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bioteknologi
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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.
BIOTECHNOLOGY AND THE PHARMACIST
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 metodaTujuan dan penggunaannya
Creation of adam
CELL FUSION
FUSI SEL :1. FERTILISASI2. KEADAAN PATOLOGIK : 18313. INDUKSI VIRUS : 19624. INDUKSI KIMIA ( NaNO3, Ca++ pH.10) : 19705. INDUKSI PEG : 19746. ELECTROFUSION : 1982
TURUNAN BARU :1. HIBRIDA SOMATIK2. 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
ALIGNMENTDIELECTRO-PHORESIS
FUSION PULSE HETERO-KARYONPHASE
NUCLEI AREFUSED 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.
ANTIBODIMONOKLONAL
PERLUKAH SAYA BELAJAR TENTANG ANTIBODI MONOKLONAL ???
HAL APA YANG PENTING SAYA KETAHUI TENTANG ANTIBODI MONOKLONAL ???Difinisi, metoda pembuatan/produksi nya, penggunaannya
IgGIgAIgDIgEIgM
ANTIBODI MONOKLONAL”protein produk hibridomaYaitu sel hasil fusi antara
sel B-Limfosit dan sel tumor Myeloma
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
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.
ANTIBODIANTIBODI
LIMFOSIT-B
AgAg
AbAb
1. TEORI INSTRUKSI2. TEORI MATRIKS3. TEORI SELEKSI KLONAL
SISTEM IMMUNSISTEM IMMUN
MONOKLONALMONOKLONAL
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 antibody-secreting 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
LIMFOSITMYELOMA
2. FUSI SEL2. FUSI SEL
BANK KULTUR SEL
1. IMUNISASI1. IMUNISASI
TUMBUH CEPAT(TUMOR)
(SULIT DIKULTURKANNAMUN MENGHASILKAN ANTIBODI)
+
PEMBUATAN ANTIBODI MONOKLONAL
LLMMLM
Kombinasi 2 , 3, sel dst
DISELEKSI ( L + M ) YANG MENGHASILKAN ANTIBODI
44HIBRIDOMAHIBRIDOMA
KULTUR SELKULTUR SEL
DITUMBUHKANRONGGA PERITONEAL
( TUMBUH CEPAT,MENGHASILKAN ANTIBODI )
3. SELEKSI SEL3. SELEKSI SEL
5. PRODUKSI ANTIBODI-MONOKLONAL5. PRODUKSI ANTIBODI-MONOKLONAL
Yang menghasilkan antibodi
MEDIA KULTURKHUSUS
ANTIBODI-MONOKLONALANTIBODI-MONOKLONAL
PENGGUNAAN ANTIBODI-MONOKLONAL
IKATANIKATANANTIGEN-ANTIBODIANTIGEN-ANTIBODI
TERKAITKEFARMASIAN
1. IMMUNOSKINTIGRAFI 2. DIAGNOSA DAN TERAPI INFEKSI 3. TRANSPLANTASI 4. TERAPI PENYAKIT AUTOIMMUN 5. TERAPI TUMOR 6. DRUG (LEVEL) MONITORING 7. DRUG CARRIER
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
O=AGO=AG
PERMUKAAN “MICROTITERPLATE”PERMUKAAN “MICROTITERPLATE”
E
1. RIA1. RIA2. ELISAcara langsung
o
W
PENGGUNAAN ANTIBODI MONOKLONAL
SEL TUMORSEL TUMOR
Eo
W
PERMUKAAN MICROTITERPLATEPERMUKAAN MICROTITERPLATE
3. ELISAcara tak langsung
SEL TUMORSEL TUMOR
SEL TUMORSEL TUMOR
OO
4. IMUNOTOKSIN
O=AGO=AG
xx
DRUGS CARRIER
Deliver :1.cytotoxic compounds2.radiologicals
SEL TARGETSEL TARGET
R
• CELL RESPONSE• CELL CYCLE
• CELL SIGNALING• SECOND MESSENGER
ANTIBODY DRUGS = ANTIBODY ANTI-RECEPTOR
AbMn
Monoclonal Antibody Drug IMC-C225 Shows Promise Against a Variety of CancersHow C225 WorksThe 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.
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 ErbituxColorectal cancer
Head & neck cancers20042006
Bevacizumab Avastin Colorectal cancer 2004
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
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 SystemTherapy by Combining CellsThe 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 andmonoclonals 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 25–30% 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).
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 pre-B-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 yttrium-90. 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 ever-increasing role in the management of the administration and supportive care related to therapeutic monoclonal antibodies
Monoclonal Antibodies as Anticancer Agents
1. ANTIBODI MONOKLONAL
APA TERAPANNYA DALAM KEFARMASIAN
APA TUJUAN STRATEGIK FUSI SEL DALAMTAHAPAN PEMBUATAN ANTIBODI MONOKLONAL
2. KULTUR SEL MAMALIA
APA TERAPANNYA DALAM KEFARMASIAN
KULTUR SEL MAMALIA TERKAIT DENGAN METODA ALTERNATIP PERCOBAAN HEWAN
APA KONDISI UTAMA DALAM MENGKULTURKAN SEL MAMALIA
APA DIFINISI / ARTINYA MONOKLONAL
