Protein Cell 2010, 1(4): 319–330
`DOI 10.1007/s13238-010-0052-8
`
`Protein & Cell
`
`REVIEW
`Monoclonal antibodies – a proven and rapidly
`expanding therapeutic modality for human
`diseases
`
`Zhiqiang An ✉
`
`Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030,
`USA
`✉ Correspondence: Zhiqiang.an@uth.tmc.edu
`Received March 10, 2010 Accepted March 31, 2010
`
`ABSTRACT
`
`The study of antibodies has been a focal point in modern
`biology and medicine since the early 1900s. However,
`progress in therapeutic antibody development was slow
`and intermittent until recently. The first antibody therapy,
`murine-derived murononab OKT3 for acute organ rejec-
`tion, was approved by the US Food and Drug Adminis-
`tration (FDA) in 1986, more than a decade after César
`Milstein and Georges Köhler developed methods for the
`isolation of mouse monoclonal antibodies from hybri-
`the scientific,
`doma cells in 1975. As a result of
`technological, and clinical breakthroughs in the 1980s
`and 1990s, the pace of therapeutic antibody discovery
`and development accelerated. Antibodies are becoming
`a major drug modality with more than two dozen
`therapeutic antibodies in the clinic and hundreds more
`in development. Despite the progress, need for improve-
`ment exists at every level. Antibody therapeutics pro-
`vides fertile ground for protein scientists to fulfill the
`dream of personalized medicine through basic scientific
`discovery and technological innovation.
`
`KEYWORDS
`monoclonal antibodies, personalized
`medicine, therapeutic antibodies
`
`INTRODUCTION
`
`The pioneering research by Robert Koch, Kitasato Shibasa-
`buro, Emil von Behring, and Paul Ehrlich in late 19th and the
`early 20th centuries on the treatment of infectious diseases
`with serum from patients who had recovered from the same
`disease was the first use of antibodies as therapeutics. The
`
`active components in the serum were described as “anti-
`bodies” “antitoxins” and “magic bullets” (Ehrlich, 1908; Winau
`et al., 2004). This crude “serum therapy” was later modified by
`isolating antibodies from the serum for the treatment of
`infectious and immune diseases, known as intravenous
`immune globulin (IVIG) (Stangel and Pul, 2006). Despite the
`early success of serum therapy and IVIG treatment, no
`significant progress was made in therapeutic antibody
`discovery and development until César Milstein and Georges
`Köhler developed methods for isolating mouse monoclonal
`antibodies (mAbs) from hybridoma cells in 1975 (Köhler and
`Milstein, 1975). Since then, mAbs have not only fueled
`breakthrough discoveries in basic research, but have also
`been developed as clinical diagnostics, reagents for high
`throughput drug screening, and more importantly, life-saving
`medicines. The first therapeutic mAb murononab, a murine-
`derived antibody for acute organ rejection, was approved by
`the US Food and Drug Administration (FDA) in 1986, a
`decade after the discovery of the mouse hybridoma technol-
`ogy (Thistlethwaite et al., 1987). As a result of technological
`breakthroughs in the 1980s and 1990s, progress in ther-
`apeutic mAbs field has been accelerated. Therapeutic
`antibodies have shown desirable safety profiles, high target
`specificity and affinity, and efficiency in disrupting protein/
`protein interactions. They are becoming a major drug
`modality with more than 25 therapeutic antibodies in clinical
`use and hundreds more in development (Reichert and Valge-
`Archer, 2007; An, 2009).
`
`ANTIBODY STRUCTURE
`
`An antibody of the IgG isotype is a homodimer composed of
`two heterodimers of one light chain and one heavy chain.
`
`© Higher Education Press and Springer-Verlag Berlin Heidelberg 2010
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`Protein & Cell
`
`Zhiqiang An
`
`Both the heterodimers and homodimers are linked by inter
`chain disulphide bonds (Stanfield and Wilson, 2009) (Fig. 1A).
`The light and heavy chains each contain variable and
`constant
`regions. The antigen binding complementarity
`determining regions (CDRs) are short hypervariable amino
`acid sequences found in the variable domains of both light
`(variable light or VL) and heavy (variable heavy or VH) chains.
`Each VH and VL contains three pairs of non-identical CDRs
`(CDR1, CDR2 and CDR3). CDRs are termed hypervariable
`domains because the majority of the sequence variations
`associated with antibodies is found in the CDRs. Among the
`six CDRs in an IgG molecule, CDR3s have the greatest
`variability. The Fc-region (fragment crystalizable region) of a
`mAb, residing in the constant regions of the heavy chains, can
`recruit effector cells such as natural killer cells, macrophages
`or neutrophils to activate the complement system to destroy
`the target-associated cells. These functions are referred to as
`antibody-dependent cellular cytotoxicity (ADCC) and comple-
`ment-dependent cytotoxicity (CDC). Four additional antibody
`isotypes are found in humans, IgA, IgD, IgE, and IgM. All five
`isotypes share a common theme of a core heterodimer
`building unit of a heavy and light chain. In IgG, IgA and IgD
`antibody isotypes, the Fc region is composed of two identical
`protein fragments, derived from the second (CH2) and third
`(CH3) constant domains of the antibody's two heavy chains.
`The Fc regions in IgM and IgE contain three heavy chain
`
`constant domains in each polypeptide chain. The IgG isotype
`is most commonly used in therapeutic applications.
`
`ANTIBODY THERAPEUTIC HISTORY
`
`The progress of antibody therapeutics is driven by both
`scientific and technological breakthroughs (Fig. 2). Thera-
`peutic antibody development also parallels the desire of the
`industry to reduce immunogenicity.
`Immunogenicity can
`reduce the efficacy of therapeutic mAbs. In severe cases,
`immunogenicity can cause anaphylaxis and hypersensitivity
`reactions. Soon after the approval of
`the murine-derived
`monoclonal antibody murononab for acute organ rejection in
`1986 (Thistlethwaite et al., 1987), it was realized that murine-
`derived monoclonal antibodies are less than ideal therapeu-
`tics due to their high immunogenicity in humans. Several
`strategies to make antibodies more human, such as chimeric
`mAb (Morrison et al., 1984) and CDR grafting (Kettleborough
`et al., 1991), were devised to reduce the human anti-mouse
`antibody (HAMA) responses. It took a decade for the first
`chimeric mAb, abciximab for hemostasis, to be approved by
`FDA in 1994 (Faulds and Sorkin, 1994). The first humanized
`mAb, Zenapax for kidney transplant rejection, was approved
`for clinical use by FDA in 1997 (Vincenti et al., 1998).
`Humanization alleviated the HAMA response to various
`degrees, but many other drawbacks became evident. For
`
`Figure 1. Diagrams of various antibody structures. (A) A generic IgG molecule. (B) A scFv fragment. (C) A Fab fragment. (D) A
`F(ab’)2 fragment. (E) A mouse IgG molecule. (F) A murine:human chimeric IgG molecule. (G) A humanized IgG molecule. (H) A
`human IgG molecule.
`
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`
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`Therapeutic monoclonal antibodies
`
`Protein & Cell
`
`example, the humanization process is technically demanding
`and the process may result in reduced antigen binding affinity
`and decreased efficacy. To avoid the human immune
`response to murine-derived mAbs and to overcome the
`technical challenges associated with humanizing murine
`mAbs, two major approaches were developed for generating
`fully human mAbs. The first approach was to express human
`antibody fragments on bacteriophage surfaces. The resulting
`libraries contain billions of unique human antibody fragments
`which can be screened for leads (Vaughan et al., 1996).
`Humira, the first fully human mAb derived from a bacterioph-
`age displayed antibody library, was approved by the FDA in
`2003 for the treatment of rheumatoid arthritis (Weinblatt et al.,
`2003). The second approach was to use transgenic mice to
`produce fully human antibodies (Russell et al., 2000; Lonberg,
`2005). This is achieved by replacing the mouse native antibody
`genes with their human counterparts. Vectibix, an anti-EGFR
`antibody approved for colorectal cancer therapy in 2006, was
`the first
`fully human antibody therapeutic derived from a
`transgenic mice system (Chua and Cunningham, 2006). The
`industry trend is to develop more human like antibodies for
`clinical use. However, immunogenicity is a complex biological
`
`process and it cannot be predicted solely on human content of
`an antibody. For example, Humira, a fully human antibody, has
`a relatively high incidence of immunogenicity (Bender et al.,
`2007). Surprisingly, there is little difference in immunogenicity
`(anti-antibody response) between humanized and chimeric
`mAbs in clinical use today (Table 1). Clearly more basic and
`clinical research is needed to develop reliable parameters to
`predict immunogenicity of therapeutic antibodies prior to their
`reaching the clinic.
`
`SOURCES OF THERAPEUTICS ANTIBODIES
`
`Accessing diversified antibody sources are paramount to the
`success in the discovery and development of antibody
`therapies. Most therapeutic antibodies in the clinic today are
`of murine origin largely due to the early availability of the
`mouse hybridoma technology; however, entirely mouse
`antibodies have poor pharmacokinetics in humans due to
`human anti-mouse antibody immune responses (Fig. 1E). To
`reduce immunogenicity, murine antibodies are commonly
`modified to murine/human chimeric antibodies or humanized
`antibodies for therapeutic applications (Carter, 2006; Reichert
`
`Figure 2. History of antibody therapeutics. Green boxes represent scientific and technology milestones. Blue boxes are
`antibody therapeutics developed as a result of the scientific and technology breakthroughs.
`
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`Protein & Cell
`
`Zhiqiang An
`
`Ferrajolietal.,2001
`
`Sorokin,2000
`
`1999
`
`AlbanellandBaselga,
`
`Storch,1998
`
`OnrustandLamb,1998
`
`Nashanetal.,1997
`
`Vincentietal.,1998
`
`Maloneyetal.,1997
`
`1994
`
`FauldsandSorkin,
`
`Cohenetal.,1989
`
`IV
`
`IV
`
`IV
`
`IM
`
`IV
`
`IV
`
`IV
`
`IV
`
`IV
`
`IV
`
`humanizedIgG1
`
`CD52
`
`leukemia
`
`chroniclymphocytic
`
`oncology
`
`2001
`
`gatedwithozogamicin
`humanizedIgG4conju-
`
`CD33
`
`leukemia
`
`acutemyelogenous
`
`oncology
`
`2000
`
`humanizedIgG1
`
`Her2
`
`breastcancer
`
`oncology
`
`chimericIgG1
`
`RSVF-protein
`
`virus
`
`respiratorysyncytial
`
`ID
`
`chimericIgG1
`
`TNFalpha
`
`rheumatoidarthritis
`
`AIID
`
`chimericIgG1
`
`CD25
`
`transplantrejection
`
`AIID
`
`humanizedIgG1
`
`CD25
`
`transplantrejection
`
`AIID
`
`chimericIgG1
`
`CD20
`
`phoma
`
`Non-Hodgkin’slym-
`
`oncology
`
`chimericFab
`
`CD41
`
`cardiovasculardisease
`
`CV
`
`murineIgG2a
`
`CD3
`
`transplantrejection
`
`AIID
`
`1998
`
`1998
`
`1998
`
`1998
`
`1997
`
`1997
`
`1995
`
`1986
`
`Bayer-Schering
`Campath
`Alemtuzumab
`
`Wyeth
`Mylotarg
`cin
`Gemtuzumab/ozogami-
`
`Genentech
`Herceptin
`Trastuzumab
`
`MedImmune
`Synagis
`Palivizumab
`
`Centocor
`Remicade
`Infliximab
`Novartis
`Simulect
`Basiliximab
`
`Roche
`Zenapax
`Daclizumab
`
`Genentech/Roche
`Rituxan/MabThera
`Rituximab
`
`EliLilly
`ReoPro
`Abciximab
`
`Johnson&Johnson
`Orthoclone/OKT3
`Muromonab
`
`reference
`
`delivery
`
`proteinform/isotype
`
`target
`
`majorindication
`
`manufacturer
`tradename
`genericname
`Table1Monoclonalantibodytherapeuticsapprovedforclinicaluse
`
`therapyarea
`
`launchdate
`
`322
`
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`

`Therapeutic monoclonal antibodies
`
`Protein & Cell
`
`Paul-Pletzer,2006
`
`2004
`
`RudickandSandrock,
`
`Kerr,2004
`
`Chenetal.,2005
`
`KiesandHarari,2002
`
`Weinblattetal.,2003
`
`Davies,2004
`
`Gauvreauetal.,2003
`
`Davis,2004
`
`2001
`
`KrasnerandJoyce,
`
`reference
`(Continued)
`
`IV
`
`IV
`
`IV
`
`IV
`
`IV
`
`SC
`
`IV
`
`SC
`
`SC
`
`IV
`
`humanizedIgG1
`
`IL-6R
`
`Castleman'sdisease
`
`AIID
`
`humanizedIgG1
`
`VLA4
`
`multiplesclerosis
`
`CNS/AIID
`
`humanizedIgG1
`
`VEGF
`
`smallcelllungcancer
`colorectalandnon-
`
`oncology
`
`gatedwithiodine-131
`chimericIgG1conju-
`
`tumors
`
`intracellularDNAin
`
`advancedlungcancer
`
`oncology
`
`chimericIgG1
`
`EGFR
`
`colorectalcancer
`
`oncology
`
`humanIgG1
`
`TNFalpha
`
`rheumatoidarthritis
`
`AIID
`
`gatedwithiodine-131
`murineIgG2aconju-
`
`CD20
`
`phoma
`
`Non-Hodgkin’slym-
`
`oncology
`
`humanizedIgG1
`
`CD11A
`
`psoriasis
`
`AIID
`
`2005
`
`2004
`
`2004
`
`2003
`
`2003
`
`2003
`
`2003
`
`2003
`
`humanizedIgG1
`
`IgE
`
`asthma
`
`respiratory
`
`2003
`
`gatedwithYttrium90
`murineIgG1conju-
`
`CD20
`
`phoma
`
`Non-Hodgkin’slym-
`
`oncology
`
`2002
`
`delivery
`
`proteinform/isotype
`
`target
`
`majorindication
`
`therapyarea
`
`launchdate
`
`Roche/Chugai
`Actemra
`Tocilizumab
`
`BiogenIDEC/Elan
`Tysabri
`Natalizumab
`
`Genentech
`Avastin
`Bevacizumab
`
`BiotechCo.
`ShanghaiMedipharm
`I-131ch-TNT
`
`ImClone/BMS
`Erbitux
`Cetuximab
`
`Abbott
`Humira
`Adalimumab
`
`GSK
`Bexxar
`Tositumomab
`
`Genentech
`Raptiva
`Efalizumab
`
`Genentech/Novartis
`Xolair
`Omalizumab
`
`Biogen/Idec
`Zevalin
`Ibritumomabtiuxetan
`
`manufacturer
`tradename
`genericname
`
`© Higher Education Press and Springer-Verlag Berlin Heidelberg 2010
`
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`

`Protein & Cell
`
`Zhiqiang An
`
`Pappasetal.,2009
`
`SC
`
`humanIgG1
`
`AIID,arthritis,immuneandinflammatorydisorders;CNS,centralnervoussystem;CV,cardiovascular;ID,infectiousdisease;IM,intramuscular;IV,intravenous;SC,subcutaneous
`Johnson&Johnson
`Simponi
`Golimumab
`
`TNFalpha
`
`rheumatoidarthritis
`
`AIID
`
`2009
`
`Keatingetal.,2010
`
`Rotheretal.,2007
`
`Rutgeertsetal.,2007
`
`2007
`
`CohenuramandSaif,
`
`IV
`
`IV
`
`SC
`
`IV
`
`humanIgG1
`
`CD20
`
`leukemia
`
`chroniclymphocytic
`
`oncology
`
`2009
`
`hybrid
`
`humanizedIgG2/IgG4
`
`C5a
`
`sis)
`
`PNH(chronichemoly-
`
`hematology
`
`2007
`
`PEGylatedfragment
`
`TNFalpha
`
`rheumatoidarthritis
`
`AIID
`
`humanIgG2
`
`EGFR
`
`colorectalcancer
`
`oncology
`
`2007
`
`2006
`
`2006
`
`KennethandKertes,
`
`injectionintotheeye
`
`mentofAvastin
`
`humanizedmabfrag-
`
`VEGF
`
`lardegeneration
`
`wetage-relatedmacu-
`
`ophthalmology
`
`2006
`
`reference
`(Continued)
`
`delivery
`
`proteinform/isotype
`
`target
`
`majorindication
`
`therapyarea
`
`launchdate
`
`GSK
`Arzerra
`Ofatumumab
`
`Alexion
`Soliris
`Eculizumab
`
`UCB-Schwarz
`Cimzia
`Certolizumabpegol
`
`Amgen
`Vectibix
`Panitumumab
`
`Genentech/Novartis
`Lucentis
`Ranibizumab
`
`manufacturer
`tradename
`genericname
`
`324
`
`© Higher Education Press and Springer-Verlag Berlin Heidelberg 2010
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`

`Therapeutic monoclonal antibodies
`
`Protein & Cell
`
`and Valge-Archer, 2007) (Fig. 1F–G). Later, transgenic mice
`and in vitro phage display were employed to generate fully
`human therapeutic antibodies to circumvent the immunogeni-
`city issue associated with murine sequences (Fig. 1H)
`(Hoogenboom, 2005; Lonberg, 2005; Jakobovits et al.,
`2007; Lee et al., 2007). In addition to phage display, antibody
`fragments can also be displayed on yeast (Feldhaus et al.,
`2003), bacteria (Harvey et al., 2004), mammalian cells (Smith
`and Zauderer, 2009) and other in vitro systems such as
`ribosomes (Hanes et al., 1998). The pros and cons of the
`various antibody platforms have been broadly reviewed
`recently (An, 2009). The ever
`increasing demand for
`improved tools for antibody drug discovery will lead to new
`platforms and technologies. For example, humanized rabbit
`mAbs are being developed as therapeutics (News, 2010; Yu
`et al., 2010).
`Another important source of antibodies is the human
`antibody B cell repertoire. The isolation of human mAbs has
`been a labor-intensive endeavor, either through EBV immor-
`talization or hybridoma fusion, or by constructing phage-
`displayed antibody libraries (Vaughan et al., 1996; Traggiai
`et al., 2004; Li et al., 2006b; Rothe et al., 2007). Significant
`technological breakthroughs in B lymphocyte culture and
`cloning were reported recently including the analysis of HIV
`and flu mAbs in naturally infected or vaccinated humans
`(Wrammert et al., 2008; Jin et al., 2009; Ogunniyi et al., 2009;
`Scheid et al., 2009; Walker et al., 2009; Kwakkenbos et al.,
`2010). It is now possible to isolate human memory B cells
`(CD27 + , sIgG + , IgD-) from peripheral blood mononuclear
`cells (PBMC), and more importantly, culture them where they
`proliferate and differentiate to IgG secreting cells (ISC) (Smith
`et al., 2009; Walker et al., 2009). Single cell culturing vessels
`have been engineered,
`thus enabling for high-throughput
`screening of functional mAbs (Jin et al., 2009; Ogunniyi et al.,
`2009). New methods of B cell immortalization other than EBV
`infection or hybridoma, such as Bcl-6/Bcl-xL, or hTERT, have
`been reported (Kwakkenbos et al., 2010).
`In addition,
`methods for cloning of IgG encoding genes from single B
`cells have been optimized (Wrammert et al., 2008; Jin et al.,
`2009; Ogunniyi et al., 2009; Scheid et al., 2009; Walker et al.,
`2009; Kwakkenbos et al., 2010). These technical and
`engineering accomplishments make it
`feasible to isolate
`human mAbs with broad coverage of therapeutic targets.
`
`FORMATS OF ANTIBODY THERAPEUTICS
`
`Most therapeutic antibodies are full length IgG molecules and
`IgG1 is the most commonly used sub-isotype (Table 1). This
`is because IgG1 molecules possess several
`favorable
`characteristics: they are structurally stable; they have a long
`in vivo half life; and IgG1 confer Fc-mediated biological effects.
`In designing antibody therapeutics, it is sometimes desirable
`to diminish or abolish the ADCC and CDC functions while
`retaining its pharmacokinetic profile, in the case of a “benign
`
`blocker” antibody. For this purpose, both IgG2 and IgG4 have
`been used in antibody therapeutics (Table 1). Protein
`engineering has been applied to create Fc with altered
`properties. For example, IgG2m4, a novel engineered IgG
`isotype with reduced Fc functionality was recently reported
`(An et al., 2009). The engineered IgG2m4 is based on the
`IgG2 isotype with four key amino acid residue changes
`derived from IgG4 (H268Q, V309L, A330S and P331S). An
`IgG2m4 antibody has an overall reduction in complement and
`Fcg receptor binding in in vitro binding analyses while
`in vivo serum half-life in rhesus
`maintaining the normal
`monkeys.
`In addition to IgG molecules, antibody fragments (e.g.,
`Fab) have also been developed as therapeutics (Sandborn
`et al., 2007). Relative to IgG molecules, antibody fragments
`have more extensive penetration of tissues (particularly of
`solid tumors) due to their smaller size. The smaller size of
`antibody fragments has the advantage of accessing ther-
`apeutically important epitopes that may be sterically hindered.
`In addition, antibody fragments may be manufactured more
`cost effectively in a microbial
`fermentation system. The
`shorter half life of antibody fragments can be extended by
`modifying the molecules such as through PEGylation. The
`absence of the Fc region in an antibody fragment may lessen
`side effects caused by the interaction between Fc and the
`immune system. ReoPro, an anti-GPIIb/IIa chimeric Fab for
`the prevention of blood clots in angioplasty, was the first
`antibody fragment approved for clinic use in the US (Faulds
`and Sorkin, 1994). Lucentis, a Fab fragment of Avastin, is
`used for the treatment of wet age-related macular degenera-
`tion (Kenneth and Kertes, 2006). More recently, Certolizumab
`pegol
`(Cimzia), a PEGylated antibody fragment, was
`approved for the treatment of rheumatoid arthritis in Europe
`(Rutgeerts et al., 2007). Currently, about 19 antibody
`fragment based therapeutics are in active clinical develop-
`ment (Nelson and Reichert, 2009).
`Other antibody formats such as domain antibodies and
`single chain antibodies are also being explored for diagnostic
`and therapeutic applications (Holt et al., 2003; Holliger and
`Hudson, 2005; Enever et al., 2009). A PEGylated human anti-
`IL-1R domain antibody is in clinical testing for the treatment of
`rheumatoid arthritis (Vk or VH dABs) (Enever et al., 2009). A
`llama nanoantibody targeting the von Willebrand factor is
`being developed for
`the treatment of
`thrombosis (Van
`Bockstaele et al., 2009).
`Antibodies can also be used as carrier agents of small
`molecule toxins or radiolabeled isotopes, guiding drugs to
`specific disease sites and limiting undesired effects on
`healthy cells. This application is most commonly employed
`in oncology. At least two radiolabeled antibodies, Zevalin and
`Bexxar, are approved for clinical use (Table 1). These drugs
`are difficult
`to administer because a radiologist and an
`oncologist are needed to oversee the administration. Mylo-
`targ, a humanized anti-CD33 IgG4 antibody conjugated to
`
`© Higher Education Press and Springer-Verlag Berlin Heidelberg 2010
`
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`

`Protein & Cell
`
`Zhiqiang An
`
`calicheamicin, is an example of antibody used to carry a
`cytotoxic payload (Table 1). Many challenges still exist in
`designing antibody:drug conjugates such as choice of linker,
`stoichiometry, and conjugation chemistry. Recent advances
`have resulted in linkers having increased stability in the
`bloodstream while allowing efficient payload release within
`the tumor cell (Ducry and Stump, 2010). Increasing evidence
`suggests that conjugated antibodies remain an effective
`alternative to mAb, small molecule or radiolabeled isotope
`monotherapies.
`It is interesting to note that the early antibody therapeutics
`started as crude polyclonals (serum therapy and IVIG) and
`the majority of today’s antibody therapeutics is target-specific
`monoclonals. Progress is being made in developing recom-
`binant polyclonal antibodies (rpAb) for the treatment or
`prophylaxis of human diseases (Pedersen et al., 2010). The
`difference is between IVIG and rpAbs is that rpAbs are
`mixtures of carefully selected monoclonal antibodies. Recom-
`binant polyclonal antibodies (rpAb) mimic the natural human
`immune response in which the human body produces
`different types of antibodies targeting different epitopes of
`an antigen. This polyclonal response may have a better
`chance to neutralize the disease target than a single antibody
`does. One of the major challenges facing this approach is to
`manufacture the antibody cocktail consistently in both
`quantity and quality.
`Today’s monoclonal antibody therapeutics functions on a
`single disease target.
`It
`is advantageous if one antibody
`molecule can bind to two or more different targets since many
`complex diseases are the result of multiple mediators.
`Multispecificity has shown in naturally isolated antibodies,
`for example, the monoclonal IgE antibody SPE7 binds not
`only to its intended antigen 2,4-dinitrophenyl (DNP) hapten,
`but it also binds to several unrelated compounds with a broad
`range of affinities (James et al., 2003). More recently,
`antibodies that binds to both HER2 and VEGF were reported
`(Bostrom et al., 2009). In these cases, the multispecificity is
`conferred by a single binding pocket (James et al., 2003;
`Bostrom et al., 2009). Multispecifity antibodies are not
`common in nature, but they can be constructed by recombi-
`nant DNA methods (Kufer et al., 2004; Wu et al., 2007).
`Design of bispecifc antibodies is an active research area and
`the anti-IL-12/IL-18 dual-variable-domain immunoglobulin
`DVD-Ig molecule is an example of
`the many designs of
`bispecific antibodies (Wu et al., 2007).
`
`IMPACT OF POST-TRANSLATIONAL
`MODIFCATION ON THE PHYSICAL AND BIOLOGICAL
`PROPERTIES OF THERAPEUTIC ANTIBODIES
`
`Antibodies are large proteins which are subjected to
`extensive and complex posttranslational modifications, such
`as deamidation, glycosylation, N-terminal pyroglutamation,
`C-terminal lysine truncation, and methionine oxidation; and
`
`these posttranslational modifications profoundly impact the
`physical, chemical, and pharmacological properties of ther-
`apeutic antibodies (Wang et al., 2009). Oxidation of methio-
`nine residues is one of the most common protein degradation
`pathways including antibodies.
`In addition, methionine
`oxidation of recombinant monoclonal antibodies can alter
`their interaction with protein A and protein G resulting in a
`decrease in binding affinity (Gaza-Bulseco et al., 2008). The
`spontaneous nonenzymatic deamidation of glutaminyl and
`asparaginyl residues can alter the structure and function of
`therapeutic antibodies, potentially resulting in decreased
`bioactivity, as well as alterations in pharmacokinetics and
`antigenicity of antibody therapeutics (Huang et al., 2005).
`Among the various posttranslational processes, glycosylation
`has the broadest effect on biologic activity, protein conforma-
`tion, stability, solubility, secretion, pharmacokinetics and
`immunogenicity of
`therapeutic antibodies (Arnold et al.,
`2007). For example, differential IgG sialylation may provide
`a switch from innate anti-inflammatory activity in the steady-
`state to generating adaptive pro-inflammatory effects upon
`antigenic challenge (Kaneko et al., 2006). Low fucose levels
`on antibodies enhance neutrophil- and mononuclear cell-
`mediated ADCC (Peipp et al., 2008). Antibody glycoengineer-
`ing is one of most active areas of research in therapeutic
`antibody discovery and development today (Mimura et al.,
`2009).
`
`MANUFACTURING
`
`Manufacturing of mAbs is expensive. A large scale facility can
`take multiple years and hundreds millions of dollars to build.
`Mammalian cell culture is the dominant production platform
`for mAb therapeutics. About half of the current marketed
`mAbs are expressed in Chinese hamster ovary (CHO) cell
`lines. Recombinant myelomas or hybridomas are still being
`used for antibody production, but their utility as a production
`platform is limited due to the low expression titer and
`instability of the cell
`lines. To reduce the cost of antibody
`production, other methods of antibody expression, such as
`bacteria, plants, transgenic animals (milk), eggs, and yeast,
`are being developed. Certolizumab pegol is an example of
`mAb (fragment) therapeutic made in a bacteria (Rutgeerts
`et al., 2007). However, antibodies produced in E. coli are not
`glycosylated and this severely limits its use as a manufactur-
`ing platform. Antibodies with specific human N-glycan
`structures have been expressed in glycoengineered lines of
`the yeast Pichia pastoris and its utility as a general platform
`for producing recombinant antibodies with human N-glycosy-
`lation is being developed (Li et al., 2006a; Lin et al., 2010).
`Antibody has been expressed in engineered chicken eggs
`and in plants (Zhu et al., 2005; Cox et al., 2006). Despite
`significant effort, cost saving alternative antibody manufactur-
`ing platforms is still lacking. This is in part due to the effect of
`the various expression hosts on antibody posttranslational
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`Therapeutic monoclonal antibodies
`
`Protein & Cell
`
`modifications and the low production titers. There is a clear
`need for innovation and technology breakthroughs in redu-
`cing the manufacturing cost of therapeutic antibodies. This is
`not limited to the choice of expression hosts. Purification,
`formulation, storage, and other steps in the entire manufac-
`turing process need to be improved to bring antibody
`therapeutics in a more cost competitive position against
`small molecule drugs.
`
`ANTIBODY THERAPEUTIC TARGETS
`
`Antibodies can engage a wide range of extracellular drug
`targets such as membrane bound proteins or circulating
`ligands and cytokines (Table 1). Even though antibodies do
`not readily cross cell membranes or the brain blood barrier
`(BBB), about 80% of
`the current druggable targets are
`accessible to antibodies (Strohl, 2009). Extracellular signaling
`(ECS) drug targets generally are not modulated by small
`molecules as ECS targets typically function through protein-
`protein interactions. ECS proteins have been successfully
`targeted by antibodies. The therapeutic areas in which
`antibodies have the strongest presence, in terms of marketed
`products and developmental research, are oncology and
`Arthritis, immune and inflammatory disorders (AIID). Of the
`more than 200 monoclonal antibodies in clinical use and
`development
`today, about half are being developed for
`oncology. The second largest therapeutic category is in the
`AIID area, and infectious disease is fast becoming a major
`disease area for antibody therapeutics. While the emphasis
`on oncology and AIID therapeutic areas will continue,
`antibody therapeutics are being developed in almost all
`disease areas such as central nervous system, cardiovas-
`cular, women's health, diabetes/endocrinology, hematology,
`ophthalmology, and respiratory diseases (Strohl, 2009).
`
`SUMMARY
`
`Antibody therapeutics represent a major breakthrough in
`combating human diseases and the improvement of human
`health. This is reflected by the recent trend in drug discovery
`and development. In 2000, nine of the top 10 medicines were
`small molecules while only one was a recombinant protein but
`by 2008, a short eight years later, half of the top 10 medicines
`are recombinant proteins and antibodies. This trend will
`continue as about 50% of the new drugs in various stages of
`clinical development are antibodies. Despite the remarkable
`progress, many scientific, technological, and clinical chal-
`lenges remain in the area of therapeutic antibody discovery
`and development. Opportunities for innovation exist at every
`level: accessing difficult antibody targets (such as G protein-
`coupled receptors), novel antibody sources and formats,
`crossing the BBB and cell membranes, modified effector
`functions, improved formulation and delivery methods, and
`lower cost manufacturing, to name a few.
`
`ABBREVIATIONS
`
`AIID, arthritis, immune and inflammatory disorders; ADCC, antibody-
`dependent cellular cytotoxicity; BBB, brain blood barrier; CDC,
`complement-dependent cytotoxicity; CDRs, complementarity deter-
`mining regions; DNP, 2,4-dinitrophenyl; ECS, extracellular signaling;
`FDA, Food and Drug Administration; HAMA, human anti-mouse
`antibody; IM, intramuscular; ISC, IgG secreting cells; IV, intravenous;
`IVIG,
`intravenous immune globulin; mAbs, mouse monoclonal
`antibodies; PBMC, peripheral blood mononuclear cells;
`rpAb,
`recombinant polyclonal antibodies; SC, subcutaneous; VL, variable
`light; VH, variable heavy
`
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