7. REFERENCES

1
PRODUCTION OF MONOCLONAL ANTIBODIES AND ITS USE
IN THE TREATMENT OF CANCER
6. MONOCLONAL ANTIBODIES (MAbs) AND CANCER The
hybridoma technique entered clinical trials in
1980, where the first patient with relapsed
lymphoma was treated. The antibody which had
shown to lyse the patients tumour cells in vitro
turned out to be clinically ineffective due to
large amounts of circulating target antigen.
However there was an exception in a patient with
B-cell lymphoma achieving a complete remission
after treatment with murine antibodies
recognising the unique immunoglobulin expressed
on the patients malignant B-lymphocytes. Today
there are various approaches developed in efforts
to produce monoclonal antibodies for diagnosis
and treatment of cancer. One way involves binding
of the variable region to the cell surface
molecules that regulate cell survival,
proliferation or death, by activating cell
signaling cascades or blocking access to growth
factors. Another by targeting oncogenic cell
surface receptors with MAbs could prevent tumor
growth. There are two types of MAbs used in
cancer treatment naked MAbs and Conjugated MAbs.
Naked MAbs are used without any radioactive drug
material attached to them. Conjugated MAbs are
joined to a chemotherapeutic drug, radioactive
particle or a toxin. Monoclonal antibodies used
in cancer therapy are often attached to different
drugs/toxins which are then delivered to the
target cells without harming the other cells.
These antibodies act by directly inducing
apoptosis, blocking growth factors and/or
stimulating antibody- dependent cellular
cytotoxicity or complement dependent cytotoxicity.
1. INTRODUCTION Biotechnology has created
unprecedented opportunities in recent years for
the benefit of mankind and for the understanding
of the fundamental life processes. The immune
system is composed of components that help to
protect the body against foreign pathogens. These
foreign substances (antigens) are recognized by
the immune system as invaders. Antibodies are a
specialized protein class produced by
B-lymphocytes which are able to interact
specifically with antigens and destroy them.
Antibodies are highly specific therefore each
antibody binds to and helps destroy one specific
antigen. Some antibodies, once activated by the
occurrence of a disease, continue to confer
resistance against that disease, examples are the
childhood diseases chickenpox and measles. This
property of antibodies allows for the production
of vaccines. Given such diversity for these
antibodies in fighting diseases, their production
in pure quantities has been the prime focus of
scientific investigation. Scientists recognized
that if antibodies could be created in the lab,
then they could potentially be used as a means
for the treatment of many diseases and to
diagnose a wide variety of diseases. In 1975, a
new era in immunology was launched with the
discovery of the hybridoma technique which has
given an insight into how currently incurable
diseases may be treated. The hybridoma technique
is a biotechnological tool for achieving the goal
of complete immunization of the human body from
infections. Human medicine has greatly benefited
from the advances of biotechnology. The first
area of medicine to see tangible progress from
this hybridoma technique was diagnostics where
more specific and improved methods for detecting
diseases have been made possible by the
development of MONOCLONAL ANTIBODIES which was
first described by Kohler and Milstein in 1975.
So important was this approach to antibody
production, that Kohler and Milstein received the
Nobel Prize for their discovery in 1984. WHAT
ARE MONOCLONAL ANTIBODIES? Monoclonal antibodies
are antibodies which have been artificially
produced against a specific antigen. They are
called monoclonal because that are produced in
the lab from multiple, identical copies (i.e.
clones) of a cell called a hybridoma. Hybridoma
cells are created by the fusion of a myeloma
(cancer) cell with an antibody producing B-cell
from a lab mouse. Monoclonal antibodies are of
exceptional purity and specificity they are
components of the immune system and are able to
bind to a specific antigen.
Figure 2 Production of monoclonal
antibodies Hurrell, J.G.R. (2000)
3. EVOLUTION OF ANTIBODY PRODUCTION
Figure 1 Evolution of antibody
production http//www.medarex.com/Development/Evol
ution.htm
Figure 5 Cancer treatment with MAbs by blocking
tumor growth http//cal.man.ac.uk/student_project
s/2001/MNQB8MJG/cancer.html
Figure 3 Monoclonal antibodies www.fostermed.com
Rituximab rituxumab was the first anti-cancer
antibody approved by the FDA (Food and drug
administration) for Non-Hodgkin lymphoma (NHL)
.Non-Hodgkin lymphoma was the first malignant
disease where single patients were successfully
treated with monoclonal antibodies. The
development of rituximab, a chimeric lgG1
monoclonal antibody against the CD20 antigen,
displays the importance of choosing an optimal
target in antibody-based therapy of cancer. CD20
is trans-membrane phosphoprotein which binds to
rituximab. Binding occurs by antibody-dependent
cellular cytotoxicity and complement-mediated
cytotoxicity. This then leads to a calcium influx
and apoptosis. Administration of rituximab causes
rapid depletion of normal and malignant CD20
positive B-lymphocytes from blood, bone marrow
and lymph nodes. Side effects include fever,
chills, hypotension and nausea. The intensity of
the adverse reactions correlates with the number
of circulating malignant cells, suggesting the
lysis of normal and malignant B-lymphocytes and
the ensuing release of cytokines is responsible
for infusion associated toxicity. Trastuzmab -
Trastuzmab (Herceptin) is used in TK-Beast
carcinoma. The action of Trastuzmab is directed
against the HER2/neu surface protein that is over
expressed in breast carcinoma. HER2/neu
glycoprotein is a receptor for Tyrosine Kinase
(TK) involved in growth and survival of breast
carcinoma cells. Trastuzmab interferes with
signaling through this receptor.
2. METHOD Production of monoclonal antibodies
involves the following steps - Immunization -
This can occur both in vivo and in vitro. The in
vivo method involves injecting an animal (e.g. a
mouse) with an antigen. The mouse spleen cells
then react to the antigen leading to antibody
production. Antibodies can also be produced in
vitro when antibody-secreting cells are removed
from a donor and then exposed to the antigen in
vitro. The in vitro method is more effective in
that fewer antigens is required and more
antibody-producing cells are stimulated however
these antibodies are of lower affinity than those
produced with in vivo methods. Fusion - The
antibody cells are then fused with myeloma cells
that have been produced in culture. This results
in the formation of hybridomas. The fusion
process is promoted by the addition of a fusion
agent such as polyethelene glycol
(PEG). Selection of hybrids - This ensures that
only fusion of myeloma cells with
antibody-producing cells is promoted. Other cell
fusion combinations do not survive. This occurs
by growth in selective media such as the HAT
(Hypoxanthine Aminopterin Thymidine)
medium. Propagation - After fusion, the hybridoma
cells are exposed to specific conditions that
promote propagation. Screening test -This test
allows for the selection of the cells that
produce the specific antibodies desired.
Therefore only the cells that produce antibody in
adequate amounts are selected. They are many
different screening methods that can be used
depending on the antigen. The screening test must
be selected to ensure that the conditions of
sensitivity, specificity, time and scale are
satisfied. Cloning -This involves the isolation
of a single specific antibody secreting cell
from the other cells in culture. Cloning can
occur via three different techniques cloning by
limiting dilution, cloning using semi-solid agar
and cloning by fluorescence activated cell
sorting (FACS). Cloning results in colonies of
cells that have a single origin and are thus
monoclonal. Cells that are not monoclonal in
origin should be re-cloned. Some of the cells
should then be cryopreserved. This ensures that
the original cell line is preserved should the
clones be lost or destroyed. Propagation -
Propagation of the selected clones can occur in
vivo or in vitro. In vivo propagation involves
ascites production from rodent hybridomas.
During this process, the monoclonal cells are
injected into the peritoneal cavity of a mouse
and allowed to grow. After approximately two
weeks, there is a build-up of ascitic fluid,
which contains the antibodies. These antibodies
can then be removed form the peritoneal cavity
using a needle and syringe. In vitro propagation
involves growing cells in culture such that the
cells increase in density and apoptosis occurs.
This increases antibody yield. This can also be
done using spinner flasks, roller bottles and
fermenters such as air-lift fermenters and
stirred tank fermenters Perfusion bioreactors and
hollow fibre reactors are also being used to
increase antibody yield. Although these methods
result in high antibody yield, they are highly
expensive and also affect the stability of the
antibody. Characterization - The monoclonal
antibodies must now be characterized such that
stability, specificity, binding affinity and
other conditions are satisfied.
4. APPLICATIONS OF MONOCLONAL ANTIBODIES Diagnosis
and treatment of cancer and infectious
diseases Measuring protein and drug levels in the
serum Transplantation Identifying tumor
antigens and auto-antibodies They are involved in
combating graft rejection auto-immune disease.
Monoclonal antibodies are also used to detect
allergies, carry out hormone tests, diagnose
viral diseases and to detect certain types of
cancers (by distinguishing cancer cells from
normal cells) Used in the preparation of very
specific vaccines, particularly against certain
viral strains and other parasites. The most
important utilization of the hybridoma-based
antibodies is their specific utilization in the
target orientated therapy so as to attain cell
specific delivery of the drugs like anti-cancer
and anti-HIV drugs
5. ETHICS Adjuvants are used to maximize the
immune response of a mouse and increase
production of antibody. Freunds complete
adjuvant (FCA) includes killed mycobacteria which
serve to increase inflammation upon injection and
thereby enhances the mouses immune response.
This inflammation often results in painful
lesions at the injection site. The resulting
discomfort is the subject of widespread concern.
Due to the pain that FCA causes, large scale FCA
has been banned. During Ascites Production
accumulation of fluid in the peritoneal cavity
causes respiratory distress. Ascites fluid volume
in mice can exceed the total blood volume of the
animal increasing physiological distress from
removal of the fluid due to hypovolemia.
Guidelines have listed based on procedures known
or assumed to decrease pain and distress.
Deviations from these guidelines must be
justified.
Figure 5 Cancer treatment with MAbs by blocking
tumor growth http//cal.man.ac.uk/student_project
s/2001/MNQB8MJG/cancer.html

• 7. REFERENCES
• Book references
• Purohit, S.S., Kakrani, H.N. and Saluja, A.K.
  (2003) Pharmaceutical biotechnology, 1st edition.
  Agrobios India. pp 15,133-140
• Liddell, J.E. and Cryer, A. (1991) A practical
  guide to Monoclonal Antibodies, 1st edition. John
  Wiley and Sons Ltd, England. pp 2-102
• Hurrell, J.G.R. (2000) Monoclonal hybridoma
  antibodies Techniques and Applications, 6th
  edition. CRC Press Inc, United States. pp 4-39
• Boss, B.D., Langman, R., Trowbridge., I. and
  Dulbecco R. (1983) Monoclonal antibodies and
  Cancer, 1st edition. Academic Press Inc. pp 86-92
• Roth, J.A. (1986) Monoclonal antibodies in
  cancer Advances in diagnosis and treatment, 1st
  edition. Futura Publishing Company Inc, New York.
  pp 5-69
• Internet refernces
• http//www.accessexcellence.org/RC/AB/IE/Monoclona
  l_Antibody.html -DATE ACCESSED 14/08/2007
• http//www.biobasics.gc.ca/english/View.asp?x787
  - DATE ACCESSED 16/08/2007
• http//grants.nih.gov/grants/policy/antibodies.pdf
  -DATE ACCESSED 16/08/2007
• http//www.vetmed.ucdavis.edu/Animal_Alternatives/
  mabs.htm - DATE ACCESSED 20/08/2007
• http//www.medarex.com/Development/Evolution.htm
  - DATE ACCESSED 20/09/07
• http//www.fostermed.com - DATE ACCESSED
  20/09/07
• Journal references
• Animal Welfare Information Center Newsletter,
  Winter 1997/1998, Vol. 8, no. 3-4
• Cersosimo, R.J. (2003) Monoclonal antibodies in
  the treatment of cancer, Part 1. American Journal
  of Health System Pharmacy 60 (15) 1531-1548
• Kozlowski, S. and Swann, P. (2006) Current and
  future issues in the manufacturing and
  development of monoclonal antibodies. Advanced
  Drug Delivery Issues 58 (5-6) 707-722
• Kohler, G. and Milstein, C. (1975) Continuous
  cultures of fused cells secreting antibody of
  predefined specificity. Nature. 256 495-497

DONE BY Zulfiah Mohamed Moosa Raeesa B.
Syed Preston Moodley

• Figure 4 Mouse showing swollen
  abdomen typical of ascites
• Animal Welfare Information Center Newsletter,
  Winter 1997/1998, Vol. 8, no. 3-4