complete description about monoclonal antibodies...

2. Monoclonal Antibodies
GROUP: 09
PRESENTED TO: MA’AM HUMA
DILSHAD

3. INTRODUCTION & DISCOVERY
IMMUNE SYSTEM!!!
Latin term "IMMUNIS" means EXEMPT,
referring to protection against foreign
agents.
An integrated body system of organs,
tissues, cells & cell products that
differentiates self from non-self &
neutralizes potentially pathogenic
organisms.

4. CELLS OF IMMUNE SYSTEM

5. WHAT ARE ANTIBODIES OR
IMMUNOGLOBULIN ?
Immunoglobulin (Ig) are
structurally related
glycoproteins that
function as antibodies.
An antibody is a protein
used by the immune
system to identify &
neutralize foreign objects
like bacteria & viruses.

6. ANTIBODIES...
Monoclonal antibodies
(mAb) are identical
because they were
produced by one type
of immune cell, all
clones of a single
parent cell.
Polyclonal antibodies
are derived from
different cell lines.
They differ in amino
acid sequence.

7. DIFFERENCE!!!
Monoclonal
Derived from a single B cell clone.
mAb offer reproducible & potentially
inexhaustible supply of Ab with
exquisite specificity.
Enable development of secure
immunoassay systems.
Polyclonal
Derived from different B
lymphocytes cell lines.
Batch to batch variation affecting Ab
reactivity & titre.
NOT powerful tools for clinical
diagnostic tests.

8. DISCOVERY!!!
The idea of a "magic bullet"
was first proposed by Paul
Ehrlich at beginning of 20th
century.
He postulated that if a compound
could be made that selectively
targeted a disease-causing organism,
then a toxin for that organism could
be delivered along with agent of
selectivity.

9. DISCOVERY!!!
George Kohler & Cesar Milstein in
1975 shared the Nobel Prize in
Physiology or Medicine in 1984
for discovery of hybridoma
technology.

10. The structure of antibodies

11. Definitions
Fab = Fragment, antigen binding
Fc = Fragment, crystalline
The Fc fragment specifies other biological activities of the
molecule. For example, the Fc fragment may determine whether
the antibody simply prevents signaling through a receptor, or
alternatively, causes the cell’s destruction through complement
fixation or targeting immune effector cells.

12. HOW MONOCLONAL ANTIBODY WORKS??
Make the cancer cell more visible to the immune system. rituximab (Rituxan) attaches to a
specific protein (CD20) found only on B cells, one type of white blood cell. Certain types of
lymphomas arise from these same B cells. When rituximab attaches to this protein on the B cells, it
makes the cells more visible to the immune system, which can then attack.
Block growth signals. Cetuximab (Erbitux), a monoclonal antibody approved to treat colon
cancer and head and neck cancers, attaches to receptors on cancer cells that accept a certain
growth signal (epidermal growth factor). Blocking this signal from reaching its target on the
cancer cells may slow or stop the cancer from growing.
Stop new blood vessels from forming. bevacizumab (Avastin) targets a growth signal called
vascular endothelial growth factor (VEGF) that cancer cells send out to attract new blood vessels.
Bevacizumab intercepts a tumor's VEGF signals and stops them from connecting with their targets.
Deliver radiation to cancer cells. Ibritumomab (Zevalin), approved for non-Hodgkin's lymphoma,
combines a monoclonal antibody with radioactive particles. The ibritumomab monoclonal
antibody attaches to receptors on cancerous blood cells and delivers the radiation.
Deliver chemotherapy to cancer cells. Ado-trastuzumab emtansine (Kadcyla) is one such drug
approved to treat HER2-positive breast cancer. Ado-trastuzumab emtansine contains an antibody
that attaches to the HER2 receptors on the breast cancer cells. The cancer cells then ingest the
antibody, which releases a few molecules of chemotherapy.

13. THE TYPES OF MONO-CLONAL ANTIBODIES
1- According To Evolution 2- According to design
Throughout
the
progression of
monoclonal
drug
development
there have
been four
major
antibody
types
developed:
First
generation
murine
Second
generation
Chimeric
Humanised
human.
Naked
Monoclonal
Antibody
Conjugated
Monoclonal
Antibody
Immune-Toxin
Monoclonal
Antibody

14. Murine monoclonal antibodies (suffix -omab)
• Murine derived
from mice,
these proteins
are purified
after
immunization
with antigens
Major problems
associated with
murine
antibodies
included
reduced
stimulation
of cytotoxicity
and the
formation
complexes
after repeated
administration,
which resulted
in mild allergic
reactions and
sometimes ana
phylactic
shock.
Chimeric monoclonal antibodies (suffixes -ximab, -
zumab respectively)
• Chimeric
antibodies are
composed of
murine variable
regions fused
onto human
constant
regions.
Human gene
sequences,
taken from the
kappa light
chain and the
IgG1 heavy
chain, results in
antibodies that
are
approximately
65% human.
This reduces
immunogenicity
, and thus
increases seru
m half-life.
Humanized monoclonal antibodies (suffixes -zumab)
• Humanised
antibodies are
produced by
grafting murine
hypervariable
regions on
amino acid
domains into
human
antibodies. This
results in a
molecule of
approximately
95% human
origin.
Human monoclonal antibodies (suffix -umab)
• Human
monoclonal
antibodies are
produced by
transferring
human
immunoglobulin
genes into the
murine
genome, after
which the
transgenic
mouse
is vaccinated a
gainst the
desired
antigen,
leading to the
production of
monoclonal
antibodies

15. ‘Naked’ Monoclonal Antibodies

16. Conjugated/ labelled/ loaded
Monoclonal Antibodies
 Conjugated monoclonal antibodies are those joined to a
chemotherapy drug, radioactive particle, or cancer cell killing agent.
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
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.

17. Immune-Toxin Monoclonal
Antibody
• Involves the application of cancer
associated monoclonal
antibodies which are linked to a
drug-activating enzyme.
• Subsequent systemic
administration of a non-toxic
agent results in its conversion to
a toxic drug, and resulting in a
cytotoxic effect which can be
targeted at malignant cells
Conjugate
with pro-drug
or enzyme
• An Mab against the CD25
antigen on B cells (and
lymphomas)
• Conjugated to either the
radio-active iasotope indium-
111 or yittrium-90 for
treatment of lymphoma
patients.
Radio-immune
Antibody e.g.
ibritumomab

18. PRINCIPLE
Hybridoma Technology Creates Monoclonal
antibodieS
Hybridoma technology is used to produce a hybrid cell. These hybrid cells
are produced by fusing B-lymphocyte with tumour cell and they are called
as myeloma cells. Thus these hybrid cells have got the ability to produce
antibodies due to the B-lymphocyte genetic material and also capacity to
divide indefinitely in the culture due to the presence of tumour cell or
myeloma cells involved in the production of hybrid cells. Therefore, these
hybrid cells produced from hybridoma technology are cultured in
laboratory or passaged or subcultured using mouse peritoneal cavity and
these cells produces monoclonal antibodies, and this technology is called
as hybridoma technology.
Monoclonal antibodies are typically made by fusing
myeloma cell with the spleen cells from a mouse that has
been immunized with desire antigen. However recent
advances have allowed the use of rabbit B cells.

19. Inject the protein into the
mouse
Remove the spleen
Identify which spleen cells
are producing antibodies
Separates these cells and
grow in tissue culture tubes
containing HAT medium
Screen each antibody for
cross reactivity
Select antibody which does
not cross react with any
other protien
Principles in
Hybridoma
technology

20. Cont’d

21. •Mice 2 weeks old are immunized with the antigen against with
monoclonal antibodies are to be raised by subcutaneous injection.
Later B Cells are isolated from the spleen of an imunized mouse.
Myeloma cell are isolated from bone marrow. MYELOMA CELLS HAVE
LOST the ability to synthesize hypoxanthine-guanine phosphoribosyl
transferase (HGPRT), an enzyme necessary for the salvage synthesis of
nucleic acids, Which enables cells to synthesize purines by the salvage
pathway here using an extracellular source of hypoxanthine as a
precursor. Myeloma cells are immortalized cells that are cultured with 8-
azaguanine to ensure their sensitivity to the hypoxanthine-aminopterin-thymidine
(HAT) selection medium.1 week before cell fusion, myeloma
cells are grown in 8-azaguanine. Cells must have high viability and rapid
growth. The HAT medium allows only the fused cells to survive in culture.
Isolation Of B Cells
and myeloma
cells
•HAT medium is used for selection of hybride cell. Nucleotide
synthesis is essential for cell survival .in HAT medium ,aminopterine
block the synthesis of purine and pyrimidine from simple sugsar
(denovo pathway). But can thrive by utilizing hypoxanthine and
thymidine present in the medium by salvage pathway using the
enzyme hypoxanthine guanine phosphoribosyl transferace
(HGPRT)
Selection of
hybride cell in HAT
meduium (
hypoxanthine
aminopterine
thymidine)

22. How HAT medium work in
selection of hybride cell.
Here myeloma cell
are HGPRT deficient ,
so these cell can not
survive in HAT
medium as
aminopterine block
denovo pathway
B cells are HGPRT
+ and can survive
in HAT
medium.after
some cell division,
B cell undergo
normal cell death.
Hybride cell has
HGPRT enzymes
from B cells. So
only hybride cell
can survive in HAT
medium.

23. Unfused
normal
spleen cells
cannot grow
indefinitely
because of
their limited
life span
Unfused
myeloma
cells cannot
grow
because they
lack HGPRT.

24. •Process by which bulk
quantities of targeted antibodies
against a specific antigen are
produced.Monoclonal antibodies
are produced via
multiple/identical copies of a
certain cell called a hybridoma.
HYBRIDOMA
TECHNOLOGY

25. HGPRT=Hypoxanthineguanine
phosphoribosyltransferase

26. PRODUCTION OF MONOCLONAL ANTIBODY
INVOLVE’S:

27. Step 1: - Immunization Of Mice & Selection Of Mouse
Donor For Generation Of Hybridoma cells
The first step in making a hybridoma is
to generate antibody producing B
cells.This is done by immunizing a
mouse against the antigen of
interest.Intraperitoneal(IP)injections
are the most common method for
delivering the antigens in to mice.

28. Step 2: - Screening Of Mice For Antibody Production
It must be determined
if the mouse is
producing antibodies
of interest.Test bleeds
are performed and
examined for the
presence of
antibodies.
If the host is producing the
desired antibody,the spleen is
removed and dissociated in
culture medium to release the
resident B cells.The culture
medium also includes cells
from a special mouse myeloma
cell line.These tumor cells can
divide indefinitely,but do not
produce antibodies

29. Step 3: - Preparation of Myeloma Cells
+ 8 - Azaguanine
Immortal Tumor Of Lymphocytes
Myeloma Cells
HGPRT
Myeloma Cells

30. Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells
&
Selection of Hybridoma Cells
PEG
FUSION
HAT Medium
1. Plating of Cells in
HAT selective
Medium
2. Scanning of Viable
Hybridomas

31. STEP 5:- SEPARATION OF FUSED HYBRIDOMA
The next step is to separate
the fused hybridoma cells
from the unfused B cells
and myeloma cells.Unfused
B cells will die because they
lack the ability to survive in
culture.
CELL AND SCREENING
Surviving hybridomas are separated and
individually cultured—one cell per
well.These cells are described as clonal
cultures because all of the cells from
each well are derived from a single
cell,and are therefore identical,or clonal.

32. SCREENING
After culturing for a few weeks,when the growing
cells can be seen,the culture fluid can be
screened for the presence of desirable
antibodies.Screening is the most labor-intensive
step in the production of monoclonal antibodies
since fusion can result in thousands of individual
hybridoma colonies.Note that different antibodies
may react with different epitopes on the same
antigen.

33. Step 6: - Cloning of Hybridoma Cell Lines by “ Limiting
Dilution” or Expansion

34. DIAGNOSTIC APPLICATION OF
MONOCLONAL ANTIBODIES
Monoclonal antibodies are used widely in the
diagnostic laboratory.
Monoclonal antibodies allow rapid diagnosis of
hepatitis, influenza, herpes, streptococcal and
Chlamydia infections.
They are also very useful in immunohistochemistry,
which detect antigen in fixed tissue sections and
immunofluorescence test, which detect the substance
in a frozen tissue section or in live cells.

35. Diagnosis of HIV Infection
HIV antigen is attached to the plate.
Patients serum passed over the plate. Any HIV antibody in the patients
serum will attached to the antigen already on the plate.
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.
Chromagen dye is passed over the complex of concentrated HIV
antibody/conjugated antibody.
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.

36. Pregnancy Tests
A breakthrough in Diagnostics a monoclonal antibody
can be used to detect pregnancy in only 14 days after
conception.
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.

37. Pregnancy Tests

38. In animal disease diagnosis, they are very useful for
identification and antigenic characterization of pathogens.
They are also used in the diagnosis of lymphoid and myeloid
malignancies, tissue typing, enzyme linked immunosorbent
assay, radio immunoassay, serotyping of microorganisms,
immunological intervention with passive antibody,
antiidiotype inhibition, or magic bullet therapy with cytotoxic
agents coupled with anti mouse specific antibody.

39. Purification of proteins
Monoclonal antibodies can also be used
to purify a substance with techniques
called immuno-precipitation and affinity
chromatography
• The Western blot test and immuno dot blot tests
detect the protein on a membrane.
• . monoclonal antibodies, allow successful
diagnosis and treatment of human
neuroectodermal tumors
• MAbs were found extremely useful in the rapid
outbreak of East Coast Fever (ECF)

40. Therapeutic Uses
Monoclonal antibodies are protein molecules made in
the laboratory from hybridoma cells (stable cell lines
derived by fusing antibody‐producing cells from
immunised animals with cells that confer immortality
and high‐yield antibody production) or by recombinant
deoxyribonucleic acid (DNA) technology.
They are originally derived from the immune system
and have a number of unique properties that have
stimulated their use in medicine, including the ability to
bind specifically and with high affinity to
almost any molecular structure.

41. Therapeutic Uses
They can be made in various expression systems such as bacteria or
mammalian cells and modified for enhanced affinity, immune
function or half‐life in blood.
They are made in a homogeneous and reproducible form that allows
comparisons across laboratories for diagnosis as well as
therapeutics. Current major therapeutic applications of monoclonal
antibodies include cancer, chronic inflammatory disease, and
infection and they constitute the largest and fastest growing sector
of the biological pharmaceutical industry.

42. Therapeutic Uses
Monoclonal antibodies are proteins with high specificity towards
targets.
Monoclonal antibodies have multiple utilities in therapy as they can
recognise specific structures in targets such as bacteria, viruses, cancer
cells, etc.
Monoclonal antibodies can be produced in large amounts in
homogeneous and reproducible form for diagnostic and therapeutic
purposes.
Monoclonal antibodies have a clear regulatory path for their approval
as therapeutics.

43. Therapeutic Uses
Monoclonal antibodies can be derived from B‐cells from
immunised animals, humans with autoimmune diseases and de
novo by phage display.
Therapeutic monoclonal antibodies can be engineered to be
more human‐like proteins, to increase their affinity, reduce their
immunogenicity and increase their half‐life in the circulation, and
can be conjugated with toxin for better lytic effect.
Monoclonal antibodies can mediate antibody‐mediated
cytotoxicity by linking the target cells to cytotoxic cells through
their binding sites and Fc sites.

44. New Research
Researchers have discovered a unique monoclonal antibody that can
effectively reach inside a cancer cell, a key goal for these important
anticancer agents, since most proteins that cause cancer or are associated
with cancer are buried inside cancer cells. Scientists from Memorial Sloan-
Kettering Cancer Center and Eureka Therapeutics have collaborated to create
the new human monoclonal antibody, which targets a protein associated
with many types of cancer and is of great interest to cancer researchers.
Unlike other human therapeutic monoclonal antibodies, which
can target only proteins that remain on the outside of cancer
cells, the new monoclonal antibody, called ESK1, targets a
protein that resides on the inside of the cell.

45. The ESK1 monoclonal antibody was engineered to recognize
WT1 peptides brought to the surface of cancer cells.

46. SIDE EFFECTS OF MONOCLONAL ANTIBODIES:
All treatments have side effects. Some side effects depend on the type
of cell the MAB is targeting. They may also depend on whether the
MAB has a drug or radioactive substance attached to it.Monoclonal
antibodies are given intravenously (injected into a vein). The antibodies
themselves are proteins, so giving them can sometimes cause
something like an allergic reaction.
The most common side effect of all monoclonal antibodies is an
allergic reaction to the drug. This reaction is most likely to happen when
you first have the treatment. You will have paracetamol and an
antihistamine drug before you have the treatment to prevent a
reaction. If you have a reaction, your doctor or nurse can usually
control it by slowing down or stopping the drip for a while.

47. CONT;
• * Chills * fever * an itchy rash *
feeling sick * breathlessness
• * Wheezing
• * Headaches
• * Flushes and faintness
• * Changes in blood pressure
• * Nausea
• * Diarrhea
An allergic
reaction can
include these
symptoms,
though you
may not
have all of
them

48. SERIOUS SIDE EFFECTS
Serious, but rare, side effects of monoclonal antibody therapy may
include:
• * Infusion reactions. Severe allergy-like reactions can occur and, in very
few cases, lead to death. You may receive medicine to block an allergic
reaction before you begin monoclonal antibody treatment. Infusion
reactions usually occur while treatment is being administered or soon
after, so your health care team will watch you closely for a reaction.
• * Dangerously low blood cell counts. Low levels of red blood cells, white
blood cells and platelets may lead to serious complications.
• * Heart problems. Certain monoclonal antibodies may cause heart
problems, including heart failure and a small risk of heart attack.
• * Skin problems. Sores and rashes on your skin can lead to serious
infections in some cases. Serious sores can also occur on the tissue that
lines your cheeks and gums (mucosa).
• * Bleeding. Some of the monoclonal antibody drugs are designed to stop
cancer from forming new blood vessels. There have been reports that
these medications can cause bleeding.

49. Compared with chemotherapy drugs, naked mabs tend
to have fewer serious side effects. But they can still cause
problems in some people. Some mabs can have side
effects that are related to the antigens they target. For
example:
•* Bevacizumab (avastin®) is an mab that targets a protein called VEGF
that affects tumor blood vessel growth. It can cause side effects such
as high blood pressure, bleeding, poor wound healing, blood clots,
and kidney damage.
•* Cetuximab (erbitux®) is an antibody that targets a cell protein called
egfr, which is found on normal skin cells (as well as some types of
cancer cells). This drug can cause serious rashes in some people.
•Conjugated antibodies can be more powerful than naked mabs, but
they can also cause more side effects. The side effects depend on
which type of substance they’re attached to.

50. Advantages
Homogeniety: monoclonal antibody represents a single antibody molecule that
binds to antigen with the same affinity and promote the same effectors function
Specificity: the product of a single hybridoma reacts with the same epitope on
antigens
Immunizing agent: need to be characterized and is ultimately not needed in large
quantities of antibody
Selection: it is impossible to select for specific epitope specificities and generate
antibodies against a wide range of antigenic determinants
Antibody production: unlimited quantities of a single well defined monospecific
reagent

51. Dis- Advantages
Affinity: monoclonal antibodies average affinity is lower than polyclonal antibodies
Specificity: monoclonal against conformational epitopes on native proteins may lose reactivity
with antigen
Cross reactions: antibodies sometimes display unexpected cross reactions with un-related
antigen
Effector function: antibody is monoclonal, it may not produce the desired biological response
Time and effort commitment is very large
Immune rejection

52. Research 1
Recent advances in molecular biology have lead
to the ability to generate molecules with the
recognition properties of antibodies by
completely in vitro processes.
In addition to being more humane, molecules
such as recombinant antibodies rAb and
aptamers have certain advantages over animal-generated
antibodies, including the fact that
they can be made to recognize a wider variety of
targets with selected specificity and affinity.

53. Recombinant antibody engineering involves
the use of viruses or yeast to create
antibodies, rather than using mice.
Advances in molecular biology have lead to
the ability to synthesize antibodies de novo in
vitro – completely without the use of animals.

54. • (1) Creation of an antibody gene
library.
• (2) Display of the library on phage
or cell surfaces.
• (3) Isolation of antibodies against
the antigen of interest.
• (4) Modification of the isolated
antibodies; and (5) Scaled up
production of selected antibodies in
a cell culture expression system.
General
production
methods for
the
manufacture
of non-animal
recombinant
antibodies can
be broken
down into five
general steps:

55. Research 2
Classical therapeutic modalities such as surgery,
radiation, and chemotherapy not only fail to cure the
great majority of malignant tumors, but their
employment often leads to severe and debilitating
side effects.
The severe cancer related morbidity is also in direct
correlation with the use of x-radiation and
chemotherapy, making them less than ideal forms of
therapy.

56. The development of hybridoma
technology and the advances in
monoclonal antibody (MoAB) production
have revitalized the initial concept of
Ehrlich concerning the existence of cancer
cell-targeted, specific "magic bullets".
Entirely new approaches to cancer therapy
that are neoplastic cell-directed, and
specifically lethal to malignant cells and
less toxic to normal tissues are being
observed and developed, adhering to the
old prayer: "Destroy the diseased tissues,
preserve the normal."

57. • Immune reaction directed
destruction of cancer cells.
• Interference with the growth and
differentiation of malignant cells.
• Antigen epitope directed transport
of anti-cancer agents to malignant
cells.
• Anti-idiotype vaccines.
• Development of engineered
(humanized) mouse monoclonals
for anti-cancer therapy.
Immunotherapy
as a modality of
cancer therapy
has already been
developed and
proven to be
quite effective.
Strategies for the
employment of
antibodies for
anti-cancer
immunotherapy
include:

58. In addition, a variety of different agents (e.g.
toxins, radionuclides, chemotherapeutic
drugs) have been conjugated to mouse and
human MoABs for selective delivery to cancer
cells.
Finally, we propose that MoAB-based
immunotherapy be accepted as a conventional
form of therapy and employed not only in
terminal cancer patients but also, for instance,
during and following surgical resection.