Monoclonal antibodies - PowerPoint PPT Presentation

1 / 51
About This Presentation
Title:

Monoclonal antibodies

Description:

Monoclonal antibodies Anticancer therapy * * * * * * * * * * * * * * Cell culture Enzymatic Feedstock Bioprocessing Product GAS LIQUID SOLID PRODUCT LINES ... – PowerPoint PPT presentation

Number of Views:644
Avg rating:3.0/5.0
Slides: 52
Provided by: SharedC
Category:

less

Transcript and Presenter's Notes

Title: Monoclonal antibodies


1
  • Monoclonal antibodies
  • Anticancer therapy

2
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 American
Heritage Stedman's Medical Dictionary)
  • The Immune System consists of
  • Innate Immunity
    Primary Response
  • Acquired Immunity
    Secondary Response

3
ANATOMY OF THE IMMUNE SYSTEM
4
FUNCTIONING OF THE IMMUNE SYSTEM
HUMORAL (ANTIBODY MEDIATED) IMMUNE RESPONSE
CELL MEDIATED IMMUNE RESPONSE
ANTIGEN (1ST EXPOSURE)
ENGULFED BY
MACROPHAGE
ANTIGENS DISPLAYED BY INFECTED CELLS ACTIVATE
FREE ANTIGENS DIRECTLY ACTIVATE
BECOMES
APC
STIMULATES
HELPER T CELLS
CYTOTOXIC T CELL
B CELLS
STIMULATES
STIMULATES
MEMORY HELPER T CELLS
GIVES RISE TO
GIVES RISE TO
STIMULATES
STIMULATES
STIMULATES
ANTIGEN (2nd EXPOSURE)
ACTIVE CYTOTOXIC T CELL
MEMORY B CELLS
PLASMA CELLS
MEMORY T CELLS
STIMULATES
SECRETE ANTIBODIES
Defend against extracellular pathogens by binding
to antigens and making them easier targets for
phagocytes and complement
Defend against intracellular pathogens and cancer
by binding and lysing the infected cells or
cancer cells
5
Lymphocytes
  • Produce antibodies
  • B-cells mature in bone marrow then concentrate in
    lymph nodes and spleen
  • T-cells mature in thymus
  • B and T cells mature then circulate in the blood
    and lymph
  • Circulation ensures they come into contact with
    pathogens and each other

6
B -Lymphocytes
  • There are 10 million different B-lymphocytes,
    each of which make a different antibody.
  • The huge variety is caused by genes coding for
    abs changing slightly during development.
  • There are a small group of clones of each type of
    B-lymphocyte

7
B -Lymphocytes
  • At the clone stage antibodies do not leave the
    B-cells.
  • The abs are embedded in the plasma membrane of
    the cell and are called antibody receptors.
  • When the receptors in the membrane recognize the
    antigen on the surface of the pathogen the B-cell
    divides rapidly.
  • The antigens are presented to the B-cells by
    macrophages

8
B -Lymphocytes
9
B -Lymphocytes
  • Some activated B cells ? PLASMA CELLS these
    produce lots of antibodies, lt 1000/sec
  • The antibodies travel to the blood, lymph, lining
    of gut and lungs.
  • The number of plasma cells goes down after a few
    weeks
  • Antibodies stay in the blood longer but
    eventually their numbers go down too.

10
B -Lymphocytes
  • Some activated B cells ? MEMORY CELLS.
  • Memory cells divide rapidly as soon as the
    antigen is reintroduced.
  • There are many more memory cells than there were
    clone cells.
  • When the pathogen/infection infects again it is
    destroyed before any symptoms show.

11
What are antibodies?
  • An antibody is a protein used by the immune
    system to identify and neutralize foreign objects
    like bacteria and viruses. Each antibody
    recognizes a specific antigen unique to its
    target.
  • Monoclonal antibodies (mAb) are antibodies that
    are identical because they were produced by one
    type of immune cell, all clones of a single
    parent cell.
  • Polyclonal antibodies are antibodies that are
    derived from different cell lines.
  • Isotypes
  • According to differences in their heavy
    chain constant domains, immunoglobulins are
    grouped into five classes, or isotypes IgG, IgA,
    IgM, IgD, and IgE.
  • IgG IgG1 (66), IgG2 (23), IgG3 (7) and IgG4
    (4) , blood and tissue liquid.
  • IgAIgA1 (90) and IgA2 (10), stomach and
    intestines
  • IgM normally pentamer, ocassionally hexamer,
    multiple immunoglobins linked with disulfide
    bonds
  • IgD1 of proteins in the plasma membranes of
    B-lymphocytes, function unknown
  • IgE on the surface of plasma membrane of mast
    cells, play a role in immediate hypersensitive
    and denfensive for parasite

12
The structure of antibodies
  • http//www.path.cam.ac.uk/mrc7/igs/mikeimages.htm
    l

13
Antibodies
  • Also known as immunoglobulins
  • Globular glycoproteins
  • The heavy and light chains are polypeptides
  • The chains are held together by disulphide
    bridges
  • Each ab has 2 identical ag binding sites
    variable regions.
  • The order of amino acids in the variable region
    determines the shape of the binding site

14
ANTIBODIES
POLYCLONAL.
MONOCLONAL.
Derived from different B Lymphocytes cell lines
Derived from a single B cell clone
mAb offer Reproducible, Predictable Potentially
inexhaustible supply of Ab with exquisite
specificity
Batch to Batch variation affecting Ab reactivity
titer
Enable the development of secure immunoassay
systems.
NOT Powerful tools for clinical diagnostic tests
15
How Abs work
  • Some act as labels to identify
  • antigens for phagocytes
  • Some work as antitoxins i.e. they block toxins
    for e.g. those causing diphtheria and tetanus
  • Some attach to bacterial flagella making them
    less active and easier for phagocytes to engulf
  • Some cause agglutination (clumping together) of
    bacteria making them less likely to spread

16
Different Immunoglobulins
17
Type Number of ag binding sites Site of action Functions
IgG 2 Blood Tissue fluid CAN CROSS PLACENTA Increase macrophage activity Antitoxins Agglutination
IgM 10 Blood Tissue fluid Agglutination
IgA 2 or 4 Secretions (saliva, tears, small intestine, vaginal, prostate, nasal, breast milk) Stop bacteria adhering to host cells Prevents bacteria forming colonies on mucous membranes
IgE 2 Tissues Activate mast cells ? HISTAMINE Worm response
18
History of Mab development
  • 1890 Von Behring and Kitasato discovered the
    serum of vaccinated persons contained certain
    substances, termed antibodies
  • 1900 Ehrlich proposed the side-chain theory
  • 1955 Jerne postulated natural selection theory.
    Frank Macfarlane Burnet expended.
  • Almost the same time, Porter isolated fragment of
    antigen binding (Fab) and fragment crystalline
    (Fc) from rabbit y-globulin.
  • 1964 Littlefield developed a way to isolate
    hybrid cells from 2 parent cell lines using the
    hypoxanthine-aminopterin-thymidine (HAT)
    selection media.
  • 1975 Kohler and Milstein provided the most
    outstanding proof of the clonal selection theory
    by fusion of normal and malignant cells
  • 1990 Milstein produced the first monoclonal
    antibodies.

19
The types of mAb designed
  1. Murine source mAbs rodent mAbs with excellent
    affinities and specificities, generated using
    conventional hydrioma technology. Clinical
    efficacy compromised by HAMA(human anti murine
    antibody) response, which lead to allergic or
    immune complex herpersensitivities.
  2. 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)
  3. Humanized mAbs contained only the CDRs of the
    rodent variable region grafted onto human
    variable region framework

20
Chemotherapy
  • Shortcomings
  • Nature of cytotoxin
  • Lack of in vivo selectivity
  • The mechanism of anti-proliferation on cells
    cycle, rather than specific toxicity directed
    towards particular cancer cell
  • Host toxixity treatment discontinued, most of
    them had bad side-effects, such as no appetites,
    lose hair etc.

21
Monoclonal antibodies for cancer treatment
  • Three mechanisms that could be responsible for
    the cancer treatment.
  • mAbs act directly when binding to a cancer
    specific antigen and induce immunological
    response to cancer cells. Such as inducing cancer
    cell apoptosis, inhibiting growth, or interfering
    with a key function.
  • mAbs was 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

22
mAbs treatment for cancer cells
ADEPT, antibody directed enzyme prodrug therapy
ADCC, antibody dependent cell-mediated
cytotoxicity CDC, complement dependent
cytotoxicity MAb, monoclonal antibody scFv,
single-chain Fv fragment. Carter P Improving
the efficacy of antibody-based cancer therapies.
Nat Rev Cancer 20011118-129
23
EVOLUTION OF MONOCLONAL ANTIBODY
1. TRANSGENIC DNA SPLICING / GENE KNOCK OUT
2. LIBRARIES a.BACTERIOPHAGE b. mRNA c. Cell
Surface
24
Conventional production of mAbs
  • The hybridoma technology
  • spleen cells from immunized mice are fused
    with the murine myeloma cells.
  • The several process had been developed at large
    scale.
  • According to the different cell culture methods,
    it can calisifed into four fields
  • Robottle cell culture process.
  • Membrane binded cell culture process
  • Microcarrier cell culture process
  • Suspended cell culture process

25
(No Transcript)
26
(No Transcript)
27
Applications of Monoclonal Antibodies
  • Diagnostic ApplicationsBiosensors Microarrays
  • Therapeutic ApplicationsTransplant rejection
    Muronomab-CD3Cardiovascular disease Abciximab
    Cancer RituximabInfectious Diseases
    PalivizumabInflammatory disease Infliximab
  • Clinical ApplicationsPurification of drugs,
    Imaging the target
  • Future Applications Fight against Bioterrorism

28
  • REPORT HIGHLIGHTS
  • The global market for therapeutic monoclonal
    antibodies (mAbs) was estimated at 44.6 billion
    in 2011.  With the rollout of at least eight new
    therapeutic mAb products and expanded indications
    for existing products expected during the
    forecast period, the global mAb market is
    expected to rise at a compound annual growth rate
    (CAGR) of 5.3 to nearly 58 billion in 2016.
  • The U.S. is projected to be the largest single
    market for therapeutic mAbs from 2011 to 2016.
    This particular market was nearly 19.8 billion
    in 2010 and reached 20.1 billion by 2011. BCC
    projects this market will grow to 27.4 billion
    by 2016, a CAGR of 6.4.
  • Sales of mAbs in the rest of the world will
    remain higher than in the U.S.despite a higher
    CAGR in the U.S. versus the rest of the world.
    This market is expected to grow from 24.6
    billion in 2011 to 30.3 billion in 2016 at a
    CAGR of 4.3.

29
Why should we be interested ?
  • mAbs drive the development of multibillion dollar
    biotechnology industry.
  • Many of the leading pharmaceutical companies have
    entered the mAb sector, attracted by quicker and
    less costly development, higher success rates,
    premium pricing, and a potentially reduced threat
    from generics
  • The outlook for monoclonal antibody therapeutics
    is healthy. 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

30
  • Anything other than Antibodies? Major
    application areas
  • Health care / Diagnostics
  • - Development of therapeutics efficacy,
    toxicity
  • - Diagnosis early detection and prevention
    of diseases
  • Agriculture Crop production with high yield and
    quality
  • Bio-based process Pollution, CO2 emission,
  • global warming
  • Alternative energy (Bio-energy)
  • - Depletion of fossil fuels
  • - Use of renewable sources Corn, sugar cane,
    cellulose
  • - Cost (?)

31
Key technologies and fields
  • Protein engineering Design of proteins/enzymes
    based on structural and mechanistic knowledge,
    molecular evolution, computational design
  • Metabolic pathway engineering Design of more
    efficient metabolic pathways high yield of
    target product, low by-product
  • Computational modeling and optimization Systems
    Biology,

  • Genome-wide analysis
  • Nano-biotechnology Use of NPs for diagnosis
    and imaging
  • Cell culture engineering Microorganisms and
    mammalian cells
  • - Hybridoma technology A technology of
    forming hybrid cell lines (called hybridomas) by
    fusing a specific antibody-producing B cell with
    a myeloma (B cell cancer) cell that is selected
    for its ability to grow in tissue culture

32
Branches of Biotechnology
  • Separation technology Recovery and purification
    of a target product
  • Synthetic biology Creation of new biosystems
    (Cells and biomolecules) Systematic,
    hierarchical design of artificial, bio-inspired
    system using robust, standardized and
    well-characterized building block
  • Blue biotechnology Marine and aquatic
    applications of biotechnology
  • Green biotechnology Agricultural applications
  • Red biotechnology Medical applications
  • White biotechnology Industrial applications

33
Typical examples of Bio-Products
Company Products
BASF Vitamin B-2 Methoxy isopropyl amine (chiral intermediate) Styrene oxide Amino acids
Eastman Chemical / Genencor Ascorbic acid
Degussa Acrylamide Fatty acid derived esters Polyglycerine ester Organo modified silicones and oleochemicals
Celanese / Diversa Acetic acid Polyunsaturated fatty acids Non-digestible starch Polylactic acid (PLA)
Cargill Polylactic acid (PLA) (140,000 MT/yr)
DuPont / Genencor 1,3-Propanediol Terephthalic acid Adipic acid
Chevron / Maxygen Methanol
34
General Bioprocess
Feedstock
Bioprocessing
Product
PRODUCT LINES
Cell culture Enzymatic
GAS
Biocatalyst
Bioreactor
Recovery product
LIQUID
SOLID
Feedstock
Bioprocessing
Product
  • Gas
  • Syn. Gas
  • CO2
  • Organic vapor
  • Liquid
  • Organic
  • Sugar solution
  • Solid
  • Biomass
  • Consumer Waste
  • Immobilized Enzymes
  • Ambient to Extreme
  • Fermentation
  • Immobilized
  • Free cell
  • Ambient to Extreme
  • Bioreactors
  • Continuous Systems
  • Membrane
  • Batch or Fed-batch
  • Separation
  • In situ
  • Secondary
  • Media
  • Gaseous
  • Aqueous
  • Organic
  • Pharmaceuticals
  • Fine chemicals
  • Specialty Chemicals
  • Feedstock
  • Bulk chemicals

35
Bio-based economy Impact on global economy
  • Shift from petroleum-based economy
  • - Exhaustion and soaring price of petroleum
    (gt 100 /gallon)
  • - Environmental issue
  • Global warming (greenhouse gas, CO2 ,
    emission)
  • Pollution
  • Development of renewable source-based Bioprocess
  • Replacement of chemical processes with Bio-based
    ones

White Biotechnology
36
Value chains from renewable sources
37
Key role of enzymes in Bio-based economy
Energy and Environmental issues - Depletion
of fossil fuels - Limitation to CO2
emission (Kyoto protocol)
Renewable source-based economy
Petrochemical-based economy
Chemical process
Bio-based process

Use of enzymes in Biofuel production from
renewable biomass such as starch and cellulose ?
amylase, cellulase etc.
38
Enzymes
  • Most proficient catalysts with high specificity
  • Competitive and cost-effective processes
  • Cleaning (Detergents)
  • Textiles
  • Starch Processing
  • Brewing
  • Leather
  • Baking
  • Pulp and Paper
  • Food and Specialties
  • Animal feeds
  • Cosmetics

Industrial use for specialty chemicals
  • Chiral drugs
  • Chiral intermediates
  • Semi-synthetic antibiotics
  • Organic acids

Therapeutics
  • Treatment of Gauchers disease

39
Chemical company devoting to Biotechnology BASF
Emphasis on Bio-products mainly using Enzymes
Ecoflex
40
Therapeutic proteins
  • High specificity and less toxicity ? high safety
    and efficacy
  • Therapeutic proteins
  • - Antibodies, proteins, enzymes, peptides etc.
  • ex) EPO, Interferon, Insulin, Avastin,
    Enbrel, Remicade, Herceptin,
  • EPO (Erythropoietin)
    Stimulating the proliferation of red blood cells
  • Herceptin Mab against
    EGFR2(Epidermal growth factor receptor 2)
  • Avastin Mab against VEGF
    (Vascular endothelial growth factor)
  • Remicade Mab against TNF-a
    (Tumor necrosis factor- a)
  • World market
  • - EPO alone 11 Billion per year
  • - 50 Billion (2007)? 190 Billion
    (2015)
  • - Antibodies gt 50
  • - Intensive investment in monoclonal antibodies
    Biosimilar

Therapeutic proteins will form the back-bone of
future medicinal therapy
41
Structural and functional features of antibodies
42
Blockbuster Therapeutic Antibodies
Approved Year Product Target Indication Company Market size(07) Antibody Type
1997 Rituxan CD20 Non-Hodgkin's lymphoma Genentech 4,603 Chimeric
1998 Herceptin Her2/neu Breast cancer Genentech 4,047 Humanized
1998 Synagis RSV RSV prophylaxis MedImmune 1,100 Humanized
1998 Remicade TNF- ? RA, Chrons disease JJ 5,234 Chiemric
2002 Humira TNF-? RA Abbott 3,064 Human
2003 Raptiva CD11a Psoriasis Genentech Xoma 211 Humanized
2004 Erbitux EGFR Colorectal cancer Imclone Bristol-Myers 1,336 Chiemric
2004 Avastin VEGF Colorectal cancer Genentech 3,335 Humanized
2006 Vectibix EGFR Colorectal cancer Amgen 170 Human
Million (Data Monitor Monoclonal 2008)
43
Drawbacks of immunoglobulin antibodies
  • Complicated process for selecting cell lines and
  • the production using mammalian cells ? very
    expensive
  • Intellectual property barriers
  • Tend to aggregate due to large size ( 150 KDa)
  • Difficult to penetrate inside the cells
  • Limited binding affinity due to confined binding
    surface

Ideal scaffold for alternative therapeutics
  • High-level soluble expression in bacteria
  • High stability (thermodynamic, pH)
  • Easy design of binders with high affinity for a
    target
  • Low immunogenicity and cytotoxicity

44
Therapeutics based on non-antibody scaffold
New paradigm in therapeutic proteins
  • Development of new therapeutics with high
    efficacy and
  • low side effect from non-antibody protein
    scaffold
  • Designer therapeutic proteins
  • IP issue and cost-effectiveness

GlaxoSmithKline, Amgen Bristol-Myers-Squibb,
Boehringer Ingelheim Eli Lilly, Roche, Avidia,
Ammunex. Affibody, Ablynex, Adnexus Therapeutics
.
Strategic alliance or merger between big pharma
and biotech companies
45
Non-antibody scaffolds
Human lipocalin
Ankyrin
Human fibronectin
Z domain of Staphylococcal protein A
46
Therapeutic Enzymes
Disease Product Developer Sales (USMillions) Sales (USMillions) Features
Disease Product Developer 2004 2007 Features
Gauchers Ceredase Genzyme 443 N/A Glucocerebrosidase Purified from human placenta
Gauchers Cerezyme Genzyme 932 (2005) 1,048 Produced in CHO cells 3 Exoglycosidases process for Terminal Mannose
Fabrys Fabrazyme Genzyme 209 397 alpha-galactosidase Mannose-6-phosphate for Glycotargeting
Fabrys Replagal TKT 57 168 alpha-galactosidase Mannose-6-phosphate for Glycotargeting
MPS-1 Aldurazyme Genzyme 12 204 alpha L-iduronidase
Pompe Myozyme Genzyme Approved (2006) Approved (2006) alfa-glucosidase
Treatment of Gauchers disease by Cerezyme costs
up to 550,000 annually
Most of therapeutic enzymes glycoproteins
47
Gauchers Disease Lysosomal Storage Disease
Caused by a recessive mutation in a gene located
on chromosome 1, affecting both males and females.
- Found by Phillipe Gaucher in 1882 - Biochemical
basis for the disease in 1965 by Brady et al..
Glucosyl
Ceramide
Glucocerebrosidase (b-glucosidase)
Autosomal recessive inheritance
Ceramide
Glucose
48
Lysosomal storage diseases (LSDs) Lysosomal
Enzymes
  • Lysosomes Cellular organelles containing acid
    hydrolase enzymes to break down waste materials
    and cellular debris
  • Cells garbage disposal system
  • Digestive organelle in the cell
  • Contains 40 hydrolytic enzyme
  • Acidic pH (about pH4.8) within the lysosome is
    required for lysosomal enzymes to be active

(1) The ER and Golgi apparatus make a
lysosome (2) The lysosome fuses with a digestive
vacuole (3) Activated acid hydrolases digest the
contents
(LSD)
Lysosome with substrate accumulation
Lysosome
Nucleus
Mitochondria
(Normal cell)
(LSD cell)
49
Gauchers disease Occurrence and symptoms
  • 1/ 40,00060,000 (Jew 1/500)
  • Swollen vacuoles ? Gaucher cells
  • Accumulation in spleen, liver, kidney, brain
  • Enlarged spleen and liver, liver malfunction,
  • neurological complications etc..

Distended abdomen
50
Diagnostics
  • Diagnosis of disease as early as possible
  • Best solution compared to treatments
  • Prediction and treatment of diseases based on
    individual genomes
  • - personalized medicine
  • - treatment with appropriate therapeutic
    agents
  • Analysis / Detection of disease biomarkers
  • - Invasive or non-invasive analysis

51
Perspectives
  • Biotechnology will have the greatest impact on
    humans in the future in terms of health,
    life-style, and economy.
  • - Therapeutic proteins
  • - Bio-based economy Bioprocess and
    Bio-Energy
  • - Diagnostics
  • Modern Biotechnology constitutes a variety of
    diverse areas and technologies, requiring
    interdisciplinary collaborations.
Write a Comment
User Comments (0)
About PowerShow.com