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.

Lecture 12 Animal Cell Biotechnology

Hybridomas and the production of antibodiesMonoclonal 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) YYYY

Y

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.

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

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.

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

Lecture 12 Animal Cell Biotechnology Hybridomas and the production of antibodies

Kuby, J. 1997. Immunology 3rd ed. New W.H. Freeman and Company. P131.

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.

Kuby, J. 1997. Immunology 3rd ed. New W.H. Freeman and Company. P134.

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

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

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)

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

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

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)

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.

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

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.

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.

Ways to humanize an antibody... Fig. 8.8

Lecture 12 Animal Cell Biotechnology Hybridomas and the production of antibodies

Kuby, J. 1997. Immunology 3rd ed. New W.H. Freeman and Company. P137.

Monoclonal antibodies approved by the FDA for clinical use

Antibody Type Therapeutic treatment Company Date approved

Orthoclone (OKT3)

Murine Ig2a Allograft rejection Ortho Biotech 1986

ReoPro Chimeric (Fab) Coronary angioplasty Centocor/ Lilly 1994

Zenapax Humanized IgG1 Allograft rejection Protein Design/ Hoffman-La Roche

1997

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/ Crohn’s disease

Centocor 1998-1999

Butler, M. (2005) Applied Microbiology and Biotechnology 68: 283-291.

The demand for mammalian cell culture products