This document discusses hybridomas and the production of monoclonal antibodies. It describes how monoclonal antibodies are produced through cell fusion, where antibody-producing B-lymphocytes are fused with myeloma cells to create a hybridoma. This allows for the mass production of antibodies that target a single antigen. The document outlines the key steps of immunization, cell fusion, genetic selection of hybridomas, and clonal selection. It also discusses how mouse antibodies can be "humanized" for therapeutic use in humans to reduce immunogenicity. Major applications of monoclonal antibodies include diagnosis and treatment of diseases.
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Lecture 10 hybridomas and the production of antibodies
1. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies
Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P145.
2. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies
Monoclonal antibody (Mab) production
• also known as immunoglobulins (Ig)
• glycoproteins found in the blood plasma, lymph and
secretions (tears, saliva, and gastrointestinal fluid)
• five classes of antibodies found in humans:
→ IgM, IgG, IgA, IgE, and IgD
• synthesized by B-lymphocytes (white blood cells)
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4. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies:
The antibody
Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P136.
5. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies
• consists of two heavy chains and two light chains
• isotypes differ in heavy chain structure –
length, number of domains, and glycan structures
• each Ig has unique antigen binding region, binds to
specific antigen with high affinity
6. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies
Major uses:
1. diagnostic
→ blood typing, detection of pathogenic
microorganisms and viruses, levels of drugs in blood
stream, pregnancy, contaminants in food, antigens shed
by tumors
2. therapeutic
→ treatment of disease
3. protein purification
Kuby, J. 1997. Immunology 3rd ed. New W.H. Freeman and Company. P135.
7.
8. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies
• in 1975 George Kohler and Cesar Milstein developed a
technique to produce hybrid cells of B-lymphocytes and
myelomas
→ myelomas are transformed lymphocytes that grow
indefinitely
• used to produce large scale quantities (kilograms) of
monoclonal antibodies (Mab)
→ antibody specific for one type of epitope
• hybrid cells were called hybridomas
• grown in suspension in large bioreactors, produce large
quantities of Mab’s
9. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies
Kuby, J. 1997. Immunology 3rd ed. New W.H. Freeman and Company. P131.
10. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies:
Cell fusion
Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P141.
11. Kuby, J. 1997. Immunology 3rd ed. New W.H. Freeman and Company. P134.
12. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies:
Cell fusion
Generation of hybridomas involves four steps:
1. immunization
2. cell fusion
3. genetic selection
4. clonal cell selection
13. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies:
Cell fusion
1. Immunization
i) in vivo immunization
• antigen injected into spleen of mouse or rat
• incubation time for antibody synthesis 3-4 weeks
→ larger molecules provoke stronger response in
shorter amount of time
→ smaller molecules may need carrier proteins
(albumin), with multiple injections over time
• spleen then removed and homogenized, lymphocytes
collected by centrifugation
14. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies:
Cell fusion
ii) in vitro immunization
• cells removed from spleen of non-immunized mouse
• cells suspended in medium containing antigen and
growth and differentiation factors
• activation may be shorter (3-4 days)
• low concentrations of weak antigens can be used
• certain Ig types produced preferentially (IgM)
15. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies:
Cell fusion
2. Cell fusion
• ab-secreting B-lymphocyte fused with myeloma
• choice of myeloma partner selected for two
characteristics:
→ does not produce antibodies
→ possesses a genetic marker
• cells induced to fuse if two populations are brought
close together at high cell concentration
16. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies:
Cell fusion
• cell membranes destabilized, eventually fuse to form a
hybrid cell
• initially form a heterokaryon (two distinct nuclei),
eventually fuse to form a stable hybrid
• fusion assisted by:
→ UV-inactivated Sendai viruses
→ polyethylene glycol (PEG)
→ electrofusion
17. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies:
Cell selection
3. Genetic selection
• cell fusion results in a heterogenous population of cells
→ unfused parental cells, lysed cells, hybrid cells
• lymphocyte cells have limited life span, will eventually
die – hybrids, myeloma parent survives
• select for hybridomas using Hypoxanthine Aminopterin
Thymidine medium (HAT)
→ myelomas are HGPRT- (hypoxanthine guanine
phosphoribosyl transferase), B-lymphocytes are
HGPRT+
• Only hybridomas will survive, myeloma parent will not
grow (afer a few days only hybridomas survive)
18. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies:
Cell selection
Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P144.
19. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies:
Cell selection
4. Clonal cell selection
• only 10% of hybridomas secrete antibody
• clones diluted and dispensed into multi-well plates (one
cell per well)
• growth supported by feeder cells
• after 1-2 weeks medium in each well tested for
antibody content
→ ELISA – enzyme-linked antibody assay
→ RIA – radioimmunoassay
→ Affinity chromatography
20. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies:
ELISA
Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P146.
21. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies
• mouse produced Ig may induce human immune response
• must “humanize” mouse antibody before human
therapeutic use
→ mRNA taken from hybridomas, expressed as
cDNA, spliced with human genes to form humanized
antibodies
• mouse variable regions linked to human constant regions
(chimeric antibody)
• replace V-regions not required for antigen binding – only
murine-derived complimentarity determining regions (CDR)
bound to human antibody (CDR-grafted antibody)
→ may have loss of affinity for antigen
→ may still have immune response against antigen binding
site Kuby, J. 1997. Immunology 3rd ed. New W.H. Freeman and Company. P136.
24. Lecture 12 Animal Cell Biotechnology
Hybridomas and the production of antibodies
Kuby, J. 1997. Immunology 3rd ed. New W.H. Freeman and Company. P137.
25. Monoclonal antibodies approved by the FDA for clinical use
Antibody Type Therapeutic treatment Company Date
approved
Orthoclone Murine Ig2a Allograft rejection Ortho Biotech 1986
(OKT3)
ReoPro Chimeric (Fab) Coronary angioplasty Centocor/ Lilly 1994
Zenapax Humanized IgG1 Allograft rejection Protein Design/ 1997
Hoffman-La Roche
Rituxan Chimeric IgG1 Non-Hodgkin’s lymphoma Genentech 1997
Synagis Humanized IgG1 Respiratory synctial virus Medimmune 1998
Herceptin Humanized IgG1 Breast cancer Genentech 1998
Simulect Chimeric IgG1 Allograft rejection Novartis Pharm 1998
Inflixmab Chimeric IgG1 Rheumatoid arthritis/ Centocor 1998-1999
Crohn’s disease
26. The demand for mammalian cell culture products
Butler, M. (2005) Applied Microbiology and Biotechnology 68: 283-291.
Notas do Editor
Dark blue – constant heavy chainDark pink – constant light chainLight blue – heavy chain variable regionLight pink – light chain variable regionLight blue and light pink domains contain the antigen binding site, contain hyper variable regions, 3 in each chain – antigen binding sites