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Title:  Regulated autocrine growth of mammalian cells

United States Patent:  6,797,515

Issued:  September 28, 2004

Inventors:  Hunt; Sybille Marie (Epping, AU); Gray; Peter Philip (Edgecliff, AU); Sleigh; Merilyn Joy (Neutral Bay, AU)

Assignee:  Unisearch Limited (Kensington, AU); Commonwealth Scientific and Industrial Research Organisation (Campbell, AU)

Appl. No.:  964338

Filed:  September 28, 2001

Abstract

Methods of production of desired recombinant proteins, polypeptides and peptides are disclosed which utilize mammalian host cells engineered for autonomous and regulated growth in low cost protein/serum-free culture media. Preferred host cells express insulin or an insulin-like growth factor, and/or transferrin and are engineered so that addition of an inducer (e.g., ZnCl2 +CdCl2) halts growth and simultaneously induces the expression of the desired recombinant protein, polypeptide or peptide.

Description of the Invention

This invention relates to methods of production of recombinant bio-pharmaceuticals and other desirable proteins, polypeptides and peptides using mammalian cell cultures. More particularly, the methods of the invention involve the use of specially bioengineered mammalian cell lines for the production of complex proteins in low cost media. These cell lines have the acquired ability for autonomous and regulated growth in cheap, reproducible, fully-defined protein-free medium, with the cells supplying all of their own growth factor requirements.

Mammalian cells have become the host cells of choice for the production of many of the new biopharmaceuticals, specifically those recombinant proteins requiring complex post-translational modifications and folding which bacterial cells cannot carry out. The production costs of these cells are far higher than with bacterial cells, with the fermentation cost accounting for an estimated 30% of the total production costs. The need to use a serum source in the growth and often production phases of the fermentation, is one of the contributing factors to the high costs of the fermentation.

Removing the need for added serum or growth factors in the fermentation medium will reduce media costs considerably. In addition, media free of added serum or growth factors should also be free of any viral or other contaminants. Further, the purity of the expressed protein will be maximized thus minimizing the steps in purification stages and maximising the recovery yield. This will allow production of complex processed proteins in mammalian cell hosts but with many of the cost advantages associated with bacterial cell hosts. Also, in future years, commercial and regulatory pressures may well dictate a requirement for such purity in the production of mammalian cell derived recombinant protein.

Furthermore, with the rising cost of serum due to more stringent testing, there is a great demand for the development of cell lines that can grow on fully defined media. Indeed, several methods have been developed for growth of cultured cells in serum-free medium (Barnes & Sato, 1980), including CHO-K1 (Mendiaz, E., et al., 1986). Media claiming to sustain growth in the absence of serum are available commercially. These are based on the fact that the serum requirement by cells in culture can be replaced by a combination of growth factors which is unique for each cell type. It is still unclear whether any of these media can sustain growth indefinitely. More recently, CHO-K1 cells have been used for high level expression of protein in serum-free medium (Ogata, M., et al., 1993), but these cells were maintained with serum during the growth phase and growth factors were added to the serum-free medium during the production phase. Although this system offers increased ease of product recovery, other factors cited above as making protein/serum-free growth media desirable, including cost, are not satisfied by this approach.

Australian patent specification No. 22120/88 (Genentech, Inc.) describes an attempt at engineering CHO cells to grow autonomously in protein-free medium. It is unclear whether these cells, which carry genes encoding insulin, transferrin and a desired protein product, are capable of continuously growing in the absence of serum. It is also unclear whether these cells are capable of expressing the desired protein product at satisfactory levels. Further, if cells such as CHO cells are engineered to produce growth factors in a constitutive fashion, then the mitogenic agents causing cell division would be present all the time and uncontrolled growth of the cells would result. Thus, in fermentation situations, it would be expected that cell numbers would increase in an uncontrolled fashion, causing in the case of attached cell cultures, multilayering of the cells and in the case of cells self-immobilised or growing as flocs, continuing division resulting in cells in the centre of the flocs becoming anaerobic and necrotic. In the case of suspension cultures, it would be expected that cell densities would increase as would toxic metabolic byproducts having a negative effect on the viability and metabolism of the cells and the culture.

Thus the present inventors have now identified and developed a method of producing recombinant proteins utilising mammalian cell lines engineered for autonomous and regulated growth in low cost, protein/serum-free media.

In a first aspect, the present invention provides a method for producing a desired recombinant protein, polypeptide or peptide comprising the step of:

culturing a mammalian host cell in culture medium, wherein said host cell includes:

(i) at least one introduced DNA sequence encoding the desired protein, polypeptide or peptide expressibly linked to a first promoter sequence, and

(ii) at least one introduced DNA sequence encoding a protein, polypeptide and/or peptide factor(s) required for growth of the host cell in said culture medium, expressibly linked to a second promoter sequence, said second promoter sequence being inducible and/or regulated by a transcription regulatory sequence(s).

The invention thereby enables the use of low cost, protein/serum-free medium by utilising a host cell which is able to produce the protein, polypeptide and/or peptide growth factor(s) required for its growth in such medium. The culture medium used in the method of the invention is, therefore, preferably serum-free or otherwise free of protein, polypeptide and/or peptide growth factor(s) necessary for the growth of the particular host cell type. However, methods wherein the culture medium includes one or more of the required growth factor(s) and the host cell itself expresses one or more of the same and/or other required growth factor(s), is also to be regarded as falling within the scope of the invention.

By expressibly linking the DNA sequence(s) encoding the protein, polypeptide and/or peptide factor(s) required for growth of the host cell to an inducible or regulated promoter, the expression of the protein, polypeptide and/or peptide growth factor(s) can be controllably regulated so that production of the factor(s) may be limited only to the stage of culturing where growth is required. As indicated, controllable regulation may be achieved by using an inducible promoter sequence (e.g. the Metallothionein IIA promoter) or by including a transcription-regulatory sequence (e.g. a repressor binding region such as from the lac repressor/operator system as modified for mammals: Hu and Davidson, 1987, and Kozak, 1986). Such transcription regulatory sequence(s) may be located at any location where it can exert a regulatory effect on expression from the second promoter. For example, the transcription regulatory sequence(s) may be located between the TATAA (SEQ ID NO:4) box and the transcription start site or, alternatively, between the transcription start site and the AUG start codon.

Where regulation of growth factor(s) production is through the use of an inducible promoter, the method of culturing may comprise a first stage of culturing in the presence of an inducer to a desired cell confluence and a second stage of culturing in the absence of an inducer. This may be achieved by replacing the first stage medium with medium without inducer. Where regulation of growth factor(s) production is through the use of a repressor binding region, the method of culturing may comprise a first stage of culturing to a desired cell confluence and a second stage of culturing in the presence of a repressor.

The first promoter, which is expressibly linked to the introduced DNA sequence encoding the desired protein, polypeptide or peptide, may be a constitutive promoter (e.g. CMV and SV40 promoters) or an inducible promoter (e.g. a metallothionein IIA promoter).

The mammalian host cell may be any of those commonly used in the art for expressing recombinant proteins or peptides. For example, the host cell may be a Chinese Hamster Ovary (CHO) cell such as CHO-K1.

The introduced DNA sequence(s) may be present on plasmids or otherwise integrated into the host cell chromosomes (e.g. by homologous recombination).

The DNA sequence(s) encoding the protein, polypeptide and/or peptide factor(s) required for growth of the host cell, may be selected from DNA sequences encoding insulin, modified insulins (e.g. to improve stability --see, for example, Brems, D. N, et al., 1992) insulin-like growth factors (e.g. IGF-1), transferrin, platelet derived growth factor (PDGF), cytokines, mitogenic proteases such as Trypsin, Thrombin and Cathepsin I, other growth factors and mixtures thereof. Where the host cell is CHO it is preferable that the host cell includes DNA sequences encoding insulin or insulin-like growth factors, and transferrin.

In one preferred embodiment of the invention, the host cell includes a DNA sequence(s) encoding the protein, polypeptide and/or peptide growth factor(s), the expression of which is regulated by a repressor binding region, and further includes a DNA sequence encoding the repressor, the expression of which is regulated by an inducible promoter sequence. The inducer may therefore be added or applied to the host cell culture, thereby causing the expression of repressor and subsequent downregulation of growth factor(s) production. In such an embodiment, it is also especially preferred to control the expression of the desired recombinant protein or peptide by the use of an inducible promoter. If the same inducible promoter is used as that controlling the expression of the repressor, then adding or applying inducer will cause the downregulation of growth factor(s) production and simultaneous expression of the desired protein or peptide. This enables an efficient method, which does not require changing the medium, wherein the host cell culture grows with minimal protein production, and then cell growth is minimised whilst protein production occurs. The effective separation of the growth and protein production phases may also be desirable if special ingredients (e.g. different sugars, etc) were to be preferentially incorporated into the product.

Alternatively, the use of different inducible promoters may allow for fine control of relative expression levels.

In a second aspect, the invention provides a host cell for use in the method according to the first aspect, wherein the host cell includes:

(i) at least one introduced DNA sequence encoding a desired protein, polypeptide or peptide expressibly linked to a first promoter sequence, and

(ii) at least one introduced DNA sequence encoding a protein, polypeptide and/or peptide factor(s) required for growth of the host cell in said culture medium, expressibly linked to a second promoter sequence, said second promoter sequence being inducible and/or regulated by a transcription-regulatory sequence(s).

In a most preferred embodiment, the host cell includes:

(i) at least one introduced DNA sequence encoding a desired protein, polypeptide or peptide expressibly linked to a first, inducible promoter sequence,

(ii) at least one introduced DNA sequence encoding a protein, polypeptide and/or peptide factor(s) required for growth of the host cell, expressibly linked to a promoter sequence, the expression of which is regulated by a repressor binding region, and

(iii) at least one introduced DNA sequence encoding a repressor molecule which binds to the repressor binding region, expressibly linked to a second, inducible promoter sequence,

wherein the first and second, inducible promoter sequence(s) may be the same or different.

Host cells capable of autonomous and regulated growth in low cost protein/serum-free media may also be useful in other applications, e.g. in the production of viruses.

Thus, in a third aspect, the invention provides a method for the regulated growth of a mammalian host cell in a culture medium, comprising the step of:

culturing said mammalian host cell in said culture medium, wherein said host cell includes at least one introduced DNA sequence encoding a protein, polypeptide and/or peptide factor(s) required for growth of the host cell in said culture medium, expressibly linked to a promoter sequence, said promoter sequence being inducible and/or regulated by a transcription regulatory sequence(s).

In a preferred embodiment of this third aspect, the invention provides a method for the regulated growth of a mammalian host cell in a culture medium, comprising the step of:

culturing said mammalian host cell in said culture medium, wherein said host cell includes:

(i) at least one introduced DNA sequence encoding a protein, polypeptide and/or peptide factor(s) required for growth of the host cell in said culture medium expressibly linked to a promoter sequence, the expression of which is regulated by a repressor binding region; and

(ii) at least one DNA sequence encoding a repressor molecule which binds to the repressor binding region, expressibly linked to an inducible promoter sequence.

In a fourth aspect, the invention provides a host cell including at least one introduced DNA sequence encoding a protein, polypeptide and/or peptide factor(s) required for growth of the host cell in a protein/serum-free culture medium, expressibly linked to a promoter sequence, said promoter sequence being inducible and/or regulated by a transcription regulatory sequence(s).

In a preferred embodiment of this fourth aspect, the invention provides a host cell including:

(i) at least one introduced DNA sequence encoding a protein, polypeptide and/or peptide required for growth of the host cell in a protein/serum-free culture medium, expressibly linked to a promoter sequence, the expression of which is regulated by a repressor binding region; and

(ii) at least one introduced DNA sequence encoding a repressor molecule which binds to the repressor binding region, expressibly linked to an inducible promoter sequence.

Conveniently laboratories would be supplied with samples (e.g. frozen stock) of a cell line wherein the cells include DNA sequences encoding their growth factor requirements for protein/serum-free media. The samples may then be transformed to include DNA sequences encoding the desirable protein(s), polypeptide(s) and/or peptide(s) (or infected with virus), and then cultivated in a defined protein/serum-free medium to satisfactory numbers and with satisfactory growth rates without any added protein.

Claim 1 of 19 Claims

What is claimed is:

1. A method for the regulated growth of a mammalian host cell in a culture medium, comprising the step of:

Culturing said mammalian host cell in said culture medium, wherein said host cell includes:

(i) at least one introduced DNA sequence encoding a protein, polypeptide and/or peptide factor(s) required for growth of the host cell in said culture medium operably linked to a promoter sequence, the expression of which is regulated by a repressor binding region; and

(ii) at least one introduced DNA sequence encoding a repressor molecule which binds to the repressor binding region, operably linked to an inducible promoter sequence;

wherein cell growth occurs in the absence of an inducer of said inducible promoter and cell growth is inhibited in the presence of an inducer of said inducible promoter.



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