Cancer Immun
`
` 1424-9634Cancer Research Institute
`
`(1 May 2012) Vol. 12, p. 14
`Cancer Immunity
`Copyright © 2012 by Andrew M. Scott
`
`120114
`
`Monoclonal antibodies in cancer therapy
`
`Andrew M. Scott1,2, James P. Allison3,4,5 and Jedd D. Wolchok3,6,7,8
`
`1Ludwig Institute for Cancer Research, Melbourne, Australia
`2University of Melbourne and Centre for PET, Austin Hospital, Melbourne, Australia
`3Ludwig Center for Cancer Immunotherapy at Memorial Sloan-Kettering Cancer Center, New York, NY, USA
`4Howard Hughes Medical Institute
`5Department of Immunology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA
`6Weill Medical College of Cornell University, New York, NY, USA
`7Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA
`8Ludwig Institute for Cancer Research at Memorial Sloan-Kettering Cancer Center, New York, NY, USA
`
`Keywords: antibody identification, tumor surface antigens,
`antigenic targets, tumor therapy
`Introduction
`Monoclonal antibody-based treatment of cancer has been
`established as one of the most successful therapeutic strategies
`for both hematologic malignancies and solid tumors in the last
`20 years. The initial combining of serological techniques for
`cancer cell surface antigen discovery with hybridoma
`technology led to a series of landmark clinical trials that paved
`the way for new generation antibodies and subsequent clinical
`success. Optimization of anti-tumor immune responses through
`Fc modifications has also made a major contribution to clinical
`efficacy. The modulation of immune system interplay with
`tumor cells through targeting of T cell receptors has emerged as
`a powerful new therapeutic strategy for tumor therapy and to
`enhance cancer vaccine efficacy. This commentary will provide
`an overview of the history of antibody identification of tumor
`surface antigens, antigenic targets suitable for antibody-based
`therapy, antibody mechanisms of action, and recent successes of
`antibodies in the clinic.
`Cancer serology - the prelude to antibody
`therapeutics
`The concept that antibodies could serve as ‘magic bullets’ in
`the diagnosis and therapy of cancer dates back to their discovery
`in the late 19th century. A considerable effort over the ensuing
`decades involved immunization of a variety of animal species
`with human cancer in the hope of generating antisera with some
`degree of cancer specificity (1). Unfortunately, this approach
`had limited early success, with the notable exception of the
`discovery of carcinoembryonic antigen (CEA), a marker for
`colon and other cancers, and -fetoprotein, a marker for
`hepatocellular cancer (1, 2).
`The development of inbred mice initiated a new era of
`serological investigation of cancer with the emergence of the
`cytotoxic test as a powerful tool to analyze the cell surface
`reactivity of alloantibodies. This subsequently led to the
`recognition that the cell surface is a highly differentiated
`structure. During the 1960s and 1970s, Lloyd Old made a series
`of discoveries that revolutionized our understanding of the
`immune system. In collaboration with Ted Boyse, he introduced
`the concept of cell surface differentiation antigens that could
`
`distinguish lineage and functional subsets of leukocytes in mice
`(3). This led to major contributions at the time which include
`the discovery of the thymus-leukemia (TL) antigen, the linking
`of the major histocompatibility complex (MHC) and leukemia,
`and subsequently the Ly series of antigens (4). These discoveries
`led to the precise and systematic identification of cell surface
`antigens that distinguished normal cells from malignant cells,
`and directly to the cluster of differentiation (CD) classification.
`Following the development of hybridoma technology by
`Köhler and Milstein (5), combined with serological techniques
`and analytical tools such as fluorescence-activated cell sorting
`(FACS), monoclonal antibodies (mAbs) were used to dissect the
`surface structure of human cancer cells, thus paving the way for
`the identification of cancer cell surface antigens suitable for
`targeting by antibodies. The characterization of the cancer cell
`“surfaceome” has been enhanced
`in recent times with
`proteomic, genomic, and bioinformatic approaches
`to
`identifying antigen targets on cancer cells, as well as in cancer
`stroma and vasculature.
`Tumor antigens as targets for antibody therapy
`The selection of tumor antigens suitable for antibody targeting
`and therapy requires a comprehensive analysis of tumor
`expression (including homogeneity of expression) and normal
`tissue expression, as well as an understanding of the biologic role
`of the antigen in tumor growth. If the desired mechanism of
`action is engagement with cell surface receptors (to either
`activate or inhibit signaling), or to activate antibody-dependent
`cell-mediated cytotoxicity (ADCC) or complement-dependent
`cytotoxicity (CDC), then it is desirable that the antigen-mAb
`complex should not be rapidly internalized. This allows the
`maximization of the availability of the Fab region to
`appropriately engage with surface receptors, and of the Fc region
`to immune effector cells and complement proteins. In contrast,
`internalization is desirable for antibodies or proteins delivering
`toxins into the cancer cell and for antibodies whose action is
`primarily based on downregulation of cell surface receptors (2).
`Tumor-associated antigens recognized by therapeutic mAbs
`are outlined in Table 1. Hematopoietic differentiation antigens
`are glycoproteins usually associated with CD groupings and
`include CD20, CD30, CD33, and CD52 (2, 6-8). Cell surface
`differentiation antigens
`represent a diverse group of
`glycoproteins and carbohydrates that are found on the surface of
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`Cancer Immunity (1 May 2012) Vol. 12, p. 14
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`both normal and tumor cells. Growth factors that are targets for
`antibodies in oncology patients include CEA (2), epidermal
`growth factor receptor (EGFR; also known as ErbB1) (9), ErbB2
`(also known as HER2) (10), ErbB3 (11), MET (12), insulin-like
`growth factor 1 receptor (IGF1R) (13), ephrin receptor A3
`(EphA3) (14), TNF receptor apoptosis-inducing ligand receptor
`1 (TRAIL-R1), TRAIL-R2, and receptor activator of nuclear
`factor 
`ligand (RANKL) (15). Antigens
`involved
`in
`angiogenesis are usually proteins or growth factors that support
`the formation of new microvasculature, including vascular
`endothelial growth factor (VEGF), VEGF receptor (VEGFR),
`and integrins V3 and 51 (16). Stromal and extracellular
`matrix antigens that are therapeutic targets include fibroblast
`activation protein (FAP) and tenascin (17-19).
`Antibody engineering and mechanisms of
`action
`The development of hybridoma technology led to the first
`generation of murine antibodies against tumor cell surface
`antigens. Following initial clinical trials in the 1980s with
`murine antibodies against CEA and CD3, a range of antibodies
`against solid tumor and hematologic malignancies were
`developed and entered clinical trials (20). The development of
`Table 1
`Tumor-associated antigens targeted by monoclonal antibodies
`
`immune responses against these murine antibodies (human
`anti-mouse antibodies, HAMA) significantly limited their
`clinical utility, and as a consequence, apart from the FDA-
`approved 131I-anti-CD20 antibody tositumomab, and 90Y-anti-
`CD20 antibody ibritumomab tiuxetan, murine antibodies were
`not further pursued (20).
`The development of humanization approaches by Winter and
`colleagues (21), whereby murine Fc and Fv framework regions
`of murine antibodies were replaced by human germline amino
`acids, revolutionized the field of antibody therapeutics. Through
`this technology, minimal immune responses to antibodies were
`observed, allowing the multiple
`infusions of engineered
`antibodies, leading to the successful entry of multiple antibodies
`into the clinic (6, 22). Additional strategies to generate fully
`human antibodies through phage display techniques, as well as
`the use of transgenic mice that produce fully human antibodies,
`have also been successfully implemented (17). More recently,
`innovative antibody engineering approaches to produce smaller
`antibody variants, fusion proteins, and bispecific antibodies
`have been utilized (6, 7, 22). Combined with improved cell line
`generation and
`larger scale production
`techniques,
`the
`transition from laboratory scale preclinical testing to large
`clinical trial batch production has been considerably enhanced.
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`Table 2
`Mechanisms of tumor cell killing by antibodies
`
`The mechanisms of tumor cell killing by antibodies are
`outlined in Table 2. These can be due to direct cell killing, such
`as through receptor blockade or agonist activity, induction of
`apoptosis, or delivery of a drug, radiation, or cytotoxic agent;
`immune-mediated cell killing mechanisms; regulation of T cell
`function; and specific effects on tumor vasculature and stroma.
`The Fc function of antibodies is particularly important in
`
`Figure 1
`
`Scott et al.
`
`mediating tumor cell killing through CDC, and immune cell
`activation (e.g., NK cells) and tumor cell killing through ADCC.
`The abrogation of tumor cell signaling (e.g., cetuximab and
`trastuzumab) (9, 10), effector function primarily through
`ADCC (e.g., rituximab) (23), and immune modulation of T cell
`function (e.g., ipilimumab) (24) are the approaches that have
`been most successful and have led to approval of antibodies
`using these effector mechanisms.
`“In vivo veritas” - translation of antibodies into
`the clinic
`While the identification of novel target antigens expressed in
`tumors and the generation of antibodies against these antigens
`with optimal functional characteristics is the important initial
`step in developing a potential therapeutic antibody, there are
`many issues to address before embarking on clinical trials in
`cancer patients. These include the physical and chemical
`properties of the antibody, a detailed specificity analysis of
`antigen expression using panels of normal and malignant
`tissues, and immune effector function and signaling pathway
`effects of antibodies. In addition, antibody humanization and in
`vivo therapeutic activity of the antibody, either alone or
`conjugated with radioactive isotopes or other toxic agents, are
`essential in the preclinical evaluation of antibodies (6, 8, 17, 25-
`29).
`
`Biodistribution of 131I-huA33 in a patient with metastatic colorectal carcinoma. Anterior whole body gamma camera images are shown following infusion of 131I-
`huA33 at (A) day 0, (B) day 1, and (C) day 5. A standard for quantitation of 131I-huA33 uptake is present, adjacent to the left shoulder. Initial (day 0) images (A) show
`blood pool appearance only, with large metastatic lesions in the liver demonstrating an initial hypovascular appearance. (B) Excellent targeting of the metastatic lesions
`in the liver by 131I-huA33 is clearly seen (arrow) as early as day 1, and increasing rapidly with time to day 5. Some central necrosis in the larger tumors is also evident
`(arrow), also seen on CT scan (D). Gradual bowel uptake (double arrow) of 131I-huA33 is also seen, which gradually decreases with time. No other normal tissue uptake
`of 131I-huA33 is evident.
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`One of the most essential steps in the clinical evaluation of a
`in vivo specificity—
`potential
`therapeutic antibody
`is
`determining
`the
`biodistribution
`of
`antibody
`(often
`radiolabeled) in patients to assess the ratio of antibody uptake in
`the tumor in relation to normal tissues (18, 25, 29) (Figure 1).
`This information is essential for the design for clinical trials, as
`knowledge about the targeting of normal tissues is critical for
`predicting toxicity and determining optimal antibody dose and
`schedule (8, 29). Under the leadership of Lloyd Old, at the
`Ludwig Institute for Cancer Research (LICR), we developed a
`model of a phase I antibody clinical trial that incorporates
`biodistribution, pharmacokinetics, and pharmacodynamics
`analyses with toxicity assessment (25). This trial design has been
`successfully applied to first-in-human clinical trials of more
`than 15 antibodies in cancer patients (18, 19, 25, 29-34). This
`approach
`can
`identify
`subtle
`changes
`in
`antibody
`physicochemical properties (28) that affect biodistribution,
`which can significantly impact efficacy. In addition, normal
`tissue and tumor distribution can be quantitated, thus allowing
`the relationship of the loading dose to tumor concentration to
`be accurately assessed, rather
`than relying on plasma
`concentration and clearance rates to establish an optimal dose.
`Examples of where this approach was successfully used include
`Table 3
`Monoclonal antibodies currently FDA-approved in oncology
`
`the early biodistribution studies of mouse anti-colon cancer
`antibody A33 (33), the anti-CD33 antibody M195 (30), anti-
`CAIX antibody G250 (34), anti-FAP antibody F19 (19), anti-
`GD3 antibody KM871 (31), and anti-Ley antibody hu3S193
`(32). This approach has also been applied to recent studies of
`trastuzumab (which targets ErbB2) biodistribution and in vivo
`assessment of ErbB2 expression by tumors (35). In non-
`Hodgkin lymphomas, assessment of the biodistribution of a
`radioconjugate in both the tumor and through whole body
`dosimetry was essential in initial trials exploring patient
`suitability for treatment and treatment dose for the United States
`Food and Drug Administration (FDA)-approved anti-CD20
`radioimmunoconjugates
`tositumumab
`and
`ibritumomab
`tiuxetan (8, 28, 36).
`The use of patient biopsies can also be utilized to assess the in
`vivo effect of antibody abrogation of signaling pathways (36).
`The evaluation of pharmacodynamics in early-phase clinical
`trials can also involve biological effector function of antibodies,
`such as ADCC (through optimized FcR binding) and
`cytotoxicity (26). The assessment of antibodies as delivery
`vehicles for toxic agents can also be assessed using this clinical
`trial design approach (8, 26-29).
`
`Success of antibodies in the clinic
`There have been twelve antibodies that have received approval
`from the FDA for the treatment of a variety of solid tumors and
`hematological malignancies (Table 3). In addition, there are a
`large number of additional therapeutic antibodies that are
`currently being tested in early- and late-stage clinical trials. The
`
`use of therapeutic antibodies in patients with solid tumors has
`been most successful with classes of antibodies targeting the
`ErbB family (which includes EGFR) and VEGF (9, 16, 20, 37-
`39). Recent evidence shows that patients with colorectal cancer
`bearing wild-type KRas tumors who were treated with anti-
`EGFR antibodies have improved responses (9, 40), disease
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`control (40), and survival (41, 42). These findings have resulted
`in the FDA-approved use of these agents restricted to patients
`with colorectal cancer in which KRas is not mutated. The use of
`trastuzumab has also been restricted to patients with high levels
`of ErbB2 expression, as studies have shown that this is the group
`that derives maximum benefit from trastuzumab treatment (6,
`10). As a result of the clinical success of these antibodies and
`preclinical data demonstrating the improved tumor response
`(and reversal of resistance to single agent) of combined signaling
`blockade with antibodies to different receptors, or to different
`epitopes on
`the same receptor (e.g.,
`trastuzumab and
`pertuzumab), numerous clinical
`trials of antibodies as
`combination therapies are currently under way (20).
`A number of antibodies have also been approved for the
`treatment of hematological malignancies, both as unconjugated
`antibodies and for delivery of isotopes and drugs or toxins to
`cancer cells (Table 3). Antibody-drug or -toxin conjugates have
`been
`shown
`to have high potency
`in hematological
`malignancies, and there have been two approved by the FDA:
`gemtuzumab ozogamicin in elderly patients with CD33-positive
`AML (although this drug was voluntarily withdrawn in June
`2010 following a post-marketing phase III trial), and more
`recently brentuximab vedotin in patients with CD30-positive
`Hodgkin lymphoma (27, 43). A similar approach in patients
`with advanced ErbB2-positive breast cancer with the antibody-
`drug conjugate trastuzumab-emtansine (T-DM1) (44)
`is
`currently being explored in phase III trials.
`There are other antibodies approved for cancer indications
`outside the U.S. Catumaxomab, a mouse bispecific antibody
`against CD3 and EpCAM, is approved in the European Union
`for use in patients with malignant ascites generated by an
`EpCAM-positive tumor (45). Nimotuzumab, a humanized IgG
`antibody against EGFR, is approved for use in some countries in
`Asia, South America, and Africa for the treatment of head and
`neck cancer, glioma, and nasopharyngeal cancer (46). Vivatuxin
`(131I-chTNT), a radiolabeled IgG1 chimeric monoclonal
`antibody against intracellular DNA-associated antigens, has also
`been approved by the Chinese drug regulator for the treatment
`of malignant lung cancer (47).
`Immune regulation by antibodies
`In addition to targeting antigens involved in cancer cell
`physiology, antibodies can also
`function
`to modulate
`immunological pathways
`that are critical
`to
`immune
`surveillance. Antigen-specific immune responses result from a
`complex dynamic interplay between antigen presenting cells, T
`lymphocytes, and target cells. Immunologic signal 1, the
`recognition of specific antigenic peptides bound to MHC by the
`T cell receptor (TCR) is insufficient for T cell activation. Signal
`2, ligation of CD28 by a member of the B7 family of
`costimulatory molecules (CD80, CD86),
`initiates T cell
`activation via signaling pathways resulting in autocrine IL-2
`production. Just after T cell activation, CTLA-4, a molecule
`normally found in intracellular stores, translocates to the
`immunologic synapse, where it serves to inhibit the activated T
`cell by binding with high avidity to the same B7 molecules and
`stopping activation signals mediated by CD28. The role for
`blockade of CTLA-4 with an antibody as a means to potentiating
`T cell activation and initiating responses to targets on tumor
`cells was proposed in 1996 (48) and provided the scientific
`foundation for the development of two fully human monoclonal
`antibodies blocking CTLA-4 (ipilimumab and tremelimumab).
`Ipilimumab was approved by the U.S. FDA, European Medicines
`Agency (EMA), and a number of other national regulatory
`
`Scott et al.
`
`agencies for treatment of patients with metastatic melanoma
`after a pivotal phase III trial demonstrated significant
`improvement in overall survival resulting from its use, making it
`the first treatment to be shown to enhance survival and the first
`newly approved medicine in 13 years for melanoma (24). CTLA-
`4 blockade does present new paradigms in terms of treatment-
`related toxicity. The
`immune-related adverse events are
`inflammatory and
`largely confined
`to
`the
`skin and
`gastrointestinal tract but can more rarely affect the liver and
`endocrine glands. With prompt diagnosis, these events are
`generally manageable with immunosuppressive medications
`such as corticosteroids, which fortunately do not seem to
`interfere with the anti-tumor effect (24).
`The therapeutic success of ipilimumab has led to enthusiasm
`for the development of other immune modulating antibodies.
`The next most advanced products target PD-1, a marker of
`activated or exhausted T cells that can trigger apoptosis when
`bound by its ligand, PD-L1 (B7-H1) (49). Interestingly, this
`ligand is found not only on antigen presenting cells, but also on
`many tumor cells. PD-1 blockade has been shown in early
`clinical trials to result in durable responses in patients with
`melanoma, renal cell carcinoma, non-small cell lung cancer, and
`colorectal cancer (49). Several antibodies that target the PD-1
`axis are in development. Agonistic antibodies are also being
`explored, including two fully human antibodies to CD137 (4-
`1BB), an activator of T cells, from Pfizer and Bristol-Myers
`Squibb (BMS). The BMS antibody has been in phase I trials,
`demonstrating anti-tumor efficacy across a wide dose range.
`Trials were temporarily suspended due to severe hepatic toxicity
`at high doses but are now opening again using low doses. This
`highlights an important aspect of antibody drug development as
`higher doses of a blocking antibody may yield better therapeutic
`results while low doses of agonistic antibodies may allow for a
`better risk-benefit profile. Other pathways of interest for
`agonistic antibodies include CD40, where favorable preclinical
`and clinical results have been noted particularly in pancreatic
`cancer (50), and the glucocorticoid-induced TNF receptor
`(GITR).
`Antibody therapeutics might also have a role in generation of
`de novo immune responses to the antigen targeted by the
`antibody through promoting antigen presentation to Fc
`receptor-bearing cells (51-53). De novo induction of secondary
`immune responses may therefore allow for the effects of
`antigen-specific antibodies to persist after the dosing is
`completed.
`Conclusion
`The use of monoclonal antibodies for the therapy of cancer is
`one of the major contributions of tumor immunology to cancer
`patients. This success is built on decades of scientific research
`aimed at serological characterization of cancer cells, techniques
`for generating optimized antibodies to tumor targets, detailed
`investigation of signaling pathways relevant to cancer cells, and
`an understanding of the complex interplay between cancer cells
`and the immune system (20, 54). The clinical development of
`antibodies is inextricably linked to disciplined and detailed
`exploration of the properties of antibodies in vivo and
`assessment of functional effects on cancer cells. One of our
`major challenges is now to fully exploit antibody therapies in
`cancer patients by combining the two major immune-based
`treatment
`approaches—antibodies
`and vaccines. Trials
`combining ipilimumab with vaccines have shown mixed results
`thus far (24, 55). The Cancer Vaccine Collaborative, a joint
`academic clinical trials infrastructure established by LICR and
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`the Cancer Research Institute (CRI), is about to embark on a
`series of trials exploring NY-ESO-1 vaccines along with
`ipilimumab to further investigate this important area. In this
`way, the full promise of tumor immunology in controlling and
`treating cancer will hopefully be realized.
`Abbreviations
`ADCC, antibody-dependent cell-mediated cytotoxicity; MHC,
`major histocompatibility complex
`Acknowledgements
`A.M.S. is supported by the Ludwig Institute for Cancer
`Research (LICR), NHMRC grants 487922 and 1030469, and OIS
`funding from the Victorian Government. J.P.A. has been
`supported by the Ludwig Center at MSKCC and the National
`Institutes of Health. J.D.W. is supported by LICR, the Cancer
`Research Institute (CRI), National Institutes of Health, Swim
`Across America, and the Melanoma Research Alliance.
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