Submitted to : Dr. Gautam KumarAssistant Professor Centre of Biological Science Central University Bihar

Presented by : P.K. AdityaM.Sc.Life science Roll no. - 09

Some termsAntibodies :-It is also called immunoglobulins, large Y-shaped proteins, a specialized immune protein, produced because of the introduction of an antigen into the body, and which possesses the remarkable ability to combine with the very antigen that triggered its production Antigens :- An antigen is any substance that causes your immune system to produce antibodies against it. An antigen may be a foreign substance from the environment, such as chemicals, bacteria, viruses, or pollen. An antigen may also be formed inside the body, as with bacterial toxins or tissue cells.Plasma cell :- is the pale yellow liquid component of blood that normally holds the blood cells in whole blood in suspension, this makes plasma the extracellular matrix of blood cells. It makes up about 55% of the body's total blood volume Plasma = Blood – ( RBC + WBC + platelets )Serum :-The clear liquid that can be separated from clotted bloodSerum = Blood - (RBC + WBC + platelets + fibrinogen + prothrombin) serum is what is left behind after blood clots

Immunoglobulin structure

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. They differ in amino acid sequence.

History of mAb development

• 1964 Littlefield developed a way to isolate hybrid cells from 2 parent cell lines using the hypoxanthine-aminopterin-thymidine (HAT) selection media.

• 1975 Kohler and Milstein provided the most outstanding proof of the clonal selection theory by fusion of normal and malignant cells

• 1990 Milstein produced the first monoclonal antibodies

Discovery of Monoclonal Antibodies

• Monoclonal antibodies were produced in mice using a technique described by Köhler and Milstein et al.. They were awarded the Nobel Prize in 1984 (along with Jerne) for their work.

Nobel prize in Medicine and Physiology was awarded to Köhler, Milstein and Jerne in 1984

Characters of Monoclonal Antibodies

• Monoclonal antibodies (mAb) are a single type of antibody that are identical and are directed against a specific epitope (antigen, antigenic determinant) and are produced by B-cell clones of a single parent or a single hybridoma cell line. A hybridoma cell line is formed by the fusion of a one B-cell lymphocyte with a myeloma cell. Some myeloma cells synthesize single mAb antibodies naturally

Principles in Hybridoma technology

• Inject the protein into a mouse.

• - remove the spleen. • - identify which

spleen cells are producing antibodies.

• - separate these cells and grow in tissue culture tubes.

• - screen each Ab for cross reactivity.

• - select the Ab which doesn't cross react with any other protein.

Strategy to generate monoclonal antibodies

HAT selection is used to select for growth of hybrids and against the growth of the parental myeloma.

• HAT medium (Hypoxanthine Aminopetrin Thymidine) is used for preparation of monoclonal antibodies,because it contains hypoxanthine, aminopterin, and thymidine. This medium is selective for fused (hybridoma) cells. Unfused myeloma cells and unfused spleen cells cannot grow because they lack HGPRT ( hypoxanthine-guanine-phosphoribosyl transferase), and thus cannot replicate their DNA and because of their limited life span respectively.

Types of Monoclonal Antibodies

Murine antibody

• Whole of the antibody is of murine origin

• Major problems associated with murine antibodies include

reduced stimulation of cytotoxicity

Formation of complexes after

repeated administration allergic reactions anaphylactic shock

Chimeric antibodies

Chimeric antibodies are composed of murine variable regions fused onto human constant regions.

Antibodies are

approximately 65% human.

This reduces

immunogenicity and thus increases serum half-life.

Humanised Mab

• Humanised antibodies are produced by grafting murine hypervariable amino acid domains into human antibodies.

• This results in a molecule of approximately 95% human origin

Applications of Monoclonal Antibodies

Diagnostic Applications

- Detects protein of interest either by blotting or immunofluorescence

Therapeutic ApplicationsTransplant rejectionCancerAutoimmune disordersInflammatory disease

• A monoclonal antibody can be used to detect pregnancy only 14 days after conception. Other monoclonal antibodies allow rapid diagnosis of hepatitis, influenza, herpes, streptococcal, and Chlamydia infections.

• Monoclonal antibodies can also be used to purify a substance with techniques called immunoprecipitation and affinity chromatography.

Advantages :

• 1) Homogeneity: • Monoclonal antibody represents a single antibody molecule that binds to antigens with

the same affinity and promote the same effectors functions.

• 2) Specificity: • The product of a single hybridoma reacts with

the same epitope on antigens.

• 3) Selection: • It is possible to select for specific epitope specificities and generate antibodies against

a wider range of antigenic determinants.

• 4) Antibody Production:• Unlimited quantities of a single well-defined

monospecific reagent.

Disadvantages:• 1) Affinity:

• Average affinity of monoclonal antibodies are generally lower than polyclonal antibodies.

• 2) Effector Functions: • Because antibody is monoclonal, it may not produce

the desired biologic response.

• 3) Specificity: • Monoclonals against conformational epitopes on

native proteins may lose reactivity with antigens.

• 4) Cross reactions: • Antibodies sometimes display unexpected

crossreactions with unrelated antigens.

• 5) Time and effort commitment: • Very large

Monoclonal antibodies for cancer treatment

There are three mechanisms that could be responsible for the cancer treatment:

1. mAbs act directely when binding to a cancer specific antigens and induce immunological response to cancer cells. Such as inducing cancer cell apoptosis, inhibiting growth, or interfering with a key function.2.mAbs may be modified for delivery of a toxin, radioisotope [RADIOIMMUNOTHERAPY], cytokine or other active conjugates.3.it is also possible to design bispecific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cell.

When a monoclonal antibody attaches to a cancer cell, it can:

• Make the cancer cell more visible to the immune system.

ex. Rituximab

• Block growth signals ex. Cetuximab used for colon cancer and block epidermal growth factors.

• Stop new blood vessels formation (angiogenesis) Ex. Devacizumab (Avastin) acts on vascular endothelial growth factor (VEGF).

• Deliver radiation to cancer cells by binding to radioactive element ex. Zevalin used for non-hodgkin lymphoma.

• Slip powerful drugs into cancer cells and kills the cancer cell. Ex. Kadcyla in breast cancer.