REVIEWS

Building better monoclonal antibody-based therapeutics George J. Weiner

Abstract | For 20 years, monoclonal antibodies (mAbs) have been a standard component of cancer therapy, but there is still much room for improvement. Efforts continue to build better cancer therapeutics based on mAbs. Anticancer mAbs function through various mechanisms, including directly targeting the malignant cells, modifying the host response, delivering cytotoxic moieties and retargeting cellular immunity towards the malignant cells. Characteristics of mAbs that affect their efficacy include antigen specificity, overall structure, affinity for the target antigen and how a mAb component is incorporated into a construct that can trigger target cell death. This Review discusses the various approaches to using mAb-based therapeutics to treat cancer and the strategies used to take advantage of the unique potential of each approach, and provides examples of current mAb-based treatments. Cluster of differentiation numbers Numbers assigned to cell surface molecules on the basis of immunophenotyping. They are often used to identify different monoclonal antibodies that bind to the same antigen.

Holden Comprehensive Cancer Center, University of Iowa Hospitals and Clinics, 200 Hawkins Drive, 5970Z‑JPP, Iowa City, Iowa 52242, USA. e-mail: [email protected] doi:10.1038/nrc3930

More than two centuries have passed since we began to understand the remarkable characteristics and potential of antibodies1. Indeed, polyclonal antisera have been used in the treatment of certain infectious diseases for decades2. Soon after the first description of monoclonal antibodies (mAbs) in 1975 (REF. 3) (a discovery that led to a Nobel Prize 10 years later 4), mAbs were recognized as unique biological tools and quickly became invaluable in pathological diagnosis and basic laboratory investigation. Their ability to bind to specific antigenic epitopes allowed for the rapid assessment of the molecular pheno­type of blood cells and, subsequently, of other tissues. Molecules that were identified by mAb binding were given cluster of differentiation numbers5 that are still used extensively in diagnosis. mAbs are now used widely in immunohistochemistry, flow cytometry and related technologies. Aside from the use of mAbs as tools in diagnosis, at the time they were first described there was equal excitement about their therapeutic potential based on the ability to manufacture mAbs of defined specificity and class in essentially unlimited amounts. Theoretically, this would allow for highly specific targeting of cancer cells on the basis of their molecular phenotype. However, early clinical results exploring mAb-based therapeutics were disappointing 6, and until only 20 years ago, some experts considered cancer treatment with antibody-­based therapy a failed hypothesis. The first mAbs evaluated in the clinic as cancer treatments were murine mAbs. Although there were intriguing hints that mAb therapy could be successful7, problems associated with administering murine mAbs to humans limited

their clinical utility. These problems included the develop­ ment of an immune response against the therapeutic mAb itself, the rapid clearance of the mAb and the suboptimal ability of the murine mAb to interact with the human immune system in a manner that led to immune destruction of the cancer. Fortunately, persistent investigators continued to explore how mAbs could be used in cancer treatment. They evaluated various strategies, including using immunoglobulin G (IgG) to target cancer directly, alter the host response to cancer, deliver cytotoxic substances to cancer and retarget the cellular immune response towards cancer (TABLE 1). The current era of successful mAb therapy began with the development of techniques that allowed genetic modification of murine mAbs to produce chimeric mouse–human mAbs that behave immunologically in most ways like naturally occurring human IgG8,9. Such mAbs are less likely to be recognized by the host immune system as a foreign antigen, have half-lives similar to those of natural human IgG (generally 2–4 weeks) and interact well with the effector arm of the human immune system. They can be administered on a schedule that is practical for patients (in many cases weekly or monthly), and they are present in the circulation of patients at therapeutic levels for months at a time. They distribute to both the intravascular and the extravascular compartments and are present within the tumour mass for long periods of time where they interact with malignant cells, stromal cells, benign lymphocytes, the extracellular matrix and the vasculature.

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REVIEWS Table 1 | Monoclonal antibody-based therapeutics mAb-based therapeutic

Structure

Characteristics of target antigen

Example of major ongoing research questions

Antitumour mAbs

Unmodified IgG or IgG modified to mediate enhanced ADCC

Tumour-associated surface antigen

Are IgGs with enhanced affinity for Fc receptors more clinically effective than unaltered IgG?

Angiogenesis inhibition

Unmodified IgG

Host molecules that control angiogenesis

What is the best way to evaluate clinical response in patients treated with angiogenesis inhibitors?

T cell checkpoint blockade

IgG1 (blocks checkpoint and mediates ADCC) or IgG4 (blocks checkpoint without mediating extensive ADCC)

Molecules that limit the anticancer T cell response

How should we combine checkpoint blockade mAbs with each other, with other immunotherapeutics and with other anticancer agents?

Radioimmunotherapy Unmodified IgG or mAb fragment

Tumour-associated antigen that is not shed or present in the circulation

How can the logistics of administering successful radioimmunotherapeutic agents be simplified to enhance their clinical utility?

Antibody–drug conjugate

IgG modified with cleavable linker and drug

Highly specific tumour-associated What is the best combination of linkers and antigen that can internalize when drugs with each mAb and target antigen? bound by a mAb

Bispecific antibody

Variable regions from cancer-specific mAbs linked to variable regions specific for activating receptors on T cells

Tumour-associated antigen that is not commonly absent in antigenloss-resistant cancer variants

Can effective bispecific constructs that have modified kinetics (thereby avoiding the logistic complexities of continuous infusion) be developed?

Chimeric antigen receptor T cell

Gene therapy approach to modifying T cells by inserting DNA coding for the mAb variable region fused to DNA coding for signalling peptides

Highly tumour-specific antigen that is not commonly absent in antigen-loss-resistant cancer variants

Can very promising preliminary results be extended to solid tumours, or will toxicity be associated even with low levels of target antigen expression by benign cells?

ADCC, antibody-dependent cellular cytotoxicity; IgG, immunoglobulin G; mAb, monoclonal antibody.

Transmembrane signalling The process by which an extracellular signal, mediated by a natural ligand or an alternative agent such as a monoclonal antibody, binds to a membrane receptor and generates an intracellular signal that can affect a broad range of cellular functions, including cell growth, cell differentiation and cell death.

Complement-mediated cytotoxicity (CMC). Also known as complement-­dependent cytotoxicity. Cell death resulting from the activation of the complement cascade by a monoclonal antibody that leads to the formation of a membrane attack complex on the surface of the cell.

Antibody-dependent cellular cytotoxicity (ADCC). Lysis of a target cell by an immune effector cell (such as a natural killer cell, monocyte, macrophage or granulocyte) induced by the recognition of an antibody bound to the surface of the target cell.

After many years of research, a variety of mAb-based approaches to cancer therapy are being used with considerable success, and progress using mAb therapeutics that are based on various different strategies (FIG. 1) is continuing at a remarkable pace. Oncologists now see mAb-based cancer therapies as a vital component of state‑of‑the-art cancer care, and even better mAb-based treatments are on the horizon. This Review provides an overview of recent progress in the development and application of mAb-based approaches to cancer therapy, including a description of mAbs and mAb-based constructs that directly target the cancer, alter the host response to the cancer, deliver cytotoxic moieties to the cancer or redirect T cells towards the cancer.

Targeting the cancer cell mAbs have been used to target a wide variety of antigens expressed on the surface of cancer cells10. Characteristics that make antigens attractive as targets for mAb therapy include the density and consistency of expression of the target molecule by malignant cells, limited expression of the target molecule on physiologically vital benign cells, lack of high levels of soluble target and limited tendency of antigen-negative tumour variants to emerge. Desirable characteristics of target tumour antigens vary depending on the mAb construct being considered, the nature of the malignancy (for example, haematological versus solid tumours) and the mechanism of action of such constructs. In vitro and animal model data indicate that some mAbs that target antigens on the surface of malignant cells are able to induce apoptosis by direct transmembrane signalling11. There is also evidence that mAbs kill

target cells by complement-mediated cytotoxicity (CMC)12 and by inducing antibody-dependent cellular cytotoxicity (ADCC)13. Determining which of these mechanisms (FIG. 2) is most important for a given mAb in a given clinical scenario remains a challenge. mAb-mediated cell signalling. Some mAbs can induce the death of malignant cells in vitro, and presumably in vivo, in the absence of immune effector mechanisms. The strength of this effect varies considerably depending on the mAb, the target antigen and the target cell14,15. Assessment of whether a given mAb can mediate signalling-­induced death in the target cell in  vitro sometimes requires crosslinking with secondary antibodies, such as those specific to human IgG16. Secondary crosslinking results in enhanced aggregation of receptors that can lead to a more robust signal. This is not necessarily an artificial way of replicating the crosslinking and signalling observed in vivo, in which crosslinking of mAbs bound to malignant cells probably occurs to a certain extent through the binding of the constant region of IgG to Fc receptors (FcRs) expressed by a variety of cells in the tumour microenvironment. It is not clear whether artificial crosslinking of mAbs speeds up and strengthens the physiological effect of mAb signalling so that it can be measured in a quick laboratory assay, or by contrast, whether it provides such a strong signal that non-physiological changes are induced that may not be clinically relevant. In vitro measurement of signalling needs to be interpreted with caution as it varies greatly from the clinical environment17. In vitro studies usually use cells that have been selected to grow rapidly and consistently outside the body — that is, independent of their normal environment.

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REVIEWS a Tumour-specific IgG Tumour cell

b Angiogenesis inhibition

c Checkpoint blockade T cell

Receptor

Tumour antigen

PDL1

Complement

VEGFR VEGF

CTLA4

Effector cell

PD1

PDL1specific

Ipilimumab Nivolumab IgG

d Radioimmunotherapy

g CAR T cells

Immunoconjugates

Antigen-based retargeting of cellular immunity

e Antibody–drug

f Bispecific

conjugate therapy

antibody therapy

CD3

Figure 1 | Monoclonal antibody-based cancer therapeutic strategies.  Successful monoclonal antibody (mAb) therapeutics have been based on a number of strategies. Nature Reviews | Cancer Immunoglobulin G (IgG) molecules that bind to target cancer cells (part a) can mediate antibody-dependent cellular cytotoxicity (ADCC) by immune effector cells, induce complement-­mediated cytotoxicity (CMC) or result in the direct signalling-induced death of cancer cells (for example, herceptin and rituximab). IgG mAbs can also be used to inhibit angiogenesis (part b) (for example, bevacizumab) or to block inhibitory signals (part c), thereby resulting in a stronger antitumour T cell response (for example, ipilimumab and nivolumab). Radioimmunoconjugates (part d) (for example, 131I tositumomab and ibritumomab tiuxetan) deliver radioisotopes to the cancer cells, whereas antibody–drug conjugates (part e) (for example, brentuximab vedotin and trastuzumab emtansine) deliver highly potent toxic drugs to the cancer cells. mAb variable regions are also used to retarget immune effector cells towards cancer cells through the use of bispecific mAbs that recognize cancer cells with one arm and activating antigens on immune effector cells with the other arm (part f) (for example, blinatumomab) or through a gene therapy approach in which DNA for a mAb variable region fused to signalling peptides is transferred to T cells, thereby rendering them chimeric antigen receptor (CAR) T cells (part g) specific for the tumour. CD3, T cell surface glycoprotein CD3 ε-chain; CTLA4, cytotoxic T lymphocyte-associated antigen 4; PD1, programmed cell death protein 1; PDL1, PD1 ligand; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.

Fc receptors (FcRs). A family of protein receptors specific for an epitope on the constant region of an antibody. When FcRs on immune effector cells come into contact with an antibody-coated target cell, this can result in immune effector cell activation (FcRs with immunoreceptor tyrosine-based activation motifs (ITAMs)) or inhibition (FcRs with immunoreceptor tyrosine-based inhibitory motifs (ITIMs)).

This can affect cell sensitivity to a wide variety of signals, and studying primary cells obtained directly from patients can avoid this problem. However, the evaluation of primary cells requires extensive previous manipulation, including mincing, filtering and washing. This obliterates the normal architecture of the microenvironment, alters cell growth properties and often leads to the activation of apoptotic signalling pathways independently of additional therapy. In vitro signalling assays are usually carried out over a timescale of minutes to hours, whereas clinical responses to mAbs are measured over weeks and months. Despite these limitations, the detection of signallinginduced apoptosis has become a common approach to the in vitro analysis of potential signalling effects of mAbs18. Even when a mAb is known to alter signalling properties and is effective clinically, it is difficult to use in vitro assays to determine whether the therapeutic effect of

the mAb results from the mAb interrupting the inter­ action between an activating ligand and the receptor, from inhibiting the dimerization of the receptor, or from having a direct effect on receptor signalling 19 (FIG. 2b). This complexity has been most extensively studied with the ERBB tyrosine kinase family of receptors20 and their various ligands, and with mAbs such as trastuzumab and pertuzumab that recognize various receptor family members21. Individual receptors in this family can have multiple ligands, mAbs can alter dimerization properties and a mAb can have different signalling properties depending on whether it is targeting a homodimeric or a heterodimeric receptor 22. Understanding this complexity is not just an academic exercise, as it can have a major impact on the development and clinical testing of novel therapeutics, including mAb combinations23. Resistance to small molecules that inhibit signalling pathways can develop with the emergence of cells that activate alternative or compensatory signalling pathways24. Similar processes can lead to the emergence of resistance to mAbs that mediate their therapeutic effects by signalling 25. As with small molecules, the use of combination therapy is one strategy to overcome this mechanism of resistance. Complement. The first studies demonstrating the very potent ability of complement fixation to enhance antibodymediated cytolysis were carried out more than a century ago. They involved the evaluation of the effect of cobra venom on the lysis of red cells and bacteria26, and took place when our understanding of both antibodies and complement was obviously fairly limited. We have since learned an amazing amount about complement in general and about how complement can have an impact on the efficacy of mAbs in particular12. We now know that some (but not all) mAbs can mediate CMC27. The ability of a given mAb to fix complement and to induce CMC is partly dependent on antigen concentration, the orientation of the antigen in the membrane, and whether the antigen is present on the surface as a monomer or a polymer. CMC can also depend on the mAb isotype and the characteristics of the target cell, including whether the cell expresses complement-neutralizing molecules. Even mAbs that are the same isotype and that target the same antigen can vary in their ability to fix complement 28. Type I human IgG1 CD20-specific mAbs (for example, rituximab) crosslink CD20 tetramers and also fix complement. By contrast, type II IgG1 CD20‑specific mAbs (such as obinutuzumab) do not crosslink tetramers or fix complement well29. Antibody engineering that alters either the constant region protein sequence or the glycosylation of the mAb, gives developers control over a number of characteristics, including the ability to fix complement 30,31 (FIG. 3). Many studies exploring CMC use serum as a source of complement, which highlights the importance of CMC in the circulation. Indeed, CMC seems to contri­ bute most to the therapeutic effect of mAb in haematological malignancies, in which target cells are exposed to complement in the circulation32. However, complement binding to target cells does not necessarily result in the lysis of the target cell. When a CD20‑specific mAb binds

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REVIEWS a Immune-mediated effects of tumour-specific IgG NK cell ADCC

ITAM

Tumour cell

Tumour antigen

Complement CMC Lysis MAC

FcR Granulocyte

Opsonization

Monocyte

b Direct effects of tumour-specific IgG Inhibit receptor dimerization Block ligand Induce apoptotic signalling

Figure 2 | Mechanisms of action of monoclonal antibodies that target cancer cells.  Monoclonal antibodies (mAbs) that bind directly to cancer cells can mediate Nature Reviews | Cancer their antitumour effects through various mechanisms. These mechanisms are routinely identified in vitro but their relative contributions to clinical responses to mAb therapy is difficult to determine. a | mAbs can mediate antibody-dependent cellular cytotoxicity (ADCC) by immune effector cells that express immunoreceptor tyrosine-based activation motifs (ITAMs), such as natural killer (NK) cells, monocytes, macrophages and granulocytes. Fixation of complement can trigger the opsonization of the target cell and thereby enhance phagocytosis and lysis by monocytes and granulocytes. Complement-mediated cytotoxicity (CMC) can directly result in target cell death through the development of a membrane attack complex (MAC). b | mAbs can also have direct effects on target cells by blocking the binding of an activating ligand that is responsible for the survival of the cancer cell, inhibiting the dimerization of a receptor, thereby blocking an activation signal or inducing an apoptotic signal by crosslinking a receptor. Such crosslinking of the receptor can be enhanced when a mAb is bound to Fc receptor-expressing cells. IgG, immunoglobulin G.

Complement fixation Initiation of the complement cascade by an antibody bound to an antigen. It can lead to complement-mediated cytotoxicity of the target cell, as well as other complex effects mediated by the activation of various complement components.

mAb isotype The subtype of a monoclonal antibody (mAb) based on the amino acid sequence of the constant region. Isotypes (IgG1, IgG2, IgG3 and IgG4) vary in their ability to mediate antibody-dependent cellular cytotoxicity and complementmediated cytotoxicity.

to a circulating chronic lymphocytic leukaemia (CLL) cell and fixes complement, the entire antigen–mAb– complement complex may be sheared off the surface of the leukaemic cell as it circulates through the liver and spleen in a process known as ‘shaving’, or trogocytosis, which is mediated by FcR-expressing cells including monocytes33. This results in circulating malignant cells that temporarily lack the target antigen and therefore evade mAbs specific for that antigen. The relative concentration of various complement components in the extravascular fluid is not well understood and may be inadequate to mediate CMC34. It is generally accepted that CMC has a limited role in the efficacy of mAbs that recognize target antigens on malignancies outside the vascular compartment: that is, solid tumours. ADCC. mAbs can induce ADCC by binding to FcRs, which are expressed by a variety of immune effector cells, including natural killer (NK) cells, granulocytes, monocytes and macrophages 35–38 (FIG.  2) . Some of these effector cells express FcRs with immunoreceptor tyrosine-based activation motifs (ITAMs). To trigger ADCC, mAbs bound to the target cell interact with

FcRs that then signal through ITAMs and induce effector cell activation13. Other cells express immuno­receptor tyrosine-based inhibitory motifs (ITIMs), which can inhibit ADCC39. mAb-coated target cells can induce the production and release of cytokines by immune effector cells that express FcRs40. These cytokines can then activate other immune effector cells in the tumour microenvironment 41. Thus, immune cell activation via FcRs can contribute to direct ADCC, as well as to the production of cytokines that contribute to the control of tumour growth in other ways. Interacting mechanisms of mAb-induced cytotoxicity. There is growing evidence that there are extensive interactions — both synergistic and antagonistic — between various mechanisms of action that can affect the antitumour effects of a single type of mAb. A mAb may be developed with one mechanism in mind, but other mechanisms may also be important 42. Complement fixation has very complex effects43. On the one hand, the CD20‑specific mAbs rituximab and ofatumumab, and the CD52‑specific mAb alemtuzumab, rapidly kill target cells in vitro via CMC; on the other hand, complement fixation can block the interaction between mAbs and activating FcRs on NK cells, and reduce ADCC as a consequence44. Thus, in some circumstances, complement fixation may induce CMC and enhance the response to mAbs, but in others it could inhibit ADCC, thereby blocking the response to mAbs. Signalling that results from a mAb binding to a receptor on a cancer cell can alter the sensitivity of that target cell to ADCC11. mAb-induced cancer cell lysis can, at least in preclinical models, lead to enhanced uptake of the target antigen and subsequent crosspresentation by cells — such as dendritic cells — thereby leading to an enhanced T cell response 45. Malignant B cells that express FcRs without activating motifs (that is, FcRs without ITAMs) may actually positively contribute to the antitumour effect of a mAb. These FcRs can allow for autocrine crosslinking of B cell-specific mAbs that bind both to target antigens, through the variable region, and to the FcRs expressed by the same cell or by neighbouring malignant cells46. An antigen that can be effectively targeted by a cancer-­specific mAb needs to be found on the surface of the cancer cell in concentrations high enough to trigger one or more of the effector mechanisms outlined above once the mAb has bound to the surface. However, the antigen should not be expressed to a similar degree by a vital population of benign cells47 or found in high concentrations as a soluble antigen in the circulation48. Efforts to identify mAbs that recognize novel tumour-associated antigens continue, although it could be argued that most of the highly promising antigens that can be effectively targeted by unmodified IgG have already been identified. Ongoing efforts to enhance the efficacy of IgGs that directly target cancer cells involve the identification of mAbs with unique signalling properties — such as HER2 (also known as ERBB2) mAbs that vary in their ability to interfere

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REVIEWS Glycosylation The post-translational attachment of a carbohydrate moiety to a protein. The Fc region of immunoglobulin G includes carbohydrate moieties. Altering the enzymes responsible for glycosylation in a cell line that produces a monoclonal antibody (mAb) can alter the glycosylation of the mAb, thereby altering its ability to activate immune effector cells.

with receptor heterodimerization49 — or the modification of the IgG constant region to enhance its ability to interact with the human immune system (FIG. 3). Approaches to enhancing this interaction include changing the amino acid sequence50 or the glycosylation pattern of the IgG in a way that enhances interaction with FcRs on effector cells51. mAbs with such modifications can have enhanced in vitro efficacy and seem to be safe in clinical trials52. One glycomodified mAb, obinutuzumab, was recently approved by the US Food and Drug Administration (FDA) for the treatment of CLL53. Obinutuzumab is unique in other ways, such as the manner by which it crosslinks its target antigen CD20, and the actual therapeutic contribution of changing IgG glycosylation remains to be determined. There have also been studies exploring mAbs that are based on other antibody isotypes, such as IgA54. These agents have some intriguing properties but are not currently in wide clinical development.

Heavy chain Light chain

Variable region Constant region

Carbohydrate

g Crosslink regions

a Glycomodified mAb

from two mAbs

f Insert DNA for mAb

b Alter amino acids in constant region

c Use different human mAb isotype (e.g. IgG4)

d Link isotope to mAb with stable linker

e Link drug

variable region fused to signalling peptide into T cell to induce expression of CAR

to mAb with cleavable linker

Figure 3 | Modifying monoclonal antibody structure.  Monoclonal antibody (mAb) structure can be modified on the basis of the desired mechanism of action. Nature Reviews | Cancer Immunoglobulin G1 (IgG1) is the most effective naturally occurring human IgG isotype at mediating antibody-dependent cellular cytotoxicity (ADCC). Glycomodified afucosylated mAbs (part a) (such as obinutuzumab) demonstrate enhanced binding to IgG Fc receptors (FcγRs) and enhanced ADCC. Afucosylated mAbs are produced using cell lines that lack the enzymes that are responsible for fucosylation. Modifying the amino acid sequence of mAb Fc (part b), as was done to produce ocaratuzumab50, can also result in enhanced binding to FcγRs and enhanced ADCC. For mechanisms of action in which ADCC is not desirable, IgG4 is a more appropriate isotype, as IgG4 mAbs do not mediate ADCC to the same degree as IgG1 (part c). Nivolumab, an IgG4 mAb that blocks programmed cell death protein 1 (PD1) on T cells, is one such example. Producing radioimmunoconjugates involves linking the radioisotope to the mAb. A stable linker is most desirable (part d) to limit the leakage of the free radioactive isotope. Conversely, optimal antibody–drug conjugates (ADCs) use a cleavable linker (part e). To avoid nonspecific toxicity, it is desirable for drugs used in ADCs to be cytotoxic once inside the target cell but non-toxic when bound to the mAb in the circulation. Linkers that are pH-sensitive or enzymatically cleaved are now a standard component of ADCs. Chimeric antigen receptor (CAR) T cells get their specificity from mAb variable regions but are a form of gene, not protein, therapy. They are produced by inserting DNA coding for the mAb variable region fused to DNA coding for signalling peptides into T cells (part f). Bispecific antibodies require the removal of a functional constant region so that they do not nonspecifically crosslink activating receptors and activate T cells (part g). The lack of a constant region on such constructs results in a short half-life, thus requiring continuous infusion to achieve the desired exposure.

T cell receptor-like mAbs. Many tumour-associated antigens are not expressed on the surface of the cancer cell. This has led to the generation of mAbs that bind to peptides expressed by human leukocyte antigen (HLA) molecules55. These mAbs recognize peptides, such as PR1 and WT1, that are derived from intracellular oncoproteins56,57. Such mAbs are HLA-restricted (that is, they only bind to antigen expressed by cells from patients with a given HLA genotype) and are in early development; however, they represent a novel approach to broadening the potential targets for mAb-based therapy.

Altering the host response Inhibiting angiogenesis. In the early 1970s, Folkman and others hypothesized that tumours require new blood vessels to grow, and that the growth of such vessels is stimulated by pro-angiogenic factors produced by the cancer 58. A number of substances that stimulate intratumoural blood vessel growth were subsequently identified, including vascular endothelial growth factor (VEGF). mAbs (including bevacizumab) were produced to inhibit angiogenesis by interfering with the ability of VEGF to stimulate new intratumoural blood vessel growth (FIG. 1). This, in turn, has an antitumour effect by depriving a growing tumour of nutrients and oxygen provided by the new blood vessels59. As such angiogenesis inhibitors do not directly target the cancer and are generally assumed to inhibit cancer growth rather than induce cancer cell death, they are most often used in combination with cytotoxic agents60. An important advantage of this approach is that it is not dependent on expression of a specific target antigen by the tumour cell, but is instead based on the role of VEGF in neo­ angiogenesis, which is vital for the growth of tumours in general. Bevacizumab has been shown to be effective, at least in some clinical trials and scenarios, in various cancers, including colorectal, lung, breast, renal, brain and ovarian cancer61. The unique mechanism of action of angiogenesis inhibition creates a particular challenge in evaluating efficacy 62. A standard approach to assess the response to most anticancer agents involves radiological determination of tumour shrinkage. It has been proposed that angiogenesis inhibitors may temporarily shrink the size of a cancer by affecting vascularity, without having a significant impact on the growth of the malignant cells. Thus, although it remains controversial, some experts claim that the evaluation of efficacy of angiogenesis inhibitors should be based on overall survival rather than on radiographical determination of tumour shrinkage63. Trials with survival as an end point are more challenging, and robust debate continues over the clinical value of angiogenesis inhibition in various cancer types. T cell checkpoint blockade. Years of basic immunology research exploring the exquisite control of T cell immunity have demonstrated that co‑regulatory ligand–receptor pairs have a central role in the balance between activation and inhibition of antigen-specific (and tumour-specific) T cells64. Such precise control is central to the ability of the immune system to respond

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REVIEWS Opsonization The process by which a target is marked for phagocytosis or for destruction by phagocytes. This term is often used to describe phagocytosis of microbial pathogens. In the case of monoclonal antibody therapy of cancer, the target is a cancer cell.

Checkpoint blockade Inhibitory pathways limit T cell activation in order to maintain self-tolerance and prevent autoimmunity. Checkpoint blockade involves blocking these inhibitory pathways, and thereby allowing for to a more robust T cell response.

Breakthrough therapy designation US Food and Drug Administration (FDA) designation if a new drug is intended to treat a serious or life-threatening disease and preliminary clinical evidence suggests that it provides a substantial improvement over existing therapies.

to infection and to avoid the autoimmunity that could result from an uncontrolled T cell response. Over the past decade, mAbs that interfere with the inhibitory signals responsible for limiting T cell activation have been developed. These mAbs, called checkpoint blockade mAbs, can maintain the T cell activation phenotype and enhance T cell-mediated lysis (FIG. 1). In the case of cancer, such mAbs can induce a more robust and sustained antitumour T cell response64,65. Cytotoxic T lymphocyte-associated antigen 4 (CTLA4; also known as CD152) is a receptor expressed by activated T cells that results in the downregulation of the T cell response. Physiologically, CTLA4 and other negative regulators of T cell activation have a role in blunting the T cell response. Such blunting of the T cell response is important for avoiding auto­ immunity but is undesirable when it comes to the generation and maintenance of an effective antitumour T cell response66. Blocking this negative signal would be expected to enhance and maintain such a response, and this has proven to be the case. Ipilimumab, a mAb that blocks CTLA4, has been approved by the FDA and has had a major impact on the treatment of patients with melanoma67. Clinical responses to ipilimumab can be profound and long-lasting. Ongoing studies in various cancers are exploring regimens that incorporate ipilimumab, both alone and in combination with other agents, particularly when there is reason to think that such therapy could be inducing a T cell response, such as in the case of vaccines or agents that induce antigen release68. The toxicity of checkpoint blockade mAbs is also unique and, as would be expected, includes autoimmunity 69. Several mAbs that interfere with a different T cell regulatory pathway, the programmed cell death protein 1 (PD1) receptor–PD1 ligand 1 (PDL1) axis, are also generating considerable excitement. These include mAbs that bind to the ligand and others that bind to the receptor 70,71. Both approaches have been very promising in early clinical trials, and we are sure to learn much more about the relative value of these approaches in the years ahead. Indeed, the FDA has recently approved two PD1‑specific mAbs (pembrolizumab and nivolumab) for the treatment of melanoma. A mAb against PDL1 has been granted breakthrough therapy designation by the FDA on the basis of its promise in early phase clinical trials70. Whereas CTLA4 regulates de novo immune responses, the PD1–PDL1 axis has a greater influence on ongoing T cell immune responses. Thus the concept of checkpoint blockade is similar, but the efficacy and toxicity of mAbs that target these two pathways are likely to be different. For instance, a PD1‑specific mAb has been found to be effective in patients with melanoma refractory to CTLA4‑specific therapy 72. Combination trials exploring the inhibition of CTLA4 and PD1 together are promising 73, although blocking both pathways does raise concerns about enhanced autoimmunity. The PD1–PDL1 axis seems to be particularly important in classic Hodgkin lymphoma in which Reed– Sternberg cells have recently been shown to express PDL1 and PDL2, which allows them to turn off T cells.

This helps to explain the hallmark of Hodgkin lymphoma that has intrigued haematologists for decades: how malignant Reed–Sternberg cells are able to survive despite being surrounded by benign immune cell infiltrates. An early phase clinical trial of single-agent PD1 blockade in relapsed or refractory Hodgkin lymphoma demonstrated very positive results74.

Delivering cytotoxic moieties Military analogies have been a part of immunology since the days of Paul Ehrlich and his description of anti­bodies as “magic bullets” (REF. 1). In more recent years, the phrase ‘smart bombs’ has been used to describe mAbs that deliver cytotoxic moieties to cancer cells. The types of payload that have been successfully delivered by mAbbased smart bombs are quite varied and include radio­ active molecules, cytotoxic small molecules and cellular components of the immune system (FIG. 3). Radioimmunoconjugates. Treatment of thyroid cancer with 131I was the first highly effective targeted cancer treatment and was successful because of the specificity of elemental iodine for the thyroid. 131I is a β-particle emitter that also emits a modest amount of γ-radiation and so can be used in both diagnosis (imaging) and treatment. This success led investigators to explore approaches to delivering radioisotopes directly to target molecules expressed by other cancers using various carrier molecules, including antibodies. Attempts to use such radioimmunoconjugates to treat cancer began with polyclonal antibodies before mAb technology was available. Order and colleagues75 produced polyclonal antisera by immunizing various species (including rabbits, pigs, monkeys and cows) with human ferritin. Antisera from these animals were labelled with 131 I and administered to patients with tumours that were known to express high levels of ferritin receptor 75. The results, particularly the images demonstrating evidence for some targeting of the agent to the tumour, were intriguing, but the use of polyclonal antisera from various species demonstrated little clinical efficacy and posed many problems. These included significant consistency and quality-control issues, and the development of host immune responses against the foreign proteins (similar to classic serum sickness)76. mAb technology solved some of these problems and resulted in a renewed interest in radioimmunoconjugates and radioimmunotherapy. However, challenges have persisted and have limited the clinical utility of radioimmunotherapy. Radioisotopes continually decay and cause nonspecific radiation damage to normal tissue when the agent is in the circulation or is taken up nonspecifically by normal tissues77. Bone marrow is particularly radiosensitive and is therefore affected by circulating radioimmunoconjugate. The kidney and the liver receive high doses of radiation because of their role in clearing the radioimmunoconjugate and free radioisotope, and only a small fraction (typically 0.01–0.001% per gram of tumour) of the injected radiation dose ends up in the tumour, even for the most specific agents77. Optimizing the very narrow therapeutic window of

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REVIEWS radioimmunotherapy requires careful choosing of the mAb, the radioisotope and the chemistry that links the two. Experience over the past 25 years has taught us that radioimmunoconjugates directed towards solid tumours can be useful as diagnostic agents, but that they have limited therapeutic impact owing to radioresistance of the tumours78. A radioimmunoconjugate consisting of a radioiodinated mAb that targets necrotic tumour cells has been approved for clinical use in China79. Ongoing studies are evaluating radioimmunotherapy combined with chemotherapy. Lymphoma is uniquely suited for radioimmuno­ therapy because of the availability of highly specific target antigens and its relative radiosensitivity. Two radio­immunoconjugates, ibritumomab tiuxetan80 and tositumomab81, have been approved by the FDA as therapeutic agents for the treatment of lymphoma. They are both based on CD20‑specific mAbs but use different isotopes (90Y for ibritumomab tiuxetan and 131I for tositumomab). The logistics of administration, need for dosimetry and pharmacokinetics of these two agents are different, but their clinical efficacies and toxic effects are similar. Despite the clear clinical efficacy and limited toxicity of lymphoma radioimmunotherapy, clinical use of this modality has been surprisingly limited. This seems to be due to the complex logistics of providing radioimmunotherapy (it requires experienced nuclearmedicine physicians), and the emergence of a number of other new therapies for lymphoma that are less complex to deliver. Recent and ongoing studies are exploring additional clinical questions related to lymphoma radioimmunotherapy, including the timing of therapy (at diagnosis versus at relapse), which lymphoma subtypes should be treated (given that it is most effective for follicular lymphoma) and how therapy should be combined with other treatments, including bone marrow transplantation82 and novel agents83. Ongoing studies are also exploring radioimmunoconjugates that contain novel radioisotopes, including α-emitters84. The advantage of α-emitters is that the ionizing radiation is delivered very locally (within just a few cell diameters), which limits radiation exposure to cells that express the target antigen and spares benign neighbouring cells. This highly specific delivery of radiation allows the treatment of malignancies such as leukaemia that are not geographically isolated but that are highly radiosensitive. The radiochemistry of working with these isotopes is challenging because of their very short half-life and because the production of isotopes with promising properties, such as 211At, requires a cyclotron85. However, not all α-particle-emitting isotopes are short-lived. Preliminary preclinical and clinical results using α-particles are intriguing 86 and studies are ongoing. Antibody–drug conjugates. In the 1970s and 1980s, a number of investigators began developing and testing immunotoxins composed of mAbs linked to a variety of very potent protein toxins, such as ricin, pseudomonas exotoxin and diphtheria toxin. For an immunotoxin to be effective, it needs to bind to a surface antigen on

a cancer cell, enter the cell by endocytosis and traffic within the cell to the site where it can kill the cell. The toxins need to be modified by removing the moieties responsible for nonspecific binding to normal cells, so that cellular targeting and uptake is controlled by mAb specificity. Initial studies of immunotoxins were carried out using protein conjugation techniques, such as the use of heterobifunctional reagents to link the mAb to the toxin. Subsequent efforts involved the development of recombinant immunotoxins. These agents were found to be highly potent, but the immunogenicity and nonspecific toxicity of protein toxins posed major problems and limited further development 87. Many of the important lessons learned from the study of immunotoxins were applied to the development and testing of antibody–drug conjugates (ADCs), a field that is showing great promise88. ADCs combine the cytotoxic potential of drugs with the specifıcity of mAbs and theor­ etically overcome the limitations of both nonspecifıc cytotoxic drugs and specifıc but often ineffective mAbs. When choosing the target antigen and the mAb for ideal ADCs, desirable characteristics are different from those used for unlabelled mAbs89. Tumour antigens for ADCs can be expressed at lower concentrations compared with unlabelled mAbs because the delivery of a highly toxic drug by a small number of ADC molecules can result in the death of the target cancer cell. Depending on the drug used, only a few molecules delivered intracellularly may be capable of killing a target cell. Internalization properties are also important. For unlabelled mAbs, it is helpful for the mAb–antigen complex to remain on the surface of the cell so that it can be recognized by the host immune system. For ADCs, internalization needs to take place so that the drug can be released inside the cancer cell and mediate its toxic effect. ADCs use small molecules instead of protein toxins and thereby reduce immunogenicity. Drugs used as components of ADCs include potent drugs such as calicheamicin, which binds to the minor groove in DNA and causes strand scission90; monomethyl auristatin E, which blocks polymerization of tubulin91; maitansine, which inhibits the assembly of microtubules92; and, most recently, pyrrolobenzodiazepines, which crosslink DNA93. These are extremely potent drugs; indeed, some were evaluated as stand-alone chemotherapy agents and rejected because of their toxicity at very low doses90,94. The linker that connects the mAb to the drug is vital. It needs to attach the drug to the mAb in a manner that does not alter the specificity of the mAb, to render the drug non-toxic while bound to the mAb, to remain stable in the circulation and to release the drug in the appropriate intracellular compartment when the ADC is internalized, so that the drug can kill the target cell95. Linker motifs include disulfides, hydrazones, peptides and thioethers. Some are cleavable, whereas others are not, based on the properties of the mAb and the desired distribution of the cytotoxic agent within the cancer cell. As with radioimmunoconjugates, ADCs seem to be particularly effective in lymphoma, but they are also showing promise in solid tumours. Brentuximab vedotin (for lymphoma)96,97 and ado-trastuzumab emtansine

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REVIEWS (for breast cancer)98 were the first ADCs to be approved by the FDA. The field is rapidly expanding with more than 30 ADCs currently in development and in clinical trials99, and many have shown encouraging preliminary results100,101. The ADC field is currently in its infancy, and major advances based on the design of new ADCs, and a better understanding of how to use them, are sure to come. An approach that combines targeting of the cancer cell directly with altering the tumour microenvironment is the use of immunocytokines that can mediate their antitumour effect through both the direct effect of the mAb and the ability to deliver a cytokine, such as interleukin (IL‑2) or granulocyte–macrophage colonystimulating factor (GM‑CSF), to the tumour. Most notable in this area of research have been immunocytokines based on ganglioside-­specific GD2, with clinical development mostly centred on the treatment of neuroblastoma102,103. These agents have shown remarkable efficacy in this rare childhood malignancy. A modified version of this immuno­cytokine, designed to mediate reduced complement fixation that is thought to decrease its neurotoxicity, has also shown promise in an early phase clinical trial104.

Bispecific antibody (Also known as bifunctional antibody). An engineered monoclonal antibody (mAb) that is composed of fragments of two different mAbs that bind to two different antigens. In cancer therapy, it typically binds to an activating antigen on an immune effector cell with one arm and to a tumourassociated antigen on a cancer cell with the other arm, thereby retargeting the immune effector cell towards the target cancer cell.

Chimeric antigen receptor (CAR). An engineered receptor that grafts an alternative specificity onto an immune effector cell, most often a T cell. Most of the current constructs for engineered receptors include a single-chain monoclonal antibody variable region that recognizes the target cell and is linked to activating transmembrane domains that activate the T cell when it comes into contact with a target cell.

Retargeting T cells Tumour immunology has taught us that T cell immunity is key to the immune rejection of many cancers. The initial step of cell-mediated cytolysis involves the formation of conjugates between T cells and target cells. The appropriate engagement of receptors and co‑­receptors on T cells is followed by the triggering of cytotoxic responses that result in the death of the cancer cell. Two broad approaches to combining the specificity of mAbs with the power of T cell immune responses have been explored with the hope of enhancing immune rejection of cancer (FIG. 3). Both of these approaches are designed to retarget large numbers of T cells towards the tumour by bypassing the need for the T cell receptor to recognize target antigens as processed and expressed by target cell major histocompatibility complexes (MHCs). Bispecific antibodies. The first approach involves the creation of bispecific antibody-like molecules that have one arm that binds to the target cell and one arm that binds to activating receptors on cytotoxic cells, such as T cells or NK cells. Most bispecific antibodies involve constructs that bind to an antigen on a cancer cell with one arm and to T cell surface glycoprotein CD3 ε-chain (CD3) on T cells with the other arm. This, as with many of the strategies based on using mAbs to treat cancer, is not a new concept, but it has benefited from a steady improvement in technology and lessons learned from earlier preclinical and clinical studies105. For example, early studies demonstrated that bispecific antibodies with intact Fc activate T cells nonspecifically and result in unacceptable toxicity. Smaller bispecific molecules lacking Fc have short halflives and need to be given by continuous infusion, but these molecules result in less nonspecific immune activation than do intact bispecific mAbs. Recent positive clinical trials with the bispecific mAb blinatumomab led to its approval by the FDA. Additional bispecific antibodies

are under development, including some with different structures that have longer half-lives and that may not require continuous infusion106,107. Chimeric antigen receptor T cells. Another example of how persistence has paid off in mAb-based cancer immuno­therapy is demonstrated by the decades of research that preceded the current high level of excitement surrounding chimeric antigen receptor (CAR) T cells108. CAR T cells are genetically modified to respond to target cells expressing a given antigen, and consist of a mAb variable region linked to a T cell-activating motif. A number of investigators worked for many years to refine various antigen-specific constructs and approaches to transferring those constructs into primary T cells109. Recent clinical trials based on some of the newer constructs show that CAR T cells can lead to robust therapeutic responses110,111. They also result in significant, but increasingly manageable, toxicity owing to cytokine storm that occurs as a consequence of massive activation and proliferation of CAR T cells; activation and proliferation are induced when CAR T cells come into contact with a large tumour burden. CAR T cells are unique among mAb-based treatment strategies in that the antitumour effect can expand over time without additional therapy as the CAR T cells divide and the tumour specificity of the CAR T cells is passed on to daughter cells. Indeed, long-lived CAR T memory cells have been observed in a number of patients 112. This memory response represents both an advantage and a challenge. On the one hand, it probably contributes to the very gratifying long-term clinical responses that are seen in some patients treated with CAR T cells. On the other hand, such long-term immune memory can result in long-term, perhaps even permanent, depletion of any cells that express the target antigen. In the case of B cell malignancies, longterm loss of benign B cells that express CD19 may be an acceptable toxicity. However, most tumour antigens are not as specific as CD19, and long-term toxicity to vital normal tissues can result if such tissues express even low levels of the target antigen recognized by CAR T cells. This may limit the number and types of cancers that can be treated with this approach. Bispecific antibodies and CAR T cells each have advantages and disadvantages. Bispecific antibodies are ‘off-theshelf ’ reagents and can be stopped if autoimmune toxic effects are observed, but they are challenging to administer. CAR T cells need to be produced individually for each patient, but they expand in response to tumour and can result in long-term antitumour immune responses. Both approaches can result in the rapid activation of large numbers of T cells interacting with large numbers of cells expressing the target antigen113. The resulting massive release of cytokines can result in a clinical cytokine storm with potentially devastating physiological effects, including cardiovascular collapse114. Cytokine storms can be limited by stopping infusion for bispecific antibodies or by using mAbs against certain cytokines such as IL-6‑specific mAbs for CAR T cells. Accumulating experience with CAR T cells is leading to the development of protocols to prevent or treat this challenging complication114.

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REVIEWS Additional studies are needed to determine the relative efficacy, toxicity and value of these two exciting new strategies for using mAbs to treat cancer.

Conclusion Cancer mAb therapy has come a long way since the days when unmodified murine mAbs were first explored as anticancer agents. A wide variety of mAb-based strategies have proven to be useful in treating patients with cancer, including: the use of unlabelled IgG that binds directly to cancer cells, mAbs that alter the active host response to the cancer, immunoconjugates that deliver cytotoxic moieties to the cancer and constructs that use the specificity of mAbs to retarget cellular immunity towards the cancer cell. The demonstration of clinical efficacy for each of these approaches was made possible by the dedication and persistence of investigators who would not give up on a good idea.

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Indeed, investment in mAb-based therapy of cancer from both the public and the private sectors and the speed of progress have never been greater. Recent advances and positive clinical results in checkpoint blockade, ADCs and retargeting T  cells via CAR T cells and bispecific antibodies are particularly exciting. We are in a period of unprecedented progress in mAb-based therapy with the rapid development of new therapeutic agents and constructs, enhanced understanding of their biological effects, and growing clinical experience based on both clinical trials and community use of FDA-approved drugs. With dedicated attention to basic, translational and clinical research geared towards additional progress, we will certainly be able to build even better anticancer mAbs in the years ahead. Equally important will be studies evaluating how best to use these agents, alone and in combination, to better serve our patients.

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Acknowledgements

The author would like to acknowledge support from the US National Institutes of Health grants P30 CA86862 and P50 CA97274.

Competing interests statement

The author declares no competing interests.

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