Sneha shreeM.Pharmacy2nd sem.

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.

• 1900 Ehrlich proposed the “ side-chain theory”

• 1990 Milstein produced the first monoclonal antibodies

• Paul Ehrlich at the beginning of the 20th century theorized that a cell under threat grew additional side-chains to bind the toxin, and that these additional side chains

broke off to become the antibodies that are circulated

through the body. It was these antibodies that Ehrlich first described as "magic bullets" in search of toxins.

Monoclonal Antibody Production technology was developed in 1975 by two scientists, Georges Kohler of West Germany and Cesar Milstei.

The previous picture is showing a mouse being immunized against a target cell “X”.(antigens) This will allow the mouse to produce antibodies for that will target against the “X” antigen.

Once the mouse has formed antibodies to the “X” antigen the Antiserum (blood serum rich in antibodies) extracted from mouse’s spleen.

Monoclonal antibodies are produced by fusing single antibody-forming cells to tumor cells grown in culture. The resulting cell is called a hybridoma.

Hybridoma cells are continuously growing cell line generated by the fusion of a myeloma cell and a normal cell that are capable of producing antibodies.

Each hybridoma will produce relatively large quantities of identical antibody molecules. Because the hybridoma is multiplying in culture, it is possible to produce a population of cells, each is producing identical antibody molecules. These antibodies are called "monoclonal antibodies" because they are produced by the identical offspring of a single, cloned antibody producing cell.

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.

The application of monoclonal antibodies can be broadly categorized as:

a) Diagnostic application b) Therapeutic application c) Catalytic MAb (Abzymes)

MAbs are utilized in diagnostic kits for the diagnosis of various infectious diseases, detecting pregnancy, monitoring drug levels, matching histocompatibility antigen,cancer and in immunoscintigraphy.

 FDA licensed a new diagnostic imaging agent that can determine the extent of disease in patients diagnosed with small cell lung cancer (SCLC). Because these agents can detect tumor in different part of the body at one time, it can help physician to advice certain patients with advanced forms of the disease about treatment option without requiring further diagnostic tests.   

      For ex.the new agent, Nofetumomab, is a fragment of a monoclonal

antibody that when tagged with the radioisotope technique, can detect a protein found on the surface of most small lung cancer cells.

Other monoclonal antibodies allows rapid diagnosis of hepatitis, influenza, herpes, streptococcal, and Chlamydia infections.

A monoclonal antibody can be used to detect pregnancy in only 14 days after conception.

Urine and serum mAbs pregnancy test are easier.Their selective binding property allow detection of low levels of human corionic gonadotropin (HCG) in urine and serum.These tests approach the accuracy of serum beta HCG radioimmuno assay for confirmation of normal pregnancy or evalution of possible abnormal pregnancy.

Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence of this substance.The Western blot test and immuno dot blot tests detect the protein on a membrane.

mAbs are also very useful in immunohistochemistry, process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues and immuno fluorescence test, which detect the substance in a frozen tissue section or in live cells.

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

Earlier horses were inoculated with bacterium Coryne bacterium diphtheriae which causes diptheria in humans, and the resulting crude horse antiserum was used to treat deptheria.Due to this discovery mortality reached 45%.

C.Diptheriae infects the throat or tonsils and produces an exotoxin that is lethal to human cells. This exotoxin enters the blood stream and damage organs that are distinct from the primary site of infection.

Today with the advent of hybridoma methodology antibodies again seen as potential therapeutic agent.

Organ transplantationFor the treatment of solid organ transplant rejection, several MAbs against T cell antigens have been evaluated. 'The most successful of these is orthrochrome OKT3 which was first MAb to be licensed for human use. The murine intact IgG antibody, directed against CD3 antigen on T lymphocytes, is used unconjugated to effectively reverse acute rejection episodes with renal, hepatic, cardiac and combined kidney pancreas transplants.Acute side effects such as fever and chills are common and probably may be a consequence of T cell damage.

MAbs are being evaluated for graft versus host disease in bone marrow transplantation.

Immune cells (white blood cells) in the tissue (the graft) recognize the recipient (the host) as "foreign". The transplanted immune cells then attack the host's body cells.

A ricin toxin immunoconjugate directed against CD5 antigen of T lymphocyte is effective both in the treatment and prevention of graft-versus host disease with histocompatible marrow grafts.

OKT3 has also shown promise in the management of immune related graft rejection events.

Bone marrow transplantation:

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.

• 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.

NAME OF DRUG

Alemtuzumab (Campath)

Bevacizumab (Avastin)

Cetuximab (Erbitux)

Gemtuzumab (Mylotarg)

TYPE OF CANCER USED TO TREATChronic lymphocytic leukemiaBreast cancerColon cancerLung cancer Colon cancerHead and neck cancers

Acute myelogenous leukemia

Ibritumomab (Zevalin)

Panitumumab

(Vectibix)

Rituximab (Rituxan)

Tositumomab (Bexxar)

Trastuzumab

(Herceptin)

Non-Hodgkin's

lymphoma

Colon cancer

Non-Hodgkin's

lymphoma

Non-Hodgkin's

lymphoma

Breast cancer

Toxins or radioactive isotopes are bound to the constant region of the mAbs. When the mAb binds to the surface cells of a tumour the toxin or radioactivity will kill the cancer cells and all cells within a certain radius (a killing zone). In this way cancer cells within the tumour will be killed.

Involves the use of radioactively conjugated Murine antibodies against cellular antigens

Eg.; Tositumomab ----- non-Hodgkins lymphoma

Monoclonal antibodies used for autoimmune diseases include infliximab and adalimumab, which are effective in rheumatoid arthritis, Crohn's disease and ulcerative Colitis by their ability to bind to and inhibit TNF-α.

Basiliximab and daclizumab inhibit IL-2 on activated T cells and thereby help to prevent acute rejection of kidney transplants.

Omalizumab inhibits human immunoglobulin E (IgE) and is useful in moderate-to-severe allergic asthma

TYPE APPLICATION MECHANISM

infliximab rheumatoid arthritisCrohn's diseaseUlcerative Colitis

inhibits TNF-α

Adalimumab rheumatoid arthritisCrohn's diseaseUlcerative Colitis

inhibits TNF-α

basiliximab Acute rejection of kidney transplants

inhibits IL-2 on activated T cells

daclizumab Acute rejection of kidney transplants

inhibits IL-2 on activated T cells

Omalizumab moderate-to-severe allergic asthma

inhibits human immunoglobulin E (IgE)

The binding of an antibody to its antigen and binding of enzyme to its substrate both involves weak, non covalent interactions and exhibits high specificity and often high affinity.

But an antigen is not altered by antibody, where as an enzyme alters the substrate and as a result the binding by catalyzing a chemical change. The enzyme uses its binding energy to stabilize the transition state of substrate. thus reducing activation energy for chemical modification of bound substrate.

Similarities between antigen-antibody interaction and enzyme-substrate interaction have led Lerner and his colleagues to explore the probability of enzyme like action of some antibodies.

The patients suffering from rheumatic diseases are found to have autoantibodies that react to cellular components. These autoantibodies increase in number alter birth up to two years and then remain constant for decades.

These antibodies have unique complement which differ from person to person and hence referred to as individual specific (IS) autoantibodies. These IS autoantibodies could be physically separated comprising of an antibody fingerprinting that could serve in the identification of an individual.

The concept of using specific antibodies conjugated with toxic or isotopically labelled materials for specific sites and drug localization is not new. This concept with the development of MAbs and hybridoma technology have gained wide appreciation in their use. The use of monoclonal antibodies to target drugs to specific cell types is a promising approach.