PRESENTERMOHAMMED AMEER AHMED

M .PHARMACYROLL.NO 170215886003

(DEPARTMENT OF PHARMACEUTICS)Under the guidance of

Mrs. Pavani M PHARM, Ph. D

& Mrs Mounika Nijahwan

M PHARM, Ph. DGOKARAJU RANGARAJU COLLEGE OF

PHARMACY

MONOCLONAL ANTIBODIES

ContentsDEFINITIONTYPES OF ANTIBODIESSTRUCTURE OF ANTIBODYPRODUCTION OF MONOCLONAL

ANTIBODIESTHERAPEUTIC APPLICATIONSLIMITATIONS OF MCA

An antibody is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target.

Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell.

Polyclonal antibodies are antibodies that are derived from different cell lines.

Isotypes

According to differences in their heavy chain constant domains, immunoglobulins are grouped into five classes, or isotypes: IgG, IgA, IgM, IgD, and IgE.

IgG: IgG1 (66%), IgG2 (23%), IgG3 (7%) and IgG4 (4%) , blood and tissue liquid.IgA:IgA1 (90%) and IgA2 (10%), stomach and intestinesIgM: normally pentamer, ocassionally hexamer, multiple immunoglobins linked with disulfide bondsIgD:1% of proteins in the plasma membranes of B-lymphocytes, function unknown IgE: on the surface of plasma membrane of mast cells, play a role in immediate hypersensitive and denfensive for parasite

The structure of antibodies

• http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html

Production of Monoclonal Anti bodies

Immunization of Mice and Selection of Mouse Donors

*Mice are immunized with an antigen that is prepared for injection either by emulsifying the antigen with Freund's adjuvant or other adjuvants or by homogenizing a gel slice that contains the antigen.

*Intact cells, whole membranes, and microorganisms are sometimes used as immunogens. In almost all laboratories, mice are used to produce the desired antibodies.

*In general, mice are immunized every 2-3 weeks but the immunization protocols vary among investigators.

*When a sufficient antibody titer is reached in serum, immunized mice

are euthanized and the spleen removed to use as a source of cells for fusion with myeloma cells.

Immunization of Mice and Selection of Mouse Donors

Screening of Mice for Antibody Production

*After several weeks of immunization, blood samples are obtained from mice for measurement of serum antibodies.

*Several humane techniques have been developed for collection of small volumes of blood from mice .

*Serum antibody titer is determined with various techniques, such as Enzyme-Linked Immunosorbent Assay (ELISA).

*If the antibody titer is high, cell fusion can be performed. If the titer is too low, mice can be boosted until an adequate response is achieved, as determined by repeated blood sampling.

*When the antibody titer is high enough, mice are commonly boosted by injecting antigen without adjuvant intraperitoneally or intravenously (via the tail veins) 3 days before fusion but 2 weeks after the previous immunization.

*The mice are then euthanized and their spleens removed for in vitro hybridoma cell production.

Screening of Mice for Antibody Production

Preparation of Myeloma Cells

*Fusing antibody-producing spleen cells, which have a limited life span, with cells derived from an immortal tumor of lymphocytes (myeloma) results in a hybridoma that is capable of unlimited growth.

*Myeloma cells are immortalized cells that are cultured with 8-azaguanine to ensure their sensitivity to the hypoxanthine-aminopterin-thymidine (HAT) selection medium used after cell fusion.

* A week before cell fusion, myeloma cells are grown in 8-azaguanine. Cells must have high viability and rapid growth. The HAT medium allows only the fused cells to survive in culture.

Preparation of Myeloma Cells

Fusion of Myeloma Cells with Immune Spleen Cells

*Single spleen cells from the immunized mouse are fused with the previously prepared myeloma cells.

*Fusion is accomplished by co-centrifuging freshly harvested spleen cells and myeloma cells in polyethylene glycol, a substance that causes cell membranes to fuse.

*Only fused cells will grow in the special selection medium. The cells are then distributed to 96 well plates containing feeder cells derived from saline peritoneal washes of mice.

*Feeder cells are believed to supply growth factors that promote growth of the hybridoma cells.

*Commercial preparations that result from the collection of media supporting the growth of cultured cells and contain growth factors are available that can be used in lieu of mouse-derived feeder cells.

*It is also possible to use murine bone marrow-derived macrophages as feeder cells.

Fusion of Myeloma Cells with Immune Spleen Cells

Cloning of Hybridoma Cell Lines by “Limiting Dilution”

*At this step new, small clusters of hybridoma cells from the 96 well plates can be grown in tissue culture followed followed by selection for antigen binding or grown by the mouse ascites method with cloning at a later time.

*Cloning by “limiting dilution” at this time ensures that a majority of wells each contain at most a single clone.

*Considerable judgment is necessary at this stage to select hybridomas capable of expansion versus total loss of the cell fusion product due to underpopulation or inadequate in vitro growth at high dilution.

*In some instances, the secreted antibodies are toxic to fragile cells maintained in vitro.

*Also, it is the experience of many that a brief period of growth by the mouse ascites method produces cell lines that at later in vitro and in vivo stages show enhanced hardiness and optimal antibody production.

Cloning of Hybridoma Cell Lines by “Limiting Dilution”

Selection in HAT medium

*DNA synthesis in mammalian cells proceed through a main (de novo) pathway which requires glutamine and aspartate respectively as initial substrates for a series of reactions for the synthesis of purine-type (dATP and dGTP) and pyrimidine-type (dCTP and dTTP) dNTPs.

*Several of the reactions involved can be blocked by aminopterin, an analogue of dihydrofolate (another one is methotrexate) that binds with very high affinity and blocks the enzyme dihydrofolate reductase.

*As a result, de novo synthesis of dATP, dGTP, dCTP and dTTP is blocked.

*Mammalian cells, however, survive culture in the presence of aminopterin because they can utilise two salvage pathways. The first converts hypoxanthine, a reaction catalysed by the enzyme hypoxanthine-guanine phosporybosyl transferase (HGPRT).

*The second converts thymidine in dTMP, a reaction catalysed by thymidine kinase (TK). Thus a mutation in either the HGPRT or the TK gene would lead to normal growth in standard culture medium but to death in Littlefield's HAT medium (aminopterin, hypoxanthine and thymidine).

*The hybrid produced between such myeloma line and a B cell, however, would survive because it would utilise the normal HGPRT or TK gene of the B cell.

Selection in HAT medium

Analytical applications

*Antibody may be used to detect an antigen in forensic applications, in microbiological testing of foodstuffs and in diagnostic testing of blood samples for toxins or infectious organisms.

*The antibody-based test for the presence of antigen may be rendered quantitative, providing an assay for antigen.

*Antibody-based assays are widely used in medicine to determine

levels of growth factors, hormones, blood cells or malignant cells; the applications are essentially unlimited.

*Still in analytical mode, monoclonal antibodies may be used to locate antigen.

*Antibodies are widely used in conjunction with color forming labels and microscopy to localize antigens in tissue sections. This is known as Immunohistochemistry.

Analytical applications

*Antibodies may also be used preparatively to purify molecules or cells from crude mixtures.

*Immunoaffinity based preparative techniques are very powerful compared to more traditional biochemical purification methods.

*A successful purification procedure will combine both affinity-based and conventional methods.

Preparative applications

*Antibody-based purification methods are useful from the laboratory scale, where the aim is to purify nanogram to milligram quantities of biological substances from complex mixtures such as serum, to production of therapeutic substances, such as the blood-clotting factor VIII from large volumes of blood.

*The use of antibody to identify particular cell types in complex mixtures has been extended to preparative methods to purify these cells

Preparative applications

*One approach is to link antibody to magnetic particles.

*A magnet is then used to physically separate cells that bear the antigen from cells that do not.

*A more powerful technique uses the Fluorescence Activated Cell Sorter (FACS) , in which a cell may be identified on the basis of antibody tagged with fluorescent dye and physically separated from the other cells.

Preparative applications

*Potential therapeutic applications of antibodies include the neutralisation of toxins, the removal of infectious agents from the circulation and the destruction of body cells mediating disease, including autoimmune cells and cancer cells.

Therapeutic applications

*Polyclonal antisera against human lymphocytes were developed in the 1970s to treat patients who were rejecting organ grafts.

*The principle was that the host immune response was responsible for the organ graft rejection;

*Antibodies against key components of the immune system should suppress the rejection.

*These antisera have largely been superseded by a monoclonal antibody called OKT3, directed against a molecule expressed on human T cells and

* Involved in T-cell function.

Therapeutic applications

*OKT3 has been highly successful in reversing rejection episodes. The injected antibody is a mouse protein and is immunogenic, but the response is muted because the patients are immunosuppressed, both by the antibody itself and by other immunosuppressive therapy used to reduce the graft response.

*Nevertheless, OKT3, successful though it has been, has been perceived as limited in effectiveness partly by its immunogenicity.

Therapeutic applications

*There were highly promising results in animal models, where antibodies can destroy established tumours, induce a state of permanent tolerance to transplanted organs, reverse autoimmune disease and rescue animals from acute toxicity caused by, for example, bacterial endotoxin.

Therapeutic applications

Antibodies have been developed against :

*CD20 in B-cell malignancies and arthritis.

*against the cell-surface receptor human epidermal growth factor receptor 2 (HER2) in breast cancer and

*against the inflammatory cytokine tumour necrosis factor (TNF) in

autoimmune disease.

Therapeutic applications

*Immunogenicity;*Difficulty and cost of production on an adequate scale.*Unwanted biological activity – due, for example, to direct effects on

cells of the immune system.*Limited binding affinity, which necessitates the injection of large

amounts of antibody in order to achieve a therapeutic effect.*Lack of direct functional action, requiring conjugation of drugs or

other biologically active materials and*Limited penetration into the target tissue – especially dense, poorly

vascularised tumour tissue.

Limitations of Monoclonal Antibodies