped early after failing to demonstratesuperiority over chemotherapy as first line treatment [26,27]. It recently demonstrated interesting activity in a smallphase II study (n = 20) of advanced hepatocellular carci-noma in chronic hepatitis C infection with a disease controlrate of 76.4% and 33% of patients experiencing stabledisease of more than 12 months duration. The median OSwas 7.5 months (95% CI 4.6, 18.0) [28]. While the exactmechanism of action of anti-CTLA4 mAbs remains a matterof discussion and research, their therapeutic activity andpotential for patient benefit is now available in the clinic.

The programmed death 1 (PD-1) receptor expressed byactivated T-cells is another key immune checkpoint recep-tor with a negative regulatory role when engaged by itsligands PD-L1 (also known as B7-H1) and PD-L2 (also

Tumour cell

PD-1

CTLA4

CD28

B7-1/2

TCR

MHC

CD40

CD40

CD27

OX40

4-1BB

BTLATIM3LAG3

LAG3

PD-1

CTLA4

PD-L1

TCR

MHC

T-cellactivation

Anti-CTLA-4

Anti-4-1BB

Anti-PD1

Anti-OX40

Anti-CD40

APC

Anti-CD40

Anti-CD27

Anti-LAG3Anti-TIM3 Anti-BTLA

Effector CTL

RegulatoryT-cell Cognate ligand binding induces

secretion of imunosuppressivecytokines, e.g. TGF-b

Anti-PD-L1

Legend:• Stimulatory receptor• Inhibitory receptor• Peptide• Agonist mAb• Antagonist mAb

Figure 2Schematic representation of the multiple immunomodulatory receptors that are potential therapeutic targets as monotherapy or combination therapy withimmunomodulatory mAbs. T-cell activation refers to the eventual activity of a complex network of different T-cell lineages (e.g. Th1, Th2, Th17, Treg etc)which can have differential expression of checkpoint regulators. APC, antigen presenting cells; CTL, cytotoxic T lymphocytes



known as B7-DC) within the tumour microenvironment[29]. Aberrant PD-L1 expression by cancers to delete orsuppress activated anti-tumour T-cells has been suggestedas an immune-evasion strategy [29–31]. Two clinical trialsinvestigating blocking mAb designed to target PD-1(nivolumab, BMS-936558, MDX-1106) or its ligand (BMS-936559) (both Bristol-Myers Squibb) have recently shownexciting clinical activity in heavily pre-treated patients withadvanced melanoma, renal cell carcinoma and non-smallcell lung cancer [32, 33]. The anti-PD-1 mAb (Nivolumab)appeared the more successful of the two, producing objec-tive responses of between 20–25% of patients dependingon the tumour type. In 65% of responders, the duration ofresponse was more than 1 year.This is a significant achieve-ment given the patient cohort and that for more than 20years the ceiling of durable response rates for many immu-notherapy approaches has stood at around 10–15% [34].Tumour response to the anti-PD-1 mAb strongly correlatedwith PD-L1 expression within the tumour adding supportto the pre-clinical evidence and raising the possibility thatPD-L1 could serve as a predictive biomarker.

Several other checkpoint receptors have been identi-fied to be potential therapeutic targets in pre-clinicalstudies warranting further evaluation on their own and incombination with other immunomodulatory strategies.V-domain Ig suppressor of T-cell activation (VISTA) is arecently discovered immunoregulatory molecule that

bears a structural resemblance to PD-1. Its expression onantigen presenting cells (APCs) inhibits T-cell proliferationand function. Application of an antagonistic mAb reversedT-cell inhibition and exacerbated a mouse model of auto-immunity, indicating that it is a checkpoint blockeramenable to therapeutic mAb manipulation [35]. T-cellimmunoglobulin and mucin domain 3 (TIM-3) and itsligand galectin-9 play an important role in mediating T-cellexhaustion by deleting T-cells secreting interferon-gamma(IFNγ) which is crucial to maintain immune activity [36, 37].Its biological role in cancer and potential in immuno-therapy have been examined in a number of settings [38–40]. In a mouse model of carcinogen-induced sarcoma,anti-TIM3 mAb showed a modest effect when used aloneand showed synergy when combined with an anti-PD-1mAb [41]. Lymphocyte-activation gene 3 (LAG-3; CD223) isa member of the immunoglobulin superfamily that isclosely related to CD4 and also binds to MHC class II.However, unlike CD4 it functions as an inhibitory moleculeand promotes immune tolerance of the tumour by inhib-iting APCs and T-cell function [42]. Recent work in mousemodels suggests potential synergy when used in combi-nation with PD-1 blockade [43]. A study demonstrateddual anti-LAG-3/anti-PD-1 antibody treatment cured mostmice of established tumours that were resistant to singleantibody treatment [44]. B and T lymphocyte attenuator(BTLA; CD272) is a receptor that closely resembles CTLA4

Table 4Immunostimulatory/immunomodulatory mAbs undergoing clinical evaluation as monotherapy or in combination with other modalities

Antibody Target Mechanism of action Cancer typePhase ofclinical trial NCT identifier

Chi Lob 7/4 CD40 Activate CD40 Refractory, advanced 1 NCT01561911CDX-1127 CD40 Activate CD40 Select types of solid tumours, leukaemias and

lymphomas1 NCT01460134

BMS-936558 and chemotherapy PD-1 Inhibit PD-1Cytotoxic chemotherapy

Advanced non-small cell lung cancer 1 NCT01454102

BMS-663513 4-1BB Inhibit 4-1BB signalling Advanced solid tumours 1 NCT01471210

Tremelimumab and CP870,893 CTLA4CD40

Inhibition of CTLA4Activate CD40

Metastatic melanoma 1 NCT01103635

Ipilimumab and bevacizumab CTLA4VEGF

Inhibit CTLA4Inhibit VEGF

Unresectable melanoma 1 NCT00790010

Ipilimumab and gemcitabine chemotherapy CTLA4 Inhibit CTLA4Cytotoxic chemotherapy

Inoperable pancreatic cancer 1 NCT01473940

Ipilimumab and carboplatin & etoposidechemotherapy

CTLA4 Inhibition of CTLA4Cytotoxic chemotherapy

Extensive stage small cell lung cancer 2 NCT01331525

Ipilimumab and vemurafenib CTLA4 Inhibition of CTLA4B-Raf inhibition

Melanoma 1–2 NCT01400451

Ipilimumab and paclitaxel & carboplatinchemotherapy

CTLA4 Inhibition of CTLA4Cytotoxic chemotherapy

Extensive stage squamous cell lung cancer 3 NCT01285609

Ipilimumab and androgen suppression CTLA4 Inhibition of CTLA4Androgen suppression

Hormone-resistant prostate cancer 2 NCT01498978

Anti-OX40Stereotactic radiotherapy

OX40 Activate OX40Radiotherapy

Breast cancer with metastases to liver or lung 1–2 NCT01642290

Anti-OX40RadiotherapyCyclophosphamide

OX40 Activate OX40RadiotherapyChemotherapy

Progressive metastatic prostate cancer 1–2 NCT01303705

C. S. Lee et al.


and PD-1 [45]. It binds to the herpes virus entry mediator(HVEM). The interaction between BTLA and HVEM canfunction either as a stimulatory or inhibitory signaldepending on the context of other binding partners orcell-type expression [46, 47]. Recent work demonstrates itspotential role in maintaining immune-tolerance to self [45,48] and tumour, making it a potential target for therapy[49–51].

Agonists of positive regulators(immunostimulators)Immunostimulatory mAbs have agonistic properties onstimulatory immune receptors. Although not yet as clini-cally successful as ipilumumab, preclinical models showthat they also hold great promise, equal to that of thecheckpoint blockers. As discussed earlier, physiologicalligation of the TCR without CD28 engagement is insuffi-cient for full T-cell activation. However, a class of ‘super-agonist’ anti-CD28 mAbs has been developed which bindto a particular epitope in CD28 and can activate T-cellswithout concomitant TCR engagement. The initial clinicaltrial with superagonist anti-CD28 antibodies resulted inextremely rapid and dysregulated immune activation,leading to severe inflammatory reactions promoted by aso-called ‘cytokine storm’ (for further details see Eastwoodet al. [52]).

CD40 is expressed by APCs [B cells, dentritic cells(DCs), monocytes and macrophages], as well as non-haemopoietic tissues such as vascular endothelium andmultiple epithelia [53]. It is also often found on the surfaceof carcinoma and lymphoma cells [54]. The ligand, CD40L(CD154) is primarily expressed on activated CD4 T-cells andligand binding activates APCs [53, 54]. An intact CD40/CD40L system appears crucial for a normal B-cell humoralimmune response and DC activation. It fulfils critical func-tions in stimulating antigen presentation leading to immu-nity and priming of T-cells to drive full effector cytotoxic Tlymphocyte (CTL) responses [55, 56]. Incompletely acti-vated DCs cause T-cell anergy or depletion after an abor-tive transient activation [57]. As such, CD40 and DCactivation is a key regulator of many facets of immunestimulation (see Figure 2).

Agonist mAbs targeting CD40 have been shown tosubstitute for CD4 T-cell help by mimicking CD40L engage-ment [55, 56, 58] and can overcome T-cell tolerancein tumour-bearing mice, evoking highly effective CTLresponses, and augment the efficacy of anti-tumour vac-cines in pre-clinical models [59–62]. There are currentlyfour anti-CD40 mAbs undergoing early clinical trials;CP-870,893 (Pfizer), Chi Lob 7/4 (Cancer Research UKBiotherapeutics Development Unit), HCD122 (Novartis)and dacetuzumab (SGN-40, Seattle Genetics). All of theseappear relatively well-tolerated with some evidence ofgenerating objective or biochemical responses [63–66].However, their relative potency seems extremely wide-ranging with the maximum tolerated dose for CP-870,893

far lower than that for SGN-40 or Chi Lob 7/4. We [67] andothers [68] recently showed that interaction with theinhibitory Fc gamma receptor (FcγRIIB, CD32b) is critical forthe immunostimulatory and therapeutic activity of anti-CD40 mAb in mice. This new finding might explain thedifferences in mAb potency as immunoglobulin subclass(IgG 1, 2a, 2b, 3) have varying binding affinities to differentFc gamma receptors (FcγRI, FcγRIIB, FcγRIII and FcγRIV) [69].Currently it remains unclear how this will translate in thehuman system owing to our differences in IgG subclassand Fc receptor interactions from mice [69, 70]. This infor-mation will have significant implications for immuno-globulin selection in the design of the next generation ofimmunostimulatory mAbs if found to affect clinicalsuccess.

Interestingly, although much of the work in pre-clinicalmodels has indicated that anti-CD40 mAbs function byproducing effective CTL responses, a phase I pancreaticcancer trial of CP-870,893 in combination withgemcitabine showed therapeutic efficacy but suggestedan alternative mode of action. In this study,CD40 activationwas insufficient by itself to invoke an anti-tumour T-cellimmunity and instead the investigators concluded that themAb was re-educating macrophages to kill tumour andstromal cells [71].This highlights the role that innate immu-nity cells play in the tumour microenvironment and thepotential collateral effects these immunomodulatorymAbs might have on other cell types. For example, anti-CD40 mAbs could also directly target CD40 expressingtumour cells (e.g. lymphoma) causing apoptosis or deathvia ADCC or CDC and at the same time activate DCs whichmay be presenting tumour antigens to primed CD8 T-cells[54] (see Figure 2).

Other members of the TNFR superfamily are alsotargets with clinical potential for immunostimulatorymAbs, such as 4-1BB, OX40 and CD27. 4-1BB (CD137) is aco-stimulatory molecule that can function independentlyof CD28 [72]. It is stably expressed on activated T-cells aswell as other myeloid cells [73–76]. The cognate ligand(4-1BBL) is expressed on APCs like activated DCs, Bcells and macrophages. Signalling through 4-1BB/4-1BBLcauses T-cell expansion, cytokine induction and up-regulation of anti-apoptotic genes in order to preventactivation-induced cell death (AICD) of the expandingT-cells [77–79]. This permits downstream differentiation ofCTL as well as the establishment of long-lived memory[80]. Much remains to be understood of the biology andfunction of the 4-1BB/4-1BBL pathway as the result oftreatment with agonist anti-4-1BB mAbs is dependent onthe context. Anti-4-1BB mAbs have been shown to reduceB-cell numbers and function in an immunosuppressiverole against autoantibody production [81]. This paradoxi-cal effect has now been examined in a number of autoim-mune settings [82–85]. An early phase clinical trialexamining anti-cancer properties of an agonistic anti-4-1BB mAbs is on-going but early results testing a fully



humanized mAb, urelumab (BMS-663513, Bristol-MyersSquibb) in 83 patients with advanced cancers (54 mela-noma, 15 renal cell carcinoma, 13 ovarian cancer and oneprostate cancer) suggest activity at various dose rangeswith partial responses in three melanoma patients andstable disease in four patients in the expansion cohort. It iswell-tolerated but may cause reversible hepatic toxicity insome patients [86].

OX40 (CD134) is a TNF receptor expressed primarily onactivated CD4 and CD8 T-cells that transmits a potentco-stimulatory signal upon engagement of its ligandOX40L [87]. OX40L, the only known ligand for OX40, ismainly found on activated APCs [88–91]. In vivo, blockadeof OX40/OX40L interaction mitigates autoimmunity [92–94] and preclinical experiments confirmed enhancementof anti-tumour T-cell responses with OX40 agonist treat-ment [95]. An agonistic mouse mAb targeting OX40 hasnow undergone a first-in-man phase I dosing study in 30patients (NCT01644968). Limited data published so farsuggest the mouse mAb was tolerated in the study sub-jects (the maximum tolerated dose was not reached) withserological evidence of activation/proliferation of T-cells[96]. Clinical data published to date showed that twopatients (ovarian and melanoma) displayed stable diseasein the lowest dose cohort [97]. This approach will now betested in combination with radiotherapy in metastaticbreast cancer (NCT01642290) as well as in metastatic pros-tate cancer in combination with chemoradiotherapy(NCT01303705). As this is a mouse mAb, repeated dosingwill require a non-immunogenic humanized product ofwhich two are currently under development [96, 98].

CD27 is another TNF receptor that is expressed onT-cells, memory B cells, plasma cells and some natural killercells and engagement by its ligand CD70 leads to prolif-eration, survival and functional activation [99–102]. A fullyhuman agonistic mAb targeting CD27 (CDX-1127) hasbeen developed and has now entered a phase I study(NCT01460134).

It has become clear that the kinetics of clinicalresponses achieved with immunomodulatory mAbs areappreciably slower than those observed following chemo-therapy. Apparent initial tumour progression may beobserved, and it may be 2 to 3 months before regression isobserved. Responses, if seen, tend to be sustained. Earlyclinical trials using conventional early tumour responsetime points may underestimate the therapeutic effects ofthese agents and specific Immune Related Response Crite-ria have been proposed [103].

Toxicity: the dark side ofimmune stimulation

In comparison with most cytotoxic agents, mAbs are well-tolerated.Toxicities tend to be either acute hypersensitivityreactions at the time the mAb is infused, or delayed

immune-mediated toxicities, related to the mechanismof action of the mAb. Infusion reactions range from mildskin reactions, pyrexia and an influenza-like response, to, inrare instances, frank anaphylaxis, systemic inflammatoryresponse syndrome and life-threatening cytokine releasesyndrome. Close monitoring during infusions is required,but most such acute reactions are readily controlled withslowing or interruption of the infusion and appropriatesymptom management (see reference [104] for a clinicalmanagement review).

Immune related adverse events (IRAEs) as seen bypatients receiving ipilimumab are a new class of sideeffects particular to treatment using these novelimmunostimulatory or immunomodulatory mAbs. Osten-sibly this is due to a breakdown of immune self-tolerancealthough initial animal toxicology studies failed to predictsuch adverse effects. CTLA4 knock-out mice also didnot suffer from the common IRAEs in humans [105,106]. The most common serious manifestations includeenterocolitis with diarrhoea, hepatitis, dermatitis andendocrinopathies (hypophysitis, hypopituitarism, adrenalinsufficiency, hypothyroidism or hypogonadism). IRAEs arereported in up to 60% of patients treated with ipilimumab,with severe toxcities (grade 3 or 4) in about 10–15% ofpatients [23]. The clinical experience in the managementof IRAEs gained by investigators of ipilimumab in clinicaltrials have been published [107, 108] and managementalgorithms are also available at the FDA Risk Eliminationand Management System (REMS) website [109] or the UKElectronic Medicines Compendium website. The manage-ment strategy typically involves early detection of toxicity,interruption of mAb therapy, close clinical monitoring,early symptomatic relief and if appropriate, the timely useof corticosteroids to prevent rapid deterioration. Moderatetoxicities (grade 2) can usually be reversed successfullywith treatment interruption or with additional low dosecorticosteroids (prednisolone 0.5 mg kg−1 day−1 or equiva-lent) if symptoms do not resolve within a week. Severeor life-threatening (grade 3 or 4) toxicities require perma-nent cessation of mAb therapy and prompt treatmentwith high dose corticosteroid (prednisolone 1 to2 mg kg−1 day−1 or equivalent). The steroid should betapered gradually at least over 1 month as prematurewithdrawal may lead to a rebound inflammatory response[107]. Persistent inflammatory IRAEs may require addi-tional immune-suppression with infliximab, myco-phenolate or tacrolimus [107].

Perhaps not surprisingly, the occurrence of IRAEs hasbeen correlated with tumour reponse, with one studyreporting clinical responses in 33% of patients with grade3 or 4 IRAEs, and only 5% of patients with no evidence ofimmune toxicity [110]. Interestingly, clinical responseshave been maintained even when systemic immunosup-pression with corticosteroids has been required to treatIRAEs [111, 112]. The reason for this remains unclear and isthe subject of further investigation.

C. S. Lee et al.


Emerging effects of other immunecell types

Our discussion has so far focused on the effects on thesenovel mAbs on T-cells. However, other cell types also play arole in immune escape by interacting directly with thetumour cells or acting in tandem with other immunosup-pressive cells; the so-called immunosuppressive network[113]. These additional cell types include regulatory T-cells(Tregs), myeloid-derived suppressor cells (a heterogene-ous population of bone-marrow derived cells of myeloidlineage characterized by their potential to suppress T-cellresponses) and tumour associated macrophages. Theeffects of novel immunomodulatory mAbs on these otherimmune cell types would depend on their receptor-ligandexpression profile.CTLA4 is constitutively expressed on thecell surface of Tregs and functions as a potent mediator ofself-tolerance and immune homeostasis [114], which mayhelp explain the immune-related toxicity profile ofipilimumab. The potential activity of antagonistic CTLA4mAbs in depletion/inhibition of Tregs to remove Treg sup-pression and enhance antitumor immunity has been pro-posed. Murine models and early clinical studies proposingthis mechanism of action [115, 116] have not been borneout by more recent and larger clinical series [117–119]. Ithas also been proposed that OX-40 stimulation is requiredfor regulatory T-cell mediated control of colitis [120], whichhighlights the intriguing possibility of differential activa-tion by the agonistic OX-40 mAb on T-cell effector functionand T-cell regulation depending on organ/tumour loca-tion. As mentioned above CP-870,893 (anti CD40) acti-vated macrophages can lead to tumour cytolysis. Theexpression, distribution and function of the targets ofthese novel mAbs are still the subject of research whichwill shed further light on potential toxicities and currentlyunidentified mechanisms of actions. For example, a posi-tive co-stimulatory role for PD-L2 has been reportedwhereby PD-L2 deficient mice have impaired CTL functionwith increased tumour growth [121]. Another recentreport surprisingly demonstrated a favourable role in PD-1expressing T-cell infiltrates in human papilloma virus asso-ciated head and neck cancer [122].

Conclusion

The use of mAbs is firmly established as a treatment forcancer. The emergence of immunomodulatory mAbs as anovel class of drugs will see cancer immunotherapy takeon an increasingly important strategic role in cancer man-agement. They can be used to produce powerful tumour-specific T-cell responses with the hope of eradicatingestablished tumour and generating long term tumourimmunity. Our increasing understanding in the regulationof the immune system will inform future strategies inimmunomodulatory mAb therapies, including identifica-

tion of new targets and rational drug combinations.Already in pre-clinical models it has been demonstratedthat synergistic activity can be achieved through combina-tions of immunostimulatory and immunomodulatorymAbs [123, 124]. Potentially the more exciting role of theseagents could be in combination with small molecularinhibitors and/or cytotoxic chemotherapy agents whereclinical trials are already ongoing or are being planned[125, 126]. The activity of such agents combined with thedurable effects observed with the checkpoint blockers hasthe potential to achieve objective responses in a greaterproportion of patients than is currently the case.

Competing Interests

All authors have completed the Unified Competing Inter-est form at http://www.icmje.org/coi_disclosure.pdf anddeclare CSL has not received support from any organiza-tion for the submitted work, has no financial relationshipswith any organizations that might have an interest in thesubmitted work in the previous 3 years or any other rela-tionships or activities that could appear to have influencedthe submitted work. MC has not received support from anyorganization for the submitted work. He acts as a consult-ant to Bioinvent and Roche and has received institutionalsupport from Bioinvent for grants and patents. MG has notreceived support from any organization for the submittedwork. He receives an institutional grant from CancerResearch UK. He acts as a consultant to a number ofbiotech companies to write general antibody expertreports and he receives institutional payments and royal-ties from antibody patents and licenses. PJ has notreceived support from any organization for the submittedwork. He acts as a consultant to Roche and Pfizer and hasreceived payments for lectures from Millennium Takedaand Pfizer.

We thank Dr Juliet Gray and Dr Ruth French for theirhelpful suggestions and proof-reading of the manuscript.

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Novel antibodies targeting immune regulatory checkpoints for cancer therapy

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