Immunopharmacology and Immunotoxicology, 2010; 32(4): 533–542

i n v i t e d R EVIE W

Monoclonal antibodies used as prophylactic,

therapeutic and diagnostic agents
Marita Chakhtoura, and Alexander M. Abdelnoor
Department of Microbiology and Immunology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon

Abstract
Monoclonal antibodies can be of mouse, part mouse part human (chimeric, humanized), or of human
origin. Their preparation involves hybridoma, gene cloning, gene recombination, phage display, and
gene transfection techniques. The preparation, mechanism of action, uses, and possible adverse effects
of most of the available monoclonal antibodies used as prophylactic, therapeutic, and diagnostic agents
are reviewed.
Keywords:  Monoclonal antibodies; preparation; mechanism of action; uses; drawbacks

Introduction
Monoclonal antibodies (moab) are directed against one
epitope or specificity. Each preparation is a homogene-
ous mixture of antibodies belonging to the same class/
subclass and selectively active against the one antigenic
determinant to which they are produced.(1,2)
Moab can be of mouse origin, part mouse part
human origin (chimeric, humanized), or of human
origin. Their preparation involves hybridoma, gene
recombination, phage display, and gene transfection
techniques.(2–8) They are mainly used as therapeutic
agents to treat autoimmune diseases and cancer, and
in transplantation to control or prevent acute rejection
episodes.(9,10) In as much as cancer therapy is concerned,
they are used either naked or conjugated to a cytotoxic
agent.(3,11–15) In the clinical pathology laboratory, moab
are being used as serological reagents and in diagnostic
radiology moab–radioisotope conjugates are used in
radioimaging.(1,4,5,16–22)
This review will deal with the preparation, mechanisms
of action, uses, and major adverse effects of monoclonal
antibodies.
Preparation
Mouse hybridomas
Hybridomas are used to prepare mouse moab. As stated
by Keenan et al (1) and Elgert (23), Kohler and Milstein intro-
duced a hybridoma as a cell that results from the fusion
of a mouse myeloma cell and a normal mouse antibody-
producing cell. The hybridoma gets its ­properties from
the two cells that fused to give rise to it.(1–5,11)
The cell line used is an immortal mouse myeloma cell
line which, if grown in an appropriate nutritive medium
in vitro, can indefinitely divide and thus survive for a long
period of time. It is also a cell that is incapable of produc-
ing immunoglobulins or hypoxanthine phosphoribosyl
transferase (HPRT), an enzyme needed in the synthesis
of nucleotides from preformed bases (Salvage pathway).
However, a mouse antibody-producing cell is a normal
mortal mouse cell that cannot indefinitely divide in vitro.
It originates from a spleen of a previously immunized
mouse with the antigen (epitope) in question. Moreover,
it is HPRT-producing. Therefore, the resulting hybridoma
cell possesses properties of both cell types; it retains the

Address for Correspondence:  Alexander M. Abdelnoor, Department of Microbiology and Immunology, Faculty of Medicine, American University of Beirut,
P.O. Box 11-0236-Riad el-Solh, Beirut 11972020, Lebanon. E-mail: aanoor@aub.edu.lb

(Received 11 January 2010; accepted 23 January 2010)

ISSN 0892-3973 print/ISSN 1532-2513 online © 2010 Informa Healthcare USA, Inc.
DOI: 10.3109/08923971003646597 http://www.informahealthcare.com/ipi
534   M. Chakhtoura and A. M. Abdelnoor
myeloma cell’s immortality, and is HPRT- as well as
antibody-producing, both are properties of the spleen
cell.(1–5,11)
To prepare a moab-producing hybridoma cell, mouse
myeloma and normal spleen cells obtained from an
immunized mouse are cultured together in a medium
containing the preformed bases, hypoxanthine and
thymidine; aminopterine, an inhibitor of the de novo
synthesis of bases; and polyethylene glycol, a cell fusing
agent. Upon culture, tumor cells that do not undergo
fusion will eventually die due to their inability to syn-
thesize nucleotides (absence of HPRT and presence of
aminopterine). However, normal murine cells that fail
to fuse will also die because they lack the property of
immortality. However, hybridomas (presence of HPRT)
will possess the properties of the two fusing cells and will
thus be antibody-producing (one type) and will have the
ability to survive indefinitely.(1,4,5)
Following preparation, single hybridomas are fished
from the culture medium and the antibody specificity
of interest is tested for by ELISA. Upon its detection, the
hybridoma needed is propagated by either intraperito-
neal injection into a mouse where these cells will grow
in the ascites form and the ascites fluid-rich moab is
harvested or by propagating a single hybridoma in vitro
and the moab is released into the culture medium.(1)
When used as therapeutic agents, mouse moab present
with a number of drawbacks and limitations including
efficacy in, and foreignness to humans. These drawbacks
will be covered later. To reduce these drawbacks, the for-
eignness of mouse moab in humans was decreased by
preparing partial or total human moab.
Transgenic mice
One way of preparing partial or complete human moab
is to produce transgenic mice that carry gene seg-
ments or genes that code for part or complete human
immunoglobulin.(3) A transgenic mouse is one with extra
and/or altered copies of one of its genes. The altera-
tion can be achieved by the transformation of embry-
onic stem cells (ES) or by microinjection of the male
pronucleus.(6,24)
To transform embryonic stem cells, cloned segments
of the target human immunoglobulin gene are intro-
duced into embryonic stem cells, allowed to recombine
with the mouse genome, and the embryonic stem cell is
subsequently injected into the blastocyst and the latter
is replanted in the uterus.(6,24)
On the other hand, microinjection of the cloned gene
segment into the male pronucleus of a fertilized egg can
be done and the egg is then implanted into the uterus of
a pseudopregnant mouse (a female mouse mated with a
vasectomized male). The injected gene segment becomes
incorporated in the mouse genome.(6,24) In both methods,
the resulting progeny are transgenic mice that produce
partial or complete human immunoglobulin.
Spleen cells from an immunized transgenic mouse
are used to prepare hybridomas that would produce
hybrid (part mouse, part human) or complete human
moab. Chimeric moab produced in this manner consist
of mouse variable regions and human constant regions
(25% mouse, 75% human). Humanized moab consist
of mouse complementary determining region and the
rest of the molecule is human (5% mouse, 95% human).
Human moab are 100% human.(3,11,25)
Phage display library
Another way to prepare moab or other fusion proteins is
by making use of a phage display library.
Production of variable (V) regions
Preparation of a phage display library to produce the vari-
able V regions (Heavy and Light) of antibodies involves
cloning the gene segments coding for a number of vari-
able domains of human immunoglobulins and fusing
them to genes coding for the coat proteins of a filamen-
tous bacteriophage. Bacteria (usually Escherichia coli)
are infected with the recombinant bacteriophage and
replicated. The resulting progeny of bacteriophages
would be heterogeneous with respect to variable regions
of antibodies expressed on their protein coats. Affinity
chromatography is used to isolate the phage expressing
the V region specific for a particular epitope. E. coli is
subsequently infected with the isolated bacteriophage
and the resulting progeny would be homogeneous,
expressing the desired monoclonal V regions.(7)
Preparation of human monoclonal antibodies
The monoclonal V region genes from the bacteriophage
are cloned and recombined with human gene segments
that code for the constant regions of an immunoglobulin
molecule. A mouse myeloma cell line (the same cell line
used to produce a hybridoma) is then transfected with
the recombinant DNA. The transfected cell progeny will
secrete human moab.(3,7,8)
Fusion proteins
Some of the immunotherapeutic agents such as
etanercept, a fusion protein consisting of soluble TNF
linked to the component of human immunoglobulin
(IgG1), can be prepared by constructing a recombinant
bacteriophage.(26,27)
Classes/subclasses of monoclonal antibody
preparations
Most therapeutic moab are of the IgG1 subclass. They
have the greatest ability to bind to the gamma Fc receptor
expressed by a number of cell types, which accounts for
 Preparation and mechanisms of action of monoclonal antibodies   535

their relatively longer half-life. The efficacy of the differ- treating autoimmune diseases, as well as other miscel-
ent moab classes/subclasses is given in Table 1. laneous conditions are given in Tables 2, 3, 4, and 5,
respectively.

Mechanism of action Transplantation

The therapeutic effects of moab are achieved through Graft rejection is a major concern post-solid or non-solid
several mechanisms. A direct effect is observed when transplantation. Graft-versus-host (GVHD) reactions
apoptosis of the target cell is achieved. Moab can block occur when the graft rejects the recipient and it most
a growth factor cell receptor resulting in the cessation commonly occurs in cases of bone marrow transplanta-
of cell growth. In as much as indirect modes of action tion. T lymphocytes belonging to the graft itself mount
are concerned, these include antibody-dependent an immune response against the recipient cells. Hale
cell-mediated cytotoxicity (ADCC) and complement- et  al.(9) reported that the administration of anti-CD52
dependent cytotoxicity (CDC). In the former, the moab moab (Campath-1H, human IgG1) and Campath-1G (rat
binds to the target antigen and recruits other cells such IgG2b) in order to deplete T lymphocytes from donor
as monocytes, macrophages, and natural killer cells via cells and residual recipient lymphocytes resulted in a
binding to their Fc surface receptors. Phagocytes in turn, decline in the occurrence of GVHD. The second type of
engulf the tumor to destroy it while natural killer cells graft rejection is that mounted by recipient cells against
recruit B cells and secrete perforins and granzymes that the donor cells. Beniaminovitz et  al.(10) subjected a
lyse the target cell. In the latter mechanism, the comple- group of patients receiving a cardiac transplant to either
ment cascade is activated leading to the formation of the
membrane attack complex that eventually kills the cell. Table 1.  Classes of monoclonal antibodies.
Moab directed against a tumor cell surface antigen can Monoclonal
behave as a drug targeting agent. They can be conjugated antibody class Efficiency
to a cytotoxic agent such as a radionuclide, a toxin, or IgG1 •  Binds well Fc receptor
chemotherapeutic agent, and as such deliver the cyto- •  Long half-life
toxic agent to the tumor cell.(12) IgG2 and IgG4 •  Inefficient effector function
IgG3 •  Long hinge region
•  Susceptible to enzymatic degradation
Nomenclature IgD and IgE •  No beneficial effect
IgM •  Large molecule
Nomenclature of moab preparations depends on the •  Difficulty reaching extravascular spaces
nature of the molecule. They all share the common suf-
fix stem “mab” but differ with the substem according Table 2.  Some naked moab used to prevent or control graft rejection.
to their origin. Murine moab made up of 100% mouse Monoclonal antibody Generic and Trade name
protein end with “momab.” Chimeric moab having 25% anti-CD25 Daclizumab, Zenapax
mouse protein end with “ximab.” Humanized moab, con- anti-CD3 muromomab, Orthoclone OKT3
sisting of 5% mouse protein, have names that end with anti-ICAM
“zumab.” Moab with names ending with “mumab” are Anti-CD25 Basiliximab, Simulect
100% human. Fusion proteins where part of the hybrid
molecule is a fragment of the immunoglobulin molecule
Table 3.  Some naked moab used for cancer therapy.
and the remaining part directed against a cytokine or its
Monoclonal
receptor end with “cept.”(3,28) antibody Generic and Trade name Used for treatment of
Anti-CD52 Alemtuzumab, Campath Chronic lymphocytic
leukemia
Naked monoclonal antibodies Anti-VEGF Bevacizumab, Avastin Colorectal cancer
Anti-EGFR Cetuximab, Erbitux Colorectal & head
Naked moab are those used alone without any cytotoxic and neck cancer
agent attached to them. They are used as prophylactic Anti-EGFR Panitumumab, Vectibix Colorectal cancer
and/or therapeutic agents mainly in preventing or Anti-CD20 Rituximab, Rituxan, Mab Non-Hodgkin’s
Thera lymphoma
controlling an acute rejection episode, treatment of
Anti-ErbB2 Trastuzumab, Herceptin Breast cancer
different forms of cancer and autoimmune diseases, (anti-Her-2/neu)
and in certain instances in preventing or treating an EGFR: epidermal growth factor receptor; Her-2/neu: human epi-
infectious disease. Some naked moab in clinical use for dermal growth factor receptor 2; VEGF: vascular endothelial growth
preventing or controlling graft rejection, treating cancer, factor.
536   M. Chakhtoura and A. M. Abdelnoor

Table 4.  Some naked moab used to treat autoimmune diseases. to evaluate the antiangiogenic effect of the vascular
Monoclonal Generic and Used for the endothelial growth factor (VEGF) moab (bevacizumab),
antibody Trade name treatment of rats having breast cancer with metastasis to bone were
Anti-TNF-α Adalimumab, Humira Severe autoimmune monitored for osteolysis, surrounding soft tissue tumor
disorders growth, and angiogenesis. Treatment of the rats with this
Anti-TNF-α Certolizumab pegol, Crohn’s disease moab resulted in a significant decrease in all three proc-
Cimzia
esses as compared with control rats.
Anti-CD11a Efalizumab, Raptiva Psoriasis
In hematologic B-cell malignancies, the targets of
Anti-TNF-α Infliximab, Remicade Severe autoimmune
disorders moab are mainly CD52 and CD20. Alemtuzumab is
Anti-α-4 integrin Natalizumab, Tysabri Multiple sclerosis, a naked humanized anti-CD52 antibody used for the
Crohn’s disease treatment of chronic lymphocytic leukemia.(3,12) The
Anti-p40 Ustekinumab, Stelara Psoriasis, other chimeric naked moab rituximab is anti-CD20. Targeting
autoimmune diseases CD20, which is overexpressed in B-cell malignancies,
ultimately induces the activation of caspase 3 leading
Table 5.  Some naked moab used for miscellaneous diseases. to cell apoptosis. In follicular lymphomas, CD20 expres-
Monoclonal Generic and Used for sion is “as high as 95%.”(12) Rituximab, which was the first
antibody Trade name treatment of moab to receive FDA approval, induced apoptosis upon
Anti-glycoprotein Abciximab, ReoPro Cardiovascular caspase activation and also acted via ADCC and CDC
IIb/IIIa disease mechanisms.(12,32) It is used as a first-line combination
Anti-C5 Eculizumab, Soliris Paroxymal nocturnal and maintenance therapy in the treatment of low-grade
hemoglobulinuria
lymphomas, such as non-Hodgkin’s lymphoma, that are
Anti-IgE Omalizumab, Xolair Allergic asthma
resistant to conventional chemotherapy. Studies were
Anti-RSV* F protein Palivizumab, Synagis Respiratory syncytial
virus conducted to assess its efficacy and side effects. Results
Anti-VEGF*-A Ranibizumab, Lucentis Macular degeneration showed encouraging outcomes especially in great num-
RSV: respiratory syncytial virus; VEGF: vascular endothelial growth bers of partial responses to combination therapy, in
factor. durability of partial and complete responses as well as
in low toxicity consisting of fever and chills during the
conventional immunosuppressive therapy (controls) or first infusion.(12)
anti-interleukin-2 receptor moab (Daclizumab). Out of In as much as solid tumors are concerned, treatment
the 28 patients receiving the moab, five developed acute is more difficult since cross-reactivity of tumor antigens
rejection as compared with 17 of the 27 controls. During with normal antigens is more common.(12) Edrecolomab
the induction therapy, the frequency of acute rejection (murine) and trastuzumab (humanized) are two impor-
episodes was higher in the control group and the time tant moab worth mentioning. The former, approved for
to the occurrence of a first set rejection was less than use in Europe for colon and rectal cancers therapies, is
the daclizumab group. During the follow-up period, directed against 17-1A glycoprotein antigen, specific to
two patients from the daclizumab group experienced these two cancers.(33) Its mode of action includes ADCC,
acute rejection in contrast with nine controls. Hence, CDC, and the induction of an anti-idiotypic network.(12)
this moab was shown to have prophylactic effects with Trastuzumab is most commonly used in the United States
regard to the frequency and severity of cardiac allograft for treating breast cancer. It is directed against HER-2/
rejection during the induction period. In an animal neu antigen, found in 25–35% of breast cancers.(12) The
model, anti-CD45RB moab prolonged murine heart graft mechanisms of action of trastuzumab include the down-
survival and when given along with cyclophosphamide, regulation of HER-2 receptor expression, inhibition
prolonged survival of kidney xenografts in a rat-to- of proliferation of human tumor cells that overexpress
mouse model.(29) Another commonly used prophylactic HER-2 protein, enhancing immune cell recruitment,
moab that increases graft survival rates in humans is ADCC against tumor cells that overexpress HER-2 pro-
anti-CD3 moab (OKT3). It actually binds CD3 and blocks tein, and down-regulation of angiogenesis factors.(12) This
T-cell function. Reports on OKT3 in induction therapy monoclonal antibody is either given as monotherapy or
indicate that it is strongly associated with increased graft in combination with chemotherapy. Safety and efficacy
survival in ischemia, delayed graft function, and HLA-DR were demonstrated when trastuzumab was given in
mismatches.(30) monotherapy in patients with metastatic breast cancer.
Moreover, when given in combination with chemother-
apy, prolongation of survival and higher responses than
Cancer
those observed with chemotherapy alone were reported.
Nowadays, cancer treatment regimens with moab are It was observed that subjects with overexpression of the
gaining much attention. In a study by Bäuerle et  al.(31) HER-2/neu antigen generally had better responses.
 Preparation and mechanisms of action of monoclonal antibodies   537
Nevertheless, cardiac dysfunction is one side effect that
can be caused by trastuzumab especially in patients with
cardiac disease.(12)
Autoimmune disorders
Autoimmunity occurs when the body loses tolerance to
self-antigens and starts mounting an immune response
against them. Several factors play a triggering or con-
tributing role in developing this kind of reaction which
is certainly always accentuated whenever a genetic
predisposition exists, causing maintained and chronic
autoimmunity. Contributing factors to a self-targeted
immune response include the presence of viral and/or
bacterial infections and the action of physical agents
like exposure to ultraviolet light.(25) Immunotherapy with
moab targets two different aspects related to the devel-
opment of autoimmunity: surface molecules including
cytokine receptors and cytokines. These moab cause
either depletion or functional inhibition of the targeted
molecule. Surface molecules targeted by passive immu-
notherapy of autoimmune diseases comprise CD5, CD7,
CD20, CDw52, IgG idiotype, TCR/CD3 complex, CD4,
CD28, CTLA-4, CD40, CD2, lymphocyte function-asso-
ciated antigen (LFA-1), and very late activation antigen
(VLA-4) as well as some integrin molecules.(25) Specific
ligands for these molecules have also been targeted for
therapy. As for the cytokine targets, these are classified
according to their anti- or pro-inflammatory function.
Therapy can rely on anti-IL-1, -IL-2, -TNF-α, -IL-6, and
-IL-12 moab to treat diseases like rheumatoid arthritis,
multiple sclerosis, juvenile diabetes, and Crohn’s disease
while anti-IL-10, -IL-4, -IL-5, -IL-11, and -IL-13 moab are
used for systemic lupus erythematosus.(25) Clinical trials
have been conducted to assess the efficacy and safety of
numerous moab in rheumatoid arthritis. Of these were
the evaluations of the chimeric anti-CD20 (Rituximab);
the chimeric, murine, and humanized anti-CD4; the
chimeric anti-CD7; anti-ICAM-1; the humanized anti-
CDw52; anti-IL-1R; as well as the murine and humanized
anti-IL-6.(25) In as much as systemic lupus erythematosus
is concerned, clinical trials have included chimeric anti-
CD4, humanized anti-CD40L, and murine anti-IL-10
antibodies.(25) Humanized anti-CDw52 moab has been
assessed for vasculitis and multiple sclerosis and chi-
meric anti-CD4 moab for multiple sclerosis.(25) A series
of moab have been included in clinical trials for therapy
against psoriasis and these consist of CTLA-4–IgGFc
fusion protein targeting B7.1/B7.2; humanized anti-
CD25, anti-CD4, and anti-CD11a; as well as LFA-3–IgGFc
fusion protein against CD2.(25) Reports have indicated the
efficacy of Rituximab in the treatment of autoimmune
thrombocytopenia, systemic lupus erythematosus, der-
matomyositis, rheumatoid arthritis,(25) and relapsing-
remitting multiple sclerosis.(32,34) Furthermore, the FDA
approved the use of infliximab to treat Crohn’s disease
(August 1998), etanercept and infliximab for the treatment
of rheumatoid arthritis (November 1998 and November
1999, respectively), and etanercept in cases of juvenile
rheumatoid arthritis (September 1999),(25) psoriatic
arthritis, ankylosing spondylitis, and plaque psoriasis.(3)
Moreover, alongside its efficacy in the treatment of B-cell
chronic lymphocytic leukemia,(35) Campath-1 moab was
effective in the treatment of Wegener’s granulomatosis,
vasculitis, rheumatoid arthritis, progressive systemic
sclerosis, multiple sclerosis, and idiopathic thrombocy-
topenia purpura.(3,25)
Th17 is a recently identified subpopulation of T-helper
lymphocytes and is now thought to play a major role in
the pathogenesis of autoimmune diseases.(36,37) IL-23 pro-
duced by dendritic cells binds to its receptor to induce
the rapid proliferation of Th17 lymphocytes. IL-23 con-
sists of two subunits; p19 and p40, the latter being shared
by IL-12. Moab against the p40 subunit (Ustekinumab,
Stelara) is being used to treat certain autoimmune dis-
eases such as psoriasis.
Infectious diseases
As far as infectious diseases are concerned, only one
moab has been approved by the FDA for the treatment
of the respiratory syncytial virus (RSV). Palivizumab
(Synagis) was approved in1998 for the purpose of pre-
venting RSV in infants.(3,38)
However, studies performed using an animal model
indicated that moab produced against certain infectious
agents proved to be efficient as therapeutic or prophylactic
agents. Simmons et al.(39) generated neutralizing human
moab against H5N1 avian influenza virus and tested their
efficacy in a murine model. Results indicated that these
moab conferred protection against the viral infection in
mice. Another report by ter Meulen et al.(40) showed that
the preparation of a neutralizing human moab against
the severe acute respiratory syndrome (SARS)-causing
coronavirus conferred protection in ferrets. The moab
was effective in terms of reducing the replication of the
virus in infected ferret lungs, preventing the develop-
ment of SARS-induced lung pathology, and abolishing
viral shedding in secretions. The intranasal administra-
tion of a moab against a common epitope to mouse and
human Pneumocystis carinii to SCID mice, resulted in
more than 99% reduction in pathogen number in mice
challenged with the organism. Thus, topical application
of moab was proven effective for prophylaxis against
infection at mucosal sites.(41) Neonatal rhesus macaques
orally infected with the chimeric simian-human immu-
nodeficiency virus were given a combination of four
moab against HIV envelope proteins. This immunization
showed promising results for the prevention of HIV trans-
mission pre- and postpartum through breastfeeding.(42)
538   M. Chakhtoura and A. M. Abdelnoor
Conjugated monoclonal antibodies
Moab directed against a target cell receptor can be
conjugated to a radioisotope, a chemotherapeutic
drug, or a toxin and behave as a drug delivery agent.
These conjugates are called poison arrows or magic
bullets(23) and when conjugated to a toxin they are called
immunotoxins.(11,23) The moab plays here the role of a
vehicle that directs the conjugated material to the dis-
eased cell. Hence, the damage is limited to the marked
cells with less harm to the normal ones.(11,12,23) To be active,
these conjugates must have a relatively long plasma half-
life and they must be capable of binding tightly to the
target cell surface antigen. A moab–drug or moab–toxin
conjugate must be capable of releasing the drug or toxin
from the moab and internalizing it within the cytosol of
the target cell to reach its intracellular target.(23) Some
conjugated moab in clinical use are listed in Table 6.
Preparation
Radiolabeled moab: Radioisotopes are covalently linked
to moab. The Boulton and Hunter method makes use of
an acetylating agent (N-succinimidyl-3[4-hydroxyphenyl]
propionate) to link iodine I125 to the moab.(1,43) Bailey(44)
has described the Chloramine T method for radiola-
beling proteins. Perk et  al.(45) reported on the labe-
ling of moab with zirconium-89 or gallium-68 using
p-isothiocyanatobenzyl-desferrioxamine.
Chemolabeled moab: Drugs are linked to moab
by disulfide bonds, a peptide linker, or a hydrazine
linker.(46)
Immunotoxins: Immunotoxins can be prepared either
by chemical or by genetic means. Chemical means
involves linking the toxin via a disulfide or thioether
bond to the F(ab)2 fragment of the moab. Genetic means
involves the usage of hybrid genes that code for moab–
toxin fusion protein.(47,48)
Radiolabeled monoclonal antibodies
These deliver a radioactive substance to a target cell
resulting in its destruction. They are used mainly in can-
cer therapy.(11,23) Zevalin (ibritumomab tiuxetan) and
Bexxar (tositumomab) are the two radiolabeled moab
approved by the FDA for the treatment of non-Hodgkin’s
lymphoma. The moab of both medications is directed
against CD20 that is expressed on all B lymphocytes.(3,23)
Table 6.  Some conjugated moab and their use.
Monoclonal antibody Conjugated to Generic & Trade name
Anti-CD20 Yttrium90 Ibritumomab, Tiuxetan, Zevalin
Anti-CD20 Iodine131 Tositumomab, Bexxar
Anti-CD33 Calicheamicin Gemtuzumab, Ozogamicin, Mylotarg
Anti-CD22 Pseudomonas BL22, Exotoxin A
In the case of Zevalin, the radioisotope used in imaging
is indium In111 while yttrium Y90 is used in therapy(49) and
that of Bexxar is iodine I131.(3,23)
Chemolabeled antibodies
Moab conjugated with chemotherapeutic agents used
for cancer therapy are still under study. Cytotoxic drugs
conjugated with moab that are being evaluated include
enediyne antibiotics, maytansinoids, dolastin analogues,
taxoids, and anthracyclines. Gemtuzumab ozogamicin
(Mylotarg) is a chemolabeled moab that has been
approved by the FDA for use in patients over the age of
60 with relapsed acute myelogenous leukemia. Mylotarg
is a moab directed against CD33 that is conjugated with
calicheamicin γ1. CD33 is expressed by most leukemic
blast cells.(3,13) Calicheamicin γ1 is an enediyne antibi-
otic produced by Micromonospora echinospora. It acts
by cleaving DNA.(13,23,50)
In as much as treating infectious diseases is con-
cerned, Singla(51) hypothesized that the use of a moab
directed against an infectious agent conjugated with an
antimicrobial agent would require lower dose for, and
shorter duration of, treatment with less side effects.
Immunotoxins
Immunotoxins (IT) are moab linked to bacterial or
plant toxins which, once delivered to the cell, destroy
it via interfering with its protein synthesis at the ribos-
omal level and other vital processes or by modifying the
tumor cell surface membrane.(11,14,15) They mostly target
hematologic neoplasms. A limited number of toxins have
been cloned for research studies including diphtheria
toxin (DT), pseudomonal exotoxin (PE40), ricin A, and
saporin.(11,14,15) However, their side effects such as toxicity
to non-target sites as well as their restricted therapeutic
window have limited their use. BL22 consists of the vari-
able portion of the anti-CD22 antibody RFB4, conjugated
with a portion of Pseudomonas exotoxin-A which pos-
sesses anticancerous effects. Since CD22 is expressed in
B-cell malignancies, the binding of the IT will lead to the
destruction of the tumor cells via apoptosis or blockage of
translational elongation.(52) BL22 is administered in cases
of chronic leukemias irresponsive to chemotherapy. In
early clinical trials, two of three patients no longer had
evidence of cancer upon treatment with BL22.(11) A phase
II study of BL22 immunotoxin which was started in 2003
Used for treatment of
Non-Hodgkin’s lymphoma
Non-Hodgkin’s lymphoma
Acute myelogenous leukemia
Chronic leukemias
 Preparation and mechanisms of action of monoclonal antibodies   539
is being conducted in patients with cladribine-resistant
hairy cell leukemia along with another trial involving a re-
treatment protocol for BL22 immunotherapy in relapsed
or refractory hairy cell leukemia.(53) Both studies have
shown a good response rate. BL22 has induced complete
remissions in many cases of hairy-cell leukemia.(54,55)
Drawbacks of therapeutic monoclonal
antibodies
Despite the many therapeutic applications of moab,
their uses have shown a number of drawbacks. Adverse
effects have been claimed to be rare, but when they occur,
they could be serious. Adverse effects are classified as
class-specific or target-specific. One of the class-specific
side effects includes the immunogenicity of the moab
that leads to hypersensitivity reactions in humans.(1,3,56)
Hypersensitivity reactions are mostly encountered with
the Fc portion of non-human, chimeric, and human-
ized moab. However, Cowden et al.(3) stated in one study
that “even 100% human proteins can be immunogenic
because the technology introduces novel peptide frag-
ments in the IgG hinge region that may be perceived as
foreign.” Anaphylaxis, serum sickness, leukocytoclastic
vasculitis, Stevens–Johnson syndrome, and injection-site
reactions could all be hypersensitivity reactions mani-
festations. Another class-specific drawback involves the
efficacy of the moab.
Cross-reactivity with similar antigens could decrease
specific binding of the moab. Moreover, the interaction
of the moab Fc portion as well as the deposition of anti-
gen/antibody complexes and subsequently their clear-
ance could modulate specific uptake and alter the fate
of bound antibodies.(1)
As for target-specific side effects, Cowden et  al.(3)
achieved immunosuppression with the administra-
tion of Alemtuzamab, but this was accompanied by an
increased susceptibility to infection. This moab depletes
T & B cells following binding to CD52 expressed on these
cells, hence making the body prone to infection. More
than 30% of Alemtuzumab recipients have been reported
to experience severe infections, with more than 15% of
them being fatal.(3) Another important moab thoroughly
studied for its side effects is anti-TNF or TNF inhibitor. Its
use blocks the proinflammatory cytokine, tumor necrosis
factor, resulting in increased susceptibility to intracellular
pathogens. Patients on anti-TNF therapy are threatened
by tuberculosis, aspergillosis, histoplasmosis, coccidi-
oidomycosis, listeriosis, legionellosis, cytomegalovirus,
and Pneumocystis jirovecii infections.(3,25) Infliximab and
etanercept are both TNF-α blocking agents and both
have caused an increased susceptibility to infection.(3)
Other rare adverse effects in patients on TNF-α inhibitors
include the appearance of anti-nucleic acid antibodies,
juvenile onset diabetes in a patient with rheumatoid
arthritis, fatal aplastic anemia, increased mortality in car-
diac patients, and demyelinating disease on infliximab
therapy.(25) All these side effects have occurred during the
course of treatment rather than recorded in clinical trials
which makes clinical trials not always the best method to
assess risk profiles of drugs.(25)
In conclusion, despite their several drawbacks, moab
still constitute valuable therapeutic agents. Future tri-
als and studies should investigate the potential efficacy
and safety of current as well as new moab preparations.
In addition, new strategies must be developed such
as combination therapies involving multiple moab or
moab conjugated with chemotherapeutic agents. Other
targeted therapies are also worth investigation. Those
include toxins conjugated with growth factors which
deliver the toxin upon binding to their corresponding
receptor. So far, only one growth factor/toxin is FDA-
approved. It consists of interleukin-2 linked to Diphtheria
toxin. Denileukin diftitox (Ontak) is a product used for
the treatment of a rare type of skin lymphoma.(11,57)
Potential replacements of therapeutic moab include
gene therapy and the use of small molecules, some of
which can penetrate cells and are more selective in their
action.(37) Until such therapeutic approaches are estab-
lished, it is of capital importance to thoroughly expand
the family of approved moab to cure debilitating and fatal
diseases.
Diagnostic reagents
Moab are being used as serological reagents and in
radioimaging.
Serological reagents
Include the determination of blood groups,(5) HLA
profiles,(58) pregnancy tests,(4,5) identification and
classification of pathogens,(5,16) classification of
blood cell malignancies,(5,12) identification of tumor
markers,(4,12) identification and counting of cells by flow
cytometry,(12,59–61) and studying the development of differ-
ent cell types.(5) They have also been used in immunoaf-
finity chromatography.(62–65)
Imaging techniques
Among diagnostic imaging techniques, nuclear medicine,
also referred to as nuclear scintigraphy, has become very
useful for its valuable contribution to diagnostic proce-
dures of various conditions and diseases.(1) In fact the
patient’s cells are often themselves labeled with a radio-
nuclide such as the case of leukocyte scintigraphy where
the radioisotope penetrates by inhalation, ingestion,
540   M. Chakhtoura and A. M. Abdelnoor
or injection and labels the target cells. However, this
branch of medicine can also be characterized by the use
of moab labeled with a radioisotope to be administered
to the patient and the radiation emission is subsequently
detected.(19) Moab have been used with various radio-
nuclides such as Indium-111 (111In), technetium-99m
(99mTc), Gallium-67 (67Ga) citrate, and others(1,19) in order
to detect artherosclerosis, sarcoidosis, vasculitis, inflam-
matory bowel disease, rheumatoid arthritis, degenerative
joint disease, and other inflammatory/infectious diseases
such as P. carinii infection and bacterial endocarditis.(19,20)
Furthermore, cases of deep vein thrombosis,(66) heart
abscess,(21) and many cancers(17) are nowadays better diag-
nosed with nuclear scintigraphy. It was demonstrated that
a number of occult tumors, which could not be revealed
by conventional procedures, have been detected by radi-
olabeled moab.(22) Thus, labeled murine anti-granulocyte
moab were used for the detection of inflammation(19) and
total knee arthroplasty,(18) 111In-anti-fibrin moab were use-
ful in detecting deep vein thrombosis(66) and 131I-labeled
anti-carcinoembryonic antigen (CEA) detected CEA-
bearing colon and pancreatic carcinomas.(1) Another
radiolabeled moab is ProstaScint, FDA-approved for
diagnosing and most importantly staging patients with
prostate cancer.(11,67,68) It is basically an 111indium-con-
jugated murine moab (7E11-C5.3), which recognizes a
membrane glycoprotein specific to prostate epithelial
cells.(67,68) However, the initial application of this moab
was somehow limited due to problems of low specificity
and interpreter inexperience.(68)
Limitations in the use of monoclonal
antibodies as diagnostic reagents
In diagnostic immunoassays, moab have led to the detec-
tion and diagnosis of various diseases and conditions.
However, their limitations must not be disregarded. As far
as assay configuration is concerned, the method of prepa-
ration of moab is characterized by flexibility with a relative
ease of purification and the option to make use of them in
excess amounts.(5) Nevertheless, this is only possible fol-
lowing the selection of the required moab. The early steps
of the procedure are painstaking and the desired anti-
body might not be encountered before several fusions.
At that point, hybridomas must be manually separated to
come across the sought antibody.(1) In addition, the assay
configuration may limit the specificity of the moab, which
in many cases is not able to cross-link many antigens.(5)
Following the selection of the moab, it should be tested
for its binding characteristics.(1) In diagnostic immu-
noassays, it is possible to select the appropriate affinity
for high sensitivity and then produce large amounts of the
same chosen antibody. However, a number of antibod-
ies will have low affinity, a disadvantage that renders the
procedure time consuming.(5) As for the cost pertaining
to moab production, it is relatively high especially before
finding the desired antibody, although decreased quality
control measures are required and newer technologies
seem promising to lower the existing costs.(5)
Declaration of interest
The authors report no conflicts of interest. The authors
alone are responsible for the content and writing of the
paper.
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