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. 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

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 If human insulin is injected into a mouse then after a few days the blood will contain significant amounts of mouse antibody capable of binding to human insulin. The immunoglobulin fraction will contain anti-insulin as well as numerous other antibodies. Hence it is difficult to isolate particular antibody that may be required. Polyclonal antibodies includes different antibodies against insulin ie antibodies reactive to different regions (epitopes) of insulin molecule

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 Complement activation leads to activation of leukocytes and phagocytosis

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. B lymphocyte produce a single kind of antibody. Myeloma are transformed lymphocytes capable of growing indefinitely. The term hybridoma was used to describe a homogeneous clone of antibody producing hybrid cells

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 Can be used in biological research and medically important products

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 Small molecules are conjugated to carrier proteins such as albumin. 1% of cell population from spleen can secrete antibodies.

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 Growth promoting factors are released by cells

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 Two important phenotypic characterisitcs from parents. B – lymphocytes have limited period of time and myeloma have infinite growth

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 Lysis of swollen cells is possible

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 50 fused cells from an original total of 5 x 106 cells from each parental cell line

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 Newly formed hybridomas will be HGPRT+ by inheritance from normal lymphocytes where unfused myelomas carry mutant HGPRT- marker. Therefore incubation in HAT will allow survival of HGPRT+ hybridomas only (after a few days)

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 Treatment with gamma irradiation or exposure to mitomycin C halts DNA synthesis. Cellular metabolism continues and secreted growth factors can promote growth of viable hybridoma cells if inoculated at low density

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 Source of lymphocytes is limited to samples of peripheral blood

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 Hard to find a suitable fusion partner to immortalize the B-lymphocytes

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 Mouse myeloma used in fusion are nonantibody secretors. So resulting hybridomas secrete antibody antibody associated with fused Blymphocyte. Therefore the culture product will be single selected antibody type.

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: against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and 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.

Disclaimer This workforce solution was funded by a grant awarded under the President’s Community-Based Job Training Grants as implemented by the U.S. Department of Labor’s Employment and Training Administration.  The solution was created by the grantee and does not necessarily reflect the official position of the U.S. Department of Labor.  The Department of Labor makes no guarantees, warranties, or assurances of any kind, express or implied, with respect to such information, including any information on linked sites and including, but not limited to, accuracy of the information or its completeness, timeliness, usefulness, adequacy, continued availability, or ownership.  This solution is copyrighted by the institution that created it.  Internal use by an organization and/or personal use by an individual for non-commercial purposes is permissible.  All other uses require the prior authorization of the copyright owner.