monoclonal

MONOCLONALMONOCLONAL

antibodiesantibodies

Dr.T.V.Rao MDDr.T.V.Rao MD

Beginning of Monoclonal Beginning of Monoclonal EraEra

Georg KohlerGeorg Kohler and and Cesar MilsteinCesar Milstein fuse mouse lymphocytes with fuse mouse lymphocytes with neoplastic mouse plasma cells to neoplastic mouse plasma cells to yield hybridomas that produce yield hybridomas that produce specific antibodies. This offers a specific antibodies. This offers a limitless supply of monoclonal limitless supply of monoclonal antibodies. Monoclonal antibodies antibodies. Monoclonal antibodies permit diagnostic tests that are permit diagnostic tests that are specific, and function as probes. specific, and function as probes.

Discovery of Monoclonal Discovery of Monoclonal AntibodiesAntibodies

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

Nobel prize in Medicine and Nobel prize in Medicine and Physiology wasPhysiology was awarded toawarded to Köhler, Köhler,

Milstein Milstein andand Jerne in 1984Jerne in 1984

Monoclonal AntibodiesMonoclonal Antibodies

Monoclonal antibodiesMonoclonal antibodies are are Monospecific antibodiesMonospecific antibodies that are that are identical because they are produced identical because they are produced by one type of immune cell that are by one type of immune cell that are all clones of a single parent cell. all clones of a single parent cell. Given (almost) any substance, it is Given (almost) any substance, it is possible to create monoclonal possible to create monoclonal antibodies that specifically bind to antibodies that specifically bind to that substance that substance

NomenclatureNomenclature

The The nomenclature of monoclonal nomenclature of monoclonal antibodiesantibodies is a naming scheme for is a naming scheme for assigning generic, or non-proprietary, assigning generic, or non-proprietary, names to a group of medicines called names to a group of medicines called monoclonal antibodies. This scheme monoclonal antibodies. This scheme is used for both the World Health is used for both the World Health Organization’s International Non-Organization’s International Non-proprietary Names and the United proprietary Names and the United States Adopted Names States Adopted Names

Study of Myeloma leads to Study of Myeloma leads to Discovery of Monoclonal Discovery of Monoclonal

antibodiesantibodies In the In the 1970’s1970’s the B- the B-

cell cancer cell cancer myelomamyeloma was known, and it was was known, and it was understood that these understood that these cancerous B-cells all cancerous B-cells all produce a single type produce a single type of antibody. This was of antibody. This was used to study the used to study the structure of structure of antibodies, but it was antibodies, but it was not possible to not possible to produce identical produce identical antibodies specific to antibodies specific to a given antigen.a given antigen.

Fusion of Mice spleen cells with Fusion of Mice spleen cells with Myeloma cells produced Monoclonal Myeloma cells produced Monoclonal

antibodiesantibodies

Characters of Monoclonal Characters of Monoclonal AntibodiesAntibodies

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

Hybridoma creates Hybridoma creates Monoclonal antibodiesMonoclonal antibodies

Monoclonal antibodies Monoclonal antibodies are typically made by are typically made by fusing myeloma cells fusing myeloma cells with the spleen cells with the spleen cells from a mouse that has from a mouse that has been immunized with been immunized with the desired antigen. the desired antigen. However, recent However, recent advances have allowed advances have allowed the use of rabbit B-cells. the use of rabbit B-cells.

Doctorrao’s ‘e’ learning seriesDoctorrao’s ‘e’ learning series

Producing Monoclonal Producing Monoclonal antibodiesantibodies

First, a mouse is First, a mouse is inoculated with the inoculated with the antigen to which antigen to which the MA are to be the MA are to be produced. After produced. After this, the spleen is this, the spleen is removed and fused removed and fused with myeloma cells with myeloma cells in order to produce in order to produce the hybridomas the hybridomas that will be selected that will be selected according to the according to the antibody produced.antibody produced.

Hybridoma leads to Hybridoma leads to ProliferationProliferation

Principles in Hybridoma Principles in Hybridoma technologytechnology

inject the protein into ainject the protein into a mouse. mouse.

- remove the spleen. - remove the spleen. - identify which spleen - identify which spleen

cells are producing cells are producing antibodies. antibodies.

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

- screen each Ab for - screen each Ab for cross reactivity. cross reactivity.

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


Monoclonal antibodies are Monoclonal antibodies are produced by Hybridoma produced by Hybridoma

techniquetechnique

Monoclonal – Diagnostic Monoclonal – Diagnostic useuse

Although monoclonal Although monoclonal antibodies were first antibodies were first produced in 1975 as produced in 1975 as research tools, research tools, scientists quickly scientists quickly recognized their recognized their practical uses, practical uses, especially in diagnostic especially in diagnostic tests and in therapy. tests and in therapy. Several diagnostic Several diagnostic procedures that use procedures that use monoclonal antibodies monoclonal antibodies are now available are now available


A breakthrough in A breakthrough in DiagnosticsDiagnostics

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


Helps in Critical Diagnostic Helps in Critical Diagnostic decisionsdecisions

They can be used to They can be used to detect for the detect for the presence and presence and quantity of this quantity of this substance, for substance, for instance in a instance in a Western blot test Western blot test (to detect a (to detect a substance in a substance in a solution) or an solution) or an immunofluorescence immunofluorescence test.test.


Monoclonal's helps In Monoclonal's helps In Immunodiagnostic testsImmunodiagnostic tests

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

Limitations with Mouse Limitations with Mouse MonoclonalsMonoclonals

Problem in medical Problem in medical applications is that applications is that the standard the standard procedure of procedure of producing producing monoclonal monoclonal antibodies yields antibodies yields mouse antibodies, mouse antibodies, and these are and these are rejected by the rejected by the human immune human immune system system

Finding solutions for Finding solutions for Human useHuman use

In one approach, one In one approach, one takes the DNA that takes the DNA that encodes the binding encodes the binding portion of monoclonal portion of monoclonal mouse antibodies and mouse antibodies and merges it with human merges it with human antibody producing antibody producing DNA, in order to make DNA, in order to make bacteria produce bacteria produce antibodies that are half antibodies that are half mouse and half human. mouse and half human.

Doctorrao’s ‘e’ learning Doctorrao’s ‘e’ learning seriesseries

Conjugated monoclonal Conjugated monoclonal antibody therapy:antibody therapy:

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


Knowledge on Monoclonal’s Knowledge on Monoclonal’s advancesadvances

Mice have been genetically engineered to Mice have been genetically engineered to produce antibodies that have human produce antibodies that have human constant regions (this is the part of the constant regions (this is the part of the antibody that the human immune system antibody that the human immune system recognizes as being foreign (mouse)). By recognizes as being foreign (mouse)). By using these hybrid (or using these hybrid (or chimericchimeric monoclonal monoclonal antibodies with human constant regions, the antibodies with human constant regions, the immune system only "sees" a human protein immune system only "sees" a human protein and does not react against them. So, they and does not react against them. So, they can be injected many times to kill all of the can be injected many times to kill all of the cells in a tumor. cells in a tumor.

Monoclonal antibodies for Monoclonal antibodies for cancer treatmentcancer treatment

Possible treatment for Possible treatment for cancer cancer involves involves monoclonal antibodies monoclonal antibodies that bind only to that bind only to cancer cells specific cancer cells specific antigenantigen and induce and induce immunological immunological response on the target response on the target cancer cell (naked cancer cell (naked antibodies). mAb can antibodies). mAb can be modificated for be modificated for delivery of [toxin], delivery of [toxin], radioisotope, cytokine.radioisotope, cytokine.


FDA approvesFDA approves

The example described The example described in class is in class is HerceptinHerceptin. . These monoclonal These monoclonal antibodies can be used antibodies can be used against certain forms of against certain forms of breast cancer and have breast cancer and have passed clinical trials passed clinical trials and been and been approvedapproved for use by the for use by the FDAFDA..


FDA approves and Trails FDA approves and Trails onon

The first FDA-approved The first FDA-approved therapeutic monoclonal therapeutic monoclonal antibody was a murine antibody was a murine IgG2a CD3 specific IgG2a CD3 specific transplant rejectiontransplant rejection drug, drug, OKT3OKT3 (also called (also called muromonab), in 1986. This muromonab), in 1986. This drug found use in solid drug found use in solid organ transplantorgan transplant recipients recipients who became who became steroidsteroid resistant. Hundreds of resistant. Hundreds of therapies are undergoing therapies are undergoing clinical trialsclinical trials. Most are . Most are concerned with concerned with immunological and immunological and oncological targets. oncological targets.

Dr.T.V.Rao MDDr.T.V.Rao MD

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monoclonal

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structure of antibodies

single mab antibodies

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identical antibodies

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