BIOTECHNOLOGY AND THE PHARMACIST

The two commonly used techniques in the

production of pharmaceuticals are namely
(1) the recombinant DNA technology and
(2) the hybridoma technology.
The former method is used to produce peptides
or proteins while the latter is used mainly in the
production of monoclonal antibodies. Structural
modification of proteins can be executed by
basic-site directed mutagenesis which is
essentially controlled mutation of genetic
material.

The process of recombinant DNA technology.

A family of enzymes called restriction endonucleases or restriction enzymes is
used to provide the DNA fragment coding for the protein of interest. These
enzymes are able to cut DNA into pieces at recognition sites that are identified
by explicit nucleotide base sequences. DNA fragment or specific gene may
also be obtained from the mRNA of eucaryotic cells through the use of another
enzyme known as reverse transcriptase. The isolated gene is inserted into a
cloning vector by the use of DNA ligase. The cloning vector or plasmid is
essentially a ring of DNA which is usually extracted from bacterial cells. Each
plasmid has an origin of replication which ensures propagation of the plasmid
in subsequent generation of the host cell. The plasmid containing the gene of
interest is introduced into a host cell by a process called transformation. The
host used may be a bacterial cell, a yeast cell or a mammalian cell. The
transformed cells are then identified and isolated. These cells may be frozen
for future use or they may be grown in culture to induce the production of the
protein of interest.

The hybridoma technology.

In a nutshell, the production of monoclonal antibodies is based on
the immortalization and proliferation of individual antibody forming
B-cells. The process of developing a monoclonal antibody is
initiated by the injection of a given antigen in a mouse.

The antibody producing B-cells are later isolated

from the spleen of the animal and fused with cells
from an immortalized mouse B-lymphocyte myeloma
cell line. This fusion results in a population of
immortalized cells, termed hybridomas, which are
able to divide in culture and to produce antibodies.
The hybridomas are identified by the use of a medium which
consists of hypoxanthine, aminopterin and thymidine (the HAT
medium). As each of these hybridomas produces different
antibodies, isolation and propagation of the individual cells will
lead to a large number of cell clones, each capable of producing
monoclonal antibodies.

CELL
FUSION

PERLUKAH SAYA BELAJAR TENTANG FUSI SEL???

HAL APA YANG PENTING SAYA KETAHUI TENTANG FUSI SEL ???
Prinsip metoda
Tujuan dan penggunaannya

CELL
FUSION
Creation of adam

FUSI SEL :
1. FERTILISASI
2. KEADAAN PATOLOGIK : 1831
3. INDUKSI VIRUS : 1962
4. INDUKSI KIMIA ( NaNO3, Ca++ pH.10) : 1970
5. INDUKSI PEG : 1974
6. ELECTROFUSION : 1982
TURUNAN BARU :
1. HIBRIDA SOMATIK
2. HIBRIDOMA

DIELEKTROFORESIS
ELECTRIC PULSE

10 V
10MHz

250 V
100 usec

Usually two cells to be fused into one have to be

in contact before the pulsed electric field is
applied. Since cells have a negative charge
on their surface and naturally repel each other,
some external force must be applied.
There are a number of techniques that have
been reported including chemical (PEG),
centrifugation, vacuum and pressure.
Dielectrophoresis, a non-uniform electric field
based technology, induces an ion charge
separation inside the cell, forming a dipole. This
has two effects: (1) the cell moves toward the
point of highest field strength, and (2) when the
cells are in close proximity they mutually attract
and form long strings called "pearl chains".

SINGLE CELL

GIANT CELL

FUSI PROTOPLAS

ALIGNMENT
DIELECTROPHORESIS

FUSION PULSE

HETEROKARYON
PHASE

NUCLEI ARE
FUSED AS WELL

Each plant has different strong

points. Some have a strong vital
power that is tolerance of hot, cold
and UV rays. Some have a weak vital
power but produce active ingredients
for the skin. The combination of each
strong point sometimes achieves a
great effect. We tried various
botanical cell fusions and produced
GL Extract that was created by 2
kinds of cucurbitaceous plants
(Gynostemma and Luffa). It has a
great effect on preventing photo
aging caused by UV rays. The hybrid
cells created by 2 kinds of
cucurbitaceous plants are cultivated
in a controlled temperature, humidity
and germ-free environment.

ANTIBODI
MONOKLONAL

PERLUKAH SAYA BELAJAR TENTANG ANTIBODI MONOKLONAL ???

HAL APA YANG PENTING SAYA KETAHUI TENTANG ANTIBODI
MONOKLONAL ???
Difinisi, metoda pembuatan/produksi nya, penggunaannya

IgG
IgA
IgD
IgE
IgM
ANTIBODI MONOKLONAL
protein produk hibridoma
Yaitu sel hasil fusi antara
sel B-Limfosit dan sel
tumor Myeloma

Structure and Function

Monoclonal antibodies are
structurally similar to natural
antibodies, which are also known
as immunoglobulins. These are
produced by B-cells and arise in
response to exposure to antigens.
Of the five innate immunoglobulins
(IgA, IgD, IgE, IgG, and IgM),
IgG is most commonly manipulated
as the backbone of the current
therapeutic monoclonal antibodies.

The immunoglobulins consist of a variable region (Fab) that is

responsible for antigen recognition, and a constant region (Fc)
that binds to receptors on immune effector cells, such as natural
killer cells, monocytes, and macrophages. These effector cells
function to elicit phagocytosis and cytotoxicity. The Fc portion of
the immunoglobulin also functions to fix and activate complement,
another important component of the host immune defense. 4,5

ANTIBODI MONOKLONAL

Ag

Ab

LIMFOSIT-B

SISTEM IMMUN
1. TEORI INSTRUKSI
2. TEORI MATRIKS
3. TEORI SELEKSI KLONAL

Myelomas can be induced in a few strains of mice by injecting

mineral oil into the peritoneum. Many of the first examples of
these myelomas were isolated from BALB/c mice by Potter
(1972), and these cells were referred to by the abbreviation
MOPC (for mineral oil plasmacytoma).
Derivatives of BALB/c myelomas have become the most
commonly used partners for fusions. Myelomas have all the
cellular machinery necessary for the secretion of antibodies, and
many secrete these proteins. To avoid the production of
hybridomas that secrete more than one type of antibody,
myelomas that are used for fusions have been selected for
the lack of production of functional antibodies.
Hybridomas can be prepared by fusing myelomas and antibodysecreting cells isolated from different species, but the number of
viable hybridomas increases dramatically when closely related
species are used. Therefore, fusions are normally done with
cells from the same species

PEMBUATAN ANTIBODI MONOKLONAL

1. IMUNISASI

BANK KULTUR SEL

+
LIMFOSIT

(SULIT DIKULTURKAN
NAMUN MENGHASILKAN ANTIBODI)

2. FUSI SEL

MYELOMA
TUMBUH CEPAT
(TUMOR)

Kombinasi 2 , 3, sel dst

LL
MM
LM

DISELEKSI ( L + M ) YANG MENGHASILKAN ANTIBODI

MEDIA KULTUR
KHUSUS

3. SELEKSI SEL

( TUMBUH CEPAT,
MENGHASILKAN ANTIBODI )

4
HIBRIDOMA Yang menghasilkan antibodi
DITUMBUHKAN
RONGGA PERITONEAL

KULTUR SEL

5. PRODUKSI ANTIBODI-MONOKLONAL

ANTIBODI-MONOKLONAL
PENGGUNAAN ANTIBODI-MONOKLONAL
1. IMMUNOSKINTIGRAFI
2. DIAGNOSA DAN TERAPI INFEKSI
3. TRANSPLANTASI
4. TERAPI PENYAKIT AUTOIMMUN
5. TERAPI TUMOR
6. DRUG (LEVEL) MONITORING
7. DRUG CARRIER

IKATAN
ANTIGEN-ANTIBODI

TERKAIT
KEFARMASIAN

The therapeutic effect is a direct result of their

binding to cell surfaces to trigger apoptosis or
immune-system-mediated cell killing.
Alternatively, there are monoclonals that deliver
cytotoxic compounds or radiologicals to kill cells at
the site of disease

PENGGUNAAN ANTIBODI MONOKLONAL

1. RIA

2. ELISA
cara langsung
W

o
E

O=AG
PERMUKAAN MICROTITERPLATE

4. IMUNOTOKSIN

3. ELISA
cara tak langsung
W

DRUGS CARRIER

o
E

SEL TUMOR
SEL TUMOR

O=AG

SEL TUMOR
PERMUKAAN MICROTITERPLATE

Deliver :
1. cytotoxic compounds
2. radiologicals

ANTIBODY DRUGS = ANTIBODY ANTI-RECEPTOR

AbMn

CELL SIGNALING
SECOND MESSENGER
CELL RESPONSE
CELL CYCLE
SEL TARGET

Monoclonal Antibody Drug IMC-C225 Shows Promise

Against a Variety of Cancers
How C225 Works
The C225 drug focuses on cancer cells that have too many
receptors for a hormone-like substance called epidermal
growth factor (EGF). EGF sends a signal to cells to grow and
divide. The extra receptors mean those cells get more
signals to grow and divide. The C225 drug homes in on and
blocks the receptors, slowing or bringing to a halt the cells
rapid growth. Many of the cells then undergo apoptosis, a
"cell suicide" process.

Scientists can make monoclonal antibodies that react with specific antigens on
certain types of cancer cells. As researchers discover more cancer-associated
antigens, they will be able to direct monoclonal antibodies against more and
more cancers
MAb Name

Trade Name

Used to Treat:

Approved in:

Rituximab

Rituxan

Non-Hodgkin lymphoma

1997

Trastuzumab

Herceptin

Breast cancer

1998

Gemtuzumab
ozogamicin*

Mylotarg

Acute myelogenous leukemia (AML)

2000

Alemtuzumab

Campath

Chronic lymphocytic leukemia (CLL)

2001

Ibritumomab tiuxetan*

Zevalin

Non-Hodgkin lymphoma

2002

Tositumomab*

Bexxar

Non-Hodgkin lymphoma

2003

Cetuximab

Erbitux

Colorectal cancer
Head & neck cancers

2004
2006

Bevacizumab

Avastin

Colorectal cancer

2004

Rationale as Anticancer Agents

Therapeutic monoclonal antibodies possess other
mechanisms of action besides antibody-dependent
cellular cytotoxicity and complement-dependent
cytotoxicity.
Monoclonal antibodies bind to specific cell surface
receptors on cancer cells, blocking the binding of natural
ligands. These natural ligands often consist of growth
factors, whose access to the cell would be denied by the
binding of the monoclonal antibody. This action is
independent of the immune system.4,6,7 In theory,
monoclonal antibodies may also induce apoptosis;
however, the mechanism of apoptosis is unknown. 6,7

Cyto Fusion Immunotherapy System

Therapy by Combining Cells
The Cyto Fusion process is the effective fusing of healthy
immune system cells with diseased cells to form a therapeutic
hybrid.
An example is cancer immunotherapy, in which dendritic cells
are obtained from a patient (or a donor) and tumor cells are
obtained from a patient (or a donor). At least one of the two
specimens must to be obtained from the patient. The tumor cells
are first killed using radiation and then fused to the dendritic
cells.
The resultant hybrid (dead tumor cell - dendritic cell) is injected
into the patient for the therapy. The result is a stimulation of the
patient's immune system by causing it to recognize and kill the
cancer cells.

Therapeutic monoclonal antibodies may be conjugated to

radioisotopes, toxins, or chemotherapy agents, using these
antibodies to deliver cytotoxic treatments directly to targeted
tumor cells.
Immunotoxins consist of monoclonal antibodies complexed
with a lethal cellular toxin, such as calicheamicin, the plant
toxin ricin, and the Pseudomonas exotoxin, that is delivered
to the cell and internalized by the receptor, causing
cytotoxicity.
Radioimmunoconjugates are monoclonal antibodies labeled
with a beta-emitting, radioactive isotope such as yttrium-90
or iodine-131. These agents bind to receptors of cell surface
antigens on tumor cells and deliver localized radiation to
both those cells and surrounding tumor cells. Because of the
effect on neighboring cells, radioimmunoconjugates have the
advantage of not requiring either receptor internalization or a
functional immune system to be effective.4,6,7

Examples of unconjugated, humanized therapeutics are moving more and

monoclonals being investigated in a variety of clinical trials :
1. Perhaps the most famous therapeutic use of monoclonals involves Herceptin, a
drug that offers hope to some breast cancer patients. Developed in the late 1980s
by oncologist Dennis Slamon of the University of California, Los Angeles, and by
the biotech company Genentech, Herceptin binds the HER-2 protein, a growth
factor receptor found on tumor cells in 2530% of women with breast cancer.
Herceptin was approved by the FDA in 1998 and is the only commercially available
monoclonal drug thus far that seems to be effective against solid tumors.
2. Another monoclonal antibody is rituximab, or Rituxan, developed by Genentech
and IDEC Pharmaceuticals. It is a drug that binds to the CD20 molecule found on
most B-cells and is used to treat B cell lymphoma.
3. Other monoclonals approved for human treatment are OKT3 the first
monoclonal drug, used to prevent the acute rejection of organ transplants
4. Abciximab, a monoclonal that inhibits the clumping of platelets and helps
prevent reclogging of arteries in patients who have undergone angioplasty.
5. as well as those that bind to vascular ab3 integrin on tumor blood vessels, tumor
necrosis factor (infliximab),
6. the CD52 protein on white blood cells (alemtuzumab).

Monoclonal Antibodies as Anticancer Agents

Rituximab
Rituximab (Rituxan) was the first monoclonal antibody
approved in the United States for the treatment of a
malignancy. Rituximab is a chimeric human/mouse antibody
that targets the CD20 surface antigen. CD20 is expressed
on both B-cell precursors and mature B-cells but is absent
on all other cells, including normal plasma cells and stem
cells. Additionally, CD20 is expressed on more than 90% of
B-cell non-Hodgkin's lymphomas (NHL) and on 50% of preB-cell acute lymphoblastic leukemias. Rituximab is
indicated for both the initial treatment and retreatment of
patients with relapsed or refractory, low-grade or follicular,
CD20-positive B-cell NHL

Monoclonal Antibodies as Anticancer Agents

Trastuzumab
The HER2/neu protein is a proto-oncogene that is
overexpressed in approximately 25% to 30% of patients with
breast cancer and is associated with a poor prognosis.
Trastuzumab (Herceptin) is a humanized monoclonal
antibody that selectively binds to the extracellular domain of
the HER2/neu protein, causing down-regulation of HER2/neu
and inhibiting the growth of HER2/neu-overexpressing
cells.13 Trastuzumab is indicated as monotherapy for
patients with refractory metastatic breast cancer and in
combination with paclitaxel for patients with metastatic
breast cancer who have not received chemotherapy for their
metastatic disease. Patients must be evaluated for HER2/neu
overexpression with a validated assay

Monoclonal Antibodies as Anticancer Agents

Alemtuzumab
Alemtuzumab (Campath) is a humanized monoclonal
antibody targeted against the CD52 surface antigen
that is expressed on virtually all normal and
malignant lymphocytes and most natural killer cells,
monocytes, macrophages, and tissues of the male
reproductive system.16,17 Alemtuzumab is indicated
for patients with B-cell chronic lymphocytic
leukemia (B-CLL) who have been treated with
alkylating agents and have failed treatment with
fludarabine

Monoclonal Antibodies as Anticancer Agents

Gemtuzumab Ozogamicin
The CD33 surface antigen is expressed on leukemic
blast cells in more than 80% of patients with acute
myeloid leukemia (AML). It is also expressed on
myeloid progenitor cells, monocytes, and myeloid
dendritic cells but is absent from pluripotent
hematopoietic stem cells and nonhematopoietic
tissues. Upon antibody binding to CD33, the
complex is internalized and therefore is an ideal
target for a conjugated antibody that can locally
deliver chemotherapy into the cell

Monoclonal Antibodies as Anticancer Agents

Ibritumomab Tiuxetan
The first radioimmunoconjugate approved as an
anticancer agent was ibritumomab tiuxetan
(Zevalin). Ibritumomab is similar to rituximab in
that it binds specifically to the CD20 surface
antigen on B-cell precursors and mature B-cells;
however, ibritumomab is a murine monoclonal
antibody and is attached to the linker tiuxetan to
provide a chelator site for the beta-emitter yttrium90. The beta emission from the yttrium-90 forms
free radicals and induces cellular damage in both
the target cells and surrounding cells.

Monoclonal Antibodies as Anticancer Agents

Conclusion
With the realization of the importance of the immune system
in the treatment of malignancies, great strides are being
made in the fight against cancer. Numerous developments in
bioimmunotherapy have occurred during the past 50 years,
and with increasing technology and knowledge, exponentially
more progress will occur during the next 50 years. With the
mounting numbers of therapeutic monoclonal antibodies
available for cancer treatment, pharmacists will have an everincreasing role in the management of the administration and
supportive care related to therapeutic monoclonal antibodies

1. ANTIBODI MONOKLONAL
APA DIFINISI / ARTINYA MONOKLONAL
APA TUJUAN STRATEGIK FUSI SEL DALAM
TAHAPAN PEMBUATAN ANTIBODI MONOKLONAL
APA TERAPANNYA DALAM KEFARMASIAN
2. KULTUR SEL MAMALIA
KULTUR SEL MAMALIA TERKAIT DENGAN
METODA ALTERNATIP PERCOBAAN HEWAN
APA TERAPANNYA DALAM KEFARMASIAN
APA KONDISI UTAMA DALAM MENGKULTURKAN SEL MAMALIA