Monoclonal Antibodies

Types, synthesis and uses of Monoclonal antibodies

1. MD. MUSTAFEED UDDIN MONOCLONAL ANTIBODIES

2. OUTLINE Introduction History of monoclonal antibodies Production Types Nomenclature Application and Side E ffects Examples Conclusion

3. What are antibodies? 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 are 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. They differ in amino acid sequence.

4. Immunoglobulin structure

5. Monoclonal Antibodies 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. Given (almost) any substance, it is possible to create monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry , molecular biology and medicine .

6. 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.

7. History of Mab development 1890 Von Behring and Kitasato discovered the serum of vaccinated persons contained certain substances, termed antibodies 1900 Ehrlich proposed the “ side-chain theory” 1955 Jerne postulated natural selection theory. Frank Macfarlane Burnet expended. Almost the same time, Porter isolated fragment of antigen binding (Fab) and fragment crystalline ( Fc ) from rabbit y-globulin.

8. 1964 Littlefield developed a way to isolate hybrid cells from 2 parent cell lines using the hypoxanthine- aminopterin - thymidine (HAT) selection media. 1975 Kohler and Milstein provided the most outstanding proof of the clonal selection theory by fusion of normal and malignant cells. This resulted in the first monoclonal antibodies, for which they received the Nobel Prize in 1984.

9. Nobel Prize Award in Medicine and Physiology in 1984 Prof.Georges J. F. Köhler Prof.César Milstein Prof.Niels Kaj Jerne

10. THE MYELOMA THEORY 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.

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 . MYELOMA CELL

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 B cell Scanning Electron Microscopic view of a B cell

13. CELLS FUSED: S pleen cells from a mouse that has been immunized with the desired antigen M yeloma cells. FUSION AGENT: Polyethylene glycol MEDIUM: HAT Medium { Hypoxathine - Aminopterin -Thymidine} THE PROCEDURE

14. Supernatent containing mAbs for transport from Mielstein’s lab

15. The types of mAb designed Murine source mAbs : rodent mAbs with excellent affinities and specificities, generated using conventional hydrioma technology. Clinical efficacy compromised by HAMA(human anti murine antibody) response, which lead to allergic or immune complex herpersensitivities . Chimeric mAbs : chimers combine the human constant regions with the intact rodent variable regions. Affinity and specificity unchanged. Also cause human antichimeric antibody response (30% murine resource ) Humanized mAbs : contained only the CDRs of the rodent variable region grafted onto human variable region framework

16. Types of Monoclonal Antibodies

17. Whole of the antibody is of murine origin Major problems associated with murine antibodies include reduced stimulation of cytotoxicity Formation of complexes after repeated administration allergic reactions anaphylactic shock Murine antibody

18. Chimeric antibodies Chimeric antibodies are composed of murine variable regions fused onto human constant regions. Antibodies are approximately 65% human. This reduces immunogenicity and thus increases serum half-life .

19. Humanised antibodies are produced by grafting murine hyper variable amino acid domains into human antibodies. This results in a molecule of approximately 95% human origin Humanised M ab

20. Human Monoclonal antibody Human monoclonal antibodies are produced by transferring human immunoglobulin genes into the murine genome, after which the transgenic mouse is vaccinated against the desired antigen, leading to the production of monoclonal antibodies

21. Every monoclonal antibody has the following components in its name Variable - Target Substem - Source Substem - Stem - Additional words (in special cases) Ex: A lacizumab pegol is Ala – ci - zu - mab - pegol NOMENCLATURE

22. 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 Target Substem

23. Letter Meaning -a- Rat -e- Hamster - i - Primate -o- Mouse -u- Human -xi- Chimeric - zu - Humanized - axo - Rat/mouse hybrid Source Substem

24. Nomenclature

25. Rituximab Ri - Variable tu - Tumour xi- Chimeric mab - Monoclonal Antibody So Rituximab is a Chimeric Monoclonal Antibody targetting a Tumour Example 1

26. Bevacizumab Beva - Variable ci - Circulatory System zu - Humanised mab - Monoclonal Antibody So Bevacizumab is a Humanised Monoclonal Antibody targetting a protein in Circulatory System Example 2

27. 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 ( 111 In) capromab pendetide   Additional words

28. 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 ADVANTAGES

29. 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. DISADVANTAGES

30. CLINICAL SIGNIFICANCE

31. 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 DIAGNOSTIC SIGNIFICANCE - A BREAKTHROUGH IN SCIENCE

32. 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

33. MAbs in Pregnancy Testing MAbs can be used to detect pregnancy with Antibody to Beta HCG.

34. MAbs in MP Card Test 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

35. MAbs in Microalbuminuria Testing Immunoprecipitation ( Micral test) : It is based on the colour shift of monoclonal antibody to human albumin labelled with gold.

36. MAbs IN IMMUNODIAGNOSTIC TESTS Monoclonal antibodies can also be used to purify a substance with techniques called immunoprecipitation and affinity chromatography.

37. MAbs in Western Blot Mab helps to identify a specific molecule/substance in a mixture of substances

38. 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 THERAPEUTIC SIGNIFICANCE

39. 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 THERAPEUTIC SIGNIFICANCE…( contd )

40. Leukemias Lymphomas Osteoporosis Platelet Aggregation Inhibitors THERAPEUTIC SIGNIFICANCE…( contd )

41. 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. MAbs in Treatment of Cancer

42. 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 MAbs in Treatment of Cancer

43. 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 MAbs in Treatment of Cancer

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

45. DRUGS

46. 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 Bevacizumab

47. 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 Infliximab

48. 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 FDA Approved MAbs of Therapeutic Significance

49. 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

50. 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

51. 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

52. RESEARCH SIGNIFICANCE 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

53. Fever Chills Weakness Headache Nausea Vomiting Diarrhea Rashes Hypo/Hypertension ADVERSE EFFECTS DRUG/ TARGET RELATED SIDE EFFECTS

54. 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. PROBLEMS WITH MONOCLONAL ANTIBODIES

55. 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: Infli xi mab ,  ritu xi mab , and  abci xi mab   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:Dacli zu mab ,  Gemtu zu mab ,  Transtu zu mab HAMA Solved

56. Fully Human Monoclonal Antibody Parts of Human Antibody Parts of Mouse Antibody

57. 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

58. THE LATEST UPDATE

59. 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  RECOMBINANT MONOCLONAL ANTIBODIES

60. NEWER TYPES 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

61. 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). MONOCLONAL `T` CELL RECEPTORS

62. 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. Monoclonal TCR

63. 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 ECONOMICS

64. 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. CONCLUSION

65. References Katzung's - Basic and Clinical Pharmacology 12th edition Roitt’s Essential Immunology 11 th edition medscape www.whatisbiotechnology.org/exhibitions/milstein/monoclonals

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