Journal of Life Science and Biomedicine  
J Life Sci Biomed, 10 (5): 59-69, 2020  
ISSN 2251-9939  
Therapeutic application of monoclonal antibodies:  
a review  
Haben FESSEHA1, Tadesse DEGU2 and Dereje ENDASHAW2  
1School of Veterinary Medicine, Wolaita Sodo University, P. O. Box 138, Wolaita Sodo, Ethiopia  
2College of Veterinary Science, Mekelle University, Mekelle, Ethiopia  
Corresponding author’s Email:;  
Review Article  
Introduction. Antibodies are an important class of proteins produced when a foreign entity  
elicits an immune response in the body. There are 5 major classes of antibodies, IgM, IgG,  
IgA, IgD, and IgE. The structure and immune function of immunoglobulins differ. An  
antibody contains two light and two heavy chains, which are linked by multiple disulphide  
bonds. Variable regions are found on light and heavy chains, known as the fragment  
antigen-binding (Fab) region, and a constant region, which is also known as the fragment  
crystallizable (Fc) region. Antibodies are, as a class, broad-spectrum antimicrobial agents  
with activity against all classes of pathogens. However, individual antibodies are usually  
pathogen-specific. Monoclonal antibodies, which specifically recognize one epitope of the  
cognate antigen, can be generated by using antibody engineering techniques such as  
hybridoma, phage display, and transgenic technologies. Removal of the entire constant  
region or part of the whole Fc portion generates antibody fragments such as Fab, scFv, and  
diabodies. Better tissue or tumor penetration characteristic of antibody fragments make  
them suited for the therapy overusing the whole antibody, hence most suited for therapy.  
Aim. Nowadays, it is possible to use antibodies for different therapeutic applications by  
modifying either their structural or functional properties.  
PII: S225199392000008-10  
Rec. 01 July 2020  
Rev. 20 September 2020  
Pub. 25 September 2020  
Antibody engineering,  
Monoclonal antibodies,  
Therapeutic antibodies  
The introduction of a trail to treat infectious human diseases using animal antibodies dates back a century [1].  
In the 1930s and 1940s antibiotics such as sulfonamides and penicillin replaced serum therapy. The decline of  
therapeutic horse and rabbit antisera is caused by the associated toxicity of serum sickness [2]. Polyclonal  
immunoglobulin and human antisera preparation were better tolerated; however, lot-to-lot variation, low  
content of specific antibodies and infectious agents such as HIV and hepatitis viruses are the limitations [3].  
In the late 1970s and early 1980s, the discovery and diffusion of monoclonal antibody (MAb) technology  
become a new promising area of human therapeutics. MAbs could be selected with exquisite specificity. They  
function on various components of the immune system such as antibody-dependent cell cytotoxicity (ADCC)  
and complement, and they showed a high biological half-life in blood and tissues, rendering them effective for  
prophylactic use. Due to their biological nature, the toxicity of infused MAbs is expected to be low. Which  
enthusiastically hailed in press accounts at the time as the solution to cancer. This concept was proven by  
successful clinical results of mouse anti-idiotypic MAbs in the treatment of lymphomas and leukemias [1] and by  
FDA approval in 1986 of the OKT3 anti-CD3 mouse MAb for acute renal transplant rejection [4].  
The adverse clinical and laboratory effects make the dark age of skepticism real. Therapy given by using  
rodent MAbs leads to the development of human anti-murine antibody (HAMA) response in more than 50% of  
treated patients. Also, the effector functions of mouse antibodies have proven to be less efficient in the human  
context. The human immunoglobulins half-life is longer than the mouse, the factor that limits usefulness.  
Therefore human MAbs are theoretical solutions despite human MAbs from hybridomas and lymphocyte cell  
lines have proven too difficult to generate easily [5].  
In 1994 the spell was broken when the FDA approved the anti-platelet mouse MAbs ReoProÔ (Centocor) to  
treat the complications of angioplasty [6]. Then between 1997 and 1999, FDA approved another engineered  
Citation: Fesseha H, Degu  
T and Endashaw D. Therapeutic application of monoclonal antibodies: A review. J Life Sci Biomed, 2020; 10(5): 59-69.  
antibodies such as RituximabÔ, a mouse/human chimeric anti-CD20 antibody (IDEC/ Genentech, San Francisco,  
CA, USA) to combat follicular non-Hodgkin’s B cell lymphoma [7]; ZenapaxÔ, a humanized anti-IL2 TAC  
receptor antibody develop by Protein Design Labs/Hoffmann- La Roche (Basel, Switzerland) to manage the  
acute rejection of kid [8], and HerceptinÔ, then Genentech developed the first antibody-based drug for adjuvant  
therapy of solid tumor (breast cancer) [9].  
High target specificity and their organization into distinct structural and functional domains make  
antibodies preferable therapeutic candidates. When an antibody is designed as a drug, features including  
immunogenicity, affinity, stability, effector functions, half-life, and tissue penetration and distribution should be  
considered and optimized accordingly. Antibody engineering uses antibody characteristics domain and a  
variety of methods to develop a suited particular therapeutic use. Nineteen therapeutic antibodies have been  
approved by the FDA in clinical use and over 150 antibodies in clinical trials. MAbs and their recombinant  
derivatives are being evaluated for the prophylaxis, therapy, and control of allergic and autoimmune diseases;  
complications of angioplasty; sepsis; a variety of inflammatory diseases; many viral and bacterial infections;  
organ transplant rejection; and solid and hematological tumors [10]. Therefore, this review is organized to give  
insight on antibody engineering and its therapeutic application areas.  
Antibody Structure  
An antibody contains two light and two heavy chains, which are linked by multiple disulphide bonds  
(Figure 1). The light and heavy chains contain a variable region, also known as the Fab (fragment antigen  
binding) region, and a constant region, which is known as the Fc (fragment crystallizable) region. Also, short  
hypervariable amino acid sequences found in the variable domains of both light and heavy chains are known as  
antigen-binding complementarity determining regions (CDRs), the majority of the sequence variations  
associated with antibodies found in CRDs. Among the six CDRs in an IgG molecule, CDR3s have the greatest  
variability. In IgG, IgA, and IgD antibody isotypes, the Fc region (the tail region of antibody) is composed of two  
identical protein fragments, derived from the second and third 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  
(Figure 2). The IgG isotype is most commonly used in therapeutic applications [11].  
Functions of antibody  
Antibodies recognize specific antigens on target pathogens and have protective effects. Some of the  
protective effects are virus and toxin neutralization, binding of the antibody alone provides sufficient steric  
interference to disrupt the interaction between the cellular receptor and the antigen, thereby abrogating virus  
uptake and replication or intoxication. But other protective effects, such as complement activation or antibody-  
dependent cellular cytotoxicity (ADCC) and phagocytosis activity lies in the bound antibody recruiting other  
components of the immune system. This occurs through Fc receptors (FcR) on the surface of immune cells to  
the Fc region of the antibody or the binding of complement proteins in the serum. Direct antifungal and in vitro  
antibacterial effect of antibody [12] and cell-mediated immune and inflammatory response regulative  
characteristics [13] were found recently (Figure 3).  
Classification of antibodies  
There are 5 major classes of antibodies, IgM, IgG, IgA, IgD, and IgE. These immunoglobulins differ in both  
their structure and immune function. IgG antibodies are monomer in structure and are the dominant form of  
immunoglobulin found in the serum. IgM is pentameric in structure and is clinically significant because they  
are predominant in early immune responses to most antigens. IgAs are polymeric and are the predominant  
immunoglobulin in saliva, tears, nasal mucosa, prostatic fluid, and many other bodily fluids. IgD antibodies are  
monomeric and trace amounts are present in serum and are found on the surface of human B lymphocytes. IgE  
antibodies are present in serum in a monomeric form and represent only a small fraction of total antibodies in  
the blood. They are involved in the production and release of vasoactive mediators e.g. histamine and other  
chemicals that cause an inflammatory reaction. In healthy adults, the four-polypeptide chain IgG monomer  
constitutes approximately 75% of the total serum immunoglobulins [14] (Figure 2).  
Citation: Fesseha H, Degu  
T and Endashaw D. Therapeutic application of monoclonal antibodies: A review. J Life Sci Biomed, 2020; 10(5): 59-69.  
Figure 1. The modular structure of antibody molecule (A) and the CDR1, 2, and 3 within the heavy and light  
chain variable domains (B); [10].  
Figure 2. Prototype structure of IgG, showing chain structure, and interchain disulfide bonds. The fragments  
produced by enzymatic digestion with pepsin or papain or by cleavage of the disulfide bonds with  
mercaptoethanol are indicated; [15].  
Citation: Fesseha H, Degu  
T and Endashaw D. Therapeutic application of monoclonal antibodies: A review. J Life Sci Biomed, 2020; 10(5): 59-69.  
Figure 3. Overview of the natural function of antibodies; [16].  
Monoclonal antibodies production by Hybridoma Technology  
Over a century ago Paul Ehrlich (19th Century) coined the phrase magic bullets to explain how antibodies  
might target and interact with their respective antigens. Seventy-five years later, Georges Kohler and Cesar  
Milstein invented the technology for cloning individual antibodies. Monoclonal antibodies specifically recognize  
one epitope of the cognate antigen. They can be generated using hybridoma technology which involves fusing  
lymphocytes from the spleen of an immunized mouse with an immortal cancer cell line (myeloma). The  
myeloma cell lines are selected so that they do not possess the capability to produce immunoglobulins unless  
fused to lymphocytes. This fusion is performed using polyethylene glycol (PEG) which is a poly wax solution  
that enhances adjacent cell fusions and the exchange of nuclei [17].  
A mixed population of fused cells, unfused myelomas, and lymphocytes results, and these are incubated for  
7 days before screening. The fused hybridoma cells are selected using HAT (Hypoxanthine, Aminopterin, and  
phosphoribosyltransferase) enzyme and when the de novo synthesis of purine and pyrimidines is blocked by  
HAT addition the cells die, while lymphocytes which do not grow in this culture media eventually die.  
Therefore, in the presence of HAT supplemented media only the hybridoma cells will proliferate. Secreted  
antibodies in conditioned media from each hybridoma are tested against the immunized antigen by ELISA.  
Positive hybridomas are scaled up and subsequently cloned (by limiting 21 dilutions) to select for a single  
monoclonal antibody-producing hybridoma cell. The hybridoma cells can be stored indefinitely in liquid  
nitrogen [18] (Figure 4).  
Citation: Fesseha H, Degu  
T and Endashaw D. Therapeutic application of monoclonal antibodies: A review. J Life Sci Biomed, 2020; 10(5): 59-69.  
Figure 4. Production of monoclonal antibody by hybridoma technology. The hybridoma technology outline  
involves the isolation of spleen cells from immunized mice, their fusion with immortal myeloma cells, and the  
production and further propagation of monoclonal antibodies from the hybrid cells; [18].  
Recombinant monoclonal antibodies  
Chimerization. Chimeric antibody refers to the replacement of murine constant region with equivalent  
human regions contributes significantly to the immunogenicity. Also, it allows for better interaction with the  
complement system and human effector cells (Figure 5). This strategy led to therapeutic successes such as  
basiliximab (Simulect: IgG1 anti-CD25, Novartis) or cetuximab (Erbitux: IgG1 anti-EGFR, ImClone) [19].  
Humanization of murine antibodies. Antibody humanization technology was developed and made possible  
by the transfer of all xenogeneic CDRs (complementary determining regions) onto the framework of a human  
Ab. The first humanized Abs was constructed based on human sequences with the known crystal structure,  
which permits the identification of residues contributing to the antigen-binding. In the “best fit” strategy, the  
closest human sequence, usually rearranged, is used as a framework to receive the murine CDRs [20] (Figure 5).  
Generation of fully human monoclonal antibodies. Human antibodies have been generated by a combined  
polyethylene glycol/electrofusion method. Recent technologic advances allowed the production of new  
monoclonal antibodies Moreover; alternative approaches based on transgenic mice or in phage display were  
developed [21].  
Human antibodies by phage display technology. Antibody phage display technology consists of the selection  
of antibody fragments from combinatorial libraries displayed on the surface of filamentous phage. The principle  
is to generate antibodies by cloning immunoglobulin variable genes using recombinant DNA technology into  
the phage genome. This technique allows displaying on the surface of bacteriophages the antibody fragment of  
interest in an scFv or Fab fragment format [22] (Figure 6).  
Citation: Fesseha H, Degu  
T and Endashaw D. Therapeutic application of monoclonal antibodies: A review. J Life Sci Biomed, 2020; 10(5): 59-69.  
Figure 6. The display and screening system of  
antibody libraries; [10].  
Figure 5. Antibody engineering for humanization;  
Immune library: Humans exposed to specific desired antigens through diseases or vaccination have high  
levels of circulating antibodies. Small libraries (~105) from immunize are enough to give rise to specific  
antibodies. Due to antibody genes in vitro experienced affinity maturation, antibodies that do not require  
further affinity maturation can potentially be isolated. Human antibodies cannot be generated by immunization  
to give human MAbs because of ethical issues. Immunological tolerance makes it difficult to isolate against self-  
antigens, many of which are potentially important therapeutic targets, particularly in cancer [10].  
Naïve library: stands for the V-gene repertoire created by cloning the antibody genes found in non-  
immunized individuals. Peripheral lymphocytes, bone marrow, tonsils, and cadaver spleens are sites for mRNA  
isolation. From the mRNA, IgM and/or IgG variable regions are amplified by PCR using degenerate  
oligonucleotide primer sets and cloned into vectors suitable for screening. Subsequently, from mRNA IgM  
repertoires are preferred to IgG because they have not been subjected to antigen selection or tolerance they are  
more diverse. In contrast, IgG chains can be biased by host immune responses will not be reactive with self-  
antigens. Once a library has been made, it can be propagated and used repeatedly to isolate antibodies against  
numerous antigens. Even though, host bacteria toxicity and poor expression are often issues with antibodies  
isolated from naïve libraries. This condition can be circumvented by using synthetic antibody repertoire  
libraries [23] (Figure 5). Synthetic library: refers to fully synthetic repertoires, germline antibody gene segments,  
VH, DH, and JH or Vκ/λ and Jκ/λ are cloned and arranged combinatorially in vitro to reconstitute genes  
encoding complete VH and VL chains. These synthetic libraries of 107-1010 clones gave rise to antibodies with  
specificity to self-antigens [23].  
Antibody library screening is the most frequently used technique. It is based on the display of antibodies on  
the surface of filamentous bacteriophages. The antibody library in the Fab or scFv format is fused to a surface  
protein of phages, displaying antigen-specific antibodies. This can readily be enriched by selective adsorption  
onto immobilized antigen, a process known as panning. The bound phage is eluted from the surface and  
amplified through infection of E. coli cells. They are 5-8 rounds of panning, elution, and amplification are  
sufficient to select for phages displaying specific antibodies, even they are up to 1011 clones large libraries [24].  
Cell display antibodies are antibodies that are displayed on the surface of microbial cells such as E. coli and  
Saccharomyces cerevisiae. A library of cells, each displaying multiple copies of a different antibody variant, is  
incubated with a fluorescently tagged ligand in a buffer for screening. Cells displaying antibodies become  
fluorescently labeled after binding with the ligand and isolated by fluorescence-activated cell sorting (FACS).  
The binding of each clone in the library to a particular ligand is quantified by flow cytometry. Parameters such  
as ligand concentration or time for the dissociation of antibody ligand complexes can be easily optimized to  
ensure the isolation of only the highest affinity antibodies [25]. Ribosome display stands for the formation of a  
ternary complex between ribosomes, mRNA, and the polypeptide. From the ribosome-mRNA-protein  
complexes, the mRNA is reverse transcribed to produce the DNA encoding the antibodies responsible for the  
binding of the complexes to the immobilized ligand. Another cycle of ternary complex formation and selection  
was done after transcribing the DNA by RNA polymerase [26].  
Citation: Fesseha H, Degu  
T and Endashaw D. Therapeutic application of monoclonal antibodies: A review. J Life Sci Biomed, 2020; 10(5): 59-69.  
Human antibodies from transgenic mice. The creation of transgenic mice expressing human  
immunoglobulin genes required two major genetic manipulations, the inactivation of the mouse antibody  
production machinery and the introduction of human immunoglobulin loci in their germline configuration.  
This complex process required the separated generation of mouse strains with inactivated mouse  
immunoglobulin genes or newly introduced human immunoglobulin loci and their successive crossbreeding. At  
the end of the process, it was possible to obtain mouse strains with a large and diverse V gene repertoire that, in  
a full immunocompetent context and upon immunization, can produce high-affinity human IgGk and IgG  
antibodies [27].  
Antibody produced by transgenic technology  
Antibodies produced in transgenic plants. In 1989 the first functional antibodies were produced from  
tobacco plants [28]. Independent cloning of heavy and light chain antibody genes in Agrobacterium tumefaciens  
vectors, the transformation of plant tissue in vitro with the recombinant bacterium, the reconstitution of whole  
plants expressing individual chains and their sexual cross was used to produce to generate plant antibodies. A  
fully assembled and functional antibody was recovered from plant tissue in some double transgenic plants  
found in Mendelian fashion. The progressive improvement of vectors for plantibodies, purification strategies,  
and the increase in transformable crop species could lead to the nearly limitless availability of inexpensive (even  
edible forms of) recombinant immunoglobulins for human and animal therapy and industrial applications of  
antibodies (e.g., catalytic antibodies) [29].  
Antibodies produced in transgenic animals. In the field of recombinant immunoglobulins, there are at least  
three examples of the application of transgenic animal technology. The first application is related to the use of  
transgenic animals as bioreactors with the production of antibodies in milk. Many human proteins of  
therapeutic importance found in the milk of transgenic animals have been well known for years, with average  
yields that amount to grams per liter in different domestic species (e.g., cattle, goats, sheep, and pig) were  
expressed. Antibody production from mammary tissue-specific promoters with correct assembly and function  
of the foreign bodies in milk [30]. Creating animals that constitutively produce recombinant antibodies or  
antibody fragments capable of neutralizing common pathogens of the species was the second application of  
transgenic animal technology. The other technology was to produce mice that carry human constant and  
variable gene segments in germline configuration. The animal transgenesis technology uses mice produced  
rearranged human antibodies in their B cells and produce human antibodies after conventional immunization  
procedures [31].  
Engineering of Antibody fragments  
The whole antibody with a molecular weight of about 150 kDa diffuses poorly from the vascular bed into a  
solid tumor mass and clears slowly from the body. Antibody fragments such as Fab, scFv, diabodies, and mini  
bodies can be generated by removing the entire constant region or part or whole of the Fc portion (Figure. 7).  
These antibody fragments are known to have better clearance from the whole body and tissue/tumor  
penetration characteristics. Antibody fragments are thought to be easy to produce in bacteria in large amounts  
and are therefore considered to bypass the hurdles associated with mammalian cell-based production of whole  
antibodies. Thus, antibody fragments are better suited for imaging and/or radiotherapy [32].  
The smallest fragment of an antibody that retains the antigen-binding specificity of the whole antibody is  
the Fv, in which single V domain binding to antigens is possible but commonly the VH and VL domains are  
noncovalently associated. Because of its instability at low concentrations, the VH and VL domains of Fv are  
linked by a flexible peptide linker to make a single-chain Fv (scFv) (Figure. 7B). The most common peptide linker  
is flexible (Gly4Ser). Also, Fv is engineered to form a disulfide bond by introducing two cysteine residues in the  
framework regions of VH and VL to yield a disulfide stabilized Fv (dsFv) (Figure 7C) [10].  
Diabodies are scFvs homodimers are covalently linked by a short peptide linker of four amino acids which  
forces the V domains to make inter-molecular complexes with their cognate domains (Figure 7D) [33]. In a  
slightly different format called (dsFv) (Figure 7E) , the VH and VL are engineered to form a dsFv but the VH is  
expressed twice in tandem separated by a flexible linker. When the VH and VL are co-expressed, a divalent  
molecule is formed (Figure 7F) [34]. Minibodies are scFvs-CH3 fusion protein homodimers (Figure 7G). In a  
different variant called Flex mini body (Figure 7H), the scFv is fused to the hinge region of IgG1 which in turn is  
fused via an additional 10 residues to the CH3 domain [35].  
Citation: Fesseha H, Degu  
T and Endashaw D. Therapeutic application of monoclonal antibodies: A review. J Life Sci Biomed, 2020; 10(5): 59-69.  
Figure 7. Schematic representation of antibody fragments; [10].  
Bispecific antibodies are artificially designed molecules, capable of simultaneously binding two different  
antigens; hence, they can be applied to redirect effector cells to tumor cells. Technologically, bsAbs can now be  
produced in various ways in more than 50 formats, including quadromas, chemical conjugates, genetically  
linked antibody fragments, and engineered constant regions that enable heterophilic association of two heavy  
chains (so-called “knobs-into-holes” technology) [36], (Figure 8).  
A key advantage of using bsAbs for therapy is that they make it possible to block multiple therapeutic  
targets with a single agent, and the effects are more pronounced when they provide pharmacological effects  
that a simple combination of each single-specific antibody cannot achieve. Removab (catumaxomab), the first  
bsAb was approved for the treatment of malignant ascites in 2009. This antibody can simultaneously bind to  
CD3 and EpCAM, and thus, can activate and redirect T cells to EpCAM-positive tumors [37].  
Figure 8. Schematic illustration of different formats of IgG-like bispecific antibodies with (A) Quadroma  
approach, (B) “Knobs-into-holes approach”, (C) Cross MAb approach. (D) Dual-Variable-Domain immunoglobulin  
(DVD-Ig) bispecific antibody with two variable domains; [16].  
Citation: Fesseha H, Degu  
T and Endashaw D. Therapeutic application of monoclonal antibodies: A review. J Life Sci Biomed, 2020; 10(5): 59-69.  
Improvement of antigen-binding affinity and effector functions  
Improvement of antigen-binding affinity. Improving the antigen-binding activity of antibodies is one of the  
most promising study areas in antibody engineering. There are two common approaches. Creating a very large  
library of randomly mutated CDRs or the entire variable domains and selecting higher affinity variants from  
this large collection of mutants. The large libraries are created by chain shuffling, error-prone, random CDR  
mutagenesis, and DNA shuffling [38]. The other approach is focused on or hot spot mutagenesis mimicking in  
vivo affinity maturation in small libraries [39].  
Improvement of effector functions. To improve effector functions therapeutic antibodies follow two basic  
mechanisms. The first mechanism is triggering intracellular signals. In this mechanism, the antibodies are  
largely dependent on their antigen-binding function rather than effector function in apoptosis and blocking  
ligand interaction. Recruiting immune system components following antigen-binding ability as well as their  
ability to trigger effector activity is the second mechanism of therapeutic antibodies. Antigen binding ability  
and engineering antibodies improve antibody binding to FcγRs or the complement factors determine the  
therapeutic efficacy of the antibodies. Glycoengineering of IgG Fc and mutating the residues that contribute to  
FcγR binding which improves ADCC activity [40]. The molecules of C1qs to the hinge and CH2 domain of  
antibodies in which the CDC depends on. Employing domain switching and site-directed mutagenesis of IgG  
constant domains indicated that the CH2 domain had an important contribution to the CDC [10].  
Altering pharmacokinetics  
Increased FcRn binding. FcRn receptor binding with a mapped and well-characterized site on IgG  
determines the plasma half-life of IgG1. Binding to FcRn is increased by mutating the FcRn binding site such  
that More recently two mutations, T250Q and M428L, have been discovered that caused respectively a 3- and a  
7-fold increase in FcRn binding and when combined together caused a 28-fold increase in FcRn binding [41].  
PEGylation. An increase in the half-lives of antibody fragments is an important area of research in the  
development of the methods. PEGylation of proteins and liposomes has been a time tested and successful  
technique that offered the advantage of reducing immunogenicity, increasing the plasma half-life, increasing  
solubility, and reducing protease sensitivity. This process is conjugating polyethylene glycol chains to the  
antibody fragments. Prolongation of the phase, a phase that represents the redistribution of a molecule in the  
extravascular environment increase in half-life observed with PEGylated antibody fragments. PEGylation slows  
the redistribution of the molecules from the plasma to the interstitial compartment. Therefore, the science of  
antibody PEGylation has two focus areas (a) to preserve the antigen-binding activity completely and (b) to link  
the PEG molecule to the antibody in a stable manner [42].  
Other antibody constructions  
Antibodies can be produced genetic fusion proteins with toxins, drugs, enzymes, and other functional  
groups and modified in their constant domains to alter the original effector mechanisms and properties of the  
antibody molecule [43]. Immunoadhesins are fusion proteins that combine the hinge and Fc regions of an  
antibody with domains of a ligand-specific cell surface receptor. These molecules are used as laboratory  
experimental tools and as promising applications in medicine [44]. Antigenized antibodies are made up of  
peptide epitopes derived from antigens that are different from immunoglobulins in the place of the CDR loops  
of immunoglobulin by grafting. The conformationally restricted exposure of short foreign peptides using the V  
region framework and the characteristics of the constant antibody domains create promising combinations for  
immunoprophylaxis or immunotherapy. They can also be extended to the peptide hormone field and the  
rational design of new drugs [43].  
Antibody engineering as an opportunity for selection and optimization of anti-HIV therapeutic agents  
Clinical need for new therapeutic approaches  
Human Immunodeficiency Virus type 1 (HIV-1) is the causal agent of the acquired immunodeficiency  
syndrome (AIDS). The introduction of highly active antiretroviral therapy (HAART), has greatly improved the  
quality of life of many infected individuals, and mortality for AIDS has dropped dramatically. However,  
currently available antiretroviral drugs have three major shortfalls: 1) the drugs have relatively high toxicities  
that cause undesirable side effects, including myocardial infarction; 2) the drug activity of the Reverse  
Citation: Fesseha H, Degu  
T and Endashaw D. Therapeutic application of monoclonal antibodies: A review. J Life Sci Biomed, 2020; 10(5): 59-69.  
Transcriptase inhibitors is cell-dependent since they are active post-infection, and 3) HIV has a high mutation  
rate leading to the rapid development of drug-resistant viral variants. For these reasons, many patients treated  
with HAART regimens still fail to achieve or maintain optimal control of the infection and development of HIV-1  
drug resistance due to the failure of available Anti-retroviral drugs. Therefore, other methods of intervention  
through control viral replication are needed. Drugs that target the cellular receptor complex are promising  
areas in HIV therapy and are a good area of interest for multi-drug resistant viruses [45].  
Anti-HIV antibodies currently in clinical trial. The major characteristics of the antibodies already entered in  
clinical trials as HIV entry inhibitors are summarized in Table 1. The monoclonal antibody Ibalizumab, formerly  
named TNX-355 and Hu5A8, is a humanized IgG4 mAb that binds to the second (C2) domain of CD4. In contrast  
to attachment inhibitors, Ibalizumab decreases the flexibility of CD4, thereby hindering the access of the CD4-  
gp120 complex to the co-receptors CCR5 and CXCR4 rather than preventing gp120 binding to CD4. This mAb is  
a potent inhibitor of HIV-1 in vitro and shows synergy when combined with other anti-HIV drugs or the fusion  
inhibitor enfuvirtide [46].  
Table 1. Antibodies who have Completed Clinical Trials for HIV  
Target Ag  
Trial status  
Humanized mAb  
Human fusion protein  
Humanized mAb  
Human mAb  
Antibody therapeutics for cancer therapy  
Monoclonal Antibodies (mAbs) comprise a class of therapeutic biologics that have been increasingly used  
over the last decades. The concept of using antibodies to selectively target tumors was proposed by Paul Ehrlich  
over a century ago [50].  
Mechanism of action of monoclonal antibodies for the treatment of cancer  
Altering signal transduction in the downstream intracellular pathways. Various cell surface receptors that  
activate intracellular pathways are expressed by cancer cells leading to growth. Amongst these, EGFR or ErbB1,  
ErbB2 or HER-2/Neu, HER-3, and HER-4 are of the same family and are over-expressed in epithelial  
malignancies originating from the colon, breast, lung, and head and neck resulting in rapidly proliferating  
disease and increased metastatic potential. Downstream activation of the receptor and increasing receptor  
internalization inhibited by Anti-EGFR antibodies bind to the receptor domain of the EGFR receptor. The cancer  
cell cycle inhibited by antibodies and cause apoptosis [51].  
Antibody-dependent cell cytotoxicity (ADCC). The cancer cells that are coated by antibodies resulted in  
immune-mediated destruction. The effector cells in the antibody-dependent cytotoxicity include macrophages,  
NK cells, and neutrophils. ADCC depends on the Fc portion of the antibody that binds an Fc gamma receptor  
(FcgR) on the effector cells. ADCC occurs when the Fab and Fc portions of the mAb engage both tumor cell  
antigen and an activating FcgR, respectively, thus creating a bridge from the tumor cell to the effector cell.  
Target cell recognition is then coupled to a lytic attack on the target cell mounted by effector cells [52].  
Complement-mediated cytotoxicity (CDC). CDC results from a cytolytic cascade mediated by a series of  
complement proteins, resulting in lysis of the antibody-bound cell [51].  
Soluble ligand neutralization. Antibodies can bind to circulating proteins and interfere with their ability to  
find their targets to help facilitate the growth of the tumors. One important example of this mechanism is  
bevacizumab which is a fully-humanized monoclonal antibody against VEGF-A. Bevacizumab binds and  
inactivates the biological activity of VEGF-A, inhibiting angiogenesis, and thus, tumor growth and proliferation  
Cytotoxic drug delivery. Tumor-targeted monoclonal antibodies are linked to Cytotoxic agents to deliver  
them specifically to the tumor cells which is preferable for its limited systemic side effect [54]. Two exciting  
samples of this technology are trastuzumab-DM1 T-DM1, a HER2 directed antibody-drug conjugate, and  
brentuximab vedotin, a CD30 directed antibody-drug conjugate [51]. Few antibody therapies are developed for  
cancer in animal besides human cancer therapy. for instance, Aratana Therapeutics, a pet health company  
Citation: Fesseha H, Degu  
T and Endashaw D. Therapeutic application of monoclonal antibodies: A review. J Life Sci Biomed, 2020; 10(5): 59-69.  
based in Kansas City, USA is developing antibody therapies for canines. Canine lymphoma monoclonal  
antibody- AT-005 (against CD52) has been approved in 2012, for the treatment of T-cell lymphoma in dogs [55].  
Monoclonal antibodies approved for clinical use in oncology. The major characteristics of monoclonal  
antibodies approved for oncology therapy are summarized in Table 2.  
Table 2. Monoclonal antibodies approved for clinical use in oncology  
Antibody Name  
Antibody format  
Colorectal, breast, and lung cancer  
Colorectal cancer  
Head and neck cancer  
Non-Hodgkin lymphoma  
Breast cancer  
Chronic lymphocytic leukemia  
Colorectal and lung cancer  
Chronic lymphocytic leukemia  
Metastatic melanoma  
Breast cancer  
RANK Ligand  
Solid tumor bony metastases  
Brentuximab vedotin  
Hodgkin’s or systemic anaplastic cell lymphoma  
Acute myelogenous  
90Y-Ibritumomab tiuxetan  
Low grade or transformed B cell Non-Hodgkin’s  
Tositumomaband 131I-  
Source: [56]  
The use of antibodies in the treatment of infectious diseases  
Anti-bacterial antibodies. Antibiotic therapy targets bacterial exotoxins. for instance, B. anthracis produces  
a potent bioweapon, a tripartite exotoxin consisting of a protective antigen (PA), lethal factor (LF), and edema  
factor (EF). Post-exposure prophylaxis with mAb against PA protected against a lethal inhalational anthrax  
challenge in rabbits and monkeys and a mAb against LF protected rats against a challenge with a lethal toxin, a  
mixture of PA and LF [57]. An anti-PA mAb acts synergistically with the antibiotic ciprofloxacin for cover  
against inhalational anthrax [58].  
Escherichia coli, Shiga toxin-producing significant exotoxin-producing pathogen causes severe  
gastrointestinal disease. Complications like hemolytic uraemic syndrome, acute kidney failure, and death may  
result from toxin entry into the bloodstream. Currently, only supportive treatments are available. a human IgG1  
mAb generated in transgenic mice against Shiga toxin subunit A prevented fatal systemic complications in  
piglets following administration after the onset of diarrhea [59]. Examples of other exotoxins against which  
mAbs are shown to possess some efficacy include Pseudomonas aeruginosa exotoxin A, eubacteria Clostridium  
perfringens epsilon toxin, and Clostridium botulinus neurotoxin [60]. Even though targeting of exotoxin requires  
prior knowledge of the pathology of the infectious agent and initial characterization of the exotoxin. Therefore  
the genetic nature, surface carbohydrates regions that show relatively little variability between bacteria  
subtypes and are explored as potential target antigens. Antibody targets against shared or invariant epitopes  
like the core carbohydrate backbone, as many bacterial species often exhibit variability in their carbohydrate  
side-chain residues. Also, targets preventing septic shock by promoting the clearance of LPS endotoxin within  
the bloodstream. Carbohydrates targeting mAbs mixed results and MAbs raised against the inner core LPS of  
varied Neisseria meningitides serotypes have shown poor phagocytic activity despite their avidity for whole-cell  
bacteria and showed poor binding to full-length LPS. MAbs produced against the deacetylated core  
carbohydrate backbone of the S. aureus surface carbohydrate, poly-N-acetylglucosamine (PNAG), conferred  
protection from a bacterial challenge in mice and performed better than mAbs against a totally acetylated wild-  
type PNAG [61].  
In another study, mAbs raised against Streptococcus pneumoniae serotype 6B capsular polysaccharide with  
strong cross-reactivity for serotype 6A showed avidity-dependent in vitro opsonization and in vivo protection  
against a bacterial challenge with either subtype. Thus, while a generic target for bacteria is attractive, much  
Citation: Fesseha H, Degu  
T and Endashaw D. Therapeutic application of monoclonal antibodies: A review. J Life Sci Biomed, 2020; 10(5): 59-69.  
work remains needed for it to be applied within the clinical setting [62]. Treatment of antibiotic-resistant  
bacteria, such as S. aureus is another area of interest during which an antibody that has reached the clinical test  
stage is tefibazumab (Aurexis), a humanized mAb that binds clumping factor A (ClfA), a serious virulence  
determinant in S. aureus. Tefibazumab has been shown to induce phagocytosis of ClfA-coated beads by human  
polymorphonuclear cells in vitro, protect against an intravenous challenge with S. aureus during a rabbit model  
of infective endocarditis and enhance the efficacy of vancomycin therapy within the rabbit therapeutic model  
Antibodies and viral disease. Palivizumab, the sole mAb currently on the marketplace for the treatment of  
infectious diseases, was developed as a prophylactic treatment against the viral disease Respiratory Syncytial  
Virus (RSV). Although mAbs are shown to be ready to neutralize many viral pathogens in vitro, the utility of  
mAb therapy in viral diseases remains a matter of contention because it is unclear to what extent viral  
clearance depends on antibody-mediated immunity. The clearance of a virus infection is typically related to T  
cell-mediated adaptive immunity. CD8+ T cells act by killing virus-infected cells, thus preventing viral  
replication and reducing the viral load. However, in acute infections, neutralizing therapeutic antibodies should  
be ready to help by suppressing viral replication and viremia, giving the host system time to develop an  
efficient response for viral clearance. Additionally, antibodies can promote the killing of infected cells  
expressing viral proteins on their surface through the activation of natural killer (NK) cells that mediate ADCC,  
additionally to their viral neutralization properties [64].  
For viral infections where the host system is unable to completely clear the virus, resulting in chronic  
infection, the administration of neutralizing antibodies might not be ready to achieve complete clearance. In  
two separate clinical trials using human mAbs against a hepatitis B virus S antigen to treat patients with  
chronic hepatitis B infection, the viral DNA and S antigen load in serum were significantly and rapidly reduced  
after antibody administration. It has been also shown the upkeep of a 90% reduction in S antigen levels 15 days  
after cessation of therapy in half the patients, and this correlated with the persistence of the administered mAb  
in serum. However, in both studies, DNA and S antigen levels eventually recovered once antibody levels within  
the serum declined following the cessation of therapy. Nonetheless, regular administration of therapeutic  
antibodies should prove useful by preventing disease transmission, the infection of healthy cells, or the event of  
pathology through the continued suppression of viral levels [65].  
Production costs  
Monoclonal antibodies are large (150 kDa) multimeric proteins containing numerous disulphide bonds and  
post-translational modifications like glycosylation. They have classy eukaryotic machinery to be produced in  
active form. Moreover, most studies have shown that these molecules need to be injected in large amounts to  
realize clinical efficacy (e.g. 8-16 doses of 375 mg·m-2, that is, a complete amount of 6-12 g per patient for  
Rituximab. Consequently, the assembly of therapeutic antibodies necessitates extensive purification steps;  
under good manufacturing practice conditions, resulting in extremely high production costs and limiting the  
wide use of those drugs [66].  
Pharmacokinetics versus tissue penetration  
Monoclonal antibodies against tumor-specific antigens only 20% of the administered dose typically  
interacts with the tumor cells in murine xenograft models. Largely remain in the blood is the major limitation  
observed [67]. Favorable pharmacokinetics and efficient penetration and retention in the targeted tissue, and  
various characteristics of mAbs, such as molecular size, shape, affinity, and valency determine tumor cell  
antibody uptake. The large size of the monoclonal antibody prevents them from the renal clearance threshold  
(70 kDa), preventing them from being eliminated through the kidney glomeruli and the half-life is prolonged.  
Fc portion of IgG molecules can interact with various receptors expressed at the surface of several cell types,  
which increase their retention in the circulation. Moreover, the Fc portion of IgG molecules can interact with  
various receptors expressed at the surface of several cell types, which increases their retention in the  
circulation. Most importantly, the Fc portion can interact with the neonatal Fc receptor (FcRn) expressed at the  
surface of several cell types, including vascular endothelium cells, monocytes, and macrophages as well as with  
barrier sites such as the blood-brain interface, the glomerular filter in the kidneys and the intestinal epithelium  
Citation: Fesseha H, Degu  
T and Endashaw D. Therapeutic application of monoclonal antibodies: A review. J Life Sci Biomed, 2020; 10(5): 59-69.  
Mode of action and associated limitations  
Binding of the antibody to its antigen, thereby interfering with its activity and interaction with binding  
partners is the simplest way MAbs to function. ADCC plays a major role in the in vivo efficacy of mAbs.  
However, they trigger ADCC by therapeutic antibodies that have several limitations. The first limitation arises  
from, 80% of the population expresses a low-affinity variant of the receptor as a major issue and the affinity  
between the Fc and its receptors plays a crucial role. The other one that arises from IgG1 molecules are  
glycosylated in the CH2 domain (Asn 297) of the Fc region. Modification is extremely important as it modulates  
the affinity of the Fc for FcγRIIIa, thereby modifying the in vivo efficacy of antibodies. The third limitation  
because of the competition of therapeutic antibodies with a high concentration of patient's IgGs for binding to  
FcγRIIIa. Even though, 66% of those molecules and the serum concentration of IgG is 817 mg·mL−1 capable of  
interacting with FcγRIIIa are IgG1. To get the therapeutic effect and to reach a serum concentration between 10  
and 100 µg·mL−1 much high doses of an antibody are required. Whereas, 10 ng·mL−1 in vitro is the level of  
antibodies saturating ADCC, in the absence of competing IgGs [69].  
Antibodies are an extremely versatile class of antimicrobial proteins that are effective against a variety of  
diverse microorganisms. Modern biotechnology by using a variety of in vivo and in vitro screening methods  
enables researchers to produce fully human antibodies against a specific target. Antibody engineering is applied  
in the therapeutic industry to improve the current therapeutic nature of antibodies so that they will be effective  
in the treatment of different diseases. Hybridoma technology, recombinant antibody fragments, Transgenic and  
phage technology, and improvement of effector functions are some of the technologies that are applied to  
antibodies to improve their therapeutic capability. Genetic engineering can be used to harness and to reformat  
individual antibody obtained from hybridoma, transgenic mouse, or phage selection. Currently, diseases that  
are global issues such as Cancer, HIV-AIDS, and many infectious bacterial and viral livestock diseases are being  
treated using this new technology. Relatively expensiveness to manufacture, the requirement of systemic  
administration, and specificity to a particular pathogen or serotype are among the challenges in the application  
of antibody therapeutics. Besides researches are ongoing to improve the efficacy of antibody, to reduce the cost  
of production and improving affinity and specificity, with considerable success. Therefore, further investigation  
is needed on antibody engineering to extend the application beyond therapy. Furthermore, the technology for  
the antibody production should be adopted in developing countries since it is less labor demanding and used to  
treat a wide range of infectious agents.  
Authors’ contributions  
All authors contributed equally to this review.  
Conflicts of interest  
The Authors declare no conflict of interest.  
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