Monoclinal antibodies and gene therapt 1
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Transcript of Monoclinal antibodies and gene therapt 1
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MONOCLONAL ANTIBODIES AND GENE THERAPY
BY
B.ALEKHYA
M.PHARM
256212886037
UNDER GUIDANCE OF
Mrs.YASMIN BEGUM
Assistant professor (Ph.D)
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CONTENTS
INTRODUCTION
DISCOVERY
PRODUCTION
TYPES OF MABs
PURIFICATION
APPLICATIONS IN THERAPY
ADVANTAGES
DISADVANTAGES
GENE THERAPY
CONCLUSION
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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.
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STRUCTURE OF IMMUNOGLOBULIN
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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
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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).
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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.
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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
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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
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Step 3: - Preparation of Myeloma Cells
Immortal Tumor Of Lymphocytes
+ HAT Medium
Myeloma Cells
High Viability & Rapid Growth
HGPRT-
Myeloma Cells
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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
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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
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Cont’d
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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.
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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
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Types of Monoclonal Antibodies
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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
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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.
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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
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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
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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
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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
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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
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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
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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.
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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.
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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
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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.
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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
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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.
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Mechanism of antitumor effect
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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
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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
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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.
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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
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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.
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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.
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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
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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
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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.
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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.
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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.
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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
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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)
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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)
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MECHANISM:
DNA SYNTHESIS
NUCLEOSIDE NUCLEOTIDE
Thymidine kinase
phosphates
Gancyclovir False nucleotide Inhibits DNA
polymerase
DNA synthesis
blockedCancer cell
dies
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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
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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
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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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