monoclonal antibodies

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Dr. Mayur M. Maybhate JR III, Dept of Pharmacology :

Dr. Mayur M. Maybhate JR III, Dept of Pharmacology Monoclonal Antibodies in therapy

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Introduction Discovery Production and purification Types and Nomenclature Applications in therapy Conclusion

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The Perfect World The Real World COLD FLU CHICKEN POX M. TB HELP ME ! HELP ! HELP ME!

Introduction:

Introduction Our body constantly exposed to antigens Taken care by IMMUNE SYSTEM 1) Innate – Skin and mucosal barriers, complement, lysozymes , interferons , neutrophills , monocytes , natural killer cells 2) Acquired – T and B cells

Structure of immunoglobulin:

Structure of immunoglobulin

Monoclonal Antibodies:

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 e.g. Human gamma globulins

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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

Monoclonal antibodies:

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 mab 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).

Discovery:

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

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Production of monoclonal antibodies involving human–mouse hybrid cells was described by Jerrold Schwaber in 1973 In 1988, Greg Winter and his team pioneered the techniques to humanize monoclonal antibodies

Production of monoclonal antibodies:

Production of monoclonal antibodies By HYBRIDOMA TECHNIQUE

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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 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

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

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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

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

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

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

Chimeric antibodies:

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 .

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

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 Applications - Detects protein of interest either by blotting or immunofloroscence Therapeutic Applications Transplant rejection Cancer Autoimmune disorders Inflammatory disease

Diagnostic applications:

Diagnostic applications

Arcitumomab:

Arcitumomab Anti-carcinoembryonic antigen (CEA) antibody labelled with technetium 99 ( 99 Tc) used for imaging patients with metastatic colorectal carcinoma

Capromab pendetide:

Capromab pendetide Murine mab specific for prostate specific antigen (PSA) coupled with radioisotope indium ( 111 In) Used in diagnosis of biopsy confirmed prostate cancer and also post prostacte- ctomy to determine extent of disease

Nofetumumab:

Nofetumumab Mice mab coupled with 99 Tc for diagnosis to determine extent and stage of disease of pts of small cell lung cancer

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mabs as immunosupressants and antiinflammatory mabs as antitumor agents mabs used to deliver radioisotopes to tumors Adalimumab , Etanercept , Infliximab Alentuzumab Arcitumomab Abciximab Bevacizumab Capromab pendetide Palivizumab Cetuximab Ibritomumab tiuxetan Abatacept Gemtuzumab Nofetumumab Alefacept Panitumumab Satumomab Bacilliximab Rituximab Tositumomab Daclizumab Trantuzumab Efalizumab Omalizumab

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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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

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Anti-inflammatory and immunosuppressant mabs

Muromonab (OKT-3):

Muromonab (OKT-3) first FDA-approved therapeutic monoclonal antibody was a murine CD3 specific in 1986 CD3 receptors present on T cells, and their blockage suppresses activity of T cells Indicated in prevention of transplant rejection in pts receiving kidney, lung transplants

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S/E – Cytokine release syndrome - Due to initial activation of T cells and release of cytokines before T cell clearance by macrophages - Fever, chills, nausea, vomiting, hypotension - As soon as T cells eliminated, symtoms improve - Pre-treatment with antihistaminics and glucocorticoids reduces risk

Adalimumab, Etanercept and infliximab:

Adalimumab , Etanercept and infliximab Anti TNF alpha mab binds to TNF- α and prevents its binding to its receptor on inflammatory cells Results in suppression of release of inflammatory cytokines IL-1,IL-6 etc. Adalimumab 20-40 mg IV/SC once weekly Infliximab 100 mg IV once weekly

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Rheumatoid arthritis Crohn’s disease, UC Psoriasis Ankylosing spondilosis

Abatacept, Alefacept,Efalizumab:

Abatacept , Alefacept,Efalizumab Antibodies against various receptors on T cells, hence blocking will prevent T cell activation CD 11

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Used for various autoimmune disorders Abatacept – 250 mg IV for severe rh. Arthritis not responding to DMARDs Alfetacept – 15 mg IV for psoriasis, closely monitor CD 4 count Efalizumab – Anti CD-11 approved for treatment of severe psoriasis

Omalizumab:

Omalizumab Acts by binding to free IgE in the circulation and prevent them from binding to mast cells and from further degranulation Indicated in asthma refractory to corticosteroids

Abciximab:

Abciximab Chimeric antiboby binds to GP IIb/IIIa receptors on platelets and prevents their aggregation

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Antiplatelet agent approved 2 mg IV - In patients undergoing PCI - In patients with unstable angina not responding to conventional medical therapy when PCI is planned within 24 hours

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Antitumor MABs

Mechanism of antitumor effect:

Mechanism of antitumor effect Antibody dependent cellular cytotoxicity (ADCC) Complement dependent cytotoxicity (CDC) Direct induction of apoptosis mab may be conjugated with a toxin e.g. gemtuzumab-ozogamicin mab can also be conjugated with radioisotope

Mechanism of antitumor effect:

Mechanism of antitumor effect

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

Alemtuzumab:

Alemtuzumab Humanised anti CD52 antiboby that binds to normal and malignant B and T cells, NK cells Approved 30 mg IV once weekly for treatment of B-cell CLL failed to respond to alkylating agents S/E – neutropenia, thrombocytopenia, pt should be closely monitored for opportunistic infections

Bevacizumab, Ranibizumab:

Bevacizumab , Ranibizumab Humanized antibody against VEGF, thus preventing angiogenesis Approved 5 mg/kg IV every 2 weekly till disease progression for use in combination therapy with fluorouracil-based regimens for metastatic ca.colon and non–small-cell lung cancer.

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S/E – Since it is antiangiogenic, pt should be watched for hemorrhage, GI perforations and wound healing problems Off labely, it is used IV for neovascular macular degeneration

Cetuximab, Panitumumab:

Cetuximab , Panitumumab MAB that targets epidermal growth factor receptor (EGFR) Binding of cetuximab to EGFR inhibits tumor growth by variety of mechanisms, including decrease in kinase activity, MMP activity, growth factor production and increased apoptosis

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Approved for metastatic colorectal carcinoma whose tumor overexpresses EGFR Cetuximab 400 mg/kg loading followed by 200 mg/kg weekly IV alone or in combination with irinotecan

Gemtuzumab:

Gemtuzumab Humanized MAB specific against CD33, protein found on leukemic blast cells in AML Gemtuzumab alone has some antiblast activity, clinical formulation has coupled to cytotoxic agent ozogamicin Internalization of both by tumor cells releases the cytotoxic agents, binds to DNA leading to cell death

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Approved for pts of AML not responding to cytotoxic agents in 2 doses of 9 mg/m 2 IV separated by 2 weeks S/E – severe myelosupression, hepatotoxicity, hypersensitivity reactions

Rituximab, Ofatumumab:

Rituximab , Ofatumumab mab binds to CD20 protein on normal and malignant B lymphocytes Rituximab approved in dose of 375 mg/m 2 IV weekly for 4 weeks for treatment of non hodgkins lymphoma, CLL S/E – severe infusion reactions, steven johnson’s syndrome

Trastuzumab:

Trastuzumab Humanized mab that binds to human epidermal growth factor receptor HER2/neu Prevents binding of natural ligand and down regulate the receptor Approved for metastatic Ca breast with overexpressed HER2/neu

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As a single agent, 4 mg/kg loading followed by 2 mg/kg weekly, induces remission in 30% cases In combination with chemotherapy, increases the response rates as well as survival rates S/E - Cardiomyopathy

Denileukin diftitox:

Denileukin diftitox Immunotoxin made from genetic recombination of IL-2 and active fragment of diphtheria toxin Selectively binds to cells overexpressing IL 2 and toxin inactivates elongation factor EF 2, thus inhibits protein synthesis

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FDA approved for treatment of recurrent or refractory cutaneous T-cell lymphomas Given 18 mcg/kg/day IV for 5 consecutive days every 21 days for 8 cycles, with 30-40% improvement in response

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MABs conjugated with radioisotopes

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Provide targeted delivery of radionucleotides to tumor cells I 131 generally preferred because it is readily available, inexpensive and easily conjugated Can be used for diagnosis as well as therapeutic purpose

Tositumumab:

Tositumumab Anti CD20 complexed with 131 I Used for refractory non hodgkins lymphoma not responding to rituximab and chemotherapy agents

Ibritumomab:

Ibritumomab Anti CD20 murine antibody coupled with isotopic yttrium ( 90 Y) Approved for refractory B cell non hodgkins lymphoma , not even responding to rituximab therapy S/E – common to both ibritumomab and tositumomab are severe cytopenias

Drugs in pipeline:

Drugs in pipeline Ibalizumab : an anti-CD4 monoclonal antibody for the treatment of HIV-1 infection HN125 : A Novel Immunoadhesin Targeting MUC16 with Potential for ovarian cancer Therapy. AMU32901 - Anti-phospholipid human monoclonal antibodies inhibit CCR5-tropic HIV-1

Conclusion:

Conclusion Antigen specific, can be produced against any type of antigen, hence vast diagnostic applications Target specificity, a novel therapeutic approach particularly in cancer

References:

References Douglas Lake, Immunopharmacology; Bertram G. Katzung , Basic and clinical pharmacology:11:963-85;2009 Bruce A. Chamber, Jeol Neal et.al, Targeted therapies, Tyrosine kinase inhibitors, monoclonal antibodies; Goodman and Gilmans , Pharmacological basis of therapeutics; 12;1731-50; 2010.

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Ronald V.M.,Immunomodulation ; Harrisons Principle of internal medicine; 17; 1600-20;2009. HL Sharma and KK Sharma , Immunomodulation and immunotherapy, Principles of Pharmacology;2;871-91;2010 Tripathi K.D .,Immunotherapy ; Essentials to medical pharmacology; 6;710-18;2009

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Raben D, Helfrich B,The effects of cetuximab alone and in combination with radiation and/or chemotherapy in lung cancer, PubMed ; 2010 Uehara K, Ishiguro S, Bevacizumab for Locally Advanced Rectal Cancer, Pub Med ; 2009 Bruno CJ, Jacobson JM, Ibalizumab : an anti-CD4 monoclonal antibody for the treatment of HIV-1 infection; J Antimicrob Chemother ;1839-41; 2010

Vaccines:

Vaccines Impart active immunity Antigens are given which stimulates production of antibodies Gives long lasting immunity

Antisera and immunoglobulins:

Antisera and immunoglobulins Impart passive immunity Readymade antibodies either from animals (antisera) or other person (immunoglobulins) are given to the recipient Less effective and short lasting

Costly Affair !!!:

Costly Affair !!! Drug Cost Abciximab $400for 2 mg Infliximab $1600 for 100 mg Omalizumab £ 260 for 150 mg vial Trastuzumab $1000 for 2 mg/kg Bevacizumab $1100for 5 mg/kg Rituximab $486for 100 mg vial

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