Presented by : SHIKHA SINGHUnder the guidance of : Dr. Srinivas MutalikDepartment of PharmaceuticsRegistration No. 160617009

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MONOCLONAL ANTIBODIES

CONTENTS

• Introduction

• Structure of monoclonal antiboby

• Types of monoclonal antibodies

• Methods for preparation of monoclonal antibodies

• Applications of monoclonal antibodies

• Marketed preparations

• Conclusion

• ReferencesMan

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INTRODUCTION

• Antibodies are glycoprotiens(globulins) present in the serum, also known as

immunoglobulins(Ig’s) & are produced by B-lymphocytes

• Monoclonal antibodies are the monospecific antibodies & are made by identical immune cells

that are all clone of a parent cell

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• Antibodies function as markers, binding to the antigen so that the antigen molecules can be

recognized & destroyed by phagocytes

• Naked mAbs are antibodies that work by themselves which are non-cytotoxic, drug or

radioactive

• Conjugated mAbs are linked to a chemotherapeutic drug, radioactive particle, or a cytotoxin,

work by take these substances directly to the cells

Types of mAbs

Naked mAbs Conjugated mAbs

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STRUCTURE OF ANTIBODY

• Y-shaped structure heterodimeric molecules

• Heavy chain and light chain

• Fab Fragment and Fc Fragment

• Paratope and Epitope Light chain

Heavy chain

FAB region

FC region

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TYPES OF ANTIBODIESM

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Advantages

Homogeneity: mAbs represents single antibody molecule that binds to antigens with the same

affinity & promote the same effector functions

Specificity: The product of a single hybridoma reacts with the same epitope on antigens

Selection: It is possible to select for specific epitope specificities

Antibody Production: Unlimited quantities of a single well-defined mono-specific antibody can

be generated

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Disadvantages

Affinity: Average affinity of monoclonal antibodies is generally lower than polyclonal

antibodies.

Cross-reactions: Antibodies sometimes display unexpected cross-reactions with unrelated

antigens.

Time and effort commitment: Very large.

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PREPARATION METHODS OF mAbs

Preparation methods of

mAbs

Main methods

Hybridoma technology

Large scale production

Encapsulated hybridoma cells

Alternative methods

Using Bacteria as precursor

Polymerisation Chain Reaction

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Hybridoma technology• Production of mAbs involved:

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Immunization

Cell fusion

Selection of

hybridomas

Screening of hybridomas

Cloning & propagation

Characterization & storage

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Immunization

Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells

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ANTIGEN + ADJUVANT

SPLEEN REMOVED

(source of cells)

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Cell fusion

Step 2: - Fusion of Myeloma Cells with Immune Spleen Cells & Selection of Hybridoma Cells

FUSIONHGPRT

MYELOMA CELLSSPLEEN CELLS

Fused cells, Free myeloma & spleen cells

PEG added & washed (3mins)

HAT Medium

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Selection of hybridoma

Myeloma cell- HGPRT- &

cannot survive in HAT medium

B cell (spleen)- HGPRT+ &

survive in HAT medium but

after some division undergoes

normal cell death

Hybrid cell (fused)- survive in

HAT medium

Step 3: - Formation & selection of Hybridoma Cells is based on 2 pathways De-novo pathway & salvage pathway

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Screening of hybridoma

Step 4: - Screening of product

ELISA RIA

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Cloning and propagation of hybridomaStep 5: - Cloning & propagation of product

Two techniques are commonly employed for cloning hybrid cells

1. Limiting dilution method or expansion

2. Soft agar method

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Characterization and storage of hybridomaStep 6: - Characterization & storage of product

The monoclonal antibody has to be subjected to biochemical & biophysical characterization for

the desired specificity

The stability of the cell lines & the MABs are important

The cells (and MABs) must be characterized for their ability to withstand freezing & thawing

The desired cell lines are frozen in liquid nitrogen at several stages of cloning & culture

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Large scale production Production of mabs in culture bottles is rather low- 5-10g/ml

Yield can be increased by growing the hybrid cells as ascites in the peritoneal cavity of mice

which yields about 5-20 mg of mabs/ml

It is superior than the in vitro cultivation techniques

Ascitic fluid in mice may have high risk of contamination by pathogenic organism of animal & in

addition, several animals have to be sacrificed to produce mabs

Hence, many workers prefer in vitro techniques rather than the use of animals

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Encapsulated hybridoma cells

Production of mAb can be substantially

increased by increasing the hybridoma cell

density in suspension culture

By this approach, a much higher

concentration of Monoclonal antibody

production (10-100g/ml) can be achieved

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Alternative methodsUsing Bacteria as a precursor:

Bacteria Antibodies

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Polymerase chain reaction:

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APPLICATION

Applications of mAbs:

1. mAbs as Target drug delivery

2. Therapeutic uses of mAbs

3. Diagnostic use

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mAbs as Target drug delivery

1. Dissolution clot

2. Immunoliposomes

3. Immunomicrospheres

4. Radio immunotheraphy

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Dissolution clot

Mechanism

mAb is coupled with tPA & used for degradation of blood clots

mAb-tPA complex due to a high affinity gets attached to fibrin

conversion of plasminogen to plasmin which is in turn dissolves blood clot (fibrin)

Clot lysis has been reported by using mAbs-tPA complex in animal

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Immunoliposomes

Antigens expressed by tumor cells are present in higher ratio than the normal cells

Immunoliposomes are expected to bind more to high antigen density tumor cells than to low antigen density normal cells

Low valency immune-liposomes were found to allow better discrimination between target and normal cells

High drug loading potential that a small number of antibody molecules conjugated to the surface of an immune-liposome can deliver many more drug molecules to the target

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Immunomicrospheres

• Biocompatible & biodegradable polymers, antibodies

have been conjugated to polymeric microparticles for

controlling their in vivo deposition

• Another study has evaluated the in vivo drug delivery

potential of albumin immune-microspheres in mice,

microspheres bearing Lewis lung carcinoma MABs

demonstrated slightly higher localization in lung

carcinoma at 24 h after its administration

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Radio immunotheraphy

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Therapeutic uses of mAbs

• Pregnancy

• HIV

• Cancer

• Autoimmuno diseases

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Pregnancy A monoclonal antibody can be used to detect pregnancy only 14 days after conception A pregnant woman has the hormone human chorionic gonadotrophin (HCG) in her urine mAb have been produced against HCG to which enzymes is attached, which can later interact

with a dye & produce a colour change

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HIV

The test of HIV infection is based on detecting the presence of HIV antibody in the patient’s blood serum

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Cancer

Cancer cells carry specific tumour-associated antigens (TAA) on their plasma membrane.

Monoclonal anti-TAA antibodies have been produced

Drugs which kill tumour cells or inhibit key proteins in tumour cells are attached to monoclonal anti-TAA antibodies

Cancer cells are specifically targeted, avoiding damage to healthy host cells

mAbs are being used to track cancer antigens & alone or linked to anticancer agents, to attack cancer metastases

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Autoimmuno diseases

Monoclonal antibodies used for autoimmune diseases include infliximab and adalimumab,

which are effective in rheumatoid arthritis, Crohn's disease & ulcerative Colitis

Basiliximab & daclizumab activated T cells & thereby help preventing acute rejection of kidney

transplants

Omalizumab inhibits IgE which is useful in moderate-to-severe allergic asthma

The monoclonal antibody known as OKT3 is saving organ transplants threatened with rejection

& preventing bone marrow transplants from setting off graft-versus-host disease

mAbs neutrilize the action of lymphocytes responsible for the rejection of grafts & destroy the

auto-antibodies produced in auto-immune disease.Man

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Diagnostic use mAbs can be used to detect for the presence & quantity of this substance, for instance in a

Western blot test (to detect a substance in a solution) or an immunofluorescence test mAbs can also be used to purify a substance with techniques called immunoprecipitation &

affinity chromatography mAbs allow rapid diagnosis of hepatitis, influenza, herpes, streptococcal & Chlamydia infections

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Antibody Brand name Type Indication

Palivizumab Synagis humanized Respiratory Syncytial Virus

Panitumumab Vectibix human Colorectal cancer

Ranibizumab Lucentis humanized Macular degeneration

Rituximab Rituxan, Mabthera chimeric Non-Hodgkin lymphoma

Tocilizumab Actemra Humanised Rheumatoid arthritis

Tositumomab Bexxar murine Non-Hodgkin lymphoma

Trastuzumab Herceptin humanized Breast cancer

Conclusion Monoclonal antibodies are new biological agents that have good clinical effects & an

extended choice in the treatment spectrum to the patients who were not responding to the

existent treatments

New therapeutic approaches are rapidly emerging and further studies may help in designing

more specific Mabs that would spare the normal tissue, have less adverse effects and

improve the patient’s quality of life

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References

1. AM Carpbell, monoclonal antibody and immunosensor, edited by P.C. van der VLIET -

Department for Physiological Chemistry, University of Utrecht, Utrecht, Volume 23, 1991

Elsevier

2. http://www.biotecharticles.com/Others-Article/Hybridoma-Technology-A-Biotechnology-Technique-378.html,25th

Jan 2017

3. http://pathology.wustl.edu/research/hybridoma.php?page=advantages, 30th Jan 2017

4. http://en.wikipedia.org/wiki/Monoclonal_antibodies,27th Jan 2017

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Thank you