Transcript Hybridoma - PharmaStreet

I. Overview: - The Immune System
II. Introduction: - Monoclonal
Antibodies
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
1. Innate Immunity
Primary Response
2. Acquired Immunity
Secondary Response
ANATOMY OF THE IMMUNE SYSTEM
CELLS OF THE IMMUNE SYSTEM
FUNCTIONING OF THE IMMUNE SYSTEM
HUMORAL (ANTIBODY MEDIATED) IMMUNE RESPONSE
CELL MEDIATED IMMUNE RESPONSE
ANTIGEN (1ST EXPOSURE)
ENGULFED BY
MACROPHAGE
FREE
ANTIGENS
DIRECTLY
ACTIVATE
ANTIGENS
DISPLAYED
BY
INFECTED
CELLS
ACTIVATE
BECOMES
APC
STIMULATES
B CELLS
STIMULATES
HELPER
T CELLS
STIMULATES
MEMORY
HELPER T
CELLS
GIVES RISE TO
STIMULATES
STIMULATES
CYTOTOXIC
T CELL
GIVES RISE TO
STIMULATES
ANTIGEN (2nd EXPOSURE)
PLASMA
CELLS
MEMORY
B CELLS
SECRETE ANTIBODIES
STIMULATES
MEMORY
T CELLS
ACTIVE
CYTOTOXIC T
CELL
IMMUNOTHERAPY
Treatment of the disease by Inducing, Enhancing or
Suppressing the Immune System.
Active Immunotherapy: -
Passive Immunotherapy: -
It stimulates the body’s own
immune system to fight the
disease.
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.
HISTORY OF IMMUNOLOGY
•1926 Lloyd Felton & GH Bailey, Isolation of pure antibody
preparation
•1958-62 Jean Dausset et al., Human leukocyte antigens
•1862 Ernst Haeckel, Recognition of phagocytosis
•1934-8 John Marrack, Antigen-antibody binding hypothesis
•1959-62 Rodney Porter et al., Discovery of antibody
structure
•1877 Paul Erlich, recognition of mast cells
•1936 Peter Gorer, Identification of the H-2 antigen in mice
•1959 James Gowans, Lymphocyte circulation
•1879 Louis Pasteur, Attenuated chicken cholera vaccine
development
•1940 Karl Lansteiner & Alexander Weiner, Identification of
the Rh antigens
•1961-62 Jaques Miller et al., Discovery of thymus
involvement in cellular immunity
•1883 Elie Metchnikoff Cellular theory of vaccination
•1941 Albert Coons, Immunofluorescence technique
•1885 Louis Pasteur, Rabies vaccination development
•1942 Jules Freund & Katherine McDermott, Adjuvants
•1961-62 Noel Warner et al., Distinction of cellular and
humoral immune responses
•1888 Pierre Roux & Alexandre Yersin, Bacterial toxins
•1942 Karl Landsteiner & Merill Chase, Cellular transfer of
sensitivity in guinea pigs (anaphylaxis)
•1798 Edward Jenner,
Smallpox vaccination
•1888 George Nuttall, Bactericidal action of blood
•1891 Robert Koch, Delayed type hypersensitivity
•1894 Richard Pfeiffer, Bacteriolysis
•1895 Jules Bordet, Complement and antibody activity in
bacteriolysis
•1900 Paul Erlich, Antibody formation theory
•1901 Karl Landsteiner, A, B and O blood groupings
•1901-8 Carl Jensen & Leo Loeb, Transplantable tumors
•1944 Peter Medwar, Immunological hypothesis of allograft
rejection
•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
•1963 Jaques Oudin et al., antibody idiotypes
•1964-8 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
•1975 Kohler and Milstein,
Monoclonal antibodies used in
genetic analysis
•1902 Paul Portier & Charles Richet, Anaphylaxis
•1903 Almroth Wright & Stewart Douglas, Opsonization reactions
•1906 Clemens von Pirquet, coined the word allergy
•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-versushost reaction
•1984 Robert Good, Failed treatment of severe combined
immunodeficiency (SCID, David the bubble boy) by bone
marrow grafting. 1985 Tonegawa, Hood et al., Identification
of immunoglobulin genes
•1985-7 Leroy Hood et al., Identification of genes for the T
cell receptor
•1907 Svante Arrhenius, coined the term immunochemistry
•1953 James Riley & Geoffrey West, Discovery of histamine
in mast cells
•1910 Emil von Dungern, & Ludwik Hirszfeld, Inheritance of ABO
blood groups
•1953 Rupert Billingham, Leslie Brent, Peter Medwar, &
Milan Hasek, Immunological tolerance hypothesis
•1990 Yamamoto et al., Molecular differences between the
genes for blood groups O and A and between those for A
and B
•1910 Peyton Rous, Viral immunology theory
•1955-1959 Niels Jerne, David Talmage, Macfarlane
Burnet, Clonal selection theory
•1990 NIH team, Gene therapy for SCID using cultured T
cells.
•1957 Ernest Witebsky et al., Induction of autoimmunity in
animals
•1993 NIH team, Treatment of SCID using genetically altered
umbilical cord cells.
•1957 Alick Isaacs & Jean Lindemann, Discovery of
interferon (cytokine)
•1985-onwards Rapid identification of genes for immune
cells, antibodies, cytokines and other immunological
structures.
•1914 Clarence Little, Genetics theory of tumor transplantation
•1915-20 Leonell Strong & Clarence Little, Inbred mouse strains
•1917 Karl Landsteiner, Haptens
•1921 Carl Prausnitz & Heinz Kustner, Cutaneous reactions
•1924 L Aschoff, Reticuloendothelial system
ANTIBODIES
STRUCTURE
CLASS
Fab
Ag
Ag
Hybridoma Technology
Fa
IgL k,l
IgH m,d,g,e,a
Fb
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 predetermined specificity.
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.
Monoclonal antibodies:
Antibodies produced from a single clone of B cells.
Monoclonal antibodies all have identical antigenbinding 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.
ANTIBODIES
POLYCLONAL.
MONOCLONAL.
Derived from different B
Lymphocytes cell lines
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
NOT Powerful tools for
clinical diagnostic tests
Enable the development of
secure immunoassay systems.
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)
• 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.
PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
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)
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)
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 too low
Titre High
2 weeks
BOOST
(Pure antigen)
BOOST
(Pure antigen)
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
 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.
PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells
&
Selection of Hybridoma Cells
PEG
FUSION
SPLEEN CELLS
MYELOMA CELLS
Feeder Cells
Growth Medium
1.
HYBRIDOMA CELLS
2.
ELISA PLATE
HAT Medium
Plating of Cells in
HAT selective
Medium
Scanning of Viable
Hybridomas
•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
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
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
PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
Sr.no
MAIN APPLICATION
EXAMPLES
1
Diagnosis
a) DIAGNOSTIC REAGENTS
Infectious Diseases,pregnancy,tumours,diabetes
b) DIAGNOSTIC IMAGING
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.
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
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
Non-Hodgkin lymphoma
2003
Cetuximab
Erbitux
Colorectal cancer
Head & neck cancers
2004
2006
Bevacizumab
Avastin
Colorectal cancer
2004
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
THANK YOU
-PHARMA STREET