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biopharmaceuticals biochemistry and biotecnology - Walsh G.

Walsh G. biopharmaceuticals biochemistry and biotecnology - John Wiley & Sons, 2003. - 572 p.
ISBN 0-470-84327-6
Download (direct link): biochemistryandbiotechnology2003.pdf
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• administration of soluble forms of the IL-2 receptor, which would complete with the native (cell surface) receptor for binding of IL-2;
• administration of monoclonal antibodies capable of binding the IL-2 receptor (it must be confirmed that the antibody used is not itself capable of initiating signal transduction upon binding the receptor);
• administration of IL-2 variants which retains their ability to bind the receptor but fail to initiate signal transduction;
• administration of IL-2 coupled to bacterial or other toxins. Binding of the cytokine to its receptor brings the associated toxin into intimate contact with the antigen-activated T cells
232 BIOPHARMACEUTICALS
Figure 5.4. Structure and mode of action of the engineered fusion protein ‘Ontak’. Refer to text for details
(and other cells, including activated B cells), leading to the destruction of these cells. This would induce selective immunotolerance to whatever specific antigen activated the B/T cells.
Ontak is the tradename given to an IL-2 toxin fusion protein first approved in 1999 in the USA for the treatment of cutaneous T cell lymphoma (Figure 5.4). It is produced in an engineered E. coli strain housing a hybrid gene sequence coding for the diphtheria toxin fragments, A and B, fused directly to IL-2. The 58 kDa product is extracted, purified and marketed as a solution (stored frozen), which contains citric acid buffer, the chelating agent EDTA and polysorbate 20. The protein targets cells displaying the IL-2 receptor, found in high levels on the surface of some leukaemic and lymphoma cells, including cutaneous T cell lymphomas. Binding appears to trigger internalization of the receptor-fusion protein complex. Sufficient quantities of the latter escapes immediate cellular destruction to allow diphtheria toxin-mediated inhibition of cellular protein synthesis. Cell death usually results within hours.
INTERLEUKIN-1 (IL-1)
IL-1 is also known as lymphocyte-activating factor (LAF), endogenous pyrogen and catabolin. It displays a wide variety of biological activities and has been appraised clinically in several trials.
Two distinct forms of IL-1 exist: IL-1a and IL-1b. Although different gene products, and exhibiting only 20% amino acid sequence homology, both of these molecules bind the same receptor and induce similar biological activities. The genes coding for IL-1a and -1b both reside on human chromosome No. 2, and display similar molecular organization, both containing seven exons.
IL-1 a and -1b are expressed as large (30 kDa) precursor molecules from which the mature polypeptide is released by proteolytic cleavage. Neither IL-1 a or -1b possess any known secretory signal peptide and the molecular mechanism by which they exit the cell remains to be characterized. Neither IL appears to be glycosylated.
CYTOKINES: INTERLEUKINS AND TUMOUR NECROSIS FACTOR 233
Table 5.5. The range of cells capable of producing IL-1
T lymphocytes B lymphocytes
Vascular endothelial cells
Fibroblasts
Astrocytes
Monocytes/macrophages NK cells
Large granular lymphocytes
Dendritic cells Microglia Glioma cells
Keratinocytes
Chondrocytes
IL-1 a is initially synthesized as a 271 amino acid precursor, with the mature form containing 159 amino acids (17.5 kDa). This molecule appears to remain associated with the extracellular face of the cell membrane. IL-1b, initially synthesized as a 269 amino acid precursor, is released fully from the cell. The mature form released contains 153 amino acids and displays a molecular mass in the region of 17.3 kDa.
X-ray diffraction analysis reveals the 3-D structure of both IL-1 molecules to be quite similar. Both are globular proteins, composed of six strands of anti-parallel b-pleated sheet forming a ‘barrel’, which is closed at one end by a further series of b-sheets.
A wide range of cells are capable of producing IL-1 (Table 5.5). Different cell types produce the different IL-1s in varying ratios. In fibroblasts and endothelial cells, both are produced in roughly similar ratios, whereas in monocytes IL-1b is produced in larger quantities than IL-1 a. Activated macrophages appear to represent the major cellular source for IL-1.
The IL-1s induce their characteristic biological activities by binding to specific cell surface receptors present on sensitive cells. Two distinct receptors, types I and II, have been identified. Both IL-1 a and IL-1b can bind both receptors. The type I receptor is an 80 kDa transmembrane glycoprotein. It is a member of the IgG superfamily. This receptor is expressed predominantly on fibroblasts, keratinocytes, hepatocytes and endothelial cells. The type II receptor is a 60 kDa transmembrane glycoprotein, expressed mainly on B lymphocytes, bone marrow cells and polymorphonuclear leukocytes. It displays a very short (29 amino acid) intracellular domain and some studies suggest that IL-1s can induce a biological response only upon binding to the type I receptor.
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