Monoclonal Antibody: Production, Mechanisms, and Applications

This essay provides a comprehensive overview of monoclonal antibodies (MAbs), starting with their production through hybridoma technology, involving the fusion of B-cells and myeloma cells. It details the mechanisms of action, focusing on how MAbs target specific epitopes and discusses their role in immunotherapy, including examples like anti-VEGF, anti-EGFR, and anti-CD20 antibodies. The essay also explores the disadvantages of MAbs, such as specificity limitations and challenges related to antigen variation and antibody penetration into tissues. Furthermore, the essay examines the evolution and future of monoclonal antibody-based therapeutics.

Running head: MONOCLONAL ANTIBODY
MONOCLONAL ANTIBODY
Name of the Student
Name of University
Author’s note

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Answer 1
Each lymphocyte present in mammals is capable of producing a single antibody directed
towards a single epitope of antigenic determinant present over the antigen molecule. However,
an antigen contains several epitopes such that and if an individual is challenges with a single
antigen then each epitope will be recognised by single B-cells leading to its activation and
production of antibodies. This activated B-cells will then form a clone of cells with each clone
producing identical antibodies known as monoclonal antibodies (MAb) (Ecker, Jones and Levine
2015).
Monoclonal Antibodies were first invented by George Kohler and Cesar Milstein in the
year 1975. The technique for the production of the MAb is based on the fact that B-cell produces
antibody of specific specificity. Since normal B-cells are unable to grow for an indefinite period
of time it is important to immortalize the B-cells. This is done by cell fusion or somatic cell
hybridization between defective myeloma cells and normal antibody producing B-cell. This is
followed by a process of selection of the fused cells that is capable of secreting antibody of the
required epitope specificity derived from the normal B-cell (Weiner 2015). The fusion of the
normal B-cell with the defective myeloma cells provides the required genes to the normal cells in
order to multiply for an indefinite period to time. Thus hybridization between two somatic cells
under a specific selection medium leads to the production of a new cells line that is capable of
producing antibody and at the same time is immortal. In order words it can be said that the
antibodies generated form this newly hybridized cell lines has the ability to multiply rapidly and
for an indefinite period under in-vitro condition along with the production of antibodies of
predisposed specificity (Weiner 2015).

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Under the laboratory techniques, Swiss albino mice is charge with a specific antigen then
the serum of the mice is extracted and assayed at a specific concentration antibodies of desired
specificity at various intervals. When the concentration of the antibodies in the serum is found be
optimal, the animal is sacrificed. The spleen of the scarified animal, which contains a significant
number of plasma cells (antibody producing B-cells) is dissociated by mechanical or enzymatic
method into single cell culture. Once the spleenocytes are isolated from the animal, the B-cells
(HGPRT+ hypoxanthine – guanine phosphoionositol transferase][/TK+) is fused with myeloma
cells line with defective gene (HGPRT-/TK- [thymaidine kinase]) (Yokoyama et al. 2013).
During cell fusion under the action of polyethylene glycol (PEG), the plasma of the cell fuse
together enabling the cytoplasm and other organelle to intermingle. The fused cells undergo cell
division producing hybrid cell line with single nucleus and chromosomes from both the parents.
This hybrid cells proliferate to hybridoma cells which have immortality like myeloma cell ands
and capable of producing nomoclonal antibody like B-lymphocyte (Yokoyama et al. 2013).
After the completion of cell fusion, the cells are incubated in HAT (hypoxanthine
Aminopterin Thymidine) medium. The unfused myeloma cells die as they are HGPRT-/TK- and
unable to use the savage pathway. On the other hand, B-cell though HGPRT+/TK+ die
eventually because they have limited lifespan. The hybrid cells are selected and diluted in multi-
well plates in such a way that each well of the multi-well plate contains single cell with desired
antibody specificity (MAb) (Zola and Brooks 2018).
Answer 2
The molecular target of the antibodies is the epitopes. The immune cells or the B-cells
do not recognize the entire immunogen or antigen molecule. The lymphocytes instead recognize

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certain discrete sites on the antigenic marco-molecule known as antigenic determinants or
epitopes. Epitopes are defined as immunologically active region of the immunogen. It has the
capability to bind to the antigen-specific membrane receptors present on the lymphocytes or the
secreted antibodies. Numerous studies have highlighted that B-cells and T-cells recognise
different epitopes on the same antigenic molecule (Suzuki, Kato and Kato 2015).
Monoclonal antibody mainly works as immunotherapy. Immunotherapy is also known as
biological therapy. The main mechanism of the action of the mono-clonal antibodies is defined
by the production of the antigen with specific antigenic determinants. When the body’s immune
system detects foreign particles, it generates B-cell response. The activated B-lymphocytes in the
presence of the antigen (foreign body) lead to the production of the antibodies. The monoclonal
antibodies which are produced through the somatic cell hybridization mainly used as the targeted
therapy in order to block the harmful protein or the antigen produced from either the cancerous
cell line or due to the infusion of the microbial organisms (Bellanti 2013).
The efficacy of the monoclonal antibodies or the therapeutic antibodies stems from
numerous natural functions of the antibodies and there include neutralization, complement-
dependent cytotoxic activity (CDC) and antibody dependent cell mediated cyto-toxicity
(ADCC). In some times monoclonal antibodies are used for the targeted delivery of the certain
drugs (missile therapy). Some of the examples of the monoclonal antibodies and their
mechanisms of actions are highlighted below (Bellanti 2013).
Anti-VEGF antibody
Bevacizumab is an anti-VEGF jumanized monoclonal antibody. This monoclonal
antibody binds to VEGF and blocks VEGF from binding with its respective receptors (VEGFR-

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1/2) which then blocks the downstream signal transduction of VEGF and thereby inhibiting
angiogenesis. VEGF is the principal co-factor that controls the process of angiogenesis and the
expression is increased in the majority of the human tumours during proliferation process. Thus
blocking of the VEGF by monoclonal antibody (Bevacizumab) helps in the effective treatment of
the colorectal cancer, non-small cell lung cancer and breast cancer (Chinot et al. 2014).
Anti-EGFR antibody
Cetuximab is a recombinant human/mouse chimeric anti-EGFR monoclonal antibody. It
binds t EGFR (Epidermal growth factor receptor) selectively and thereby preventing the binding
of EGFR with its ligand EGF (epidermal growth factor) and thereby helping to block the
downstream signal transduction. EGFR is defined as a transmembrane glycoprotein and is
mainly expressed in the epithelial cells or tissues and acts as receptor for EGF. Binding of EGFR
with EGF induces the mechanism of the receptor dimerization followed by autophosphorylation
and thereby promoting cell proliferation and subsequent differentiation. Thus, binding of
Cetuximab helps in inhibiting the proliferation of the tumorous epithelial cells under the action
of the EGFR (Van Cutsem et al. 2015).
Anti-CD20 antibody
Rituximab is a monoclonal antibody. It is targeted against the pan-B-cell marker CD-20.
It binds to the B-cell expressing CD20 and prmotes cell death by CDC or ADCC. CD20 is
mainly expressed in the non-neoplastic B-cells (immature, premature or activated) and neoplastic
cells derived from B-cells. Rituximab is used for the treatment of the non-Hodkin’s lymphoma,
rheumatoid arthritis and chronic lymhocytic leukemia (Furman et al. 2014).

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Suzuki, Kato and Kato (2015), stated that the changes induced by the monoclonal
antibody is mainly thought of as biological reactions dependent on the target molecule. For
example, it can be said that with the help of the blocking antibody the changes occurs in the
tissues and organs which is targeted pathways. With antibodies that are specific for the ligands,
changes occur in the organs and the tissues that can express the receptor of the targeted ligands.
With the antibodies that are target specific receptors, the desired set of changes are mainly
expressed in the organs or the tissue system that expressed the targeted receptor molecules. The
cytotoxic monoclonal antibody the changes are identified in the tissues or the organs expressing
that particular target molecule (Suzuki, Kato and Kato 2015).
Answer 3
The main disadvantage of the monoclonal antibodies include monoclonal antibodies are
specific to the antigenic determinants and thus their application are specific. Any change in the
antigenic determinants like antigenic drift or shift (change in the epitopes of the antigen), mainly
common among the bacterial cells leads to non-functioning of the monoclonal antibodies.
Moreover, monoclonal antibody are not suitable for using in the assays like the hemagglutination
involving the antigen cross-linking. Any minor modifications and affect the binding site of the
antibody leading to the inactivation of the monoclonal antibody (Sela-Culang, Kunik and Ofran
2013). Yokoyama et al. (2013) reported that though these shortcomings can be overcome by
increasing the pool of the multiple monoclonal antibodies of different specificities. However,
pool of a large pool of antibody of different specificities are expensive, laborious and also time
consuming. Liu (2014) stated that MAb directed against the tumour specific antigens mainly
remains within the blood circulation and only 20% or less amount of the administered dosage
typically reacts with the tumour. This is probably regarded as one of the major limitations of the

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MAb therapy. The uptake of the antibody by tumour mainly depends on the subtle balance
between the favourable pharmacokinetics along with retention and penetration inside the target
tissue. The uptake of the antibody is also guided by the size, shape, affinity and valency controls
of the MAb. MAbs are large molecules with limited serum half-life and also exceeds the
threshold of the renal clearance. This prevents them from being eliminated through the glomeruli
of the kidneys. The Fc portion of the IgG molecules interact with receptors expressed on the cell
surface molecules and thereby increasing tenure for which MAbs stay in the blood and thereby
preventing tissue penetration (Liu 2014).

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References
Bellanti, J. ed., 2013. Immunology (Vol. 6). Springer Science & Business Media.
Chinot, O.L., Wick, W., Mason, W., Henriksson, R., Saran, F., Nishikawa, R., Carpentier, A.F.,
Hoang-Xuan, K., Kavan, P., Cernea, D. and Brandes, A.A., 2014. Bevacizumab plus
radiotherapy–temozolomide for newly diagnosed glioblastoma. New England Journal of
Medicine, 370(8), pp.709-722.
Ecker, D.M., Jones, S.D. and Levine, H.L., 2015, January. The therapeutic monoclonal antibody
market. In MAbs (Vol. 7, No. 1, pp. 9-14). Taylor & Francis.
Furman, R.R., Sharman, J.P., Coutre, S.E., Cheson, B.D., Pagel, J.M., Hillmen, P., Barrientos,
J.C., Zelenetz, A.D., Kipps, T.J., Flinn, I. and Ghia, P., 2014. Idelalisib and rituximab in relapsed
chronic lymphocytic leukemia. New England Journal of Medicine, 370(11), pp.997-1007.
Liu, J.K., 2014. The history of monoclonal antibody development–progress, remaining
challenges and future innovations. Annals of Medicine and Surgery, 3(4), pp.113-116.
Sela-Culang, I., Kunik, V. and Ofran, Y., 2013. The structural basis of antibody-antigen
recognition. Frontiers in immunology, 4, p.302.
Suzuki, M., Kato, C. and Kato, A., 2015. Therapeutic antibodies: their mechanisms of action and
the pathological findings they induce in toxicity studies. Journal of toxicologic pathology, 28(3),
pp.133-139.
Van Cutsem, E., Lenz, H.J., Köhne, C.H., Heinemann, V., Tejpar, S., Melezínek, I., Beier, F.,
Stroh, C., Rougier, P., van Krieken, J.H. and Ciardiello, F., 2015. Fluorouracil, leucovorin, and

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irinotecan plus cetuximab treatment and RAS mutations in colorectal cancer. Journal of clinical
oncology, 33(7), pp.692-700.
Weiner, G.J., 2015. Building better monoclonal antibody-based therapeutics. Nature Reviews
Cancer, 15(6), p.361.
Yokoyama, W.M., Christensen, M., Dos Santos, G., Miller, D., Ho, J., Wu, T., Dziegelewski, M.
and Neethling, F.A., 2013. Production of monoclonal antibodies. Current protocols in
immunology, 102(1), pp.2-5.
Yokoyama, W.M., Christensen, M., Dos Santos, G., Miller, D., Ho, J., Wu, T., Dziegelewski, M.
and Neethling, F.A., 2013. Production of monoclonal antibodies. Current protocols in
immunology, 102(1), pp.2-5.
Zola, H. and Brooks, D., 2018. Techniques for the production and characterization of
monoclonal hybridoma antibodies. In Monoclonal Hybridoma Antibodies (pp. 1-57). CRC Press.