SEMINAR : SEMINAR TOPIC: MONOCLONAL
PRESENTER: Dr. Ramakrishna J
Junior resident OUTLINE : OUTLINE CONCEPT OF MONOCLONAL ANTIBODIES
Structure of immunoglobulin
Polyclonal Vs monoclonal antibodies
PRODUCTION AND PURIFICATION TECHNICHES
Purification by chromatographic techniques
1. Murine 2.Chimeric 3.Humanised 4.Human
Therapeutic : Rhematologic , hematological ,oncologic
INDIVIDUAL MAb s Slide 3: The Perfect World The Real World COLD FLU CHICKEN POX STOMACH UPSET HELP ME ! HELP ! HELP ME! Slide 4: THE IMMUNE SYSTEM DEFINITION: - The integrated body system of organs, tissues, cells & cell products that differentiates self from non – self & neutralizes potentially pathogenic organisms The Latin term “IMMUNIS” means EXEMPT, referring to protection against foreign agents. The Immune System consists of
Innate Immunity Primary Response
Acquired Immunity Secondary Response Slide 5: Initiation of the humoral immune response can occur when a pathogen is recognized by receptors on B cells. Antigen-derived peptides are then displayed on the B cell surface bound to class II molecules.
CD4+ T cells, also called T helper (Th) cells, have their own receptors that recognize the peptide bound to the B cell class II molecule. Slide 6: When the CD4+ T cell recognizes the antigen, it becomes activated and releases stimulatory molecules called Type II cytokines.
Type II cytokines, in turn, activate the B cell to produce and secrete antibodies, which can protect against infection by the same pathogen in the future. Some B cells become “memory” cells Structure of immunoglobulin's & types : Structure of immunoglobulin's & types Slide 8: IgG Ab are large heterodimeric molecules approximately 150 kDa and are composed of two different kinds of polypeptide chain, called the heavy (~50kDa) and the light chain (~25kDa).
There are two types of light chains, kappa (κ) and lambda (λ). Monoclonal &Polyclonal : Monoclonal &Polyclonal Monoclonal antibodies are 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
Ex: IV Immunoglobulin Slide 10: ANTIBODIES Derived from different B Lymphocytes cell lines POLYCLONAL. MONOCLONAL. Derived from a single B cell clone Batch to Batch variation affecting Ab reactivity & titre mAb offer Reproducible, Predictable & Potentially inexhaustible supply of Ab with exquisite specificity Enable the development of secure immunoassay systems. NOT Powerful tools for clinical diagnostic tests : The idea of a "magic bullet" was first proposed by Paul Ehrlich who at the beginning of the 20th century postulated that if a compound could be made that selectively targeted a disease-causing organism, then a toxin for that organism could be delivered along with the agent of selectivity. Discovery Slide 12: Georges Köhler César Milstein, and Niels Kaj Jerne in 1975 who shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery hybridoma technology Slide 13: In 1988 Greg Winter and his team pioneered the techniques to humanize monoclonal antibodies, removing the reactions that many monoclonal antibodies caused in some patients. Hybridoma technology : Hybridoma technology Principle : Principle MYELOMA CELLS HAVE LOST the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for the salvage synthesis of nucleic acids
Which enables cells to synthesize purines by the salvage pathway here using an extracellular source of hypoxanthine as a precursor Slide 16: The selective culture medium is called HAT medium because it contains Hypoxanthine, Aminopterin, and Thymidine
Unfused myeloma cells cannot grow because they lack HGPRT.
Unfused normal spleen cells cannot grow indefinitely because of their limited life span. Slide 18: 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 Slide 19: Step 2: - Screening Of Mice For Antibody Production HYBRIDOMA TECHNOLOGY After several weeks of immunization Serum Antibody Titre Determined
(Technique: - ELISA / Flow cytometery) Titre too low BOOST(Pure antigen) Titre High BOOST(Pure antigen) 2 weeks PRODUCTION OF MONOCLONAL ANTIBODY Slide 20: Step 3: - Preparation of Myeloma Cells HYBRIDOMA TECHNOLOGY Immortal Tumor Of Lymphocytes + 8 - Azaguanine Myeloma Cells High Viability & Rapid Growth HGPRT- Myeloma Cells PRODUCTION OF MONOCLONAL ANTIBODY Slide 21: Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells &
Selection of Hybridoma Cells HYBRIDOMA TECHNOLOGY 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 PRODUCTION OF MONOCLONAL ANTIBODY Slide 22: Step 5: - Cloning of Hybridoma Cell Lines by “ Limiting Dilution” or Expansion HYBRIDOMA TECHNOLOGY A. Clone Each +ve Culture B. Test Each Supernatant for Antibodies C. Expand +ve Clones Mouse Ascites Method Tissue Culture Method PRODUCTION OF MONOCLONAL ANTIBODY Slide 23: HYBRIDOMA TECHNOLOGY PRODUCTION OF MONOCLONAL ANTIBODY Cont’d : Cont’d Purification techniques : Purification techniques Cells, cell debris, lipids, and clotted material are first removed, typically by filtration with a 0.45 um filter. Chromatography : Chromatography Most of the charged impurities are usually anions such as nucleic acids and endotoxins. These are often separated by ion exchange chromatography. contd : contd A much quicker method of separation is Protein A affinity chromatography. The antibody selectively binds to Protein A, so a high level of purity is obtained.
However, this method is not advisable for antibodies that are easily damaged Types of Monoclonal Antibodies : Types of Monoclonal Antibodies Murine antibody : 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
Anaphylactic shock Slide 30: 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. Humanised Mab : Humanised Mab Humanised antibodies are produced by grafting murine hypervariable amino acid domains into human antibodies.
This results in a molecule of approximately 95% human origin
These bind weakly to the antigens Human Monoclonal antibody : 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 Nomenclature : Nomenclature Applications of Monoclonal Antibodies : Applications of Monoclonal Antibodies Diagnostic ApplicationsBiosensors & Microarrays
Clinical ApplicationsPurification of drugs, Imaging the target
Future Applications Fight against Bioterrorism WESTERN BLOT : WESTERN BLOT IMMUNOFLOURESENCE : IMMUNOFLOURESENCE Side effects : Side effects more common side effects
Allergic reactions, such as hives or itching
Flu-like symptoms, including chills, fatigue, fever, and muscle aches and pains
Skin rashes contd : contd Rare ---- more serious side effects
Infusion reactions. Severe allergy-like reactions can occur and, in very few cases, lead to death
Dangerously low blood cell counts. Decreased red blood cells, white blood cells and platelets
Cardiac complications Certain monoclonal antibodies may cause heart failure and a small risk of MI
Bleeding. Some of the monoclonal antibody drugs are designed to stop cancer from forming new blood vessels. There have been reports that these medications can cause bleeding OKT3 : OKT3 Prevents acute rejection of kidney transplants
Prevents autoimmune destruction of islet cells in type I Diabetes mellitus DACLIZUMAB: ANTI IL-2 : DACLIZUMAB: ANTI IL-2 Daclizumab binds specifically to the Tac subunit of the human high-affinity interleukin-2 (IL-2) receptor
It functions as an IL-2 receptor antagonist inhibiting IL-2-mediated stimulation of lymphocytes, a critical event in the process of allograft rejection.
It is indicated for the prophylaxis of acute organ rejection in patients receiving renal transplants ABCIXIMAB : ABCIXIMAB Anti platelet factor(GP IIb-IIIa inhibitor) is the Fab fragment of the chimeric human-murine monoclonal antibody (Reopro)
Adjunct to PCI for the prevention of cardiac ischemic complications
In patients undergoing PCI
In patients with unstable angina not responding to conventional medical therapy when PCI is planned within 24 hours INFLIXIMAB: ANTI-TNF AGENT : INFLIXIMAB: ANTI-TNF AGENT Rheumatoid arthritis
Crohns disease OMALIZUMAB: ANTI IgE : OMALIZUMAB: ANTI IgE Acts by binding to free IgE in the circulation and prevent them from binding to mast cells and from further degranulation Palivizumab( synagis) : Palivizumab( synagis) It is a humanized monoclonal antibody (IgG1κ) produced by recombinant DNA technology, directed to an epitope in the A antigenic site of the F protein of respiratory syncytial virus (RSV)
Synagis is a composite of human (95%) and murine (5%) antibody sequences
It works by preventing the growth of RSV Monoclonal antibodies for cancer treatment : Monoclonal antibodies for cancer treatment Mechanisms that could be responsible for the cancer treatment
Binding to a critical receptor and blocking down stream
Down regulation of receptors
CDCC Slide 53: mAbs can modified for delivery of a toxin, radioisotope, cytokine or other active conjugates.
it is also possible to design bispecific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cell PRINCIPLES OF CANCER THERAPY : PRINCIPLES OF CANCER THERAPY RADIOIMMUNOTHERAPY : RADIOIMMUNOTHERAPY Involves the use of radioactively conjugated Murine antibodies against cellular antigens
Ex; Tositumomab ----- non-Hodgkins lymphoma. ADEPT (Antibody Directed Enzyme Prodrug Therapy) : 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. IMMUNOLIPOSOMES : IMMUNOLIPOSOMES Immunoliposomes are antibody-conjugated liposomes.
Liposomes can carry drugs or therapeutic nucleotides and when conjugated with monoclonal antibodies RITUXIMAB:ANTI CD-20 : RITUXIMAB:ANTI CD-20 Rituximab (Rituxan) is a humanized anti-CD20 monoclonal antibody that was the first MAb to be approved by the FDA for use in human malignancy
This antibody can sensitize chemotherapy-resistant cell lines.
Acts by causing B cell apoptosis
Used in B cell lymphomas
Rheumatoid arthritis Alemtuzumab (Campath-1H) : Alemtuzumab (Campath-1H) Alemtuzumab targets the CD52 glycopeptides, which is highly expressed on T and B lymphocytes.
It has been tested as a therapeutic agent for chronic lymphocytic leukemia and promyelocytic leukemias, as well as other
non-Hodgkin's lymphomas. gemtuzumab ozogamicin (Mylotarg) : gemtuzumab ozogamicin (Mylotarg) Approved for the t reatment of acute myeloid leukemia.
Gemtuzumab ozogamicin is a humanized, calicheamicin conjugate that focuses the DNA-damaging act ivity to CD33+ cells.
CD33 is an antigen expressed on the surface of
85% to 90% of leukemia progenitor cells, but not on mature granulocytes or non hematopoiet ic cells. trastuzumab : trastuzumab RhuMAb HER-2,93 also known as trastuzumab (Herceptin), is a humanized antibody derived from a murine monoclonal antibody that recognizes HER-2/neu
HER-2/neu (c-erbB-2) , a member of the EGFR family, has been targeted for antibody therapy
Myocardial dysfunction --- trastuzumab is not recommended in combination with anthracyclines bevacizumab : Bevacizumab is a recombinant humanized anti-VEGF- A monoclonal antibody
Bevacizumab binds VEGF and prevents the interact ion of VEGF to its receptors (Flt -1 and KDR) on the surface of endothelial cells.
Approved for use in combination therapy with fluorouracil-based regimens for metastatic ca.colon
Non–small-cell lung cancer.
grade 1 or 2 proteinur ia,
slight increase in bleeding, and
impaired surgical wound healing.
Potentially life-threatening events (arterial thrombotic events, gastro intestinal perforation, hemoptysis) have occurred
in a small number of patients bevacizumab Cetuximab (Erbitux) : Cetuximab (Erbitux) I t is a recombinant, human/mouse chimeric monoclonal antibody that binds specifically to the extracellular domain of the human epidermal growth factor receptor (EGFR)
Cetuximab is used to treat cancers of the colon and rectum
It is also used to treat head and neck cancer.