Monoclonal Antibodies

Miss Afshan Arshad

What is antibodies
An antibody is a protein used by the immune system to identify and neutralize foreign objects like
bacteria and viruses. Each antibody recognizes a specific antigen unique to its target.

Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by
one type of immune cell, all clones of a single parent cell.

Polyclonal antibodies are antibodies that are derived from different cell lines.

Isotypes
According to differences in their heavy chain constant domains, immunoglobulins are grouped
into five classes, or isotypes: IgG, IgA, IgM, IgD, and IgE.
IgG: IgG1 (66%), IgG2 (23%), IgG3 (7%) and IgG4 (4%) , blood and tissue liquid.
IgA:IgA1 (90%) and IgA2 (10%), stomach and intestines
IgM: normally pentamer, ocassionally hexamer, multiple immunoglobins linked with
disulfide bonds
IgD:1% of proteins in the plasma membranes of B-lymphocytes, function unknown
IgE: on the surface of plasma membrane of mast cells, play a role in immediate
hypersensitive and denfensive for parasite
Antibodies
• Antibodies bind other molecules strongly and specifically and are therefore
useful as reagents in research, diagnosis and therapy. Antibodies taken from the
blood of immunized animals are a mixture of different antibodies produced by
different cells. Monoclonal antibodies are antibodies with a unique specificity,
generally made by cloning cells containing a particular antibody gene set to
produce population of identical cells, derived from a single cell, which all
produce the same antibody. Monoclonal anti-bodies are therefore much more
specific than polyclonal antibodies. Monoclonal antibodies can be made in cell
culture and are therefore also more reproducible from batch to batch than
polyclonal antibodies. Mono clonal antibodies have become the preferred
reagents in many research and diagnostic applications and are increasingly used
in therapy of cancer and immunological disorders.
Structure Of Antibody
What is a monoclonal antibody
• A monoclonal antibody is a laboratory-produced molecule that
carefully engineered to attach to specific defects in your cancer cells.
Monoclonal antibodies are proteins produced in the laboratory from a
single clone of a B cell, the type of cells of the immune system that
make antibodies
Immune System
• The immune system acts as defense against various infectious agents
that cause different forms of diseases. Two major components are the
humoral (antibody-mediated) and cellular (cell-mediated) immune
responses. The humoral immune system which comprises B-
lymphocytes recognizes the type of foreign invading antigens and
produces specific antibodies against them.
• The two important characteristics of an antibody are its specificity to
the antigen, and its assurance to provide continual resistance to that
particular type of antigen .
Hybridoma
• Inject a mouse with a specific antigen to stimulate its immune system
to produce necessary antibodies.
• Extract mouse spleen cells (containing B-lymphocytes) and culture
them in the lab.
• Extract mouse tumor cells, which grow continuously, and culture
them in the lab.
• Mix spleen cells and tumor cells on the same plate and culture
• Add polyethylene glycol – this causes some B-lymphocytes to fuse
with tumour cells to produce a hybrid cell called a hybridoma.
• Grow the cells under conditions that allow only hybridoma cells to
survive.
• Extract the cells, culture them separately and test the medium around
each cell for the specific antibody of interest.
• Culture the cells making the desired antibody and use as needed
 Diagnosis & Types of
Monoclonal Antibodies
Pregnancy Tests
• A pregnant woman has the hormone human chorionic
gonadotrophin (HCG) in her urine.
• Monoclonal antibodies to HCG have been produced. These
have been attached to enzymes which can later interact
with a dye molecule and produce a colour change.
Pregnancy Tests
• Pregnancy test contains three regions – reaction region, test
region and control region
– Reaction region: contains monoclonal anti-HCG antibodies linked to
enzyme
– Test region: contains polyclonal anti-HCG antibodies, which will bind to
HCG molecules bound to monoclonal anti-HCG antibodies. Also
contains dye molecules which will be activated if monoclonal
antibodies bind to polyclonal anti-HCG antibodies
– Control region: contains anti-mouse antibodies and dye molecules
which will be activated if monoclonal antibodies bind here
Pregnancy Tests
• When pregnant woman’s urine travels up the pregnancy test, HCG will
bind to monoclonal antibodies in reaction region
• Movement of the urine will move the monoclonal antibodies up to
the test region
• Monoclonal antibodies with bound HCG will bind to antibodies in test
region and activate dye molecules, producing a colour change
• Any unbound monoclonal antibodies will continue to travel to control
region and will bind to anti-mouse antibodies, activating dye
molecules and producing a colour change
Test
Diagnosis of HIV Infection
• The test of HIV infection is based
on detecting the presence of HIV
antibody in the patient’s blood
serum
a) HIV antigen is attached to the plate.
b) Patients serum passed over the plate. Any HIV antibody in
the patients serum will attached to the antigen already on
the plate.
c) A second antibody which is specific to the HIV antibody is
passed over the plate. This antibody will attach to the
concentrated HIV antibody on the plate. This second
antibody has an enzyme attached to its structure.
d) Chromagen dye is passed over the complex of concentrated
HIV antibody/conjugated antibody.
e) The enzyme will turn the chromagen to a more intense
colour. The more intense the colour, the greater the HIV
antibody level. This would be the a positive result for a HIV
test.
Conjugated monoclonal antibodies
• Monoclonal antibodies (mAbs) joined to a chemotherapy drug or to a
radioactive particle are called conjugated monoclonal antibodies. The
mAb is used as a homing device to take one of these substances
directly to the cancer cells. The mAb circulates throughout the body
until it can find and hook onto the target antigen. It then delivers the
toxic substance where it is needed most. This lessens the damage to
normal cells in other parts of the body.
• Conjugated mAbs are also sometimes referred to as tagged, labeled,
or loaded antibodies.
Radiolabeled antibodies
• Radiolabeled antibodies have small radioactive particles attached to
them. Ibritumomab tiuxetan (Zevalin®) is an example of a
radiolabeled mAb. This is an antibody against the CD20 antigen, which
is found on lymphocytes called B cells. The antibody delivers
radioactivity directly to cancerous B cells and can be used to treat
some types of non-Hodgkin lymphoma.

• Treatment with this type of antibody is sometimes known as radio

immunotherapy (RIT)
Chemo labeled antibodies
• These mAbs have powerful chemotherapy (or other) drugs attached
to them. They are also known as antibody-drug conjugates (ADCs).
(The drug is often too powerful to be used on its own – it would cause
too many side effects if not attached to an antibody.)

• Chemo labeled antibodies used to treat cancer include:

• Brentuximab vedotin (Adcetris®), an antibody that targets the CD30

antigen (found on lymphocytes), attached to a chemo drug called
MMAE. This drug is used to treat Hodgkin lymphoma
• Ado-trastuzumab emtansine (Kadcyla®, also called TDM-1), an antibody
that targets the HER2 (human epidermal receptors ) protein, attached to a
chemo drug called DM1. It’s used to treat some breast cancer patients
whose cancer cells have too much HER2.
• A related drug known as denileukin diftitox (Ontak®) is an immune system
protein known as interleukin-2 (IL-2) attached to a toxin from the germ that
causes diphtheria. Although it’s not an antibody, IL-2 normally attaches to
certain cells in the body that contain the CD25 antigen, which makes it
useful for delivering the toxin to these cells. Denileukin diftitox is used to
treat lymphoma of the skin (also known as cutaneous T-cell lymphoma). It’s
also being studied for use against a number of other cancers
Bispecific monoclonal antibodies
• These drugs are made up of parts of 2 different mAbs, meaning they
can attach to 2 different proteins at the same time. An example is
blinatumomab (Blincyto), which is used to treat some types of acute
lymphocytic leukemia . One part of blinatumomab attaches to the
CD19 protein, which is found on some leukemia and lymphoma cells.
Another part attaches to CD3, a protein found on immune cells called
T cells. By binding to both of these proteins, this drug brings the
cancer cells and immune cells together, which is thought to cause the
immune system to attack the cancer cells
ANTIBODY FRAGMENTS
• Antibody fragments were originally derived from enzyme digestion of
intact antibodies producing the Fc, Fab products. Production of
antibody fragments has become more sophisticated. In some
diagnostic and therapeutic situations, antibody fragments, namely
Fab and F(ab’)2 fragments, are attractive. These fragments can be
made from enzyme digests of intact antibodies or synthesized using
recombinant DNA technology, circumventing hybridoma . Fragments
are easier to manipulate genetically and express in bacterial systems
• The smaller fragments penetrate tissue better and faster, clear the
general circulation faster, and are eliminated more completely with
less hepatic binding than whole antibodies. These smaller molecules
tend to have better penetration into solid tumors than whole
antibodies. Generally there is faster systemic clearance than that of
intact monoclonal antibodies. While this is a desirable feature, it
should be noted that faster clearance sometimes comes with a price,
such as increased renal exposure. Fragments are often useful when
specific faster tissue penetration and rapid clearance are preferred, as
in tumor imaging. Fragments entirely of human peptide sequences
present less of an immunogenic target for the patient.
Catumaxomab
• Catumaxomab has two different antigen-binding specificities: one for
EpCAM (epithelial cell adhesion molecule) on tumor cells and one for
the CD3 antigen on T-cells. Catumaxomab exerts its anti-tumor effects
via T-cell-mediated lysis, ADCC, and phagocytosis via activation of
FcγR-positive accessory cells Its anti-tumor activity is assisted by the
induction of T-cell-secreted cytokines, such as interferon (IFN)-γ and
tumor necrosis factor (TNF) .An important aspect of catumaxomab’s
mode of action is that no additional activation of immune cells is
required for effective tumor eradication, so it is a self-supporting
system
Malignant Ascites
• MA is an increased accumulation of protein-containing fluid within the
peritoneal cavity that is caused by spread of cancer. It is associated with
advanced ovarian cancer, gastrointestinal malignancies, and other
carcinomas, and leads to abdominal pain and swelling, dyspnea, nausea,
vomiting, malnutrition, and anorexia. Patients with MA have a poor
quality of life and a poor prognosis, with median overall survival of
approximately 1–6 months. The causes of MA are independent of the
origin of the primary tumor . Tumor-secreted factors lead to tumor
neovascularization and increased capillary permeability, resulting in
increased plasma inflow into the peritoneal cavity. Tumor cells obstruct
lymphatic drainage, leading to decreased fluid efflux from the peritoneal
cavity
Rationale for use of Catumaxomab in the
Treatment of MA
• Prior to the approval of catumaxomab, no agents were specifically
approved for the treatment of MA and treatment options, such as
peritoneovenous shunts, paracentesis, and diuretics, are only palliative.
There was thus a need for an effective treatment for MA. The rationale
for the use of catumaxomab for the imp treatment of MA epithelial
tumors spreading into the peritoneal cavity play a major role in the
development of MA epithelial tumors frequently express EpCAM in the
peritoneal cavity, EpCAM is a tumor-specific antigen; and immune
effector cells are present in MA. Targeting EpCAM by i.p. administration
of catumaxomab leads to a depletion of epithelial tumor cells in the
peritoneal cavity and a sustained reduction of MA production.
Catumaxomab mechanism of action. ADCC = antibody-dependent cell-mediated cytotoxicity, CK =
cytokine, DC = dendritic cell, EpCAM = epithelial cell adhesion molecule, Fcγ R = Fcγ receptor, GM-CSF =
granulocyte–macrophage colony-stimulating factor, IL = interleukin, IFN = interferon, LFA = lymphocyte
function-associated antigen, NK = natural killer, TNF = tumor necrosis factor.
Pathophysiology of malignant ascites.
Monoclonal antibodies for cancer treatment
• Three mechanisms that could be responsible for the cancer treatment.
• mAbs act directly when binding to a cancer specific antigens and induce immunological
response to cancer cells. Such as inducing cancer cell apoptosis, inhibiting growth, or
interfering with a key function.

• mAbs was modified for delivery of a toxin, radioisotope, cytokine or other active
conjugates.

• It is also possible to design bispecific antibodies that can bind with their Fab regions
both to target antigen and to a conjugate or effector cell
mAbs treatment for cancer cells

ADEPT, antibody directed enzyme prodrug therapy;

ADCC, antibody dependent cell-mediated cytotoxicity;
CDC, complement dependent cytotoxicity; MAb,
monoclonal antibody; scFv, single-chain Fv fragment
• mAbs target growth factor
receptors to exert a direct effect
on the growth and survival of
the cancer cells by antagonizing
ligand-receptor signaling.

• mAbs can target to cell surface

antigens and directly elicit
apoptotic signaling
Oncolytic virus therapy
• Oncolytic virus therapy uses genetically modified viruses to kill cancer
cells. First, the doctor injects a virus into the tumor. The virus then
enters the cancer cells and makes copies of itself. As a result, the cells
burst and die. As the cells die, they release specific substances called
antigens. This triggers the patient’s immune system to target all the
cancer cells in the body that have those same antigens. The virus does
not enter healthy cells.
chimeric antigen receptor (CAR) T-cell
therapy
• T cells are immune cells that fight infection. In T-cell therapy, some T
cells are removed from a patient’s blood. Then, the cells are changed
in a laboratory so they have specific proteins called receptors. The
receptors allow those T cells to recognize the cancer cells. The
changed T cells are grown in large numbers in the laboratory and
returned to the patient’s body. Once there, they seek out and destroy
cancer cells. This type of therapy is called chimeric antigen receptor
(CAR) T-cell therapy
• The use of T cells for CAR therapy has been very effective in treating
certain blood cancers. Researchers are still studying this and other
ways of modifying T cells to treat cancer