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OUTLINE CONCEPT OF MONOCLONAL ANTIBODIES Structure of immunoglobulin Types Polyclonal Vs monoclonal antibodies DISCOVERY PRODUCTION AND PURIFICATION TECHNICHES Hybridoma technology Purification by chromatographic techniques TYPES 1. Murine 2.Chimeric 3.Humanised 4.Human NOMENCLATURE APPLICATIONS Diagnostic Therapeutic : Rhematologic , hematological ,oncologic INDIVIDUAL MAb s

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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

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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.

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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

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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

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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

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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

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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

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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.

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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

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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

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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

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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

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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

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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 Allergic reactions Anaphylactic shock

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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 : 


Applications of Monoclonal Antibodies : 

Applications of Monoclonal Antibodies Diagnostic ApplicationsBiosensors & Microarrays Therapeutic Applications Transplant rejection Cardiovascular disease Cancer Infectious Diseases Inflammatory disease Clinical ApplicationsPurification of drugs, Imaging the target Future Applications Fight against Bioterrorism





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 Nausea Diarrhea Skin rashes

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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 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 Anti platelet factor(GP IIb-IIIa inhibitor) is the Fab fragment of the chimeric human-murine monoclonal antibody (Reopro) INDICATIONS: 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 Rheumatoid arthritis Psoriasis Ankylosing spondylitis Crohns disease


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 signaling Down regulation of receptors Immunomodulation ADCC CDCC

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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




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 are antibody-conjugated liposomes. Liposomes can carry drugs or therapeutic nucleotides and when conjugated with monoclonal antibodies


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 Immune thrombocytopenia 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 Other uses Non–small-cell lung cancer. Side effects hypertension, 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.

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