Antibody Therapeutics in Cancer Mark X. Sliwkowski and Ira Mellman Science 341, 1192 (2013); DOI: 10.1126/science.

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Antibodies
REVIEW

Antibody Therapeutics in Cancer

Mark X. Sliwkowski and Ira Mellman In a relatively short period of time, monoclonal antibodies have entered the mainstream of cancer therapy. Their first use was as antagonists of oncogenic receptor tyrosine kinases, but today monoclonal antibodies have emerged as long-sought vehicles for the targeted delivery of potent chemotherapeutic agents and as powerful tools to manipulate anticancer immune responses. With ever more promising results from the clinic, the future will likely see continued growth in the discovery and development of therapeutic antibodies and their derivatives. ince the discovery in 1948 that cytotoxic folate antimetabolites could be used in the treatment of childhood leukemia, our basic approach to cancer therapy has remained fundamentally the same: surgery followed by sublethal administration of various cytotoxic compounds or radiation. However, over the past 16 years the situation has started to change substantially with the advent of targeted cancer therapies: the use of drugs developed to inhibit oncogenic proteins or survival factors selectively expressed by tumors. Although targeted agents are often thought of as low molecular weight inhibitors that can be given orally and gain access to cytoplasmic targets by diffusion across the plasma membrane, the first and perhaps most effective targeted therapies involve the use of biotherapeutics, recombinant proteins (usually antibodies) that modulate targets expressed at the cancer cell surface. This is quite remarkable because, not so long before the introduction of the first effective therapeutic antibodies in cancer (the CD20 antibody rituximab and the HER2 antagonist trastuzumab), there were severe doubts that antibodies could ever be safely used in humans. One major impediment was that monoclonal antibodies were typically produced in mice and therefore would be expected to be immunogenic in humans. The discovery that one could humanize murine antibodies by grafting complementarity-determining regions (CDRs) from the desired mouse antibody onto a recombinant human immunoglobulin backbone changed everything (1, 2). Today, a variety of sophisticated strategies exist to engineer humanized antibodies and to produce human antibodies by using rodents partially reconstituted with human immunoglobulin genes, human immune cells, or combinatorial phage or yeast display libraries that can yield high-affinity antibodies without requiring animal immunization at all (3). These approaches have led to an explosion of therapeutic antibodies both in cancer and in other diseases (4). Thus far, the only Food and Drug Administration (FDA)/European

Medicines Agencyapproved agents for oncology are conventional immunoglobulin G (IgG) molecules or their conjugates (Fig. 1), but engineered antibodies and novel antibody-like variants are increasingly making their way into clinical trials. In this review we will discuss the general classes of antibody therapeutics that are commonly in use, provide a detailed analysis of two specific examples of oncogene-targeted antibody drugs, and consider some of the most promising new strategies, namely the use of antibodies for targeted delivery of conventional chemotherapeutics and to enlist the power of the immune system. Classes of Antibody Therapeutics in Cancer There are currently 13 antibodies approved by the FDA for various oncology indications (Table 1), and many more are currently being evaluated in clinical trials. Antibodies for the treatment of hematologic cancers, such as non-Hodgkins lymphoma or chronic lymphocytic leukemia, and antibodies to B cellassociated targets (CD20, CD52) act, at least in part, because of their ability to induce apoptosis after binding to B cell tumors (5). The first of the approved antibodies directed against targets expressed on solid tumors were selected in cell culture as antagonists of the oncogenic receptor tyrosine kinases epidermal growth factor receptor (EGFR) and HER2 (6). One antibody, bevacizumab, binds and sequesters vascular endothelial growth factorA (VEGF-A), a key factor that is required for the growth of blood vessels and exerts its antitumor effect by functionally altering or slowing the formation of the tumor vasculature (7). Although this is at present the only approved antibody in oncology that targets a secreted protein, this class of therapeutic is common in the case of chronic inflammatory diseases, where multiple approved agents target proinflammatory cytokines such as tumor necrosis factora (4). Two antibodies are now approved that are covalently modified with cytotoxic microtubule antagonists. These antibody-drug conjugates (ADCs), one of which is a modified version of trastuzumab for HER2-positive breast cancer patients (8), enable a form of targeted chemotherapy. These recent VOL 341 SCIENCE

Genentech, Incorporated, 1 DNA Way, South San Francisco, CA 94080, USA. E-mail: mellman.ira@gene.com (I.M.); marks@ gene.com (M.X.S.)

successes have led to a plethora of ADCs in clinical development (see below). Last, the recent approval of ipilimumab has introduced yet another therapeutic strategy for anticancer antibodies, namely active immunotherapy (9). Ipilimumab blocks the activity of the negative regulator of T lymphocytes, CTLA4, resulting in the activation of a patients immune response to cancer. This exciting approach has already demonstrated long-term, durable benefit. Most of the approved antibodies in oncology are of the human IgG1 subclass, the subclass that is the most effective at engaging Fcg receptors (FcgRs) on natural killer (NK) cells, macrophages, and neutrophils (Table 1). Antibody engagement of these receptors leads to the killing of antibodybound target cells by antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent phagocytosis (ADP) (1012). This is an important consideration given that ADCC or ADP likely contributes to the efficacy of these antibodies, above and beyond any direct effect on modulating signal transduction. FcgR-mediated cross-linking of surface-bound antibodies may be important in some settings, for example, to induce apoptosis (13). The next generation of antibody therapeutics will likely extend beyond the IgG1 isotype and/or include modifications to the Fc domain, the region that binds to FcRs. Where it may be beneficial to reduce ADCC, the IgG4 subclass has been used, although here the hinge region of the antibody must be engineered to prevent premature dissociation and reassembly of heavy chainlight chain half molecules with endogenous IgG4s in the plasma (14). Complete elimination of ADCC can be achieved by modifying specific residues in the Fc domain that bind to FcgR or by producing recombinant antibodies that lack the N-glycosylation of heavy-chain Fc regions, required for recognition by FcgRs (15). Conversely, alterations in glycosylation have been identified that enhance FcgR binding (effector domainenhanced antibodies), thereby increasing the potential for ADCC or FcgR cross-linkingdependent signaling. Still other Fc domain mutations are being tested that either enhance or reduce binding the neonatal FcR for IgG (FcRn), the receptor responsible for returning IgGs to the circulation after their internalization by endothelial cells. Thus, enhanced FcRn binding will serve to increase the half-life of injected antibodies, whereas reduced FcRn binding will decrease halflife (16). These may prove to be important considerations in controlling the pharmacokinetic exposure levels of a given antibody, with a potential toxicity possibly mitigated by faster clearance. New formats are also being developed, such as two types of bispecific antibodies. In the first, single heavy chainlight chain pairs derived from antibodies with distinct specificities can be covalently or noncovalently linked to yield a single IgG molecule bearing arms targeted to two different antigens. To overcome the issue of monovalent binding that comes with this approach, phage

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display has been used to make single CDRs that exhibit dual specificities. Such dual-action Fabs are otherwise normal antibodies that retain high affinity and bivalent affinity for two antigens simultaneously. One such antibody (MEHD7945A) that detects EGFR and the HER3 co-receptor simultaneously is currently in phase II clinical trials (17). In other cases, one-armed antibodies (i.e., an intact IgG missing one Fab domain, onartuzumab) have been deployed where it was desirable to avoid the cross-linking of a potentially activating receptor (c-Met) (18). Even less traditional formats are being evaluated, involving attenuated single-chain antibodies that have either single or dual specificities (19). These antibodies have the limitation that smaller antibody constructs have shorter in vivo halflives after injection, which necessitates frequent or continuous injections. Larger, multiple-armed antibodies or antibodies genetically fused to other mediators (e.g., cytokines, hormones, and toxins) as well as a variety of synthetic minibodies and antibody mimetics are also on the horizon and, if successful, could increase the potential range of therapeutic strategies. However, one caution is that the farther one strays from the conventional, the greater the chances are for unanticipated behavior, adverse effects, or inherent immunogenicity. HER2 Therapeutics Three mAbs (trastuzumab, pertuzumab, and adotrastuzumab emtansine) and a small-molecule TKI (lapatinib) are approved for the treatment of HER2-positive breast cancer (Table 1), yet interpretation of the clinical data is less complex than that observed with EGFR antagonists. Lapatinib, a low molecular weight HER2 and EGFR TKI, effectively blocks HER2 signaling and was approved on the basis of compelling clinical data generated in patients whose tumors had progressed on trastuzumab-based therapy (26). Thus, HER2 tyrosine kinase antagonism alone is an effective treatment for this form of breast cancer. Indeed, trastuzumab and pertuzumab, antibodies against HER2, were selected for their ability to block proliferation (27) and signaling activity (28). Although these antibodies were also humanized to IgG1, suggesting that ADCC might add to their clinical activity, human FcgR polymorphisms that enhance binding are not linked to improved responses in patients (29). Yet the relative importance of ADCC remains an active area of debate. In contrast to EGFR antagonists, HER2directed antibodies and small molecules are very effective in the treatment of HER2-positive breast cancer in combination with conventional chemotherapy. Because of favorable tolerability profiles, HER2 targeting can also be combined with standard cytotoxic chemotherapy. Indeed, pertuzumab EGFR Therapeutics is approved only in combination with trastuzumab and docetaxel (30). As is often the case in cancer The EGFR family (ErbB/HER) has proved to be therapy, the ability to combine multiple drugs a fruitful area for the development of cancer produces better clinical outcomes; combinations therapeutics. To date, eight agents are approved are enabled by better safety profiles. for the treatment of various solid tumors, and at Because trastuzumab and pertuzumab bind to least a dozen other compounds are currently in different regions of HER2 (Fig. 2), dual antibody clinical development. therapy should allow for simultaEGFR therapeutics are divided neous antagonism of both activated into two classes, both of which informs of HER2. Support for this hibit EGFR: monoclonal antibodies Antigen hypothesis was initially obtained in (mAbs), cetuximab and panitumumab, binding Light chain a phase II clinical trial that tested and low molecular weight tyrosine combination antibody therapy in pakinase inhibitors (TKIs), gefitinib and tients whose tumors progressed on erlotinib. mAbs directed against EGFR trastuzumab and cytotoxic chemowere first developed in advanced cotherapy. The findings in this trial demlorectal cancer. The pivotal trial for onstrated an objective response (tumor cetuximab demonstrated tumor shrinkshrinkage) of 25% and a clinical benage when used in combination with the efit rate of 50% (31). A neoadjuvant Heavy chain topoisomerase I inhibitor irinotecan in Fab' region (i.e., treatment before surgery) trial patients whose tumors were progressfurther confirmed that the antibody ing on irinotecan-based therapy (20). combination was also effective in paDiscernable single-agent cetuximab actients who were nave to therapy (32). tivity was also observed. The murine Equipped with the knowledge that parent mAb, 225, was reengineered dual HER2 antibody blockade was as a human chimera, which allows for active in early breast cancer and lateADCC. In contrast, panitumumab was stage metastatic breast cancer, a pivderived from a transgenic mouse caotal trial validated the approach for pable of producing human antibodies the treatment of first-line metastatic and exhibits considerably higher bindbreast cancer patients and led to the ing affinity for EGFR than cetuximab. accelerated, full approval of pertuzumab Panitumumab is a human IgG2, which is less effective in mediating ADCC Fig. 1. Space-filling model of IgG1. Light chain, magenta; heavy chain, dark in June 2012 (30). Indeed the magnitude of patient benefit, as measured than IgG1 (21). Both antibodies are blue; carbohydrate, yellow. [Credit: C. Eigenbrot, Genentech, Incorporated] restricted to use in patients whose tumors overexpress EGFR and are wild type for the protooncogene KRAS but have not yet been compared directly, making it impossible to judge the contribution of ADCC. Although both EGFR mAbs and smallmolecule inhibitors have been commercially available for more than a decade, there is remarkably little overlap in how the two classes of antagonists are used clinically. Combining mAbs and small molecules is ineffective, despite preclinical data suggesting increased efficacy (22). Similarly, antagonism of EGFR was originally predicted to synergize with cytotoxic chemotherapy. Combination therapy of irinotecan with cetuximab was key to cetuximab FDA approval. Yet erlotinib or gefitinib failed to show clinical benefit in combination with several chemotherapies (23, 24). Indeed, there is a suggestion that patient outcome may be worse when treated with the combination (25). Many factors contribute when preclinical observations fail to predict clinical responses, including differences in pharmacokinetics and, most importantly, differences in tolerability. Although both mAb and chemical inhibitors of EGFR cause skin rash, erlotinib and gefitinib cause additional toxicities that further limit tolerability when combined with conventional chemotherapies. Of interest in this regard is the recent clinical demonstration (and FDA approval) that erlotinib is more effective against tumors expressing an activated mutant allele of EGFR (24). Although both wild-type and mutant kinases are inhibited, selectivity for the mutant kinase might be expected to increase therapeutic index by sparing, relatively, the activity of the wild-type kinase in normal cells and tissues.
Fc region

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Antibodies
Table 1. Antibody therapies and their indications.
Antibody type Cetuximab (Erbitux; Bristol-Myers Squibb): chimeric human-murine IgG1 Target EGFR Tumor types Colon, head, and neck Dx test Yes Indication Indicated for the treatment of KRAS wild-type, EGFR-expressing, metastatic colorectal cancer (mCRC) in combination with FOLFIRI [irinotecan, 5-fluorouracil (5-FU) leucovorin] for first-line treatment, in combination with irinotecan in patients who are refractory to irinotecan-based chemotherapy, as a single agent in patients who have failed oxaliplatin- and irinotecan-based chemotherapy or who are intolerant to irinotecan. In combination with radiation therapy for the initial treatment of locally or regionally advanced squamous cell carcinoma of the head and neck and in combination with platinum-based therapy with 5-FU for the first-line treatment of patients with recurrent locoregional disease or metastatic squamous cell carcinoma of the head and neck. As a single agent, is indicated for the treatment of patients with recurrent or metastatic squamous cell carcinoma of the head and neck for whom prior platinum-based therapy has failed. Indicated for the treatment of EGFR-expressing and KRAS wild-type mCRC with disease progression after fluoropyrimidine-, oxaliplatin-, and irinotecan-containing regimens. Indicated for adjuvant treatment of HER2overexpressing node-positive or node-negative breast cancer. As part of a treatment regimen containing doxorubicin, cyclophosphamide, and either paclitaxel or docetaxel. Also with docetaxel and carboplatin. As a single agent after multimodality anthracycline-based therapy. For metastatic breast cancer in combination with paclitaxel for the first-line treatment of HER2-overexpressing metastatic breast cancer. As a single agent for treatment of HER2-overexpressing breast cancer in patients who have received one or more chemotherapy regimens for metastatic disease. In combination with cisplatin and capecitabine or 5-FU, for the treatment of patients with HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma, who have not received prior treatment for metastatic disease. In combination with trastuzumab and docetaxel for the treatment of patients with HER2-positive metastatic breast cancer who have not received prior anti-HER2 therapy or chemotherapy for metastatic disease. Treatment of patients with HER2-positive metastatic breast cancer who have received prior treatment with Herceptin and a taxane chemotherapy. mCRC for first- or second-line treatment in combination with intravenous 5-FUbased chemotherapy. It is also approved to treat mCRC for second-line treatment when used with fluoropyrimidine-based (combined with irinotecan or oxaliplatin) chemotherapy after cancer progresses following a first-line treatment that includes Avastin. Advanced nonsquamous nonsmall cell lung cancer in combination with carboplatin and paclitaxel in people who have not received chemotherapy for their advanced disease. Metastatic kidney cancer when used with IFN-a. Glioblastoma when taken alone in adult patients whose cancer has progressed after prior treatment.

Panitumumab (Vectibix; Amgen): human IgG1

EGFR

Colon

Yes

Trastuzumab (Herceptin; Genentech): humanized IgG1

HER2

Breast, gastric

Yes

Pertuzumab (Perjeta; Genentech): humanized IgG1

HER2

Breast

Yes

Ado-trastuzumab emtansine (Kadcyla, Genentech): HER2 trastuzumab, derivative of DM1, 4-(Nmaleimidomethyl)cyclohexane-1-carboxylate linker Bevacizumab (Avastin; Genentech/Roche): VEGF humanized IgG1

Breast Colon, nonsmall cell lung, glioblastoma, kidney

Yes

No

Continued on next page

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Antibody type Ipilimumab (Yervoy; Bristol-Myers Squibb): IgG1 Target CTLA4 Tumor types Melanoma Dx test Yes Indication Treatment of unresectable or metastatic melanoma in patients who have failed or do not tolerate other systemic therapy for advanced disease. As a single agent, in patients with relapsed or refractory, low-grade or follicular, CD20-positive, B cell non-Hodgkins lymphoma (NHL). Previously untreated follicular, CD20-positive, B cell NHL in combination with first-line chemotherapy and in patients achieving a complete or partial response to Rituxan in combination with chemotherapy, as single-agent maintenance therapy. Nonprogressing (including stable disease), low-grade, CD20-positive, B cell NHL as a single agent after first-line CVP chemotherapy. Previously untreated diffuse large B cell, CD20-positive NHL in combination with CHOP or other anthracycline-based chemotherapy regimens. Previously untreated and previously treated CD20-positive chronic lymphocytic leukemia in combination with fludarabine and cyclophosphamide. As a single agent for the treatment of patients with chronic lymphocytic leukemia refractory to fludarabine and alemtuzumab. Treatment of B cell NHL (relapsed or refractory, low grade, follicular, transformed, or rituximabrefractory). Patients with previously untreated follicular NHL who achieve a partial or complete response to first-line chemotherapy.

Rituximab (Rituxan/Mabthera; Roche): chimeric human-murine IgG1

CD20 NHL, chronic lymphocytic leukemia

No

Ofatumubab (Arzerra; Genmab): human IgG1

90

CD20

Chronic lymphocytic leukemia

No

Y-labeled ibritumomab tiuxetan (Zevalin; IDEC Pharmaceuticals): murine IgG1, linker-chelator: tiuxetan (N-(2-Bis(carboxymethyl)amino)3(Pisothio-cyanatophenyl)~propyl)N-(2-Bis(car-boxymethyl)-amino)2-(Methyl)ethyl)glycine 131 I-labeled tositumomab (Bexxar; GlaxoSmithKline): murine IgG2, direct covalent linkage to tositumomab

CD20

Low-grade or follicular B cell NHL

Yes

CD20

Low-grade or follicular B cell NHL

Yes

Alemtuzumab (Campath; Genzyme): humanized IgG1 Brentuximab vedotin (Adcetris; Seattle Genetics): chimeric IgG1, MMAE, maleimidocaproyl valine-citrulline-PAB linker

CD52 CD30

Chronic lymphocytic leukemia Hodgkins lymphoma

No No

Gemtuzumab ozogamicin (Mylotarg; Wyeth): CD33 humanized IgG2), N-acetyl gamma-calichaemicin dimethyl hydrazide, AcBut hydrazone linker

Acute myelogenous leukemia

Yes

Treatment of patients with CD20-positive relapsed or refractory, low-grade, follicular or transformed NHL who progressed during or after rituximab therapy, including patients with rituximabrefractory NHL. As a single agent for the treatment of B cell chronic lymphocytic leukemia For the treatment of patients with Hodgkin lymphoma after failure or autologous stem cell transplant (ASCT) or after failure of at least two prior multiagent chemotherapy regimens in patients who are not ASCT candidates. The treatment of patients with systemic anaplastic large cell lymphoma after failure of at least one prior multiagent chemotherapy regimen Withdrawn from the market in June 2010. Previously indicated for the treatment of patients with CD33-positive acute myeloid leukemia in first relapse who are 60 years of age or older and who are not considered candidates for other cytotoxic chemotherapy.

by progression-free survival (3.9 versus 6.1 months) (33) and overall survival (34), is greater than what was observed for the initial approval of trastuzumab. In summary, the advent of mAbs to oncogenic receptor tyrosine kinases of the EGFR family has represented a major advance in cancer therapy. Yet, there have also been attempts that have not yet led to success in the clinic, using mAbs to target other signaling receptors. Notable among these are antibodies to insulinlike growth factor 1 receptor, despite this receptor having a mechanistic link to many types of cancer. On the other hand, antibodies to c-Met

continue in clinical development. The number of mAbs tested in clinical trials is small, which makes it difficult to draw any clear lessons yet. However, it should be noted that effective antibodies against solid tumors tend to recognize targets that are either subject to mutation (EGFR, c-Met) or overexpression (HER2) in cancer, even if the antibody itself is inactive against the mutant allele. Conceivably, mAb therapy against signaling receptors may prove most effective in cases where targets can be shown genetically to be important drivers of cancer or clear drivers of resistance to other forms of therapy. SCIENCE VOL 341

Antibody-Drug Conjugates (ADCs) The concept that antitumor antibodies could be used as vehicles for the selective delivery of cytotoxic agents to tumors has been around nearly as long as mAbs. However, until very recently, the idea has eluded successful implementation, probably for three reasons: (i) the use of antibodies against targets that were not sufficiently restricted to tumor cells, (ii) the use of drugs with insufficient potency or (in the case of bacterial or plant toxins) that were immunogenic, and (iii) the linker chemistry used to attach drugs to antibodies was not optimized. The latter consideration is

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one that required an enormous empirical effort to solve. A linker that was too labile allowed too much of the toxic drug to dissociate from the antibody in the blood. This then exposed normal tissues to the freed drug and, as a result, reduced the doses of the ADC that could be used. Stable linkers require complete proteolytic digestion of the ADC, releasing the derivatized amino acid, linker, and cytotoxic drug as the active metabolite (35). Similarly, the choice of drug required a great deal of trial and effort, with current successes being seen by using microtubule antagonists. Gemtuzumab ozogamicin was the first ADC to reach the market in 2000. It is directed against CD33, a surface glycoprotein characteristic of acute myeloid leukemia (36). The conjugate is composed of a humanized IgG4, a pH labile linker, and calicheamicin, a very potent DNA minor groove binder. Unfortunately, the drug was withdrawn from the market in 2010 because of the failure of subsequent confirmatory studies to reproduce the initial clinical benefit profile (37, 38). More recently, brentuximab vedotin, a mAb against CD30 that was covalently modified with the microtubule antagonist monomethyl auristatin E (MMAE), was approved for use in advanced Hodgkins lymphoma given its efficacy and good safety profile (39). The linker used to tether MMAE to the antibody contains a peptidic moiety that was designed as a substrate for cathepsin B; the antibody was delivered to cathepsin Bcontaining endosomes and lysosomes after internalization by the CD30-positive lymphoma cells (40). Another ADC that has also recently gained approval is T-DM1, which is composed of trastuzumab (T) conjugated to a maytansine derivative (DM1) (41). In this instance, the linker is designed not to be cleaved by lysosomal proteases, suggesting that the drug is released only after limited digestion of the entire antibody. T-DM1 was found to prolong progression-free survival (9.6 versus 6.4 months) and overall survival (30.9 versus 25.1 months) in late-stage HER2-positive breast cancer patients who progressed on multiple other treatments (42). The clinical successes of T-DM1 and brentuximab vedotin have stimulated a great deal of activity in developing ADCs to different solid and hematologic tumor targets using mAbs to a variety of antigens, most often conjugated to microtubule antagonists (MMAE and DM1 or their derivatives). In addition, new toxic payloads and linkers are under development in an effort to further increase therapeutic index and provide for combination therapy. Novel engineered antibody platforms are also being investigated. For example, the typical cleavable MMAE linker drug (vc-MMAE) is coupled to antibodies after the reduction and then the derivatization of endogenous cysteine residues normally engaged in interchain disulfide bonds. Methods are being developed to engineer specific conjugation sites (e.g., unpaired cysteine residues) and defined sites on the heavy or light chains, leading to greater ease of conjugation and control over the number of drug molecules per antibody molecule (43). Other modifications are also being investigated in an effort to increase specificity of cancer cell binding (e.g., use of bispecific antibodies that recognize two antigens at once) or decrease the nonspecific uptake of the ADCs by normal cells that do not express the targets. Use of antibody-conjugated nanocarriers loaded with cytotoxic agents is also under investigation. Although this would increase the payload delivered after endocytosis, a potential limitation is the nonspecific loss of the cytotoxic in the plasma. We anticipate that the next several years will witness explosive growth and innovation in this area. Antibodies and Cancer Immunotherapy As was true for ADCs, the concept that a patients immune system could be activated to generate anticancer responses has been actively explored for >30 years. For most of this time, however, little progress was made in the clinical arena, largely because of our incomplete knowledge concerning the functional organization of the immune system and how tumors manipulate the immune response. Moreover, much of the field was long fascinated by anticancer vaccines, an approach limited by the immunosuppressive tumor microenvironments (44). Many cancer patients make anticancer T cell responses because tumors nearly always express mutant proteins or antigens to which the immune system is not tolerized and therefore are immunogenic. Yet these responses rarely manifest in clinical benefit. An appreciation has now emerged that overcoming immunosuppression and enhancing the quality of the immune response may be key to developing effective cancer immunotherapies (3). This realization has generated enormous interest and excitement fueled by the early clinical successes of two classes of mAbs. The first of these is ipilimumab, which inhibits CTLA4, a negative regulator expressed on the surface of all T cells (Fig. 3A) (45). CTLA4 normally binds to CD80/CD86 on the surface of antigen-presenting cells, providing a powerful brake or checkpoint that limits the activation and proliferation of

A
um ab

Pertuzumab

HER2

s Tra

tuz

HER2 HER3

HER3 Ligand

Kin
P P P P

Kin
P P

C
Cetuximab Panitumumab

D
M1
HER3

HER2

T-D

EGFR

*
Kin
P P P

**

Fig. 2. Epitope binding of HER2 and EGFR therapeutic antibodies. (A) Trastuzumab. (B) Pertuzumab. (C) Cetuximab or panitumumab. (D) T-DM1 (ado-trastuzumab emtansine). In the case of HER2 signaling, a functional and physical association with a second related receptor, HER3, is often important (60). HER2 kinase phosphorylates the HER3 cytoplasmic domain, activating it as a scaffold to promote the phosphatidylinositol 3-phosphate kinase cascade. Conversely, activation of HER3 by binding ligands such as heregulin can allosterically activate HER2 kinase. Pertuzumabs likely mechanism of action is to block heterodimerization of HER2 and HER3. Asterisks indicate a cytotoxic agent, derivative of maytansine 1 (DM1). VOL 341 SCIENCE www.sciencemag.org

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T cells after recognizing their cognate antigen. Ipilimumab blocks this interaction, thereby releasing the checkpoint and amplifying T cell responses. CTLA4 blockade also induces the death of regulatory T cells, which contribute to the immunosuppressive tumor microenvironment, by a mechanism that is still being characterized (possibly ADCC in the tumor bed; ipilimumab is an IgG1) (46, 47). In the clinic, ipilimumab was found to provide robust and long-term survival benefit or cures to patients with late-stage metastatic melanoma who otherwise had no other treatment options (9). Although the percentage of patients receiving benefit was small (3-year survival of 23.5% versus ~10% in a control arm) and cannot yet be diagnostically defined, the results are impressive and led to ipilimumab being approved by the FDA in 2011. Subsequently, ipilimumab has been reported to exhibit varying levels of clinical activity in other indications, but its use is complicated by the fact that it also induces occasionally serious immune-related adverse events (e.g., autoimmune inflammation). In any event, these studies established critical proof of concept for checkpoint inhibitors specifically and for other immunomodulators in general. Following closely behind the success of ipilimumab are mAbs that antagonize the interaction of programmed death1 (PD-1), another negative regulator of T cells, with its ligands PD-L1 and PD-L2 (Fig. 3B) (48). Two antibodies against PD-1 (nivolumab and lambrolizumab) and one to PD-L1 (MPDL3280A) are the most advanced in the clinic. Although not as powerful of a brake as CTLA4, PD-1s role is to prevent the unrestrained activation of T cells that have been previously activated. PD-L1 is often expressed by tumor cells or immune cells infiltrating into tumors. Evolutionarily, its role probably developed as a regulatory node in viral immunity, preventing T cell hyperactivation and bystander killing of noninfected cells during virus infection: For example, normal cells react to interferon-g (IFN-g) release by local effector T cells by expressing PD-L1 to protect themselves against T cell killing. In cancer, tumor cells are thought to react in much the same way upon entry of tumor-specific IFN-g secreting cytotoxic T cells. Thus, the binding of tumor PD-L1 to PD-1 on activated T cells inhibits their effector function. Mechanistically, this appears to occur by the recruitment of a phosphatase to the PD-1 cytoplasmic domain that inactivates the phosphatidylinositol 3-kinase pathway in T cells, exhausting their ability to generate or release cytotoxic granules (49). Thus, the PD-1/PD-L1 axis serves as a front-line mechanism of immune suppression in the tumor bed. PD-L2 in particular is also expressed by dendritic cells and may play a role in maintaining peripheral tolerance. Both preclinically and clinically, the addition of mAbs to either PD-1 or PD-L1 blocks their interaction, thereby rescuing T cell cytotoxic activity; often, this results in rapid and substantial tumor shrinkage coupled with long-term, durable responses (48). Presumably because the primary mode of action is to overcome immunosuppression of antitumor T cells in the tumor bed (as opposed to anti-CTLA4, which amplifies the production of Tcells of all specificities in lymph nodes),

T cell

B IFN-

CTLA4 CD80 CD86

CD28

PD-1

CD8

PD-1

T cell receptor PD-L2 Tumor antigen

PD-L1

PD-L1

Dendritic cell

Tumor cell

Dendritic cell

Tumor cell

Anti-CTLA4 Ipilimumab Anti-PD-L1 MPDL3280A

Anti-PD-1 Nivolumab Lambrolizumab

Anti-CD-3

Fig. 3. The use of antibodies in active and passive immunotherapy of cancer. (A) Checkpoint blockade by anti-CTLA4. CD80 and CD86 are ligands expressed on the surface of activated dendritic cells during the presentation of MHC [human lymphocyte antigen (HLA)]peptide complexes to T cell receptors. CD80/86 binds to the costimulatory molecule CD28 to help activate T cell proliferation and then to the checkpoint inhibitor CTLA4 to attenuate T cell proliferation. The antibody ipilimumab blocks the interaction of CTLA4 with its ligands, thereby releasing the checkpoint inhibitor and favoring T cell proliferation. (B) Checkpoint blockade and inhibiting immune suppression by anti-PD1 or antiPDL1. (Left) T cell influx into tumors results in the release of IFN-g, which up-regulates PD-L1 expression by tumor cells. PD-L1 binds to PD-1, which is expressed by activated T cells, generating a negative signal that causes T cell exhaustion (inhibiting the ability of T cells to recognize and kill their targets). (Right) During antigen presentation by dendritic cells, PD1 can also act as a checkpoint inhibitor www.sciencemag.org SCIENCE

where a negative signal can be sent by its binding to either PD-L1 or the closely related (and dendritic cellspecific) negative regulatory ligand PD-L2. Generally, inhibition of both PD-L1 and PD-L2 (e.g., by antiPD-1) is required to block negative regulation by dendritic cells, whereas only PD-L1 inhibition (by antiPD-1 or antiPD-L1) should relieve immunosuppression (immune rheostat) activity in the tumor bed. Note that, for clarity, only the primary interactions of PD-1, PD-L1, and PD-L2 are illustrated. (C) Bispecific antibodies against CD3 passively recruit cytotoxic T cells to tumor cells. Blinatumomab is a single-chain bispecific antibody that is composed of an anti-CD3 arm that recognizes the T cell receptor and an anti-CD19 arm that recognizes a surface antigen on the surface of B cell lymphoma cells; diagrammed is a conventional bispecific IgG for clarity. Recruitment of the T cells to the tumor cells in this way results in efficient tumor cell killing, as if the T cell had recognized its cognate peptideMHC on the tumor cell target. VOL 341 13 SEPTEMBER 2013

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adverse events associated with PD-1/PD-L1 blockade appear substantially less serious than with ipilimumab. Although registrational trials are just now getting under way, preliminary suggestions are that mAbs against both PD-1 and PD-L1 are active (5054); it is too soon to know whether one or the other approach will distinguish itself on the basis of safety or efficacy. In this respect, it is interesting to note that PD-1 blockade will also inhibit interactions of T cells with PD-L2 on antigen-presenting cells (especially in the lung), which may or may not increase chances for toxicity; pneumonitis is a risk factor in patients treated with nivolumab, anantibody against PD-1. In addition, the PD-1 antibodies in the clinic are of the human IgG4 subclass with reduced potential for ADCC compared with the PD-L1 antibody, which has been engineered to completely eliminate FcgR binding and has yet to elicit serious pneumonitis (53). This difference or the difference in target (PD-1 versus PD-L1) may or may not correlate with increased efficacy or safety. Given the complementary mechanisms of action of ipilimumab and the antiPD-1/L1 antagonists, a recent clinical study has demonstrated that the two agents can show impressive additive activity, as well as additive toxicity, in melanoma (55). Because different criteria were used to judge clinical responses in this versus single-antibody trials [e.g., use of Response Evaluation Criteria in Solid Tumors (RECIST) versus World Health Organization (WHO) criteria for tumor shrinkage], further study will be required to understand the degree of enhanced benefit. Antibodies that block immune checkpoints and immune suppression in the tumor bed have produced long-term, durable patient responses rarely seen with other therapeutics and, as such, may again change the face of cancer therapy similar to the initial antibodies against EGFR and HER2 15 years ago. It is likely that the coming years will see many more antibodies in this space as additional regulatory targets, both cell-associated and secreted, are identified and investigated. Other Related Approaches At some level, all therapies involving mAbs are immune therapies in that the immune system was required to produce and engage the therapeutic. However, it is useful to regard immunotherapies as ones that seek to actively manipulate the immune system, most often the T cell compartment, as is the case for antibodies to CTLA4, PD-1, and PD-L1. Hybrid approaches are already being introduced. One example of note is a specific singlechain antibody, blinatumomab, comprising tandem single-chain Fv fragments that bind CD19 on lymphoma and normal B cells and CD3, the antigen receptor of T cells (56). Treatment of patients with this small protein is effective at depleting nonHodgkins lymphoma cells by recruiting T cells to tumor cells and activating their cytotoxic effector function regardless of the T cells own inherent specificity. Although active in early clinical trials, it is rapidly cleared from the circulation because of its small size, necessitating continuous infusion. Future work will no doubt investigate modifying the platform to enable more convenient dosing (Fig. 3C). Another approach accomplishes the same task, except by fusing the anti-CD3 specificity to soluble, recombinant T cell receptors that can be selected to detect tumor-specific peptidemajor histocompatibility complex (MHC) class I molecules (57). As above, T cells are recruited regardless of their inherent specificity and triggered to become antitumor effectors. A third approach involves genetically modifying a patients own T cells to express a membranebound antibody against tumors fused to signaling molecules that trigger T cell killing when the modified T cell detects a cognate tumor cell. The T cells (CARs) have yielded a promising result, at least in hematologic cancer in early clinical trials (58). Last, conventional antibodies can be generated with engineered Fc domains that increase, rather than decrease, FcgR binding, in order to recruit macrophages and NK cells to mediate ADCC (59). The Past, Present, and Future Antibody therapeutics in cancer have a rich history, an exciting present, and a promising future. Although it is uncertain what new platforms will emerge as being efficacious and useful, it is certain that many new approaches will be tried in the years to come. The use of antibody therapeutics in combination with each other is also emerging. Indeed, compelling phase I data have recently been reported for dual immune checkpoint therapy using ipilimumab and nivolumab (55). These combinations have the potential to significantly lower or, in some cases, eliminate the amount of cytotoxic chemotherapy that is still currently the backbone of most oncology treatments. Systemic treatment of patients, which includes targeted mAb therapy, before surgery is also emerging (32). Moreover, one could imagine scenarios where these approaches are likely to reduce the need for extensive surgery. New delivery platforms, new approaches to ADCs, and new ideas to manipulate the immune response or the tumor microenvironment are certainly on the horizon and bode well for a renaissance of interest in antibody biochemistry and function, as well as for cancer patients.
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