Monoclonal antibodies and gene therpy

1. MONOCLONAL ANTIBODIES AND GENE THERAPY BY B.ALEKHYA M.PHARM 256212886037 UNDER GUIDANCE OF Mrs.YASMIN BEGUM Assistant proffessor ( Ph.D )

2. CONTENTS INTRODUCTION DISCOVERY PRODUCTION TYPES OF MABs PURIFICATION ADVANTAGES APPLICATIONS IN THERAPY CONCLUSION

3. INTRODUCTION Antibodies are glycoprotein molecules present in serum,produced against antigens. Antibodies are secreted by a class of blood cells known as B-lymphocytes. These are produced when body comes in contact and is invaded by a foreign particle or organism. Composed of two identical heavy chains and two identical light chains.

4. STRUCTURE OF IMMUNOGLOBULIN

5. MONOCLONAL ANTIBODIES Monoclonal antibodies: are the antibodies that are identical because they were produced by one type of immune cell (B cell), all clones of a single parent cell . Polyclonal antibodies - represent the antibodies from multiple clones of B lymphocytes, and therefore bind to a number of different epitopes e.g. Human gamma globulins

6. MONOCLONAL ANTIBODIES specifically bind to target cells . This may then stimulate the patient's immune system to attack those cells. It is possible to create a MABs specific to almost any extracellular/ cell surface target , and thus there is a large amount of research and development currently being undergone to create monoclonals for numerous serious diseases (such as rheumatoid arthritis, multiple sclerosis and different types of cancers).

7. DISCOVERY The idea of a " magic bullet " was first proposed by Paul Ehrlich , who, at the beginning of the 20th century, postulated that, a compound can be made that selectively targeted a disease-causing agent . Kohler and M ilstein in 1975 were the first to report on production of monoclonal antibodies.Awarded with the nobel prize

8. PRODUCTION OF MONOCLONAL ANTIBODY Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells HYBRIDOMA TECHNOLOGY ANTIGEN ( Intact cell/ Whole cell membrane/ micro-organisms ) + ADJUVANT (emulsification ) Ab titre reached in Serum

9. Step 2: - Screening Of Mice For Antibody Production After several weeks of immunization Serum Antibody Titre Determined (Technique: - ELISA / Flow cytometery ) Titre too low BOOST (Pure antigen) Titre High Cell fusion performed

10. Step 3: - Preparation of Myeloma Cells Immortal Tumor Of Lymphocytes + HAT Medium Myeloma Cells High Viability & Rapid Growth HGPRT - Myeloma Cells

11. Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells & Selection of Hybridoma Cells FUSION PEG MYELOMA CELLS SPLEEN CELLS HYBRIDOMA CELLS ELISA PLATE Feeder Cells Growth Medium HAT Medium Plating of Cells in HAT selective Medium Scanning of Viable Hybridomas

12. Step 5: - Cloning of Hybridoma Cell Lines by “ Limiting Dilution” or soft agar. A. Clone Each + ve Culture B. Test Each Supernatant for Antibodies C. Expand + ve Clones Mouse Ascites Method Tissue Culture Method

13. Cont’d

14. Concept of drug targeting by monoclonal antibody : Targeting antibodies with drugs involve the following steps: 1. Identification of the antigen produced by the tumor cells. 2. Production of antibody monoclonally against the identified antigen. 3. Formation of drug antibody conjugate or complexes. These complexes concentrate at the tumor site and deliver the drug.

15. PURIFICATION TECHNIQUES Cells, cell debris, lipids, and clotted material are first removed, typically by filtration with a 0.45 um filter. Chromatography Affinity chromatography: IgG antibodies using protein A agarose Anion exchange chromatography: Endotoxins and DNA Gel filtration:high and low molecular wt MABs such as aggregates and small fragments

16. Types of Monoclonal Antibodies

17. Murine antibody 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

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 MABs Humanised antibodies are produced by grafting murine hypervariable amino acid domains into human antibodies. This results in a molecule of approximately 95% human origin

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. Applications of Monoclonal Antibodies Diagnostic Applications Detects protein of interest either by blotting or immunoflouroscence Cardiovascular diseases Deep vein thrombosis Location of 1 and 2 metastatic tumours Immunosuppressive therapy Pregnancy testing kits Therapeutic Applications Radioisotope immunoconjugates Toxin and drug immunoconjugates Immunoliposome based kits In cancer

22. Location of 1 and 2 metastatic tumours can be located with help of radiolabelled MABs (specific to tumour associated membrane proteins) MABs specific to breast cancer- labelled with I 131 detects tumour in regional lymphnodes . Similarly MABs specific to breast cancer-by Gadolinium( Gd ) detected by MRI Pin head size metastases can be located & visualised

23. Immuno suppressive therapy MABs suppress T-cell activity.injection of MABs results in rapid depletion of T-cells Mechanism: binding of antibody coated T-cell to F C receptors on phagocytic cells phagocytose & clear T-cells from circulation

24. Mechanism of antitumor effect Antibody dependent cellular cytotoxicity (ADCC ) Eg : Rituximab ADEPT (Antibody mediated Enzyme prodrug therapy) Radioimmunotherapy eg : Tositomomab MAB may be conjugated with a toxin MAB can also be conjugated with radioisotope Immunoliposomes

25. Antibody dependent cellular cytotoxicity (ADCC) Immunoglobulin's clustered on the surface of the targeted cells and exposes its tail { Fc } region, to be recognized by the Fc receptors present on the surface of the macrophages and neutrophils . This causes Lysis of tumor cell.

26. ADEPT (Antibody Directed Enzyme Prodrug Therapy) 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.

27. RADIOIMMUNOTHERAPY By conjugating a radioactive isotope to a murine antibody, targeted immunotherapy is possible. ca More applicable to lymphomas as they are highly radiosensitive malignancies. Antibody with radio isotope Cancer cell Destruction of cancer cell by emmitted beta particles

28. IMMUNOTOXINS Immunotoxins are proteins that contain a toxin along with an antibody that binds specifically to target cells. All protein toxins are work by enzymatically inhibiting protein synthesis. Various plant & bacterial toxins have been genetically fused/chemically conjugated with the antibodies that bind to cancer cells . Plant toxins: ricin,abrin,modecin Bacterial toxins: diptheria and pseudomanas aeruginosa toxin A.

29. THERAPY FOR GLIOMAS FORM OF BRAIN THERAPY Isolation-indicates that patient with glioma do produce antibodies against their own tumours and are secreted by lymphocytes. These Abs may be isotope labelled and used for localisation of intracerebral disease and also used as immunotoxin Fusion of lymphocytes extracted from glioma with human myeloma Human hybridomas secreting antiglioma antibodies

30. IMMUNOLIPOSOMES This class of monoclonal antibody are those conjugated to liposomes or another form of nanotechnology drug delivery system. By attaching antibodies to the outside of a nanosized drug delivery device, large quantities of therapeutic drug can be delivered to a targeted environment. Many new nanotech devices including liposomes , nanotubes and other such containers have been developed.

31. Mechanism of antitumor effect

32. PREGNANCY TESTING KITS Sample containing HCG Antibody specific for HCG mixture of samples+ latex microspheres Positive test: No agglutination Negative test: Agglutination If HCG present,it binds to antibodies preventing from agglutinating microspheres

33. advantages Specificity for one antigenic determinant. Antiserum titer values obtained are very high. Antibodies with high avidity are produced. High reproducibility. Radiolabelling & fluorescent conjugation or enzyme marking of MABs are easy. Ideal agents for drug targeting in chemotherapy

34. disadvantages Monoclonal antibodies production, a time consuming process because entire process requires 3-4 months for one fusion experiment. Average affinity of Monoclonal antibodies are generally lower. Any physical/chemical treatment will affect all Monoclonal antibodies in that production .

35. Problems with monoclonal therapy The main difficulty is that mouse antibodies are “seen” by human immune system as foreign and mounts an immune response against them producing HAMA(human anti-mouse antibodies). These not only causes rapid elimination from the host,but also form immune complexes that causes damage to kidneys. Two approaches are used to reduce the problem: Chimeric antibodies Humanised antibodies eg:infliximab and absiximab

36. GENE THERAPY It is the process of replacement of a defected gene with a new gene,to treat diseases. Newly introduced gene will encode proteins and correct deficiencies that occur in genetic diseases. Therefore gene therapy primarily involves genetic manipulations in animals or humans to correct a disease and keep the oraganism in good health.

37. APPROACHES FOR GENE THERAPY Somatic cell gene therapy: Somatic means non-reproductive cells of an organism,other than sperm and egg cells eg:bonemarrow cells,blood cells,skin cells etc Inolves insertion of fully functional and expressible gene into a target somatic cellto correct genetic disease permanently. Germ cell gene therapy: Germ cells are reproductive cells Involves introduction of DNA into germ cells,which is passed onto next generations Genetic alterations in somatic cells are not carried to next generations.therefore somatic is prefered .

38. TWO TYPES OF GENE THERAPY: Ex vivo gene therapy: transfer of genes into cultured cells-which are then reintroduced into the patient. eg : bonemarrow cells Technique involves following steps: Isolate cells with genetic defect Grow the cells in culture Introduce therapeutic gene to correct defect Select genetically corrected genes and grow Transplant the modified cells to the patient

39. VECTORS: Viruses: RNA is the genetic material As retrovirus enters Host cell Synthesise DNA from RNA ( by reverse transcription ) Viral DNA formed ( provirus ) Gets incorporated into the DNA of host cells

40. HUMAN ARTIFICIAL CHROMOSOMES HAC is a synthetic chromosome that can replicate with other chromosomes. HAC are used to avoid heavy risk with viruses. BONE MARROW CELLS: Contains totipotent embryonic stem cells(ES) capable of divide and differentiate into various cell types( eg:RBC,platelets,macrophages ) Most widely used technique.

41. INVIVO GENE THERAPY Direct delivary of therapeutic gene into target cells of a particular tissue( eg:liver,muscle,skin,spleen etc) Depends on-efficiency of uptake of genes by target cells. Intracellular degradation of gene & its uptake by nucleus. Expression capability of gene gene delivary by viral/non-viral systems By non-viral systems: viral proteins often induce inflammatory responses in host.

42. NON-VIRAL DELIVARY Pure DNA constructs-can be introduced directly into target tissues Lipoplexes -lipid DNA complexes-have DNA surrounded by lipid layers HAC-can carry large DNA (one or more genes) VIRAL DELIVARY By retrovirus,adenovirus,herpes simplex virus.

43. GENE THERAPY STRATEGIES FOR CANCER Tumour necrosis factor gene therapy: TNF-protein produced by human macrophages Provide defence against cancer cells-brought about by enhancing cancer fighting ability of Tumour Infiltrating Lymphocytes (TILs),a special type of immune cells. TILs transformed with a TNF gene used to treat malignant melanoma

44. SUICIDE GENE THERAPY: Thymidine kinase-refered as suicide gene (used to treat certain cancers) TK- phosphorylates nucleosides to nucleotides synthesis of DNA during cell division Drug Gancyclovir (GCV)-bears close structural resemblance to certain nucleosides ( thymidine )

45. By mistake,TK phosphorylates Gancyclovir Triphosphate -GCV (false & unsuitable nucleotide for DNA synthesis) triphosphate -GCV inhibits DNA polymerase Results is that elongation of DNA molcule abruptly stops at a point containing false nucleotide(of Gancyclovir )

46. MECHANISM: DNA SYNTHESIS NUCLEOSIDE NUCLEOTIDE Thymidine kinase phosphates Gancyclovir False nucleotide Inhibits DNA polymerase DNA synthesis blocked Cancer cell dies

47. Triphosphate -GCV: enter and kill the neighbour cancer cells,this phenomenon called as- bystander effect. Ultimate result – cancer cells cannot multiply & therefore die Gancyclovir -treat brain tumours ( eg : glioblastoma , cancer in glial cells frequently refered as prodrug -approach is called prodrug activation gene therapy

48. conclusion The future of monoclonal antibodies in the treatment of cancer is bright. Rituximab and trastuzumab have established roles in the treatment of lymphoma and breast cancer, respectively. Radioimmunoconjugates are close to gaining approval for use and will likely impact significantly on the treatment of lymphomas

49. references Monoclonal antibodies: Powerful new tool in biology and medicine,Annual review of biochemistry,vol:50,page no:657-680 Fundamentals of medical biotechnology: Author:Aparna rajagopalan,page no: 209-253 www.genetics.com Biotechnology: U.Sathyanarayana ,page no:652-657 Biochemistry: Gene therapy,author U.Sathyanarayana,page no:413

50. THANK you