Monoclonal antibodies and gene therpy
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Transcript of Monoclonal antibodies and gene therpy
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
THANK you