Immunology and antibody.... Engg. ppt.ppt

Immunology

1. Monoclonal antibodies

2. THE IMMUNE SYSTEM
DEFINITION: -The integrated body system of organs, tissues,
cells & cell products that differentiates self from non –self &
neutralizes potentially pathogenic organisms.
(The American Heritage Stedman's Medical Dictionary)
The Latin term “IMMUNIS”means EXEMPT, referring to
protection against foreign agents.
The Immune System consists of
1.Innate Immunity Primary Response
2.Acquired Immunity Secondary Response

3. CELLS OF THE IMMUNE SYSTEM

4. BECOMES
FUNCTIONING OF THE IMMUNE SYSTEM
HUMORAL (ANTIBODY MEDIATED) IMMUNE RESPONSE CELL MEDIATED IMMUNE RESPONSE
ANTIGEN (1
ST
EXPOSURE)
ANTIGENS
DISPLAYED
BY
INFECTED
CELLS
ACTIVATE
CYTOTOXIC
T CELL
GIVES RISE TO
ACTIVE
CYTOTOXIC T
CELL
ENGULFED BY
STIMULATES
MACROPHAGE
APC
HELPER
T CELLS
STIMULATES
MEMORY
HELPER T
CELLS
MEMORY
T CELLS
MEMORY
B CELLS
PLASMA
CELLS
STIMULATES STIMULATES
B CELLS
FREE
ANTIGENS
DIRECTLY
ACTIVATE
STIMULATES
GIVES RISE TO
SECRETE ANTIBODIES
STIMULATES
ANTIGEN (2
nd
EXPOSURE)
STIMULATES

5. IMMUNOTHERAPY
Treatment of the disease by Inducing, Enhancing or
Suppressing the Immune System.
Active Immunotherapy: -
It stimulates the body’s own
immune system to fight the
disease.
Passive Immunotherapy: -
It does not rely on the body to
attack the disease, instead they
use the immune system
components ( such as
antibodies) created outside the
body.

6. Lymphocytes
Produce antibodies
B-cellsmature in bone marrowthen concentrate in
lymph nodes and spleen
T-cellsmature in thymus
B and T cells mature then circulate in the blood and
lymph
Circulation ensures they come into contact with
pathogens and each other

7. B -Lymphocytes
There are approximately 10 million different B-
lymphocytes, each of which make a different antibody
at any given time in the body.
The huge variety is caused by genes coding for abs
changing slightly during development.
There are a small group of clones of each type of B-
lymphocyte

8. B -Lymphocytes
At the clone stage antibodies do not leave the B-
cells.
The abs are embedded in the plasma membrane of
the cell and are
called antibody receptors.
When the receptors in the membrane recognise
and antigen on the surface of the pathogen the B-
cell divides rapidly.
The antigens are presented to the B-cells by
macrophages

9. B -Lymphocytes

10. B -Lymphocytes
Some activated B cells PLASMA CELLSthese
produce lots of antibodies, < 1000/sec
The antibodies travel to the blood, lymph, lining of gut
and lungs.
The number of plasma cells goes down after a few
weeks
Antibodies stay in the blood longer but eventually
their numbers go down too.

11. B -Lymphocytes
Some activated B cells MEMORY CELLS.
Memory cells divide rapidly as soon as the antigen is
reintroduced.
There are many more memory cells than there were
clone cells.
When the pathogen/infection infects again it is
destroyed before any symptoms show.

12. What are antibodies
An antibodyis 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 antibodiesare 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

13. The structure of antibodies
http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html

14. Antibodies
Also known as immunoglobulins
Globular glycoproteins
The heavy and light chains are polypeptides
The chains are held together by disulphide bridges
Each ab has 2 identical ag binding sites –variable regions.
The order of amino acids in the variable region
determines the shape of the binding site

15. How Abs work
Some act as labelsto identify
antigens for phagocytes
Some work as antitoxinsi.e. they block toxins for e.g.
those causing diphtheria and tetanus
Some attach to bacterial flagella making them less active
and easier for phagocytes to engulf
Some cause agglutination (clumping together)of bacteria
making them less likely to spread

16. ANTIBODIES
STRUCTURE CLASS

17. Type Number of
ag binding
sites
Site of actionFunctions
IgG 2 •Blood
•Tissue fluid
•CAN CROSS
PLACENTA
•Increase
macrophage activity
•Antitoxins
•Agglutination
IgM 10 •Blood
•Tissue fluid
Agglutination
IgA 2 or 4 •Secretions (saliva,
tears, small intestine,
vaginal, prostate,
nasal, breast milk)
•Stop bacteria
adhering to host
cells
•Prevents bacteria
forming colonies on
mucous membranes
IgE 2 Tissues •Activate mast cells
HISTAMINE
•Worm response

18. ANTIBODIES
Derived from different B
Lymphocytes cell lines
POLYCLONAL. MONOCLONAL.
Derived from a single B cell
clone
Batch to Batch variation
affecting Ab reactivity &
titre
mAb offer Reproducible,
Predictable & Potentially
inexhaustible supply of Ab
with exquisite specificity
Enable the development of
secure immunoassay systems.
NOT Powerful tools for
clinical diagnostic tests

19. History of Mab development
1890 Von Behring and kitasato discovered the serum of vaccinated persons
contained certain substances, termed antibodies
1900 Ehrlich proposed the “ side-chain theory”
1955 Jerne postulated natural selection theory. Frank Macfarlane Burnet
expended.
Almost the same time, Porter isolated fragment of antigen binding (Fab) and
fragment crystalline (Fc) from rabbit y-globulin.
1964 Littlefield developed a way to isolate hybrid cells from 2 parent cell lines
using the hypoxanthine-aminopterin-thymidine (HAT) selection media.
1975 Kohler and Milstein provided the most outstanding proof of the clonal
selection theory by fusion of normal and malignant cells
1990 Milstein produced the first monoclonal antibodies.

20. PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY

21. PRODUCTION OF MONOCLONAL ANTIBODY
Step 1: -Immunization Of Mice & Selection Of Mouse
Donor For Generation Of Hybridoma cells
HYBRIDOMA TECHNOLOGY
ANTIGEN ( Intact cell/
Whole cell membrane/
micro-organisms) +
ADJUVANT
(emulsification)
Ab titre reached in Serum
Spleen removed
(source of cells)

22. PRODUCTION OF MONOCLONAL ANTIBODY
Step 2: -Screening Of Mice For Antibody Production
HYBRIDOMA TECHNOLOGY
After several
weeks of
immunization
Serum Antibody Titre Determined
(Technique: -ELISA / Flow cytometery)
Titre too low
BOOST
(Pure antigen)
Titre High
BOOST
(Pure antigen)
2 weeks

23. PRODUCTION OF MONOCLONAL ANTIBODY
Step 3: -Preparation of Myeloma Cells
HYBRIDOMA TECHNOLOGY
Immortal Tumor Of Lymphocytes
+
8 -Azaguanine
Myeloma Cells
High Viability & Rapid Growth
HGPRT
-
Myeloma Cells

24. PRODUCTION OF MONOCLONAL ANTIBODY
Step 4: -Cloning of Hybridoma Cell Lines by “ Limiting
Dilution” or Expansion
HYBRIDOMA TECHNOLOGY
A. Clone Each +ve Culture
B. Test Each Supernatant for Antibodies
C. Expand +ve Clones
Mouse
Ascites
Method
Tissue
Culture
Method

25. PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY

28. The types of mAb designed
A.Murine source mAbs: rodent mAbs with excellent affinities and
specificities, generated using conventional hydrioma technology.
Clinical efficacy compromised by HAMA(human anti murine
antibody) response, which lead to allergic or immune complex
herpersensitivities.
B.Chimeric mAbs: chimers combine the human constant regions with
the intact rodent variable regions. Affinity and specificity unchanged.
Also cause human antichimeric antibody response (30% murine
resource)
C.Humanized mAbs: contained only the CDRs of the rodent variable
region grafted onto human variable region framework

29. EVOLUTION OF MONOCLONAL ANTIBODY
1. TRANSGENIC
DNA SPLICING / GENE KNOCK
OUT
2. LIBRARIES
a.BACTERIOPHAGE
b. mRNA
c. Cell Surface

30. ENGINEERED ANTIBODIES

32. Applications of Monoclonal
Antibodies
•Diagnostic Applications
Biosensors & Microarrays
•Therapeutic Applications
Transplant rejectionMuronomab-CD3
Cardiovascular diseaseAbciximab
CancerRituximab
Infectious DiseasesPalivizumab
Inflammatory diseaseInfliximab
•Clinical Applications
Purification of drugs, Imaging the target
•Future Applications
Fight against Bioterrorism

33. Chemotherapy
Shortcomings:
A.Nature of cytotoxin
B.Lack of in vivoselectivity
C.The mechanism of anti-proliferation on cells cycle, rather than specific
toxicity directed towards particular cancer cell
D.Host toxixity: treatment discontinued, most of them had bad side-effects,
such as no appetites, omit, lose hair

34. Monoclonal antibodies for cancer treatment
Three mechanisms that could be responsible for the cancer treatment.
A.mAbs act directly when binding to a cancer specific antigen and induce
immunological response to cancer cells. Such as inducing cancer cell
apoptosis, inhibiting growth, or interfering with a key function.
B.mAbs was modified for delivery of a toxin, radioisotope, cytokineor other
active conjugates.
C.it is also possible to design bispecificantibodies that can bind with their
Fab regions both to target antigen and to a conjugate or effector cell

35. 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.
Carter P: Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer
2001;1:118-129

36. Dale L Ludwig, etal. Oncogene(2003) 22, 9097-9106
Strategy of a direct or in direct induction
of apoptosis in targeted cancer cells
1.mAbs target growth factor receptors
to exert a direct effect on the growth
and survival of the cancer cells by
antagonizing ligand-receptor
signaling.
2.mAbs can target to cell surface
antigens and directly elicit apoptotic
signaling.

37. 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

38. 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