Monoclonal Antibodies and their role in Pharmacology


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  • The functional groups of the paratope (Fab) interact with the epitope (antigen) Hydrogen bonding Van der Waals forces Ionic interactions The CDRs are necessary for antigen binding The tertiary structure of this region can contain pockets, undulating flatter surfaces, and even protrusions Small antigens typically bind in deep pockets
  • Antibodies have two major functions: • They recognize and bind antigens • They induce immune responses in the host organism after binding an antigen 14 The two functions of an antibody correlate with the two major regions of the antibody. Variable Region: The amino acid sequence in the tips of the “Y” varies greatly among different antibodies. This variable region, composed of 100-110 amino acids, gives the antibody its specificity for binding antigen. The variable region includes the ends of the light and heavy chains. Treating the antibody with a protease can cleave this region, producing Fab (fragment, antigen binding) that include the variable ends of an antibody. The specificity that an antibody shows for a given antigen is absolute. To date, nothing else developed for therapeutic purposes has shown this kind of specificity. 14 Constant Region: The amino acid sequence in the rest of the antibody is conserved and exhibits low variability among different antibodies. Different classes of constant regions in the stem of the antibody generate different isotypes with differing properties based on their amino acid sequence. Antibodies are divided into five major classes, IgM, IgG, IgA, IgD and IgE, based on their constant region structure and immune function. These classes differ in the mechanism used to destroy antigen. 14
  • The ongoing success of existing products, combined with a bulging pipeline of new products awaiting approval and limited generic erosion, point towards robust growth in this segment
  • 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 Recombinant monoclonal antibodies . Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice. Phage display library: construction of V H and V L gene libraries and expression of them on a filamentous bacteriophage. The phage expressing an antigen-bonding domain specific for a particular antigen to screen the mAbs
  • ICAM -1 intercel adhesion molecule rogressive multifocal leukoencephalopathy (PML) LFA-1 leukocyte fxn associated ag
  • cytokine syndrome: skin reactions,  fatigue ,  fever ,  chills ,  myalgia ,  headaches ,  nausea  and  diarrhea T cells recognise  antigens  primarily via the  T cell receptor . This receptor needs various co-receptors to function, one of which is CD3. The T cell receptor-CD3 complex transduces the signal for the T cell to  proliferate  and attack the antige
  • rash rarely leads to dose reductions or termination of therapy. It is generally reversible after treatment is finished and may also be associated with a good response to therapy. [5]
  • pretreatment with  diphenhydramine  30-60 min. before administration is standard of care
  • Monoclonal Antibodies and their role in Pharmacology

    1. 1. MONOCLONAL ANTIBODIES AND THEIR ROLE IN PHARMACOLOGY Dr.Harmanjit Singh PG Resident (pharmacology) GMC, Patiala
    2. 2. Monoclonal Antibodies <ul><li>Monoclonal antibodies are Monospecific antibodies that are identical because they are produced by one type of immune cell that are all clones of a single parent cell. </li></ul><ul><li>Class of highly specific antibodies </li></ul><ul><ul><li>Produced by clone of single hybrid cells or hybridoma </li></ul></ul><ul><ul><li>Fusing B lymphocytes with a tumour cell </li></ul></ul>
    3. 3. The Structure of an Antibody <ul><li>2 identical light chains (~220 amino acids long) </li></ul><ul><ul><li>Variable domain: V L </li></ul></ul><ul><ul><li>Constant domain: C L </li></ul></ul><ul><li>2 identical heavy chains (~440 amino acids long) </li></ul><ul><ul><li>Variable domain: V H </li></ul></ul><ul><ul><li>3 Constant domains: C H 1, C H 2, C H 3 </li></ul></ul><ul><li>Covalent, disulfide bonds between cysteine residues </li></ul><ul><li>Flexible “hinge region” </li></ul>
    4. 4. <ul><li>Antibodies have two major functions: </li></ul><ul><ul><li>Recognize and bind antigen </li></ul></ul><ul><ul><li>Induce immune responses after binding </li></ul></ul><ul><li>The variable region mediates binding </li></ul><ul><ul><li>Affinity for a given antigen is determined by the variable region </li></ul></ul><ul><ul><li>The variable region confers absolute specificity for an antigen </li></ul></ul><ul><li>The constant region mediates immune response after binding </li></ul><ul><ul><li>Different classes of constant regions generate different isotypes </li></ul></ul><ul><ul><li>Different isotypes of antibody have differing properties </li></ul></ul>Antibody Function Constant region Variable region
    5. 5. History <ul><li>1975 : </li></ul><ul><li>Hybridoma Technology </li></ul><ul><li>George Kohler and Cesar Milstein devised a method to obtain large amounts of a mAb </li></ul><ul><li>1984 : </li></ul><ul><li>The Nobel Prize for Medicine </li></ul><ul><li>- In 1988, Greg Winter et al pioneered the techniques to humanize monoclonal antibodies </li></ul>
    6. 6. Hybridoma production <ul><li>Healthy B lymphocyte s </li></ul>Fusion of mouse myeloma cells Murine Mabs
    8. 8. Transgenic mice <ul><li>Transgenic mammalian cells (e.g. Chinese hamster ovary cells ) grown in culture are the industry standard for producing full-length mAb </li></ul>
    9. 9. Why should we be interested ? <ul><li>The outlook for monoclonal antibody therapeutics is healthy </li></ul><ul><li>mAbs drive the development of multibillion dollar biotechnology industry. </li></ul><ul><li>US$26 billion by the end of the decade. </li></ul><ul><ul><li>Quicker and less costly development </li></ul></ul><ul><ul><li>higher success rates </li></ul></ul><ul><ul><li>premium pricing and </li></ul></ul><ul><ul><li>potentially reduced threat from generics </li></ul></ul><ul><li>100 mAb were Expected By 2010 </li></ul>
    10. 10. Origin <ul><li>First generation </li></ul><ul><li>Murine, rabbit or rat proteins purified after immunisation with antigen </li></ul><ul><li>Abs to these proteins (Ag) generated in patients human antimouse antibody ( HAMA) </li></ul><ul><ul><li>Block effectiveness of therapy </li></ul></ul><ul><ul><li>Adverse events serum sickness or anaphylaxis </li></ul></ul>
    11. 11. Origin <ul><li>Second generation </li></ul><ul><li>DNA technology or genetic engineering used to construct hybrids composed of human Abs regions with murine </li></ul><ul><ul><li>Chimeric Abs </li></ul></ul><ul><ul><li>Humanized </li></ul></ul><ul><ul><li>Human </li></ul></ul>
    12. 12. EVOLUTION OF MONOCLONAL ANTIBODY 1. TRANSGENIC 2. LIBRARIES a.BACTERIOPHAGE b. mRNA c. Cell Surface Ist generation mab 2 nd generation mab daclizumab
    13. 13. Types of mAbs Purple denotes human component orange murine component
    14. 14. Murine <ul><li>Derived from mice </li></ul><ul><li>Patients treated with murine mAbs develop a human antimouse antibody (HAMA) response </li></ul><ul><ul><li>Rapid clearance of the mAb </li></ul></ul><ul><ul><li>Poor tumour penetration </li></ul></ul><ul><ul><li>Hypersensitivity reactions </li></ul></ul><ul><li>90 Y-ibritu momab </li></ul><ul><li>131 I -Tositu momab </li></ul>
    15. 15. Chimeric Abs <ul><li>Antigen binding parts (variable region) of mouse </li></ul><ul><li>Ab with effector parts (constant region) of human </li></ul><ul><ul><li>Infli xi mab </li></ul></ul><ul><ul><li>Abci xi mab </li></ul></ul><ul><ul><li>Ritu xi mab </li></ul></ul>
    16. 16. Humanized <ul><li>Human Ab with complimentary determining region </li></ul><ul><li>(CDR) or hypervariable region from non human source </li></ul><ul><ul><li>Dacli zu mab </li></ul></ul><ul><ul><li>Trastu zu mab </li></ul></ul>
    17. 17. Human Abs <ul><li>Recombinant DNA technology : </li></ul><ul><li>Genes for variable Fab portion of human Abs is inserted in genome of bacteriophages & replicated </li></ul><ul><li>Mixed with Ag & complementary Ab producing </li></ul><ul><li>phages selected </li></ul><ul><ul><li>e.g. Adalim umab </li></ul></ul>
    18. 18. Nomenclature of Monoclonal Antibodies Prefix Target Source Suffix variable -o(s)- bone -u- human -mab -vi(r)- viral -o- mouse -ba(c)- bacterial -a- rat -li(m)- immune -e- hamster -le(s)- infectious lesions -i- primate -ci(r)- cardiovascular -xi- chimeric -mu(l)- musculoskeletal -zu- humanized -ki(n)- interleukin -axo- rat/murine hybrid -co(l)- colonic tumor -me(l)- melanoma -ma(r)- mammary tumor -go(t)- testicular tumor -go(v)- ovarian tumor -pr(o)- prostate tumor -tu(m)- miscellaneous tumor -neu(r)- nervous system -tox(a)- toxin as target
    19. 19. Nomenclature <ul><li>Suffix </li></ul><ul><ul><li>Human: -umab </li></ul></ul><ul><ul><li>Humanized: -zumab </li></ul></ul><ul><ul><li>Murine: -momab </li></ul></ul><ul><ul><li>Chimeric: -ximab </li></ul></ul>
    20. 20. Examples <ul><li>ab- + -ci- + -xi- + -mab : chimeric monoclonal antibody used on the cardiovascular system. </li></ul><ul><li>tras- + -tu- + -zu- + -mab : humanized monoclonal antibody used against a tumor. </li></ul><ul><li>Pali- + -vi- + -zu- + -mab </li></ul><ul><li>humanized mab used against a virus (RSV) </li></ul>
    21. 21. Pharmacokinetics: mAbs <ul><li>Routes of administration : </li></ul><ul><ul><li>Subcutaneously (Rituximab, Trastuzumab, Adalimumab) </li></ul></ul><ul><ul><li>Intramuscularly ( Palivizumab) </li></ul></ul><ul><ul><li>Intravenously </li></ul></ul><ul><li>IV route: preferred because of 100% bioavailability </li></ul><ul><li>Route for elimination of antibodies </li></ul><ul><ul><li>Via uptake & catabolism by reticuloendothelial system & target tissue. </li></ul></ul>
    22. 22. P/K: mAbs <ul><li>Half-life </li></ul><ul><ul><li>Chimeric : 4 –15 days </li></ul></ul><ul><ul><li>Humanized: 3 - 24 days </li></ul></ul><ul><ul><li>Recombinant human: 11– 24 days </li></ul></ul><ul><li>Human antimouse antibody (HAMA) response develops 7–10 days following exposure to murine antibody </li></ul>
    23. 23. List of some important Mabs Therapeutic agent Indication Alemtuzumab B cell CLL Bevacizumab Met. Colon cancer Ranibizumab Neovas. Macular degenration Cetuximab Met. Colon cancer Gemtuzumab AML Panitumumab Colorectal cancer Rituximab Low grade NHL Trastuzumab Met. Breast cancer
    24. 24. Therapeutic agent Indication Abatacept Severe RA Basiliximab, Renal, heart transplant Daclizumab Renal transplant, M.Sclerosis Efalizumab Psoriasis Adalimumab RA Abciximab ACS Omalizumab Allergic asthma Palivizumab RSV infection Natalizumab M.Sclerosis
    25. 25. Therapeutic uses <ul><li>Immunosuppression </li></ul><ul><li>Autoimmune diseases </li></ul><ul><li>Malignancies </li></ul><ul><li>Antiplatelet therapy </li></ul><ul><li>Infectious diseases </li></ul><ul><li>Asthma </li></ul><ul><li>Osteoporosis </li></ul>
    26. 26. 1.Immunosuppression <ul><li>Muromonab CD-3: Murine mAb </li></ul><ul><li>  1 st mAb  approved for clinical use in humans </li></ul><ul><li>Act on CD3 receptors </li></ul><ul><ul><li>Apoptosis of the T cells </li></ul></ul><ul><li>Prevents graft rejection in renal transplant </li></ul><ul><li>ADRs: skin reactions, fatigue, fever, chills, myalgia, headaches, nausea & diarrhea </li></ul>
    27. 27. Autoimmune diseases <ul><li>Therapeutic uses of Infliximab: </li></ul><ul><li>Blocks TNF- α </li></ul><ul><ul><li>Crohn’s disease & Ulcerative colitis </li></ul></ul><ul><ul><li>RA & AS </li></ul></ul><ul><ul><li>Psoriatic arthritis </li></ul></ul><ul><li>ADR- </li></ul><ul><ul><li>Reactivation of TB, H epatitis B </li></ul></ul><ul><ul><li>Leukopenia, Neutropenia, Thrombocytopenia & Pancytopenia  </li></ul></ul>
    28. 28. Autoimmune diseases <ul><li>Epratuzumab: </li></ul><ul><ul><li>Humanized Ab </li></ul></ul><ul><ul><li>Binds to the glycoprotein CD 22 of mature and malignant B-cells </li></ul></ul><ul><ul><li>Therapeutic use: </li></ul></ul><ul><ul><ul><li>NHL </li></ul></ul></ul><ul><ul><ul><li>SLE </li></ul></ul></ul>
    29. 29. Autoimmune diseases <ul><li>Efalizumab: </li></ul><ul><li>Humanized Ab </li></ul><ul><li>MoA </li></ul><ul><ul><li>  binds wth CD11, a subunit of lymphocyte function-associated antigen 1 </li></ul></ul><ul><li>Therapeutic uses </li></ul><ul><ul><li>Plaque psoriasis </li></ul></ul><ul><ul><li>ADRs </li></ul></ul><ul><ul><li>Bacterial sepsis, viral meningitis, invasive fungal disease and progressive multifocal leukoencephalopathy (PML) </li></ul></ul><ul><ul><li>Withdrawn from market </li></ul></ul>
    30. 30. 2.Malignancies <ul><li>Rituximab: </li></ul><ul><li>First chimeric IgG-I mAb </li></ul><ul><li>MoA: </li></ul><ul><ul><li>Directed against CD 20 on B cells </li></ul></ul><ul><li>P/K </li></ul><ul><ul><li>Bioavailability: 100% (IV) </li></ul></ul><ul><ul><li>Half life: 30 to 40 hours </li></ul></ul><ul><ul><li>Excretion: Phagocytosis and catabolism in RES </li></ul></ul>
    31. 31. Malignancies <ul><li>Therapeutic uses of Rituximab: </li></ul><ul><ul><li>Non hodgkins lymphoma </li></ul></ul><ul><ul><li>Multiple myeloma </li></ul></ul><ul><li>ADRs: </li></ul><ul><ul><li>Severe infusion reactions </li></ul></ul><ul><ul><li>Cardiac arrest </li></ul></ul><ul><ul><li>Tumor lysis syndrome </li></ul></ul>
    32. 32. Malignancies <ul><li>Alemtuzumab </li></ul><ul><li>Humanized IgG1 </li></ul><ul><li>MoA: </li></ul><ul><ul><li>Binds to CD52 B & T cells </li></ul></ul><ul><li>Therapeutic uses </li></ul><ul><ul><ul><li>  Chronic lymphocytic leukemia (CLL) in  failed fludarabine therapy </li></ul></ul></ul><ul><ul><ul><li>Cutaneous T-cell lymphoma(CTCL) </li></ul></ul></ul><ul><li>ADRs: </li></ul><ul><ul><li>H ypotension, rigors, fever, shortness of breath, bronchospasm, chills, </li></ul></ul><ul><ul><li>Lymphopenia, Anaemia, Thrombocytopenia </li></ul></ul>
    33. 33. Malignancies <ul><li>Cetuximab </li></ul><ul><li>Chimeric Ab </li></ul><ul><li>Directed against EGFR </li></ul><ul><ul><li>Inhibits tumor growth </li></ul></ul><ul><li>Therapeutic use </li></ul><ul><ul><li>Metastatic colorectal cancer (EGFR over expressive) </li></ul></ul><ul><ul><li>Head and neck cancer </li></ul></ul>
    34. 34. Malignancies <ul><li>ADRs of Cetuximab: </li></ul><ul><ul><li>Acne-like rash </li></ul></ul><ul><ul><li>Fevers, chills, rigors, urticaria, pruritis, rash, hypotension, bronchospasm, dyspnea, wheezing, angioedema, dizziness, anaphylaxis </li></ul></ul>
    35. 35. Malignancies <ul><li>Trastuzumab </li></ul><ul><li>Humanized Ab </li></ul><ul><li>MoA: Binds to HER-2/neu, Downregulation of receptor </li></ul><ul><li>Therapeutic use </li></ul><ul><li>Metastatic breast ca (over express HER-2/neu) </li></ul><ul><li>ADR : Cardiomyopathy </li></ul>
    36. 36. Malignancies <ul><li>Bevacizumab </li></ul><ul><li>Humanized mAb </li></ul><ul><li>MoA: </li></ul><ul><ul><li>Directed against VEGF </li></ul></ul><ul><li>Blocks VEGF in neovascularisation </li></ul><ul><li>Therapeutic uses </li></ul><ul><ul><li>Metastatic colorectal ca, Pancreatic ca, Breast ca </li></ul></ul><ul><ul><li>Prostate ca </li></ul></ul><ul><li>ADRs </li></ul><ul><ul><li>Risk of bleeding, Bowel perforation, Nasal septum perforation </li></ul></ul>
    37. 37. 3.Antiplatelet therapy <ul><li>Abciximab </li></ul><ul><li>Chimeric mAb </li></ul><ul><li>MoA </li></ul><ul><ul><li>Glycoprotein IIb/ IIIa receptor antagonist </li></ul></ul><ul><ul><li>Prevents platelet aggregation </li></ul></ul><ul><ul><li>Inhibits fibrinogen, von Willebrand factor </li></ul></ul><ul><li>Therapeutic uses </li></ul><ul><ul><li>Acute coronary syndrome </li></ul></ul><ul><ul><li>PCI </li></ul></ul><ul><li>ADRs: ↑ risk of bleeding, Thrombocytopenia </li></ul>
    38. 38. 5.Asthma <ul><li>Omalizumab </li></ul><ul><li>Humanized Ab </li></ul><ul><li>MoA </li></ul><ul><ul><li>Binds IgE </li></ul></ul><ul><ul><li># interaction with basophils & mast cells </li></ul></ul><ul><li>Therapeutic use </li></ul><ul><ul><li>B. Asthma in adults & adolescents refractory to inhaled corticosteroid therapy </li></ul></ul>
    39. 39. Osteoporosis <ul><li>Denosumab </li></ul><ul><li>- Human mab </li></ul><ul><li>- Act against RANK Ligand </li></ul><ul><li>-  Approved by FDA for use in postmenopausal women with risk of osteoporos </li></ul><ul><li>- ADRs </li></ul><ul><li>UTI  and RTI </li></ul><ul><li>Cataract , constipation ,  rashes  and joint pain </li></ul>
    40. 40. Future aspects <ul><li>Zanolimumab </li></ul><ul><ul><li>CD4 specific mAb </li></ul></ul><ul><ul><li>Cutaneous T cell lymphoma </li></ul></ul><ul><ul><li>Phase III clinical trial </li></ul></ul><ul><ul><ul><li>Daclizumab: Humanized mAb ( Phase II clinical trials ) </li></ul></ul></ul><ul><li>Binds to IL – 2 </li></ul><ul><li>Therapeutic use : Prophylaxis of acute organ rejection in renal transplant </li></ul><ul><ul><ul><li>Multiple Sclerosis </li></ul></ul></ul>
    41. 41. Future aspects <ul><li>Ipilimumab </li></ul><ul><ul><li>Human monoclonal antibody </li></ul></ul><ul><ul><li>  Undergoing clinical trials treatment of melanoma </li></ul></ul><ul><li>Tremelimumab </li></ul><ul><ul><li>Human IgG2 monoclonal antibody </li></ul></ul><ul><ul><li>Phase III clinical trial for advanced melanoma  </li></ul></ul><ul><li>Ruplizumab </li></ul><ul><ul><li>  Humanized monoclonal antibody </li></ul></ul><ul><ul><li>  Systemic lupus erythematosus  </li></ul></ul><ul><ul><li>Lupus nephritis </li></ul></ul>
    42. 42. Future aspects <ul><li>Efungumab : for Invasive Candida Infection </li></ul><ul><li>Panobacumab : for Pseudomonas Infection </li></ul><ul><li>Solanezumab : for Alzheimer’s Disease </li></ul><ul><li>Natalizumab : for Multiple Sclerosis </li></ul>
    43. 43. OTHER USES <ul><li>IMAGING : </li></ul><ul><li>Arcitumomab : colorectal carcinoma </li></ul><ul><li>Nofetumomab : small cell lung cancer </li></ul><ul><li>DIAGNOSTICS : </li></ul><ul><li>HIV Diagnostic kit </li></ul><ul><li>A monoclonal antibody can be used to detect pregnancy only 14 days after conception. </li></ul><ul><li>Other monoclonal antibodies allow rapid diagnosis of hepatitis, influenza, herpes, streptococcal, and Chlamydia infections . </li></ul><ul><li> </li></ul>
    44. 44. <ul><li>Ethical issues : </li></ul><ul><li>Freund’s complete adjuvant (FCA) (to enhance the immune response): painful lesions at the injection site. </li></ul><ul><li>Pristane as a &quot;priming&quot; agent - granulomatous reactions </li></ul><ul><li>Respiratory distress: due to ascites. </li></ul><ul><li>Shock - rapid fluid loss. </li></ul><ul><li>FCA and pristane should not be used where it is possible to use no adjuvant or less irritant adjuvants. </li></ul><ul><li>FCA should not be used more than once in individual mice. </li></ul><ul><li>The volume of FCA and pristane used should not exceed 0.1ml and 0.2ml respectively. </li></ul><ul><li>Individual mice should not be inoculated with adjuvant more than 3 times. </li></ul><ul><li>A priming agent should not be used in individual mice more than once. </li></ul><ul><li>Ascites fluid should only be harvested once at the time of euthanasia. </li></ul>
    45. 45. <ul><li>TO SUMMARISE : </li></ul><ul><li>MAbs are highly specific Abs produced by a clone of single hybrid cells formed by fusion of B cell with the tumor cell. </li></ul><ul><li>The hybridoma formed yields higher amount of MAbs. </li></ul><ul><li>MAbs can be produced  in vitro  and  in vivo  . </li></ul><ul><li>Animals are utilized to produce MAbs, but these antibodies are associated with immunogenicity and ethical problems. </li></ul><ul><li>Recombinant DNA technology, genetic engineering and transgenic animals are used to produce humanized MAbs or pure human MAbs, with fewer ADRs </li></ul><ul><li>Used for treatment of cancer, autoimmune disorders, graft rejections, infections, asthma etc. </li></ul>