R E V I E W S
`
`THERAPEUTIC ANTIBODIES FOR
`HUMAN DISEASES AT THE DAWN OF
`THE TWENTY-FIRST CENTURY
`
`Ole Henrik Brekke* and Inger Sandlie ‡
`
`Antibodies are highly specific, naturally evolved molecules that recognize and eliminate
`pathogenic and disease antigens. The past 30 years of antibody research have hinted at
`the promise of new versatile therapeutic agents to fight cancer, autoimmune diseases and
`infection. Technology development and the testing of new generations of antibody reagents
`have altered our view of how they might be used for prophylactic and therapeutic purposes.
`The therapeutic antibodies of today are genetically engineered molecules that are designed
`to ensure high specificity and functionality. Some antibodies are loaded with toxic modules,
`whereas others are designed to function naturally, depending on the therapeutic application.
`In this review, we discuss various aspects of antibodies that are relevant to their use as as
`therapeutic agents.
`
`IMMUNOGLOBULIN DOMAIN
`Compactly folded globular units
`of approximately 110 amino
`acids that comprise immuno-
`globulin heavy and light chains.
`
`CYTOKINES
`A class of small proteins
`released by one cell that affects
`the physiology of other cells
`locally and systemically in a
`particular fashion through
`binding to a specific receptor.
`
`Antibody structure
`The typical antibody — or immunoglobulin (Ig) —
`consists of two antigen-binding fragments (Fabs),
`which are linked via a flexible region (the hinge) to a
`constant (Fc) region (FIG. 1). This structure comprises
`two pairs of polypeptide chains, each pair containing a
`heavy and a light chain of different sizes. Both heavy
`and light chains are folded into IMMUNOGLOBULIN DOMAINS.
`The ‘variable domains’ in the amino-terminal part of the
`molecule are the domains that recognize and bind anti-
`gens; the rest of the molecule is composed of ‘constant
`domains’ that only vary between Ig classes (BOX 1). The
`Fc portion of the Ig serves to bind various effector mole-
`cules of the immune system, as well as molecules that
`determine the biodistribution of the antibody.
`
`*Affitech AS, Gaustadalleen
`21, N-0349 Oslo, Norway.
`‡Department of Biology,
`University of Oslo, N-0349,
`Norway. Correspondence
`to O.H.B. e-mail:
`o.h.brekke@affitech.com
`doi:10.1038/nrd984
`
`Mechanisms of in vivo action
`In antibody-based therapies, the goal is to eliminate or
`neutralize the pathogenic infection or the disease target,
`for example, bacterial, viral or tumour targets.
`Therapeutic antibodies can function by three principal
`modes of action: by blocking the action of specific mole-
`cules, by targeting specific cells or by functioning as
`
`signalling molecules. The blocking activity of therapeutic
`antibodies is achieved by preventing growth factors,
`CYTOKINES or other soluble mediators reaching their tar-
`get receptors, which can be accomplished either by the
`antibody binding to the factor itself or to its receptor.
`Targeting involves directing antibodies towards specific
`populations of cells and is a versatile approach; anti-
`bodies can be engineered to carry effector moieties,
`such as enzymes, toxins, radionuclides, cytokines or
`even DNA molecules, to the target cells, where the
`attached moiety can then exert its effect (for example,
`toxins or radionuclides can eliminate target cancer
`cells). The natural EFFECTOR FUNCTIONS of antibodies are
`associated with binding to Fc receptors or binding
`to complement proteins and inducing COMPLEMENT-
`DEPENDENT CYTOTOXICITY (CDC). Targeting antibodies can
`retain such effector functions intact or they can be
`abolished during the design of the antibody, depending
`on the therapeutic strategy. The signalling effect of anti-
`bodies is predicated on either inducing crosslinking of
`receptors that are, in turn, connected to mediators of
`cell division or PROGRAMMED CELL DEATH, or directing them
`towards specific receptors to act as agonists for the
`
`52 | JANUARY 2003 | VOLUME 2
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`
`© 2002 Nature Publishing Group
`
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`
`

`

`R E V I E W S
`
`that are responsible for antigen binding are inserted into
`the human variable-domain framework. The ability to
`manipulate antibodies into more human variants
`finally made antibodies useful for clinical use. With the
`isolation of genes encoding human variable regions,
`their successful expression in Escherichia coli 8,9 and the
`introduction of phage-display technology10, the task of
`selecting fully human variable domains has been
`greatly simplified.
`Selection from phage-display libraries of human
`antibody fragments is today the most used and well-
`established technology for the development of new
`human antibodies (FIG. 3a). Another approach is to use
`mice that are transgenic for the human Ig locus11.
`Immunization of such a transgenic mouse results in a
`human antibody response, from which hybridomas that
`produce human antibodies can be generated (FIG. 3b).
`Today there are approximately 200 antibodies in
`clinical trials and the US Food and Drug Admin-
`istration has approved several antibodies against
`cancer12,13, transplant rejection14, rheumatoid arthritis
`and Crohn’s disease15,16, and antiviral prophylaxis17
`(TABLE 1). So far, 20% of all biopharmaceuticals in clini-
`cal trials are monoclonal antibodies (examples are
`shown in TABLE 2,and see clinical trials web site in online
`links box), making this the second largest biopharmaceu-
`tical product category after vaccines. As the development
`of potential new therapeutic agents into commercial
`products takes about 10 years, the FDA-approved anti-
`bodies, and some of those in the end-stages of develop-
`ment pipelines, are chimeric or humanized antibodies
`that were developed with early antibody engineering
`technologies. The more recently developed reagents,
`on the other hand, are completely human antibodies
`that are derived from phage antibody libraries and
`transgenic mice17–20.
`
`Antibodies against infectious agents
`In the pre-antibiotic era, SERUM THERAPY was widely used
`as treatment for infectious diseases such as anthrax,
`smallpox, meningitis and the plague. With the introduc-
`tion of vaccines and antibiotics, the use of serum therapy
`declined. However, with the reduction and eradication
`of certain diseases (for example, smallpox), which has
`had the result that populations are no longer routinely
`vaccinated against these infectious agents, and the
`increasing emergence of antibiotic-resistant bacteria,
`humans are susceptible to acute outbreaks of disease or
`to biological terrorism. Antibiotics are obviously ineffec-
`tive at eliminating viral infections, and the antiviral
`drugs in use today are often associated with a short
`serum half-life and resistance often emerges after
`repeated use21. Passive antibody serum therapy is today
`used merely in replacement therapy for patients with
`immune disorders, for POST-EXPOSURE PROPHYLAXIS against
`several viruses (for example, rabies, measles, hepatitis A
`and B, varicella and respiratory syncitial virus (RSV))
`and for toxin neutralization (for example, diphtheria,
`botulism and tetanus). Passive immunization has sub-
`stantial advantages over the administration of anti-
`microbial agents, including low toxicity and high specific
`
`Antigen binding
`
`VH
`
`VL
`
`CH
`1
`
`CL
`
`Fab
`
`CDR loops
`
`1
`
`3
`
`2
`
`N
`
`s
`
`s
`
`Complement and
`Fc receptor binding
`
`C
`
`CH
`
`2 C
`
`H
`3
`
`Fv
`
`Fc
`
`Figure 1 | The modular structure of immunoglobulins. This figure shows a single
`immunglobulin (Ig) molecule. All immunoglobulin monomers are composed of two identical light
`(L) chains and two identical heavy (H) chains. Light chains are composed of one constant domain
`(CL) and one variable domain (VL), whereas heavy chains are composed of three constant
`domains (CH1, CH2 and CH3) and one variable domain (VH). The heavy chains are covalently linked
`in the hinge region and the light chains are covalently linked to the heavy chain. The variable
`domains of both the heavy and light chains compose the antigen-binding part of the molecule,
`termed Fv. Within the variable domains there are three loops designated complementarity-
`determining regions (CDRs) 1, 2 and 3, which confer the highest diversity and define the
`specificity of antibody binding. The Fc portion is glycosylated and contains the sites for interaction
`with effector molecules, such as the C1 complex of the complement system and a variety of Fc
`receptors including the neonatal Fc receptor (FcRn).
`
`activation of specific cell populations. Another
`approach is to use antibodies as delivery vehicles for
`DNA and, more recently, to deliver antigens to certain
`immune cells that present processed antigenic pep-
`tides, or epitopes, to T cells, to activate a specific
`immune response against that antigen.
`
`The development of therapeutic antibodies
`Target specificity in the treatment and prophylaxis of
`diseases such as infection, cancer and autoimmune dis-
`orders has become more viable through the develop-
`ment of monoclonal antibodies. The mouse HYBRIDOMA
`technology described by Köhler and Milstein was an
`important step in the development of antibody technol-
`ogy and paved the way for the emergence of therapeutic
`monoclonal antibodies1. During the 1980s, resources
`were directed towards the evaluation of the in vivo use
`of mouse monoclonal antibodies in humans, aimed at
`both imaging and therapy2. Mouse monoclonal anti-
`bodies were shown to have limited use as therapeutic
`agents because of a short serum half-life, an inability to
`trigger human effector functions and the production of
`human antimouse-antibodies3 (the HAMA response).
`In an attempt to reduce the immunogenicity of mouse
`antibodies, genetic engineering was used to generate
`chimeric antibodies, that is, antibodies with human
`constant regions and mouse variable regions4,5.
`However, although chimeric antibodies were perceived
`as less foreign, and therefore less immunogenic, than
`mouse monoclonal antibodies, human anti-chimeric
`antibody responses (HACAs) have nonetheless been
`observed6. Further minimization of the mouse com-
`ponent of antibodies was achieved through CDR
`(complementarity-determining region) grafting7 (FIG. 2).
`In such ‘humanized’ antibodies, only the CDR loops
`
`EFFECTOR FUNCTIONS
`The antigen-elimination
`processes mediated by
`immunoglobulins and initiated
`by the binding of effector
`molecules to the Fc part of the
`immunoglobulin. The common
`effector functions are
`complement-dependent
`cytotoxicity (CDC), phagocytosis
`and antibody-dependent cellular
`cytotoxicity (ADCC).
`
`COMPLEMENT-DEPENDENT
`CYTOTOXICITY
`Once bound to antigen, both IgM
`and IgG can trigger a sequence of
`reactions by which serum
`proteins called complement
`factors are cleaved. One of the
`results is destruction of the target
`cell through complement-
`dependent cytotoxicity.
`
`PROGRAMMED CELL DEATH
`Programmed cell death infers
`that cells are determined to die at
`a specific stage of development
`or having received a specific
`signal. The process is known as
`apoptosis. The cells shrivel and
`are engulfed by nearby
`phagocytic cells without eliciting
`any inflammatory response.
`
`NATURE REVIEWS | DRUG DISCOVERY
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`VOLUME 2 | JANUARY 2003 | 5 3
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`R E V I E W S
`
`Box 1 | Immunoglobulin classes
`
`Antibodies belong to either one of five immunoglobulin (Ig) classes: IgA, IgD, IgE, IgG
`or IgM. Each class has a distinct structure and biological activity. Some of the classes
`are further divided into subclasses — for example, there are four IgG subclasses and
`two IgA subclasses. IgM is the first antibody to be produced in an immune response
`and forms a pentameric complex comprised of Ig monomers. IgA is the main class of
`antibody in external secretions, where it is found as a dimer that protects the body’s
`mucosal surfaces from infection; it is also found as a monomer in serum. IgD is the
`main antibody on the surface of B cells. IgE is found bound to cells that secrete
`histamines after antigen binding. IgG is the main antibody in serum. The IgG class is
`the most stable and has a serum half-life of 20 days, whereas IgM and IgA persist for
`only 5–8 days. Both IgM and IgG can mediate complement fixation, whereas only IgG
`can promote antibody-dependent cellular cytotoxicity (ADCC). IgA, and to a certain
`degree IgM, can mediate trancytosis to mucosal surfaces, whereas only IgG can be
`transported across placenta for fetal protection.
`
`activity, as well as an immediate effect compared with
`vaccines and even antibiotics. Given these issues,
`biotechnology companies and institutions working
`within the field of infectious disease protection are
`certain to direct efforts towards developing highly
`effective and functional antibody candidates against
`specific disease targets.
`The isolation of protective toxin-neutralizing
`human monoclonal antibodies was described in 1993
`in a study in which several human monoclonal anti-
`bodies against tetanus toxin were isolated and a pro-
`tective effect against tetanus toxin was observed22.
`However, when combinations of these specific mono-
`clonal antibodies were administered, an extra potent
`(that is, synergistic) effect was observed. Such an effect
`has also recently been described in the neutralization
`
`Mouse hybridoma
`
`In vitro antibody libraries
`Transgenic mouse
`Human hybridomas
`
`Mouse
`
`Chimeric
`
`Humanized
`
`Human
`
`Genetic engineering
`V gene cloning
`CDR grafting
`Eukaryotic expression
`
`Figure 2 | Antibody engineering. Mouse hybridoma technology generates mouse monoclonal
`antibodies. Genetic engineering has fostered the generation of chimeric, humanized and human
`antibodies. Cloning of mouse variable genes into human constant-region genes generates
`chimeric antibodies. Humanized antibodies are generated by the insertion of mouse
`complementarity-determining regions (CDRs) onto human constant and variable domain
`frameworks; however, additional changes in the framework regions have, in several cases, been
`shown to be crucial in maintaining identical antigen specificity75,76. Fully human antibodies can be
`generated by the selection of human antibody fragments from in vitro libraries (see BOX 2 and
`FIG. 3a), by transgenic mice (FIG. 3b) and through selection from human hybridomas.
`
`of botulinum neurotoxin. The anti-botulinum toxin
`antibodies were derived from different phage-display
`libraries obtained from humans or immunized mice
`(J. Marks, presented at Cambridge Healthtech
`Institute conference on Recombinant Antibodies,
`Cambridge, Massachusetts, USA, 24–25 April 2002).
`Recently, Maynard and colleagues described high-
`affinity antibodies against Bacillus anthracis23. These
`antibodies were fragments derived from variable-
`chain genes of a mouse monoclonal antibody and
`expressed in E. coli as single-chain Fv fragments
`(scFvs). By administering the antibody fragments to
`mice before injection of anthrax toxin, protection
`against the toxin was observed. Protective human
`antibodies against Shiga-toxin1 were recently isolated
`by the immunization of transgenic mice20. The panel
`of ten different antibodies of the IgM and IgG1 class
`that showed specificities to different subunits of the
`toxin effectively neutralized the toxin.
`Compared with toxin neutralization, the use of
`antibodies in the prevention of viral disease is a more
`complex prospect. Palivizumab (Synagis; MedImmune
`Inc) is a humanized IgG1 monoclonal antibody
`approved for the prevention of RSV infections in high-
`risk infants17,24. Palivizumab was the first monoclonal
`antibody approved for an infectious pathogen and is,
`so far, the only antiviral monoclonal antibody in clini-
`cal use. The development of sevirumab (Protovir;
`Protein Design Labs) — a human anti-cytomegalo-
`virus (CMV) antibody — was, by contrast, halted in
`Phase III clinical trials as a supplemental treatment for
`CMV-induced retinitis because of a lack of evidence of
`efficacy. The elimination of a viral infection requires
`that a number of events occur, including inhibition of
`cell infection, mediation of cell killing of infected cells,
`inhibition of viral replication, inhibition of viral
`release and inhibition of cell–cell transmission25,26.
`Within the multitude of antibody specificities gener-
`ated in the typical human polyclonal response, it is
`likely that one or several effective antibodies against
`one or more of the particular processes listed will be
`found. Some of these antibodies bind neutralizing epi-
`topes, whereas others bind non-neutralizing epitopes.
`The overall outcome might indeed be effective protec-
`tion due to the combination of the blocking, neutral-
`izing and eliminating effect of human antibodies.
`Recently, XTL Pharmaceuticals reported on Phase I/II
`clinical trials with two human monoclonal antibodies
`against hepatitis B virus28. The two monoclonal anti-
`bodies were combined with lamivdudine (Epivir-
`HBV; GlaxoWellcome) — an antiviral drug that
`inhibits DNA replication — and showed significant
`reduction in serum viral titre. If monoclonal antibod-
`ies are to be used as prophylactics or therapeutics
`against infectious diseases, it is likely that their efficacy
`will be increased when a polyclonal passive human
`serum therapy is mimicked — that is, when a pool of
`highly specific and high-affinity monoclonal antibod-
`ies are administered. There are, however, limitations
`to this approach, including the production costs asso-
`ciated with manufacturing intact human antibodies.
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`54 | JANUARY 2003 | VOLUME 2
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`R E V I E W S
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`Figure 3 | In vitro and in vivo human antibody techniques exemplified by phage display and transgenic mouse
`technologies. a | The in vitro process is based on panning the library of antibodies against an immobilized target. The non-
`binding phage antibodies are washed away and the recovered antibodies are amplified by infection in Escherichia coli. The
`selection rounds are subsequently repeated until the desired specificity is obtained. The antibody format for screening is either
`Fab or single-chain Fv. The expression of antibodies in E. coli and recent developments in screening technologies77 have made
`it possible to screen tens of thousands of clones for specificity. The antibody fragments themselves can be used as therapeutic
`agents as discussed in this review, but they can also be converted into intact immunoglobulins by the cloning of the variable
`genes into plasmids incorporating the constant-region genes of immunoglobulins. The genes are transfected into cell lines and
`therefore produce fully human immunoglobulins. b | The in vivo process is based on the immunization of a transgenic mouse.
`The mouse has been genetically engineered and bred for the expression of human immunoglobulins. The B cells harvested after
`immunization can be immortalized by fusion with a myeloma cell line, as in traditional hybridoma technology. The hybridomas
`can then be screened for specific antibodies.
`
`A possible solution to this problem is to instead use
`blocking antibody fragments synthesized in E. coli,
`which could be used to combat, for example, viral
`infections. A further difficulty in developing such
`multi-antibody therapeutics might arise from regula-
`tory concerns regarding the administration of multiple
`monoclonal antibodies. The experience obtained
`from XTL Pharmaceuticals’ use of combined antibod-
`ies might be an important step in resolving — or
`exacerbating — these concerns.
`
`Anti-inflammatory antibodies
`Antibodies with high specificity and affinity can be
`developed to bind specific cytokines or their recep-
`tors. In both cases, the purpose is to inhibit the detri-
`mental effect of the cytokine. Cytokines associated
`
`with inflammation and autoimmunity include
`tumour-necrosis factor-α (TNF-α), interleukins and
`complement proteins. Modulation of
`immune-
`responses, such as immune-cell depletion by the target-
`ing of antibodies to cell-surface receptors, for example,
`CD20 and CD4 on B or T cells, has also been shown as
`a viable therapeutic strategy in autoimmune diseases.
`Together with cancer, inflammatory and autoimmune
`diseases are an important focus for companies devel-
`oping antibody therapies. In patients with rheumatoid
`arthritis (RA), TNF-α accumulates in the joints and
`contributes to the inflammation and joint destruction
`that is associated with the disease. Marketed products
`directed towards the regulation of TNF-α include
`the soluble TNF-α receptor eternacept (Enbrel;
`Amgen Inc/Wyeth) and the antibody infliximab
`
`HYBRIDOMA
`An antibody-secreting B-cell line
`that is generated by fusing
`splenic-derived B cells with a
`plasmacytoma. A hybridoma
`produces the same antibody as
`the parent B cell and divides and
`grows in culture like the parent
`cancer cell. The antibody
`produced is monoclonal.
`
`SERUM THERAPY
`The treatment of an infectious
`disease with the serum from an
`immunized animal or individual,
`and which contains antibody.
`
`POST-EXPOSURE PROPHYLAXIS
`A treatment that is designed
`to protect an individual against
`a disease agent to which the
`individual has been recently
`exposed.
`
`NATURE REVIEWS | DRUG DISCOVERY
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`VOLUME 2 | JANUARY 2003 | 5 5
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`R E V I E W S
`
`Table 1 | Approved monoclonal antibodies
`Product
`Year
`Type of molecule
`approved
`1986
`1994
`
`OKT-3
`ReoPro
`
`Panorex
`(Germany only)
`Rituxan
`Zenapax
`
`Herceptin
`
`Remicade
`Simulect
`
`Synagis
`
`Mylotarg
`
`Campath
`
`Zevalin
`
`1995
`
`1997
`1997
`
`1998
`
`1998
`1998
`
`1998
`
`2000
`
`2001
`
`2002
`
`Murine (anti-CD3)
`Chimeric
`(anti-platelet gpIIb/IIIa)
`Murine (anti-EpCAM)
`
`Chimeric (anti-CD20)
`Humanized
`(anti-IL-2 receptor)
`Humanized
`(anti-ERBB2)
`Chimeric (anti-TNF-α)
`Chimeric
`(anti-IL-2 receptor)
`Humanized
`(anti-F-protein)
`Humanized
`(anti-CD33)
`Humanized
`(anti-CD52)
`Mouse (anti-CD20)
`
`2002
`
`Xolair
`(Australia only)
`Adapted and updated from REF. 81.
`
`Humanized
`(anti-IgE Fc)
`
`(Remicade; Centocor Inc). Infliximab is a chimeric
`antibody with mouse variable domains and human
`constant domains of the IgG1 subclass. There are a
`number of anti-TNF-α antibodies in clinical trials,
`including CDP571 and CDP870 (Celltech Plc) and
`adalimumab (D2E7; Abbot Laboratories/Cambridge
`Antibody Technology). Adalimumab is the first
`phage-display-derived human antibody brought into
`the clinic, and was generated by ‘guided selection’
`using a mouse monoclonal antibody28. The method is
`based on the selection of a human variable-domain
`repertoire coupled to one of the original mouse vari-
`able domains, so as to ‘guide’ the human variable-
`domain repertoire towards the same specificity as the
`original mouse variable domains. The antibody is
`affinity optimized by iterative rounds of selection and
`mutagenesis (FIG. 3; BOX 2, 3). Adalimumab has com-
`pleted Phase III clinical trials and is currently in regis-
`tration for FDA approval. Recently, the anti-CD20
`antibody rituximab (Rituxan; IDEC Pharmaceuticals;
`see below) completed Phase II clinical trials in RA and
`showed promising results compared with the anti-
`TNF-α antibodies adalimumab and infliximab29, indi-
`cating that depletion of B cells might be an effective
`treatment in RA and other autoimmune diseases30.
`Eculizumab (5G1.1; Alexion Pharmaceuticals) is a
`humanized monoclonal antibody that prevents the
`cleavage of human complement component C5 into
`its pro-inflammatory components31, whereas J695
`(Cambridge Antibody Technology) is a human anti-
`body derived from a phage-display library against
`interleukin-2 (see online links box). Both antibodies
`
`Disease indication
`
`Company
`
`Organ transplant rejection
`Coronary intervention
`and angioplasty
`Colorectal cancer
`
`Ortho Biotech Products LP
`Centocor Inc
`
`Centocor Inc
`
`Non-Hodgkin’s lymphoma
`Refractory unstable angina
`
`IDEC Pharmaceuticals Corp
`Centocor Inc
`
`Metastatic breast cancer
`
`Genentech Inc
`
`Crohn’s disease
`Kidney transplant rejection
`
`Centocor Inc
`Novartis AG
`
`Respiratory syncitial viral
`disease
`Chemotherapy for acute
`myeloid leukemia
`B-cell chronic lymphocytic
`leukemia
`B-cell non-Hodgkin’s
`lymphoma
`Allergy
`
`MedImmune Inc
`
`Celltech Group plc/Wyeth
`
`Millennium Pharmaceuticals/
`Ilex Oncology Inc
`IDEC Pharmaceuticals Corp
`
`Tanox Inc/Genentech Inc/
`Novartis AG
`
`are in clinical trials at present and have shown poten-
`tial in the treatment of inflammatory diseases such as
`RA and nephritis.
`
`Antibodies targeting cancers
`In cancer therapy, the purpose of antibody adminis-
`tration is to induce the direct or indirect destruction of
`cancer cells, either by specifically targeting the tumour
`or the vasculature that nourishes the tumour. Indeed,
`new technologies for the panning of antibody libraries
`on intact cells have made it possible to isolate antibod-
`ies against novel and promising cancer-associated
`antigens32. However, the most common cancer targets
`are the carcinoembryonic antigen (CEA), which is
`associated with colorectal cancers, MUC1, epidermal
`growth factor receptor (EGFR) and ERBB2 (also
`known as HER2/neu, associated with lung and breast
`cancer) and CD20 on B cells, which is a marker for
`non-Hodgkin’s lymphoma (NHL). Examples of
`approved therapeutic antibodies directed towards
`ERBB2 and CD20 include the humanized IgG1 anti-
`body trastuzumab (Herceptin; Genentech) for breast
`cancer and the chimeric IgG1 antibody rituximab
`(Rituxan; IDEC Pharmaceuticals) for NHL.
`Today, specific antibody therapy is used in combi-
`nation with classical chemotherapy, but the remaining
`challenge is to develop specific and highly cytotoxic
`drugs against cancer cells. Recently, the FDA approved
`two new antibody chemotherapies: ibritumomab
`tiuxetan (Zevalin; IDEC Pharmaceuticals) and gem-
`tuzumab ozogamicin (Mylotarg; Wyeth). Ibritu-
`momab tiuxetan is a mouse anti-CD20 antibody
`
`56 | JANUARY 2003 | VOLUME 2
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`R E V I E W S
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`attached to the radioisotope 90yttrium that targets the
`surface of the mature B cells and B-cell tumours,
`inducing cellular damage in the target and neighbour-
`ing cells. Ibritumomab tiuxetan is the first radio-
`immunotherapeutic antibody approved by the FDA
`and is approved for use in the treatment of NHL. The
`antibody reacts with the same antigen as rituximab,
`but is a mouse antibody, which results in beneficial
`rapid clearance to decrease the undesired effect of total
`body irradiation. Gemtuzumab ozogamicin is a
`humanized monoclonal antibody that is linked to the
`antitumour agent calicheamicin, a bacterial toxin33.
`The antibody is targeted to CD33, which is expressed
`in about 90% of all acute myelogenic leukaemia
`(AML) cases, and has been approved for administra-
`tion to patients who have relapsed AML. Both ibritu-
`momab tiuxetan and gemtuzumab ozogamicin are
`examples of antibodies designed to specifically deliver
`their toxic load directly to cancer cells. Antibodies
`directed against major histocompatibility complex
`(MHC) class II proteins specifically target and elimi-
`nate cancer cells. The mouse monoclonal antibody
`Oncolym (Lym-1; Peregrine Pharmaceuticals)34 and
`the antibody Hu1D10 (Remitogen; Protein Design
`Labs)35 are two examples of such antibodies. Whereas
`
`one form of Oncolym is radiolabelled with 131iodine
`(REF. 36), the humanized IgG1 Hu1D10 eliminates cancer
`cells by virtue of its natural effector functions (FIG. 4 and
`see below), and also induces apoptosis. Recently, an anti-
`MHC class II human antibody derived from an antibody
`phage-display library was shown to induce apoptosis of
`activated lymphoid cells19. Vascular endothelial growth
`factor (VEGF) is a potent cytokine for angiogenesis (the
`formation of blood vessels). Inhibitory agents, including
`specific antibodies, have been developed to block VEGF-
`stimulated angiogenesis as a strategy to inhibit tumour
`growth. One example of such an antibody is the human-
`ized anti-VEGF antibody bevacizumab (Avastin; Genen-
`tech), which is in Phase III clinical trials for the treatment
`of breast cancer and colorectal cancer. Monoclonal anti-
`bodies are being continuously developed for cancer ther-
`apy, and most new antibody formats have demonstrated
`utility within this field.
`
`Naked antibodies
`Both trastuzumab and rituximab are ‘naked antibodies’,
`meaning that they do not have a radioisotope or toxin
`attached to them. The elimination of the target of these
`antibodies depends entirely on the recruitment of the
`body’s own effector mechanisms, namely complement
`
`Table 2 | A selection of antibodies in clinical development
`Product
`Stage
`Type of molecule
`Disease indication
`(2002)
`Phase II
`Phase II
`
`ABX-EGF
`ABX-IL8
`
`Human (anti-EGF-R)
`Human (anti-IL-8)
`
`Eculizumab
`(5G1.1)
`D2E7
`
`Phase IIb
`
`Humanized (anti-C5)
`
`Phase III
`
`Human (anti-TNF-α)
`
`CAT-152
`
`Phase II/III
`
`Human (anti-TGF-β2)
`
`J695
`
`Phase II
`
`Human (anti-IL-2)
`
`Antegren
`(natalizumab)
`
`Phase III
`
`Humanized
`(anti-α-4 integrin)
`
`Phase III
`(anti-VEGF)
`
`Humanized
`
`Non-small-cell lung cancer
`Pulmonary disease, chronic
`obstructive bronchitis
`Rheumatoid arthritis,
`lupus nephritis
`Rheumatoid arthritis
`
`Scarring following glaucoma
`surgery
`Autoimmune diseases
`including rheumatoid arthiritis
`
`Crohn’s disease,
`multiple sclerosis,
`inflammatory bowel disease
`Metastatic breast cancer,
`non-small-cell lung cancer
`
`Company
`
`Abgenix Inc/Immunex Corp
`Abgenix Inc/Immunex Corp
`
`Alexion Pharmaceutical Inc
`
`Cambridge Antibody
`Technology/
`Abbot Laboratories
`Cambridge Antibody
`Technology
`Cambridge Antibody
`Technology/Abbot/
`Wyeth Genetics Institute
`Elan Pharmaceuticals
`Corp/Biogen Inc
`
`Genentech Inc
`
`Avastin
`(rhuMAb-
`VEGF)
`IDEC-151
`(Clenoliximab)
`MEDI-507
`(Siplizumab)
`XTL 001
`(pairs of
`monoclonals)
`CDP870
`
`Phase II
`
`Primatized (anti-CD4)
`
`Rheumatoid arthritis
`
`IDEC Pharmaceuticals Corp
`
`Phase II
`
`Phase I/II
`
`Humanized
`(anti-CD2 Receptor)
`Human (anti-HBV)
`
`Suppresses NK and T-cell
`function
`Hepatitis B virus neutralization
`
`MedImmune Inc
`
`XTL Bioharmaceuticals Ltd
`
`Phase II
`
`Fab fragment
`(anti-TNF-α)
`Humanized
`(anti-TNF-α)
`Humanized
`Hu1D10
`(anti-MHC class II)
`(Remitogen)
`Adapted from REFS 34, 81 and selected company web sites.
`
`CDP571
`
`Phase III
`
`Phase II
`
`Rheumatoid arthritis
`
`Celltech Group plc
`
`Crohn’s disease
`
`Celltech Group plc
`
`Non-Hodgkin’s Lymphoma
`
`Protein Design Labs Inc
`
`NATURE REVIEWS | DRUG DISCOVERY
`
`VOLUME 2 | JANUARY 2003 | 5 7
`
`© 2002 Nature Publishing Group
`
`Lassen - Exhibit 1057, p. 6
`
`

`

`R E V I E W S
`
`Box 2 | In vitro human antibody libraries
`
`The strategy of selecting antibodies from large repertoires is dependent on the coupling between genotype and
`phenotype — that is, the displayed protein (the antibody) carries its own encoding gene. As a result, the process
`enables the easy recovery of the DNA encoding the selected protein. The power of such antibody libraries lies in the
`fact that selection takes place in vitro, thereby making them excellent systems for isolating antibodies to certain drugs,
`potent toxins and haptens that would normally be impossible to raise within in vivo systems such as the mouse
`hybridoma technology.
`In phage display, the variable genes encoding the antibody variable domains are fused to genes encoding
`bacteriophage coat proteins. The most commonly used phage protein is the pIII protein located at the tip of the long,
`thin filamentous phage M13. The system is highly effective and is used to isolate single-chain Fv or Fab fragments with
`specificity to almost any kind of antigen. The plasmid encoding the variable genes is packaged within the viral capsid
`and the expressed antibody is presented on the bacteriophage surface. Phage libraries are made from the human naive
`repertoire (that is, the IgM and IgD class) from pre-immunized donors or totally synthetic variable genes. As the
`repertoire is created in vitro, the technology also generates high-affinity human antibodies against human proteins. The
`process is based on panning the library against an immobilized target in a test tube (FIG. 3a). The non-binding phage
`antibodies are washed away and the recovered antibodies are amplified by infection in E. coli. The selection rounds are
`subsequently repeated until the desired specificity is obtained.
`In messenger RNA (mRNA)-display technologies, such as the ribosomal display technology, antibody domains are
`linked to ribosomes attached to the mRNA that they are translating. This process takes place entirely in vitro by
`transcripton of a DNA library followed by in vitro translation of the mRNA library. The subsequently recovered mRNA
`transcripts are reverse transcribed and amplified by polymerase chain reaction (PCR) for the subsequent round of
`selection. The method has been successfully used for the affinity maturation and stability engineering of antibody
`fragments58,70. A similar technology called ProFusion is based on the covalent linkage of