Monoclonal Antibodies & Hybridoma Technology- Dr C R Meera

 

1. What are Polyclonal & Monoclonal antibodies?

Antibodies (Ab) are immunoglobulins produced against the antigen (An) by activated B -Lymphocytes. On activation, B cells differentiate into plasma cells and memory cells. Plasma cells proliferate to form clone of cells and produce large number of antibodies targeted against the antigen that had stimulated the entire process. However, Ans have multiple antigenic determinants or epitopes. Hence, on encountering an An, multiple clones of B cells are generated which will result in the production of antisera containing Abs or Igs of different classes with specificities against different epitopes of the same An. Such Abs are called “Polyclonal Abs”.

Abs produced by clones of a single B lymphocytes and directed against single antigenic determinant or epitopes are called “Monoclonal Abs”. Means, these Abs will bind with single specific epitope on an An. Ie; they are identical Abs with same specificity. Polyclonal Abs are heterogenous population of Igs whereas Monoclonal Abs are population of single type of Ab. Monoclonal Abs are useful tools for diagnostic and research techniques. The technique used for the production of large amount of monoclonal Abs is called Hybridoma technology.

2. What is Hybridoma technology?

Hybridoma technology is a versatile and efficient method for large scale production of monoclonal antibodies against desired antigen. Hybridomas- are somatic cell hybrids produced by fusing Ab producing B lymphocytes from spleen cells and Myeloma cells. Production of desired monoclonal Abs from these hybridoma cells is called hybridoma technology. This method was developed by Georges J F Kohler and Cesar Milstein in 1975 and jointly awarded Nobel prize in Physiology & Medicine in 1984 for this work.

Step 1- Immunization of laboratory animal

Lab animal like Swiss albino mice is immunized with the An against which we have to produce Abs. This An will be having multiple epitopes on it. B lymphocytes get activated against the epitopes of the An. After few weeks, the mice is sacrificed and spleen is removed aseptically.  Spleen is the secondary lymphoid organ and we can easily harvest B lymphocytes from spleen. Spleen is subjected to mechanical and enzymatic disruption followed by density gradient centrifugation to separate activated B cells from normal B cells of spleen. Now we have B cells capable of producing Abs against the multiple epitopes of the injected An.

Step 2- Fusion of cells to produce Hybridomas

B cells have only short life span in cell culture. Hence, to increase their life span in laboratory conditions they are fused with Myeloma (Blood cancer) cells. Myeloma cells used here are mutated Myeloma cells. Myeloma cells are cancerous B cells (Plasma cells) that can divide indefinitely in culture media. Their 2 genes are mutated. One is HGPRT (Hypoxanthine phosphoribosyl transferase) gene. Hence, they are not able to synthesize nucleotides by the Salvage pathway. Second mutated gene is Ig gene. As a result of this mutation myeloma cells could not synthesize their own Abs.  These mutations are represented as HGPRT^- & Ig^-.

Activated B cells and Myeloma cells are fused in the presence of chemical fusogen Polyethylene Glycol (PEG).  As a result of cell fusion, 5 types of cells are obtained.

• ·      Unfused B cells
• ·      Fused B cells
• ·      Unfused myeloma cells
• ·      Fused myeloma cells
• ·      Hybrid cells (Hybridomas)

Hybridomas are formed by the fusion of B cells and myeloma cells. Hybridomas will be of different types depending on the type of B cell fused with myeloma cells. Next task is the selection of this hybridomas from mixture of above cells. 

Step 3- Selection of Hybridomas using HAT medium

H= Hypoxanthine

A= Aminopterin

T= Thymidine

HAT medium is a selection medium for mammalian cell cultures. During cell multiplication, nucleic acid synthesis takes place in 2 pathways. The De novo pathway & Salvage pathway. Aminopterin in HAT medium blocks the De novo pathway. Hence the only the cells that can synthesize their nucleic acid using Salvage pathway can survive in HAT medium. H & T are major metabolites in Salvage pathway and HGPRT is the key enzyme. So HGPRT^- cells die in HAT medium.

Once the hybridoma mixture is added to the HAT medium, fused and unfused B cells die out in few days as they have short life span in culture media. They can not divide indefinitely in cell cultures. Fused and unfused myeloma cells will also die as they are HGPRT^- and Aminopterin blocks De novo pathway in HAT medium. Hybrid cells survive in HAT medium as they are HGPRT⁺ due to the activated B cell and divide indefinitely, property due to the myeloma cell. Hence, what will be remaining in HAT medium will be hybrid cells that are able to produce Abs against the different epitopes of the particular An. Now these hybridomas are mixture of B cells producing Abs of different specificities. Now our aim is to select and propagate hybrid cells that produce single type of Ab. So, we have to separate these hybridomas and grow them individually. So next step is isolation of hybridomas of single specificity.

Step 4 - Isolation of Hybridomas of single specificity

Isolation of hybridomas of single specificity is achieved by limiting dilution method. In this technique cells are distributed at very low density in multi well culture plates so that each well contain a single cell only.

 Step 5 – Screening of Abs produced

Next step is the screening of Abs produced by individual hybridoma cells in the multi well culture plates. Collected supernatants could be analyzed for Abs by techniques like ELISA and RIA.

Step 6 – Cloning & Propagation

Once the hybridomas producing the desired Abs are identified they are isolated, cloned and propagated. Now we have hybridomas producing Abs of single specificity or monoclonal Abs.

Step 7 – Characterization and storage

Finally, mAbs are characterized and stored, usually in liquid nitrogen. Now they can be readily used in treating and diagnosing diseases.  

3. Advantages & applications of Hybridoma technology

Discovery of hybridoma technology was a revolution in immunology.

• ·    Hybrid cells can be maintained indefinitely in cultures for the continuous production of mAbs in vitro.
• ·     Invivo maintenance can be done by injecting hybrid cells in the intraperitoneal cavity of mice and mAbs can be harvested from the ascitic fluid produced.
• ·      Hybrid cells can be kept frozen for prolonged usage.
• ·     Powerful tool of passive immunization.
• ·     Numerous therapeutic, diagnostic (bacterial, viral & other Ans) and research applications.
• ·mAbs against various Ans are now used in commercially available immunofluorescence & ELISA kits.

T Dependent (TD) Antigens

Ø  T Dependent (TD) Antigens require the participation of T lymphocytes for the stimulation of B cells to produce Abs. T cells are of two types. Helper T cells (T_H) or CD 4+ cells and cytotoxic T cells (T_C) or CD 8+ cells. T_H cells are involved in B cell activation by TD antigens.

Ø  TD antigens are structurally complex molecules like serum proteins, protein-hapten complex, erythrocytes etc. Activation of B cells by TD antigens is more complex, but results in stronger response with immunological memory. TD antigens have to be first processed by the An processing/ presenting cells (APC).

Ø  Macrophages, dendritic cells and B cells usually act as An processing/presenting cells. These cells capture An and present them to T lymphocytes. APCs have 2 unique properties.

1.      They express MHC Class II molecules on their surface.

2.      They can also produce co-stimulatory signals necessary to activate T_H cells

APCs first internalize the An by phagocytosis or endocytosis. Then APCs display part of An bound to MHC Class II molecules on their surface membrane.  T_H cells can recognise only those Ans that are presented or attached to MHC class II.

·         I signal - If B cell is acting as the APC, then the first signal is binding of An and BCR. B cell will then internalise the An, process it and present part of An on it surface along with MHC Class II which will be recognized by  T_H cells.

·         II signal - T Cell Receptor (TCR)-CD3 complex on the surface of T Cell recognize and bin to the MHC Class II + An expressed on the surface of B cells. CD4 molecule of T cell also bind to a specific portion on the MHC molecule of B cell to make the binding more  effective. This binding of B cell/APC to T_H cell is called “linked recognition”.

Ø  Additional activation of T_H cells are brought about co-stimulatory signals produced by the APCs. A number of co-stimulatory molecules are involved in T cell activation.  

Co-stimulatory signals

·      CD 28 on T_H cells binds to CD 80 (B7-1) or CD86 (B7-2) membrane proteins on APC. This interaction will activate the T_H cells to secrete various cytokines.

·       T_H cells also start expressing CD40 Ligand (CD40L) which will interact with complementary CD40 of the B cell. This interaction is essential for the survival of B cells and also for germinal centre formation for the secretion of memory cells.

Ø Activated T_H cells secrete T cell growth factor or Interleukin-2 (IL-2) which will act on the T_H cell itself and initiate its proliferation. Proliferated T_H cells are called naïve T cells or T_H0 cells.

Ø Proliferated T_H cells secrete IL-2, IL-4, IL-5, IFN-γ etc. B cells begin to express receptors for various cytokines, bind to cytokines released by T_H cells and thus B cells get activated.

Ø The activated B-cell clonally proliferates to produce a population of plasma cells (Effector B cells) and secrete Abs against the An which had initiated all these process. Initially IgM are the Abs produced by plasma cells. After several rounds of proliferation, B cells differentiate to form memory B cells which will act during secondary response.   

Ø After initial secretion of IgM, cytokines secreted by T_H2 cells stimulate the plasma cells to switch from IgM production to production of IgG, IgA, or IgE. This process is called class switching or isotype switching. By class switching, plasma cells produced from the same B cell could produce a variety of antibody classes with the same epitope specificity. Class switching is due to genetic rearrangement of gene segments encoding the constant region of Abs which determines the antibody type or class.

Ø Differences between TI & TD Antigens

TI Antigens	TD Antigens
Structurally simple & composed of limited number of repeating epitopes	Structurally complex
Molecules like bacterial capsular polysaccharides, bacterial lipopolysaccharides and  some polymeric proteins like flagellar protein flagellin	Molecules like serum proteins, protein-hapten complex, erythrocytes etc
Immune response is dose dependent. Too little An is non- immunogenic & too much An cause immunological tolerance	Immunogenic over wide dose range. Do not readily cause tolerance
Do not require preliminary processing by APCs	Require preliminary processing by APCs and presented along with MHC class II molecules on the surface of APC
Ab response limited to IgM and no isotype switching	Isotype switching occurs and induce formation of almost all isotypes like IgM, IgG, IgA &IgE
Week response & no immunological memory	Strong response with immunological memory
Metabolized slowly and remain in body for long periods	Metabolized rapidly in the body

T Independent Antigens

During humoral immune response, Ab production is brought about by B lymphocytes. Based on the ability to induce Ab formation, antigens can be classified into T independent and T dependent antigens. Some antigens can directly induce the B cells to produce the Abs and are called T Independent Ans. However, some Ans require the help of T lymohocytes for the production of Abs from B cells. These Ans are called T Dependent Ans.

T Independent (TI) Antigens

Most of the protein Ans require the help of T cells for the production of Abs by B lymphocytes. However, Ans like bacterial capsular polysaccharides and bacterial lipopolysaccharides and some polymeric proteins like flagellar protein flagellin, can directly stimulate the B Cells to produce Abs, without the involvement of T cells. They can directly activate B cells with out An processing and presentation to the T cells.  Such Ans are called T Cell or Thymus Independent (TI) antigens. They are structurally simple and carry repeating epitopes. These repeating epitopes cross link the BCRs and act as first signal of activation. Eventhough T lymphocytes are not involved in Ab production, they assist the B cell proliferation and differentiation. For complete activation of B cells a dual signal is required and the second signal usually comes from T cells. But here, as T cells are not, the second signal should come from other sources.

TI Ans are metabolized slowly and remain in the body for long periods. There are two types of TI Antigens. TI-1 antigens and TI-2 antigens. TI-1 are usually bacterial lipopolysaccharides (LPS) which have mitogenic properties can deliver dual signal to B cells by themselves. First signal by binding to BCR and second by binding to the lipid moiety of LPS whose nature is not well studied.

TI-2 antigens are non-PAMP compounds, such as polysaccharides, that lack mitogenic properties and activate B cells through a T cell-independent mechanism. These antigens have multiple repeating sugar units that cause extensive cross-linking of B cell receptors (BCRs), generating a strong activation signal that can partially compensate for the absence of co-stimulatory signals from T helper cells. However, a second signal is still required for full activation. This secondary signal can come from the engagement of toll-like receptors (TLRs) with pathogen-associated molecular patterns (PAMPs), such as flagellin, or interactions with components of the complement system. Here, flagellin serves as a PAMP that can provide the necessary secondary signal to support B cell activation.

Toll-like receptors are receptor proteins help in the recognition of wide array of pathogens and are found on the membranes of leukocytes including dendritic cells, macrophages, natural killer cells, immune cells like T cells and B cells, and also on non-immune cells like epithelial and endothelial cells, and fibroblasts.  PAMPs are Pathogen Associated Molecular Patterns found on infectious agents which are recognized by Toll like receptors.  

Once the B cells are activated, they undergo clonal proliferation and daughter cells differentiate into plasma cells. Plasma cells are antibody factories that secrete large quantities of antibodies. Abs produced by TI Ans are mainly IgM Abs. Type switching to IgG can take place with the help of T lymphocytes. Here, there is no production of memory cells and no immunological memory. Hence immune response is effective in primary infection and not effective in secondary infection and also short lived. Also, immune response by TI Ans are dose dependent. Means, too little An is not immunogenic and too much An cause immunological tolerance than immunity.

B Cell Receptor and Antiboby Production

Anything that is non-self or foreign to the body is considered as  antigens.  Once antigens enter our body, lymphocytes produce specific immunological reactions against the antigens in order to remove them and to prevent the development of  an infection or disease due to that particular antigen. This immune response can be humoral or cell mediated. Humoral immune response results in the production of antibodies (Ab) by B lymphocytes. Cell-mediated immunity does not involve antibodies. Rather, it involves antigen-specific cytotoxic T-lymphocytes, the activation of phagocytes and the release of various cytokines in response to antigen.

Antigens (An) are usually  bacteria, bacterial products, fungi, viruses or other parasites.  Entire organisms  usually do not function as the antigen. Particular macromolecules of these infectious agents usually act as antigens. Proteins are the major antigens and polysaccharides coming next to it. Lipids and nucleic acids of these microorganisms do not act as antigens unless they are coupled with proteins or polysaccharides.  Antigens are also called antibody generators as they induce the immune system to produce antibodies. Hence, Antibodies are glycoproteins produced in response to  antigens by the host immune system whose function is to eliminate the entered antigen. 

In order to produce antibodies, first the antigen should activate the B lymphocytes. Activated B cells are called Plasma cells or Plasma B cells.  Plasma cells are white blood cells that could secrete large volumes of antibodies or immunoglobulins. These secreted immunoglobulins are transported by blood plasma and lymphatic system to destroy the entered antigens. 

The B cells, a type of white blood cells, are produced by multipotent hematopoietic stem cells (HSCs) in the bone marrow.  Mature B-cells that have not been exposed to antigens are called naive B cells.

Naive B cells carry special receptors on their cell membrane to which the An binds. These receptors are called B Cell Receptors or BCRs. BCRs are made up of two parts. First part is the transmembrane antibody attached to the cell membrane of the B cell, to which the  antigen binds. Antibodies of  any one isotype like IgD, IgM, IgA, IgG, or IgE are acting as BCRs.  These antibodies are also called membrane immunoglobulin (mIg). This region is oriented outward, away from the cell. The second part is the signal transduction transmembrane heterodimer proteins embedded in the cell membrane of B cells called Igα & Igβ. These heterodimers are held together by disulfide bonds. The transmembrane immunoglobulin and the heterodimer proteins together make up the so called BCRs. 

Each individual B cell has around 50,000 BCRs on its surface. But one B cell carries BCRs specific for only one particular antigen. Thus, the B cell population of an adult individual carries BCRs for as many as 10 ¹³    different antigens. Means 10 ¹³   different and undifferentiated B cells are present in an adult human body. They circulate in blood, awaiting the activation by specific antigen. 

When an An is captured, the membrane receptor communicates with the nucleus through the signal transduction heterodimer proteins and B Cell is activated. Upon activation,  these B-cells immediately undergo clonal proliferation and differentiate into antibody producing plasma cells (effector B cells) and memory cells.  Plasma cells will secrete soluble antibodies and these Abs circulate through the bloodstream to identify the antigen that had induced its synthesis.  Such circulating antibodies are also present in the serum, tissue fluid and mucosal surfaces of vertebrates. Memory cells remain in circulation for very longer periods and produce secondary response. When the same An enters the body for a second time, memory cells  proliferate and differentiate into plasma cells, which then clear the antigen.

                                                    Image Courtesy: immunology.org

Antigens-Types-Hapten, Complete antigen, Epitope (antigenic determinants)

An antigen is defined as any substance which, when introduced parenterally into the body,stimulates the production of an antibody with which it reacts specifically and in an observable manner. Some antigens may not induce antibodies but may sensitize specific lymphocytes leading to cell mediated immunity or may cause immunological tolerance.

The word “parenteral” means “outside the intestinal tract”. When antigens are orally given, they are denatured by the intestinal enzymes and their antigenicity is destroyed, so no antibody formation takes place. When given parentarally, they are not denatured, hence induce antibody production.  However, some antigens given as oral vaccines are exceptions.  The word “specifically” is important as specificity is the hallmark of all immunological reactions.  An antigen introduced into the body reacts only with those particular immunocytes (B or T lymphocytes) which carry the specific marker for that antigen and which produce an antibody or cells complementary to that antigen only.  The antibody so produced will react only with that particular antigen and with no other, however cross reactions may occur between closely related antigens.

The two attributes of antigens are:

1. Induction of an immune response (immunogenicity)

2. Specific reaction with antibodies or sensitized cells (Antigenicity/immunological reactivity)

Immunogenicity and antigenicity are related, but distinct. Immunogenicity is the ability to induce a humoral or cell mediated immune response.

B cells + antigen→ effector B cells + memory B cells

                          (plasma cells)

T cells + antigen→effector T cells + memory T cells

Although a substance that induces a specific immune response is usually called an antigen, it is more appropriately called an immunogen.

Antigenicity is the ability to combine specifically with the final products of the above responses (ie., antibodies or cell-surface receptors).  All molecules that have the property of immunogenicity also have the property of antigenicity, but the reverse is not true. Based on the ability to carry out the above mentioned attributes, antigens may be classified into different types.  A complete antigen is able to induce antibody formation and produce a specific and observable reaction with the so produced antibody.  Haptens are substances that are incapable of inducing antibody formation by themselves but can react specifically with antibodies.  In other words, haptens are antigenic, but lack immunogenicity.  The term hapten is derived from the Greek haptein which means “to fasten”.  Haptens become immunogenic on combining with a larger molecule carrier. Or they are low molecular weight molecules that can be made immunogenic by conjugation to a suitable carrier.  Haptens may be complex or simple.  Complex haptens can precipitate with specific antibodies, simple haptens are nonprecipitating. Complex hapten is polyvalent and simple hapten is univalent, since it is assumed that the precipitation requires the antigen to have two or more antibody combining sites.

The smallest unit of antigenicity is known as the antigenic determinant or epitope.  Epitope is the portion of an antigen that is recognized and bound by an Ab or TCR/MHC complex.  The epitope is that small area on the antigen, usually consisting of four or five aminoacid or monosaccharide residues, possessing a specific chemical structure, electric charge and spatial configuration, capable of sensitizing an immunocyte and of reacting with its complementary site on the specific antibody or T cell receptor.  Epitopes when present as a single linear segment of the primary sequence is called sequential or linear epitope.  When formed by bringing together on the surface residues from different sites of the peptide chain during its folding into tertiary structure, it is called conformational epitope.  T cells recognize sequential epitopes where as B cells recognize conformational epitopes.  The combining area on the antibody molecule, corresponding to the epitope, is called the paratope.  Epitopes and paratopes determine the specificity of immunological reactions.

Adjuvants

Adjuvants (from Latin adjuvare-to help) are substances that, when mixed with an antigen and injected with it, enhance the immunogenicity of that antigen.  Adjuvants are often used to boost the immune response when an antigen has low immunogenicity or when only small amounts of an antigen are available.   They are known to exert one or more of the following effects:

·         Prolong antigen persistence

·         Enhance co-stimulatory signals

·         Induce granuloma formation

·         Stimulate lymphocyte proliferation nonspecifically

Examples- Freund’s complete adjuvant, Aluminium potassium sulfate (alum), Mycobacterium tuberculosis, Bacterial lipopolysaccharide (LPS) etc.