Therapeutic monoclonal

antibodies

Dr. Md. Mahbubur Rahman

Department of Pharmaceutical Sciences
North South University
 Antibody
➢ An antibody (Ab), also known as an immunoglobulin (Ig) is a
large protein produced mainly by plasma cells that is used by the
immune system to neutralize antigen such as pathogenic bacteria
and viruses.
➢ The antibody recognizes a unique molecule of the pathogen,
called an antigen, via the fragment antigen-binding (Fab) variable
region.
➢ It is an Y shaped molecule.
➢ Each tip of the "Y" of an antibody contains a paratope (analogous
to a lock) that is specific for one particular epitope (analogous to
a key) on an antigen,
➢ Paratope and epitope allows antibody and antigen binding with
specificity and precision.
Structure of antibody
 Structure of antibody
➢ There are 4 polypeptide chains: 2 identical heavy chains and 2
identical light chains connected by disulfide bonds.
➢ Light chain consists polypeptides of around 22 kDa and heavy chain
consists larger polypeptides of around 50 kDa or more.
➢ There are five types of Ig heavy chain (in mammal) denoted by the
Greek letters: α, δ, ε, γ, and μ.
➢ There are two types of Ig light chain (in mammal), which are called
lambda (λ) and kappa (κ).
➢ Region that changes to various structures depending on differences
in antigens is called the variable region, and the other part that does
not is called the constant region.
➢ The fragment crystallizable region (Fc region) is the tail region of an
antibody that interacts with cell surface receptors called Fc receptors
and some proteins of the complement system.
Classification of antibody
 Immunological functions of antibody
➢ IgG provides long term protection (for months and years) after the
antigen that has triggered their production.
➢ IgG protect against bacteria, viruses, neutralize bacterial toxins,
trigger complement protein systems and bind antigens to enhance
the effectiveness of phagocytosis.
➢ Main function of IgA is to bind antigens on microbes before they
invade tissues.
➢ IgA are also first defense for mucosal surfaces such as the
intestines, nose, and lungs.
➢ IgM enhance ingestions of cells by phagocytosis.
➢ IgE bind to mast cells and basophils which participate in the
immune response.
➢ IgD is present on the surface of B cells and plays a role in the
induction of antibody production.
 More about IgG
➢ Immunoglobulin G (IgG) is the most common type of antibody
representing approximately 75% of serum antibodies in humans.
➢ IgG molecules are created and released by plasma B cells.
➢ Each IgG antibody has two paratopes.
➢ By binding many kinds of pathogens such as viruses, bacteria,
and fungi, IgG protects the body from infection.

Complement
Name Percentage Half life
activator
IgG1 66% second-highest 21 days
IgG2 23% third-highest 21 days
IgG3 7% highest 7 days
IgG4 4% no 21 days
 Cluster of differentiation
➢ The cluster of differentiation (also known as classification
determinant, CD) is a protocol used for the identification and
investigation of cell surface molecules.

➢ The CD system is commonly used for defining cells based on the

molecules present on their surface.

➢ CD molecules can act in numerous ways including receptors and

ligands.

➢ Two commonly used CD molecules are CD4 and CD8, which are,
in general, used as markers for helper and cytotoxic T cells,
respectively.

➢ Both CD4 and CD8 are glycoprotein.

 Polyclonal Antibody
➢ Polyclonal antibodies (pAbs) are antibodies that are secreted by
different B cell lineages within the body.
➢ They are a collection of immunoglobulin molecules each of which
reacts against a specific antigen, and thus identifying different
epitopes.
➢ These antibodies are typically produced by inoculation of a
suitable mammal, such as a mouse, rabbit or goat.
➢ Larger mammals are often preferred as the amount of serum that
can be collected is greater.
➢ The animal must be in good health, free of infections and kept
under veterinary supervision.
➢ To prepare an immune serum, horses or other animals are injected
with a sequence of spaced doses of an antigen.
 Production of polyclonal antibody
➢ This induces the production of IgG by B-lymphocytes against the
antigen injected.
➢ Injection is continued until the blood sample shows that the
injections have induced a high titre of antibody to the injected
antigen.
➢ An adjuvant may be used if required.
➢ This polyclonal IgG is purified from the mammal’s serum.
➢ The blood cells are allowed to settle and the supernatant plasma
is drawn off. Alternatively, the blood can be mechanically
defibrinated.
➢ By contrast, monoclonal antibodies are derived from a single cell
line.
Production of polyclonal antibody
 Monoclonal Antibodies
➢ Monoclonal antibodies (mAb) are monospecific antibodies that are
made by identical immune cells that are all clones of a unique
parent cell, in contrast to polyclonal antibodies, which are made
from several different immune cells.
➢ Monoclonal antibodies have monovalent affinity, in that they bind to
the same epitope (the part of an antigen that is recognized by the
antibody).
➢ Given almost any substance, it is possible to produce monoclonal
antibodies that specifically bind to that substance; they can then
serve to detect or purify that substance.
➢ This has become an important tool in immunology, molecular
biology and medicine.
 Monoclonal Antibodies
➢ A monoclonal antibody (MoAb) is an antibody made by cloning a
unique white blood cell.
➢ All subsequent antibodies derived this way trace back to a unique
parent cell.
➢ Monoclonal antibodies can have monovalent affinity, binding only
to the same part of an antigen that is recognized by the antibody.
➢ In contrast, polyclonal antibodies bind to multiple epitopes and are
usually made by several different antibody secreting plasma cells.
 Production of monoclonal antibodies
Spleen B cells are obtained from an Myeloma cells are cultured
animal immunized with an antigen separately

Hybridoma cells are produced

Culture in selection media: Only hybridoma cells grow and the myeloma
cells die due to the lack of enzyme

Isolation of clones derived from single hybridoma cells

Screening of supernatant of each clone for the desired antibody

Production of monoclonal antibodies
Production of monoclonal antibodies
 Myeloma Cell Lines
➢ Multiple myeloma cells or plasmacytoma cells are abnormal
plasma cells (a type of WBC) that build up in the bone marrow and
form tumors in many bones of the body.
➢ They can be generated experimentally in mice and rats (the Balb/c
mouse strain being particularly susceptible).
➢ It is very difficult to generate myeloma cells secreting antibody of
predetermined specificity.
➢ Normal plasma cells make antibodies to help the body fight
infection and disease.
➢ As the number of multiple myeloma cells increases, more
antibodies are made.
 Recombinant monoclonal antibodies (rAbs)

➢ rAbs are constructed in vitro, outside the constraints of the

immune system, using recombinant DNA technologies.
➢ The antibody genes are isolated and then incorporated into
plasmid DNA vectors.
➢ The resulting plasmids are transfected into expression hosts such
as bacteria, yeast, or mammalian cell lines.
➢ For recombinant antibody production the stable cell lines such as
Chinese hamster overy (CHO) cells and human embryonic kidney
(HEK293) are most frequently used.
➢ Once the cell culture produces the desired recombinant antibody,
it is regularly collected, purified and analyzed or used for further
experimentation.
 Advantages of rAbs compared to mAbs

➢ Producing rAbs is cheaper than generating mAbs.

➢ Indeed, rAbs required less purified antigen to produce than
classical mAbs.
➢ Moreover, the production time is shorter (weeks vs. months).
➢ Mass production of rAbs does not required the use of animals
(overcomes ethical concerns).
➢ rAbs can be produced in several formats like Fab fragments,
single-chain variable region fragments (ScFv), diabodies (Dimeric
ScFvs).
➢ rAbs can be produced in several hosts ranging from bacteria to
human cells.
➢ We can switch the antibodies of species (from mouse to human),
of classes (from IgG to IgE).
Generic name of therapeutic antibodies
➢ The names of the antibodies are given based on their origin and their target.
➢ Each mAb generated follows a rule determined by the WHO International
Nonproprietary Names (INN) system of recombinant monoclonal antibodies.

Substem A = Target* Substem B = Source¶

Prefix Suffix
Substem Definition Substem Definition
-ami Amyloid protein -a Rat
-ba Bacterial protein -axo Rat-mouse
-ci Cardiovascular -e Hamster
-fung Fungal protein -i Primate
-gros Skeletal muscle -o Mouse
-ki Interleukin -u Human
Random and -li Immuno-modulating -xi Chimeric -mab
distinctive
-os Bone -zu Humanized
-toxa Toxin
-ta Tumor antigen
-vi Viral antigen
 Therapeutic monoclonal antibodies
➢ In 1975, the first monoclonal antibody was produced.
➢ In 1986, the first mAb, muromonab-CD3, was approved for treating
steroid-resistant acute allograft rejection in renal transplant
recipients.
➢ A majority of mAb therapeutics have been approved for oncology,
rheumatoid and autoimmune diseases.
➢ The first fully human mAb (Adalimumab) was approved for treating
rheumatoid arthritis in 2002.
➢ Around the world, at least 600 therapeutic mAbs have been
studied in clinical trials.
➢ Till May 2021, 100 therapeutic mAbs have been approved by the
US FDA and are currently in the market, including 30 mAbs for the
treatment of cancer.
 Top therapeutic monoclonal antibody brands

Product MoAb Company Revenue

Humira Adalimumab AbbVie Inc., USA 20 B USD
Keytruda Pembrolizumab Merck & Co., Inc., USA 7.2 B USD
Herceptin Trastuzumab Hoffmann-La Roche, Switzerland 7.0 B USD
Opdivo Nivolumab Bristol-Myers Squibb, USA 7.6 B USD
Rituxan Rituximab Hoffmann-La Roche, Switzerland 6.8 B USD
Remicad Infliximab Johnson & Johnson, USA 5.9 B USD
Avastin Bivacizumab Hoffmann-La Roche, Switzerland 6.7 B USD
Stelara Ustekinumab Johnson & Johnson, USA 5.2 B USD
 Types of therapeutic MAbs

Ian N. Foltz. Circulation. Evolution and Emergence of Therapeutic Monoclonal

Antibodies, Volume: 127, Issue: 22, Pages: 2222-2230
 Murine monoclonal antibody
➢ Murine monoclonal antibodies were the first antibodies ever to be
produced (in 1975) at lab-scale by the hybridoma technology.
➢ Now-a-days, a very few of these antibodies are in use for therapy
due to their low compatibility with the human immune system.
➢ These antibodies are still in high demand for analytical applications.
➢ But, for therapeutic applications, these antibodies serve mostly as
the framework for antibody development and engineering
techniques consisting in antibody chimerization, humanization and
bispecific antibody development from antibody fragments.
➢ This process generates murine antibodies that serve as
frameworks for further antibody humanization processes.
➢ Patients treated with native murine antibodies develop an allergic
reaction termed as human anti-murine antibody (HAMA) response.
 Chimeric monoclonal antibody
➢ Chimeric antibodies consist of an antibody’s original antigen-
binding variable domains with the constant domains from a
different species.
➢ Chimeric antibodies retain the original antibody’s antigen
specificity and affinity and are valuable tools for in vivo and in
vitro research, as well as diagnostic assay development.
➢ Many therapeutic monoclonal antibodies were originally generated
in mice and chimerized to reduce immunogenicity in humans,
before humanization was widely adopted.
➢ Examples of chimeric antibodies in the clinic include infliximab,
rituximab and abciximab.
➢ Chimeric antibodies containing human constant domains and
mouse variable domains are substantially cheaper than fully
humanized antibodies.
 Humanized monoclonal antibody
➢ Humanized antibodies are antibodies from non-human species
whose protein sequences have been modified to increase their
similarity to antibody produced naturally in humans.
➢ The process of "humanization" is usually applied to monoclonal
antibodies developed for administration to humans.
➢ Humanization may be necessary when the process of developing a
specific antibody involves generation in a non-human immune
system (such as that in mice).
➢ The International Nonproprietary Names of humanized antibodies
end in –zumab.
➢ Humanized antibodies are distinct from chimeric antibodies.
➢ The latter also have their protein sequences made more similar to
human antibodies, but carry a larger stretch of non-human protein.
 Chimeric vs humanized antibody
Chimeric antibody
A chimeric antibody is an antibody
which has its original antigen-binding
variable domain with constant domain
from a different species.
Produced by fusing mouse derived
variable regions to human constant
regions
Carries a larger stretch of non-human
proteins
Possesses higher risk of inducing
immune response in human
Cheaper than fully humanized
antibodies
Higher research uses less therapeutic
uses
Humanized antibody
Humanized antibody is an antibody from
non-human animal whose protein
sequences are modified to increase
their similarity to antibody variants
produced naturally in humans.
Produced by post-translational
modification
Carries a shorter stretch of non-human
proteins
Possesses lower risk of inducing
immune response in human
Costly than chimeric antibodies
Higher therapeutic uses than chimeric
 Monoclonal antibodies for cancer treatments
➢ Monoclonal antibody-based immunotherapy is now considered to
be one of the main strategy of cancer therapy, alongside surgery,
radiation, and chemotherapy.
➢ Some monoclonal antibody drugs may be used in combination
with other treatments, such as chemotherapy or hormone therapy.
➢ The main mechanism by which many antibodies induce tumor cell
death is the blockade of growth factor receptor signaling.
➢ Programmed cell death protein 1 (PD-1) is the most important
target of MoAbs for cancer treatment.
 How do monoclonal antibodies fight
against cancer?
➢ Flagging cancer cells: Cancer cells that are coated in
monoclonal antibodies may be more easily detected and targeted
for destruction.
➢ Triggering cell-membrane destruction: Some monoclonal
antibodies can trigger immune response that can destroy the
membrane of a cancer cell.
➢ Blocking cell growth: Some monoclonal antibodies block the
connection between a cancer cell and proteins that promote cell
growth: activity that is necessary for cancer growth and survival.
➢ Preventing blood vessel growth: In order for a cancerous tumor
to grow and survive, it needs a blood supply. Some monoclonal
antibody drugs block protein-cell interactions necessary for the
development of new blood vessels.
 How do monoclonal antibodies fight
against cancer?
➢ Blocking immune system inhibitors: Our body controls the
immune system from being overactive by some proteins.
Monoclonal antibodies can interfere that process and our immune
system cells can work against cancer cells without control.
➢ Directly attacking cancer cells: Certain monoclonal antibodies
can attack the cell directly which causes the activation of a series
of events inside the cell that may cause it to self-destruct.
➢ Delivering chemotherapy: Some monoclonal antibodies are
combined with a chemotherapy drug in order to deliver the
treatment directly to the cancer cells affecting healthy cells.
➢ Binding cancer and immune cells: Some drugs combine two
monoclonal antibodies, one that attaches to a cancer cell and one
that attaches to a specific immune system cell. This connection
may promote immune system attacks on the cancer cells.
 Rituximab
➢ Some monoclonal antibodies mark cancer cells so that the immune
system will better recognize and destroy them.
➢ An example is rituximab, which binds to a protein called CD20 on B
cells and some types of cancer cells, causing the immune system to
kill them.
➢ CD20 is a surface antigen present on B cells.
➢ Interaction of rituximab with CD20 receptor causes 3 responses:
➢ (1) antibody dependent cellular cytotoxicity (ADCC)
➢ (2) complement mediated cytotoxicity (CMC), and
➢ (3) apoptosis; subset panel illustrates a schematic view of CD20
structure and rituximab.
➢ Rituximab acts by depleting normal as well as pathogenic B cells
while sparing plasma cells and hematopoietic stem cells as they do
not express the CD20 surface antigen.
 Trastuxumab
➢ Trastuzumab, (Brand name: Herceptin) is a monoclonal antibody
used to treat breast cancer and stomach cancer.
➢ It is specifically used for cancer that is HER2 (human epidermal
growth factor receptor) receptor positive.
➢ The HER2 pathway promotes cell growth and division when it is
functioning normally.
➢ By the same token, when HER2 is overexpressed, cell growth
accelerates beyond its normal limits.
➢ Trastuzumab may be used alone or together with other
chemotherapy medication.
➢ One of the more serious complications of trastuzumab is its effect
on the heart, although this is rare.
➢ In 2-7% of cases, trastuzumab is associated with cardiac
dysfunction, which includes congestive heart failure.
 Bevacizumab
➢ Bevacizumab (brand name Avastin) is a medication used to treat
several types of cancers.
➢ For example it is given by slow IV injection in colon cancer, lung
cancer, and renal-cell carcinoma.
➢ In many of these diseases it is used as a first-line therapy.
➢ It is also used for the treatment of age-related macular
degeneration (ARMD) and diabetic retinopathy.
➢ Bevacizumab is a monoclonal antibody that functions as an
angiogenesis inhibitor.
➢ It works by slowing the growth of new blood vessels by inhibiting
vascular endothelial growth factor A (VEGF-A).
➢ VEGF-A is a growth factor protein that stimulates angiogenesis in a
variety of diseases, especially in cancer.
 MoAbs for cancer treatments

MoAb Brand Company Market size Indication

name (2018)

Rituximab Rituxan Roche $ 6.8 Billion Non-Hodgkin’s

lymphoma
Bevacizumab Avastin Roche $ 6.8 Billion Colorectal carncer
Lung cancer,
Breast cancer
Trastuzumab Herceptin Roche $ 7.0 Billion Breast cancer
Stomach cancer
Cetuximab Erbytux Ely Lilly Colorectal cancer
and Co. Head and neck
cancer
Nivolumab Opdivo Bristol $ 7.6 Billion Lung cancer
Myers Renal cell carcinoma
Squibb
Panitumumab Vectibix Amgen Colorectal cancer
 Treatment of autoimmune and rheumatic
diseases by MoAbs
➢ An autoimmune disease is a condition arising from an abnormal
immune response to a functioning body part.
➢ There are at least 100 types of autoimmune diseases.
➢ Some common autoimmune diseases are celiac disease, diabetes
mellitus type 1, Graves' disease, inflammatory bowel disease,
multiple sclerosis, psoriasis, and rheumatoid arthritis.
➢ NSAIDs and immuno-suppressants are often used.
➢ MoAbs are the latest addition to this class.
➢ About 24 million (~7.5%) people in the US are affected.
➢ Women are more commonly affected than men.
➢ Having an autoimmune disease increases the risk or likelihood of
developing certain cancers.
 Adalimumab: A magical solution for
autoimmune and rheumatic diseases
➢ Adalimumab, is a disease-modifying anti-rheumatic drug and
monoclonal antibody that works by inactivating tumor necrosis
factor-alpha (TNFα).
➢ It is a medication used to treat rheumatoid arthritis, psoriatic
arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis,
psoriasis, uveitis, and juvenile idiopathic arthritis.
➢ Use is generally only recommended in people who have not
responded to other treatments.
➢ It is administered by injection under the skin.
➢ Adalimumab was approved for medical use in USA in 2002.
➢ It is on the World Health Organization's List of Essential Medicines.
➢ It is available as a biosimilar medication.
 Mechanism of action of TNFα inhibitors
➢ Activated T-cells & macrophages produce several proinflammatory
cytokines including tumor necrosis factor α (TNFα).
➢ TNFα is a naturally occurring cytokine that is involved in normal
inflammatory and immune responses.
➢ High level of TNFα is found in the synovial fluid of rheumatoid
arthritis patients.
➢ TNFα plays an important role in both the pathologic inflammation
and the joint destruction that are hallmarks of rheumatoid arthritis.
➢ The drugs infliximab and adalimumab bind to TNFα.
➢ Thus adalimumab and infliximab block the interaction of TNFα
with the p55 and p75 cell surface TNFα receptors.
➢ Therefore, these so-called TNFα inhibitors can be used as anti-
inflammatory medications.
Mechanism of action of TNFα inhibitors
Adalimumab
Etanercept
Infliximab
 Mechanism of action of IL6R inhibitors

➢ Interleukin 6 (IL-6) is involved in the development of

immunological and inflammatory reactions.
➢ Some autoimmune diseases like rheumatoid arthritis are
associated with abnormally high IL-6 levels.
➢ Tocilizumab and sarilumab binds to soluble as well as membrane
bound interleukin-6 receptors.
➢ Thus tocilizumab hinders IL-6 from exerting its pro-inflammatory
effects.
➢ It has been reported that the soluble form of the IL-6 receptor
contributes more in the pathogenesis of rheumatoid arthritis than
the membrane bound form.
 Monoclonal antibodies for Alzheimer’s
disease treatment
➢ Amyloid beta (Aβ) are peptides of 36–43 amino acids that are the
main component of the amyloid plaques found in the brains of
people with Alzheimer's disease.
➢ Amyloid beta plaques accumulate and damage synapses in the
brain, preventing neurons from signaling each other, and ultimately
killing them.
➢ This loss of signaling corresponds to a loss of neural function, most
commonly memory loss.
➢ New research also indicates that amyloid beta may damage
synapses before plaques are detected.
➢ How this protein destroys neurons is still unclear but it is known to
be the primary factor in initiating Alzheimer’s disease.
 Aducanumab
➢ Aduhelm (aducanumab) is a human, immunoglobulin gamma 1
(IgG1) monoclonal antibody directed against aggregated soluble
and insoluble forms of amyloid beta.
➢ The accumulation of amyloid beta plaques in the brain is a defining
pathophysiological feature of Alzheimer's disease.
 Monoclonal antibodies for the
treatment of Alzheimer’s disease

Generic Type Target Use

Name
Aducanumab Humanized IgG1 Beta-amyloid Alzheimer’s disease
plaques
Bapineuzumab Humanized IgG1 Beta-amyloid Alzheimer’s disease
(Aβ) and glaucoma
Solanezumab Humanized IgG1 Beta-amyloid Alzheimer’s disease
(Aβ) Down’s syndrome
Gantenerumab Human IgG1 Beta-amyloid Investigational
(Aβ)

Common side effects: Dizziness, brain swelling, vomiting,

confusion, tremor.
 Monoclonal antibodies recently approved
for the treatment of covid-19
Generic Name Target Company Status

Itolizumab CD6 Biocon In July 2020, Biocon received

authorization in India for its use
in the treatment of COVID-19
Tocilizumab IL-6 Roche It was granted an emergency
use authorization (EUA) for the
treatment of COVID-19 in the
United States in June 2021
Casirivimab/ Spike protein Regeneron In November 2020, FDA issued
imdevimab of SARS-CoV-2 an EUA for casirivimab and
imdevimab together for the
treatment of COVID-19
Sotrovimab Spike protein GSK In May 2021, FDA issued an
of SARS-CoV-2 EUA for sotrovimab for the
treatment of COVID-19
 Further reading
•https://www.alliedmarketresearch.com/monoclonal-antibodies-market-A11789
•https://jbiomedsci.biomedcentral.com/articles/10.1186/s12929-019-0592-z
•https://www.fortunebusinessinsights.com/monoclonal-antibody-therapy-market-
102734
•https://www.accessdata.fda.gov/drugsatfda_docs/label/2002/adalabb123102LB.htm
•https://www.uptodate.com/contents/overview-of-therapeutic-monoclonal-antibodies
•https://www.proteogenix.science/scientific-corner/antibody-production/fully-human-
monoclonal-antibody-discovery-technologies-and-trends/
•https://www.researchgate.net/publication/338343963_Development_of_therapeutic_a
ntibodies_for_the_treatment_of_diseases/figures?lo=1
•https://www.cancer.org/treatment/treatments-and-side-effects/treatment-
types/immunotherapy/monoclonal-antibodies.html
•https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4622599/