MONOCLONAL ANTIBODIES AND GENE THERAPY

BY

B.ALEKHYAM.PHARM

256212886037UNDER GUIDANCE OFMrs.YASMIN BEGUMAssistant proffessor (Ph.D)

CONTENTS

INTRODUCTION DISCOVERY PRODUCTION TYPES OF MABs PURIFICATION ADVANTAGES APPLICATIONS IN 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

HYBRIDOMA TECHNOLOGY

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 CELLSELISA PLATE

Feeder CellsGrowth 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

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.

Antibodies are approximately 65% human.

This reduces immunogenicity and thus increases serum half-life.

Humanised MABs

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

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

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 mediated 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 are 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 cellto 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

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