Hybridomas - sources of antibodies

Chapter 8 from ‘The Basics’

Objectives

• What are monoclonal antibodies (Mabs)?

• What are polyclonal antibodies (Pabs)?

• What are hybridomas?

• How hybridomas produce Mabs?

Antibody production in vivo

• Found in specific protein fraction of blood – gammaglobulin or immunoglobulin fraction

• Synthesized by subset of white blood cells – the B-lymphocytes

• Abs have two heavy + two light chains

• Heavy chain – variation in length, number of domains and glycan structures.

Antibodies are valuable

• Abs are found in all body fluids including blood, milk and mucous secretions and serve an essential role in immune system that protects animals for infection or cytotoxic effects of foreign compounds

• Bind antigens at their epitopes

• Valuable – specific recognition and affinity for antigen

Structure of Antibodies

• IgG – major class of immunoglobulin found in blood serum – molecular mass of 150KDa

• The heavy chain of IgG has four domains – VH-CH1-CH2-CH3 and light chain has two domains VL-CL

• Constant C region of any Ig class varies with species

Structure of Antibodies

• Digestion of molecule with papain cleaves the heavy chain in hinge region and forms three fragments (2 Fab + Fc)

• Two fab (antibody-binding fragments) contain N-terminal end of a heavy chain with disulfide linked light chain

- Variable sequences of amino acids – allows to bind to antigen with high affinity

• Fc consists of C-terminal end of two heavy chains

Antibody production in vivo

• Each B-lymphocyte – produce one type of antibody in response to particular antigen – Monoclonal antibody

• Immunoglobulin fraction of blood will contain numerous other antibodies – Polyclonal antibody

• The variety of antibodies present in any animal reflect the population of B-lymphocytes – exposure to previous range of antigens

Glycosylation of antibodies

• Ab are glycoproteins containing variable glycan structures

• Glycosylation of Fc region is essential for effector functions of antibody such as complement binding, binding to Fc receptors, and induction of ADCC

• 20% glycosylation is present on Fab fragment in human ab

• Glycan structures – role in immune response

Hybridomas- Kohler and Milstein-1975

• Hybridomas are hybrid cells capable of continuous production of monoclonal ab

• Hybrid of B-Lymphocytes and myelomas

• Can be grown in bioreactors- produc. of kilograms of Mabs.

Hybridomas

• Produce monoclonal antibodies for diagnosis and testing in applications such as blood typing, detection of virus, pregnancy testing or for detection of contaminants in food

• Kohler and Milstein work – four stages – Immunization, Cell Fusion, Genetic Selection and cell selection

Immunization in vivo

• Injection of a chosen antigen into mice/rat• Synthesis of antibodies will depend upon

antigen (3-4 weeks for good response)- Large molecules – produce stronger response

in short period of time- Small molecules - multiple injections spaced

over several days - Spleen is homogenized - B-lymphocytes

isolated by centrifugation

Immunization in vitro

• Obtain spleen from nonimmunized mouse• Cells suspended in medium containing selected

antigen + factors stimulating growth and differentiation + incubation with mixed lymphocytes = conditioned media

• Growth promoting factors – cytokines like interleukins, B-cell growth factor and B-cell differentiation factor

• Antigens at lower conc + activation of B-lymphocytes takes 3-4 days rather than few weeks in vivo

Cell Fusion/Hybridization

• Fusion between B-lymphocyte (ability to synthesize antibodies) + Myeloma (ability for infinite growth)

• Suitable myeloma fusion partners selected for two important characteristics

- Nonproduction of antibodies – resulting hybridoma produces not more than one antibody

- Myelomas deficient in HGPRT (hypoxanthine guanine phosphoribosyl transferase) are used. Allows selection in HAT (hypoxanthine, aminopterin, thymidine

Cell Fusion/Hybridization

• Cells can be induced to fuse if two cell populations are brought close

• Destabilization of adjacent cell membranes• Two distinct nuclei fuse to form heterokaryon –

produce a stable hybrid cell

Methods of Cell Fusion

• Fusion by PEG at 4000-6000 KDa is suitable for cell fusion – within 1-2 minutes

• Swelling accompanies fusion

• Allows adjacent cells to approach closely

• Plasma membrane becomes permeable to small ions

Methods of Cell Fusion

• Electrofusion – two populations are introduced into a small sterile chamber

• Electric current is applied in high-voltage pulses (200 V/cell pellet) for short time periods

• Allows cells to orient along the line of current and fuse

• Produces high % of viable hybrid cells

Selectable gene markers for cell selection

• Heterogeneous population of cells – unfused parental cells, lysed cells and hybrid cells

• Two stages of selection

- Isolation of hybrid cells from parental cells

- Selection of antibody-secreting cells within hybrid cell population

Selectable gene markers for cell selection

• Selective medium contains HAT – hypoxanthine, aminopterin and thymidine

• Allows selection and growth of hybridomas which are HGPRT+

• Unable to support growth of HGPRT- myelomas because denovo pathway is inhibited and salvage pathway cannot function because of defective enzyme

Clonal selection of Mab-secreting hybridomas

• Only some hybridomas (10%) from HAT culture will secrete antibodies

• To select Mab-secreting hybridomas – dispense suspension into 96-well plate so that each well has one cell

• Growth supported by feeder layer of cells (thymocytes, macrophages or splenocytes treated to prevent growth/DNA synthesis)

• 1-2 weeks for growth – medium of each well tested for antibody by suitable assay

• Promising ones cultured in suspension to produce 1-2 x106 cells/ml and Mab conc. Of 100-200 μg/ml

Assay of Mabs Detection

• ELISA – enzyme-linked immunosorbent assay

- Most commonly used, done in multi-well plates for analyzing multiple samples

• RIA-radioimmunoassay

- More time consuming and expensive

• Affinity chromatography

- Is ideal if an HPLC is available and hybridomas – grown in serum-free media

Open book exam questions

• Why do murine-derived monoclonal antibodies have limited success in human therapy?

• What is the disadvantage of using microbial fermentation for production of antibodies? How do usage of plant cells win over them?

• Mention one limitation of plant-derived antibodies.

• What is glycosylation? What is its importance to therapeutic antibodies?

What are the major difficulties in producing human hybridoma cells?

• Source of antibody-secreting lymphocytes - Spleen of immunized mouse is used – not possible with humans. Can be taken from patients – acquired immunity against particular compound or disease

What are the major difficulties in producing human hybridoma cells?

• Immortalization and chromosome instability –

Human myeloma cell lines are difficult to grow in culture.

Human lymphoblastoid cell lines – used as fusion partners – cell fusion and genetic stability is low

What are the major difficulties in producing human hybridoma cells?

• Antibody secretion of human parental fusion partners

• Mouse myeloma used in fusion are nonantibody secretors

• Human myeloma or lymphoblastoid cells – immunoglobulin secretors

- Will secrete two kinds of antibodies – associated with parental cells

Why is antibody production in plants very successful?

• Mabs in plants – absence of animal pathogens, ease of genetic manipulation, ability of post translational modification and potential for scale up to an economic production level.

• Low cost of large scale produc. – plantibodies

• Directly eat plants without purification

• This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal opportunity employer and does not discriminate on the following basis:

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• against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity.

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