monoclonal Antibidy ppt

1. Presented By -
MOUSAM BHOWMIK
M. PHARM, 1ST YEAR 2ND SEM
ACHARYA & BM REDDY COLLEGE OF PHARMACY
MONOCLONAL ANTIBODIES Presented To
Dr. Joysa Ruby J
Assistant professor

2.  What is Antibody?
• Antibodies are glycoprotein molecule present in
the serum. They are produced in response to
antigen which are either protein or polysaccharide
molecules which may be foreign to the body.
Antibodies are secreted by a class of blood cells
known as B-lymphocytes. Each antibody produced
is specific to that particular antigen which has
stimulated its production.

3. • Antibodies are also called as Immunoglobulin (Ig) and
the structure of antibody contains two chain such as –
Heavy chain, Light chain and constant region ,variable
region and they also contain di sulphide bond etc.

4.  What is Monoclonal Antibodies?
• Monoclonal antibodies (mAbs) are defined as
“laboratory-produced molecules engineered to serve as
substitute antibodies that can restore, enhance, or mimic
the immune system's attack on cancer cells” by binding
to antigens found on the surface of cancer cells.
• In the 18th century, it was discovered by Dr Edward
Jenner that “fluid obtained from a smallpox pustule
when injected into a recipient provided immunity from
acquiring the disease.

5. Structure of monoclonal antibody

6.  Mechanism of action
• The understanding of how these antibodies target cancer
cells has helped to revolutionize the methods used to
treat cancer and resulted in a more tolerable toxicity
profile than standard chemotherapy. When utilizing
mAbs in oncology, several mechanisms of action exist
to destroy the cancer cells. These mechanisms include
impeding tumor cell survival cascades, inhibiting tumor
growth by interfering with tumor angiogenesis, eluding
programmed cell death, and evading immune
checkpoints. Some of the different mechanisms of
action include the following:

7. 1. Direct tumor cell killing
• This action is stimulated by receptor agonist activity,
such as an antibody binding to a tumor cell surface
receptor and activating it, leading to cell death.
• It can also be stimulated by receptor antagonist activity
“such as an antibody binding to a cell surface receptor
and blocking dimerization, kinase activation, and
downstream signaling, leading to reduced proliferation
and apoptosis.” A mAb binding to an enzyme can lead
to neutralization and apoptosis, while conjugated
antibodies can be used to deliver a payload, such as a
drug, to the tumor cell.

8. 2. Immune-mediated tumor cell killing
• In this setting, the immune system will seek out cancer cells
and destroy them. This type of cell death may be carried out
by the initiation of one of several mechanisms. These
mechanism.
a) Phagocytosis;
b) complement activation;
c) antibody-dependent cellular cytotoxicity (ADCC);
d) genetically modified T cells being targeted to the tumor by
single-chain variable fragment;
e) T cells being activated by antibody-mediated cross-
presentation of antigen to dendritic cells; or
f) inhibition of T-cell inhibitory receptors.

9. 3. Vascular and stromal cell ablation
• This action is also initiated by one of many options.
These include:
a) Vasculature receptor antagonism or ligand trapping;
b) Stromal cell inhibition;
c) Delivery of a toxin to stromal cells; or
d) Delivery of a toxin to the vasculature.

11.  Classification and Types of mAbs
There are four classifications of mAbs:
1. Murine
2. Chimeric
3. humanized
4. human

12. a. Murine
• The first mAb to be discovered and reproduced was the murine monoclonal
antibody.
• This type of mAb arises from harvesting B lymphocytes from the spleen of a mice
and then fused with an immortal myeloma cell line lacking the hypoxanthine-
guanine-phosphoribosyl transferase (HPTR) gene.
• All of these mAbs are identified with a name that ends in -omab (ie,
muromonab-CD3, blinatumomab, capromab).
• Allergic reactions are common when used in humans and often result in the
induction of anti-drug antibodies.
• Murine mAbs also have a short half-life when used in humans because of a
relatively weak binding to the human FcR.
• For oncology, these mAbs may not be the most beneficial as they are “relatively
poor recruiters of effector function, antibody-dependent cellular cytotoxicity, and
complement-dependent cytotoxicity,” essential functions needed for tumor
destruction.

13. b. Chimeric
• Chimeric mAbs utilize the murine antigen-specific variable
region, but the remaining heavy and light chains are human.
This was accomplished using genetic engineering
techniques, which resulted in mAbs that are approximately
65% human and 35% murine.
• The chimeric mAbs are identified with names ending in
-ximab (ie, rituximab, infliximab, cetuximab).
• Compared with their murine counterparts, these mAbs
“exhibit an extended half-life in man and show reduced
immunogenicity, but nevertheless, the propensity of
chimeric mAbs to induce anti-drug antibodies is still
considerable”.

14. c. Humanized
• Humanized mAbs are created by grating the murine
hypervariable regions of the light and heavy chains onto
a human Ab framework. This results in molecules that
are approximately 95% human. This resulted in
decreased production of anti-drug antibodies. However,
the process to create these molecules is hard and has
limitations.
• These mAbs are identified with names ending in -
zumab (ie, trastuzumab, alemtuzumab,
bevacizumab).

15. d. Human
• With the development of new technology, fully human mAbs were
able to be created.
• These are created utilizing animals carrying human Ig genes. These
transgenes include parts of the variable regions, which enable the
recombination of the human antibodies.
• The animal’s own endogenous Ig genes have been inactivated,
enabling the generation of fully human mAbs.
• These mAbs are less antigenic and better tolerated compared with
the other classes of mAbs.
• Additionally, they appear to remain present in the human body’s
circulation compared with the other classes.
• These mAbs are identified with names ending in -umab (ie,
ofatumumab, daratumumab, denosumab).

16. There are three types of mAbs that are
dependent upon how they are administered or
used:
1. unconjugated or naked
2. conjugated
3. bispecific

17. 1. Unconjugated mAbs
• Unconjugated mAbs or “naked” mAbs are those antibodies
that function by themselves.
• These are the most commonly used in the treatment of cancer.
• In most cases, these molecules attach to antigens on cancer
cells. This can illicit one of several responses, which can be
that the person’s natural immune response against the cancer
cells is enhanced.
• The mAb attracts immune cells and aids in enhancing the
recognition of cancer cells by the immune system, leading to
increased apoptosis.
• Another mechanism is the targeting of immune system
checkpoints, while other unconjugated mAbs block antigens
on cancers that help them to expand and proliferate

18. 2. Conjugated mAbs
• When an mAb is combined with a chemotherapy agent
or with a radioactive particle it is referred to as a
conjugated monoclonal antibody.
• The mAb serves as a delivery mechanism for the
chemotherapy or radioactive particle, circulating
through the patient’s body until it finds the intended
target antigen.
• This method helps to minimize harm to normal cells by
the chemotherapy agents or radioactive particle attached
to the mAb.

19. 3. Bispecific mAbs
• This unique type of mAb is a combination of two
different mAbs which allows for the mAb to attach to
two different antigens at the same time.
• One target is the protein found on cancer cells, and the
other target is a protein found on immune cells.
• This combination allows for the immune cell and cancer
cell to be brought together, in hopes of inciting an
increased immune response and the destruction of
cancer cells.

20.  Production of monoclonal antibody
Basic steps involved in the production of a monoclonal antibody
1. Immunization (Immunize the animal)
2. Cell fusion process
3. Selection of hybridomas
4. Screening the products
5. Cloning and propagation
6. Characterization and storage

21. 1. Immunize the animal
The first step in hybridoma technology is to immunize an animal (mouse), with
appropriate antigen. The antigen, along with an adjuvant like Freund’s complete
or incomplete adjuvant is injected subcutaneously.
Three days prior to killing of the animal, a final dose of antigen is intravenously
administered.
The concentration of the desired antibodies is assayed in the serum of the animal
at frequent intervals during the course of immunization, when optimal the animal
is sacrificed.

22. The spleen is aseptically removed and disrupted by mechanical or
enzymatic methods to release the cells.
The lymphocytes of the spleen are separated from the rest of the cells by
density gradient centrifugation.

23. 2. Cell Fusion process
The thoroughly washed lymphocytes (spleen cell) are mixed with HGPRT
negative myeloma cells.
The mixture of cells is exposed to polyethylene glycol (PEG) for a short period
(a few minutes), since it is toxic
PEG is removed by washing and the cells are kept in a fresh medium. These cells
are composed of a mixture of hybridomas (fused cells), free myeloma cells and
free lymphocytes.

24. 3. Selection of Hybridomas
When the cells are cultured in HAT medium only the hybridoma cells grow, while the
rest will slowly disappear.
Selection of a single antibody producing hybrid cells is very important. This is possible
if the hybridomas are isolated and grown individually
The suspension of hybridoma cells is so diluted that the individual aliquots contain on
an average one cell each. These cells, when grown in a regular culture medium,
produce the desired antibody.

25. 4. Screening the Products
The hybridomas must be screened for the secretion of the antibody of desired
specificity
The culture medium from each hybridoma culture is periodically tested for the
desired antibody specificity.
In both the assays (ELISA, RIA) the antibody binds to the specific antigen
(usually coated to plastic plates) and the unbound antibody and other
components of the medium can be washed off

26. Thus, the hybridoma cells producing the desired antibody can be identified by
screening. The antibody secreted by the hybrid cells is referred to as monoclonal
antibody.

27. 5. Cloning and Propagation
The single hybrid cells producing the desired antibody are
isolated and cloned. Two techniques are commonly employed for
cloning hybrid cells:
 limiting dilution method
 soft agar method

28.  Limiting dilution method
In this procedure, the suspension of hybridoma cells is
serially diluted and the aliquots of each dilution are put
into micro culture wells. The dilutions are so made that
each aliquot in a well contains only a single hybrid
cell. This ensures that the antibody produced is
monoclonal.

29.  Soft agar method
In this technique, the hybridoma cells are cultured in
soft agar. It is possible to simultaneously grow many
cells in semisolid medium to form colonies. These
colonies will be monoclonal in nature. In actual
practice, both the above techniques are combined and
used for maximal production of Mabs.

31.  Characterization and Storage
• The monoclonal antibody has to be subjected to biochemical
and biophysical characterization. It is also important to
elucidate the MAb for the immunoglobulin class or sub-class,
the epitope for which it is specific and the number of binding
sites it possesses. The stability of the cell lines and the MAbs
are important. The cells (and MAbs) must be characterized
for their ability to withstand freezing, and thawing. The
desired cell lines are frozen in liquid nitrogen at several
stages of cloning and culture.

32.  Applications
 Therapeutic Applications:- Monoclonal antibodies have a wide
range of therapeutic applications. MAbs are used in the treatment of
cancer, transplantation of bone marrow and organs, autoimmune
diseases, cardiovascular diseases and infectious diseases.
The therapeutic applications of MAbs are broadly grouped into 2 types:
 Direct use of MAbs as therapeutic agents
 MAbs as targeting agents

33.  Direct use of MAbs as therapeutic agents
• Monoclonal antibodies can be directly used for enhancing the
immune function of the host. Direct use of MAbs causes
minimal toxicity to the target tissues or the host.
• In destroying disease-causing organisms: MAbs promote efficient
opsonization of pathogenic organisms (by coating with antibody)
and enhance phagocytosis. In fact, MAbs were found to protect
chimpanzees against certain viral (hepatitis B-virus) and bacterial
(E. coli Haemophilus influenza, Streptococcus sp and Pseudomonas
sp) infections.

34. • In the treatment of cancer: MAbs, against the antigens on the
surface of cancer cells, are useful for the treatment of cancer. The
antibodies bind to the cancer cells and destroy them. This is brought
out by antibody—dependent cell-mediated cytotoxicity,
complement-mediated cytotoxicity and phagocytosis of cancer cells
(coated with MAbs) by reticuloendothelial system.

35. • The patients suffering from leukemia, colorectal cancer, lymphoma
and melanoma have been treated with MAbs. However, there was a
wide variation in the success rate. A monoclonal antibody specific to
the cells of leukemia is used to destroy the residual leukemia cells
without affecting other cells. MAbs are used in vitro to remove the
residual tumor cells prior to autologous bone marrow transplantation
(transplantation of the patient’s own bone marrow cells, due to non-
availability of a suitable donor).

36. Limitations for direct use of MAbs in cancer:
1. The MAbs produced in mice and directly used for therapeutic
purposes may lead to the development of antimouse antibodies and
hypersensitivity reactions
2. All the cancer cells may not carry the same antigen for which MAb
has been produced. Thus, MAbs may not be attached to some cancer
cells at all.
3. The free antigens (of target cells) present in the circulation may bind
to MAbs and prevent them from their action on the target cells

37. • In the treatment of AIDS: Immunosuppression is the hall mark of
AIDS. This is caused by reduction in CD4 (cluster determinant
antigen 4) cells of T-lymphocytes. The human immunodeficiency
virus (HIV) binds to specific receptors on CD4 cells by using
surface membrane glycoprotein (gp120). Genetic engineers have
been successful to attach Fc portion of mouse monoclonal antibody
to human CD4 molecule. This complex has high affinity to bind to
membrane glycoprotein gp120 of virus infected cells.

38. • In the treatment of autoimmune diseases: Autoimmune
diseases like rheumatoid arthritis and multiple sclerosis are of
great concern. Some success has been reported in the clinical
trials of rheumatoid arthritis patients by using MAbs directed
against T-lymphocytes and B-lymphocytes.

39.  MAbs as Targeting Agents in Therapy
Toxins, drugs, radioisotopes etc., can be attached or conjugated
to the tissue-specific monoclonal antibodies and carried to target
tissues for efficient action. This allows higher concentration of
drugs to reach the desired site with minimal toxicity. In this way,
MAbs are used for the appropriate delivery of drugs or isotopes.

40. • MAbs in drug delivery:- In general, the drugs are less
effective in vivo (in the living body) when compared to in
vitro (in laboratory when tested with cultured cells). This is
mainly due to the fact that sufficient quantity of the drug does
not reach the target tissue. This problem can be - solved by
using tissue-specific MABs. MABs. The drugs can be
coupled with MAb (directed against a cell surface antigen of
the cells, say a tumor) and specifically targeted to reach the
site of action.

41. • MAbs in radio immunotherapy (RAIT): The radioisotopes
can be coupled to MAbs that are directed against tumor cells.
This allows the concentration of radioactivity at the desired
sites and a very efficient killing of target cells (tumor cells).
The advantage with radio immunotherapy is that conjugated
complex need not penetrate the cells, as is required in
immunotoxin therapy. The limitation is that the neighboring
normal cells may also get damaged or killed. This can be
minimized by using radioisotopes with short half-lives.
Yttrium-90 with a half-life of 64 hours is a suitable isotope to
be employed in RAIT. Due to shortage in the supply of
yttrium-90, indium-111 is more commonly used.

42.  ADVANTAGE
• HOMOGENEITY : Monoclonal antibody represents a single
antibody molecule that binds to an antigen with the same affinity
and promote the same effectors.
• SPECIFICITY: The product of a single hybridoma reacts with the
same epitope on antigen.
• SELECTION: It is possible to select for specific epitope
specificities and generated antibodies against a wider range of
antigenic determination.
• ANTIBODY PRODUCTION: Unlimited quantities of a single
well defined monospecific.

43.  DISADVANTAGE
1. AFFINITY: average affinity of monoclonal antibodies are
generally lower than polyclonal antibodies
2. Effector Function: because antibody is monoclonal, it may
produce the desire biologic response.
3. SPECIFICITY: Monoclonal against conformational
epitopes on negative proteins may lose reactivity with
antigens.
4. CROSS REACTION: Antibodies sometime display
unexpected cross reaction with unrelated antigens.

44. FDA approved MABs for Oncology

46.  Reference
• Talreja S, Pandey S, Kumar S. A review on Monoclonal
antibody and its application in biotechnology. Journal of
Pharmaceutical Sciences and Research. 2020;12(1):49-53.
• Bayer V. An overview of monoclonal antibodies. InSeminars
in oncology nursing 2019 Oct 1 (Vol. 35, No. 5, p. 150927).
WB Saunders.
• Modjtahedi H, Ali S, Essapen S. Therapeutic application of
monoclonal antibodies in cancer: advances and challenges.
British medical bulletin. 2012 Dec 1;104(1):41-59.