Monoclonal

Antibodies

 Keynotes

Nobel Prize Presented
Principle of monoclonal antibodies
Development
Uses
Recent market antibodies
 Nobel Prize 1984

Georges J.F. Khler Csar Milstein Niels K. Jerne

 Antibodies

Also known as immunoglobulins

Functions as a protective mechanism

for the body

Specific antigen-binding site

Each antibody produced by

specific B cells
 Mechanism of
Antibodies

Specific Antigen-Binding Site:
Each tip of the Y of an antibody contains a
paratope specific to a particular epitope on an
antigen

Mechanism 1: Tagging
Mechanism 2: Neutralization
 Mechanism of
Antibodies
(Tagging)

3)
Stimulates
2) Antibody
other 4)Antigen is
binds to the
1) Antigen immune removed
antigen
is detected responses from the
(Antigen is
such as body
tagged)
complement
pathway
Mechanism of
Antibodies
http://www.youtube.co
m/watch?v=KpNFAEbLcvk
&feature=related
Development of

Monoclonal
Antibodies
 ANTIGENS

IMMUNISATION
HOST The first step in making a
ANIMAL hybridoma is to generate
(MOUSE) antibody-producing B
lymphocytes (immune cells of
a mouse). Multiple
immunizations are performed
until an appropriate level of
antibodies are achieved.
 TEST
BLEEDS

To determine if the mouse is

producing antibodies of
interest, test bleeds are
performed. (Use of ELISA
method)
Some B cells will produce
antibodies that bind to epitopes
on the antigen of interest whilst
others produce antibodies that
 HGPRT+ HGPRT-

Myeloma
cells
Spleen of
mouse

Polyethylene glycol
(PEG) is added to
culture to promote
membrane fusion.

Hybridomas

Separate hybridomas from

unfused cells.
ccess rate of fusion of cells is low so a selective medium is us
HAT Hypoxanthin
Medium e

Aminopterin

Thymidine

Unfused myeloma
cells
THEREFORE, only the
Unfused spleen hybridomas survive as
cells
the myeloma cells
Hybridomas cannot replicate their
DNA ( because they
lack HGPRT) and
unfused spleen cells
have limited lifespan.
Surviving
Hence the
hybridomas
name,
are
monoclonal,
separated
as the cells
and
are derived
individually
from one
cultured.
single cell.
(one cell per
wall)
 After a few weeks of
culture, the culture fluid
is screened for presence
of desirable antibodies.

Antibodies secreted by the different clones

are assayed for their ability to bind to the
antigen.
This is done by ELISA (Enzyme-
linked Immunosorbent Assay)
The most stable and productive clone is
then selected for future use.
 PURIFICATION STEP

Protein A/G affinity chromatography
Antibody selectively binds to protein A/G so a high
level of purity is obtained.
But harsh conditions are used and may damage
some antibodies. (eg low pH)
Hence they immediately neutralize it against a
buffer of pH 7
Recombinant versions of protein-A and protein-G
developed that elute at milder conditions (pH 6.0)
 CHIMERIC
ANTIBODIES
Methods used to produce
monoclonal antibodies yielded
mouse antibodies, not human.
This led to an immune response.

Using recombinant DNA, the

expression of this chimeric DNA
through cell culture yielded
partially-mouse, partially-human
monoclonal antibody
 Human Monoclonal
Antibodies
Generate in-vitro.

1) Phage display-generated
antibodies
Use of bacteriophage
and E. coli
2) Transgenic Mice
Insert human antibody
gene loci
knocking out genes
for making antibodies
Uses of monoclonal

antibodies
 Diagnostic tests

Used in several diagnostic tests to detect small
amounts of drugs, toxins, hormones or infectious
diseases.
1) Immunoassay procedures
2) Radioimmunodetection - attachment of MAbs to
radioactive atoms
3) Fluorescent molecules or metal atoms such as
copper and gold coupled to the antibody to
assist in imaging the target.
 Diagnostic tests

Monoclonal antibodies to human chorionic
gonadotropin (HCG) are used in pregnancy
test kits.
Diagnosis of AIDS by the ELISA (enzyme-linked
immunosorbent assay) test
 Monoclonal antibody
therapy

Use ofmonoclonal antibodiesto specifically
bind to antigens of target cells or proteins.
This may then stimulate the patient'simmune
systemto attack those cells.

Used in the treatment of cancer.

 Cancer treatment

Naked monoclonal antibodiesare those
without any drug or radioactive material
attached to them.
Conjugated monoclonal antibodiesare those
joined to a chemotherapy drug, radioactive
particle, or a toxin
 Naked monoclonal antibodies

Markers for destruction
act as a marker for the body's immune system to destroy
them.
Rituximab (Rituxan) - treat B-cell non-Hodgkin lymphoma
Activation blockers
attach to the specific antigens that are working parts of
cancer cells or other cells that help cancer cells grow,
and stop them from working.
Cetuximab (Erbitux)- blocks the activation of EGFR. It is
used to treat some advanced colorectal cancers as well
as some head and neck cancers
 Conjugated monoclonal antibodies

Monoclonal antibodies that are attached to
drugs, toxins, or radioactive substances.
Used as homing devices to take these
substances directly to the cancer cells.
It then delivers the toxic substance where it is
needed most. This lessens the damage to
normal cells in other parts of the body.
 Conjugated monoclonal antibodies

MAbs with radioactive particles attached are
referred to asradiolabeled, and therapy with
this type of antibody is known
asradioimmunotherapy(RIT).
MAbs with chemotherapy drugs attached are
often referred to aschemolabeled.
MAbs attached to toxins are
calledimmunotoxins.
 Other uses

OKT3, an antibody to the T3 antigen of T cells,
is used to alleviate the problem of organ
rejection
MAbname Tradename Usedto treat: Approvedin:

rituximab Rituxan non-Hodgkin lymphoma 1997

trastuzumab Herceptin breast cancer 1998

acute myelogenous leukemia

gemtuzumab ozogamicin* Mylotarg (AML) 2000**

chronic lymphocytic leukemia

alemtuzumab Campath (CLL) 2001

ibritumomab tiuxetan* Zevalin non-Hodgkin lymphoma 2002

tositumomab* Bexxar non-Hodgkin lymphoma 2003

colorectal cancer 2004

cetuximab Erbitux head & neck cancers 2006

colorectal cancer 2004

non-small cell lung cancer 2006

breast cancer 2008

glioblastoma 2009

bevacizumab Avastin kidney cancer 2009

panitumumab Vectibix colorectal cancer 2006

chronic lymphocytic leukemia

ofatumumab Arzerra (CLL) 2009
The Future of Monoclonal
Antibodies

Monoclonal antibodies may soon be extended
to the therapeutic treatment of diseases such
as infectious diseases and ophthamology.
With sales exceeding 32 billion dollars in 2008,
MAbs are set to become an important sector
of the pharmaceutical industry.