Monoclonal Antibody
By
Dr. Adel Gabr
Terminology
Target
therapy
Immunotherapy
Monoclonal
antibody
Biotherapy
Biological
therapy
TKIs
Cancer
vaccine
Types of targeted therapy used today
• Today many different types of targeted
therapies are used to treat cancer.
• There are 2 main types of targeted therapy
drugs: antibody drugs Large molecule
and small-molecule drugs. Some
targeted therapy drugs are actually
antibodies—man-made versions of immune
system proteins
immunotherapy
• Immunotherapy is also sometimes called biologic therapy
or biotherapy. It is treatment that uses certain parts of the
immune system to fight diseases such as cancer. This can
be done in a couple of ways:
• Stimulating your own immune system to
work harder or smarter to attack cancer cells
• Giving you immune system components,
such as man-made immune system
proteins
Monoclonal
Antibody
Man Made immunity
MONOCLONAL ANTIBODY
One way the immune system normally attacks
foreign substances in the body is by making
large numbers of different antibodies. An
antibody is a “sticky” protein that targets a
specific antigen. Antibodies circulate in the
body until they find and attach to the antigen.
Once attached, they recruit other parts of the
immune system to destroy the cells containing
the antigen.
Hybridomas Technique
mABs Production
- B lymphocytes can mutate into tumor cells
that result in a type of cancer termed
myeloma.
- Myeloma cells become “immortal” and will
grow indefinitely in culture.
- Fusion of a single activated B cell and a
myeloma cell will create a hybridoma that can
grow indefinitely in culture.
Hybridoma Selection
The “HAT Trick”
Myeloma cells have been genetically 
engineered such that they can not use
hypoxanthine, aminopterin, and thymidine
(HAT medium) as a source for nucleic acid
biosynthesis and will die in culture.
Only B cells that have fused with the 
engineered myeloma cells will survive in
culture when grown in HAT medium.
Producing Monoclonal Antibodies
1. Inject a mouse with a specific antigen to
stimulate its immune system to produce
necessary antibodies.
2. Extract mouse spleen cells (containing Blymphocytes) and culture them in the lab.
3. Extract mouse tumour cells, which grow
continuously, and culture them in the lab.
4. Mix spleen cells and tumour cells on the
same plate and culture.
Producing Monoclonal Antibodies
5. Add polyethylene glycol – this causes some Blymphocytes to fuse with tumour cells to produce a
hybrid cell called a hybridoma.
6. Grow the cells under conditions that allow only
hybridoma cells to survive.
7. Extract the cells, culture them separately and test the
medium around each cell for the specific antibody of
interest.
8. Culture the cells making the desired antibody and
use as needed.
Secrete antibody but don't grow
in tissue culture
Grow indefinitely in
cell culture but don't
secrete the desired
antibody
Myeloma cells
FUSE
Grow indefinitely in
cell culture AND
secrete antibody
Hybridoma cells
Harvest Ab
Monoclonal antibodies
Polyclonal antibodies
(Polyclonal antiserum)
Harvest Ab
Monoclonal antibodies
B
B
B
B
B
B
B
B
The structure of antibodies
http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html
•
Protein
Immunize
Antibodies
Immune Response
Epitopes
A mixture of antibodies - all bind to epitopes of
the original antigen. Some bind with higher
affinity than others.
Polyclonal antibodies
Polyclonal antibodies
(Polyclonal antiserum)
Harvest Ab
Monoclonal antibodies
B
B
B
B
B
B
B
B
Nature of Antibody
Omab
Ximab
Zumab
umab
Hybridoma
technology has been
replaced by
recombinant DNA
technology, transgenic
mice and phage
Types of monoclonal antibodies
• Two types of monoclonal antibodies are used in
cancer treatments:
• Naked mAbs : are antibodies that work by
themselves. There is no drug or radioactive material
attached to them. These are the most commonly
used mAbs at this time.
• Conjugated mAbs : are those joined to a
chemotherapy drug, radioactive particle, or a toxin
(a substance that poisons cells). These mAbs work,
at least in part, by acting as homing devices to take
these substances directly to the cancer cells.
Naked monoclonal antibodies :
• Naked mAbs can work in different ways.
• A- Some may boost a person’s immune response against
cancer cells.
• B- Others work by blocking specific proteins that help
cancer cells grow. (Some may do both.)
• C- Some naked MAbs attach to cancer cells to act as a
marker for the body's immune system to destroy them. An
example of this is alemtuzumab
• D- Some naked mAbs work mainly by attaching to and
blocking specific antigens that are important signals for
cancer cells (or other cells that help cancer cells grow or
spread).
Conjugated monoclonal antibodies
• Monoclonal antibodies attached to a radioactive
substance, drug, or toxin, are called conjugated mAbs.
The mAb is used as a homing device to take one of
these substances directly to the cancer cells. The mAb
circulates in 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. They can be
divided into groups depending on what they are linked
to.
• mAbs with radioactive particles attached
• are referred to as radiolabeled, and treatment with
this type of antibody is known as
radioimmunotherapy (RIT).
• Radiolabeled antibodies: Radiolabeled
antibodies have small radioactive particles attached
to them. Ibritumomab tiuxetan (Zevalin®) and
tositumomab (Bexxar®) are examples of
radiolabeled mAbs. Both of these are antibodies
against the CD20 antigen, but they each have a
different radioactive particle attached. They deliver
radioactivity directly to cancerous B cells and can be
used to treat some types of non-Hodgkin
lymphoma.
mAbs with chemotherapy drugs attached
(chemolabeled).
Chemolabeled antibodies: These mAbs have powerful
chemotherapy drugs attached to them. (The
chemotherapy drug is often too powerful to be used on its
own – it would cause too many side effects if not attached
to an mAb.)
• The only chemolabeled antibody approved by the FDA to
treat cancer at this time is brentuximab vedotin
(Adcetris). This drug is made up of an antibody that
targets the CD30 antigen (found on B cells and T cells),
attached to a chemo drug called MMAE. It is used to treat
Hodgkin lymphoma and anaplastic large cell lymphoma
that is no longer responding to other treatments.
mAbs attached to cell toxins.
• Immunotoxins: These mAbs have cell poisons (toxins)
attached to them, which makes them similar in many ways to
chemolabeled mAbs. At this time no immunotoxins are
approved to treat cancer, although many are being studied.
• However, a related drug known as denileukin diftitox
(Ontak®) is being used to treat some cancers. It consists of 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 mAb, 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 is also being studied to be
used against a number of other cancers.
Does mAbs
work only for
treatment
only?
Uses
Measuring protein and drug levels in serum
Typing tissue and blood
Identifying infectious agents
Identifying clusters of differentiation for the
classification and follow-up therapy of leukemias
and lymphomas
Identifying tumor metastasis
Identifying and quantifying hormones
Immunoaffinity
Purification
Possible side effects of monoclonal
antibodies
Monoclonal antibodies are given intravenously (injected
into a vein). Compared with the side effects of
chemotherapy, the side effects of naked mAbs are
usually fairly mild and are often more like an allergic
reaction. These are more common while the drug is first
being given. Possible side effects can include:
Fever, Chills, Weakness, Headache, Nausea
Vomiting, Diarrhea, Low blood pressure,
Rashes
Possible side effects of monoclonal
antibodies
Some mAbs can also have other side effects that are
related to the antigens they target. For example,
bevacizumab (Avastin®), an mAb that targets tumor
blood vessel growth, can cause side effects such as
high blood pressure, bleeding, poor wound healing,
blood clots, and kidney damage.
Conjugated antibodies may pack more of a punch
than naked mAbs, but they often cause more side
effects. The side effects depend on which type of
substance they're attached to.
Thank
you
Hybridoma Selection
The “HAT Trick”
Myeloma cells have been genetically 
engineered such that they can not use
hypoxanthine, aminopterin, and thymidine
(HAT medium) as a source for nucleic acid
biosynthesis and will die in culture.
Only B cells that have fused with the 
engineered myeloma cells will survive in
culture when grown in HAT medium.
Producing Monoclonal Antibodies
1. Inject a mouse with a specific antigen to
stimulate its immune system to produce
necessary antibodies.
2. Extract mouse spleen cells (containing Blymphocytes) and culture them in the lab.
3. Extract mouse tumour cells, which grow
continuously, and culture them in the lab.
4. Mix spleen cells and tumour cells on the
same plate and culture.
Producing Monoclonal Antibodies
5. Add polyethylene glycol – this causes some Blymphocytes to fuse with tumour cells to produce a
hybrid cell called a hybridoma.
6. Grow the cells under conditions that allow only
hybridoma cells to survive.
7. Extract the cells, culture them separately and test the
medium around each cell for the specific antibody of
interest.
8. Culture the cells making the desired antibody and
use as needed.
The structure of antibodies
http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html
•
Protein
Immunize
Antibodies
Immune Response
Epitopes
A mixture of antibodies - all bind to epitopes of
the original antigen. Some bind with higher
affinity than others.
Polyclonal antibodies
Polyclonal antibodies
(Polyclonal antiserum)
Harvest Ab
Monoclonal antibodies
B
B
B
B
B
B
B
B
What Diseases to Target and How?
Cancer cells express a variety of antigens that •
are attractive targets for monoclonal
antibody-based therapy.
The development of monoclonal antibodies •
against specific targets has been largely
accomplished by immunizing mice against
human tumor cells and screening the
hybridomas for antibodies of interest.
Unfulfilled Promise?
• The early promise of the use of antibodies in the
treatment of disease initially went unfulfilled (more
than two decades) for two reasons:
1. Early antibodies displayed insufficient activation
of human effector functions (i.e. the antibodies did
not kill the infecting organism or cell)
2. The early antibodies were of murine (mouse)
origin, and thus triggered the production of human
anti-mouse antibodies (HAMA).
Other obstacles to the use of monoclonal
antibodies in cancer treatment
Antigen distribution of malignant cells is •
highly heterogeneous, so some cells may
express tumor antigens, while others do not.
Tumor blood flow is not always optimal •
High interstitial pressure within the tumor can •
prevent the passive monoclonal antibody
from binding.
The types of mAb designed
Murine source mAbs: rodent mAbs with excellent affinities and .A
specificities, generated using conventional hydrioma technology. Clinical
efficacy compromised by HAMA(human anti murine antibody) response,
which lead to allergic or immune complex herpersensitivities.
Chimeric mAbs: chimers combine the human constant regions with the .B
intact rodent variable regions. Affinity and specificity unchanged. Also
cause human antichimeric antibody response (30% murine resource)
Humanized mAbs: contained only the CDRs of the rodent variable region .C
grafted onto human variable region framework
Nomenclature of Therapeutic
Antibodies
• Terminate the name in –ximab for chimeric
antibodies and –umab for humanized
antibodies.
Common Chemotherapy in Treatment of Cancer
Shortcomings:
A.
Nature of cytotoxin
B.
Lack of in vivo selectivity
C.
The mechanism of anti-proliferation on cells cycle, rather than specific toxicity
directed towards particular cancer cell
D.
Host toxicity: treatment discontinued, most of them had bad side-effects, such as
no appetites, omit, lose hair
http://www.meds.com/immunotherapy/monoclonal_antibodies.html
Rituximab (Rituxan)
Rituximab is a chimeric monoclonal antibody that
targets the CD20 B-cell antigen.
This antigen is expressed on 90% of B-cell neoplasms
The precise biological functions of CD20 are uncertain,
but the antibody is believed to function by flagging the
B-cells for destruction by the body’s own immune
system, including ADCC, CDC, and apoptosis.
This antibody thus leads to the elimination of all B-cells
from the body (including cancerous ones), allowing
new, healthy B-cells to be produced from lymphoid
stem cells.
•
•
•
•
Trastuzumab (Herceptin)
Herceptin is an anti-cancer antibody that acts on
HER2/neu (erbB2) receptor, which is overexpressed in
breast cancer. Only cells overexpressing this receptor
are susceptible.
Such cells, when treated with Herceptin, undergo
arrest in the G1 phase of the cell cycle and experience
a reduction in proliferation.
This can reduce the rate of relapse of breast cancer by
50% during the first year.
The precise mechanism of action is still under
investigation.
•
•
•
•
Monoclonal antibodies which deliver a
toxin
Monoclonal antibodies can be utilized to •
selectively deliver a toxin to a malignant cell.
Gemtuzumab ozogamicin (Mylotarg)
This monoclonal antibody is conjugated to the •
cytotoxic agent calicheamycin
It is used to treat acute myelogenous leukemia •
(AML), which is a cancer of the myeloid line of blood
cells.
This monoclonal antibody attacks the CD33 receptor, •
which is found in most leukemic blast cells, but not in
normal hematopoietic stem cells
Gemtuzumab ozogamicin (Mylotarg)
Once bound to CD33, the antibody-calicheamycin •
complex is transported inside of the AML cells by
lysosomes.
To facilitate selective release inside of the cancer •
cells, calicheamycin is connected to gemtuzumab by
a chemical linker that is stable at physiologic pH but
is hydrolyzed in the acidic pH of the lysosomes that
transport the antibody-calicheamycin complex into
the cell.
Strategy of a direct or in direct induction of
apoptosis in targeted cancer cells
mAbs target growth factor receptors to .1
exert a direct effect on the growth and
survival of the cancer cells by
antagonizing ligand-receptor signaling.
mAbs can target to cell surface .2
antigens and directly elicit apoptotic
signaling.
Dale L Ludwig, etal. Oncogene(2003) 22, 9097-9106
Until Feb 28, 2005, 18 mAbs were
approved by FDA, which were applied in
the treatment of organ transplant, Cancer,
Asthma, Hematopoietic malignancies and
psoriasis.
The first approved mAbs was OKT-3, which
is a murine IgGa2 protein to deplete T cells
in patients with acute rejection of renal
allotransplant.
HAMA response
Jancie, M Recheit, etal. Nature biotechnology, 2005,
Sep,Vol. 23, No.9
Stamatis-Nick C. J Allergy Clin. Immunol, Oct. 2005
mAbs development
Phage display library: construction of VH and VL gene libaries and
.1
expression of them on a filamentous bacterophage. The phage expressing an
antigen-bonding domain specific for a particular antigen to screen the mAbs.
Transgenic plants: transgenic tobacco plants to produce IgA.
.2
Transgenic animals: transgenic mouse to make humanized
IgG. (Abgenix,CA)
.3
Conventional production of mAbs
The hybridoma technology:
spleen cells from immunized mice are fused with the murine myeloma cells.
The several process had been developed at large scale.
According to the different cell culture methods, it can calisifed into four fields
1.
Robottle cell culture process.
2.
Membrane binded cell culture process
3.
Microcarrier cell culture process
4.
Suspended cell culture process