Monoclonal antibody

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Monoclonal antibody:

Monoclonal antibody Presenter: Dr. Tina Damodar

Antibody and antigen:

Antibody and antigen An antibody ( Ab ), also known as an immunoglobulin ( Ig ), is a large Y-shaped protein produced by B-cells that is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. An antigen (Ag) is a substance that evokes the formation of antibodies in an animal immunized with the particular antigen and specifically combines with the antibody.



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The smallest unit of antigenicity which is capable of sensitizing an immunocyte and of reacting with its complementary site on the specific antibody or T cell receptor – EPITOPES The combining area on the antibody molecule, corresponding to the epitope is PARATOPE Epitopes and Paratopes determine the specificity of the immnunological reaction.

Antigens constitute a ‘mosaic ’ of antigenic determinants, hundreds or even thousands of epitopes may exist:

Antigens constitute a ‘mosaic ’ of antigenic determinants, hundreds or even thousands of epitopes may exist

Polyclonal antibodies:

Polyclonal antibodies Mixture of antibodies with different antigen binding sites that may bind to different epitopes with varying affinities . They may be of different antibody classes . When polyclonal sera ( heterogenous array of Ig produced during an immune response ) is used for immunological tests, cross reactivity may be noted due to: 1. Sharing of antigenic determinants by different species. 2. Mutations leading to evolution of epitopes close in specificity.

What is monoclonal antibody?:

What is monoclonal antibody? Identical antibodies produced by a single clone of B-cell or Plasma cell undergoing selective proliferation and directed against a desired epitope on the antigen are called MONOCLONAL ANTIBODIES. High degree molecular homogenetity Specificity for single antigenic epitope . No Cross Reactivity


history Kohler and Milsten - 1975; were awarded the Nobel Prize for medicine in 1984 for mAb . Network Hypothesis- Neils K Jerne Jerne . Nobel Prize for Medicine in 1984. 1964 Littlefield developed a way to isolate hybrid cells from 2 parent cell lines using the hypoxanthine- aminopterin - thymidine (HAT) selection media. Chimeric mABs - Boulianne et al.,1984; Morrison et al.,1984;Wright et al.,1992. The first FDA-approved therapeutic monoclonal antibody was a murine IgG2a CD3 specific transplant rejection drug, OKT3 (also called muromonab )

Conventional production of mabs:

Conventional production of mabs Hybridoma cultures can be maintained indefinitely: In vitro: in culture vessels. Conc. of Ab obtained 10-60 µg/ml. Robottle cell culture process. Membrane binded cell culture process Microcarrier cell culture process Suspended cell culture process In vivo: growing in mice. 1-10 mg/ml. Not encouraged by animal activists


hybridoma Hybridomas are hybrid cells that have inherited some characteristics of both parent cells

Hybridoma selection- the “hat trick”:

Hybridoma selection- the “hat trick” Myeloma cells have been genetically engineered ( HGPRT - ) such that they can not use H ypoxanthine, A minopterin , and T hymidine (HAT medium) as a source for nucleic acid biosynthesis and will die in culture. Only B cells that have fused with the engineered myeloma cells will survive in culture when grown in HAT medium(B cells on their own cannot divide)

HGPRT- hypoxanthine guanine phosphorybosyltransferase :

HGPRT- hypoxanthine guanine phosphorybosyltransferase Enables cells to synthesize purines using an hypoxanthine as a precursor. Ordinarily, the absence of HGPRT is not a problem for the cell because cells have an alternate pathway that they can use to synthesize purines . However, when cells are exposed to aminopterin (a folic acid analog), they are unable to use this other pathway and are now fully dependent on HGPRT for survival. Enzyme present in B-Cell

Types of mABS:

Types of mABS

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Murine mABs - (suffix - omab ) Chimeric mABS - ( - ximab ) Mouse antibodies are "seen" by the human immune system as foreign, and the human patient mounts an immune response against them, producing HAMA ("human anti-mouse antibodies"). These not only cause the therapeutic antibodies to be quickly eliminated from the host, but also form immune complexes that cause damage to the kidneys

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To address this problem, the complementary regions (CDRs), which are the responsible for antigen binding within the variable regions, have been transferred to human frameworks creating ‘‘CDR-grafted’’ or ‘‘humanized’’ antibodies. This is, in essence a human Ab with small segments containing mouse Ab genes.( - zumab ) Human mABS (- umab ) - technically much more difficult to immortalize and clone human B-cells and human hybridomas ; ethical problems of immunising human donors


uses M easuring protein and drug levels in serum T yping tissue and blood I dentifying infectious agents I dentifying clusters of differentiation for the classification and follow-up therapy of leukemias and lymphomas I dentifying tumor metastasis I dentifying and quantifying hormones I mmunoaffinity Purification

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As of 2011, 35 monoclonal antibody preparations have been approved by the U.S. Food and Drug Administration for use in humans.

Trigger the immune system:

Trigger the immune system

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Rituximab (trade name = Rituxan ®). Binds to the CD20 molecule found on most B-cells and is used to treat B-cell lymphomas Alemtuzumab ( MabCampath ). Binds to CD52 and depletes both T cells and B cells. CLL and rejection of kidney transplants . LymphoCide . Binds to CD22, a molecule found on some B-cell leukemias Daclizumab ( Zenapax ®). Binds to part of the IL-2 receptor exposed at the surface of activated T cells. Used to prevent acute rejection of transplanted kidneys . Has also showed promise against T-cell lymphoma .

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Muromonab-CD3 (OKT3) and two humanized anti-CD3 monoclonals . Bind to the CD3 molecule on the surface of T cells. Used to prevent acute rejection of organ , e.g., kidney, transplants. The humanized versions show promise in inhibiting the autoimmune destruction of beta cells in Type 1 diabetes mellitus Infliximab ( Remicade ®) and adalimumab ( Humira ®). Bind to tumor necrosis factor-alpha(TNF-α). Show promise against some inflammatory diseases such as rheumatoid arthritis Omalizumab ( Xolair ®). Binds to IgE thus preventing IgE from binding to mast cells. Shows promise against allergic asthma.

Stop cancer cells from taking up proteins:

Stop cancer cells from taking up proteins

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Trastuzumab ( Herceptin ) Blocks HER2, a growth factor receptor ; breast cancer and stomach cancer Bevacizumab ( Avastin ) for advanced bowel cancer, breast cancer and some other cancers Cetuximab ( Erbitux ) Blocks HER1 for advanced bowel cancer or in trials for other cancers Panitumumab ( Vectibix ) for advanced bowel cancer Pertuzumab ( Omnitarg ) Blocks HER2, breast and prostate cancer.

Carry cancer drugs or radiation to cancer cells:

Carry cancer drugs or radiation to cancer cells

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Still in clinical trials. Gemtuzumab ozogamicin ( Mylotarg ) – for acute leukaemia ADEPT – for bowel cancer Ibritumomab ( Zevalin ®) This is a monoclonal antibody against the CD20 molecule on B cells (and lymphomas) conjugated to either the radioactive isotope indium-111 ( 111 In) or the radioactive isotope yettrium-90 ( 90 Y) Lymphoma patient Tositumomab ( Bexxar ®). Conjugate of a monoclonal antibody against CD20 and the radioactive isotope iodine-131 ( 131 I). Lymphoma

Angiogenesis inhibition:

Angiogenesis inhibition Vitaxin . Binds to a vascular integrin found on the blood vessels of tumors but not on the blood vessels supplying normal tissues. Bevacizumab ( Avastin ® ). Binds to vascular endothelial growth factor (VEGF) preventing it from binding to its receptor. Colorectal cancers . Abciximab ( ReoPro ® ). Inhibits the clumping of platelets by binding the receptors on their surface that normally are linked by fibrinogen. Prevents reclogging of the coronary arteries in patients of angioplasty.

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Monoclonal antibodies for cancer. ADEPT, antibody directed enzyme prodrug therapy; ADCC, antibody dependent cell-mediated cytotoxicity ; CDC, complement dependent cytotoxicity ; MAb , monoclonal antibody; scFv , single-chain Fv fragment

Obstacles to the use of monoclonal antibodies:

Obstacles to the use of monoclonal antibodies Antigen distribution of malignant cells is highly heterogeneous, so some cells may express tumor antigens, while others do not. Tumor blood flow is not always optimal High interstitial pressure within the tumor can prevent the passive monoclonal antibody from binding. Coupling of mAB with radioactive atom/fluorescent molecule can be tedious.


developements Phage Display Library Transgenic Mouse Monoclonal T cell receptor Anti- Idiotypic antibodies Heteroconjugates Abzymes

Phage display library:

Phage display library Construction of V H and V L gene libraries(single chain fragment variable) obtained from population of B cell is cloned into phages used to infect E.coli . Growth of the phage infected bacteria generate a phage library that is screened . Phage of the desired antigen specificity is isolated and genes for VH and VL grafted on the constant region of the antibody using recombinant DNA technology. Monoclonal Ab of complete human origin of desired specificity obtained

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This technology has been successful to generate a human monoclonal antibody against the inflammatory Cytokine TNF . HUMIRA ( adalimumab ) Approved by FDA and is in clinical use for treatment of Rheumatoid Arthritis.

Transgenic mice:

Transgenic mice Transgenic (genetically engineered) mouse to produce more human-like antibodies ( Abgenix,CA ) Such mice have human antibody gene loci inserted into their bodies (using the embryonic stem cell method) Their own genes for making antibodies are "knocked out“ Other ways of solving the problem of HAMA .

Monoclonal t cell receptors:

Monoclonal t cell receptors Antibodies can bind to molecules expressed at the surface of target cells but are not effective against the peptide fragments that antigen-presenting cells contain tucked within their histocompatibility molecules. T-cell receptors are the ligands needed for that job. So monoclonal antibodies are not effective against intracellular antigens, e.g. virus-encoded proteins and tumor-specific antigens. But now progress is being made toward the development of monoclonal T-cell receptors ( αβ TCRs). Under clinical trials

Anti-idiotype mab:

Anti- idiotype mab

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B-cell cancers such as non-Hodgkin’s lymphoma (NHL) are tumors arising from a single malignant transformed B-cell, the tumor cells in NHL maintain on their surface the original malignant B-cell’s immunoglobulin (collectively referred to as, the “tumor idiotype ”) that are distinct from those found on normal B cells. The idiotype of a B-cell lymphoma can therefore serve as a tumor-specific antigen for therapeutic cancer vaccine development.

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Racotumomab (1E10) anti- idiotype antibody- advnced breast cancer , skin and occular malignant melanoma, SCLC Stilll under trials.

Heteroconjugates/ bispecific antibodies:

Heteroconjugates / bispecific antibodies One half of the Ab has specificity for the tumor cell and the other half for an immune effector cell(NK, CTL) promoting the destruction of the tumor cell. Bispecific Ab TGN1412 under trial


abzymes Monoclonal Catalytic antibodies that acts as enzymes . Use- 1.Structural and functional analysis of proteins. 2. Dissolve blood clots. 3. Cleave viral glycoproteins at specific site blocking viral infectivity. Disadvantage: Difficult to derive

PowerPoint Presentation:

The similarities btw ag-ab interactions and enzyme substrate interactions Haptn carries complex was syntheisd in whch hapten structurally resembled transition state of an ester under hydrolysis. Spleen cells from mice immunised with this transitions state analogue fused with myeloma cellsto generate monoclonal antihapten antibodies.when incubated with ester substrte these abs accelerated hydrolysis by 100 folds.

Side effects:

Side effects An allergic reaction can include these symptoms, though one may not have all of them Chills Fever An itchy rash Feeling sick Breathlessness Wheezing Headaches Flushes and faintness Changes in blood pressure


economics Since 2000, the therapeutic market for monoclonal antibodies has grown exponentially. The current “big 5” therapeutic antibodies on the market are bevacizumab , trastuzumab (both oncology), adalimumab , infliximab (both autoimmune and inflammatory disorders, ‘AIID’) and rituximab (oncology and AIID) accounted for 80% of revenues in 2006. In 2007, eight of the 20 best-selling biotechnology drugs in the U.S. were therapeutic monoclonal Ab.

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