2. WHAT ARE 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.
4. HISTORY :
• 1900 Ehrlich proposed the “ side-
chain theory”
• 1990 Milstein produced the first
monoclonal antibodies
• Paul Ehrlich at the beginning of
the 20th century theorized that
a cell under threat grew
additional side-chains to bind the
toxin, and that these additional
side chains
broke off to become the
antibodies that are circulated
through the body. It was
these antibodies that
Ehrlich first described as
"magic bullets" in search
of toxins.
6. PREPARATION OF MONOCLONAL ANTIBODIES
Monoclonal Antibody Production technology was developed in 1975
by two scientists, Georges Kohler of West Germany and Cesar
Milstei.
The previous picture is showing a mouse being immunized against a
target cell “X”.(antigens) This will allow the mouse to produce
antibodies for that will target against the “X” antigen.
Once the mouse has formed antibodies to the “X” antigen the
Antiserum (blood serum rich in antibodies) extracted from mouse’s
spleen.
Monoclonal antibodies are produced by fusing single antibody-
forming cells to tumor cells grown in culture. The resulting cell is
called a hybridoma.
Hybridoma cells are continuously growing cell line generated by the
fusion of a myeloma cell and a normal cell that are capable of
producing antibodies.
7. Each hybridoma will produce relatively large quantities of identical
antibody molecules. Because the hybridoma is multiplying in culture,
it is possible to produce a population of cells, each is producing
identical antibody molecules. These antibodies are called
"monoclonal antibodies" because they are produced by the identical
offspring of a single, cloned antibody producing cell.
HAT medium (Hypoxanthine Aminopetrin Thymidine) is used for
preparation of monoclonal antibodies,because it contains
hypoxanthine, aminopterin, and thymidine. This medium is selective
for fused (hybridoma) cells. Unfused myeloma cells and unfused
spleen cells cannot grow because they lack HGPRT ( hypoxanthine-
guanine-phosphoribosyl transferase), and thus cannot replicate their
DNA and because of their limited life span respectively.
8. APPLICATION OF MABS:
The application of monoclonal antibodies can be
broadly categorized as:
a) Diagnostic application
b) Therapeutic application
c) Catalytic MAb (Abzymes)
9. (A) DIAGNOSTIC APPLICATION :
MAbs are utilized in diagnostic kits for the diagnosis of various infectious
diseases, detecting pregnancy, monitoring drug levels, matching
histocompatibility antigen,cancer and in immunoscintigraphy.
FDA licensed a new diagnostic imaging agent that can determine the extent
of disease in patients diagnosed with small cell lung cancer (SCLC). Because
these agents can detect tumor in different part of the body at one time, it
can help physician to advice certain patients with advanced forms of the
disease about treatment option without requiring further diagnostic tests.
For ex.the new agent, Nofetumomab, is a fragment of a monoclonal antibody
that when tagged with the radioisotope technique, can detect a protein found
on the surface of most small lung cancer cells.
Other monoclonal antibodies allows rapid diagnosis of hepatitis, influenza,
herpes, streptococcal, and Chlamydia infections.
10. DIAGNOSTIC APPLICATION
A monoclonal antibody can be used
to detect pregnancy in only 14 days
after conception.
Urine and serum mAbs pregnancy
test are easier.Their selective
binding property allow detection of
low levels of human corionic
gonadotropin (HCG) in urine and
serum.These tests approach the
accuracy of serum beta HCG
radioimmuno assay for
confirmation of normal pregnancy
or evalution of possible abnormal
pregnancy.
11. DIAGNOSTIC APPLICATION
Once monoclonal antibodies
for a given substance have
been produced, they can be
used to detect the presence
of this substance.The
Western blot test and
immuno dot blot tests
detect the protein on a
membrane.
12. DIAGNOSTIC APPLICATION:
mAbs are also very useful in immunohistochemistry,
process of detecting antigens (e.g., proteins) in cells of a
tissue section by exploiting the principle of antibodies
binding specifically to antigens in biological tissues and
immuno fluorescence test, which detect the substance in
a frozen tissue section or in live cells.
Monoclonal antibodies can also be used to purify a
substance with techniques called affinity
chromatography.
13. (B) THERAPEUTIC APPLICATION:
Earlier horses were inoculated with bacterium Coryne
bacterium diphtheriae which causes diptheria in humans,
and the resulting crude horse antiserum was used to
treat deptheria.Due to this discovery mortality reached
45%.
C.Diptheriae infects the throat or tonsils and produces
an exotoxin that is lethal to human cells. This exotoxin
enters the blood stream and damage organs that are
distinct from the primary site of infection.
Today with the advent of hybridoma methodology
antibodies again seen as potential therapeutic agent.
14. THERAPEUTIC APPLICATION:
Organ transplantation
For the treatment of solid organ transplant rejection, several MAbs
against T cell antigens have been evaluated. 'The most successful of
these is orthrochrome OKT3 which was first MAb to be licensed for
human use.
The murine intact IgG antibody, directed against CD3 antigen on T
lymphocytes, is used unconjugated to effectively reverse acute
rejection episodes with renal, hepatic, cardiac and combined kidney
pancreas transplants.
Acute side effects such as fever and chills are common and
probably may be a consequence of T cell damage.
15. MAbs are being evaluated for graft versus host disease
in bone marrow transplantation.
Immune cells (white blood cells) in the tissue (the graft)
recognize the recipient (the host) as "foreign". The
transplanted immune cells then attack the host's body
cells.
A ricin toxin immunoconjugate directed against CD5
antigen of T lymphocyte is effective both in the
treatment and prevention of graft-versus host disease
with histocompatible marrow grafts.
OKT3 has also shown promise in the management of
immune related graft rejection events.
Bone marrow transplantation:
16. MONOCLONAL ANTIBODIES FOR CANCER TREATMENT
There are three mechanisms that could be responsible for the cancer
treatment:
1. mAbs act directely 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.
2. mAbs may be modified for delivery of a toxin, radioisotope
[RADIOIMMUNOTHERAPY], cytokine or other active conjugates.
3. 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.
17.
18. WHEN A MONOCLONAL ANTIBODY ATTACHES TO A
CANCER CELL, IT CAN:
• Make the cancer cell more
visible to the immune
system.
ex. Rituximab
• Block growth signals ex.
Cetuximab used for colon
cancer and block epidermal
growth factors.
19. Stop new blood vessels formation (angiogenesis) Ex.
Devacizumab (Avastin) acts on vascular endothelial
growth factor (VEGF).
Deliver radiation to cancer cells by binding to radioactive
element ex. Zevalin used for non-hodgkin lymphoma.
Slip powerful drugs into cancer cells and kills the cancer
cell. Ex. Kadcyla in breast cancer.
20. FDA-APPROVED MONOCLONAL ANTIBODIES FOR
CANCER TREATMENT :
NAME OF DRUG
Alemtuzumab (Campath)
Bevacizumab (Avastin)
Cetuximab (Erbitux)
Gemtuzumab (Mylotarg)
TYPE OF CANCER USED TO
TREAT
Chronic lymphocytic
leukemia
Breast cancer
Colon cancer
Lung cancer
Colon cancer
Head and neck cancers
Acute myelogenous
leukemia
21. FDA-APPROVED MONOCLONAL ANTIBODIES FOR
CANCER TREATMENT :
Ibritumomab (Zevalin)
Panitumumab (Vectibix)
Rituximab (Rituxan)
Tositumomab (Bexxar)
Trastuzumab (Herceptin)
Non-Hodgkin's lymphoma
Colon cancer
Non-Hodgkin's lymphoma
Non-Hodgkin's lymphoma
Breast cancer
22. CONJUGATED MONOCLONAL ANTIBODY THERAPY:
Toxins or radioactive isotopes
are bound to the constant
region of the mAbs. When the
mAb binds to the surface cells
of a tumour the toxin or
radioactivity will kill the
cancer cells and all cells within
a certain radius (a killing
zone). In this way cancer cells
within the tumour will be
killed.
23. RADIOIMMUNOTHERAPY:
Involves the use of
radioactively conjugated
Murine antibodies against
cellular antigens
Eg.; Tositumomab -----
non-Hodgkins lymphoma
24. AUTOIMMUNE DISEASES:
Monoclonal antibodies used for autoimmune diseases
include infliximab and adalimumab, which are effective in
rheumatoid arthritis, Crohn's disease and ulcerative
Colitis by their ability to bind to and inhibit TNF-α.
Basiliximab and daclizumab inhibit IL-2 on activated T
cells and thereby help to prevent acute rejection of
kidney transplants.
Omalizumab inhibits human immunoglobulin E (IgE) and is
useful in moderate-to-severe allergic asthma
25. MONOCLONAL ANTIBODIES FOR ANTI INFLAMMATORY:
TYPE APPLICATION MECHANISM
infliximab rheumatoid arthritis
Crohn's disease
Ulcerative Colitis
inhibits TNF-α
Adalimumab rheumatoid arthritis
Crohn's disease
Ulcerative Colitis
inhibits TNF-α
basiliximab Acute rejection of kidney transplants inhibits IL-2 on activated T
cells
daclizumab Acute rejection of kidney transplants inhibits IL-2 on activated T
cells
Omalizumab moderate-to-severe allergic asthma inhibits human
immunoglobulin E (IgE)
26. (C) CATALYTIC MONOCLONAL ANTIBODY (ABZYMES)
The binding of an antibody to its antigen and binding of enzyme to
its substrate both involves weak, non covalent interactions and
exhibits high specificity and often high affinity.
But an antigen is not altered by antibody, where as an enzyme
alters the substrate and as a result the binding by catalyzing a
chemical change. The enzyme uses its binding energy to stabilize the
transition state of substrate. thus reducing activation energy for
chemical modification of bound substrate.
Similarities between antigen-antibody interaction and enzyme-
substrate interaction have led Lerner and his colleagues to explore
the probability of enzyme like action of some antibodies.
27. AUTOANTIBODY FINGERPRINTING USING DIPSTICKS
The patients suffering from rheumatic diseases are found to have
autoantibodies that react to cellular components. These
autoantibodies increase in number alter birth up to two years and
then remain constant for decades.
These antibodies have unique complement which differ from person
to person and hence referred to as individual specific (IS)
autoantibodies. These IS autoantibodies could be physically
separated comprising of an antibody fingerprinting that could serve
in the identification of an individual.
28. MONOCLONAL ANTIBODIES IN DRUG TARGETING:
The concept of using specific antibodies conjugated with
toxic or isotopically labelled materials for specific sites
and drug localization is not new.
This concept with the development of MAbs and hybridoma
technology have gained wide appreciation in their use. The
use of monoclonal antibodies to target drugs to specific
cell types is a promising approach.
