Monoclonal Pres Final

8/7/2019 Monoclonal Pres Final

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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



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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



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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



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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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Monoclonal Pres Final

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