04/03/2023
Monoclonal Antibodies
SAI HS . BODDU
PROFESSOR OF PHARMACE UTI CS
A JMAN UNIVE RS ITY
https://www.coursera.org/videos/immunologyfundamentalsimmunitybcells/KxBvo
Introduction
▪Vertebrates inevitably die of infection if they are unable to make antibodies.
▪Antibodies defend us against infection by binding to viruses and microbial
toxins, thereby inactivating them
▪Binding of antibodies to invading pathogens also recruits various types of
white blood cells and a system of blood proteins, collectively
called complement
▪White blood cells and activated
complement components work
together to attack the invaders.
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Introduction
▪Synthesized exclusively by B cells, antibodies are produced in billions of forms, each with a
different amino acid sequence and a different antigen-binding site.
▪Collectively called immunoglobulins (abbreviated as Ig)
▪Ig is the most abundant protein components in the blood, constituting about 20% of the total
protein in plasma by weight.
▪Mammals make five classes of antibodies, each of which mediates a characteristic biological
response following antigen binding (IgG, IgM, IgA, IgD, and IgE)
▪Ig is a large, Y-shaped protein used by the immune system to identify
and neutralize foreign objects such as pathogenic bacteria and viruses.
▪Antibody recognizes a unique molecule of the pathogen, called an antigen.
Monoclonal Antibody
▪Can be defined as an identical mono-specific antibody produced by a
single immune cell and consists of a single type of immunoglobulin
that is directed against a specific antigen.
▪Immune cells are lymphocytes (B-cells & T-cells).
▪They are formed in the bone marrow & the thymus gland.
▪B-cells produce antibodies to fight bacterial infection.
▪T-cells produce antibodies to fight viral infections.
▪Both B-&T-cells sometimes inhibit tumor by producing antibodies that
may attack cancer cells
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Production of monoclonal antibodies
The production of monoclonal antibodies using hybridoma technology
▪Hybridoma technology was pioneered in 1975 by George Köhler and Cesar Milstein, who have
been awarded Nobel prize for their invention in 1983.
▪Hybridoma technology is one of the most common methods used to produce monoclonal
antibodies.
▪In hybridoma technology, antibody-producing B cells were fused with immortal cancerous cell lines
such as myeloma cells creating an immortal hybrid cell line that produces antibodies limitlessly.
▪Hybridoma cells are cultured in a lab to produce monoclonal antibodies, against a specific antigen.
▪This can be achieved by an in vivo or an in vitro method.
Production of monoclonal antibodies
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Production of monoclonal antibodies
▪Use of hybridoma produces monoclonal antibodies of high purity and are highly
sensitive and specific
▪An animal usually a mouse is repeatedly immunized by injecting an appropriate
antigen which could be a bacteria.
▪B-lymphocytes are isolated from the mouse spleen.
▪Myeloma Cells (abnormal rapidly dividing WBC formed in the bone marrow) are
cultured in a medium containing 8-azaguanine to inhibit hypoxanthine-guanine-
phospho-ribosyl-transferase enzyme (HGPRT), i.e. myeloma cells become HGPRT
deficient.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8521504/
Steps involved in production of
monoclonal antibodies
▪B-lymphocytes are then mixed with myeloma cells and grown in a culture containing poly
ethylene glycol (PEG), where the two cells fuse to form hybrid cells called hybridomas.
▪Hybridomas maintained the character of lymphocytes to secret antibodies, and of myeloma
cells to multiply in culture & produce unlimited supply of antibodies.
▪To separate hybridomas from other un-fused cells i.e. free myeloma cells, and free lymphocytes,
the mixture of cells is cultured in HAT medium containing Hypoxanthine, Aminopterin &
Thymidine for 7-10 days
▪Hypoxanthine & thymidine can be used by free lymphocytes to make Deoxyguanosine
triphosphate (dGTP) & Deoxythymidine triphosphate (dTTP) respectively, as they have HGPRT
enzyme, but still free lymphocytes having a short life span, die in culture medium within 7-10
days.
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Steps involved in production of
monoclonal antibodies
▪Free myeloma cells are HGPRT deficient, so they can not use hypoxanthine & thymidine to make
dGTP & dTTP.
▪In addition, aminopetrin blocks the synthesis of tetrahydrofolate which is a nucleotide precursor
that inhibits DNA, RNA, and protein synthesis & thus free myeloma cells will die.
▪The hybridoma cells can survive by using hypoxanthine & thymidine to make dGTP & dTTP, as
they have HGPRT enzyme (B-lymphocytes contribution) & can multiply in culture to produce
unlimited supply of antibodies (myeloma cells contribution).
▪Thus hybridoma are selected by using HAT medium.
▪Suspension of hybridomas is diluted & can be cultured in a soft agar culture medium, to multiply
& produce the desired antibodies.
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hypoxanthine-guanine-
phospho-ribosyl-
transferase enzyme
Hypoxanthine,
Aminopterin &
Thymidine
Steps involved in production of
monoclonal antibodies
▪These cell lines can also be cryopreserved for a long period of time.
▪Hybridoma technology has resulted in the production of a variety of different
monoclonal antibodies with specificity for a specific antigen.
▪Antigen molecules include enzymes, hormones, internal and external structures of
bacteria, viruses, and eukaryotic cells.
▪Monoclonal antibodies produced by this method are highly specific antibodies, which
are derived from a single parental B cell clone
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Types of Monoclonal Antibodies
There are four types of MAB, depending on the proteins they are made of:
▪Murine: made of mouse proteins & the name of the product either starts with Muro- or ends in
-omab.
▪Chimeric: made of a combination of an equal parts of mouse & human proteins & the name of
the product ends in -ximab.
▪Humanized: made of small parts of mouse proteins with large parts of human proteins & the
name of the product ends in -zumab.
▪Human: made of full human proteins & the name of the product ends in -umab.
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Application of monoclonal antibodies
▪Immunotherapy of cancer: FDA (US) approved more than 25 mAb approved for the treatment of
various type of cancer such as rituximab, cetuximab, pertuzumab etc.
▪Extensively used in disease diagnosis and testing kit such as pregnancy etc.
▪Used in vaccine production
▪Used in protein purification and other biomolecules
▪Drug targeting
▪Used as enzyme (abzyme) and many more.
antibody linked with enzyme
Some examples of monoclonal antibody
therapies
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Some examples of monoclonal antibody
therapies
More examples
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