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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
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.
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.
Table 5.5. The range of cells capable of producing IL-1
T lymphocytes B lymphocytes
Vascular endothelial cells
Monocytes/macrophages NK cells
Large granular lymphocytes
Dendritic cells Microglia Glioma cells
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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