REVIEWTherapeutic Monoclonal Antibodies
Taketo Yamada

Department of Pathology, School of Medicine, Keio University, Tokyo, Japan(Received for publication on October 5, 2010)(Revised for publication on December 3, 2010)(Accepted for publication on December 16, 2010)

Over a hundred years has passed since the discovery of the “magic bullet” serum therapy by Kitasato
and Behring, the first ever therapeutic use of antibodies. More than 80 years later, the investigation of
immunoglobulin structure and function and the development of cell and molecular biology introduced
the production of monoclonal antibodies (MoAbs). In the 35 years since the first process for creating
MoAbs was introduced, they have remained the centerpiece of the growing biotechnology and pharmaceutical industry. Herein, I review the history, development, and clinical settings of therapeutic MoAbs
that have had a significant impact on life-saving medicine. (Keio J Med 60 (2) : 37–46, June 2011)

Keywords: monoclonal antibody, molecular target therapy, immunoglobulin

￼￼￼Introduction

tubercle bacillus, assembled a team to tackle the newly
forming field of immunotherapeutics; the team included
Paul Ehrlich, Emil von Behring, Erich Wernicke, andShibasaburo Kitasato, all of whom would have a significant impact on the beginnings of antibody-based therapy.
Behring made some key observations that led to the concept of serum therapy. He noticed that the blood of thoserats resistant to anthrax was able to kill the anthrax bacterium. Together with Wernicke, he developed the firstworking serum therapy for diphtheria. Kitasato and Behring had isolated the tetanus-forming bacillus and had
determined that its pathogenesis lay in the activity of atoxin, and then demonstrated that the transfer of serumfrom a guinea pig immunized with diphtheria toxin toanother guinea pig offered protection from the toxin.1 Kitasato and Behring also obtained anti-sera against tetanus
toxin, demonstrating the breadth of the principle (Table1). Their serum therapy against diphtheria was first tested
clinically in 1891 at Charite Hospital in Berlin. A year
later, Behring started working with the pharmaceuticalmanufacturer Farbwerke Hoechst to develop the diphtheria serum treatment. In 1894, Hoechst launched the first
immunobiological therapeutic, dispatching 25,000 dosesof anti-diphtheria serum to prevent epidemic diphtheria
that took the lives of 50,000 children annually in Germany. The introduction of serum therapy decreased mortal-

Shibasaburo Kitasato and Emil von Behring showedthat the serum from human patients or horses who had recovered from an infectious disease could be used to prevent and/or treat the disease in other people or animals.
Human immunoglobulins (Igs) obtained from donors areused to treat a variety of infectious diseases; however,these applications are restricted by availability and limited potency. Monoclonal antibodies (MoAbs) resolved
many limitations in the clinical usage of Igs. The first
MoAb for clinical use, OKT3 against T lymphocytes, wasgenerated as a therapy against tissue rejection. Initially,
the field of MoAb therapy developed slowly because of
the time required to improve methods for humanizing
murine MoAbs and to develop manufacturing processes
and capability. Some novel procedures for humanizing
murine MoAbs decrease the antigenicity of Igs and provide progressively greater potency of MoAb therapy. This
article provides a comprehensive review of the development, characterization, and clinical application of therapeutic antibodies.
Origin of Serum Therapy
In the year 1882, Robert Koch, the discoverer of theReprint requests to: Taketo Yamada, MD, PhD, Department of Pathology, School of Medicine, Keio University, Shinanomachi 35, Shinjuku-ku,
Tokyo 160-8582, Japan, E-mail: taketo@sc.itc.keio.ac.jp
Copyright © 2011 by The Keio Journal of Medicine.
37

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Yamada T : Therapeutic Monoclonal Antibodies

Table 1 The development of therapeutic monoclonal antibodies

There are two major eras in the development of therapeutic antibodies: 1) the discovery of serum therapy (1890~) and 2) the development of
monoclonal antibodies (1975~). Therapeutic MoAbs are often developed 10-12 years after the establishment of novel technology.

ity in Paris from 52% to 25%. This success popularized
the term “magic bullet,” which was an apt and prescientdescription for present immunotherapy. The discovery ofserum therapy led to the awarding of the Nobel Prize in
Medicine and Physiology to Behring in 1901 (Table 1).
Behring used the funds from this Nobel Prize to set up
a new company in 1904, which today is known as Novartis (Chiron) Behring. It was likely the first example
of venture capital funding in biotechnology. The serum
for serum therapy was crude and came from immunized,
non-human sources (horses or rabbits). It contained many
foreign proteins and gave rise to a phenomenon that has
been generally called “serum sickness.” The onset of serum sickness in 100% of patients treated with serum therapy led to various efforts to improve on this approach.
Behring recognized these toxic side effects of serum
therapy and introduced improved methods for purification of serum.
Meanwhile, Ehrlich, another member of theteam,realized the standardization of serum therapy,
which led to the development of procedures for quantification of the serum therapeutic effect, including theconcept of LD50. This serum therapy was used widely

until approximately the onset of World War II for various
bacterial diseases including diphtheria, meningitis, andpneumonia. An example of how widespread serum therapy had become is that 86% of patients diagnosed with
type I streptococcal pneumonia in the late 1930s at Boston City Hospital were treated with a type-specific serum
therapy (Table 1).2 The second trial of serum therapy wasdone by isolating natural antibodies in either vaccinatedor naïve humans, followed by isolation of IgG fractions
from pooled serum for the therapy of a patient with primary immunodeficiency.3
Development of Monoclonal Antibodies (MoAbs)
Köhler and Milstein developed methods for the isolation of MoAbs from hybridoma cells in 1975 (Table 1).4
They showed that the cell fusion technique could be used
to produce hybrids between myeloma cells and antibodyproducing cells. The resulting hybrid lines were permanently adapted to grow in tissue culture and were capableof inducing antibody production in mice using Jerne’s
hemolytic plaque assay, which allows direct visualizationof antibody-producing B cells.5 This discovery led to theKeio J Med 2011; 60 (2): 37–46

awarding of the Nobel Prize in Physiology or Medicine in1984 to Köhler, Milstein, and Jerne. Köhler and Milstein
did not patent this method, which opened up the use of
their hybridoma technology to academics and pharmaceutical fields alike for generations of future potential therapy. The first therapeutic MoAb was generated as OKT3
by Ortho Biotech in 1984 (Table 1). The first step was theability to clone out, using the PCR technique, murine VH
and VL genes for recombinant expression.6 The second
step was to express both heavy and light chain antibody
genes in stable human cell lines after transfection.7 The
third step was the method of producing chimeric antibodies possessing murine VH and VL chains fused with human constant regions.8,9 Chimeric antibodies have about
one-third murine sequences (2 VH and 2 VL subunits)
and two-thirds human sequences, including a human Fc.
For MoAb expressions, the pSV2 vector and SP2/0 murine myeloma cell line were widely used in the early period.10 Recently, Chinese hamster ovary (CHO) cell lines
and a glutamine synthetase (GS) expression system wereused, as widely licensed by Lonza.11 After cell cloning
and optimization using this CHO-GS expression system,
MoAb production is 1–5 g/L. Chimeric antibodies stillretain one-third murine sequences, which may lead to enhanced immunogenicity.12
￼ Humanization is the idea of making the V chainsfrom a murine or other mammalian antibody “more human.” The complementarity-determining regions (CDRs)
were grafted from a murine antibody into the closely related human framework, followed by making amino acidchanges required to stabilize the engineered constructs.13
Queen et al. developed a detailed process for humanizing
antibodies via CDR grafting, which was the basis for humanization of many MoAbs currently on the market orundergoing clinical trials.14 Other procedures of humanization without CDR grafting have also been developed,
most notably the resurfacing of antibodies to remove
B cell epitopes.15 The first humanized MoAb used as atherapeutic agent was an anti-CD25 (IL-2 alpha subunit)
MoAb that was humanized and developed at Protein Design Labs and licensed by Roche to suppress rejection
after transplantation. In contrast to these in vitro manipulations for humanizing MoAb, two research groups
separately and independently were successful in developing functional human MoAbs directly from “humanized”
transgenic mice (Table 1).
The researchers at Cell Genesys (which later spun off
Abgenix, which was acquired by Amgen in 2007) and
GenPharm (acquired by Medarex in 1997) both engineered mice by disabling the ability of mice to produce
their own murine antibodies and replacing that function
with human antibody genes.16,17 These transgenic humanized mice were immunized with an antigen and, asa result, fully human antibodies were generated in thesemice. The first fully human MoAb to be developed andmarketed from one of these humanized systems was

39

Vectibix, a human IgG2 antibody discovered using Abgenix XenoMouse technology in 2006, 12 years after theannouncement of these engineered mice. These fully human MoAbs derived from humanized mice make up 25%of phase III clinical trials. Smith reported that peptides
were able to be displayed as fusions of P3 on the tail fibers of Escherichia coli filamentous phage M13.18 Thismethod may be available broadly for the display of proteins including MoAbs; additionally, a phagemid system
which allows for monovalent display is useful in selections of clones, especially for high-affinity binders rather
than high-avidity binders.19 This M13 P3-based phagedisplay technology has become an optimal methodology
for selection of antibody fragments capable of increased
binding capabilities because antibodies function via
binding ligands.20 McCafferty et al. reported the bypassing of immunization by building a library of antibody
genes, displayed on the P3 protein of M13 phage, using
PCR techniques to recover the human gene from either B
cells or hybridomas.21 This method was followed by theconstruction of huge human libraries from either synthetic repertoires or from multiple naïve human donors.22,23
The latter library from Cambridge Antibody Technology (CAT, a part of MedImmune, an owned subsidiaryof Astra-Zeneca) was constructed using single-chain Fv
constructs fused with a His and myc tag, produced undernM binders, and has become the prototype for many later
libraries. A wide variety of strategies for making hugelibraries or more focused libraries have been reviewed.24
The first antibody from a phage-displayed human antibody library to be approved for therapeutic use was theanti-TNF alpha antibody Humira in 2002. Interestingly,
Humira was not identified de novo from a human antibody library, but instead was identified via a guided selection method using murine antibody as the primarybinder.25
Glycosylation of Therapeutic MoAbs
Glycosylation has become a focus of interest for medical biological investigators as a means to control the efficacy and safety of biological pharmaceuticals. MoAbs
have a conserved glycosylation site at Asn297 on eachCH2 domain of Fc, and in IgG from human serum thesesites contain a family of diantennary complex-type oligosaccharides. The Fc glycans are highly heterologous
due to variable processing of outer-arm monosaccharide
residues onto the core heptasaccharide. Glycosylation at
Asn297 is essential for stabilization of the CH2 domains
and to achieve optimal antibody effector functions, including Fcg receptor and complement activation (Table1). IgG glycoforms play a role in modulation of antibody
effector functions. Recently, IgG-Fc bearing non-fucosylated oligosaccharides have been shown to enhance antibody-dependent cellular cytotoxicity (ADCC),26 while
IgG-Fc bearing sialylated oligosaccharides may modu-

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Yamada T : Therapeutic Monoclonal Antibodies

late antibody-induced inflammation.27 These properties
may be exploited when developing antibody therapeutics
to target tumors or to modulate inflammation in autoimmune diseases. Glyco-engineering of IgG provides possibilities for the production of therapeutic MoAbs with
tailored effector functions.
Antibody Databases
To arrange and use the large amounts of data on immunology and its genetics, databases have been developed by
IMGT (international ImMunoGeneTics)-ONTOLOGY.28
The IMGT databases are standardized and combine genomic, genetic, and structural approaches, providing abioinformatics of immunology essential for immunotherapy by allowing data comparison on antibodies. IMGT isavailable on the web at http://imgt.org. These databases
are queried extensively and are used by scientists fromboth academic and pharmaceutical laboratories all over
the world. The functions of the databases are 1) repertoire
analysis of IgG antibody recognition sites in normal andpathological states such as autoimmune disease, infectious diseases, immunodeficiency, or hematological malignancies; 2) Ig repertoires in domestic and wild species;3) genomic evolution research of adaptive immune responses; 4) structural evolution of Igs which interact with
antibodies such as Fc receptors; 5) antibody engineering;6) pathological diagnosis; and 7) therapeutic approaches.
Pharmacokinetics and Pharmacodynamics
The time course of therapeutic antibodies in the human body is determined by all processes involved in theabsorption, distribution, metabolism, and excretion of
antibodies. Despite the increasing number of therapeutic
MoAbs in clinical use, certain aspects of the pharmacokinetic and pharmacodynamic properties of these MoAbs
are not fully understood. Target-mediated disposition,in part, explains the characteristic non-linear pharmacokinetic and pharmacodynamic characteristics often
observed for MoAbs. The interindividual variability of
MoAb pharmacokinetics may be explained by interindividual variability with respect to target molecule expression and generation of immunological responses against
administered MoAbs; among other sources, genetics andclinical status may explain interindividual pharmacodynamic variability. Pharmacokinetics and pharmacodynamics and related variability are explored through theuse of pharmacokinetics and pharmacodynamics modeling approaches, which may be more complex than thoseof conventional small compounds. The half-life of IgG1,
2, and 4 in humans is approximately 21 days and that of
IgG3, which has a different affinity to neonatal Fc-receptor, is 7 days. The half-lives of therapeutic MoAbs generally increase with the degree of humanization: murine(1.5 days) < chimeric (10 days) < humanized (12–20 days)= or < fully human (15–20 days).29 The shorter half-life of
murine MoAbs has been attributed to the lack of bindingof murine IgG to human neonatal Fc-receptor and the generation of human anti-mouse antibodies (HAMA). The
elimination of MoAbs is a complex multifactorial process
involving protein catabolism, interaction with neonatal
Fc-receptor, target-mediated elimination, immunogenicity, proteolytic degradation, and glycosylation.
Therapeutic MoAbs in Clinical Use
Since the first therapeutic MoAb OrthoClone OKT3
was approved in 1986 for the treatment of transplant rejection, many MoAbs have been developed and have become indispensable for some diseases. At least twentyfive different MoAbs were in clinical use by May 2009,
and the current development pipeline contains more than
250 potential therapeutic MoAbs (Table 1).
1) MoAbs in oncology
CD20
CD20 antigen is expressed on normal B cells and onsome neoplastic B cells. Rituximab, a mouse-humanchimeric MoAb against CD20, was derived from mouseanti-CD20 MoAb IDEC-2B8 by engineering of human
IgG1 and kappa constant regions and the original murine
variable regions.30 CD20 may function in normal B cell
growth and activation and is expressed in more than 90%
of non-Hodgkin lymphomas and 10% of chronic lymphocytic leukemias. Anti-CD20 MoAb may be an efficient
treatment for B cell malignancies because CD20 is expressed at high levels on B cells, is relatively resistant
to internalization, and is not shed, thereby allowing the
MoAb to persist on the cell surface. Rituximab binds toan epitope on the large extracellular loop of CD20 with anaffinity of 8 nM. The binding between CD20 and rituximab induces apoptosis and cell lysis via complement-dependent cytotoxicity (CDC) and ADCC.30 Furthermore
rituximab may sensitize the cells to the effect of chemotherapeutic reagents.31 In most B cell lymphoma patients,
rituximab causes a swift and profound depletion of circulating B cells which lasts for 3–6 months.32 In a central
trial investigating the safety and efficacy of rituximab forthe treatment of relapsed or refractory non-Hodgkin B
cell lymphoma, patients receiving four weekly doses of
375 mg/m2 of rituximab intravenously had an overall response rate of 48% and a complete response rate of 6%.33
Clinical studies of rituximab in relapsed or refractory
CD20-positive non-Hodgkin B cell lymphomas showedthat combination chemo/immunotherapy is superior to either treatment alone.34 Rituximab was the first MoAb tobe approved by the United State Food and Drug Administration (FDA) for treatment of malignancy.

Keio J Med 2011; 60 (2): 37–46

Human epidermal growth factor receptor 2 (HER2)
Anti-HER2 MoAb trastuzumab, created by Genentech,
was derived from murine MoAb mumAb4D5 by humanization of human IgG1 and kappa constant regions as wellas human framework regions while retaining the complementarity-determining regions of murine mumAb4D5.35
HER2 is a 185-kDa transmembrane receptor that is amember of the epidermal growth factor receptor familyof receptor tyrosine kinases. While a ligand has not yet
been identified for HER2, the receptor has been demonstrated to heterodimerize with other family members
where it mediates signaling to the others. HER2 normallyregulates cell growth and cell cycling via cyclin D andc-myc.36 HER2 gene amplification and/or overexpressionof HER2 occurs in approximately 20–30% of primarybreast carcinomas and is associated with reduced overall
survival.37 Trastuzumab binds to an epitope in the juxtamembrane region of HER2 on breast carcinoma cellswith an approximate affinity of 5 nM.38 Trastuzumabfunctions as an anti-cancer reagent via direct inhibitionof cell growth (receptor internalization, degradation, anddecreased cell signaling) and indirect ADCC and CDC
pathways.39 Furthermore trastuzumab inhibits cleavageof an extracellular domain induced by metalloproteinase
and induces apoptosis and impairs tumor angiogenesis.39–41 In clinical study, patients who were administeredwith an intravenous loading dose of 4 mg/kg followed by
2 mg/kg weekly maintenance doses had an objective response rate of 15% and a median duration of response of
9.1 months.42 In later phase II and III trials in metastatic
breast carcinoma patients, trastuzumab combined withchemotherapy produced a better response rate, increased
time to disease progression, and overall survival as compared with either chemotherapy or trastuzumab alone.43
Interestingly, better response rates were seen in patientswith overexpression of HER2 as compared to patients withnormal expression.44 A pharmacokinetic study of 4 mg/
kg loading followed by 2 mg/kg weekly dosing showedthat the mean half-life of trastuzumab was approximately
6 days.42 However, a longer half-life of 16–18 days wasseen in patients treated with 8 mg/kg loading followed by
6 mg/kg every 3 weeks.45 Trastuzumab was approved in1998 for treatment of breast cancer with HER2 overexpression. The indication of FDA approval recently was asadjuvant treatment of patients with HER2-overexpressing tumors with lymph node metastasis as part of a regimen of doxorubicin, cyclophosphamide, and paclitaxel.
Trastuzumab is approved for use as a single reagent inpatients with metastatic breast carcinoma whose tumors
overexpress HER2 and who have received one or morechemotherapy regimens. Most adverse events in patients
receiving trastuzumab are infusion-related, usually feverand chills. Other common adverse reactions are nausea,
vomiting, diarrhea, rash, neutropenia, anemia, and myalgia. Serious toxicities of trastuzumab are cardiomyopathy, pulmonary damage, severe infusion reactions, and41

febrile neutropenia. Due to the risk of cardiomyopathy,
it is recommended that patients undergo a thorough cardiac assessment before therapy and that cardiac function
should be monitored throughout therapy.
CD33
CD33 is a type I transmembrane sialoglycoprotein thatis a member of the immunoglobulin superfamily subset
of immunoglobulin-like lectins.46 CD33 is expressed on
the cell surface of myeloid and sialic acid -binding early
multilineage hematopoietic progenitor cells, monocytes,
and blasts of acute myeloid leukemia (AML) (90%);
however, CD33 is not expressed on normal pluripotent
stem cells or non-hematopoietic cells.47 Gemtuzumab
ozogamicin (Mylotarg) is a humanized MoAb against
CD33 conjugated with cytotoxic anti-tumor antibiotic
calicheamicin. Gemtuzumab is derived from murine
IgG1 MoAb P67.6 generated with human IgG4 and kappa constant regions as well as framework regions, while
retaining complementarity-determining regions of themurine MoAb.48 Gemtuzumab ozogamicin binds to thecell surface of CD33-positive leukemic cells with an approximate affinity of 0.08 nM. This MoAb functions as
a vehicle delivering a conjugated toxic payload to CD33positive leukemic cells. Binding of the MoAb to CD33 results in endocytosis, cleavage of the link between MoAb
and calicheamicin, and release of calicheamicin.49 The
released calicheamicin is reduced by glutathione to forma reactive intermediate that binds to DNA, resulting indouble-strand breaks and inducing apoptosis.50 In a phase
II study, 277 patients with CD33-positive AML received
monotherapy with gemtuzumab ozogamicin in two doses
separated by 2 weeks. As a result, 26% achieved remission, i.e., 13% of the patients had complete remission withplatelet recovery and 13% had complete remission without platelet recovery.51 The current FDA approved indications include treatment of patients with CD33-positive
AML in first relapse who are more than 60 years old andwho are not considered candidates for chemotherapy.
Vascular endothelial growth factor (VEGF)
VEGF is an angiogenic growth factor whose activity is
mediated mainly by activation of VEGF receptor 2 [VEGFR-2 (Flk-1/KDR)], a high-affinity receptor tyrosine kinase. VEGF can also ligate other cell surface receptors
including VEGFR1 and neuropilin-1 and -2. These receptors are located on the cell surface of the endothelium of
blood vessels and lymphatic vessels. Stimulation of thesecells by VEGF leads to cellular growth, inhibition of cell
death, and new blood vessel formation.52 VEGFR-2 is themain receptor through which the mitogenic and angiogenic effects of VEGF occur. There are several isoformsof VEGF: 121-, 165-, 189-, and 206-amino-acid isoforms
can form by alternate splicing. VEGF functions in theregulation of both normal and abnormal angiogenesis,

42

including neoplasm and wound healing.52 The production of VEGF is elevated in malignant neoplasm. It issecreted by tumor cells as well as by tumor-associatedstromal cells.53 The impairment of VEGF signaling by
MoAbs, small molecules, or antisense oligonucleotides
induces growth inhibition of tumor.54,55 Bevacizumab, ahumanized MoAb against VEGF, was derived from murine MoAb A.4.6.a against human VEGF165 by Genentech. Bevacizumab has human IgG1 and kappa constantregions as well as human framework regions, while retaining murine complementarity-determining regions.56
Bevacizumab reacts and neutralizes all isoforms of
VEGF effectively.57 Bevacizumab binds an epitope of
VEGF that is distinct from the receptor-binding site withapproximate affinity of 2.2 nM.58 In a xenograft modelwith human rhabdomyosarcoma and breast carcinoma,
bevacizumab could inhibit tumor growth to under 10%of the original tumor weight and impaired tumor vasculature, decreasing microvessel density and permeability.56,59 The FDA approved bevacizumab for the first-line
treatment of metastatic colorectal carcinoma in 2004. Recent FDA approved indications include use in combination with 5-fluorouracil-based chemotherapy for first- or
second-line treatment of patients with metastatic carcinoma of the colon or rectum. It is also indicated for use incombination with carboplatin and paclitaxel for the firstline treatment of advanced, recurrent, or metastatic nonsquamous cell, non-small-cell lung carcinoma. Recently,
r84, a fully human MoAb against VEGF, was reported.
r84 binds human and mouse VEGF and selectively blocks
VEGF from interacting with VEGFR2, but it does notinterfere with VEGF-VEGFR1 interaction.60
2) MoAbs in autoimmune and inflammatory disorders
and infectious diseases
Tumor necrosis factor (TNF) alpha
TNF alpha is a proinflammatory cytokine known as amediator of chronic inflammation and cachexia. The antiTNF-alpha MoAb infliximab (Remicade) was developed
as a human/murine chimeric IgG by Centocor and may
be used for the treatment of autoimmune diseases such as
rheumatoid arthritis, Crohn’s disease, ulcerative colitis,
psoriasis, and ankylosing spondylitis.61
Infliximab ligates to TNF alpha with an affinity of 1.8
nM and strong avidity; it thus effectively prevents TNF
alpha from binding to its receptors.62 Infliximab neutralizes all forms of TNF alpha, including soluble, transmembrane, and receptor-bound forms; in contrast, theTNF-alpha antagonist Enbrel, in the format of a recombinant FC fusion protein, neutralizes only soluble TNF-alpha and beta . Infliximab is administered by intravenous
infusion and has a half-life of 8–10 days.63 The clinicaleffect of infliximab was obvious from the rapid reductionof proinflammatory cytokines and other inflammatory

Yamada T : Therapeutic Monoclonal Antibodies

markers. Infliximab has a safety profile, and infusions are
generally well tolerated. Serious adverse events include
an increased risk of tuberculosis, opportunistic infections, or worsening of preexisting heart failure. The other
MoAb against TNF alpha, adalimumab (Humira), wasdeveloped as a fully human MoAb by Cambridge Antibody Technology.64 Adalimumab was derived from murine antibody MAK195 using the guided selection phagedisplay method of Cambridge Antibody Technology. Thismethod involves a series of selections of the human heavy
chain and light chain variable region by pairing themwith the variable regions of MAK195; these variable domains were fused with the human IgG kappa constantdomain. The fully human MoAb clone D2E7, producedin CHO cells, has a binding affinity of 100 pM to TNF
alpha.65 Adalimumab is administered subcutaneously at
40 mg every 1 or 2 weeks for rheumatoid arthritis patients. The maximum serum concentration and the time
to reach the maximum concentration were 4.7 µg/mL and
131 h, respectively, following a single 40-mg subcutaneous injection to healthy adult human volunteers. The average absolute bioavailability of adalimumab, evaluatedfollowing a single 40-mg subcutaneous dose, was 64%.
The mean serum half-life was 10–20 days for intravenous
infusions.66 In patients with rheumatoid arthritis, a dramatically decreased level of inflammatory markers suchas C-reactive protein, fibrinogen, and cytokines was observed. Adalimumab is a fully human MoAb; however,
an immune response against adalimumab is still present
in 5% of infused patients. It is marketed both as preloaded
syringes and as preloaded pen devices for self-injection
by patients. Recently, a new fully human MoAb against
TNF alpha, golimumab, was developed by Centocor. Golimumab is a human IgG1 (VH3 and V kappa 3) generated using Medarex’s UltiMAb technology. Its variabledomains are almost 100% identical to the human germline sequences and its constant domain is identical to corresponding regions of infliximab in terms of amino acidsequences.67,68 It was approved by the FDA in April 2009,
alone or in combination with methotrexate, for the treatment of moderate to severe rheumatoid arthritis, active
psoriatic arthritis, and active ankylosing spondylitis. Unlike infliximab, which is administered through intravenous infusion, golimumab has been approved for subcutaneous injection once a month, which is a less frequent
dose than two other leading biologic reagents, etanercept
and adalimumab, in the class of anti-TNF alpha therapy.
Respiratory syncytial virus (RSV)RSV is classified in the genus Pneumovirus, belongs
to the Paramyxoviridae family, and caused of serious
respiratory tract diseases in children and infants. Over
70% of infants are infected in the first year after birth,and virtually every infant has experienced RSV infection by their second birthday.69 RSV infection causesKeio J Med 2011; 60 (2): 37–46

about 4500 deaths and the hospitalization of about 90,000
children every year in the United States. The humanized
MoAb against the F protein of RSV, palivizumab (Synagis), was derived from a selected murine MoAb using a
CDR-grafting-based humanization method.70 The original murine MoAb, mAb 1129, was selected because it
recognized the antigenic site A on the F protein of RSV.71
Palivizumab has a binding affinity of 3.46 nM to the Fprotein of RSV using Biacore analysis, and its half-lifeis 17 days in humans.72 Palivizumab has been shown to
cut the chance of RSV high-risk infant hospitalization by
50%. Palivizumab was the first MoAb reagent to treat an
infectious disease.
3) A candidate novel MoAb therapy in oncology and autoimmune and inflammatory disorders
CD26 is a 110-kDa surface glycoprotein with dipeptidyl peptidase IV activity able to cleave selected biologicalfactors to alter their functions.73 CD26/dipeptidyl peptidase IV is involved in T-lymphocyte costimulation andsignal transduction processes.74 CD26 is involved in thedevelopment of certain human cancers.75 It was foundthat CD26 is a collagen-binding protein using a CD26positive JMN cell line, which is derived from malignant
mesothelioma.76 Several studies have shown that antihuman CD26 murine MoAb inhibits growth of CD26positive T-cell malignancies and renal cell carcinoma.77,78
YS110, a humanized MoAb against CD26, was developed
from murine MoAb 14D10 against human CD26 by Y’s
Therapeutics. YS110 has human IgG1 and kappa constantregions as well as human framework regions, while retaining partially murine complementarity-determining
regions using an in silico humanization method. YS110
induces cell lysis of malignant mesothelioma cells via
antibody-dependent cell-mediated cytotoxicity in addition to its direct anti-tumor effect via p27kip1 accumulation. In vivo experiments with mouse xenograft models
involving human malignant mesothelioma cells showedthat YS110 treatment drastically inhibits tumor growthin tumor-bearing mice, resulting in enhanced survival.79
YS110 may have potential clinical use as a novel cancer therapeutic agent in CD26-positive cancer including
malignant mesothelioma, lymphoma, and renal cell carcinoma. Furthermore, it was shown that YS110 induced
a translocation of CD26 from cell membrane to nucleus
within 1 h, and intra-nuclear CD26 may function as a
growth inhibitor.80 On the other hand, CD26 expression
on T lymphocytes is enhanced after activation of T cells,
while it is preferentially expressed on a subset of CD4+
memory T cells in the resting state. The binding of antiCD26 MoAb inhibits human T-cell growth and proliferation in both CD26-transfected Jurkat T-cell lines andhuman T-cell clones by inducing G1/S arrest, which is associated with enhancement of p21Cip1 expression.81 YS110

43

may have potential use in the clinical setting involving
activated T cell dysregulation, including autoimmune
disorders and graft-versus-host disease.81 YS110 was approved as an Investigational New Drug by the FDA and iscurrently at the phase I clinical trial stage.
4) Adverse effect of MoAbs in a clinical trial of immunotherapy
The first phase I clinical trial of TGN1412, a novel superagonist anti-CD28 monoclonal antibody that directly
stimulates T cells, was carried out in 2006. Within 90 min
after receiving a single intravenous dose of the drug, all
six volunteers had a systemic inflammatory response
characterized by a rapid induction of proinflammatory
cytokines and accompanied by headache, myalgia, nausea, diarrhea, erythema, vasodilatation, and hypotension.
Within 12–16 h after infusion, they became critically ill,with pulmonary infiltrates and lung injury, renal failure,and disseminated intravascular coagulation. Severe andunexpected depletion of lymphocytes and monocytes occurred within 24 h after infusion. All six patients weretransferred to an intensive care unit where they received
intensive cardiopulmonary support, dialysis, steroids,
and an anti-interleukin-2 receptor antagonist antibody.
Despite evidence of multiple cytokine-release syndrome,
all six patients survived.82 Stebbings et al. reported novelin vitro procedures in which TGN1412, immobilized invarious ways, is presented to human white blood cells ina manner that stimulates the striking release of cytokinesand profound lymphocyte proliferation that occurred invivo in humans. These procedures would have predicted
the toxicity of this superagonist and are being applied toemerging immunotherapeutics.83
Summary
Thirty therapeutic MoAbs have been approved aroundthe world, including 24 in the United States, several ofwhich have attained blockbuster status with sales reaching the coveted billion-dollar mark and beyond. Five
MoAbs, rituximab (Rituxan), infliximab (Remicade),bevacizumab (Avastin), trastuzumab (Herceptin), andadalimumab (Humira), generated sales of over $4 billion each in 2008, and global sales for this entire sector
surpassed $30 billion in that year. There are more than
250 therapeutic MoAbs now undergoing clinical trials,
mostly concentrated in the areas of malignancy, autoimmune and inflammatory diseases, and infectious diseases. Beyond these, hundreds more candidates are alreadyat the preclinical stage of development. Dimitrov et al.
suggested that there have been two major eras in antibody discovery: the original serum therapy period of theearly 1900s and today, in which significant changes are
impacting the way investigators design and make thera-

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Yamada T : Therapeutic Monoclonal Antibodies

peutic proteins.84 In the next era, therapeutic MoAbs may
be broadly developed using novel concepts or methodologies such as 1) extensive searches of target molecules, 2)
new protein engineering, 3) generation of improved drugdelivery systems, and 4) research on direct functions of
MoAbs against cells other than by ADCC or CDC.
Acknowledgments
This article was supported by the Program for Promotion of Fundamental Studies in Health Sciences of the
National Institute of Biomedical Innovation (07–17) anda research grant from the Ministry of Education in Japan(20590354).
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