Since the mid‑1990s, antibodies have grown steadily into a clinically and commercially successful drug class. Over 60 antibody‑based drugs have been approved for therapeutic use and are currently marketed, with worldwide revenues of ~US$89 billion in 2016 (TABLE 1). Historically, the success rate of humanized and human antibodies from first‑in‑human studies to regulatory approval has been at least 15%1. Thus, many new anti‑body drugs are expected from the extensive pipeline of over 550 antibodies in clinical development (J. Reichert, personal communication), including more than 50 anti‑bodies in phase III clinical trials2.

Immunoglobulin G (IgG) is the predominant molecular format used in current antibody drugs. A few antibody conjugates (including those conjugated to cytotoxic drugs, radioisotopes or polyethylene glycol (PEG)), antibody fragments and bispecific antibodies have also been approved (FIG. 1). The proportion of anti‑bodies with non‑IgG formats is higher for antibodies in early clinical development, although IgG still pre‑dominates3. At least 30 antibody drugs are indicated for use in oncology, including for the treatment of many prevalent solid and haematological tumours (TABLE 1). An approximately similar number of antibodies are approved for the treatment of chronic inflammatory or autoimmune diseases. A few antibody drugs are being used to treat patients in other areas of medicine includ‑ing cardiovascular disorders, infectious and ophthalmic diseases, osteoporosis, as well as transplantation.

One consequence of the approval of so many anti‑body drugs is that most share a therapeutic target with at least one other antibody or protein therapeutic. For example, there are three to six marketed antibody drugs

for each of the most widely pursued targets, namely, B lymphocyte antigen CD20, epidermal growth fac‑tor receptor (EGFR), human epidermal growth factor receptor 2 (HER2; also known as ERBB2), programmed cell death 1 ligand 1 (PDL1) and tumour necrosis factor (TNF), not including biosimilar antibodies or the Fc fusion protein etanercept that targets TNF and lymphotoxin‑α. In several cases, different antibody drugs engage compo‑nents of the same pathway, such as a receptor or its cor‑responding ligand. Overlap in the approved indications for antibodies is even more widespread. For example, at least nine antibody drugs targeting interleukin‑17A (IL‑17A), IL‑17 receptor A (IL‑17RA), TNF, CD6 IL‑23 or a subunit shared by IL‑12 and IL‑23 are approved for the treatment of different forms of psoriasis, whereas seven or more antibodies targeting CD20, IL‑6R or TNF are indicated for rheumatoid arthritis.

This crowded marketplace for antibody drugs has created a strong incentive to develop second‑generation antibodies that are substantially improved and well differentiated from their first‑generation counterparts. The recent or pending patent expiration for several so‑called blockbuster antibody drugs has motivated the development of many biosimilar antibodies, including a few that have already been approved4. In addition, cost‑effectiveness analysis is being increasingly applied to assess the benefits of new drugs5.

Major factors affecting the development of anti‑body drugs include the selection of antibody mecha‑nisms of action (MoAs) that exploit the target biology, known target biochemistry and adequate exposure of the target to active antibody (FIG. 2). Many of the most tractable and best understood secreted or membrane

Department of Antibody Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.pjc@gene.com; lazarg@gene.com

doi:10.1038/nrd.2017.227Published online 1 Dec 2017

Biosimilar antibodiesAntibodies produced using different clones and cell lines than the original approved antibody product of identical amino acid sequence, often by a different manufacturer.

BlockbusterA term used for pharmaceuticals to refer to those drugs with annual sales of at least US$1 billion per year.

Mechanisms of action(MoAs). Specific biochemical interactions and biological processes through which a drug elicits its pharmacologic effects.

Next generation antibody drugs: pursuit of the ‘high-hanging fruit’Paul J. Carter and Greg A. Lazar

Abstract | Antibodies are the most rapidly growing drug class and have a major impact on human health, particularly in oncology, autoimmunity and chronic inflammatory diseases. Many of the best understood and most tractable cell surface and secreted targets with known roles in human diseases have been extensively exploited for antibody drug development. In this Review, we focus on emerging and novel mechanisms of action of antibodies and innovative targeting strategies that could extend their therapeutic applications, including antibody–drug conjugates, bispecific antibodies and antibody engineering to facilitate more effective delivery. These strategies could enable the pursuit of difficult to hit, less well-understood or previously undruggable targets — the ‘high-hanging fruit’.

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mailto:pjc@gene.commailto:lazarg@gene.comhttp://dx.doi.org/10.1038/nrd.2017.227

Table 1 | Antibody drugs marketed for therapeutic use or undergoing regulatory review

INN (trade name)

2016 worldwide sales (US$ million)

Companies Antigens Antibody format (dosing route)

Approved indications (or potential first indication)

Proposed MoA highlights

Oncology

Mogamulizumab (Poteligeo)

18 Kyowa Hakko Kirin

CCR4 Humanized, glyco-engineered IgG1, κ-chain (i.v.)

Adult T cell leukaemia or lymphoma

Depletes target cells by ADCC, afucosylated for increased ADCC

Blinatumomab (Blincyto)

115 Amgen CD3 and CD19

Mouse bispecific tandem scFv: BiTE (c.i.v.)

ALL Mediates formation of a T lymphocyte–tumour cell synapse that results in tumour cell lysis

Rituximab (Rituxan and MabThera)

7,482 Roche/Genentech, Pharmstandard

CD20 Chimeric IgG1, κ-chain (i.v.)

Non-Hodgkin lymphoma, CLL, rheumatoid arthritis, granulomatosis with polyangiitis and microscopic polyangiitis

Depletes target cells by ADCC, CDC and inducing apoptosis

Ofatumumab (Arzerra)

46 Novartis CD20 Human, mouse-derived IgG1, κ-chain (i.v.)

CLL Depletes target cells by CDC and ADCC

Obinutuzumab (Gazyva)

199 Roche CD20 Humanized, glyco-engineered IgG1, κ-chain (i.v.)

CLL, follicular lymphoma Depletes target cells by ADCC, CDC, ADCP and inducing apoptosis; reduced fucosylation for increased ADCC

Ibritumomab tiuxetan (Zevalin)

11 Spectrum Pharmaceuticals

CD20 Mouse IgG1, κ-chain; 90Y-containing radioimmunocon-jugate (i.v.)

Non-Hodgkin lymphoma Radiation from 90Y induces cellular damage

Tositumomab (Bexxar)

8 Novartis CD20 Mouse IgG2a, λ-chain; 131I-containing radioimmunocon-jugate (i.v.)

Non-Hodgkin lymphoma Radiation from 131I induces cell death, possibly through ADCC, CDC and inducing apoptosis

Inotuzumab ozogamicin (Besponsa)

NA Pfizer, UCB CD22 Humanized IgG4, κ-chain; ADC (i.v.)

ALL The cytotoxin, calicheamicin, induces dsDNA breaks, leading to cell cycle arrest and apoptosis

Brentuximab vedotin (Adcetris)

544 Seattle Genetics, Takeda

CD30 Chimeric IgG1, κ-chain; ADC (i.v.)

Hodgkin lymphoma, systemic anaplastic large cell lymphoma

The cytotoxin, MMAE, disrupts microtubules, leading to cell cycle arrest and apoptosis; depletes target cells by ADCP

Gemtuzumab ozogamicin (Mylotarg)

NA Pfizer, Wyeth, Takeda, UCB, Celltech Group, PDL BioPharma, Fred Hutchinson Cancer Research Center

CD33 Humanized IgG4, κ-chain; ADC (i.v.)

AML The cytotoxin, calicheamicin, induces dsDNA breaks, leading to cell cycle arrest and apoptosis

Daratumumab (Darzalex)

572 Johnson & Johnson

CD38 Human, transgenic mouse-derived IgG1, κ-chain (i.v.)

Multiple myeloma Depletes target cells by CDC, ADCC, ADCP and inducing apoptosis

Ipilimumab (Yervoy)

1,053 Bristol-Myers Squibb

CTLA4 Human, transgenic mouse-derived IgG1, κ-chain (i.v.)

Melanoma Binds and antagonizes receptor; augments T lymphocyte activation and proliferation

Cetuximab (Erbitux)

1,555 Eli Lilly & Co., Merck KGaA

EGFR Chimeric IgG1, κ-chain (i.v.)

Colorectal cancer, head and neck cancer

Binds and antagonizes receptor; inhibits cell proliferation; induces apoptosis; sensitizes cells to chemotherapy and radiotherapy; decreases VEGFA production; depletes target cells by ADCC

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Table 1 (cont.) | Antibody drugs marketed for therapeutic use or undergoing regulatory review

INN (trade name)

2016 worldwide sales (US$ million)

Companies Antigens Antibody format (dosing route)

Approved indications (or potential first indication)

Proposed MoA highlights

Oncology (cont.)

Panitumumab (Vectibix)

785 Amgen, Takeda EGFR Human, transgenic mouse-derived IgG2, κ-chain (i.v.)

Colorectal cancer Binds, antagonizes and downregulates receptor; inhibits cell proliferation; induces apoptosis; decreases pro-inflammatory cytokine and VEGF production

Necitumumab (Portrazza)

15 Eli Lilly & Co. EGFR Human, phage-derived IgG1, κ-chain (i.v.)

Squamous NSCLC Binds, antagonizes and induces internalization and degradation of receptor; depletes target cells by ADCC; increases sensitivity to chemotherapy (in in vivo models)

Nimotuzumab (TheraCIM, BIOMAb-EGFR)

NA Biocon, PT Kalbe Farma, Ferozsons Laboratories, Probiotec

EGFR Humanized IgG1, κ-chain (i.v.)

Glioma, head and neck, nasopharyngeal and pancreatic cancers

Binds and antagonizes receptor; anti-angiogenic, antiproliferative and proapoptotic effects; decreases motility, cell invasion and metastasis in tumours that overexpress EGFR

Dinutuximab (Unituxin)

63 United Therapeutics

GD2 Chimeric IgG1, κ-chain (i.v.)

Neuroblastoma Depletes target cells by ADCC and CDC

Trastuzumab (Herceptin)

6,884 Genentech/Roche

HER2 Humanized IgG1, κ-chain (i.v.)

Breast, gastric and gastroesophageal junction cancers

Inhibits tumour cell growth in vitro and in vivo; depletes target cells by ADCC

Pertuzumab (Perjeta)

1,874 Genentech/Roche

HER2 Humanized IgG1, κ-chain (i.v.)

Breast cancer Binds and antagonizes receptor; arrests cell proliferation; induces apoptosis; depletes target cells by ADCC; augments antitumour activity of trastuzumab in xenograft models

Ado-trastuzumab emtansine (Kadcyla)

843 Genentech/Roche

HER2 Humanized IgG1, κ-chain; ADC (i.v.)

Breast cancer The cytotoxin, DM1, disrupts the microtubule network in cells leading to cell cycle arrest and apoptosis; inhibits HER2 signalling; depletes target cells by ADCC; inhibits shedding of HER2

Name pending (Xilonix)

NA XBiotech, Megapharm

IL-1α Human B cell-derived IgG1, κ-chain (i.v.)

(Advanced colorectal cancer)

Binds and neutralizes ligand

Nivolumab (Opdivo)

4,735 Bristol-Myers Squibb, Ono Pharmaceutical

PD1 Human, mouse-derived IgG4, κ-chain (i.v.)

Melanoma, Hodgkin lymphoma, NSCLC and renal cell, head and neck squamous cell, urothelial and hepatocellular cancers, MSI-high or MMR-deficient colorectal cancer

Binds and antagonizes receptor; releases inhibition of the immune response, including the antitumour response

Pembrolizumab (Keytruda)

1,402 Merck & Co. PD1 Humanized IgG4, κ-chain (i.v.)

Melanoma, Hodgkin lymphoma, NSCLC and head and neck squamous cell, urothelial and gastric cancers, MSI-high or MMR-deficient cancers

Binds and antagonizes receptor; releases inhibition of the immune response, including the antitumour response

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Table 1 (cont.) | Antibody drugs marketed for therapeutic use or undergoing regulatory review

INN (trade name)

2016 worldwide sales (US$ million)

Companies Antigens Antibody format (dosing route)

Approved indications (or potential first indication)

Proposed MoA highlights

Oncology (cont.)

Atezolizumab (Tecentriq)

159 Genentech/Roche

PDL1 Human, phage-derived, aglycosylated IgG1, κ-chain (i.v.)

Urothelial cancer and NSCLC

Binds and neutralizes ligand; releases inhibition of the immune response, including the antitumour response, without inducing ADCC

Avelumab (Bavencio)

NA Pfizer, Merck KGaA, Shire

PDL1 Human, phage-derived IgG1, λ-chain (i.v.)

Merkel cell and urothelial cancers

Binds and neutralizes ligand; releases inhibition of the immune response, including the antitumour immune response; depletes target cells by ADCC

Durvalumab (Imfinzi)

NA AstraZeneca PDL1 Human, transgenic mouse-derived IgG1, κ-chain (i.v.)

Urothelial cancer Binds and neutralizes ligand; releases inhibition of immune response; Fc-engineered to attenuate effector functions

Olaratumab (Lartruvo)

12 Eli Lilly & Co. PDGFRα Human, transgenic mouse-derived IgG1, κ-chain (i.v.)

Soft tissue sarcoma Binds and antagonizes receptor; in vitro and in vivo antitumour activity

Denosumab (Prolia, Pralia, Xgeva, Ranmark)

3,459 Amgen, Daiichi Sankyo

RANKL Human, transgenic mouse-derived IgG2, κ-chain (s.c.)

Skeletal events in patients with bone metastases from solid tumours; patients with osteoporosis at high risk of bone fracture

Binds and neutralizes ligand; decreases bone resorption and increases mass and strength of some bones

Elotuzumab (Empliciti)

150 Bristol-Myers Squibb

SLAMF7 Humanized IgG1, κ-chain (i.v.)

Multiple myeloma Activates NK cells through SLAMF7 pathway and Fc receptors; depletes target cells by ADCC

Bevacizumab (Avastin)

6,885 Genentech/Roche

VEGFA Humanized IgG1, κ-chain (i.v.)

Non-squamous NSCLC and colorectal, renal cell, cervical ovarian, fallopian tube and peritoneal cancers and glioblastoma

Binds and neutralizes ligand; reduces microvascular growth and inhibits metastatic disease progression in mouse xenografts

Ramucirumab (Cyramza)

614 Eli Lilly & Co. VEGFR2 Human, phage-derived IgG1, κ-chain (i.v.)

NSCLC and gastric and colorectal cancers

Binds and antagonizes receptor; inhibits ligand-induced proliferation and endothelial cell migration; inhibits angiogenesis in an animal model

Autoimmunity & inflammation

Natalizumab (Tysabri)

1,964 Biogen α4β1 and α4β7 integrins

Humanized IgG4, κ-chain (i.v.)

Multiple sclerosis and Crohn’s disease

Binds and antagonizes receptors; inhibits leukocyte adhesion

Vedolizumab (Entyvio)

1,324 Takeda α4β7 integrin Humanized IgG1, κ-chain (i.v.)

Ulcerative colitis and Crohn’s disease

Binds and antagonizes receptor; inhibits leukocyte adhesion

Belimumab (Benlysta)

414 GlaxoSmithKline BAFF Human, phage-derived IgG1, λ-chain (i.v.)

Systemic lupus erythematosus

Binds and neutralizes ligand; inhibits survival of B lymphocytes

Itolizumab (Alzumab)

Unknown; not reported

Biocon, CIMAB, Probiotec

CD6 Humanized IgG1 (i.v.)

Plaque psoriasis Modulates T lymphocyte activation and proliferation induced by CD6 co-stimulation

Ocrelizumab (Ocrevus)

NA Genentech/Roche, Biogen, PDL BioPharma

CD20 Humanized IgG1, κ-chain (i.v.)

Multiple sclerosis Depletes B lymphocytes by ADCC and CDC

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Table 1 (cont.) | Antibody drugs marketed for therapeutic use or undergoing regulatory review

INN (trade name)

2016 worldwide sales (US$ million)

Companies Antigens Antibody format (dosing route)

Approved indications (or potential first indication)

Proposed MoA highlights

Autoimmunity & inflammation (cont.)Alemtuzumab (Lemtrada)

470 Sanofi CD52 Humanized IgG1, κ-chain (i.v.)

Multiple sclerosis Depletes target cells by ADCC and CDC

Omalizumab (Xolair)

2,356 Genentech/Roche, Novartis

IgE Humanized IgG1, κ-chain (s.c.)

Asthma and chronic idiopathic urticaria

Binds and neutralizes IgE; reduces degranulation of mast cells and basophils; downregulation of receptor (FcεRI)

Canakinumab (Ilaris)

283 Novartis IL-1β Human, transgenic mouse-derived IgG1, κ-chain (s.c.)

Periodic fever syndromes and systemic JIA

Binds and neutralizes ligand

Daclizumab (Zinbryta)

17 Biogen, AbbVie IL-2R Humanized IgG1, κ-chain (s.c.)

Multiple sclerosis Binds and antagonizes receptor

Dupilumab (Dupixent)

NA Regeneron Pharmaceuticals, Sanofi

IL-4Rα Human, transgenic mouse-derived IgG4, κ-chain (s.c.)

Atopic dermatitis Binds and antagonizes receptor

Reslizumab (Cinqair)

5 Merck & Co., Teva, UCB

IL-5 Humanized IgG4, κ-chain (i.v.)

Eosinophilic asthma Binds and neutralizes ligand

Mepolizumab (Nucala)

138 GlaxoSmithKline IL-5 Humanized IgG1, κ-chain (s.c.)

Eosinophilic asthma Binds and neutralizes ligand

Benralizumab (Fasenra)

NA MedImmune, AstraZeneca, Kyowa Hakko Kirin, Lonza

IL-5Rα Humanized, glyco-engineered IgG1, κ-chain (s.c.)

Asthma Binds and antagonizes receptor; depletes target cells by ADCC; afucosylated for increased ADCC

Sirukumab (pending)

NA GlaxoSmithKline, Johnson & Johnson

IL-6 Human (source not reported) IgG1, κ-chain (s.c.)

(Rheumatoid arthritis) Binds and neutralizes ligand

Siltuximab (Sylvant)

NA Johnson & Johnson

IL-6 Chimeric IgG1, κ-chain (i.v.)

Castleman disease Binds and neutralizes ligand

Sarilumab (Kevzara)

NA Regeneron Pharmaceuticals, Sanofi

IL-6R Human, transgenic mouse-derived IgG1, κ-chain (s.c.)

Rheumatoid arthritis Binds to membrane-bound and soluble forms of IL-6R and inhibits IL-6 binding

Tocilizumab (RoActemra, Actemra)

1,713 Chugai/Roche IL-6R Humanized IgG1, κ-chain (i.v. or s.c.)

Rheumatoid arthritis, giant cell arteritis, systemic and polyarticular JIA, cytokine release syndrome associated with CAR T cell therapy

Binds to membrane-bound and soluble forms of IL-6R and inhibits IL-6 binding

Ustekinumab (Stelara)

3,232 Johnson & Johnson

IL-12 and IL-23

Human, transgenic mouse-derived IgG1, κ-chain (i.v. or s.c.)

Psoriasis, psoriatic arthritis and Crohn’s disease

Binds and neutralizes ligand

Ixekizumab (Taltz) 113 Eli Lilly & Co. IL-17A Humanized IgG4, κ-chain (s.c.)

Plaque psoriasis Binds and neutralizes ligand

Secukinumab (Cosentyx)

1,128 Novartis IL-17A Human, mouse-derived IgG1, κ-chain (s.c.)

Plaque psoriasis, psoriatic arthritis and ankylosing spondylitis

Binds and neutralizes ligand

Brodalumab (Siliq) NA Valeant Pharmaceuticals, Amgen, AstraZeneca

IL-17RA Human, mouse-derived IgG2, κ-chain (s.c.)

Plaque psoriasis Binds and antagonizes receptor

Guselkumab (Tremfya)

NA MorphoSys, Johnson & Johnson

IL-23 p19 Human, phage-derived IgG1, λ-chain (s.c.)

Plaque psoriasis Binds and neutralizes ligand

Tildrakizumab (pending)

NA Merck & Co., Almirall, Sun Pharmaceutical Industries, Schering-Plough

IL-23 p19 Humanized IgG1, κ-chain (s.c.)

(Plaque psoriasis) Binds and neutralizes ligand

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Table 1 (cont.) | Antibody drugs marketed for therapeutic use or undergoing regulatory review

INN (trade name)

2016 worldwide sales (US$ million)

Companies Antigens Antibody format (dosing route)

Approved indications (or potential first indication)

Proposed MoA highlights

Autoimmunity & inflammation (cont.)

Infliximab (Remicade)

8,070 Merck & Co., Johnson & Johnson, Mitsubishi Tanabe Pharma

TNF Chimeric IgG1, κ-chain (i.v.)

Crohn’s disease, ulcerative colitis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and plaque psoriasis

Binds soluble and transmembrane forms of TNF; inhibits binding to TNFR; depletes TNF-positive cells

Adalimumab (Humira)

16,515 AbbVie, Eisai TNF Human, phage-derived IgG1, κ-chain (s.c.)

Rheumatoid arthritis, JIA, psoriatic arthritis, ankylosing spondylitis, Crohn’s disease, ulcerative colitis, plaque psoriasis, hidradenitis suppurativa and uveitis

Binds soluble and transmembrane forms of TNF; inhibits binding to TNFR; depletes TNF-expressing cells in presence of complement

Certolizumab pegol (Cimzia)

1,446 UCB TNF PEGylated, humanized Fabʹ, κ-chain (s.c.)

Crohn’s disease, rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis

Binds soluble and transmembrane forms of TNF; inhibits binding to TNFR

Golimumab (Simponi)

2,511 Johnson & Johnson, Merck & Co.

TNF Human, mouse-derived IgG1, κ-chain (s.c.)

Rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis and ulcerative colitis

Binds soluble and transmembrane forms of TNF; inhibits binding to TNFR

Cardiovascular diseases

Eculizumab (Soliris)

2,843 Alexion Pharmaceuticals

C5 Humanized IgG2/4, κ-chain (i.v.)

Paroxysmal nocturnal haemoglobinuria and atypical haemolytic uraemic syndrome

Binds C5 and inhibits its cleavage, thus preventing the generation of the terminal complement complex C5b–9

Idarucizumab (Praxbind)

0 Boehringer Ingelheim

Dabigatran Humanized Fab, κ-chain (i.v.)

Reversal of dabigatran-induced anticoagulation

Binds and neutralizes anticoagulant activity of dabigatran

Emicizumab (Hemlibra)

NA Chugai, Roche Coagulation factors IXa and X

Humanized bispecific IgG4, κ-chain (i.v.)

Haemophilia A Binds coagulation factors IXa and X, mimicking the cofactor function of coagulation factor VIII

Abciximab (Reopro)

87 Eli Lilly & Co. GPIIb/IIIa and αVβ3 integrin

Chimeric IgG1, κ-chain Fab (i.v.)

Prevention of blood clots in angioplasty

Binds and antagonizes receptor; inhibits platelet aggregation

Alirocumab (Praluent)

116 Sanofi PCSK9 Human, mouse-derived IgG1, κ-chain (s.c.)

Hypercholesterolaemia Binds and neutralizes ligand; lowers LDL-C levels

Evolocumab (Repatha)

151 Amgen, Astellas Pharma

PCSK9 Human, mouse-derived IgG2, λ-chain (s.c.)

Hypercholesterolaemia Binds and neutralizes ligand; lowers LDL-C levels

Caplacizumab (pending)

NA Ablynx von Willebrand factor

Humanized nanobody (i.v.)

(Acquired thrombotic thrombocytopenic purpura)

Binds von Willebrand factor and inhibits interaction with platelets; blocks clot formation

Infectious diseases

Raxibacumab (ABthrax)

1 GlaxoSmithKline Bacillus anthracis protective antigen

Human, phage-derived IgG1, λ-chain (i.v.)

Anthrax infection Binds and neutralizes antigen; prevents intracellular entry of anthrax lethal factor and oedema factor

Obiltoxaximab (Anthim)

0 Elusys Therapeutics

Bacillus anthracis protective antigen

Chimeric IgG1, κ-chain (i.v.)

Inhalational anthrax Binds and neutralizes antigen; prevents intracellular entry of anthrax lethal factor and oedema factor

Ibalizumab (pending)

NA Roche, Biogen, Theratechnologies, TaiMed Biologics, Fountain Biopharma, The Aaron Diamond AIDS Research Center

CD4 Humanized IgG4, κ-chain (i.v.)

(HIV infection) Binds CD4; prevents HIV entry and fusion

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Table 1 (cont.) | Antibody drugs marketed for therapeutic use or undergoing regulatory review

INN (trade name)

2016 worldwide sales (US$ million)

Companies Antigens Antibody format (dosing route)

Approved indications (or potential first indication)

Proposed MoA highlights

Infectious diseases (cont.)

Bezlotoxumab (Zinplava)

NA Merck & Co., Bristol-Myers Squibb, University of Massachusetts

Clostridium difficile toxin B

Human, mouse-derived IgG1, κ-chain (i.v.)

Reduce recurrence of Clostridium difficile infection

Binds and neutralizes toxin B

Rmab (Rabishield) Unknown; not reported

Serum Institute of India, MassBiologics (University of Massachusetts Medical School)

Rabies virus G glycoprotein

Human IgG1 (i.v. and s.c.)

Post-exposure prophylaxis of rabies

Binds to a conformational epitope of G glycoprotein of rabies virus

Palivizumab (Synagis)

1,055 Astra Zeneca, AbbVie

RSV gpF Humanized IgG1, κ-chain (i.m.)

Prevention of RSV infection Binds and neutralizes RSV; inhibits viral fusion and replication

OphthalmologyRanibizumab (Lucentis)

3,262 Roche/Genentech, Novartis

VEGF Humanized IgG1, κ-chain, Fab (i.v.t.)

Neovascular (wet) age-related macular degeneration, macular oedema following retinal vein occlusion, diabetic macular oedema, diabetic retinopathy and myopic choroidal neovascularization

Binds and neutralizes ligand; reduces endothelial cell proliferation, vascular leakage and new blood vessel formation

TransplantationMuromonab-CD3 (Orthoclone OKT3)

46 Johnson & Johnson

CD3 Mouse IgG2a, κ-chain (i.v.)

Prophylaxis of acute kidney-transplant rejection

Blocks function of T cell-expressed CD3; reverses graft rejection

Basiliximab (Simulect)

114 Novartis IL-2R Chimeric IgG1, κ-chain (i.v.)

Prevention of kidney transplant rejection

Binds and antagonizes receptor

OsteoporosisRomosozumab (Evenity)

NA Amgen, UCB, Astellas Pharma

Sclerostin Humanized IgG2, κ-chain (s.c.)

(Osteoporosis in postmenopausal women at increased risk of fracture)

Binds and neutralizes ligand; increases bone mineral density and bone formation and reduces bone resorption

MiscellaneousErenumab (Aimovig)

NA Amgen, Astellas Pharma, Novartis

CGRPR Human, transgenic mouse-derived IgG2, λ-chain (s.c.)

(Migraine prevention) Binds and neutralizes ligand

Burosumab (pending)

NA Kyowa Hakko Kirin, Ultragenyx Pharmaceutical

FGF23 Human , transgenic mouse-derived IgG1, κ-chain (s.c.)

(X-linked hypophosphataemia)

Binds and neutralizes ligand

This list of antibodies marketed in the USA or European Union was adapted from the Antibody Society; the list was developed and is regularly updated by Janice Reichert. Also included is mogamulizumab, which is approved in Japan, as well as itolizumab and rmab, which are approved in India. Antibodies are listed by disease area then alphanumerically by antigen. Companies listed are those that report sales revenue for the branded antibody in their financial statements, which may not be the original developers of the antibodies. Some or all of the listed sponsor companies may report future sales. Commercial data were obtained from EvaluatePharma in June 2017. Most data on target antigen, antibody format, approved indications and mechanisms of action (MoAs) were obtained and abbreviated from the corresponding drug-prescribing information or from phase III clinical trial data for antibodies in regulatory review. Rituximab and denosumab are listed under oncology although they are also approved for rheumatoid arthritis and postmenopausal osteoporosis, respectively. Excluded are biosimilar antibodies and antibodies that were approved and later withdrawn from the market unless currently undergoing regulatory review. Also excluded is Rituxan hycela, which includes the same antibody as intravenous Rituxan (rituximab) in combination with hyaluronidase human, an enzyme that helps with subcutaneous delivery of rituximab. ADC, antibody–drug conjugate; ADCC, antibody-dependent cell-mediated cytotoxicity; ADCP, antibody-dependent cell-mediated phagocytosis; ALL, acute lymphoblastic leukaemia; AML, acute myeloid leukaemia; BAFF, B cell activating factor, also known as TNFSF13B; BiTE, bispecific T cell engager; CAR, chimeric antigen receptor; CCR4, CC-chemokine receptor 4; CDC, complement-dependent cytotoxicity; CGRPR, calcitonin gene-related peptide type 1 receptor; c.i.v., continuous intravenous infusion; CLL, chronic lymphocytic leukaemia; CTLA4, cytotoxic T lymphocyte protein 4; DM1, mertansine; dsDNA, double-stranded DNA; EGFR, epidermal growth factor receptor; FGF23, fibroblast growth factor 23; GD2, disialoganglioside; GPIIb/IIIa, glycoprotein IIb/IIIa; gpF, glycoprotein F; HER2, human epidermal growth factor receptor 2; Ig, immunoglobulin; IL, interleukin; i.m., intramuscular injection; INN, international nonproprietary name; i.v., intravenous infusion; i.v.t., intravitreal injection; JIA, juvenile idiopathic arthritis; LDL-C, low-density lipoprotein cholesterol; MMAE, monomethyl auristatin E; MMR, mismatch repair; MSI, microsatellite instability; NA, not applicable; NK, natural killer; NSCLC, non-small cell lung cancer; PCSK9, proprotein convertase subtilisin/kexin type 9; PD1, programmed cell death protein 1; PDL1, PD1 ligand 1; PDGFRα, platelet-derived growth factor receptor-α; PEG, polyethylene glycol; RANKL, receptor activator of nuclear factor-κB ligand; RSV, respiratory syncytial virus; s.c., subcutaneous injection; scFv, single chain variable fragment; SLAMF7, signalling lymphocytic activation molecule family member 7; TNF, tumour necrosis factor; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2.

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www.antibodysociety.orghttp://EvaluatePharma

Effector functionsFc‑mediated antibody properties that allow for target cell depletion via immune mechanisms including antibody‑dependent cell‑mediated cytotoxicity, antibody‑dependent cell‑mediated phagocytosis and complement‑dependent cytotoxicity.

EpitopeThe part of an antigen that is contacted by an antibody.

targets with known roles in human diseases have been extensively exploited for antibody drug development. Thus, the proverbial ‘low‑hanging fruit’ of therapeutic targets has been harvested (FIG. 2). Nevertheless, oppor‑tunities still abound for antibodies by devising meth‑ods to drug more challenging therapeutic targets — the ‘high‑ hanging fruit’ — which are the focus of this Review, as well as opportunities from new biology dis‑coveries. Major themes explored here include emerging or novel MoAs for antibodies and delivery of antibod‑ies to difficult‑to‑reach sites of action. Biosimilar anti‑bodies6, Fc fusion proteins7, immunocytokines8,9 and chimeric antigen receptor (CAR) T cells10 have been reviewed elsewhere and are beyond the scope of this article.

Antibody mechanisms of actionPostulated MoAs for marketed antibody drugs are reported in their corresponding prescribing informa‑tion and are summarized in TABLE 1. Common MoAs for antibodies include blockade of receptors or their cognate ligands, target cell depletion, receptor downregulation and induction of target cell signalling11. Antitumour antibodies can target tumours either directly or indi‑rectly and exemplify many of these MoAs (FIG. 3). A plethora of different strategies are under development for enhancing the clinical potential of antibodies by improving their existing properties or by endowing them with new functions12,13.

Tumour-targeting antibodiesHere, we discuss emerging MoAs for antibodies that directly target tumours: T cell‑dependent bispecific anti‑bodies (TDBs)14, antibody–drug conjugates (ADCs)15–17 and improvement of antibody effector functions18. These strategies may improve the antitumour activity of antibodies that engage well‑validated targets or facil‑itate the pursuit of previously undruggable targets. Additionally, we consider immune system modulation as a breakthrough MoA for indirect tumour targeting, along with other up‑and‑coming MoAs such as multi‑ target or multi‑epitope strategies and agonist antibodies. Antibody combinations and oligoclonal antibodies are also being developed (BOX 1).

Bispecific antibodies for engaging T cells or other immune cells. The therapeutic use of bispecific anti‑bodies to recruit T cells to kill tumour cells independ‑ent of their underlying antigen specificity was first demonstrated in vitro in the mid‑1980s19. The first clin‑ical validation of TDBs came with the 2009 approval of catumaxomab (Removab, a bispecific IgG antibody that targets CD3 and epithelial cell adhesion mole‑cule (EPCAM)) for the treatment of malignant ascites. However, in 2013, catumaxomab was voluntarily with‑drawn from the market for commercial reasons. In 2014, blinatumomab (Blincyto; Amgen, a bispecific T cell engager (BiTE) targeting CD3 and B lymphocyte antigen CD19) was approved for the treatment of acute

Nature Reviews | Drug Discovery

C5: eculizumabCD20: rituximabHER2: trastuzumabIL-12 and IL-23: ustekinumabPD1: nivolumabRANKL: denosumabTNF: adalimumab, infliximab and golimumabVEGFA: bevacizumab

RadioimmunoconjugatesCD20: 90Y-ibritumomab tiuxetan,131I-tositumomab

FabGPIIb/IIIa, α

Vβ

3-integrin: abciximab

VEGFA: ranibizumabDabigatran: idarucizumab

Antibody–drug conjugatesCD22: inotuzumab ozogamicinCD30: brentuximab vedotinCD33: gemtuzumab ozogamicinHER2: ado-trastuzumab emtansine

F(abʹ)–PEGTNF: certolizumabpegol

T cell-dependentbispecific antibodyCD3 × CD19:blinatumomab

IgG Antibody conjugates Antibody fragments and bispecific antibodies

VH

VL

Figure 1 | Formats of approved antibody drugs. The majority of approved antibody drugs are immunoglobulin G (IgG) molecules, including the examples shown, which were the top-ten selling IgGs in 2016 (TABLE 1). All currently marketed drugs are shown for antibody conjugates, fragments and bispecific antibodies. Not shown is the bispecific antibody, emicizumab (Hemlibra; Chugai/Roche) that was very recently approved for the treatment of haemophilia A. The antigen-binding heavy (VH) and light chain (VL) variable domains are shown in light colours, whereas the constant domains are shown in dark colours. The green and blue variable domains bind to different antigens. GPIIb/IIIa, glycoprotein IIb/IIIa; HER2, human epidermal growth factor receptor 2; IL-12, interleukin 12; PD1, programmed cell death protein 1; PEG, polyethylene glycol; RANKL, receptor activator of nuclear factor-κB ligand (also known as TNFSF11); TNF, tumour necrosis factor; VEGFA, vascular endothelial growth factor A.

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Plasma half-lifeThe time taken for the plasma concentration of a drug to decrease to half of its original value. The initial half‑life and terminal half‑life refer to the distribution phase and elimination phase of biexponential pharmacokinetics, respectively.

lymphoblastic leukaemia. Blinatumomab treatment was associated with complete remission in 43% of patients in a phase II trial in adults with relapsed or refractory disease20. This striking clinical efficacy has reinvigorated the field with at least 20 TDBs in early clinical develop‑ment against a broad range of haematological and solid tumours21.

Many questions remain regarding the molecular design and clinical development of TDBs14. For exam‑ple, clinical stage TDBs employ at least six different molecular formats that vary greatly in size and antigen‑ binding valency21. Small antibody fragment formats used for TDBs include BiTEs, such as blinatumomab20, and dual‑affinity re‑targeting (DART) molecules22, both of which are ~50 kDa and monovalent for each antigen. TDBs formatted as larger antibody fragments include bispecific tandem diabodies (TandAbs)23, which are ~100 kDa and bivalent for each antigen. Full length IgG TDB formats include bispecific IgGs24, which are ~150 kDa and monovalent for each antigen, and CrossMab Fab–IgG25, which is ~200 kDa and bivalent for the tumour antigen but monovalent for CD3. Additional design choices for TDBs include selection of epitope and affinity for both the tumour‑associated antigen and

CD3. Developability characteristics are important con‑siderations in designing bispecific antibodies, including TDBs26, as they are for monospecific antibodies27 (dis‑cussed further below). For example, bispecific antibod‑ies are best assembled from components with drug‑like properties, as their properties may be compromised by the least stable component28.

The design of TDBs will determine their potency for tumour cell killing, biodistribution, tumour penetration, selectivity for tumour cells over normal cells, immuno‑genicity and pharmacokinetic (PK) properties, which together will ultimately dictate TDB clinical effectiveness and safety. For example, the small size of BiTEs can allow close proximity of target and T cells that may favour high potency killing in vitro and tumour penetration in vivo. However, the potential benefits of this small format are offset by the very rapid clearance, which necessitates dosing by continuous infusion, as is the case for blinatu‑momab (TABLE 1). In a more advanced design format, the plasma half‑life of a BiTE targeting myeloid cell surface antigen (CD33) in nonhuman primates was extended from 6 to 167 hours by fusion with human Fc, compat‑ible with once weekly dosing29. Larger Fc‑containing formats are anticipated to achieve a favourable balance

Nature Reviews | Drug Discovery

Disease driveror cell lineage

markers (e.g. TNF, HER2, EGFRand CD20)

Emerging or novel MoA (e.g. TDBs,

ADCs, agonists and improved effector

functions)

Multi-target or epitope strategies, (e.g. bispecific and

multispecific antibodies,

oligoclonals)

Multipassmembrane proteins,

marginally stable soluble protein or

ECD

Major barriers to access by antibodies (e.g. brain, ocular or

intracellular targets, ororal delivery for systemic

exposure)

Multiple MoAs

Improvingantibody

selectivity fortarget tissue Member of large

receptor or ligand family

More difficult to access by antibodies(e.g. solid tumours orlung tumours, or oral

delivery to thegastrointestinal

lumen)

Increasing systemic exposure

of antibodies(e.g. dose,

developability, PKproperties and low

immunogenicity)

Single or well established MOA (e.g. receptor or ligand blockade)

Antigen expression,

restricted to targettissue (or lineage)

Soluble protein or membrane protein

ECD with favourable physicochemical

properties

Ready accessby antibodies

(e.g. blood)

Major differences in biology between human and species used for preclinical

testing

Target exposure to active antibodyTarget biochemistryTarget biology and antibody MoA

High-hanging fruit

Low-hanging fruit

Immune system modulation

Figure 2 | Fruit tree model to represent the difficulty of antibody drug development for different therapeutic targets. Major factors that affect the difficulty of developing antibody drugs include the biology associated with the target, which is closely interrelated with the antibody mechanism of action (MoA). Additional important factors include the biochemistry of the target and exposure of the target to active antibody drug. This Review focuses on more challenging therapeutic targets for antibodies located higher up the tree — the ‘high-hanging fruit’ — with an emphasis on the

topics enclosed in solid lines. Multipass membrane proteins are of increasing interest as antibody targets. Opportunities and challenges in developing antibody drugs to G protein-coupled receptors (GPCRs)298 and ion channels299 have been extensively reviewed by others. ADCs, antibody–drug conjugates; ECD, extracellular domain; EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; PK, pharmacokinetic; TDBs, T cell-dependent bispecific antibodies; TNF, tumour necrosis factor.

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Adverse eventsUndesirable experiences associated with the use of a medical product in a patient that do not necessarily have a causal relationship with that product.

Therapeutic indexA ratio of drug doses that cause toxic versus therapeutic effects that is used to assess the relative safety of the drug for a particular treatment

between longer plasma half‑life and increased molecular weight, which may promote and impair tumour uptake, respectively30. No clearly preferred format for TDBs has yet emerged, so several of the current formats may ulti‑mately be developable as drugs.

TDBs have been associated with substantial adverse events in the clinic, and the therapeutic index can be narrow. For example, the prescribing information for blinatumomab includes boxed safety warnings for neuro toxicity and cytokine release syndrome, which can be fatal. Further clinical studies are needed to investigate how effectively strategies such as dose fractionation, or treatment with steroids or anti‑cytokine antibodies can mitigate cytokine release syndrome observed with sev‑eral TDBs. The neurotoxicity seen with blinatumomab has also been associated with CAR T cells that target CD19 (REF. 31) but not with all BiTEs32. As for on‑ target toxicity, antibodies are being engineered to improve

their tumour‑to‑normal cell selectivity (see later sec‑tion). Importantly, TDBs may synergize with checkpoint blockade. Indeed, preclinical studies have shown that the antitumour efficacy of TDBs can be increased by com‑bining them with antibodies that block the interaction between programmed cell death protein 1 (PD1) and its ligand, PDL1 (REFS 33,34), providing a rationale for evaluating such combinations in the clinic.

Bispecific antibodies and other bifunctional mole‑cules are also being used to recruit T cells to tumour cells via neoantigen peptide–major histocompatibility complex (MHC) interactions. One molecular format, known as an immune‑mobilizing monoclonal T cell receptor (TCR) against cancer (ImmTAC), comprises an engineered TCR that binds to target MHC‑presented peptides with high affinity and selectivity, connected via a peptide linker to an anti‑CD3 single chain variable fragment (scFv) to recruit T cells35. Encouragingly, an

Nature Reviews | Drug Discovery

Tumour neovasculature

Tumour cell

NK cell ormacrophage

T cell

Anti-angiogenesisVEGFA: bevacizumabVEGFR2: ramucirumab

Immune system modulationCTLA4: ipilimumabPD1: nivolumab and pembrolizumabPDL1: atezolizumab, avelumab and durvalumab

Direct tumour targeting

Indirect tumour targeting

IgGCD20: rituximab, ofatumumabCD38: daratumumab, EGFR: cetuximab, panitumumab, necitumumab and nimotuzumab GD2: dinutuximab HER2: trastuzumab and pertuzumabPDGFRα: olaratumab SLAMF7: elotuzumab

IgG with improved effector functionsCCR4: mogamulizumab CD20: obinutuzumab

T cell-dependent bispecific antibodyCD19 × CD3: blinatumomab

Antibody–drug conjugatesCD22: inotuzumab ozogamicinCD30: brentuximab vedotinCD33: gemtuzumab ozogamicin HER2: ado-trastuzumab emtansine

RadioimmunoconjugatesCD20: 90Y-ibritumomab tiuxetan and 131I-tositumomab

Emerging

Established

Figure 3 | Direct and indirect targeting of tumours with marketed antibody drugs. Anticancer antibodies can target tumours either directly or indirectly. This Review focuses on strategies used for emerging rather than established mechanisms of action with anticancer antibodies. These emerging strategies may improve the antitumour activity for antibodies engaging validated targets (‘low-hanging fruit’) or facilitate the pursuit of previously undruggable targets (‘high-hanging fruit’). Anticancer antibodies are being widely evaluated in combination with each other as well as with the full gamut of other therapeutic modalities for cancer therapy. Denosumab (not shown) utilizes an additional strategy for indirect tumour targeting. Denosumab neutralizes its target, receptor activator of nuclear factor-κB ligand (RANKL), decreases bone resorption and reduces skeletal-related events in some patients with bone metastases from solid tumours and is also indicated for the treatment of postmenopausal osteoporosis (TABLE 1). CCR4, CC-chemokine receptor 4; CTLA4, cytotoxic T lymphocyte antigen 4; EGFR, epidermal growth factor receptor; GD2, disialoganglioside; HER2, human epidermal growth factor receptor 2; IgG, immunoglobulin G; NK, natural killer; PD1, programmed cell death protein 1; PDGFRα, platelet-derived growth factor receptor-α; PDL1, PD1 ligand 1; SLAMF7, signalling lymphocytic activation molecule family member 7; VEGFA, vascular endothelial growth factor A; VEGFR2, vascular endothelial growth factor receptor 2.

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ImmTAC that targets the melanoma‑ associated anti‑gen melanocyte protein PMEL (also known as gp100), referred to as IMCgp100, was well tolerated with signs of efficacy in a phase I trial in melanoma35 and is cur‑rently in phase I/II clinical trials as a monotherapy (NCT02889861) and in combination with checkpoint inhibitors (NCT02535078). Alternatively, a TCR‑mimic antibody that is specific for a particular peptide– MHC complex can be used36. Relative to targeting conventional tumour‑associated antigens, targeting peptide–MHC complexes offers access to intracellular antigens and the potential for greater tumour specificity. Disadvantages include low copy number of target on the surface of tumour cells, MHC restriction, MHC downregulation and potential cross‑reactivity to related linear epitopes.

The feasibility of engaging other types of immune cells with bispecific antibodies to kill tumour cells is also being explored. For example, a TandAb target‑ing FcγRIIIA (also known as CD16A) and CD30 was designed to recruit natural killer cells to lyse tumour cells37. This TandAb, now known as AFM13, has reached phase II clinical development for Hodgkin disease (NCT02321592) and for cutaneous T cell lymphoma (NCT031922020). The immune modulator leukocyte surface antigen CD47 binds to the macrophage recep‑tor tyrosine‑protein phosphatase non‑receptor type substrate 1 (SIRPα; also known as SHPS1) to inhibit

phagocytosis of normal, healthy cells. CD47 is often upregulated on tumour cells, effectively providing a ‘do not eat me’ signal to macrophages38. Bispecific antibodies have been developed that bind CD47 with one arm to prevent interaction with SIRPα, and the other arm to target a tumour‑associated antigen such as CD19, CD20 or mesothelin39,40. The Fc region of the antibody recruits macrophages and other innate immune killer cells to the tumour. Low affinity for CD47 minimizes bind‑ing to the large sink of CD47 present on normal cells. An anti‑CD19–CD47 bispecific antibody (κλ‑body, NI‑1701) is in late‑stage preclinical development39.

Antibody–drug conjugates. Antibodies were first armed with toxins to selectively kill target cells in 1970 (REF. 41). The first marketed ADC was gemtuzumab ozogamicin (Mylotarg; Wyeth/Pfizer), a humanized anti‑CD33 IgG4 conjugated to calicheamicin that was approved for the treatment of acute myeloid leukaemia in 2000. Gemtuzumab ozogamicin was voluntarily withdrawn from the market in 2010 when a subsequent phase III study (NCT00085709) revealed a lack of clinical ben‑efit and excess mortality42. However, gemtuzumab ozogamicin was recently reapproved for acute myeloid leukaemia at a lower dosage range on the basis of data from an additional phase III study (NCT00927498)43 plus a meta‑analysis of multiple phase III trials. Convincing

Box 1 | Antibody combinations and oligoclonals

Combinations of antibodies have several potential advantages over individual antibodies, such as increased potency through engagement of multiple or mutating targets on pathogens or toxins, or through engagement of different epitopes on disease-associated antigens287. Oligoclonal antibodies comprise combinations of two or more antibodies that are developed as a defined mixture in a single drug product. The potential advantages of oligoclonal antibodies over their monoclonal counterparts need to be weighed against the greater complexity and uncertainty that oligoclonals bring to drug development.

At least 12 recombinant oligoclonal or polyclonal antibodies have reached clinical development, mainly for infectious diseases or oncology, but none is yet approved287. Nevertheless, clinical validation of the antibody combination concept has come with the approval of two different antibody pairs, namely, trastuzumab plus pertuzumab (antibodies targeting human epidermal growth factor receptor 2 (HER2)) for breast cancer288 and ipilimumab (an antibody targeting cytotoxic T lymphocyte antigen 4 (CTLA4)) plus nivolumab (an antibody targeting programmed cell death protein 1 (PD1)) for melanoma289.

Botulinum neurotoxin provides a striking demonstration of the superiority of an oligoclonal over individual component antibodies. A three-antibody oligoclonal neutralized 20,000 median lethal doses (LD50s) of toxin in vivo, whereas no single antibody provided significant protection290. A phase I study (NCT01357213) in healthy volunteers revealed that this oligoclonal (Xoma 3AB) was well tolerated, with plasma half-lives for the component antibodies of 10–24 days at the highest dose tested (0.33 mg per kg)291. Oligoclonal antibodies are also in early clinical development for other infectious disease targets including Staphylococcus aureus infection, rabies, HIV, hepatitis, influenza A virus, Shiga toxin and Ebola virus.

In oncology, oligoclonal antibodies may help to overcome tumour heterogeneity and plasticity. For example, an oligoclonal of three antibodies against epidermal growth factor receptor (EGFR), MM-151, showed potent antitumour activity in vivo, overcame acquired resistance resulting from the emergence of extracellular mutations and more potently blocked pathway signalling than individual anti-EGFR antibodies292,293. MM-151 has completed a phase I clinical trial (NCT01520389). An oligoclonal of two anti-EGFR antibodies (Sym004) has shown evidence for antitumour activity in phase I clinical trials294.

Hurdles in developing oligoclonal antibodies as drugs include manufacturing them with high purity and consistency, navigating previously uncharted waters with regulatory agencies, potential pharmacokinetic differences between component antibodies, overlapping toxicity profiles or mechanisms of action and translating preclinical findings to clinical development287. The most common manufacturing strategy for oligoclonals has involved separate expression and purification of component antibodies followed by mixing in a single vial or simultaneous administration at defined ratios295. This approach is resource-intensive and may be prohibitively expensive for oligoclonals comprising three or more antibodies295. An alternative strategy is single batch expression, in which cell lines produce all component antibodies of the oligoclonal296,297.

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Biparatropic antibodyAn antibody that binds to two different sites (epitopes) on the same antigen via two different sites (paratopes) on the antibody; also called a biepitopic antibody.

Antibody-dependent cell-mediated cytotoxicity(ADCC). The killing of an antibody‑coated target cell by a cytotoxic effector cell through a lytic process typically involving the release of cytotoxic granules.

Antibody-dependent cell-mediated phagocytosis(ADCP). The engulfment of an antibody‑coated target cell by a phagocytic effector cell.

Complement-dependent cytotoxicity(CDC). The killing of an antibody‑coated target cell via recruitment of the complement membrane attack complex (C5b–9).

clinical validation of ADCs came with the approval of brentuximab vedotin (Adcetris; Seattle Genetics/Takeda Pharmaceuticals), a chimeric anti‑CD30 IgG1 conjugated to monomethyl auristatin E44, for the treat‑ment of Hodgkin lymphoma45 and systemic anaplastic large cell lymphoma. The first ADC to be approved for a solid tumour indication was ado‑trastuzumab emtansine (Kadcyla; Genentech/Roche), a humanized anti‑HER2 IgG1 (REF. 46) conjugated to the maytansinoid DM1 (REF. 47) for HER2‑positive metastatic breast cancer48,49.

ADCs are complex; the multiple parameters for optimization include target antigen and epitope, anti‑body, drug, linker, attachment chemistry and drug‑to‑ antibody ratio (DAR). More than 60 ADCs are currently in clinical development17 including at least 6 that have reached pivotal clinical trials2. Nearly 50 years of expe‑rience with ADCs have yielded many insights that are guiding the design of next generation conjugates, as dis‑cussed extensively elewhere15–17. Some of these lessons may expand the range of accessible targets beyond those druggable with unarmed IgG.

Early ADCs were heterogeneous in their sites and stoichiometries of drug attachment. ADCs with higher DARs were cleared more rapidly from circulation in animal models and had a lower therapeutic index than their lower‑loaded counterparts50. The acceler‑ated clearance of high DAR ADCs has been correlated with ADC hydrophobicity51. Redesign of drug linkers to minimize hydrophobicity has enabled the generation of homo geneous eight‑drug‑loaded ADCs with PK prop‑erties similar to the parent antibody yet with superior antitumour activity to the corresponding four‑drug‑loaded ADCs51. Using a hydrophilic polymer known as a Fleximer, high‑loaded ADCs (with a DAR of 20) have been developed with favourable PK profiles and robust in vivo anti tumour activity52. High DAR conjugates may broaden the range of tumour‑ associated antigens amena‑ble to ADCs to targets with lower levels of expression on the surface of tumour cells.

Site‑specific drug conjugation with naturally occur‑ring cysteines led to the first ADCs with defined site and stoichiometry of drug attachment53. Subsequently, con‑jugation through engineered cysteines resulted in ADCs with an improved therapeutic index54, and the site of drug attachment was identified as crucial for in vivo ADC sta‑bility55. Several additional approaches to site‑specific con‑jugation have been developed, and a few site‑specifically conjugated ADCs have entered clinical development17.

Payload selection has also been a major focus area for enhancing the therapeutic potential of ADCs. Few tumours are intrinsically sensitive to antimicrotubule agents, and yet ~70% of ADCs in clinical development use the antimicrotubule agents auristatins and maytan‑sinoids16,17. An expanding repertoire of alternative drug chemotypes with different MoAs are now being incor‑porated into ADCs, including DNA damaging agents such as benzodiazepines and duocarmycins as well as camptothecin analogues that inhibit DNA topoisomer‑ase 1 (REF. 17). The most efficacious chemotherapy regi‑mens have come not from individual cytotoxics but from combinations of drugs with non‑overlapping toxicities

and resistance mechanisms — a paradigm that is being tested extensively for ADCs16. For example, clinical trials with the ADC brentuximab vedotin encompass numer‑ous different combinations with other drugs including immune system modulator antibodies, anti‑angiogenic antibodies, targeted small molecule inhibitors and cyto‑toxic chemotherapy as individual drugs or in combi‑nation (see ClinicalTrials.gov). A method was recently developed to generate ADCs that contain two different classes of auristatin drug linkers with complementary antitumour activity56. The dual‑auristatin ADCs had superior in vivo antitumour activity to corresponding mono‑auristatin ADCs56.

Parameters that affect the efficiency of ADC inter‑nalization are not well defined but presumably include target and drug‑related factors such as the antibody, epitope, payload, linker and DAR. Certainly, ADCs are sometimes inefficiently internalized by tumour cells, which may limit their antitumour activity. Bispecific antibodies could be useful here. For example, a bipara‑tropic antibody targeting two distinct HER2 epitopes induces robust target receptor cell surface clustering, internalization, lysosomal trafficking and degradation57. This biparatopic anti‑HER2 antibody was conjugated to the potent anti‑microtubule agent, tubulysin. This ADC, now known as MEDI4276, was highly efficacious in mouse tumour models and is currently in a phase I/II clinical trial for the treatment of HER2‑expressing breast or gastric cancers (NCT02576548). Increased cellular internalization of ADCs has also been achieved using bispecific antibodies in which one specificity is to a tumour‑associated antigen and the second specificity is to a lysosomal marker such as CD63 (also known as LAMP3)58 or, alternatively, to a cell surface protein that traffics efficiently to lysosomes such as amyloid‑like protein 2 (APLP2)59 or prolactin receptor60. Increased in vivo efficacy was demonstrated for an anti‑HER2–CD63 bispecific antibody relative to a monovalent anti‑HER2 comparator58.

The ADC concept is now being explored beyond oncology, including for the treatment of infectious diseases and for immunosuppression. For example, an antibody–antibiotic conjugate was developed to com‑bat methicillin‑resistant Staphylococcus aureus (MRSA) infections. The antibody–antibiotic conjugate killed intracellular reservoirs of MRSA and was more effective than the antibiotic vancomycin in treating bacterae‑mia in mice61. This antibody–antibiotic conjugate, now known as DSTA4637S or RG7861, successfully com‑pleted a phase I study of safety, tolerability and PK stud‑ies in healthy volunteers (NCT02596399). ADCs may also be used for immunosuppression: an antibody tar‑geting CXC‑chemokine receptor 4 (CXCR4) conjugated to the LCK inhibitor dasatinib inhibited TCR‑mediated T cell activation and cytokine expression62.

Improved effector functions. Depletion of target cells by effector functions such as antibody‑dependent cell‑ mediated cytotoxicity (ADCC), antibody‑dependent cell‑me‑diated phagocytosis (ADCP) and complement‑ dependent cytotoxicity (CDC) are common MoAs for antibody

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drugs (TABLE 1). The ADCC activity of antibodies can be increased by glycosylation or protein engineering of the Fc region to increase the affinity to FcγRIIIA63. For example, afucosylation of a chimeric antibody targeting CC‑chemokine receptor 4 (CCR4) increased its in vitro ADCC activity and in vivo antitumour activity64. In 2012, a humanized anti‑CCR4 antibody, mogamulizumab (Poteligeo; Kyowa Hakko Kirin), became the first mar‑keted glyco‑engineered antibody drug upon its approval in Japan for the treatment of relapsed or refractory adult T cell leukaemia or lymphoma65. More recently, a gly‑co‑engineered anti‑CD20 antibody, GA101, was devel‑oped with increased ADCC activity66. This antibody, now known as obinutuzumab (Gazyva; Roche), was approved in combination with cytotoxic chemotherapy for the treatment of chronic lymphocytic leukaemia and follicular lymphoma. Several other ADCC‑enhanced antibodies are in clin ical development including the afucosylated anti‑IL‑5Rα antibody benralizumab (Fasenra; MedImmune, AstraZeneca, Kyowa Hakko Kirin, Lonza), which was efficacious in multiple phase III clinical trials for asthma and was recently approved (TABLE 1). Antibodies have also had their Fc regions engi‑neered for increased ADCC and ADCP activity63, includ‑ing the anti‑CD19 antibody MOR00208 (XmAb5574)67,68 that is being investigated in multiple clinical trials includ‑ing a phase II/III trial (NCT02763319) for relapsed or refractory diffuse large B cell lymphoma.

In IgG molecules, the homodimeric Fc region binds asymmetrically to FcγRs such that the two Fc chains use different residues to interact with different regions of the receptor69. This observation has been exploited to engineer heterodimeric IgGs with different mutations on each Fc chain to exquisitely tailor the affinity and selec‑tivity for different FcγRs70–72. Heterodimeric IgG1 vari‑ants have been developed with similar or greater potency in ADCC‑mediated tumour cell killing compared with that of previously described homodimeric variants or afucosylation70,71.

Several antibody drugs support killing of target cells through CDC. For example, the anti‑CD20 antibody ofatumumab (Arzerra; Genmab/Novartis) mediates both CDC activity and ADCC activity and is approved for the treatment of chronic lymphocytic leukaemia. Ofatumumab was identified from a panel of human anti‑bodies for its potent CDC activity against tumour cell lines with high levels of the complement inhibitory pro‑teins, complement decay‑accelerating factor (CD55) and CD59, which renders these cell lines resistant to CDC induced by the anti‑CD20 antibody rituximab73. At least two different protein engineering strategies have been used to increase CDC activity. First, a number of substi‑tutions have been engineered that directly increase bind‑ing between the antibody Fc region and the complement protein C1q63. Second, the newly discovered weak intrin‑sic propensity of IgG to form Fc‑mediated hexamers, as suggested by X‑ray crystallographic structures74,75, has been strengthened through point mutations, for exam‑ple, the E345R mutation. Upon antibody binding to cells, such mutations facilitate hexamer formation, thereby greatly enhancing CDC activity76,77. This hexamerization

strategy has been used to convert anti‑EGFR anti bodies from having minimal to highly potent CDC activity78. IgG hexamerization warrants further exploration as a novel approach for enhancing CDC and perhaps other activities of IgG for clinical applications.

An ambitious future objective for the field is to better understand the contributions of different immuno logical activities to MoA during the preclinical development stage and to tailor selectivity accordingly. Dissecting the contributions of different Fc receptors to antibody pharmacology is key, particularly if Fc engineering can be used to promote adaptive antitumour immune responses79. Thus, it will be interesting to see whether improved effector functions can synergize with check‑point blockade and other immuno‑oncology approaches discussed below.

Improving antibody selectivity for target tissues. The ideal therapeutic target for an armed antibody is expressed on the surface of target cells but entirely absent from normal tissue. Unfortunately, some target expression on normal cells commonly, if not invariably, occurs. For a few ADCs in clinical development, adverse events have been associated with normal tissue expres‑sion of the therapeutic target15. For example, skin‑related adverse events were observed in four phase I clinical trials of bivatuzumab mertansine (an antibody that targets a splice variant of CD44 (CD44v6) and is conjugated to DM1 (also known as mertansine))80. Development of this ADC was discontinued after a fatal case of toxic epidermolysis, likely due to CD44v6 expression in the skin80. Enhancing the selectivity of armed antibodies for tumour over normal tissue may extend the range of therapeutic targets.

One way to improve the tumour selectivity of anti‑bodies is by targeting two tumour‑associated antigens through the use of bispecific antibodies39,81–84. Such dual targeting might spare normal tissue that expresses only one of the antigens. For example, a bispecific IgG antibody targeting both CD4 and CD70 showed some selectivity in binding to cells expressing both antigens over binding to cells expressing individual antigens. Greater selectivity for CD4+CD70+ cells was achieved by reducing the affinity of the anti‑CD4 arm without compromising the potency of killing by ADCC81. An anti‑HER2–EGFR bispecific IgG with carefully tuned antigen‑binding affinity eliminated tumours expressing both antigens in xenograft studies but spared tumours expressing a single antigen in the same mice83. A theo‑retical downside to using dual targeting to increase selec‑tivity is that it provides an additional route for tumours to escape therapy — loss of expression of either antigen. The potential requirement for dual companion diag‑nostics and a more limited population of dual‑positive patients are also downsides.

Another strategy to improve tumour‑targeting selectivity is through the development of an antibody prodrug, or probody, as first shown for an anti‑EGFR antibody85. The probody includes a peptide fused to one of the antibody variable domains that attenuates binding to the therapeutic target until activated locally

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by proteases. The anti‑EGFR probody was relatively stable in circulation in mice but was activated within tumour tissue and showed antitumour efficacy simi‑lar to its parental antibody, cetuximab. The probody showed improved safety and increased plasma half‑life in nonhuman primates, enabling it to be dosed safely to higher levels than cetuximab. The first phase I trial (NCT03149549) of a probody–drug conjugate, in this case, targeting CD166, recently began. A priori, ADC‑associated toxicities may be on‑target (resulting from their activity on antigen‑positive normal cells) or off‑target (due to either ADC catabolism by antigen‑negative normal cells or drug release from the ADC and subsequent expo‑sure to normal tissue)15. ADCs in the clinic often share overlapping toxicity profiles consistent with off‑target effects15. Probody–drug conjugates may reduce on‑ target — but likely not off‑target — toxicities associated with ADCs.

Immune system modulators for cancer immunotherapy. A paradigm shift in the treatment of cancer has been ushered in by the approval of antibodies that block immune checkpoint inhibitors. These antibodies do not target tumour cells directly but instead inhibit pathways that suppress T cell‑mediated antitumour responses, which evolve in parallel with the tumour. Tumour necro‑sis can release antigens that are captured and presented as peptide–MHC molecules on antigen‑presenting cells (APCs). Recognition of presented antigens by TCRs and subsequent activation of T cells can lead to adaptive tumour immunity. Multiple ligand–receptor interactions between T cells and APCs regulate T cell response by providing either co‑stimulatory or inhibitory signals86.

Cytotoxic T lymphocyte antigen 4 (CTLA4) is a powerful inhibitor that is present the surface of T cells. Interaction of this receptor with CD80 or CD86 on APCs provides a checkpoint or brake on the proliferation of antigen‑activated T cells. Allison and collaborators87 demonstrated that blockade of CTLA4 with an antibody can potentiate immune responses, leading to tumour eradication. Subsequently, a human antibody to CTLA4, ipilimumab, was generated for clinical investigation88. Ipilimumab stimulates tumour immunity by both reliev‑ing inhibition of effector T cell function and depleting regulatory T cells89. Ipilimumab, with or without a gp100 peptide vaccine, improved the overall survival of patients with previously treated and unresectable melanoma rel‑ative to patients treated with gp100 alone90. As for safety, severe (grade 3 or 4) immune‑related adverse events occurred in 10–15% of patients treated with ipilimumab. The impressive antitumour activity of ipilimumab in the context of a difficult to treat disease led to its approval (Yervoy; Bristol‑Myers Squibb) for the treatment of mel‑anoma in 2011. More recent data demonstrated increased 5‑year survival in patients with advanced melanoma treated with ipilimumab plus dacarbazine versus dacar‑bazine alone: 18.2% versus 8.8%, respectively91. Thus, durable clinical responses can be achieved with ipili‑mumab, albeit in a small fraction of patients. Ipilimumab has provided compelling clinical validation for immune checkpoint blockade by antibodies.

There are many more surface receptors and ligands that are inhibitors or stimulators of T cell function86. Several of these immunoregulators are being pursued as drug targets, including for antibodies, in the con‑text of cancer immunotherapy92. In particular, PD1 on T cells interacts with corresponding ligands, PDL1 and PDL2, on APCs. Two anti‑PD1 antibodies (nivolumab (Opdivo; Bristol‑Myers Squibb/Ono Pharmaceutical) and pembrolizumab (Keytruda; Merck & Co.)) and three anti‑PDL1 antibodies (atezolizumab (Tecentriq; Genentech/Roche), avelumab (Bavencio; Pfizer, Merck KGaA and Shire) and durvalumab (Imfinzi; AstraZeneca)) have been approved for a broad and growing array of cancers (TABLE 1).

The high expectations of antibodies for cancer immunotherapy can be seen in the ~2,000 clinical trials listed on ClinicalTrials.gov with the six aforementioned approved immunomodulatory antibodies as of October 2017 (FIG. 3). These trials include antibody dose optimi‑zation studies as well as combinations with other anti‑bodies, drugs or treatment modalities such as surgery or radiation therapy. Antibodies engaging other negative and positive regulatory checkpoint molecules, includ‑ing lymphocyte activation gene 3 protein (LAG3), hepa‑titis A virus cellular receptor 2 (HAVCR2; also known as TIM3), T cell immunoreceptor with immunoglob‑ulin and immunoreceptor tyrosine‑based inhibition motif domains (TIGIT), inducible T cell costimulator (ICOS), OX40 (also known as TNFRSF4 and CD134), 4‑1BB (also known as TNFRSF9 and CD137) and V‑type immunoglobulin domain‑containing suppressor of T cell activation (VSIR; also known as VISTA), have reached clinical development for cancer93,94.

Much research is focused on understanding why only a small subset of patients exhibit durable responses to checkpoint inhibitors, how resistance arises and which combination strategies may have curative potential94. Many tumour, host and environmental factors con‑tribute to the strength and timing of the anticancer immune response as captured in the coined phrase ‘cancer‑ immune set point’ (REF. 95). For example, mis‑match repair deficiency can predict the response of solid tumours to PD1 blockade by pembrolizumab96. Mechanistically, mismatch repair deficiency is hypoth‑esized to lead to more mutant neoantigens and greater sensitivity to immune checkpoint blockade, independ‑ent of the tissue of origin of the cancer. Pembrolizumab and nivolumab were recently approved for the treatment of microsatellite instability‑high or mismatch repair‑ deficient solid tumours.

Antibody agonistsBlockade of receptor–ligand interactions plays to the strengths of antibodies and is their most widely employed MoA (TABLE 1). A more complex mechanism, agonism, occurs when receptor engagement by the antibody promotes receptor activation and signalling within the target cell97. Although agonistic antibodies have been explored for over three decades98, interest has resurged in part from the potential for activating T cell co‑ stimulatory receptors for cancer immunotherapy99.

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Anti-drug antibodies(ADAs). Antibodies that can result from an immune response to the therapeutic administration of an antibody or other protein drug.

Agonist antibodies for therapeutic applications are commonly selected to mimic the cellular signalling properties of a natural ligand97. Many different ligands have been successfully developed as drugs including cytokines, hormones and growth factors, which fuelled the early growth of the biotechnology industry100. Antibody agonists have several advantages that may make them preferable to natural ligands in some cases: higher recombinant expression titres, greater thermal or in vivo stabilities and longer or more readily customized plasma half‑lives. In contrast to antibody drugs, anti‑drug antibodies (ADAs) elicited in response to ligand drugs can, on rare occasions, clear endogenous ligand leading to serious adverse events such as thrombocytopenia101 and red‑cell aplasia102 that had resulted from ADAs to thrombopoietin and erythropoietin, respectively.

Receptor activation through dimerization. Signalling for many receptors is promoted by ligand‑induced dimeri‑zation that can often be mimicked, at least in part, with a bivalent IgG antibody97. For example, several different antibodies have been explored as agonists for the cytokine erythropoietin103–105. However, the erythropoietin receptor agonist ABT007 did not precisely replicate erythro poietin activity based on the altered signal transducer and acti‑vator of transcription (STAT) signalling profile104, and an inverse correlation of agonist activity with affinity was observed106. Antibodies have also been discovered that agonize the thrombopoietin receptor (MPL)107–109. Such antibodies may avoid the dangerous consequences of ADAs that cross‑reacted with endogenous ligand in the initial clinical trials with PEG‑conjugated ligand101. Although several anti‑MPL anti bodies have been explored107–109, none have, to our knowledge, reached clinical development. Activity of these antibodies depends upon the distance between individual variable regions108,109, highlighting the importance of geometry and flexibility for agonist antibody mechanisms.

Fibroblast growth factor 21 (FGF21) analogues and mimetics are of high interest for the treatment of type 2 diabetes and obesity110. FGF21 activates the receptors FGFR1c, FGRF2c and FGFR3c in the presence of the obligatory co‑receptor β‑klotho. At least five different engineered versions of FGF21 with improved properties have progressed to early clinical development110 including LY2405319 (REF. 111) and PF‑05231023 (REF. 112), which have shown preliminary evidence for clinical benefit in patients with obesity and type 2 diabetes. Unfortunately, FGF21 is susceptible to proteolytic inactivation, has low stability, tends to aggregate and is rapidly cleared in vivo. Antibodies to β‑klotho are being explored to both avoid these challenges associated with the natural ligand and restrict the activity to cells that express both β‑klotho and FGFR1c113, which should avoid unwanted effects such as hypophosphataemia114. An alternative approach utilizes a bispecific antibody that binds to FGFR1 and β‑klotho to promote hetero dimerization of these two receptors into a productive signalling complex115. This bispecific antibody, now known as BFKB8488A or RG7992, is cur‑rently in phase I clinical trials for type 2 diabetes mellitus (NCT03060538) and insulin resistance (NCT02593331).

Beyond their use for receptor agonism, bispecific antibodies are also being explored to mimic the activity of other endogenous proteins. For example, the bi spe‑cific antibody ACE910 (emicizumab) brings together coagulation factors IXa and X to replace the cofactor function of coagulation factor VIII116–118. Emicizumab has been investigated for the treatment of haemophilia A — a genetic bleeding disorder that results from defi‑ciency of factor VIII. Haemophilia A is commonly treated with recombinant factor VIII, which elicits neu‑tralizing ADAs (known as inhibitors) that block factor VIII activity in ~30% of patients and consequently lead to bleeding sequelae119. Emicizumab prophylaxis was associated with significantly fewer bleeding events in a phase III clinical trial for the treatment of haemophilia A in patients who had such ADA inhibitors of factor VIII (NCT02622321)120. No ADAs were detected fol‑lowing treatment with emicizumab120, which is a major advantage over recombinant factor VIII therapy119. Emicizumab (Hemlibra; Chugai/Roche) was very recently approved for the treatment of haemophilia A (TABLE 1).

A more nuanced example of bispecific agonist anti‑bodies is the Fc engineering approach for co‑engagement of the B cell receptor with the inhibitory Fcγ receptor IIB (FcγRIIB)121,122. Rather than a classical bispecific strategy involving two distinct Fab arms, this approach uses an engineered Fc variant (that has S267E and L328F muta‑tions) to increase affinity for the inhibitory Fc receptor FcγRIIB to induce negative regulatory signals within CD19+ B cells. This antibody, now known as XmAb5871, is currently in two phase II clinical trials for systemic lupus erythematosus (NCT02725515) and IgG4‑related disease (NCT02725476). A similar approach is being taken to co‑target FcγRIIB with membrane IgE for the treatment of allergy and asthma (XmAb7195; NCT02148744 and NCT02881853)123.

The propensity of growth factor receptors to signal upon dimerization can raise challenges for the develop‑ment of bivalent antagonist antibodies, as exemplified by non‑agonist targeting of the proto‑oncogene hepatocyte growth factor receptor (MET). Proliferative signals have been avoided by monovalent engagement of MET with an engineered one‑armed antibody124 or, alternatively, by using an unusual antibody that binds in a unique manner that does not permit receptor signalling125.

Receptor activation through clustering. Some receptors require higher‑order crosslinking — not just dimeriza‑tion — to trigger their activation. Examples include receptors with multimeric cognate ligands or receptors for which clustering of ligand–receptor complexes is driven by cell–cell interactions, as occurs within the immune synapse at the interface between T cells and APCs. The most well‑characterized of this agonist class are members of the TNF receptor superfamily (TNFRSF), which are of high interest as therapeutic targets owing to their roles in apoptosis and T cell acti‑vation99,126. Agonist antibodies have been reported for many TNFRSF members including death receptor 4 (DR4; also known as TNFRSF10A), DR5 (also known

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Volume of distributionThe theoretical volume that would be necessary to contain the total amount of an administered drug at the same concentration as observed in the blood plasma.

as TNFRSF10B)127–130, FAS (also known as TNFRSF6 and CD95)131,132, lymphotoxin‑β receptor (LTβR)133, OX40 (REFS 134,135), glucocorticoid‑induced TNFR‑related protein (GITR; also known as TNFRSF18 and CD357)136, CD27 (also known as TNFRSF7)137, 4‑1BB138 and CD40 (also known as TNFRSF5)139–141. Generally, although agonist activity can be demonstrated in vitro by artificially crosslinking with secondary antibodies or by coating to plates, in vivo crosslinking typically requires engagement of antibody Fc with FcγRs142,143.

The most clinically advanced agonist antibodies against TNFRSF members target 4‑1BB, DR4 and DR5; all have reached phase II trials. Dose‑limiting liver toxicity of anti‑4‑1BB antibodies led to termination of multiple trials and has hindered advancement into phase III. Nevertheless, clinical development of 4‑1BB agonists continues, particularly in combination with other cancer immunotherapies144,145. By contrast, agonist antibodies against the pro‑apoptotic death receptor DR4 (mapatumumab) or DR5 (conatumumab, lexatumumab, tigatuzumab and drozitumab) were well tolerated but had minimal efficacy as monotherapies or in combination with chemotherapy130. In addition to the obvious differ‑ences in receptor biology, 4‑1BB is expressed broadly on FcγR‑bearing cells, whereas DR4 and DR5 are not, which could be key to antibody effectiveness. As a consequence, Fc‑mediated crosslinking of anti‑4‑1BB antibodies is pos‑sible on the surface of the same cell (cis co‑ engagement), whereas DR4 and DR5 relies on crosslinking from sepa‑rate FcγR‑bearing cells (trans co‑engagement) that must be present in the tumour microenvironment.

Several approaches have been explored to improve this mechanistic class of antibodies, with recent work focused on the role of specific FcγRs in vivo. Relevant aspects include FcγR expression and site of action, the relevance of cis versus trans cellular co‑engagement of antibody to target and FcγR, the role of activating ver‑sus inhibitory FcγRs and the potential contribution of FcγR signalling to antibody mechanism beyond simply providing a scaffold for crosslinking. The most extensive investigation has been with agonist antibodies targeting CD40 and the role of the inhibitory receptor FcγRIIB in promoting activity140,143, which has opened the door to optimization using Fc engineering146,147. Whereas the expression of CD40 on FcγRIIB+ lymphoid and myeloid lineage cells enables cis-engagement on the cell surface, trans-engagement between neighbouring CD40+ and FcγRIIB+ cells contributes to CD40 crosslinking and agonism in vitro148. Moreover, an in vivo role for FcγRIIB has been observed for antibodies that activate TNFRs on non‑FcγR‑bearing cells, for example, DR5 (REFS 143,147). The importance of FcγRIIB for in vivo crosslinking may also reflect its high expression level and broad expres‑sion profile relative to other FcγRs149. As yet, it is unclear whether lessons learned on the importance of FcγRIIB for anti‑CD40 agonism will be applicable to other agonist antibodies.

The intrinsic properties of antibodies that promote receptor agonism have also been important areas of research. Parameters such as affinity, geometry, epitope, propensity for oligomerization and valency affect

activity. An inverse correlation between activity and affinity was observed for anti‑FAS antibodies, leading to the suggestion that kinetic dissociation and receptor recruitment are mechanistic components of receptor clustering and activation131. The unique hinge region disulfide bonding patterns of human IgG2 relative to other human IgG subclasses has provided some insights into the importance of conformation for antibody agonism150. Although IgG2 antibodies against CD40, 4‑1BB and CD28 were demonstrated to be superior to IgG1, sub‑fractionation and engineering experiments elucidated that a particular structurally constrained isoform of IgG2 is responsible for the activity, at least for anti‑CD40 antibodies. The epitope can be a crucial aspect of activity, as has been demonstrated for agonist anti‑CD28 antibodies151,152. The unique ability of one anti‑DR5 antibody to oligomerize in the presence of its target receptor, mediated through dimer interactions in the Fab region, highlights the versatility of antibodies for intrinsic agonist activity129. Finally, Fc engineering has been utilized to increase both extrinsic Fc‑mediated crosslinking and intrinsic multimerization to improve the agonist activity of an anti‑OX40 antibody153.

Antibody agonism remains a clinically under‑utilized MoA, with the exception of the well‑established, albeit serendipitous, apoptotic signalling mediated by so