1. Overview: - The Immune System
II. Introduction: - Monoclonal Antibodies

2.                                                             
THE IMMUNE SYSTEM
The Latin term “IMMUNIS” means EXEMPT, referring to protection against foreign agents.
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 Immune System consists of
Innate Immunity Primary Response
Acquired Immunity Secondary Response

3. ANATOMY OF THE IMMUNE SYSTEM

4. CELLS OF THE IMMUNE SYSTEM

5. FUNCTIONING OF THE IMMUNE SYSTEM
HUMORAL (ANTIBODY MEDIATED) IMMUNE RESPONSE
CELL MEDIATED IMMUNE RESPONSE
ANTIGEN (1ST EXPOSURE)
ENGULFED BY
MACROPHAGE
ANTIGENS DISPLAYED BY INFECTED CELLS ACTIVATE
FREE ANTIGENS DIRECTLY ACTIVATE
BECOMES
APC
STIMULATES
HELPER T CELLS
CYTOTOXIC T CELL
B CELLS
STIMULATES
STIMULATES
MEMORY HELPER T CELLS
GIVES RISE TO
GIVES RISE TO
STIMULATES
STIMULATES
STIMULATES
ANTIGEN (2nd EXPOSURE)
PLASMA CELLS
MEMORY B CELLS
ACTIVE CYTOTOXIC T CELL
STIMULATES
MEMORY T CELLS
SECRETE ANTIBODIES
Defend against extracellular pathogens by binding to antigens and making them easier targets for phagocytes and complement
Defend against intracellular pathogens and cancer by binding and lysing the infected cells or cancer cells

6. 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.

7. 1798 Edward Jenner, Smallpox vaccination
HISTORY OF IMMUNOLOGY
1926 Lloyd Felton & GH Bailey, Isolation of pure antibody preparation
John Marrack, Antigen-antibody binding hypothesis
1936 Peter Gorer, Identification of the H-2 antigen in mice
1940 Karl Lansteiner & Alexander Weiner, Identification of the Rh antigens
1941 Albert Coons, Immunofluorescence technique
1942 Jules Freund & Katherine McDermott, Adjuvants
1942 Karl Landsteiner & Merill Chase, Cellular transfer of sensitivity in guinea pigs (anaphylaxis)
1944 Peter Medwar, Immunological hypothesis of allograft rejection
Jean Dausset et al., Human leukocyte antigens
Rodney Porter et al., Discovery of antibody structure
1959 James Gowans, Lymphocyte circulation
Jaques Miller et al., Discovery of thymus involvement in cellular immunity
Noel Warner et al., Distinction of cellular and humoral immune responses
1963 Jaques Oudin et al., antibody idiotypes
Anthony Davis et al., T and B cell cooperation in immune response
1965 Thomas Tomasi et al., Secretory immunoglobulin antibodies
1967 Kimishige Ishizaka et al., Identification of IgE as the reaginic antibody
1971 Donald Bailey, Recombinent inbred mouse strains
1974 Rolf Zinkernagel & Peter Doherty, MHC restriction
1975 Kohler and Milstein, Monoclonal antibodies used in genetic analysis
1984 Robert Good, Failed treatment of severe combined immunodeficiency (SCID, David the bubble boy) by bone marrow grafting Tonegawa, Hood et al., Identification of immunoglobulin genes
Leroy Hood et al., Identification of genes for the T cell receptor
1990 Yamamoto et al., Molecular differences between the genes for blood groups O and A and between those for A and B
1990 NIH team, Gene therapy for SCID using cultured T cells.
1993 NIH team, Treatment of SCID using genetically altered umbilical cord cells.
1985-onwards Rapid identification of genes for immune cells, antibodies, cytokines and other immunological structures.
1798 Edward Jenner, Smallpox vaccination
1862 Ernst Haeckel, Recognition of phagocytosis
1877 Paul Erlich, recognition of mast cells
1879 Louis Pasteur, Attenuated chicken cholera vaccine development
1883 Elie Metchnikoff Cellular theory of vaccination
1885 Louis Pasteur, Rabies vaccination development
1888 Pierre Roux & Alexandre Yersin, Bacterial toxins
1888 George Nuttall, Bactericidal action of blood
1891 Robert Koch, Delayed type hypersensitivity
1894 Richard Pfeiffer, Bacteriolysis
1798 Edward Jenner, Smallpox vaccination
1948 Astrid Fagraeus, Demonstration of antibody production in plasma B cells
1948 George Snell, Congenic mouse lines
1949 Macfarlane Burnet & Frank Fenner, Immunological tolerance hypothesis
1950 Richard Gershon and K Kondo, Discovery of suppressor T cells
1952 Ogden and Bruton, discovery of agammagobulinemia (antibody immunodeficiency)
1953 Morton Simonsen and WJ Dempster, Graft-versus-host reaction
1953 James Riley & Geoffrey West, Discovery of histamine in mast cells
1953 Rupert Billingham, Leslie Brent, Peter Medwar, & Milan Hasek, Immunological tolerance hypothesis
Niels Jerne, David Talmage, Macfarlane Burnet, Clonal selection theory
1957 Ernest Witebsky et al., Induction of autoimmunity in animals
1957 Alick Isaacs & Jean Lindemann, Discovery of interferon (cytokine)
1895 Jules Bordet, Complement and antibody activity in bacteriolysis
1900 Paul Erlich, Antibody formation theory
1901 Karl Landsteiner, A, B and O blood groupings
Carl Jensen & Leo Loeb, Transplantable tumors
1902 Paul Portier & Charles Richet, Anaphylaxis
1903 Almroth Wright & Stewart Douglas, Opsonization reactions
1906 Clemens von Pirquet, coined the word allergy
1975 Kohler and Milstein, Monoclonal antibodies used in genetic analysis
1907 Svante Arrhenius, coined the term immunochemistry
1910 Emil von Dungern, & Ludwik Hirszfeld, Inheritance of ABO blood groups
1910 Peyton Rous, Viral immunology theory
1914 Clarence Little, Genetics theory of tumor transplantation
Leonell Strong & Clarence Little, Inbred mouse strains
1917 Karl Landsteiner, Haptens
1921 Carl Prausnitz & Heinz Kustner, Cutaneous reactions
1924 L Aschoff, Reticuloendothelial system

8. ANTIBODIES
STRUCTURE
CLASS

9. Fab Ag Ag Hybridoma Technology Fa Fb IgL k,l IgH m,d,g,e,a Fc
A hybridoma is a cell line in which an antibody-producing B cell (human) is fused to an immortalized or long-lived mouse cell to produce a hybrid cell. The hybrid cell is cloned and expanded in culture to yield continuous production of an antibody of pre-determined specificity. Fa Fb
IgL k,l
IgH m,d,g,e,a Fc

10. Polyclonal antibodies:
Polyclonal antibodies are a mixture of antibodies with different antigen binding sites that may bind to different epitopes or antigens of the immunizing agent with varying affinities. They may be of different antibody classes.
The serum obtained from an immunized animal is referred to as a polyclonal antiserum.

11. Monoclonal antibodies:
Antibodies produced from a single clone of B cells.
Monoclonal antibodies all have identical antigen-binding sites. Thus they all bind to the same epitope with the same affinity. They are all of the same antibody class (isotype).
Produced by fusing a B cell secreting the desired antibody with a myeloma cell capable of growing indefinitely in tissue culture.

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

13. mAB Production
There are currently two widely adopted methods used to produce monoclonal Ab.
In vivo & in vitro
There are advantages and disadvantages for both methods.
The generation of mAb producing cells are typically done in mice.
This method involves fusing a B cell with a myeloma cell (tumor cell)

14. 1) Immunize animal (mouse or rabbit)
2) Isolate spleen cells (containing antibody-producing B cells)
3) Fuse spleen cells with myeloma cells (e.g. using PEG - polyethylene glycol)
4) Allow unfused B cells to die
5) Add HAT (hypoxanthine-aminopterin-thymidine ) culture to kill unfused myeloma cells
6) Clone remaining cells (place 1 cell per well and allow each cell to grow into a clone of cells)
7) Screen supernatant of each clone for presence of the desired antibody (ELISA)
8) Grow the chosen clone of cells in tissue culture indefinitely.
9) Harvest antibody from the culture supernatant.

15. PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY

16. Preparations of mAb Step 1: Immunization of mice
Mice are immunized with an antigen (attached to adjuvant). The antigen can be whole cells, membrane fragment, or complex molecules.
Mice serum’s are screened using various techniques such as ELISA.
When sufficient titer is reached the mice are euthanize and spleen is removed as a source of cells for cell fusion.
Step 2: Preparation of Myeloma Cells
Myeloma cells are immortalized cells that are capable of dividing indefinitely.
These cells are treated with 8-azaguanine to ensure sensitivity to HAT (hypoxanthine-aminopterin-thymidine)

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

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

19. + 8 - Azaguanine Myeloma Cells Myeloma Cells
Step 2: - Preparation of Myeloma Cells +
8 - Azaguanine
Myeloma Cells
Immortal Tumor Of Lymphocytes
Myeloma Cells
HGPRT-
(hypoxanthine guanine phosporibosyl transferase)
High Viability & Rapid Growth

20. Step 3: Fusion of myeloma cells with Spleen cells
Spleen cells harvested from mice are fused with myeloma cells.
Fusion is done through co-centrifusing in polyethylene glycol.
Cells are plated in selection medium
hypoxanthine-aminopterin-thymidine (HAT) selection medium – inhibitor of aminoterin which blocks nucleotide synthesis.
Only fused cells with grow on HAT
Cells are distributed on feeder cells (murine bone-marrow ) to promote growth of the hybridomal cells.

21. PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells &
Selection of Hybridoma Cells
PEG
FUSION
MYELOMA CELLS
SPLEEN CELLS
Feeder Cells
Growth Medium
Plating of Cells in HAT selective Medium
Scanning of Viable Hybridomas
HYBRIDOMA CELLS
ELISA PLATE
HAT Medium

22. Step 4: Cloning of Hybridoma cells.
A mouse is inoculated with the cell and thereby becomes a factory for producing the mAB.
Ascites are collected from the mouse.
Step 5: Ab are screened and Purified
Ab are screened using specific Ag binding.
Step 6: Desired Ab are cloned
This is done in vitro on culture bottles

23. PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
Step 5: - Cloning of Hybridoma Cell Lines by “ Limiting Dilution” or Expansion
A. Clone Each +ve Culture
B. Test Each Supernatant for Antibodies
C. Expand +ve Clones
Tissue Culture Method
Mouse Ascites Method

24. Advantage of in vivo process:
Relatively inexpensive and easy
Pure antibodies produced from crude antigen preparation
Ab produced are of single immunoglobulin class and specific for single epitope.
High production rate
High reproducibility with respect to specificity and avidity.
Disadvantage:
Ethical concerns with using animals.
Poor compliment fixation capabilities

25. PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY

26. Sr.no
MAIN APPLICATION
EXAMPLES 1
Diagnosis
DIAGNOSTIC REAGENTS
DIAGNOSTIC IMAGING
Infectious Diseases,pregnancy,tumours,diabetes
Cardiovascular Disease,cell Surface Markers,detection Of Circulating Antigens,cancer And Hormones. 2
THERAPEUTICS
Autoimmune Disease,cancer,cardiovascular Disease,bone Marrow And Organ Transplant,toxin –Drug Conjugate,antidotes,enzymes And Proteins. 3
INVESTIGATIONS AND ANALYTICAL
RIA,ELISA,Lymphocyte Phenotyping,autoantibody,finger Printing,purification Of Proteins. 4
DRUG TARGETING
Ab Enzyme Conjugates,immunotoxins,site Specific Modification 5
MISCELLANEOUS
Autoantibody Finger Printing,catalytic Monoclonal Antibodies.

27. Applications of Monoclonal Antibodies
Diagnostic Applications Biosensors & Microarrays
Therapeutic Applications Transplant rejection Muronomab-CD3 Cardiovascular disease Abciximab Cancer Rituximab Infectious Diseases Palivizumab Inflammatory disease Infliximab
Clinical Applications Purification of drugs, Imaging the target
Future Applications Fight against Bioterrorism

28. Examples of FDA approved treatments of certain cancers:
MAb Name
Trade Name
Used to Treat:
Approved in:
Rituximab
Rituxan
Non-Hodglymphoma
1997
Trastuzumab
Herceptin
Breast cancer
1998
Gemtuzumab ozogamicin*
Mylotarg
Acute myelogenous leukemia (AML)
2000
Alemtuzumab
Campath
Chronic lymphocytic leukemia (CLL)
2001
Ibritumomab tiuxetan*
Zevalin
Non-Hodgkin lymphoma
2002
Tositumomab*
Bexxar
2003
Cetuximab
Erbitux
Colorectal cancer Head & neck cancers
Bevacizumab
Avastin
Colorectal cancer
2004

29. Uses
Measuring protein and drug levels in serum
Typing tissue and blood
Identifying infectious agents
Identifying clusters of differentiation for the classification and follow-up therapy of leukemias and lymphomas
Identifying tumor metastasis
Identifying and quantifying hormones
Immunoaffinity Purification

30. THANK YOU -PHARMA STREET