Monoclonal antibodies Clinical Significance


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Monoclonal antibodies Clinical Significance

  1. 1. Dr.Vignesh.S Resident in Internal Medicine Guided by Prof.Dr.R.L.Meena
  2. 2. 1) History and Discovery 2) Monoclonal Antibodies 3) Production and Nomenclature 4) Advantages and Disadvantages 5) CLINICAL SIGNIFICANCE 1) DIAGNOSTIC 2) THERAPEUTIC 3) RESEARCH 6)FDA approved MAbs 7)Problems faced with old generation MAbs 8)How was it overcome 9)LATEST UPDATE on MAbs 10)Conclusion ABBREVIATION USED: MAb  Monoclonal Antibody
  3. 3.  Humans have the ability to make antibodies able to ◦ recognize (by binding to) virtually any antigenic determinant (epitope) ◦ to discriminate between even similar epitopes  The remarkable specificity of antibodies makes them promising agents for human therapy.
  4. 4. But there are problems to be solved before antibodies can be used in human therapy.  The Polyclonal Response  Limitation of the Growth Potential
  5. 5.  What was needed was a way ◦ To make antibodies of a single specificity ◦ To make antibodies all built alike manufactured by a single clone of cells ◦ To make antibodies that can be grown indefinitely.
  6. 6. To make  An antibody that will bind only to one type/group of cells in a patient  Coupling a cytotoxic agent (e.g. a strong radioactive isotope) to that antibody, and  Then giving the complex to the patient so it can seek out and destroy the only particular type of cells (and no normal cells).
  7. 7.  In the 1970’s the B- cell cancer myeloma was discovered, and it was understood that these cancerous B- cells all produce a single type of antibody.  This was used to study the structure of antibodies, but it was not possible to produce identical antibodies specific to a given antigen.
  8. 8.  This problem was solved in 1975 with a technique deviced by Köhler , Milstein and Jerene  The technique is called somatic cell hybridization.  The result is a hybridoma cell producing antibodies targeting a single epitope in large numbers
  9. 9. Prof.Georges J. F. Köhler Prof.César MilsteinProf.Niels Kaj Jerne
  10. 10.  They are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell  They have monovalent affinity, in that they bind to the same epitope.  Given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to that substance; they can then serve to detect that substance.
  11. 11.  lost the ability to synthesize hypoxanthine- guanine-phosphoribosyl transferase (HGPRT). ◦ This enzyme enables cells to synthesize purines using an extracellular source of hypoxanthine as a precursor. ◦ Ordinarily, the absence of HGPRT is not a problem for the cell because cells have an alternate (de novo)pathway that they can use to synthesize purines. ◦ However, when cells are exposed to aminopterin (a folic acid analog), they are unable to use de novo pathway and are now fully dependent on HGPRT for survival.
  12. 12.  B cell has the enzyme HGPRT  But B cells die soon  They do not have the capacity to grow indefinitely because of their limited life span Scanning Electron Microscopic view of a B cell
  13. 13.  CELLS FUSED: ◦ Spleen cells from a mouse that has been immunized with the desired antigen ◦ Myeloma cells.  FUSION AGENT: ◦ Polyethylene glycol  MEDIUM: ◦ HAT Medium {Hypoxathine-Aminopterin- Thymidine}
  14. 14. VIDEO Courtesy:Dr.Bhanu Prakash
  15. 15.  Every monoclonal antibody has the following components in its name  Variable-Target Substem-Source Substem- Stem-Additional words(in special cases)  Ex: Alacizumab pegol is  Ala –ci-zu-mab-pegol
  16. 16. OLD NEW MEANING -anibi- - Angiogenesis inhibitor -ba(c)- -ba- Bacterium -ci(r)- -ci- Circulatory system -fung- -fu- Fungus -ki(n)- -ki- Inlterleukin -les- - Inflamattory lesions -li(m)- -li- Immune system -mul- - Musculoskeletal system -ne(ur)- -n(e)-* Nervous system -os- -s(o)- bone -toxa- -tox(a)- toxin - -tu- Tumour -vi(r) -vi- virus
  17. 17. Letter Meaning -a- Rat -e- Hamster -i- Primate -o- Mouse -u- Human -xi- Chimeric -zu- Humanized -axo- Rat/mouse hybrid
  18. 18.  Rituximab ◦ Ri- Variable ◦ tu- Tumour ◦ xi- Chimeric ◦ mab- Monoclonal Antibody  So Rituximab is a Chimeric Monoclonal Antibody targetting a Tumour
  19. 19.  Bevacizumab ◦ Beva- Variable ◦ ci- Circulatory System ◦ zu- Humanised ◦ mab- Monoclonal Antibody  So Bevacizumab is a Humanised Monoclonal Antibody targetting a protein in Circulatory System
  20. 20. A second word following the name of the antibody indicates that another substance is attached. 1)An antibody can be PEGylated (attached to molecules of polyethylene glycol) ◦ to slow down its degradation by enzymes and to decrease its immunogenicity  Ex:alacizumab pegol 2)A cytotoxic agent can be linked to an anti-tumor antibody for drug targeting purposes. ◦ vedotin,(monomethyl auristatin E) which is toxic by itself but predominantly affects cancer cells if used in conjugates  Ex:glembatumumab vedotin 3)A chelator for binding a radioisotope can be attached. ◦ Pendetide, a derivative of pentetic acid, in capromab pendetide to chelate indium-111.If the drug contains a radioisotope, the name of the isotope precedes the name of the antibody.  Ex: indium (111In) capromab pendetide
  21. 21. 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) Immunizing Antigen: Need not be characterized and is ultimately not needed in large quantities to produce large quantities of antibody. 4) Selection: It is possible to select for specific epitope specificities and generate antibodies against a wider range of antigenic determinants. 5) Antibody Production: Unlimited quantities of a single well-defined monospecific reagent
  22. 22.  Average affinity of monoclonal antibodies are generally lower than polyclonal antibodies  Monoclonals against conformational epitopes on native proteins may lose reactivity with antigens.  Antibodies sometimes display unexpected crossreactions with unrelated antigens.  Immune rejections  Time and effort commitment: VERY LARGE.
  23. 23. They are used in  Western blot test  ELISA TEST  Antigen capture assays  Immuno dot blot tests to detect the specific protein on a membrane.  Naked eye dipstick tests  Immuno-histochemistry , which detect antigen in fixed tissue sections and  Immuno-fluorescence test, which detect the substance in a frozen tissue section or in live cells.  Radio immuno assays  Tissue typing  Serotyping of Microorganisms  They are also used in the diagnosis of lymphoid and myeloid malignancies
  24. 24. Number of Monoclonal Antibodies have been developed in detection and diagnosis of  Parasites like ◦ Trichomonas vaginalis ◦ Leishmania donovani ◦ Trypanosoma congolense ◦ Babesia bovis  Human Viruses like ◦ Influenza virus ◦ Rotavirus ◦ Rabies Virus etc  Animal Viruses like ◦ Bovine herpes virus, ◦ Cervine herpes virus type I
  25. 25. MAbs can be used to detect pregnancy with Antibody to Beta HCG.
  26. 26.  Capture of parasite antigen from peripheral blood using monoclonal antibodies prepared against a malaria antigen target and conjugated to either a liposome containing selenium dye or gold particles in a mobile phase.  A second or third capture monoclonal antibody applied to a strip of nitrocellulose acts as the immobile phase.  The migration of the antigen- antibody complex in the mobile phase along the strip enables the labeled antigen to be captured by the monoclonal antibody of the immobile phase, thus producing a visible colored line
  27. 27.  Immunoprecipitation (Micral test): It is based on the colour shift of monoclonal antibody to human albumin labelled with gold.
  28. 28.  Monoclonal antibodies can also be used to purify a substance with techniques called immunoprecipitation and affinity chromatography.
  29. 29.  Mab helps to identify a specific molecule/substance in a mixture of substances
  30. 30.  MAbs have tremendous application not only in the field of diagnostics but also in ◦ therapeutics and targeted drug delivery systems for infectious diseases caused by bacteria, viruses, protozoa and for cancer, metabolic and hormonal disorders. ◦ They are also used in the immunological intervention with passive antibody, magic bullet therapy with cytotoxic agents coupled with anti mouse specific antibody
  31. 31.  Auto immune Conditions ◦ Rheumatoid arthritis, ◦ Crohn's disease ◦ Ulcerative Colitis by their ability to bind and inhibit TNF Alpha  Acute rejection of organ transplants by inhibiting Interleukin-2 on activated T cells  Allergic Asthma by inhibiting IgE Antibodies  Malignancies of solid organs
  32. 32.  Leukemias  Lymphomas  Osteoporosis  Platelet Aggregation Inhibitors
  33. 33. Anti-cancer monoclonal antibodies can be targeted against malignant cells by several mechanisms: 1)Radioimmunotherapy (RIT) involves the use of radioactively conjugated murine antibodies against cellular antigens to limit radiation exposure.  Murine antibodies were especially chosen, as their high immunogenicity promotes rapid clearance from the body. ◦ Ex:Tositumomab in non-Hodgkins lymphoma.
  34. 34. 2)Antibody-directed enzyme prodrug therapy (ADEPT)  It involves the application of cancer associated monoclonal antibodies which are linked to a drug-activating enzyme.  Subsequent systemic administration of a non-toxic agent results in its conversion to a toxic drug, and resulting in a cytotoxic effect which can be targeted at malignant cells
  35. 35. 3)Immunoliposomes are antibody- conjugated liposomes.  Liposomes - carry drugs or therapeutic nucleotides - conjugated with monoclonal antibodies - directed against malignant cells.  Tissue-specific gene delivery using immunoliposomes has also been achieved in brain, and breast cancer tissue
  36. 36. DRUGS
  37. 37.  Target: ◦ VEGF-A(Vascular Endothelial Growth Factor) that stimulates neovascularisation  Indications: ◦ Metastatic Breast,Colon Renal and Brain Cancers ◦ Diabetic Retinopathy,Retinopathy of Prematurity and Choroidal neovascularisation membrane  Adv Eff: ◦ Poor wound Healing, Impaired Collateral Formation around Atherosclerotic Vessels, Hypertension and Bleeding
  38. 38.  Target: ◦ TNF-Alpha  Indications: ◦ Rheumatoid Arthritis, Ulcerative Colitis, Crohn`s disease,Psoriatic Arthritis, Ankylosing Spondylitis  Adverse Effects: ◦ Infections,Lymphomas,Reactivation of Hep B,Tuberculosis,Drug induced Lupus,Vitiligo
  39. 39. ANTIBODY TARGET USES Abciximab CD41(integrin alpha receptor) Platelet aggregation inhibitor Adalimumab TNF ALPHA RA, Crohn's, Psoriasis, Psoriatic Arthritis, Ankylosing Spondylitis, Juvenile Idiopathic Arthritis, Hemolytic disease of the newborn Alemtuzumab CD 52 CLL
  40. 40. ANTIBODY TARGET USES Basiliximab CD 25(alpha chain of interleukin 2) Prevention of Organ Transplant Rejection Belimumab BAFF NHL Bevacizumab VEGF -A Metastatic Cancer, Diabetic Retinopathy Brentuximab Vedotin CD 30 Hematologic Cancers Canakinumab IL-1 RA Cetuximab EGFR Metastatic Colorectal Cancer and H&N Cancer Certolizumab pegol TNF alpha Crohns disease Daclizumab CD 25(alpha chain of interleukin 2) Prevention of Organ Transplant Rejection
  41. 41. ANTIBODY TARGET USES Denosumab RANKL Osteoporosis & Bone Cancer Eculizumab C5 PNH Efalizumab LFA-1 (CD11a) Psoriasis Gemtuzumab CD33 AML Infliximab TNF Alpha rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis, Crohn's disease, ulcerative colitis Muromonab CD33 Prevention of Organ Transplant Rejection
  42. 42. ANTIBODY TARGET USES Natalizumab Integrin alpha 4 Multiple Sclerosis,Crohn`s Omalizumab Ig E Fc region Allergic Asthma Panitumumab EGFR Colorectal Cancer Ranibizumab VEGF-A Macular Degeneration Rituximab CD20 Lymphomas,Leukemia Tocilizumab IL-6 Receptor RA Tositumomab CD20 Follicular Lymphoma Trastuzumab HER2neu Breast Cancer
  43. 43.  MAbs are used in research purpose also in analysing ◦ Human Lymphocytes ◦ MHC Antigens ◦ HLA system  Analysis of antigenic differences between virus and viral related proteins  Antigenic Characterization of viruses, bacteria and parasites
  44. 44.  Fever  Chills  Weakness  Headache  Nausea  Vomiting  Diarrhea  Rashes  Hypo/Hypertension DRUG/ TARGET RELATED SIDE EFFECTS
  45. 45.  Immune response against monoclonal Antibodies, producing HAMA ("human anti- mouse antibodies").  Quickly eliminated  Immune complexes - Renal Damage  Monoclonal antibodies raised in humans would lessen the problem, but few people would want to be immunized in an attempt to make them, and most of the attempts that have been made have been unsuccessful.
  46. 46.  Genetic engineering  mouse-human hybrid antibodies to reduce the problem of HAMA.  Chimeric antibodies. ◦ Antigen-binding parts (variable regions) of the mouse antibody with the ◦ Effector parts (constant regions) of a human antibody. ◦ Ex: Infliximab, rituximab, and abciximab  Humanized antibodies. ◦ Only the amino acids responsible for making the antigen binding site (the hypervariable regions) of a mouse (or rat) antibody with ◦ the rest of a human antibody molecule. ◦ Ex:Daclizumab, Gemtuzumab, Transtuzumab
  47. 47. Letter Meaning -a- Rat -e- Hamster -i- Primate -o- Mouse -u- Human -xi- Chimeric -zu- Humanized -axo- Rat/mouse hybrid --
  48. 48. Fully Human Monoclonal Antibody Parts of Human Antibody Parts of Mouse Antibody
  49. 49.  Transgenic mice.  Have human antibody gene loci inserted into their bodies (using the embryonic stem cell method).  have had their own genes for making antibodies "knocked out".  The result is a mouse that ◦ can be immunized with the desired antigen ◦ produces human, not mouse, antibodies against the antigen ◦ can yield cells to be fused with myeloma cells to manufacture all-human monoclonal antibodies.  Phage display is another technique for making all- human monoclonal antibodies
  51. 51.  The production of recombinant monoclonal antibodies involves technologies, referred to as cloning or phage display/yeast display.  Involves the use of viruses or yeast to create antibodies, rather than mice.  Rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies from which antibodies with desired specificities can be selected.  These techniques can be used to enhance ◦ The specificity with which antibodies recognize antigens ◦ The stability in various environmental conditions ◦ Their therapeutic efficacy and ◦ Their detectability in diagnostic applications
  52. 52.  mab: whole monoclonal antibody  Fab: fragment, antigen-binding (one arm)  F(ab')2: fragment, antigen-binding, including hinge region (both arms)  Fab': fragment, antigen-binding, including hinge region (one arm)  Variable fragments:  scFv: single-chain variable fragment  di-scFv: dimeric single-chain variable fragment  sdAb: single-domain antibody  Bispecific monoclonal antibodies:  3funct: trifunctional antibody  BiTE: bi-specific T-cell engager
  53. 53.  Antibodies can bind to epitopes expressed at the surface of target cells (as well as to soluble molecules) 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).
  54. 54.  Preparing a fusion protein of ◦ the engineered TCR conjugated to ◦ an effector molecule ◦ Cytotoxic Agent to destroy cells expressing the target MHC-peptide complex.  These could then be introduced into a cancer patient to target the tumor-specific antigens or into an AIDS patient to target HIV-infected cells.
  55. 55.  Since 2000, the therapeutic market for monoclonal antibodies has grown exponentially.  The current “big 5” therapeutic antibodies on the market are ◦ BEVACIZUMAB ◦ TRASTUZUMAB ◦ ADALIMUMAB ◦ INFLIXIMAB ◦ RITUXIMAB accounted for 80% of revenues in 2006.  In 2007, eight of the 20 best-selling biotechnology drugs in the U.S. are therapeutic monoclonal antibodies.  This rapid growth in demand for monoclonal antibody production has been well accommodated by the industrialization of mAb manufacturing
  56. 56.  The monoclonal antibody production technology has revolutionized the world of Biotechnology.  Advances in genetic engineering over the years have provided numerous ways to design MAbs that are more robust and efficacious compared with their original murine version.  MAbs have not only been used as diagnostics, therapeutics, research reagents, drug targettor for various infectious diseases but also cancerous, metabolic and hormonal disorders.  MAb technology in conjunction with recombinant DNA technology has successfully led to the reconstruction of chimeric, humanized and fully human antibodies and has enormous potentials for therapeutic uses.
  57. 57.  Ophthalmology Dept ◦ Bevacizumab 1.25mg/0.1ml  Intravitreal injection once monthly for 3 months  Cost Rs.28,000  Radiotherapy Dept ◦ Rituximab(100mg/vial) in Non Hodgkin`s Lymphoma ◦ Dosage:375-500 mg/m*2 on day 1of R-CHOP Regimen ◦ Cost Rs.40,000
  58. 58. VIDEO 2