Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-01-21
2002-09-03
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069700, C435S325000, C435S440000, C435S455000, C435S463000, C435S464000, C536S023100, C536S023400
Reexamination Certificate
active
06444441
ABSTRACT:
DESCRIPTION
The invention concerns a process for the production of muteins of eukaryotic polypeptides in eukaryotic cells by means of homologous recombination. The invention additionally concerns a process for the production of human cells which are suitable for the production of human mutated proteins. Finally the invention concerns the human cells produced by the process and mutated human proteins obtainable therefrom as well as pharmaceutical preparations which contain these muteins.
The production of recombinant human proteins in large amounts is known in the field of biotechnology. Proteins obtained in this manner can be used as therapeutic agents. The recombinant production of mutated human proteins which differ from corresponding natural human proteins by deletion, addition or/and substitution of individual amino acids or whole peptide sections is also known.
Especially for pharmaceutical applications it is often desirable to produce human polypeptides in eukaryotic cells since, in contrast to polypeptides produced in prokaryotic cells such as
E. coli,
these are glycosylated and therefore differ less from the polypeptides that occur endogenously in the body so that the occurrence of undesired side effects such as for example increased immunogenicity or poor tolerance is less frequently observed.
Mutated human proteins have been previously produced by heterologous recombinant gene expression. For this a nucleic acid construct is introduced into the desired eukaryotic cell which contains the nucleic acid sequence coding for the mutated polypeptide under the control of a promoter and a selection marker gene. In this process the nucleic acid construct is integrated site-unspecifically into the genome of the cell.
In this heterologous recombinant gene expression undesired and disadvantageous processes can frequently occur due to the site-unspecific integration. For example mutations and especially deletions in the sequence coding for the protein can occur during the process of integration into the genome. Furthermore the integration can take place at a site in the genome at which Cis elements are located which have a repressing effect on the expression control sequence of the nucleic acid construct and as a result of which cells are obtained with a reduced production output for the recombinant protein. An integration of the expression construct into an important gene for the cell leads either to death of this cell or to a recombinant cell with functional disorders which can, among others, result in a reduced yield of the recombinant protein.
The insertion can also lead to a reduced stability of the cells obtained in this manner so that over a long period they lose their ability to express the recombinant protein.
The object of the present invention was therefore to provide a process for the production of muteins of eukaryotic polypeptides with a glycosylation which is as similar as possible to that of the natural protein, in a stable production cell and in good yields and thus at least partially eliminate the disadvantages of the prior art.
This object is achieved according to the invention by a process for the production of muteins of eukaryotic polypeptides wherein
(i) a nucleic acid molecule capable of homologous recombination is introduced into eukaryotic cells which contain a target nucleic acid sequence coding for an endogenous target polypeptide, the said nucleic acid molecule comprising
(a) at least one sequence section which is homologous to sequences in the gene locus of the target nucleic acid sequence and, compared to the endogenous target nucleic acid sequence, has a mutation in the coding region of the mature polypeptide and
(b) a nucleic acid section coding for a selection marker,
(ii) the cells are cultured under such conditions that a homologous recombination of the introduced nucleic acid molecule takes place whereby the cell contains a mutated target nucleic acid sequence after the homologous recombination which is able to express a mutein of the target polypeptide,
(iii) the cells, in which a homologous recombination has taken place, are selected and
(iv) the mutein is isolated from the cells or/and the cell supernatant.
Mutated eukaryotic proteins and in particular mutated human proteins can be produced in a homologous cell by the process according to the invention. surprisingly this enables a mutated protein to be obtained in high yields with a very similar glycosylation pattern to that of the natural protein. An advantage of the process according to the invention is that a protein can be mutated in a eukaryotic cell and this mutein is synthesized by this cell like the protein of the cell that occurs endogenously. A further advantage of the process according to the invention is that the properties of the resulting cells that produce the mutated protein are not disadvantageously altered due to a site-unspecific gene integration. Thus the genome of the cell is not changed in any manner apart from the gene locus of the protein to be expressed and hence the associated adverse effects can be excluded.
The human mutated protein produced by the process according to the invention differs from the corresponding natural protein by deletion, addition or/and substitution of individual amino acids or whole peptide sections. Muteins are preferably produced which have mutations at the N-terminus and/or at the C-terminus such as e.g. deletions, insertions, substitutions or/and fusions with other e.g. human proteins.
The muteins according to the invention are preferably non-naturally occurring polypeptides and differ from allelic variations of the polypeptide to be mutated which occur naturally in other starting cells by at least one amino acid. Non-naturally occurring muteins particularly preferably differ by deletions, additions or/and insertions of individual amino acids or peptide sections from naturally occurring allelic variations.
The cell used in the process according to the invention is an arbitrary eukaryotic cell which has at least one endogenous copy of the target gene to be mutated. The cell is preferably a human cell, particularly preferably an immortalized human cell such as a HeLa cell, a Namalwa cell or a HT1080 cell.
It was surprisingly found that when starting cells are used which contain an increased number of chromosomes on which the target gene is located, cells can be produced by homologous recombination which produce an increased yield of mutated human proteins compared with cells which only contain two copies of the target gene. Examples of such starting cells are tumour cell lines with genetic rearrangements such as HeLaS3 (Puck et al., J.Exp.Med. 103 (1996), 273-284) and Namalwa (Nadkarni et al., Cancer 23 (1969), 64-79) which contain an increased number of copies of the chromosome 7.
An endogenous gene activation of the mutated target gene can be carried out to further improve the expression of the mutated polypeptide.
For this additional sequences can be introduced into the genome which positively influence the expression yield in which for example the endogenous expression control sequence of the target nucleic acid sequence is replaced at least partially by a heterologous expression control sequence. This heterologous expression control sequence can contain a heterologous promoter or/and enhancer, the heterologous expression control sequence preferably contains a viral promoter, in particular a CMV promoter. Replacement of the endogenous promoter not only enables the expression to be increased but allows synthesis of the mutein when a suitable promoter is used. The heterologous promoter can be a regulatable or constructive promoter. In addition this can be used to inactivate Cis elements that have a repressive effect on the endogenous promoter. This can also lead to an increase in yield.
The nucleic acid molecule introduced into the starting cell comprises at least one sequence section which allows an integration by homologous recombination in the locus of the target gene and is suitable for introducing the mutation in the coding region
Honold Konrad
Stern Anne
Fulbright & Jaworski LLP
Guzo David
Leffers, Jr. Gerald G.
Roche Diagnostics GmbH
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