Monoclonal antibody and its applications

S.Tamilarasi 24711014

CONTENTS Introduction  Monoclonal antibody  Hybridoma technology  Production of monoclonal antibody  Types of monoclonal antibody  Applications of monoclonal antibody  Conclusion


Introduction


 

Antibodies are proteins produced by a type of white blood cell called a B cell (B lymphocyte) of the immune system in response to foreign proteins,called antigens. Antibodies can be triggered by and directed at foreign proteins, microorganisms, or toxins. Antibodies function as markers, binding to the antigen so that the antigen molecules can be recognized and destroyed by phagocytes

Monoclonal antibody


 

An antibody produced by a single clone of B cells (specifically, a single clone of hybridoma cells) and therefore a single pure homogeneous type of antibody. Each B cell in an organism synthesizes only one kind of antibody. In an organism, different types of B cells and their respective antibodies were produced in response to the various antigens that the organism had been exposed to. Therefore we need a method to culture a population of B cells derived from a single ancestral B cell, so that this population of B cells would allow us to harvest a single kind of antibody. This population of cells would be correctly described as monoclonal, and the antibodies produced by this population of B cells are called monoclonal antibodies. Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell.

Monoclonal antibodies were produced in mice using a technique described by Khler and Milstein et al.. They were awarded the Nobel Prize in 1984 (along with Jerne) for their work. There are three principal steps in production of monoclonal antibodies: 1) immunization, 2) hybridoma formation 3) MAb production.

HYBRIDOMA TECHNOLOGY Immunization y Cell fusion y Selection of hybridomas y Screening the products y Cloning and propagation y Characterization and storage
y

Immunize an animal usually mouse by injecting with an appropriate antigen along with Freunds complete or incomplete adjuvant. Adjuvants are non specific potentiators of specific immune responses. Injection of antigens at multiple sites are repeated several times for increased stimulation of antibodies. 3 days prior to killing of animal a final dose is given intravenously. Spleen is aseptically removed and disrupted by mechanical or enzymatic methods to release the cells. By density gradient centrifugation lymphocytes are separated from rest of the cells.

y y

y y

Lymphocytes are mixed with HGPRT deficient myeloma cells and is exposed to PEG for a short period. Polyethylene glycol is used to fuse adjacent plasma membranes The mixture contains hybridomas, free myeloma cells, and free lymphocytes.

Dihydrofolate
Aminopterin

Tetrahydrofolate Precursors Nucleotides---->DNA

Hypoxanthine Thymidine

y y

y y

y y

The above mixture is cultured in HAT medium for 7-10 days. Due to lack of HGPRT enzyme in myeloma cells, salvage pathway is not operative and aminopterin in HAT medium blocks the de novo synthesis of nucleotides. Hence free myeloma cells are dead. As the lymphocytes are short lived they also slowly dissappear. Only the hybridomas that receives HGPRT from lymphocytes are survived. Thus hybridomas are selected by using HAT medium Suspension is diluted so that each aliquot contains one cell each. These are cultured in regular culture medium, produced desired antibody.

y y

y y

Screening is done for antibody specificity. For this we need to test the culture medium from each hybridoma culture for desired antibody specificity. Common tests like ELISA and RIA are used for this. In these tests the antigens are coated to plastic plates. The antibodies specific to the antigens bind to the plates. The remaining are washed off. Thus the hybridomas producing desired antibodies are identified. The antibodies secreted by them are homogenous and specific and are referred as monoclonal antibodies.

The single hybrid cell producing the desired antibody are isolated and cloned. The hybridomas can be grown indefinitely in a suitable cell culture medium.They can also be injected into mice (in the peritoneal cavity, surrounding the gut). There, they produce tumors secreting an antibody-rich fluid called ascites fluid.
Reclone and cultivate positive clones

Tissue culture method

Mouse ascites method

Biochemical and biophysical characterization are made for desired specificity. It is important to note the monoclonal antibody is specific for which antigen MAbs must be characterized for their ability to withstand freezing and thawing.

13

(Most common screening techniques are ELISA and RIA)

Low concentration (1-20 ug/ml)

High concentration (1-10 mg/ml)

Production of monoclonal antibody from hybridomas

 I. II.  

There are currently two widely adopted methods used to produce monoclonal Ab from positive hybridomas. In vivo (mouse ascites method) in vitro (tissue culture method) There are advantages and disadvantages for both methods. For individuals needing only small quantities of MAbs, simple, inexpensive stationary cell cultures may be suitable. These involve a variety of flasks and bottles and are simple and easy to use with existing equipment and skills. Regardless of the configuration selected, the MAbs must be concentrated after production.

Invivo method (Mouse ascites method)



1.

2.

3.

4.

5.

Hybridomas are injected into mouse peritoneal cavity and after sometime the mouse cavity is filled with fluid (ascitic fluid)containing these hybridomas (therefore called ascites method). Priming - Intraperitoneal injection with pristane with a volume not to exceed 0.5 ml. Hybridomas are injected after a suitable period (from 3 days to 3-4 weeks following priming). All tumor cells should be screened prior to injection to prevent transmission of common rodent viruses. Animals should be checked every day following priming with pristane. Once abdominal distention has been noted, frequency of monitoring may need to be increased. Abdominal taps (paracentesis) or Euthanasia to harvest ascitic fluid

Invitro method(Tissue culture method)

1.

Batch Tissue-Culture Methods The simplest approach for producing mAb in vitro is to grow the hybridoma cultures in batches and purify the mAb from the culture medium. One may use either tissue culture flasks (T flasks) or other cell culture dishes for stationary culture. Cells and media are placed in the flask, maintained in a CO2 incubator, and handled as a batch culture. Incubation times are typically 7-10 days before harvest. MAb concentration is typically in the range of 10-100 g/ml. T flasks are simple to use and require minimal technical expertise, inexpensive and are stackable, disadvantage is that MAb concentrations are very low, should be considered for use when only small amounts of MAb are needed;

2. Roller Bottles and Spinner Flasks



 

MAb concentrations obtained with these two methods are typically greater than stationary culture techniques, ranging from approximately 10-220 g/ml Average culture duration is longer -- approximately 12 days Advantages and disadvantages are similar to those described for T flasks, but roller bottles and spinner flasks require more incubator space, and are more expensive than T flasks.

3. Gas permeable tissue culture bags

 

 

Gas-permeable pre-sterilized disposable cell-culture bags are commercially available for MAb production. The bags feature attached ports, and tubing with roller clamps, which are used for inoculation of cells and media, sampling during production, and harvest The bags are a closed system with no mechanical parts, they are relatively free of contamination and require little or no monitoring. Compared to standard T flasks, bags have more surface area for CO2 and oxygen diffusion, improving cell oxygenation

Other In Vitro MAb Production Methods

  

Wave bioreactors CELLine culture systems Hollow fiber bioreactors Laboratory-scale stirred tank reactors Fermenters Ceramic-matrix bioreactors and Packed-bed bioreactors50

   

Types of monoclonal antibody Murine antibody  Chimeric antibody  Humanised antibody  Fully human antibody


Murine monoclonal antibodies (suffix -omab)

 

Murine antibodies were obtained by hybridoma technology dissimilarity between murine and human immune systems led to the clinical failure of these antibodies Ibritumomab - Non-Hodgkin lymphoma Muromonab-CD3 - Transplant rejection

Chimeric antibodies (suffix -ximab)

   

Chimeric antibodies are composed of murine variable regions fused onto human constant regions. Human gene sequences, taken from the kappa light chain and the IgG1 heavy chain, results in antibodies that are approximately 65% human. This reduces immunogenicity, and thus increasesserum half-life. While structurally similar, differences between the two are sufficient to invoke an immune response occurred when murine monoclonal antibodies were injected into humans and resulted in their rapid removal from the blood, systemic inflammatory effects, and the production of human anti-mouse antibodies (HAMA). Cetuximab - Colorectal cancer, Head and neck cancer Infliximab - Several autoimmune disorders Abciximab - Cardiovascular disease

Humanized monoclonal antibodies(suffix zumab)



Humanised antibodies are produced by grafting murine hypervariable amino acid domains into human antibodies. This results in a molecule of approximately 95% human origin. However it has been shown in several studies that humanised antibodies bind antigen much more weakly than the parent murine monoclonal antibody, with reported decreases in affinity of up to several hundredfold Omalizumab - mainly allergy-related asthma Natalizumab - Multiple sclerosis and Crohn's disease Efalizumab - Psoriasis

Human monoclonal antibodies (suffix umab)

 

  

Human monoclonal antibodies are produced using transgenic mice or phage display libraries. Human monoclonal antibodies are produced by transferring human immunoglobulin genes into the murine genome, after which the transgenic mouse is vaccinatedagainst the desired antigen, leading to the production of monoclonal antibodies. allowing the transformation of murine antibodies in vitro into fully human antibodies. Ofatumumab - Chronic lymphocytic leukemia Adalimumab - Several auto-immune disorders Golimumab - Rheumatoid arthritis, Psoriatic arthritis, and Ankylosing spondylitis

Evolution of monoclonal antibody

1. TRANSGENIC
DNA SPLICING / GENE KNOCK OUT

2. LIBRARIES
a.BACTERIOPHAGE b. mRNA c. Cell Surface

mAbs development

Recombinant monoclonal antibodies. Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice. Phage display library: construction of VH and VL gene libraries and expression of them on a filamentous bacteriophage. The phage expressing an antigen-bonding domain specific for a particular antigen to screen the mAbs.

Applications


 

 

Although monoclonal antibodies were first produced in 1975 as research tools, scientists quickly recognized their practical uses, especially in diagnostic tests and in therapy. Several diagnostic procedures that use monoclonal antibodies are now available Diagnostic Applications Biosensors & Microarrays Therapeutic Applications Transplant rejection Muronomab-CD3 Cardiovascular disease Abciximab Cancer Rituximab Infectious Diseases Palivizumab Inflammatory disease Infliximab Clinical Applications Purification of drugs, Imaging the target Future Applications Fight against Bioterrorism

Purification of protiens
Monoclonal antibodies can also be used to purify a substance with techniques called immunoprecipitation and affinity chromatography. A monoclonal antibody can be used to detect pregnancy in 14 days after conception. Other monoclonal antibodies allow rapid diagnosis of hepatitis, influenza, herpes, streptococcal, and Chlamydia infections

Diagnostic tests


 

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. They are also very useful in immunohistochemistry, which detect antigen in fixed tissue sections and immunofluorescence test, which detect the substance in a frozen tissue section or in live cells.

Monoclonal antibody drugs for cancer treatment

  i. ii. iii. iv.

Monoclonal antibody drugs are a relatively new innovation in cancer treatment. When a monoclonal antibody attaches to a cancer cell, it can: Make the cancer cell more visible to the immune system. Block growth signals. Stop new blood vessels from forming. Deliver radiation to cancer cells.

Radioimmunotherapy
Involves the use of radioactively conjugated Murine antibodies against cellular antigens Eg.; Tositumomab ----non-Hodgkins lymphoma

Autoimmune diseases
 i.

ii.

iii.

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- . Basiliximab and daclizumab inhibit IL-2 on activated T cells and thereby help preventing acute rejection of kidney transplants. Omalizumab inhibits human immunoglobulin E(IgE) and is useful in moderate-to-severe allergic asthma.

FDA approved therapeutic antibodies



  

The first FDA-approved therapeutic monoclonal antibody was a murine IgG2a CD3 specific transplant rejection drug, OKT3 (also called muromonab), in 1986. This drug found use in solid organ transplant recipients who became steroid resistant. Prevents acute rejection of kidney transplants Prevents autoimmune destruction of islet cells in type I Diabetes mellitus. Hundreds of therapies are undergoing clinical trials. Most are concerned with immunological and oncological targets.

Conclusion


 

Monoclonal antibodies (mAb) are important reagents used in biomedical research, in diagnosis of diseases, and in treatment of such diseases as infections and cancer. Several different monoclonal antibodies have been licensed for the treatment of a variety of diseases, and have proved to be useful in patients with more severe disease. The use of monoclonal antibodies (mAb) in biomedical research has been and will continue to be important for the identification of proteins, carbohydrates, and nucleic acids. Due to their expense and mode of administration they tend to be reserved for when conventional drugs have failed to elicit a response. The future may see combinations of monoclonal antibodies being used to better target complex disease processes.

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