Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Patent
1995-12-18
1998-01-20
Guzo, David
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
435 6952, 435 704, 530415, 530416, C07K 1454, C07K 118, C07K 122, C07K 136
Patent
active
057102511
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
Interleukin-10 (IL-10), a recently discovered lymphokine, was originally described as an inhibitor of interferon-.gamma. synthesis and is postulated as a major mediator of the humoral class of immune response K. W., et al., Science 248:1230-1234 (1990)!. Two classes of often mutually exclusive immune responses are the humoral (antibody-mediated) and the delayed-type hypersensitivity.
It is postulated that these two differing immune responses may arise from two types of helper T-cell clones, namely Th1 and Th2 helper T-cells, Vieira, P. et al., Proc. Nat. Acad. Sci. USA Vol. 88:1172 (1991)!. Mouse Th1 cell clones secrete interferon-.gamma., and IL-2 and preferentially induce the delayed-type hypersensitivity response while Th-2 cell clones secrete IL-4, IL-5 and IL-10 and provide support for the humoral responses because interferon-.gamma. secreted by the Th1 cell clones inhibits Th2 clone proliferation in vitro, while IL-10 secreted by the Th2 cell clones supra and Moore et al., supra!. Thus the two T-helper cell types may be mutually inhibitory and may provide the underpinning for the two dissimilar immune responses.
IL-10 has been cloned and sequenced from both murine and human T cells open reading frame encoding a polypeptide of 178 amino acids with an N-terminal hydrophobic leader sequence of 18 amino acids, and have an amino acid sequence homology of 73%.
Biologically active IL-10 is a dimer as determined by analytical gel filtration. Generally, the dimer is non-covalently bonded based upon its migration as a monomer on non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant human IL-10 can be expressed both by prokaryotic and eukaryotic expression systems.
N-terminal analysis of recombinant human IL-10 produced in a eukaryotic expression system indicates that a small percentage of IL-10 polypeptides have the first two N-terminal amino acid residues missing. This truncated polypeptide is referred to as the .DELTA.2 IL-10 polypeptide, or simply .DELTA.2. The full-length chain is therefore referred to as .DELTA.0, indicating that no amino acid has been deleted. Accordingly, biologically active, eukaryotically expressed IL-10 can appear as three different dimers. The first biologically active dimer and the major form is .DELTA.0:.DELTA.0, a homodimer in which both polypeptides of the dimer have the full-length chain of amino acids. The second IL-10 dimer is .DELTA.0:.DELTA.2, a heterodimer in which one of the polypeptide chains has the full-length chain of amino acids and the second chain, .DELTA.2, has the first two N-terminal amino acids missing. The third IL-10 dimer is .DELTA.2:.DELTA.2, a homodimer in which both polypeptide chains of the dimer have the initial two N-terminal amino acid residues missing. Thus, there is a need for a process to purify IL-10, and in particular there is a need for a process which separates the different dimers of IL-10 from each other.
IL-10 contained in inclusion bodies expressed by a prokaryotic expression system must be, denatured, refolded, and purified from contaminants including host proteins, modified variants of IL-10 and heterodimers of those variants. Furthermore, in a prokaryotic system, the IL-10 monomer can be acetylated at one or more of the lysine residues. If an acetylated monomer binds to another acetylated monomer then an acetylated homodimer is produced. If however, a non-acetylated monomer binds to another non-acetylated monomer then a non-acetylated homodimer is produced. If an acetylated monomer binds to a non-acetylated monomer then a heterodimer is produced. Furthermore, IL-10 is normally produced as a non-covalently bonded homodimer. However, during denaturing of the inclusion bodies, and refolding of the IL-10 a covalently bonded homodimer can be produced, i.e. one which migrates as a dimer on non-reducing SDS-PAGE but as a monomer under reducing conditions. This is probably caused by one or more intermolecular disulfide bonds which are formed between the two monomers. Thus, there is a nee
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Cannon-Carlson Susan
Tang John
Vellekamp Gary
Degen Nancy T.
Dulak Norman C.
Guzo David
McLaughlin Jaye P.
Schering Corporation
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