The document provides a comprehensive overview of monoclonal antibodies, detailing their discovery, characteristics, and production methods through hybridoma technology. It explains the process of antibody production, the significance of hybridomas, and the development of various types of antibodies, including murine, chimeric, humanized, and bispecific antibodies. Additionally, it discusses the medical applications of monoclonal antibodies in diagnostics and targeted therapies, emphasizing their role in immunotherapy and drug delivery systems.

1. Monoclonal antibodies
Dr.S.Sethupathy,M.D.,Ph.D.,
Professor of Biochemistry,
Rajah Muthiah Medical College,
Annamalai University

2.  In 1975, Kohler and Milstein first
fused lymphocytes to produce a
cell line which was both immortal
and a producer of specific
antibodies.
 They were awarded the Nobel Prize
for Medicine in 1984 for the
development of this "hybridoma."

3. Immunity
 Substances foreign to the body, such as
disease-causing bacteria and viruses and
other infectious agents, known as
antigens
 They are recognized by the body's
immune system through antibodies
( proteins)
 These natural defenses against these
infectious agents seek out the antigens
and help destroy them.

4.  When an organism is exposed to an antigen,
the immune system produce immunological
response.
 One response -The activation of B-cells and
subsequent release of antibodies.
 Thousands of different B-cells can be
activated against different epitopes on a
single antigen.
 B-cells mature into antibody releasing plasma
cells
 Thousands of different antibodies (Polyclonal)
are released into the blood against the
invading antigen.

5. Specificity
 Antibodies are extremely specific
 Each antibody binds to and attacks
one particular antigen.
 Second, some antibodies, once
activated by the occurrence of a
disease, continue to confer
resistance against that disease.
 Eg: Antibodies to the childhood
diseases chickenpox and measles

6. Vaccines
 The second characteristic of antibodies
makes it possible to develop vaccines.
 A vaccine is a preparation of killed or
weakened bacteria or viruses that,
when introduced into the body,
stimulates the production of antibodies
against the antigens
 These antibodies protect against
particular disease causing pathogens.

7. Structure of Ig

8. Epitopes
Immune Response
Antibodies
A mixture of antibodies - all bind to
epitopes of the original antigen. Some
bind with higher affinity than others.
Polyclonal antibodies
Protein
Immunize

9. In a specific immune response, only those T and B cells that can bind to the
of the pathogen are selected to participate in the response.
Clonal selection of lymphocytes during the specific immune response
An antigen with 2 epitopes
- red epitope, blue epitope
Mixture of T and B cells
with different antigen
specificities
Proliferation of cells
with receptors capable
of binding epitopes of
the antigen

10. From http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AffinityChrom.html
Affinity chromatography - antibody
purification.
Antigen can be bound to the support
matrix in order to purify antigen-specific
antibody from a polyclonal antiserum.

11. Polyclonal antibodies
(Polyclonal antiserum)
B B B B B B B B
Harvest Ab
Monoclonal antibodies

12. Mono and poly clonal antibodies

14. - B lymphocytes can mutate into
tumor cells that result in a type of
cancer termed myeloma.
- Myeloma cells become “immortal”
and will grow indefinitely in culture.
- Fusion of a single activated B cell
and a myeloma cell will create a
hybridoma that can grow indefinitely
in culture.
Hybridomas Technique

15. Hybridoma
 In monoclonal antibody technology, tumor
cells that can replicate endlessly are fused
with mammalian cells that produce an
antibody.
 The result of this cell fusion is a
"hybridoma," which will continually
produce antibodies.
 From single clone -monoclonal
 They have ability to grow continually, and
produce large amounts of pure antibody

16. B cells
 Mice are immunized every 2-3 weeks
till. sufficient antibody titer is
reached
 After several weeks of immunization,
blood samples are obtained from
mice for measurement of serum
antibodies
 Mice are euthanized and the spleen
removed as a source of cells for
fusion with myeloma cells.

17. Myeloma cells have been genetically
engineered such that they can not use
hypoxanthine, aminopterin, and
thymidine (HAT medium) as a source for
nucleic acid biosynthesis and will die in
culture.
Only B cells that have fused with the
engineered myeloma cells will survive in
culture when grown in HAT medium.
Hybridoma Selection
The “HAT Trick”

18. Fusion
 Myeloma cells are immortalized cells that are
cultured with 8-azaguanine
 They are sensitive to the hypoxanthine-
aminopterin-thymidine (HAT) selection
medium
 A week before cell fusion, myeloma cells are
grown in 8-azaguanine
 Single spleen cells from the immunized
mouse are fused with the previously
prepared myeloma cells.
 Fusion is by co-centrifuging freshly harvested
spleen cells and myeloma cells in
polyethylene glycol , a substance that causes
cell membranes to fuse.

19. HAT medium
 The selection growth medium contains the
inhibitor aminopterin, which blocks synthetic
pathways
 So cells must use salvage pathway to
synthesize nucleic acids
 HGPRTase gene is absent in the myeloma cell
line to which the normal antibody- producing
cells are fused.
 Because neither the myeloma nor the
antibody-producing cell will grow on its own,
only hybrid cells grow in HAT medium

21.  Fused cells are incubated in HAT medium
(hypoxanthine-aminopterin-
thymidinemedium) for roughly 10 to 14 days.
 Aminopterin blocks the pathway that allows
for nucleotide synthesis.
 Unfused myeloma cells die, as they cannot
produce nucleotides by the de
novo or salvage pathways because they lack
HGPRT.
 Unfused B cells die as they have a short life
span.
 only the B cell-myeloma hybrids survive,
since the HGPRT gene coming from the B
cells is functional.

22. Harvest Ab
Monoclonal antibodies
Myeloma cells
Grow indefinitely
in cell culture but
don't secrete the
desired antibody
FUSE Hybridoma cells
Secrete antibody but
don't grow
in tissue culture
Grow indefinitely
in cell culture
AND secrete
antibody

23. Screening
 Since the antibodies in a well are produced
by the same B cell, they will be directed
towards the same epitope
 Next is a rapid primary screening process to
identify and select only those hybridomas
that produce antibodies of appropriate
specificity.
 The first screening technique used is
called ELISA.
 Alternatively, immunocytochemical, western
blot, and immunoprecipitation-mass
spectrometry screening can also be used.

25. Feeder cells
 Mouse peritoneal cells, most of which are
macrophages, have been found to be
convenient and effective feeder cells which
are a source of soluble growth factors
for hybridoma cells.
 The cells are then distributed to 96 well
plates containing feeder cells derived from
saline peritoneal washes of mice.
 Feeder cells are believed to supply growth
factors that promote growth of the
hybridoma cells .
 It is also possible to use murine bone
marrow-derived macrophages as feeder cells

26. Culture methods
 Batch Tissue-Culture Methods
 The simplest approach for producing mAb in
vitro is to grow the hybridoma cultures in
batches and purify the mAb from the culture
medium.
 Fetal bovine serum is used in most tissue-
culture media
 In most cases, hybridomas growing in 10%
fetal bovine serum (FBS) can be adapted
within four passages (8-12 days) to grow in
less than 1% FBS or in FBS-free media.

27. Hybridoma cells growth

28. Murine MAB
 Early forms of monoclonal antibodies were
mouse antibodies
 When injected into humans, the
antibodies would either be rapidly cleared
from the body or result in systemic
inflammatory effects that were harmful.
 Murine antibodies typically have a “mo”
before the “mab” in their name
 Eg: Tositumomab
 drug used to treat lymphoma.

29. Chimeric antibodies
 Chimeric anibodies are antibodies that have the Fab
regions of a mouse antibody, but have the Fc
region of a human antibody.
 Fusing the Fab region coding DNA extracted from
mice B-cells and human antibody DNA that only
codes for the Fc region.
 Fc region of an antibody is glycosylated and human
immune system determines self vs non-self
 Chimeric antibodies typically cause a minimal
immune response against them and thus have a
longer half life

30.  Chimeric antibodies typically have a “xi” before
the “mab” at the end of their name to indicate
their chain type, as is the case with “abciximab”
(ReoPro), “cutximab” (Erbitux),
or Rituximab (Rituxan).
 Chimeric antibodies further humanized through
mutagenesis to the Fab sequence (excluding
the CDR segments which are critical to antigen
binding)
 After identifying those parts which differ in humans
and mutating them
 This is a difficult process, but can create
antibodies that are unrecognizable from human
antibodies.

31. Insertion of CDR
 Insertion of only the CDRs from mice into a
full human antibody scaffold has been used
to create humanized antibodies.
 Humanized antibodies typically have a “zu”
before the “mab” to indicate their chain type.
 The drug Alemtuzumab for the treatment of
leukemia
 It is an example of a humanized antibody
with foreign CDR (Complementarity-
determining regions)regions.

32. Human Antibody Production &
Phage Display
 In phage display, DNA coding the binding
region of the antibody is ligated into the
genes of a phage which code for proteins
that are expressed on the protein coat of
the virus.
 This phage gene hybrid is then
transformed into bacterial cells.
 The virus hijacks the bacterial replication
machinery, producing vast quantities of
phage.

34.  By using many DNA fragments that are
slightly different, a library of slightly different
antibodies can be expressed on the outside
of these phages, one type of antibody per
phage.
 Phages can then be selected by running them
through a column with the protein target
immobilized on the column.
 Once the phages that bind the target protein
well are identified, they can be used to
reinfect bacterial cells
 Then isolate the DNA of antibodies that code
for those antibodies with the greatest affinity
for the target.

35. Humanized antibodies
 They can be combined with human
antibody scaffolds or human Fc
regions as above to create
humanized antibodies.
 Antibodies created through phage
display have a “u” before the “mab”
in their name to indicate their chain
origins as is the case for
Adalimumab (HUMIRA)

36. Glycosylation of MAB
 Protein glycosylation is critical for the
immune system to recognize a protein as
native
 It allows the protein to stay in circulation
in the human body without creating an
immune response
 Chinese Hamster Ovary (CHO) cells can
glycosylate antibodies appropriately, but
they are difficult to work with and
relatively expensive at an industrial scale.

37. Yeast cells for glycosylation
 Dr. Tillman Gerngross, et al. have
demonstrated that yeast can be
genetically manipulated to produce
human glycosylation on expressed
antibodies.
 Yeast replicate much faster than
CHO cells, more ideal as hosts for
industrial scale production of
therapeutic proteins.

39. Medical applications
 Eg:Prostate specific antigen, placental
alkaline phosphatase, human chorionic
gonadotrophin, α-fetoprotein
 Organ-associated antigens and the
production of monoclonal antibodies
against these antigens helps in
determining primary tumor.
 We can have increased sensitivity over
normal histopathological staining.

40. Medical Applications
 They are essential components
in immunofluorescence experiments,West
ern Blot and immuno dot blot tests to
detect protein on a membrane.
 As drugs- Rituximab binds to CD20
surface proteins on B-Cells and elicits
natural immune responses to these
cancerous cells
 Via ADCC, activating cellular apoptosis, or
by attracting compliment proteins which
kill the cell via the compliment pathway.

42. Targeted immunotherapy
 By conjugating a radioactive isotope to a
murine antibody, targeted immunotherapy
done
 The antibody binds to the targeted cancer
cell and the cell is damaged or destroyed by
emitted beta particles.
 Murine antibodies are used because they are
cleared from the body rapidly so as to avoid
extensive radiation damage from the drug.
 This is the mechanism used
by Tositumomab which uses radioactive
Iodine-131.

43. ADEPT
 The Third class of monoclonal antibody drugs are
those conjugated to a drug activating enzyme
also known as Antibody-Directed Enzyme
Prodrug Therapy (ADEPT).
 An enzyme that can activate a drug is attached
to an antibody with specificity toward a target of
choice.
 After ample time of the injected
antibody/enzyme compound to bind the target
 An inert prodrug is injected.
 Prodrug when comes into contact with the
antibody/enzyme compound bound to the target
gets activated and able to attack infected target
cells

44. Drug delivery
 Monoclonal antibody conjugated to
liposomes or another form of
nanotechnology used in 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

45.  A normal antibody-producing cell only expresses one
antibody,
 When two myeloma cells are fused, the derived hybrids are
capable of co-dominantly expressing the antibody genes of
both parents.
 Although the respective variable (V) and constant (C)
region genes remain expressed, heavy and light chains of
both parents are scrambled, and hybrid molecules are
formed.
 Fusion therefore allows the production of hybrid
immunoglobulin molecules containing two different
combining sites.
 Hybrid molecules of this type retain antigen-binding
activity and specificity.
 Bispecific monoclonal antibodies secreted by hybridomas
may have a variety of uses in biology and in medicine such
as in histochemistry.

46. Bispecific hybrid-hybridomas

47. Thank you