MONOCLONAL ANTIBODIES AND GENE THERAPY

BY

B.ALEKHYA

M.PHARM

256212886037

UNDER GUIDANCE OF

Mrs.YASMIN BEGUM

Assistant professor (Ph.D)

CONTENTS

INTRODUCTION

DISCOVERY

PRODUCTION

TYPES OF MABs

PURIFICATION

APPLICATIONS IN THERAPY

ADVANTAGES

DISADVANTAGES

GENE THERAPY

CONCLUSION

INTRODUCTION

Antibodies are glycoprotein molecules present in

serum,produced against antigens.

Antibodies are secreted by a class of blood cells known as

B-lymphocytes.

These are produced when body comes in contact and is

invaded by a foreign particle or organism.

Composed of two identical heavy chains and two

identical light chains.

STRUCTURE OF IMMUNOGLOBULIN

MONOCLONAL ANTIBODIES

Monoclonal antibodies: are the 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

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 MABs 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

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.

Kohler and Milstein in 1975

were the first to report on

production of monoclonal

antibodies.Awarded with the

Nobel prize.

PRODUCTION OF MONOCLONAL ANTIBODY

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

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

Cell fusion performed

Step 3: - Preparation of Myeloma Cells

Immortal Tumor Of Lymphocytes

+ HAT Medium

Myeloma Cells

High Viability & Rapid Growth

HGPRT-

Myeloma Cells

Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells &

Selection of Hybridoma Cells

FUSION

PEG

MYELOMA CELLSSPLEEN CELLS

HYBRIDOMA CELLS

ELISA PLATE

Feeder Cells

Growth Medium

HAT Medium

1. Plating of Cells in

HAT selective

Medium

2. Scanning of Viable

Hybridomas

Step 5: - Cloning of Hybridoma Cell Lines by “ Limiting Dilution” or soft agar.

A. Clone Each +ve Culture

B. Test Each Supernatant for Antibodies

C. Expand +ve Clones

Mouse

Ascites

Method

Tissue

Culture

Method

Cont’d

Concept of drug targeting by monoclonal antibody :

Targeting antibodies with drugs involve the following

steps:

1. Identification of the antigen produced by the tumor

cells.

2. Production of antibody monoclonally against the

identified antigen.

3. Formation of drug antibody conjugate or complexes.

These complexes concentrate at the tumor site and

deliver the drug.

PURIFICATION TECHNIQUES

Cells, cell debris, lipids, and clotted

material are first removed, typically by

filtration with a 0.45 um filter.

Chromatography

Affinity chromatography: IgG antibodies

using protein A agarose

Anion exchange chromatography:

Endotoxins and DNA

Gel filtration: High and low molecular

wt MABs such as aggregates and small

fragments

Types of Monoclonal Antibodies

Murine antibody

Whole of the antibody is of murine origin

Eg:Aflimomab

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 are

composed of murine variable

regions fused onto human

constant regions.

Eg:cetuximab

Antibodies are approximately

65% human.

This reduces immunogenicity

and thus increases serum half-

life.

Humanised MABs

Humanised antibodies are produced by grafting murine hypervariableamino acid domains into human antibodies.

Eg:Atlizumab

This results in a molecule of approximately 95% human origin

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.

Eg:Belimumab

Applications of Monoclonal Antibodies

Diagnostic Applications

Detects protein of interest either by blotting or immunoflouroscence

Cardiovascular diseases Deep vein thrombosis Location of 10 and 20 metastatic tumours Immunosuppressive therapy Pregnancy testing kits

Therapeutic Applications Radioisotope immunoconjugates Toxin and drug immunoconjugates Immunoliposome based kits In cancer

Location of 10 and 20 metastatic tumours

Can be located with help of radiolabelled MABs

(specific to tumour associated membrane proteins)

MABs specific to breast cancer-labelled with I131

detects tumour in regional lymphnodes.

Similarly MABs specific to breast cancer-by

Gadolinium(Gd) detected by MRI

Pin head size metastases can be

located & visualised

Immuno suppressive therapy

MABs suppress T-cell activity.injection of

MABs results in rapid depletion of T-cells

Mechanism: binding of antibody coated T-cell

to FC receptors on phagocytic cells

phagocytose & clear T-cells from circulation

Mechanism of antitumor effect

Antibody dependent cellular cytotoxicity (ADCC)

Eg: Rituximab

ADEPT (Antibody Directed Enzyme prodrug therapy)

Radioimmunotherapy eg: Tositomomab

MAB may be conjugated with a toxin

MAB can also be conjugated with radioisotope

Immunoliposomes

Antibody dependent cellular cytotoxicity

(ADCC)

Immunoglobulin's clustered on the surface of

the targeted cells and exposes its tail {Fc}

region, to be recognized by the Fc receptors

present on the surface of the macrophages and

neutrophils.

This causes Lysis of tumor cell.

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.

RADIOIMMUNOTHERAPY

By conjugating a radioactive isotope to a murine

antibody, targeted immunotherapy is possible.

ca

More applicable to lymphomas as they are highly

radiosensitive malignancies.

Antibody

with radio

isotope

Cancer

cell

Destruction of

cancer cell by

emmitted beta

particles

IMMUNOTOXINS

Immunotoxins are proteins that contain a toxin along

with an antibody that binds specifically to target cells.

All protein toxins work by enzymatically inhibiting

protein synthesis.

Various plant & bacterial toxins have been genetically

fused/chemically conjugated with the antibodies that

bind to cancer cells.

Plant toxins: ricin,abrin,modecin

Bacterial toxins: diptheria and pseudomanas

aeruginosa toxin A.

THERAPY FOR GLIOMAS FORM OF BRAIN

THERAPY

Isolation-indicates that patient with glioma do

produce antibodies against their own tumours

and are secreted by lymphocytes.

These Abs may be isotope labelled and used

for localisation of intracerebral disease and also

used as immunotoxin

Fusion of lymphocytes

extracted from glioma

with human myeloma

Human hybridomas

secreting antiglioma

antibodies

IMMUNOLIPOSOMES

This class of monoclonal antibody are those conjugated

to liposomes or another form of nanotechnology drug

delivery system. By attaching antibodies to the outside

of a nanosized drug delivery device, large quantities of

therapeutic drug can be delivered to a targeted

environment.

Many new nanotech devices including liposomes,

nanotubes and other such containers have been

developed.

Mechanism of antitumor effect

PREGNANCY TESTING KITS

Sample containing

HCG

Antibody specific for

HCG

mixture of

samples+

latex

microspheres

Positive test:

No agglutinationNegative test:

Agglutination

If HCG present,it

binds to antibodies

preventing from

agglutinating

microspheres

advantages

Specificity for one antigenic determinant.

Antiserum titer values obtained are very high.

Antibodies with high avidity are produced.

High reproducibility.

Radiolabelling & fluorescent conjugation or

enzyme marking of MABs are easy.

Ideal agents for drug targeting in chemotherapy

disadvantages

Monoclonal antibodies production, a time consuming

process because entire process requires 3-4 months for

one fusion experiment.

Average affinity of Monoclonal antibodies are

generally lower.

Any physical/chemical treatment will affect all

Monoclonal antibodies in that production.

Problems with monoclonal therapy

The main difficulty is that mouse antibodies are “seen”

by human immune system as foreign and mounts an

immune response against them producing

HAMA(human anti-mouse antibodies).

These not only causes rapid elimination from the

host,but also form immune complexes that causes

damage to kidneys.

Two approaches are used to reduce the problem:

Chimeric antibodies

Humanised antibodies eg:infliximab and absiximab

GENE THERAPY

It is the process of replacement of a defected

gene with a new gene,to treat diseases.

Newly introduced gene will encode proteins

and correct deficiencies that occur in genetic

diseases.

Therefore gene therapy primarily involves

genetic manipulations in animals or humans

to correct a disease and keep the oraganism

in good health.

APPROACHES FOR GENE THERAPY

Somatic cell gene therapy: Somatic means non-reproductive

cells of an organism,other than sperm and egg cells

eg:bonemarrow cells,blood cells,skin cells etc

Inolves insertion of fully functional and expressible

gene into a target somatic cell to correct genetic disease

permanently.

Germ cell gene therapy: Germ cells are reproductive cells

Involves introduction of DNA into germ cells,which is

passed onto next generations

Genetic alterations in somatic cells are not carried to next

generations.Therefore somatic is prefered.

TWO TYPES OF GENE THERAPY:

Ex vivo gene therapy: transfer of genes into cultured

cells-which are then reintroduced into the patient.

eg: bonemarrow cells

Technique involves following steps:

Isolate cells with genetic defect

Grow the cells in culture

Introduce therapeutic gene to correct defect

Select genetically corrected genes and grow

Transplant the modified cells to the patient

VECTORS:

Viruses: RNA is the genetic material

As retrovirus enters Host cell Synthesise DNA

from RNA

( by reverse

transcription )

Viral DNA formed

( provirus )

Gets incorporated

into the DNA of host

cells

HUMAN ARTIFICIAL CHROMOSOMES

HAC is a synthetic chromosome that can replicate with

other chromosomes.

HAC are used to avoid heavy risk with viruses.

BONE MARROW CELLS:

Contains totipotent embryonic stem cells(ES)

capable of divide and differentiate into various cell

types (eg:RBC,platelets,macrophages)

Most widely used technique.

INVIVO GENE THERAPY

Direct delivary of therapeutic gene into target cells of a

particular tissue(eg:liver,muscle,skin,spleen etc)

Depends on-efficiency of uptake of genes by target

cells.

Intracellular degradation of gene & its uptake by

nucleus.

Expression capability of gene

Gene delivary by viral/non-viral systems

By non-viral systems: viral proteins often induce

inflammatory responses in host.

NON-VIRAL DELIVARY

Pure DNA constructs-can be introduced directly into

target tissues

Lipoplexes-lipid DNA complexes-have DNA

surrounded by lipid layers

HAC-can carry large DNA (one or more genes)

VIRAL DELIVARY

By retrovirus,adenovirus,herpes simplex virus.

GENE THERAPY STRATEGIES FOR CANCER

Tumour necrosis factor gene therapy:

TNF-protein produced by human macrophages

Provide defence against cancer cells-brought about by

enhancing cancer fighting ability of Tumour

Infiltrating Lymphocytes (TILs),a special type of

immune cells.

TILs transformed with a TNF gene

used to treat malignant melanoma

SUICIDE GENE THERAPY:

Thymidine kinase-refered as suicide gene (used to treat

certain cancers)

TK-phosphorylates nucleosides to nucleotides

synthesis of DNA during cell

division

Drug Gancyclovir (GCV)-bears close structural

resemblance to certain nucleosides (thymidine)

By mistake,TK phosphorylates

Gancyclovir Triphosphate-GCV

(false & unsuitable nucleotide for

DNA synthesis)

triphosphate-GCV inhibits DNA polymerase

Results is that elongation of DNA molcule

abruptly stops at a point containing false

nucleotide(of Gancyclovir)

MECHANISM:

DNA SYNTHESIS

NUCLEOSIDE NUCLEOTIDE

Thymidine kinase

phosphates

Gancyclovir False nucleotide Inhibits DNA

polymerase

DNA synthesis

blockedCancer cell

dies

Triphosphate-GCV: enter and kill the neighbour cancer

cells,this phenomenon called as- bystander effect.

Ultimate result – cancer cells cannot multiply &

therefore die

Gancyclovir-treat brain tumours (eg: glioblastoma,

cancer in glial cells

frequently refered as prodrug-approach is called

prodrug activation gene therapy

conclusion

The future of monoclonal antibodies in the treatment of

cancer is bright. Rituximab and trastuzumab have

established roles in the treatment of lymphoma and

breast cancer, respectively.

Radioimmunoconjugates are close to gaining approval

for use and will likely impact significantly on the

treatment of lymphomas

references

Monoclonal antibodies: Powerful new tool in

biology and medicine,Annual review of

biochemistry,vol:50,page no:657-680

Fundamentals of medical biotechnology:

Author:Aparna rajagopalan,page no: 209-253

www.genetics.com

Biotechnology: U.Sathyanarayana ,page

no:652-657

Biochemistry: Gene therapy,author

U.Sathyanarayana,page no:413