1. ABDUL MUHEEM
M.PHARMA 2ND SEM
(PHARMACEUTICS)
JAMIA HAMDARD

2. Contents

 INTRODUCTION
 STRUCTURE OF ANTIBODIES
 HISTORY OF AB
 ANTIGEN-AB BINDING
 PRINCIPLE INVOLVED FOR MAB
 MAB FOR TUMOUR TARGETING
 LIMITATION
 REFERENCES

3. 
HOW THE ANTIBODIES ARE
PRODUCED?
3

4. INTRODUCTION

 Antibodies are produced by a specialized group of cells
called B-Lymphocytes.
 When an foreign antigen enters the body due immune
response B-Lymphocytes develops into plasma cells and
liberates antibodies or immunoglobulins of various
types(Ig A, Ig D, Ig E, Ig G, Ig M).
4

5. Classes of Igs
IgG: IgG1 (66%), IgG2 (23%), IgG3 (7%) and IgG4

(4%) , blood and tissue liquid
IgA: IgA1 (90%) and IgA2 (10%), external secretions
(stomach, intestines, saliva, tears, etc.)
IgM: 5-10% of total serum Ig [1.5mg/ml serum
conc.]
IgD: 1% of proteins in the plasma membranes of B-
lymphocytes, function unknown [30µg/ml serum
conc.] 0.2% of total serum Ig
IgE: 0.3µg/ml on the surface of plasma membrane of
mast cells, play a role in immediate hypersensitivity
and denfense for parasite

6. 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. They differ in amino acid
sequence.

7. 
• Each Antigen has specific antigen determinants
(epitopes) located on it. The antibodies have
complementary determining regions (CDRs). These
are mainly responsible for the antibody specificity.
• Each antigen has several different epitopes on it.
They are recognised by many different antibodies.
All these antibodies thus produced act on the same
antigen. Hence these are designated as polyclonal
antibodies.
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8. 
• In general naturally produced antibodies are non-
specific and heterogenous in nature. Hence there are
several limitations in the use of polyclonal antibodies
for therapeutic and diagnostic purposes.
• Thus there is a need for producing monoclonal
antibodies for different antigens.
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9. • 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.

10.  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.

11. 
Georges Köhler César Milstein, and Niels Kaj Jerne in
1975 who shared the Nobel Prize in Physiology or
Medicine in 1984 for the discovery hybridoma technology

12. The structure of antibodies

 http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html

13. Structure of MAb

13

14. Antigen- antibody
binding

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15. Types of mAbs designed
• Murin source mAbs:: rodent mAbs with excellent

affinities and specificities, generated using conventional
hybridoma technology. Clinical efficacy compromised by
HAMA (human anti murine antibody) response, which
lead to allergic or immune complex hypersensitivities.
• 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)
• Humanized mAbs:: contained only the CDRs of the
rodent variable region grafted onto human Framework
Regions [FR]

16. Types of mAbs designed . .
 Human mAb :: three currently available approaches to the

production of human monoclonal antibodies are described. These
include :-
 the hybridoma technique, based on the fusion of antibody-
producing human B lymphocytes with either mouse or human
myeloma or lymphoblastoid cells;
 the EBV immortalization technique, based on the use of Epstein-
Barr virus (EBV) to immortalize antigen-specific human B
lymphocytes;
 the EBV-hybridoma technique, based on a combination of the first
two methods.
The EBV-hybridoma system retains the advantageous features of the
other two systems while overcoming their pitfalls and may be the
current method of choice for producing human monoclonal
antibodies with a defined specificity.

17. 

18. 
 Hybridoma technology: In this B-Lymphocytes and
myeloma cells are mixed together and exposed to
PEG for a short period.
 The mixture contains hybridoma cells, myeloma cells
and lymphocytes.
 This mixture is washed and cultured in
HAT(hypoxanthine aminopterin and thymidine)
medium for 7-10 days.
 only hybridoma cells remain in the mixture.
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19. Immunise
Spleen Cell Myeloma Cell Line
HAT sensitive
FUSE
Hybridoma
HAT resistant
SELECT
Stable hybrid myeloma producing desired antibody 19

20. Nomenclature of mAbs


21. 
 Immunization
 Cell fusion
 Selection of hybridomas
 Screening the products
 Cloning and propagation
 Characterization and storage
21

22. 
 Immunize an animal usually mouse by injecting with an
appropriate antigen along with Freund’s complete or incomplete
adjuvant.
 Adjuvants are non specific potentiators of specific immune
responses.
 Injection of antigens at multiple sites are repeated several times
for increased stimulation of antibodies.
 3 days prior to killing of animal a final dose is given
intravenously.
 Spleen is aseptically removed and disrupted by mechanical or
enzymatic methods to release the cells.
 By density gradient centrifugation lymphocytes are separated
from rest of the cells.
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23. 
 Lymphocytes are mixed with HGPRT deficient
myeloma cells and is exposed to PEG for a short
period.
 The mixture is then washed and kept in a fresh
medium.
 The mixture contains hybridomas, free myeloma
cells, and free lymphocytes.
23

24. Dihydrofolate
 Aminopterin
Tetrahydrofolate
Precursors
Nucleotides---->DNA
Hypoxanthine
Thymidine
24

25. 
 The above mixture is cultured in HAT medium for 7-10 days.
 Due to lack of HGPRT enzyme in myeloma cells, salvage
pathway is not operative and aminopterin in HAT medium
blocks the de novo synthesis of nucleotides. Hence free
myeloma cells are dead.
 As the lymphocytes are short lived they also slowly dissappear.
 Only the hybridomas that receives HGPRT from lymphocytes
are survived.
 Thus hybridomas are selected by using HAT medium
 Suspension is diluted so that each aliquot contains one cell each.
These are cultured in regular culture medium, produced desired
antibody.
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26. 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.
1. Membrane binded cell culture process
1. Microcarrier cell culture process
1. Suspended cell culture process

27. 
 Screening is done for antibody specificity.
 For this we need to test the culture medium from each
hybridoma culture for desired antibody specificity.
 Common tests like ELISA and RIA are used for this.
 In these tests the antigens are coated to plastic plates. The
antibodies specific to the antigens bind to the plates. The
remaining are washed off.
 Thus the hybridomas producing desired antibodies are
identified. The antibodies secreted by them are
homogenous and specific and are referred as monoclonal
antibodies.
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28. 
 The single hybrid cell producing the desired antibody are
isolated and cloned.
 Usually two techniques are commonly employed for this
 a) Limiting dilution method: Suspension of hybridoma
cells is serially diluted so the aliquot of each dilution is
having one hybrid cell. This ensures that the antibody
produced is monoclonal.
 b) Soft agar method: In this method the hybridoma cells
are grown in soft agar. These form colonies and the
colonies are monoclonal in nature.
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29. 
 Biochemical and biophysical characterization are
made for desired specificity.
 It is important to note the monoclonal antibody is
specific for which antigen
 MAbs must be characterized for their ability to
withstand freezing and thawing.
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30. 
 Encapsulating the hybridoma cells in alginate gels
and using a coating solution containing poly-lysine is
employed.
 These gels allow the nutrients to enter in and
antibodies to come out.
 Damon biotech and cell-tech companies are using this
technique for commercial production of MAbs.
 They employ 100-litres fermenters to yield about 100g
of MAbs in about 2 weeks period.
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31. 
 MAbs derived from mouse are murine derivatives.
As they are not human origin, they show
HAMA(human antimouse antibody) response.
 To overcome this we need to cleave the antibody into
its respective Fc and Fab fragments.
 Fab fragments are less immunogenic and their
smaller molecular size may facilitate penetration into
tumor tissue and result in a longer half-life.
 Engineering is needed to reduce the immunogenicity.
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32. 
Chimeric antibodies:
 Hence the murine antibodies are immunogenic to
humans, the obvious solution for this is to clone a
fully human antibody. But it has many problems like
ethical clearance, difficult to culture, impossible to
obtain many of the appropriate antibodies.
 To over come HAMA(human antimouse antibody)
response, a chimeric antibody is prepared with Fc
region of human IgG and Fab regions of murine
origin by the use of DNA recombinant technology.
32

33. V domains
Mouse
Chimeric
Human
C domains
33

34. 
Humanized antibodies:
 Though chimeric antibodies elicit less HAMA response
than murine antibodies, they are still immunogenic due to
their murine regions(30%)
 It is came to know that a small portion(CDR) of an
antibody was actually responsible for antigen binding.
 By this humanized antibodies are prepared by
recombinant DNA technology with majority of human
antibody framework and CDR’s of murine antibody.
 Thus humanized antibodies are 95% homology with
human antibodies.
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35. hypervariable
Mouse
Humanised
Human
framework
35

36. 
Bispecific antibodies:
 These are specific to two types of antigens.
 They are constructed by r.DNA technology.
 Each arm is specific to one type of antigen.
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37. 
Immunoconjugate:
 For MAb targeted drug delivery, a drug is bound
covalently to an antibody that is chosen to target it to the
desired site of action.
 Spacer is present between the antibody and the drug.
 Polymer may be present to increase the no. of drug
molecules attached to the antibody.
 Drug is non-covalently incorporated into a liposome or
microsphere to which the targeting antibody is bound to
the surface—immunoliposome or immunomicrosphere
resp.
37

38. 
 Principle involved:
 As several classes of the drugs lack specificity for diseased
cells, they show their action on other sites of action.
Ex: cytotoxic action of chemotherapeutic agents is directed
against any rapidly proliferating cell population.
 Hence drug targeting is required to overcome this
problem.
 Targeting is classified into three categories:
1. Passive targeting
2. Physical targeting
3. Active targeting
38

39. 
 It is the natural in-vivo distribution pattern of the
drug delivery system. It is determined by the
inherent properties of the carrier like hydrophobic
and hydrophilic surface characteristics, particle
size, surface charge, particle number.
Ex: passive targeting of the lungs is made by
modulating the size of the particles to >7µm
passive targeting of the Reticuloendothelial system is
made by modulating the size of the particles to 0.2-
7µm
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40. 
 In this some characteristics of the environment are
utilized for the carrying of the drug to the specific
site.
Ex: thermal sensitive liposomes(local hyperthemia)
magnetically responsive albumin microspheres
(localized magnetic field)
40

41. 
 Active targeting is usually done by cell-specific
ligands. These are specific to specific cell types. But it
is limited to small no. of tumor types.
 Hence MAb targeting is adopted for active targeting.
MAb targeting is done by conjugating the drug
antibody of the specific targeting type.
 Hence antibody drug conjugates are used as active
targeting drug delivery systems.
41

42. 
 Toxin conjugates (immunotoxins)
EX: diphtheria toxin, Ricin have been conjugated to
the tumor specific antibodies
Ricin has two chains. Amoung these A-chain is
cytotoxic and B-chain is non-specific. Hence B-chain
is removed and the toxin is conjugated to tumor
specific antibody. Thus we increase the specificity of
the toxins by using MAbs as active drug targeting
systems.
42

43. 
 Drug immunoconjugates:
Agents like chlorambucil, methotrexate and
doxorubicin are conjugated with tumor specific
antibodies.
Ex: doxorubicin-BR96 immunoconjugate for Lewis
antigen found on the surface of tumor cells.
43

44. 
 They are homogenous in nature.
 They are specific to a particular antigen with a
particular epitope.
Ex:Rituximab (Rituxan®, anti-CD20) is a good
example – this antibody is used for the treatment of
lymphoma.
44

45. 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

46. Strategy of a Direct or Indirect
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.
1. mAbs can target to cell surface antigens and directly
elicit apoptotic signaling.
Dale L Ludwig, et. al. Oncogene(2003) 22, 9097-9106

47. FDA Approval

The first approved mAbs was OKT-3 [1986], which is a
murine IgGa2 protein to deplete T cells in patients with
acute rejection of renal allotransplant.
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.
Jancie, M Recheit, etal. Nature biotechnology, 2005, Sep,Vol. 23, No.9
Stamatis-Nick C. J Allergy Clin. Immunol, Oct. 2005

48. OKT3
 Prevents acute
rejection of kidney
transplants
Prevents
autoimmune
destruction of islet
cells in type I
Diabetes mellitus

51.  Cell Depletion
Rituxan, Campath (naked)
Myelotarg (drug)

Zevalin, Bexxar (radioisotope)
 Blocking receptors
Herceptin
 Attacking vasculature
Avastin, Erbitux
 Vaccination against idiotype
Panorex?
51

52. 
 As they are specific to a particular antigen, they cannot distinguish
molecule as a whole.
 Some times they cannot distinguish groups of different molecules.
Ex:- presence of retro viruses as a part of mammalian chromosomes
is not distinguished.
 Mice used in MAb production carry Adenovirus, Hepatic virus,
Retrovirus, reovirus, cytomegalovirus, thymic virus.
 The presence of some of these viruses is detected in hybridomas.
This poses a great danger since there is no guarantee for MAb
produced is totally virus free.
 For this reason US food and drug administration insists that MAb
for human use should be totally free from all pathogenic organisms
including viruses.
52

53. Latest News
North Carolina-based  has established a joint
PPD
venture with Taijitu Biologics to develop and
commercialize a technology platform for the
discovery of first- and best-in-class monoclonal
antibody therapies in collaboration with MAB
Discovery GmbH in Munich. The joint venture
will provide drug discovery services based on this
technology platform to global biopharmaceutical
companies, enabling them to discover best-in-class
monoclonal antibodies against both novel and
validated targets.

54. 
 Biotechnology by U. Satyanarayana
 Harper's Biochemistry 26th edition
 Carter P: Improving the efficacy of antibody-based cancer
therapies. Nat Rev Cancer 2001;1:118-129
 Dale L Ludwig, et. al. Oncogene(2003) 22, 9097-9106
 Jancie, M Recheit, etal. Nature biotechnology, 2005, Sep,Vol. 23,
No.9 Stamatis-Nick C. J Allergy Clin. Immunol, Oct. 2005
 www.wikipedia.com
 www.google.com
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55. 
THANK
U
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