This document provides an overview of monoclonal antibodies and gene therapy. It discusses the discovery of monoclonal antibodies by Kohler and Milstein in 1975. It also describes the multi-step process of producing monoclonal antibodies through cell fusion and hybridoma technology. Several types of monoclonal antibodies are outlined, along with their purification techniques and therapeutic applications in cancer treatment and other diseases. Gene therapy approaches including ex vivo and in vivo methods are briefly introduced.

1. MONOCLONAL ANTIBODIES AND GENE
THERAPY
BY
B.ALEKHYA
M.PHARM
256212886037
UNDER GUIDANCE OF
Mrs.YASMIN BEGUM
Assistant professor (Ph.D)

2. CONTENTS
 INTRODUCTION
 DISCOVERY
 PRODUCTION
 TYPES OF MABs
 PURIFICATION
 APPLICATIONS IN THERAPY
 ADVANTAGES
 DISADVANTAGES
 GENE THERAPY
 CONCLUSION

3. 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.

4. STRUCTURE OF IMMUNOGLOBULIN

5. 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

6. 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).

7. 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.

8. 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

9. 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

10. Step 3: - Preparation of Myeloma Cells
Cells
Immortal Tumor Of Lymphocytes
+ HAT Medium
Myeloma Cells
HGPRT-Myeloma
High Viability & Rapid Growth

11. Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells
&
Selection of Hybridoma Cells
PEG
FUSION
SPLEEN CELLS MYELOMA CELLS
Feeder Cells
Growth Medium
HYBRIDOMA CELLS
ELISA PLATE
HAT Medium
1. Plating of Cells in
HAT selective
Medium
2. Scanning of Viable
Hybridomas

12. 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

13. Cont’d

14. 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.

15. 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

16. Types of Monoclonal Antibodies

17. 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

18. 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.

19. Humanised MABs
 Humanised antibodies
are produced by grafting
murine hypervariable
amino acid domains into
human antibodies.
Eg:Atlizumab
 This results in a
molecule of
approximately 95%
human origin

20. 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

21. 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

22. 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

23. 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

24. 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

25. 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.

26. 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.

27. RADIOIMMUNOTHERAPY
 By conjugating a radioactive isotope to a murine
antibody, targeted immunotherapy is possible.
Antibody
with radio
isotope
 ca
Cancer
cell
 More applicable to lymphomas as they are highly
radiosensitive malignancies.
Destruction of
cancer cell by
emmitted beta
particles

28. 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.

29. THERAPY FOR GLIOMAS FORM OF BRAIN
THERAPY
Fusion of lymphocytes
extracted from glioma
with human myeloma
Human hybridomas
secreting antiglioma
antibodies
 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

30. 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.

31. Mechanism of antitumor effect

32. PREGNANCY TESTING KITS
Sample containing
HCG
Antibody specific for
HCG
mixture of
samples+
latex
microspheres
If HCG present,it
binds to antibodies
preventing from
agglutinating
microspheres
Positive test:
Negative test: No agglutination
Agglutination

33. 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

34. 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.

35. 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

37. 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.

38. 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.

39. 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

40. 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

41. 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.

42. 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.

43. 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.

44. 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

45. 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)

46.  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)

47. MECHANISM:

 DNA
SYNTHESIS
NUCLEOSIDE NUCLEOTIDE
Thymidine kinase
phosphates
Gancyclovir False nucleotide
Inhibits DNA
polymerase
DNA synthesis
blocked
Cancer cell
dies

48.  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

50. 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

51. 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