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Overview of Presentation
Introduction
History
Structure of Human and therapeutic Antibodies
Production of MAbs
Mabs Vs Polyclonal Abs
Application of Monoclonal Antibody
Market of Pharmaceutical Antibodies
EngineeredAntibodies
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Unique features ofMonoclonal Antibodies
Modification to structure and refinement in production methods have made
antibodies a viable modern drug.
Production of monoclonal antibodies(MAbs) to an almost endless variety of
antigenic substances
Can be used to treat serious diseases
like multiple sclerosis, rheumatoidarthritis and different types of cancer
High specificity in binding
Monoclonal antibodies (MAb or moAb) are monospecific antibodies that arethe same because they are all clones of a B cell and have identical paratopes.
Humans and all jawed vertebrates have the ability to make antibodies whichare able to recognize (by binding to) virtually any antigenic determinant(epitope) and to discriminate between even similar epitopes.
Introduction
Can activate and couple components ofthe immune system
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Immunization
Emil von Behringdeveloped serum
therapy as aneffective treatment
against diptheria and
tetanus
Side chain theory
Paul Ehlrich gav e side
chain theory - Toxinand antitoxins were
chemical substances.
Antitoxins were side
chains on cells thatcould bind with a toxin
like a lock and key
Discovery of
chemical structure of
antibody
In 1961, GeraldEdelman and RodneyPorter gave chemical
structure of antibody
Development of
Hybridoma
Technology
Jerne, Kohler andMilstein in 1975
developed
hybridoma technologyfor production ofmonoclonal antibdies
History
In 1986, OKT- 3 was first monoclonal antibody to be approved for use in organ
transplant rejection.
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Antibodies are a key component of the adaptive immune response,playing a central role in both in the recognition of foreign antigensand the stimulation of an immune response to them.
2 identical light chains (~220 amino acids long)Variable domain: VLConstant domain: CL
2 identical heavy chains (~440 amino acids long)Variable domain: VH3 Constant domains: CH1, CH2, CH3
Covalent, disulfide bonds between cysteine residuesFlexible hinge region
Structure of Human and therapeuticAntibodies
Heavy
Chains
LightChain
Antigen
binding site
Constant
region
Hinge
region
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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 (Complementarity-Determining Region) are necessary forantigen binding.
The tertiary structure of this region can contain pockets, undulating flattersurfaces, and even protrusions.
Small antigens typically bind in deep pockets
For some therapeutic mAbs, the affinity must be balanced so thateffective antigen binding occurs while tissue
penetration is allowed.
A Dynamic Binding Site
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Production of MAbs
Hybridoma Technology
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Step 1: - Immunization Of Mice & Selection Of Mouse
Donor For Generation Of Hybridoma cells
ANTIGEN ( Intact cell/
Whole cell membrane/micro-organisms ) +
ADJUVANT
(emulsification)
Ab titre reached in Serum
Spleen removed
(source of cells)
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Step 2: - Screening Of Mice For Antibody Production
After several
weeks of
immunization
Serum Antibody Titre Determined
(Technique: - ELISA / Flowcytometery)
Titre too low
BOOST(Pure antigen)
Titre High
BOOST(Pure antigen)
2 weeks
PRODUCTIONOF MONOCLONAL ANTIBODY
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Step 3: - Preparation of Myeloma Cells
Immortal Tumor Of Lymphocytes
+ 8 -Azaguanine
Myeloma Cells
High Viability & Rapid Growth
HGPRT-
Myeloma Cells
PRODUCTIONOF MONOCLONAL ANTIBODY
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Step 4: - Fusion of Myeloma Cells with Immune SpleenCells &
Selection of Hybridoma Cells
FUSIONMY LOMA C LLSS L N C LLS
HYBRIDOMA C LLS
LISA LAT
Feeder Cells
rowth Medium
HAT Medium
1. lating of Cells
in HAT selective
Medium
2. Scanning of
Viable
Hybridomas
PRODUCTIONOF MONOCLONAL ANTIBODY
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Step 4: - Cloning of Hybridoma Cell Lines by LimitingDilution or Expansion
A. Clone Each +ve
CultureB. Test Each Supernatant for
Antibodies
C. Expand +ve
Clones
Mouse
Ascites
Method
Tissue
Culture
Method
PRODUCTIONOF MONOCLONAL ANTIBODY
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PRODUCTIONOF MONOCLONAL ANTIBODY
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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 Vs Polyclonal Antibodies
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Diagnostic Appliations Biosensors and Microarrays
Therapeutic Applications
Transplant rejection Muronomab CD3
Cardiovascular disease - Abciximab
Cancer - Rituximab
Clinical Applications Purification of the drug
Imaging the target
Application of MAbs
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Fastest growing segment of Biopharmaceuticalmarket with an average annual growth of 18%.
Monoclonal Antibody market in 2010 was of $30Billion and Mabs, in vitro diagnostic marketwas that of $ 34 billion.
Currently there are approx 500 antibody basedtherapies under development and more than 20therapeutic Mabs on the market.
Market of Pharmaceutical Antibodies
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Small antibody fragments (Fv or Fab) are alsoeffective in blocking cytokines Benefit: More readily penetrate tissue
Coupling of antibody fragments to form dimers andtetramers Increases avidity and cross-linking
Nanobodies 1989 - Raymond Hamers
Discovered in camels
Completely lack the light chain Same antigen affinity as their four-chain
counterparts
Structure makes them more resistant to
heat and pH
May lead to development of oral
nanobody pills
Engineered Antibodies
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