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1
 Antibodies are produced by a specialized group of cells called B-
Lymphocytes.
 When a foreign antigen enters the body due
to immune response B-Lymphocytes develops
into plasma cells , liberate antibodies or
immunoglobulin's of various
types(Ig A, Ig D, Ig E, Ig G, Ig M).
2
 Monoclonal antibodies (mAb) are antibodies that are identical
because they are 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.
3
4
 Monoclonal antibodies are identical immunoglobulins, generated from a
single B-cell clone.
 These antibodies recognize unique epitopes, or binding sites, on a single
antigen.
5
 Murine monoclonal antibodies
 Chimeric monoclonal antibodies
 Humanized monoclonal antibodies
 Human monoclonal antibodies
6
7
8
 Specificity
 Side effects can be treated and reduced by using mice-human hybrid cells or by
using fractions of antibodies.
 They bind to specific diseased or damaged cells needing treatment.
 They treat a wide range of conditions.
9
 Time consuming project - anywhere between 6 -9 months.
 Very expensive and needs considerable effort to produce them.
 More than 99% of the cells do not survive during the fusion process – reducing
the range of useful antibodies that can be produced against an antigen.
 There is possibility of generating immunogenicity.
10
1. Immunization
2. Cell fusion
3. Selection of hybridoma
4. Screening
5. Cloning and propagation
11
12
13
 Immunize an animal (mouse) by injecting with an appropriate antigen
along with Freund’s adjuvant.
 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.
14
 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.
15
 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. 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 disappear.
 Only the hybridomas that receives HGPRT from lymphocytes are survive.
 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.
16
17
 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.
18
 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.
19
The application of monoclonal antibodies can be broadly categorized as:
(1) Diagnostic Applications
 Biochemical analysis
 Diagnostic Imaging
(2) Therapeutic Applications
 Direct use of MAbs as therapeutic agents
 MAbs as targeting agents.
(3) Protein Purification
20
A) Biochemical analysis:
 Antibodies are used in several diagnostic tests to detect the toxins,
hormones(insulin, human chorionic gonadotropin, growth hormone,
progesterone, thyroxine, triiodothyronine, thyroid stimulating hormone),
diseases .
 monoclonal antibodies to human chorionic gonadotropin (HCG) are used
in pregnancy test kits.
21
 The test of HIV infection is based on
detecting the presence of HIV antibody in
the patient’s serum .
22
B ) Diagnostic imaging:
 Radiolabeled—MAbs are used in the diagnostic imaging of diseases, and
this technique is referred to as immunoscintigraphy. The radioisotopes
commonly used for labeling MAb are iodine—131 and technetium—99.
The MAb tagged with radioisotope are injected intravenously into the
patients.
 These MAbs localize at specific sites (say a tumor) which can be
detected by imaging the radioactivity. In recent years, single photon
emission computed tomography (SPECT) cameras are used to give a
more sensitive three dimensional appearance of the spots localized by
radiolabeled— MAbs.
23
a) Direct use of Mabs as therapeutic agents :
i. 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 via different
pathways.
24
ii. In the treatment of AIDS: 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. The Fc fragment induces
cell-mediated destruction of HIV infected cells.
25
iii. In the immunosuppression of organ transplantation: In the
normal medical practice, immunosuppressive drugs such as
cyclosporin and prednisone are administered to overcome the
rejection of organ transplantation. In recent years, MAbs specific to
T-lymphocyte surface antigens are being used for this purpose
26
27
MAbs in cancer treatment :
 The drugs which kill tumor cells are coupled with monoclonal anti-TAA
antibodies.
 Cancer cells are specifically targeted , avoiding damage to healthy host
cells
 Example : rituximab, trastuzumab etc.
28
29
 MAbs in the dissolution of blood clots:
Fibrin is the major constituent of blood clot which gets dissolved by plasmin.
Plasmin in turn is formed by the activation of plasminogen by plasminogen
activator. Tissue plasminogen activator (tPA) can be used as a therapeutic
agent to remove the blood clots.
30
 Monoclonal antibodies can also be used to purify a substance with
techniques called affinity chromatography.
31
32
 Shivanand P. (2010). “Hybridoma technology for production of monoclonal antibodies”
International Journal of Pharmaceutical Sciences Review and Research vol.1, issue 2 (017)
 U. Marx et al. (1997)“Monoclonal Antibody Production” The Report and Recommendations of
ECVAMWorkshop ATLA 25, 121.137,
 Edward A. Greenfield, (2014) “Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory
Press, 2nd ed. Chapter 7
 Justin K.H. Liu, (2014) “The history of monoclonal antibody development Progress, remaining
challenges and future innovations” Annals of Medicine and Surgery (2014) 113-116
 Andrew S., Otavia C., (2014) “Monoclonal antibodies for the therapy of cancer” Simpson and
Caballero BMC Proceedings 2014, 8 (Suppl 4)
 WHO Guidelines on evaluation of monoclonal antibodies as similar biotherapeutic products, 2016
 Bishwjit Ghosal, Research project on “Study of Monoclonal Market and it’s potential in India”,
NIPER
33
34

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Monoclonal antibodies

  • 1. 1
  • 2.  Antibodies are produced by a specialized group of cells called B- Lymphocytes.  When a foreign antigen enters the body due to immune response B-Lymphocytes develops into plasma cells , liberate antibodies or immunoglobulin's of various types(Ig A, Ig D, Ig E, Ig G, Ig M). 2
  • 3.  Monoclonal antibodies (mAb) are antibodies that are identical because they are 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. 3
  • 4. 4
  • 5.  Monoclonal antibodies are identical immunoglobulins, generated from a single B-cell clone.  These antibodies recognize unique epitopes, or binding sites, on a single antigen. 5
  • 6.  Murine monoclonal antibodies  Chimeric monoclonal antibodies  Humanized monoclonal antibodies  Human monoclonal antibodies 6
  • 7. 7
  • 8. 8
  • 9.  Specificity  Side effects can be treated and reduced by using mice-human hybrid cells or by using fractions of antibodies.  They bind to specific diseased or damaged cells needing treatment.  They treat a wide range of conditions. 9
  • 10.  Time consuming project - anywhere between 6 -9 months.  Very expensive and needs considerable effort to produce them.  More than 99% of the cells do not survive during the fusion process – reducing the range of useful antibodies that can be produced against an antigen.  There is possibility of generating immunogenicity. 10
  • 11. 1. Immunization 2. Cell fusion 3. Selection of hybridoma 4. Screening 5. Cloning and propagation 11
  • 12. 12
  • 13. 13
  • 14.  Immunize an animal (mouse) by injecting with an appropriate antigen along with Freund’s adjuvant.  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. 14
  • 15.  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. 15
  • 16.  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. 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 disappear.  Only the hybridomas that receives HGPRT from lymphocytes are survive.  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. 16
  • 17. 17
  • 18.  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. 18
  • 19.  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. 19
  • 20. The application of monoclonal antibodies can be broadly categorized as: (1) Diagnostic Applications  Biochemical analysis  Diagnostic Imaging (2) Therapeutic Applications  Direct use of MAbs as therapeutic agents  MAbs as targeting agents. (3) Protein Purification 20
  • 21. A) Biochemical analysis:  Antibodies are used in several diagnostic tests to detect the toxins, hormones(insulin, human chorionic gonadotropin, growth hormone, progesterone, thyroxine, triiodothyronine, thyroid stimulating hormone), diseases .  monoclonal antibodies to human chorionic gonadotropin (HCG) are used in pregnancy test kits. 21
  • 22.  The test of HIV infection is based on detecting the presence of HIV antibody in the patient’s serum . 22
  • 23. B ) Diagnostic imaging:  Radiolabeled—MAbs are used in the diagnostic imaging of diseases, and this technique is referred to as immunoscintigraphy. The radioisotopes commonly used for labeling MAb are iodine—131 and technetium—99. The MAb tagged with radioisotope are injected intravenously into the patients.  These MAbs localize at specific sites (say a tumor) which can be detected by imaging the radioactivity. In recent years, single photon emission computed tomography (SPECT) cameras are used to give a more sensitive three dimensional appearance of the spots localized by radiolabeled— MAbs. 23
  • 24. a) Direct use of Mabs as therapeutic agents : i. 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 via different pathways. 24
  • 25. ii. In the treatment of AIDS: 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. The Fc fragment induces cell-mediated destruction of HIV infected cells. 25
  • 26. iii. In the immunosuppression of organ transplantation: In the normal medical practice, immunosuppressive drugs such as cyclosporin and prednisone are administered to overcome the rejection of organ transplantation. In recent years, MAbs specific to T-lymphocyte surface antigens are being used for this purpose 26
  • 27. 27
  • 28. MAbs in cancer treatment :  The drugs which kill tumor cells are coupled with monoclonal anti-TAA antibodies.  Cancer cells are specifically targeted , avoiding damage to healthy host cells  Example : rituximab, trastuzumab etc. 28
  • 29. 29
  • 30.  MAbs in the dissolution of blood clots: Fibrin is the major constituent of blood clot which gets dissolved by plasmin. Plasmin in turn is formed by the activation of plasminogen by plasminogen activator. Tissue plasminogen activator (tPA) can be used as a therapeutic agent to remove the blood clots. 30
  • 31.  Monoclonal antibodies can also be used to purify a substance with techniques called affinity chromatography. 31
  • 32. 32
  • 33.  Shivanand P. (2010). “Hybridoma technology for production of monoclonal antibodies” International Journal of Pharmaceutical Sciences Review and Research vol.1, issue 2 (017)  U. Marx et al. (1997)“Monoclonal Antibody Production” The Report and Recommendations of ECVAMWorkshop ATLA 25, 121.137,  Edward A. Greenfield, (2014) “Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, 2nd ed. Chapter 7  Justin K.H. Liu, (2014) “The history of monoclonal antibody development Progress, remaining challenges and future innovations” Annals of Medicine and Surgery (2014) 113-116  Andrew S., Otavia C., (2014) “Monoclonal antibodies for the therapy of cancer” Simpson and Caballero BMC Proceedings 2014, 8 (Suppl 4)  WHO Guidelines on evaluation of monoclonal antibodies as similar biotherapeutic products, 2016  Bishwjit Ghosal, Research project on “Study of Monoclonal Market and it’s potential in India”, NIPER 33
  • 34. 34