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INTERNATIONAL UNION OF BASIC AND CLINICAL PHARMACOLOGY REVIEW Immunotherapy of cancer: from monoclonal to oligoclonal cocktails of anti-cancer antibodies: IUPHAR Review 18 Correspondence Yosef Yarden, Department of Biological Regulation, Candiotty Building, Weizmann Institute of Science, 234 Herzl Street, Rehovot 76100, Israel. E-mail: [email protected]

Received 17 September 2015; Revised 14 January 2016; Accepted 20 January 2016 This article is an NC-IUPHAR review contributed by the Immunopharmacology Section (ImmuPhar) of the International Union of Basic and Clinical Pharmacology (IUPHAR).

Silvia Carvalho1, Francesca Levi-Schaffer3, Michael Sela2 and Yosef Yarden1 1

Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel, 2Department of Immunology, Weizmann Institute of Science, Rehovot, Israel, and 3Pharmacology and Experimental Therapeutics Unit, Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The

Hebrew University of Jerusalem, Jerusalem, Israel

Antibody-based therapy of cancer employs monoclonal antibodies (mAbs) specific to soluble ligands, membrane antigens of Tlymphocytes or proteins located at the surface of cancer cells. The latter mAbs are often combined with cytotoxic regimens, because they block survival of residual fractions of tumours that evade therapy-induced cell death. Antibodies, along with kinase inhibitors, have become in the last decade the mainstay of oncological pharmacology. However, partial and transient responses, as well as emergence of tumour resistance, currently limit clinical application of mAbs. To overcome these hurdles, oligoclonal antibody mixtures are being tested in animal models and in clinical trials. The first homo-combination of two mAbs, each engaging a distinct site of HER2, an oncogenic receptor tyrosine kinase (RTK), has been approved for treatment of breast cancer. Likewise, a hetero-combination of antibodies to two distinct T-cell antigens, PD1 and CTLA4, has been approved for treatment of melanoma. In a similar vein, additive or synergistic anti-tumour effects observed in animal models have prompted clinical testing of hetero-combinations of antibodies simultaneously engaging distinct RTKs. We discuss the promise of antibody cocktails reminiscent of currently used mixtures of chemotherapeutics and highlight mechanisms potentially underlying their enhanced clinical efficacy.

Abbreviations ADC, antibody-drug conjugate; ADCC, antibody-dependent cellular cytotoxicity; ADPh, antibody-dependent phagocytosis; bsAb, bispecific antibodies; CDC, complement-dependent cytotoxicity; CDRs, complementary- determining regions; CRC, colorectal carcinoma; CTLA-4, cytotoxic T-lymphocyte associated protein-4; EFS, event free survival; ErbB, erythroblastic leukaemia viral oncogene homolog; FcγR, Fc-γ receptor; FDA, food and drug administration; HER, human EGF receptor; mAbs, monoclonal antibodies; MAC, membrane attack complex; NHL, non-Hodgkin’s lymphoma; NK, natural killer; NSCLC, non-small cell lung cancer; OS, overall survival; PD-1, programmed cell death-1; PFS, progression free survival; PKI, protein kinase inhibitor; RTKs, receptor TKs; T-DM1, trastuzumab emtansine; TIM-3, T-cell immunoglobulin and mucin domain 3; TNBC, triple-negative breast cancer

© 2016 The British Pharmacological Society

DOI:10.1111/bph.13450

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Tables of Links LIGANDS

TARGETS a

b

Amphiregulin

Epiregulin

Polatuzumab vedotin

BCL-2 family

EGFR

Atezolizumab

Erlotinib

Ramucirumab

CD19

HER2

AZD9291, osimertinib

Ibritumomab tiuxetan RANKL, RANK ligand

CD20

HER3

Bevacizumab

Ipilimumab

CTLA-4

MET

Blinatumomab

Lapatinib

Trastuzumab

PD-1, CD279

VEGFR2

Brentuximab vedotin

Nivolumab

Trastuzumab emtansine (T-DM1)

Capecitabine

Obinutuzumab

VEGF-A

Catumaxomab

Panitumumab

Venetoclax

Other proteins

Catalytic receptors

PD-L1, CD274

Cetuximab

Pembrolizumab

Docetaxel

Pertuzumab

Rituximab

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology. org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise a,b Guide to PHARMACOLOGY 2015/16 ( Alexander et al., 2015a,b).

Introduction Since 1997–1998 and the clinical approvals of rituximab (Maloney et al., 1997; McLaughlin et al., 1998) and trastuzumab (Baselga et al., 1996; Slamon et al., 2001), the field of immunotherapy has become one of the most dynamic and rapidly expanding areas of cancer therapy. Lack of adequate preclinical models that reliably simulate disease complexity and heterogeneity and the possibility that it is critical to target the bulk of tumour cells, rather than a small population of cancer stem cells, might underlie the rather low rates of drug approval (Gupta et al., 2009). Nevertheless, the accelerating pace of clinical approvals of new monoclonal antibodies (mAbs) is impressive. Tables 1, 2 list all antibodies that are currently applied in solid and in haematological malignancies respectively. Notably, approval by regulatory agencies, such as the United States Food and Drug Administration (FDA) and the European Medicines Agency, usually requires an overall survival (OS) benefit in phase-III trials, in comparison with the respective standard therapy. In addition, due to favourable tolerability profiles, most antibodies might be safely combined with standard cytotoxic treatments or with other antibodies and low MW inhibitors.

A primer to cancer therapy Surgery is the oldest and, until recently, the only cancer treatment able to cure patients with cancer. Moreover, improvements in radiation therapy and the development of systemic therapies that can control microscopic disease have allowed surgeons to substantially limit the magnitude of surgery necessary. Chemotherapy, the main mode of systemic therapy, might cure advanced cancer, especially when 1408

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combined with surgery and radiation therapy for locally advanced disease. However, toxicity to rapidly dividing and other normal cells, especially at high doses, limit clinical application of chemotherapeutic drugs. Molecular targeted therapies might overcome these limitations because they are designed to recognize specific molecules, which are either mutated in a malignant tissue or are otherwise critical for cancer cell survival. For example, the EGF receptor (EGFR) is mutated in a fraction of non-small cell lung cancer (NSCLC) (Lynch et al., 2004; Paez et al., 2004; Pao et al., 2004), and inhibitors that preferentially bind with mutant receptors effectively inhibit lung tumours. By contrast, EGFR of colorectal carcinoma (CRC) is rarely mutated. Yet, anti-EGFR antibodies can inhibit a fraction of CRC because EGFR is essential for survival of colon cells (Cunningham et al., 2004). The majority of approved targeted therapies fall into two classes: (i) mAbs, which are either naked or conjugated to a cytotoxic compound, and (ii) protein kinase inhibitors (PKIs), which are either mono-specific or designed to inhibit several different protein kinases (Levitzki and Mishani, 2006). Notably, while mAbs are remarkably specific to an antigen, target selectivity of PKIs varies depending on the target and concentration of the drug (Karaman et al., 2008). Yet another difference entails mechanisms that confer patient resistance to these two classes of drugs. Secondary mutations confined to the target kinase domains often confer resistance to PKIs, such as a secondary replacement of threonine790 of the EGFR by a methionine (T790M), which blocks target recognition by first generation kinase inhibitors like erlotinib (Kobayashi et al., 2005; Pao et al., 2005; Oxnard et al., 2011). By contrast, resistance to mAbs frequently involves mechanisms other than secondary mutations (Wheeler et al., 2008). One notable exception is an acquired EGFR ectodomain mutation (S492R) that prevents cetuximab binding and confers resistance to the antibody

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Table 1 Monoclonal antibodies currently approved for solid malignancies and their clinical indications

Antibody

Target

Approved clinical indication(s)

Cetuximab (Erbitux, ImClone LLC): chimeric human-murine IgG1

EGFR

For use in combination with chemotherapy for first-line treatment of patients with KRAS wild-type CRC (improved PFS, OS and ORR); in combination with chemotherapy for first line treatment of patients with locoregional and/or metastatic SCCHN (improved PFS, OS and ORR); for use in combination with radiation therapy for first line treatment of SCCHN (improved OS and duration of locoregional disease control); as a single agent in second or third line for the treatment of advanced SCCHN (improved durable objective tumour response).

Panitumumab (Vectibix, Amgem, Inc.): human IgG2

EGFR

For the treatment of patients with EGFR-expressing metastatic CRC with disease progression on/or following fluoropyrimidine, oxaliplatin and irinotecan containing chemotherapy regimens (improved PFS).

Necitumab (Portrazza, Eli Lilly and Co) recombinant human IgG1

EGFR

In combination with gemcitabine and cisplatin for first-line treatment of patients with metastatic squamous NCSLC (improved PFS and OS).

Pertuzumab (Perjeta, Genentech, Inc.): humanized IgG1

HER2

For use in combination with trastuzumab and chemotherapy for the neoadjuvant treatment of patients with HER2-positive, inflammatory or early stage breast cancer (improved pathological complete response); for use in combination with trastuzumab and docetaxel for patients with HER2-positive cancer who have not received prior anti-HER2 therapy or chemotherapy (improved PFS).

Trastuzumab (Herceptin, Genentech, Inc.): humanized IgG1

HER2

For use as single agent or in combination with chemotherapy for adjuvant treatment of HER2-positive breast cancer (improved DFS); for use in combination with chemotherapy for patients with HER2-overexpressing metastatic gastric or gastroesophageal junction cancer (improved OS).

Trastuzumab emtansine (Kadcyla, Genentech, Inc.): humanized IgG1

HER2

For use as a single agent for the treatment of patients with HER2-positive, metastatic breast cancer who previously received trastuzumab and a taxane, separately or in combination (improved PFS and OS).

Ramucirumab (CYRAMZA, Eli Lilly and Co.): recombinant human IgG1

VEGF-R2

For use in combination with chemotherapy for the treatment of patients with metastatic CRC whose disease has progressed on a first-line bevacizumab, oxaliplatin and fluoropyrimidine-containing regimen (improved PFS and OS); for use in combination with docetaxel for the treatment of patients with metastatic NSCLC with disease progression (improved PFS and OS); for use in combination with chemotherapy (paclitaxel) for the treatment of patients with advanced gastric or gastroesophageal junction adenocarcinoma (improved PFS and OS).

Bevacizumab (Avastin, Genentech, Inc.): humanized IgG1

VEGF-A

In combination with chemotherapy for the treatment of patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer (improved PFS); for the treatment of recurrent or metastatic cervical cancer, in combination with chemotherapy (improved OS); for use in combination with chemotherapy for treatment of patients with CRC whose disease has progressed on a first-line bevacizumab-containing regimen (improved PFS and OS); in combination with interferon α for the treatment of patients with metastatic renal cell carcinoma (improved PFS); in combination with chemotherapy for treatment of patients with advanced NSCLC (improved OS); in combination with chemotherapy, for the second-line treatment of metastatic carcinoma of the colon or rectum (improved OS); as a first-line treatment for patients with metastatic colorectal cancer (improved OS).

Denosumab (Xgeva, Amgem, Inc.): human IgG2

RANK-L

For the treatment of adults and skeletally mature adolescents with giant tumour of bone that is unresectable or where surgical resection is likely to result in severe morbidity (improved ORR); for prevention of skeleton-related events in patients with bone metastases from solid tumours (increased the time to first skeleton-related event).

Dinutuximab (Unituxin, United Therapeutics Corporation): chimeric human-murine IgG1

GD2

In combination with GM-CSF, IL-2 and 13-cis-retinoic acid, for the treatment of paediatric patients with high risk neuroblastoma who

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Table 1 (Continued)

Table 1. (Continued) Antibody Antibody

Target Target

Approved clinical indication(s) Approved clinical indication(s) achieve at least a partial response to prior first-line multi-agent, multimodality therapy (improved EFS and OS).

Ipilimumab (Yervoy, Bristol-Myers Squibb): human IgG1

CTLA-4

For the treatment of unresectable or metastatic melanoma (improved OS); in combination with nivolumab for the treatment of patients with BRAF V600 wild-type, unresectable or metastic melanoma (improved PFS and ORR).

Nivolumab (Opdivo, Bristol-Myers Squibb Company): human IgG4

PD-1

For the treatment of metastatic squamous or non-squamous NSCLC with progression on/or after platinum-based chemotherapy (improved OS); and unresectable or metastatic melanoma and disease progression following ipilimumab and a BRAF inhibitor (improved ORR and durabilty of response); in combination with ipilimumab for the treatment of patients with BRAF V600 wild-type, unresectable or metastatic melanoma (improved PFS and ORR); for the treatment of advanced renal cell carcinoma in patients who have received prior anti-angiogenic therapy (improved OS).

Pembrolizumab (Keytruda, Merck Sharp & Dohme Corp.): humanized IgG4

PD-1

For the treatment of unresectable or metastatic melanoma with or without disease progression following ipilimumab, if BRAF V600 positive, a BRAF inhibitor (improved PFS and ORR); for the treatment of patients with metastatic NSCLC with progression on/or after platinum-based chemotherapy, whose tumours express PD-L1 (improved ORR).

CRC, colorectal carcinoma; PFS, progression free survival; OS, overall survival; ORR, overall response rate; SCCHN, squamous cell carcinoma of head and neck; NSCLC, non-small cell lung cancer; EFS, event free survival; PD-L1, programmed death ligand 1.

(Montagut et al., 2012). Other mechanisms include activating mutations affecting downstream components, such as presence of mutant K-RAS proteins, which predict resistance of colorectal

cancer patients to two anti-EGFR antibodies, cetuximab and panitumumab (Amado et al., 2008; Karapetis et al., 2008; Lievre et al., 2008).

Table 2 Monoclonal antibodies currently approved for haematological malignancies and their clinical indications

Antibody

Target

Approved clinical indication(s)

Blinatumomab (Blincyto, Amgen, Inc.): mouse bi-specific T-cell engager

CD3/CD19

For the treatment of Philadelphia chromosomenegative relapsed or refractory B-cell precursor acute lymphoblastic leukaemia (achievement of durable complete remission and response).

Ofatumumab (Arzerra, GlaxoSmithKline): human IgG1

CD20

For the treatment of previously untreated patients with CLL (improved PFS).

Obinutuzumab (Gazyva, Genentech, Inc.): humanized IgG1

CD20

For CLL patients previously untreated or no longer responding to chemotherapy (improved PFS).

Rituximab (Mabthera, Roche): chimeric human-murine IgG1

CD20

For use in the first-line treatment of patients with diffuse large B-cell, CD20-positive, non-Hodgkin’s lymphoma or patients with low grade or follicular B-cell, CD20-positive non-Hodgkin’s lymphoma (improved PFS); for use in both previously treated or untreated patients with CLL (improved PFS).

90

CD20

For the treatment of patients with relapsed or refractory low-grade follicular or transformed B-cell non-Hodgkin’s lymphoma (improved ORR).

Y-labelled Ibritumomab tiuxetan (Zevalin, IDEC Pharmaceuticals): murine IgG1

CD30

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Table 2 (Continued)

Table 2. (Continued) Antibody Antibody Brentuximab vedotin (Adcetris, Seattle Genetics): chimeric IgG1

Target Target

Approved clinical indication(s) Approved clinical indication(s) For the treatment of relapsed Hodgkin’s lymphoma and relapsed systemic anaplastic large cell lymphoma (improved ORR).

Daratumumab (Darzalex, Janssen Biotech Inc.): human IgG1

CD38

Administered as a single agent for the treatment of patients with multiple myeloma, who have received at least three prior lines of therapy, including a proteasome inhibitor (PI) and an immunomodulatory agent (IA), or who are double-refractory to a PI and an IA (improved ORR).

Alemtuzumab (Campath, Genzyme): humanized IgG1

CD52

For the treatment of B-cell CLL (improved PFS, ORR and CRR).

Elotuzumab (Emplicit, BristolMyers Squibb): humanized IgG1

CD319

In combination with lenalidomide and dexamethasone for the treatment of patients with multiple myeloma who have received one to three prior therapies (improved PFS and OS).

CLL, chronic lymphocytic leukaemia; PFS, progression free survival; ORR, overall response rate; PI, proteasome inhibitor; IA, immunomodulatory agent; CRR, complete response rate; OS, overall survival.

Active and passive immunotherapy Antibody-mediated cancer therapy comprises at least two different approaches. The first, termed active immunization, is elicited following exposure to an antigen, which stimulates an immune response and induces immunological memory. This approach has, however, some disadvantages: the generation of primary immune response is delayed, rather than immediate, and it might require multiple exposures to an antigen. Active immunizations with peptide, protein or DNA-based vaccines, as well as whole tumour cell or dendritic vaccines, are in different stages of clinical trials (Diaz-Rubio, 2004; Liu et al., 2004; Oki and Younes, 2004; Sutton et al., 2004; Ueda et al., 2004; Mellman et al., 2011; Topalian et al., 2011; Milani et al., 2013). HER2 might serve as an example of active immunization: several studies showed that breast cancer patients are able to develop spontaneous anti-HER2 responses, including both T-cell and antibody-mediated immunities (Disis et al., 1994; Peoples et al., 1995). This prompted the notion that HER2 might be a suitable target for active immunization, which led to the development of several anti-HER2 vaccines. However, although safe and tolerable, to date, none of the available active immunological agents has generated superior clinical outcome in comparison with mainstay treatments. The other approach of mAb-mediated therapy of cancer, passive immunization, entails administration of recombinant mAbs that are specific to a particular antigen of interest. This approach confers immediate, albeit short-lived and limited protection, because treated individuals do not mount their own immunological responses. For instance, membrane-bound receptors, such as EGFR or HER2, can be inhibited with anti-receptor mAbs like cetuximab or trastuzumab respectively. Alternatively, soluble ligands of specific receptors may be neutralized, before they bind to their receptors, such as the mAb bevacizumab, which targets VEGF. Passive mAb-mediated therapy of cancer, employing

primarily anti-receptor mAbs, is already well established in the clinical practice (Scott et al., 2012) and will be the focus of this review.

Development of antibodies as pharmacological agents Antibodies are highly specific targeting molecules that provide key defence against pathogenic organisms and toxins. Initially, therapeutic mAbs of murine origin were generated using hybridoma technologies (Köhler and Milstein, 1975). Because of human anti-mouse responses, murine mAbs may not be repeatedly infused to patients. Later technological advances in protein engineering, transgenic mice and phage display, have led to the development of chimeric, humanized and eventually fully human mAbs, which accelerated development of antibodies as therapeutics (McCafferty et al., 1990; Hudson and Souriau, 2003; Lonberg, 2005). In recent years, we have learned that developing an immunotherapeutic molecule must be combined with identification of suitable biomarkers, which might be (i) prognostic, indicating whether patient outcome is good or poor, (ii) predictive, stratifying patients who are likely to benefit from the treatment, or (iii) pharmacodynamic, measuring treatment effects on tumours or guiding dose selection (Sawyers, 2008).

Murine, chimeric, humanized and fully human monoclonal antibodies Due to short half-lives in serum (Ober et al., 2001), immunogenicity in humans (Kuus-Reichel et al., 1994; Ober et al., 2001) and ineffective recruitment of human immune effector British Journal of Pharmacology (2016) 173 1407–1424

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responses (Kipps et al., 1985), only two radio-labelled murine antibodies, ibritumomab tiuxetan and tositumomab, have been approved for use in cancer therapy (Cersosimo, 2003; Stern and Herrmann, 2005). Grafting the entire murine variable regions or only the murine complementary-determining regions (CDRs) into a human IgG backbone will create a chimeric or a humanized antibody, respectively (Jones et al., 1986; Riechmann et al., 1988). Rituximab provides an example of a chimeric antibody. In 1997, it became the first recombinant monoclonal antibody approved for cancer therapy (Jazirehi and Bonavida, 2005). It targets CD20, a transmembrane protein expressed primarily by B-cells. Rituximab was initially approved for use against indolent Bcell non-Hodgkin’s lymphoma (NHL), but its use was later expanded to additional, more aggressive, clinical indications (Jazirehi and Bonavida, 2005). Cetuximab, another chimeric antibody, targets EGFR (Mendelsohn and Baselga, 2006). Trastuzumab and pertuzumab are two recombinant humanized monoclonal antibodies that target HER2 by binding to extracellular sub-domains IV and II, respectively (Cho et al., 2003; Baselga, 2010). Human mAbs have been generated using either transgenic mice, phage or yeast display technologies (Hudson and Souriau, 2003; Carter, 2006). Panitumumab and ipilimumab are just two examples of fully human mAbs. Panitumumab, an anti-EGFR antibody, blocks ligand binding to the receptor, hence preventing activation of downstream signalling and kinase cascades (Yang et al., 2001). Ipilimumab belongs to an emerging

therapeutic approach that blocks immune checkpoints and enhances anti-tumour immunity (Hodi et al., 2010).

Mechanisms of action of anti-cancer therapies harnessing mAbs: ADCC and ADPh Therapeutic monoclonal antibodies, other than immune checkpoint inhibitors, might contribute to the elimination of tumour cells by mobilizing two major classes of cellular mechanisms (Ben-Kasus et al., 2007; Scott et al., 2012; Shuptrine et al., 2012): (i) Immune mechanisms, such as induction of antibodydependent cellular cytotoxicity (ADCC), antibodydependent phagocytosis (ADPh) and complementdependent cytotoxicity (CDC; Figure 1). (ii) Non-immune mechanisms that directly intercept specific pathways essential for tumorigenesis (Figure 2). Class G immunoglobulins (IgGs) might activate ADCC through recognition of their Fc-domain by Fc-γ receptors (FcγRs) located on the surface of specific immune effector cells. The effector cells are recruited to an antibody-coated tumour cell and cause its lysis through release of granzymes and

Figure 1 Immunological mechanisms of action of therapeutic antibodies. Binding of monoclonal antibodies to antigens on the surface of target cells might induce complement binding (via C1q), for example, following binding of trastuzumab to HER2-overexpressing cancer cells. Similarly, antibodydependent cellular cytotoxicity (ADCC) requires interaction between the Fc portions of the antibody, for example cetuximab, with FcγR molecules expressed on the surface of effector cells, such as natural killer (NK) cells. A third mechanism, antibody-dependent phagocytosis (ADPh), enables macrophages to phagocytose tumour cells decorated by an antibody. Binding of an antigenic peptide to MHC molecules of cancer cells permits presentation to T-cells. The latter might be activated by antibody-mediated cross-presentation of an antigenic peptide to dendritic cells or inhibited through inhibitory receptors, such as CTLA-4 or PD-1. This inhibition can be blocked by the monoclonal antibodies ipilimumab (antiCTLA-4) or nivolumab (anti-PD-1), thus favouring T-cell activation. 1412

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Figure 2 Non-immunological mechanisms of action of anti-cancer therapeutic antibodies. Monoclonal antibodies able to recognise specific surface molecules of tumours, for example EGFR, might directly intercept pathways essential for tumorigenesis. One potential mechanism involves intracellular degradation of the respective surface antigens, which is preceded by slow endocytosis of antigen-mAbs complexes and leads to antigen degradation in lysosomes. Note that EGF binding to EGFR similarly induces rapid degradation of EGFRs, but unlike mAb-induced endocytosis, this is accompanied by receptor phosphorylation (shown by encircled P letters) and activation of downstream signalling. Therapeutic antibodies might also block growth factor binding or inhibit receptor dimerization, which would block receptor activation and downstream signalling pathways, leading to growth arrest and/or apoptosis. Angiogenesis can be inhibited by a monoclonal antibody (bevacizumab) to VEGF, or by a soluble VEGF-receptor (aflibercept). Note that also trastuzumab, an anti-HER2 antibody, might inhibit the ability of tumours to generate new blood vessels.

perforin (Kubota et al., 2009). This mechanism was previously associated with anti-tumour effects, in animals, of both trastuzumab and rituximab (Clynes et al., 2000). Moreover, specific polymorphic isoforms of FcγRs have been associated with altered patient response rates to mAb treatment (Cartron et al., 2002; Musolino et al., 2008). Large clinical studies have broadened these findings by associating the occurrence of polymorphisms with responses to several different antibodies: patients with B-cell non-Hodgkin’s lymphoma treated with rituximab (Weng and Levy, 2003), CRC patients treated with cetuximab (Zhang et al., 2007; Bibeau et al., 2009) and breast cancer patients treated with trastuzumab (Musolino et al., 2008). ADCC is primarily attributed to NK cells, but it has been suggested that monocytes and macrophages might also mediate ADCC (Ferris et al., 2010; Hubert et al., 2011). In addition, macrophages (and monocytes) can phagocytose tumour cells in the presence of mAbs. This mechanism is known as ADPh (Jiang et al., 2011). ADPh is enhanced after opsonization with antibodies, because macrophages express specific Fc receptors (Braster et al., 2014).

Complement-dependent cytotoxicity induced by anti-cancer mAbs CDC requires formation of a proteolytic cascade that lyses foreign cells through assembly of a membrane attack

complex (MAC) (Dunkelberger and Song, 2010). When antibodies bind to a cell, the classical complement pathway is activated through binding of the C1 complex, a serine protease consisting of C1q, C1r and C1s, to the antibody’s Fc-domain. MAC formation and subsequent release of anaphylins and opsonins results in cell lysis and phagocytosis (Dunkelberger and Song, 2010; Stoermer and Morrison, 2011). Reduced efficacy of rituximab in a lymphoma model was observed in animals lacking C1q, the first component of the complement pathway (Di Gaetano et al., 2003). Likewise, complement inhibitory proteins have been shown to inhibit rituximabmediated cell killing (Golay et al., 2001).

Antibody-mediated interception of pathways essential for tumourigenesis Tumour signalling might be disrupted when antibodies intercept growth and survival pathways, either through directly binding to angiogenic (Ferrara et al., 2004; Ellis, 2006) and other growth factors (Lindzen et al., 2010; Carvalho et al., 2016) or by blocking surface-localized receptors (Baselga, 2006; Arteaga et al., 2012) (Figure 2). Growth factor receptors are often overexpressed in tumours, and antagonistic antibodies, like cetuximab or trastuzumab, might inhibit their ability to mediate mitogenic signalling (Lane et al., 2000; Baselga, 2001; Yakes et al., 2002; Nagata et al., 2004). British Journal of Pharmacology (2016) 173 1407–1424

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Cetuximab and its murine parent, C225, function in model systems by preventing binding of activating ligands (Sunada et al., 1986) and by inhibiting receptor dimerization (Li et al., 2005). The findings that CRC patients with tumours that have high gene expression levels of epiregulin and amphiregulin, two EGFR-specific ligands, are more likely to have disease control on cetuximab treatment (KhambataFord et al., 2007) support receptor blockade models. Notably, no growth factor binds with high affinity to HER2 (Klapper et al., 1999), and accordingly, trastuzumab exerts its antitumour effects through mechanisms other than blocking ligand binding. These might include inhibition of receptor dimerization, accelerated endocytosis and degradation of HER2 in lysosomes (Klapper et al., 2000; Hudis, 2007; Ben-Kasus et al., 2009) or inhibition of angiogenesis (Izumi et al., 2002). Another class of anti-HER2 antibodies, which engage a site distinct from the domain recognized by trastuzumab, might inhibit tumours by preventing heterodimerization with EGFR and with other HER/ERBB family members (Klapper et al., 1997; Agus et al., 2015a). Malignant tumours secrete factors, such as VEGF, that enable them to control their own blood supply (angiogenesis), and blocking the action of these factors can inhibit tumour growth. For example, trastuzumab inhibits angiogenesis in a breast cancer model (Izumi et al., 2002) and bevacizumab, which blocks binding of VEGF-A to its receptor, inhibits formation of tumour vasculature (Ferrara et al., 2004; Ellis, 2006). Several therapeutic antibodies, including rituximab, have been implicated in the induction of apoptosis via the intrinsic (or mitochondrial) pathway, which leads to release of cytochrome C from mitochondria and down-regulation of anti-apoptotic BCL-2 family members (Bubien et al., 1993). In contrast, cetuximab increases expression of a pro-apoptotic protein, called BAX, and decreases abundance of BCL-2 (Huang et al., 1999). A promising new class of anticancer agents directly inhibits BCL-2. Venetoclax, a oral drug that mimics physiological antagonists of BCL-2, induced substantial responses in patients with relapsed chronic lymphocytic leukaemia (Roberts et al., 2015). A combination of venetoclax with obinutuzumab, a glycosylation engineered anti-CD20 mAb, is being evaluated in several clinical trials.

Resistance to anti-tumour antibodies The therapeutic utility and widespread use of mAbs is often limited by mechanisms of resistance. In primary resistance, a drug with proven efficacy fails to elicit visible response upon initial treatment, although the antigen is present on tumour cells. In acquired resistance, patients that were initially sensitive to treatment no longer respond. Impairment of immune effector mechanisms, such as ADCC (Levy et al., 2009; Ferris et al., 2010), CDC (Jurianz et al., 1999; Fishelson et al., 2003; You et al., 2011) or down-regulation of the target antigen, might confer resistance. Similarly important is adaptation of signalling circuitry. One comprehensively described example entails the KRAS mutational status of CRC tumours and sensitivity to two anti-EGFR antibodies, cetuximab and panitumumab (Amado et al., 2008; Karapetis et al., 2008; 1414

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Lievre et al., 2008). Similarly, activating PIK3CA mutations and PTEN loss were shown to mediate resistance of breast cancer to trastuzumab (Nagata et al., 2004; Berns et al., 2007). In addition, trastuzumab resistance may be associated with decreased levels of the cyclin-dependent kinase inhibitor, p27 (kip1) (Nahta et al., 2004b). Promiscuous signalling due to compensatory interactions between cell surface receptors (e.g. activation of MET, HER3 and VEGF-receptors) may also play major roles in loss of response to mAb treatment (Wheeler et al., 2008; Beck et al., 2010; Brand et al., 2011; Pillay et al., 2011). In conclusion, understanding the dynamic rewiring of intracellular pathways may provide insights into possible sequential or combinatorial treatments able to overcome resistance.

Antibody-drug conjugates Although the concept of antibody-drug conjugates (ADCs) is not new, it has recently become an especially active arena of new cancer therapies. ADCs combine the specificity of a mAb and the cell killing action of a cytotoxic agent. Using monoclonal and conventional antibodies, Sela and coworkers originally demonstrated enhanced anti-tumour effects of a mixture containing an antibody and chemotherapy (Aboud-Pirak et al., 1988), and other efforts exemplified efficacy of covalent drug-antibody conjugates (Tsukada et al., 1982; Tsukada et al., 1983). The features underlying clinical success of some new ADCs include target antigen selection, humanized antibody structures, conjugate internalization by tumour cells, drug potency and stability of the linker between drug and antibody. These improved agents are typically internalized and transported to intracellular organelles, where the drug is released. Upon release, the cytotoxic drug interferes with various cellular processes, leading to cell death (Sievers and Senter, 2013; Hamilton, 2015). Currently, there are two approved ADCs, brentuximab vedotin and trastuzumab emtansine. The former has been approved for treatment of patients with relapsed or refractory Hodgkin’s lymphoma or those with relapsed or refractory systemic anaplastic large cell lymphoma (Younes et al., 2010; de Claro et al., 2012). The antibody, an anti-CD30 monoclonal, is covalently coupled via a valine-citrulline peptide linker to the synthetic tubulin inhibitor monomethyl auristatin-E (MMAE). The approval for Hodgkin’s lymphoma was based on a trial that enrolled 102 patients who had CD30-positive Hodgkin’s lymphoma that relapsed after autologous stemcell transplantation (Younes et al., 2012). The approval for systemic anaplastic large cell lymphoma was based on a single-arm multicenter clinical trial that enrolled patients who had CD30-positive systemic anaplastic large cell lymphoma and had previously received front-line multi-agent chemotherapy (Pro et al., 2012). Of the 58 patients treated, 57% achieved a complete remission, and 17% achieved a partial remission. The HER2-targeted trastuzumab emtansine (T-DM1) further exemplifies the clinical potential of ADCs. Through a stable linker, trastuzumab is conjugated to the microtubule-inhibitory agent called DM1 (a derivative of maytansine) (Lewis Phillips et al., 2008; Junttila et al., 2011). This combination allows intracellular drug delivery specifically to HER2-overexpressing cells, in a way that not only retained all modes of action of trastuzumab, including

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ADCC, but also inhibited xenograft models resistant to trastuzumab and lapatinib (a HER2/EGFR-specific PKI). The EMILIA clinical trial compared T-DM1 with lapatinib plus chemotherapy and demonstrated significantly improved OS (12.6 months in the T-DM1 group vs. 6.3 months in the lapatinib plus capecitabine group) among patients with HER2-positive metastatic breast cancer (Verma et al., 2012). These results led to the approval of T-DM1 for metastatic breast cancer. Another trial compared T-DM1 with physicians’ treatment of choice in patients with progressive HER2positive advanced breast cancer, who had received two or more HER2-directed regimens. Patients receiving T-DM1 had a progression free survival (PFS) of 6.2 months compared with 3.3 months in patients from the control group (Krop et al., 2014). Taken together, these findings identify T-DM1 as a new standard option for HER2-positive, advanced-stage breast cancer patients who have previously received one or more HER2-directed agents. Currently, several ADCs are available, or they are being examined in clinical trials. For example, polatuzumab vedotin, an ADC containing an anti-CD79B mAb, has already been tested in relapsed or refractory B-cell non-Hodgkin’s lymphoma and chronic lymphocytic leukaemia (Palanca-Wessels et al., 2015).

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inhibitors have thus far been approved. An antibody against the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), ipilimumab, was approved in 2011, and two antibodies against the programmed cell death-1 (PD-1), pembrolizumab and nivolumab, were approved in 2014. Ipilimumab’s approval was based on two phase-III clinical trials that demonstrated improved OS in a group of advanced melanoma patients (Hodi et al., 2010; Robert et al., 2011). Furthermore, evidence supporting the durability of long-term survival in a proportion of ipilimumab-treated patients was obtained from a recent pooled analysis of OS (Schadendorf et al., 2015). Similarly, antibodies targeting the PD-1/PD-L1 axis have shown clinical responses in multiple tumour types. Both PD-1 ligands, PD-L1 and PD-L2, engage the PD-1 receptor and induce immune escape (Wherry, 2011). Blocking this interaction through PD-1 inhibitory antibodies, like pembrolizumab and nivolumab, or through the anti-PD-L1 antibody atezolizumab, might lead to enhanced tumour recognition by cytotoxic T-cells. Pembrolizumab was granted an accelerated approval for melanoma patients, based on results from clinical trials demonstrating an ability to improve PFS of ipilimumabrefractory melanoma patients (Hamid et al., 2013; Robert et al., 2014; Ribas et al., 2015). Approval of nivolumab for melanoma and for squamous-cell NSCLC followed phase-III trials that demonstrated improvements of OS in comparison with chemotherapy (Brahmer et al., 2015; Robert et al., 2015).

Immune checkpoint inhibitors Antibodies that restore T-cell responses against tumours represent a rapidly emerging anti-cancer strategy. Tumour cells usually escape the host immune system by up-regulating immune inhibitory signals. Importantly, the corresponding checkpoint inhibitors target host T-cells, rather than malignant cells; hence, mutation-driven mechanisms of resistance might not limit their clinical application (Hawkes et al., 2015; Sharma and Allison, 2015). Three immune checkpoint

Synergistic combinations of monoclonal antibodies The field of mAb-induced therapy of cancer has overcome many hurdles over the past 20 years. Although the development of patient resistance tainted the initial excitement, it also prompted the research community to gain insights into the

Figure 3 Mechanisms of action for homo-combination and hetero-combination of monoclonal antibodies. Combining two or three monoclonal antibodies that engage distinct, non-overlapping epitopes of the same receptor is termed a homo-combination mixture. Applying homo-combination mixtures on receptor tyrosine kinases, such as EGFR and HER2, might be associated with synergistic anti-tumour effects due to acceleration of receptor degradation or because of enhanced ADCC. By contrast, combinations of antibodies specific to distinct, yet functionally collaborating receptors (termed hetero-combinations), might similarly enhance ADCC and receptor degradation, but in addition, it might inhibit compensatory signals that often contribute to emergence of resistance to a single monoclonal antibody. British Journal of Pharmacology (2016) 173 1407–1424

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mechanisms of action of antibodies and ways to overcome this impediment. At a later point, we discuss an emerging resistance-overcoming strategy that utilizes concurrent treatment with two or more mAbs (Figure 3). The departure from a monoclonal scenario was initiated by attempts that combined mAbs able to engage two or more non-overlapping antigenic determinants of EGFR (Friedman et al., 2005) and HER2 (Spiridon et al., 2002). We will refer to such cocktails as homocombinations. Treatments making use of two or more antibodies targeting distinct, yet complementary receptors will be called hetero-combinations. A related technology employing engineered antibody molecules, called bispecific antibodies, is reviewed in the next section, prior to a fuller description of homo-combination and hetero-combination of mAbs.

Bispecific anti-cancer antibodies The idea behind construction of bispecific antibodies (bsAb) is to combine specificities of at least two antibodies in order to simultaneously engage different antigens or epitopes (reviewed by (Kontermann, 2012; Weidle et al., 2014; Kontermann and Brinkmann, 2015). Catumaxomab has been the first bsAb to receive approval, for i.p. treatment of patients with malignant ascites (Linke et al., 2010). It is an IgG-like rat-mouse hybrid targeting both EpCAM, an epithelial cell adhesion molecule, and CD3 of T-cells (Zeidler et al., 2000; Heiss et al., 2005; Seimetz et al., 2010). Although most patients develop anti-mouse (or anti-rat) antibody responses, surprisingly, this correlated with favourable clinical outcome (Ott et al., 2012). Another bsAb, blinatumomab, was granted an accelerated approval for treatment of acute lymphoblastic leukaemia (Topp et al., 2015), but patient treatment might be associated with neurotoxic and other adverse effects. Blinatumomab is a bispecific T-cell engager, which targets the CD19 antigen of B-cells and CD3 of T-cells. More than 30 investigational bsAbs are currently in clinical development (Spiess et al., 2015). They include agents intended to interact with two different soluble ligands, such as vanucizumab, a bsAb simultaneously blocking angiogenesis induced by angiopoietin-2 and VEGF-A (Spiess et al., 2015). Besides recruiting immune effector cells, bsAbs might also be directed at two different RTKs, for it is already established that cancer cells often escape growth inhibition caused by blockage of individual signalling pathways, by switching to another (Yarden and Sliwkowski, 2001). Two relevant examples, which have been discontinued, are MM-111 (McDonagh et al., 2012) and MEHD7945A (Schaefer et al., 2011; Huang et al., 2013b), which target HER2/HER3 and EGFR/HER3 respectively.

Homo-combinations of antibodies targeting RTKs Antibodies to EGFR. Combining two or more mAbs to EGFR (or HER2) enhanced receptor degradation and cancer cell inhibition, provided that the antibodies engaged distinct, non-overlapping epitopes (Friedman et al., 2005). Importantly, receptor internalization and subsequent degradation in lysosomes naturally inactivate RTKs, and oncogenic mutants of several RTKs frequently display relatively slow internalization rates (Mosesson et al., 2008; Abella and Park, 2009). Like other mAbs, cetuximab induces down-regulation of EGFR, and this might cause growth inhibition (Fan et al., 1993). Furthermore, experiments that 1416

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employed a radiolabelled cetuximab confirmed endocytosis of the mAb, but the internalized mAb recycled more effectively than internalized EGF (Jaramillo et al., 2006). Using a xenograft model of glioblastoma multiforme, it was found that a mixture of two antibodies, mAb 806, which recognizes a conformational epitope of EGFR and the truncated EGFRvIII mutant, and mAb 528, which acts as a ligand antagonist, resulted in additive and, in some cases, synergistic, anti-tumour activity (Perera et al., 2005). Interestingly, neither mAbs 806 nor mAb 528 induced down-regulation of the mutant EGFR when used as single agents. However, the combination of antibodies elicited a dramatic decrease in total cell surface receptors, both in vitro and in xenografts. This and similar observations have been translated to cancer therapy by Symphogen, a Danish pharmaceutical entity. Initially, they generated 24 anti-EGFR antibodies and tested dual and triple mixtures for inhibition of cancer cell growth (Koefoed et al., 2011). As a result, they identified a mixture of two mAbs (denoted Sym004) that displayed synergy in several assays: receptor degradation, anti-tumour effects in animals (Pedersen et al., 2010), ability to overcome resistance to cetuximab (Iida et al., 2013) and augmented response to radiation (Huang et al., 2013a). A parallel effort performed in our laboratories found that a combination of the clinically approved mAbs to EGFR, cetuximab and panitumumab, displayed no synergy in terms of receptor internalization (Ferraro et al., 2013). Hence, we selected a pair of synergistic mAbs that was active in an animal model of triple negative breast cancer (TNBC) (Ferraro et al., 2013), a fraction of which overexpresses EGFR (Reis-Filho et al., 2006). The last chapter of this avenue of research is a phase-I trial that enrolled patients with metastatic CRC and acquired EGFR inhibitor resistance. The results reported by this study confirmed marked Sym004induced EGFR down-regulation and partial response in 13% of patients (Dienstmann et al., 2015). Clearly, further clinical development of homo-combinations of anti-EGFR antibodies is warranted. It is important, however, to consider the mutational status of EGFR: perturbing mutant forms in NSCLC, using either third-generation inhibitors like AZD9291 (Janne et al., 2015) or cetuximab plus a PKI (Janjigian et al., 2014), yielded stronger therapeutic effects than the antitumour effects observed when intercepting the wild type form of EGFR. Further, enhancement of ADCC, by using glycoengineered monoclonals like imgatuzumab (GA201) (Gonzalez-Nicolini et al., 2015), might face both high toxicity and low efficacy. Conceivably, targeting mutant forms of EGFR using homo-combinations of mAbs would elicit stronger effects than attempts to target the un-mutated form. Antibodies to HER2. Early animal studies that tested a set of mAbs to the rodent form of HER2 indicated that individual mAbs cause partial tumour eradication, whereas the administration of certain antibody mixtures resulted in synergistic effects (Drebin et al., 1988). Similar effects on the human HER2 protein were later confirmed (Kasprzyk et al., 1992; Spiridon et al., 2002). In vitro, the more effective mAb mixture was also more effective than the single mAbs in inducing receptor degradation (Kasprzyk et al., 1992) and ADCC (Spiridon et al., 2002). Synergistic anti-tumour effects were confirmed, as well as associated with receptor

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degradation, using another set of mAbs to HER2 (Ben-Kasus et al., 2009). Similarly, testing two humanized anti-HER2 antibodies, trastuzumab and pertuzumab, alone and in combination, both in vitro (Nahta et al., 2004a) and in HER2-positive breast xenografts, revealed strongly enhanced inhibition of growth (Scheuer et al., 2009). Although the authors observed that trastuzumab and pertuzumab potently activated ADCC, they concluded that synergy was mainly due to the differing but complementary mechanisms of action, namely, inhibition of HER2 dimerization and prevention of HER2 cleavage. It is worthwhile noting, however, that pertuzumab affects endocytosis of both EGFR and HER3 (Sak et al., 2013). These pre-clinical studies were followed by several clinical tests. The CLEOPATRA trial investigated a combination of trastuzumab and docetaxel chemotherapy without or with addition of a second mAb to HER2, pertuzumab, in patients with HER2-positive metastatic breast cancer. The median PFS was significantly longer in the pertuzumab group than in the control group (18.5 months vs. 12.4 months respectively) (Baselga et al., 2012). Follow-up data at a median of 50 months indicated that the median overall survival was 56.5 months in the group receiving the pertuzumab combination, as compared with 40.8 months in the group receiving the placebo combination, an impressive difference of 15.7 months in favour of the pertuzumab group (Swain et al., 2015). These results led to approval of the homo-combination (pertuzumab and trastuzumab) plus docetaxel for treatment of HER2-positive breast cancer. To extend the label, the subsequent NeoSphere trial evaluated the homo-combination of mAbs, without or with docetaxel, in women with HER2-positive breast cancer in the neoadjuvant setting (Gianni et al., 2012). The results obtained clearly showed that patients receiving pertuzumab, trastuzumab and docetaxel had a significantly improved pathological response (46%) compared with the group receiving trastuzumab plus docetaxel (29%). However, contrary to predictions, the MARIANNE trial, which evaluated T-DM1 plus placebo versus T-DM1 plus pertuzumab, showed no added benefit of adding two antibodies.

Hetero-combinations of antibodies targeting receptor tyrosine kinases (RTKs) The rationale underlying high-order combinations of mAbs directed against different RTKs, including members of the HER/ERBB family, is rooted in the well-understood features of network biology and feedback control (Amit et al., 2007; Kholodenko et al., 2010). Inactivation of any single member of the family is expected to evoke compensatory pathways engaging other untargeted HER/ERBB proteins. Hence, simultaneous interception of both the primary receptor and the compensatory receptor(s) would be beneficial in terms of avoiding resistance to a single mAb. For example, resistance of CRC to cetuximab might be due to amplification of HER2 (Yonesaka et al., 2011), whereas resistance of lung cancer to an EGFR-specific PKI might involve amplification of MET and activation of HER3 (Engelman et al., 2005). In line with these observations, hetero-combinations of anti-EGFR and anti-HER2 mAbs synergistically inhibited pancreatic and other xenografts (Larbouret et al., 2012; Maron et al., 2013; Assenat et al., 2015). However, discouraging results were

BJP

obtained in a small clinical trial that recruited patients with metastatic pancreatic cancer (Assenat et al., 2015), probably due to the advanced state of disease and lack of patient selection on the basis of p53 or KRAS mutations. Signalling in trans often involves HER3, a kinase-defective member of the family, which undergoes compensatory shifts in phosphorylation–dephosphorylation equilibrium and increased delivery to the plasma membrane when EGFR is blocked (Sergina et al., 2007). HER3 involvement in acquirement of resistance to trastuzumab and other cancer drugs has been amply supported (Ritter et al., 2007; Narayan et al., 2009; Campbell et al., 2010; Schoeberl et al., 2010), and although several anti-HER3 mAbs entered clinical trials, currently no mAb has progressed to clinical approval. For example, lumretuzumab, a glycoengineered anti-HER3 monoclonal antibody (Meulendijks et al., 2016), failed to show added benefit when combined with the EGFR inhibitor erlotinib in a phase I/II NSCLC trial. In addition, it is still unclear whether homo-combinations of anti-HER3 antibodies are endowed with synergistic anti-tumour effects (D’Souza et al., 2014; Gaborit et al., 2015). Nevertheless, several nonclinical studies have attributed an advantage to heterocombinations containing an anti-HER3 component. For instance, our laboratory has shown that treatment of PKIresistant NSCLC with cetuximab elicits up-regulation of both HER2 and HER3, which over-activate ERK/MAPK, but a cocktail of three mAbs, against EGFR, HER2 and HER3, prevented activation of downstream signalling cascades, accelerated receptor degradation and markedly reduced growth of tumours in animal models (Mancini et al., 2015). A higher-order combination was introduced by Symphogen (Jacobsen et al., 2015). Their strategy entails simultaneous targeting of EGFR, HER2 and HER3 by using pairs rather than single mAbs, which translates into the application of a mixture of six mAbs. This pan-HER antibody mixture demonstrated potent activity in a variety of cancer animal models. In summary, future studies will need to resolve the clinical potential of pan-HER strategies making use of cocktails of 3–6 mAbs, multi-specificity kinase inhibitors like dacomitinib or combinations of mAbs and PKIs. Moreover, with the increasing availability of two antibodies approved for the same clinical indication, we might witness more examples of sequential/combinatorial mAb treatments, apart from T-DM1 (Kadcyla) following progression on trastuzumab/taxane. Examples might include administration of trastuzumab and ramucirumab (an anti-VEGFR antibody) in gastric cancer and either cetuximab and bevacizumab or cetuximab followed by Sym004 for metastatic CRC patients who acquired EGFR inhibitor resistance (Dienstmann et al., 2015).

Hetero-combinations of immune checkpoint inhibitors The CTLA-4 and PD-1 receptors regulate two non-redundant T-cell signalling pathways; hence, simultaneous dual blockade might be additive or even synergistic (Mahoney et al., 2015). This may be especially important for some tumour types, such as prostate cancer, in which single agents have a low level of activity (Callahan et al., 2014). Consistent with this scenario, combining CTLA-4 and PD-1 blockade had synergistic anti-tumour activity in a mouse model of colon British Journal of Pharmacology (2016) 173 1407–1424

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adenocarcinoma, as well as expanding infiltrating T-cells and reducing regulatory T and myeloid cells in a melanoma model (Curran et al., 2010). According to a recently proposed mechanism, CTLA-4 therapy drives T-cells into tumours and indirectly induces expression of PD-L1, which primes the tumour microenvironment to anti-PD-1/PD-L1 therapy (Sharma and Allison, 2015). Another mechanism might involve depletion of regulatory T-cells (Suarez et al., 2011). A phase-I clinical trial using ipilimumab in combination with nivolumab demonstrated tumour regression in a substantial proportion of patients with advanced melanoma when compared with monotherapy (Wolchok et al., 2013). Interestingly, the combination of antibodies might also be associated with an earlier onset of patient response. These results were supported by another study that compared a combination group (ipilimumab plus nivolumab) and an ipilimumab monotherapy group: among patients with BRAF wild-type tumours, the rate of objective response was 61% in the combination group versus 11% in the ipilimumab-monotherapy group (Postow et al., 2015). The increase in ORR, prolonged response duration and improvement in PFS led to the approval of the combination treatment, namely ipilimumab plus nivolumab, for patients who were previously untreated for unresectable or metastatic BRAF V600 wild-type melanoma. Similarly, encouraging results were reported by another randomized phase-3 study, which recruited untreated patients with metastatic melanoma (Larkin et al., 2015). However, treatmentrelated adverse events occurred in 16.3% of the patients in the nivolumab group, 55.0% of those in the nivolumabplus-ipilimumab group and 27.3% of those in the ipilimumab group. Further studies testing this combination in a variety of tumour types are ongoing. Moreover, tests of similar combinations and antibodies against other checkpoints are underway. For example, the antitumor activity of the anti-4-1BB/ anti-PD-1 combination has been compared with that of the anti-PD-1/anti-LAG-3 combination in a poorly immunogenic melanoma model (Chen et al., 2015). Another study combined in a murine ovarian cancer model antagonistic antiTIM-3 (T-cell immunoglobulin and mucin domain 3) antibodies and agonistic antibodies against CD137, a costimulatory receptor, which is transiently upregulated on Tcells following activation (Guo et al., 2013). In conclusion, more studies will need to confirm synergy and examine potential increased burden of toxicity associated with combined checkpoint blockade. In addition, it will be essential to identify suitable biomarkers indicative of pharmacodynamic activity and select clinical indications most responsive to specific combinations. Nevertheless, it is already clear that hetero-combinations of immune checkpoint inhibitors offer improved clinical outcome in patients with melanoma and possibly in additional clinical indications.

which are rarely achieved by low MW drugs. In addition, within mutation-prone tumour environments that rapidly generate resistance to low MW drugs, antibodies might not be as frequently evaded because their binding sites are relatively shallow and conformation-dependent. These reasons underlie, in part, the relatively low attrition rate of engineered antibodies, as compared with other drug families, in clinical trials (Kola and Landis, 2004). Although therapeutic antibodies are associated with several limitations, such as restriction to cell surface targets, i.v. delivery and high cost (DiMasi and Grabowski, 2007), this review exemplifies their enormous potential, from bispecific and mAb fragments to drug conjugates and adjuvant administration. Specifically, we focused upon a feature that might change the landscape of cancer therapy in the near future, namely concurrent delivery of several distinct antibodies. Individual antibodies in such cocktails might share an antigen molecule (homocombinations), or they might engage distinct, yet complementary antigens (hetero-combinations). Additive or synergistic effects observed when applying mAb mixtures might not be limited to RTKs and regulators of immune checkpoints. Moreover, in contrast to admixing other classes of drugs, because most clinically approved mAbs are human immunoglobulins of type G1 (with the exception of panitumumab and denosumab, both are IgG2 and two IgG4 antibodies, nivolumab and pembrolizumab), the components of mAb mixtures are highly similar (>90% identity). Thus, for their high efficacy, acceptable safety and favourable pharmacological features, we predict that cocktails of carefully selected mAbs will occupy an important domain in the future armamentarium of medical oncologists.

Acknowledgements We thank Nadege Gaborit, Ruth Maron, Dan Aderka and Bilha Schechter for their collaboration. Our laboratories are supported by the European Research Council, the Israel Cancer Research Fund the Rosetrees Foundation, the Israel Science Foundation, the Israel Cancer Association and the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation. Y.Y. is the incumbent of the Harold and Zelda Goldenberg Professorial Chair, and M.S. is the incumbent of the W. Garfield Weston Chair. Yarden’s team is located at the Marvin Tanner Laboratory for Research on Cancer. NC-IUPHAR receives financial support from the Wellcome Trust.

Conflict of interest Y.Y. served as consultant of Symphogen and received grants from Merck-Serono. All other authors declare no conflicts of interest.

Concluding remarks The current accelerating pace of approval of new anti-cancer antibodies promises an avalanche of novel drugs that are safe and highly effective in defined populations of patients. Due to the nature of biological recognition, antibodies offer high affinity and avidity, along with selectivity to target molecules, 1418

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Immunotherapy of cancer: from monoclonal to oligoclonal cocktails of anti-cancer antibodies: IUPHAR Review 18.

Antibody-based therapy of cancer employs monoclonal antibodies (mAbs) specific to soluble ligands, membrane antigens of T-lymphocytes or proteins loca...
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