Mammalian expression vector with a highly efficient secretory signal sequence

Mammalian protein expression systems are the best choice for the production of eukaryotic proteins, especially when correct folding and post-translational modification is required. They produce eukaryotic recombinant proteins in the most natural state, with native tertiary structure, physiochemical characteristics and bioactivities. They have been successfully applied in the biopharmaceutical production of cytokines, monoclonal antibodies, growth factors and so on. Email:marketing@medicilon.com.cn web:www.medicilon.com

The present invention relates to a mammalian cell based expression and secretion system and the expression and secretion of recombinant proteins by using secretory signal peptides. The present invention also relates to an expression cassette useful for the secretion of a heterologous gene from a mammalian cell, in particular a CHO cell. The present invention is also directed to a method of secreting a heterologous protein from mammalian cells such as CHO cells.

The present invention relates to mammalian cell based expression systems and the expression and secretion of recombinant proteins by using secretory signal peptides. The present invention also relates to an expression cassette useful for the secretion of a heterologous gene from a mammalian cell, in particular a CHO cell. The present invention is also directed to methods of secreting a heterologous protein from mammalian cells such as CHO cells and methods for the production of secreted recombinant proteins in mammalian host cells.

Recombinant polypeptides for medical, research and veterinary applications such as antibodies are produced using a wide variety of genetically engineered organisms that include prokaryotic and eukaryotic cells. However, a lot of these proteins are glycoproteins requiring post-translational modifications. Thus, prokaryotic host cells such as bacterial cells are not suitable. For this reason other protein expression systems were developed using the cells of higher eukaryotes, e.g. insect cells or mammalian cells. Viral expression systems can produce recombinant proteins both in insects and mouse cell lines but suffer from several serious drawbacks, in particular that purification of recombinant proteins from virus infected systems is very problematic.

A major problem in biotechnology exists in the production and recovery of recombinant polypeptides that are not readily secreted such as intracellular proteins or protein subunits, from genetically engineered organisms. Often these intracellular proteins or protein subunits can be expressed at only moderate levels inside a cell and their purification must first include steps to lyse the cells, followed by several procedures to isolate the desired polypeptides from the other intracellular proteins.

One approach to solve these problems is to have recombinant proteins secreted into the periplasmic space or culture medium. Whenever possible, secretion is the preferred strategy since it permits easy and efficient purification from the extracellular medium. In addition, the secretory production of recombinant proteins has the advantage that proteolytic degradation may be avoided and that there is a better chance of correct protein folding. Successful protein secretion requires effective translocation of the protein across the endoplasmatic reticulum or plasma membrane. Proteins that are secreted from a cell through a cell membrane are generally produced within the cell in a precursor form, referred to as a "preprotein". This "preprotein" form includes an additional peptide sequence at the amino-terminus which is required to target the nascent peptide chain to the endoplasmatic reticulum to enable its entry into the secretory pathway. This additional peptide sequence is referred to as a signal sequence.

However, it is known that secretion frequently does not function to the degree desired, for example because the native signal sequence of the recombinant protein often does not operate well in the host cell. To date, each expression system needs specific tailoring to meet the requirements for each protein product to ensure correct folding, activity and desired yield. Although quite a lot of signal sequences have been identified which might be useful for the secretion of particular recombinant proteins, there is still a need in the art for additional signal sequences that can promote efficient secretion of recombinant proteins, in particular immunoglobulins, in mammalian host cells.

Thus, the technical problem underlying the present invention is to provide a method to efficiently secrete non-secretion competent polypeptides, in particular antibody chains from a eukaryotic host cell such as a Chinese Hamster Ovary (CHO) cell.

The present invention solves this technical problem by providing an expression cassette for the secretion of a heterologous protein from a mammalian host cell, in particular a CHO cell comprising a promoter, functionally linked to a DNA sequence encoding a signal peptide which is linked in frame to a DNA sequence encoding a heterologous protein, wherein the DNA sequence encoding the signal peptide is selected from the group consisting from SEQ ID No. 2, SEQ No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No.10 or a DNA sequence encoding an amino acid sequence depicted in SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7 or SEQ ID No. 9.

It was found by the inventors of the present invention that the signal sequences employed in the expression cassettes provide for a proper processing and an efficient secretion of operably linked polypeptide sequences in mammalian host cells. As an initial screen, nineteen different signal sequences have been tested that were derived from different secreted proteins of different species to the CHO cells routinely used for recombinant protein production whereby only 5 of them resulted in an improved secretion of the polypeptides tested. Thus, the experiments, demonstrated that it is generally unpredictable whether a signal sequence from one species would be functional in another species.

Two of the five signal sequences, namely V19 (SEQ ID No. 9) and Vl 7 (SEQ ID No. 7), resulted in cell lines with a particularly strong increase in mean antibody concentration over control cell lines.

On the basis of the amino acid sequence of signal peptide Vl 9 the following consensus amino acid sequence of a signal peptide was derived: MMRP [hydrophobic amino acid]nTSALA. On the basis of the amino acid sequence of signal peptide Vl 7 the following consensus amino acid sequence of a signal peptide was derived: MKT[hydrophobic amino acid]nCATVHC.

Thus, the present invention solves the technical problem also by providing a signal sequence with the general amino acid sequence MMRP [hydrophobic amino acid]nTSALA and also a signal sequence with the general amino acid sequence MKT[hydrophobic amino acid] nC AT VHC. Both signal sequences provide for an efficient secretion of operably linked polypeptide sequences, e.g. antibody chains, in mammalian host cells such as CHO cells. Preferably the hydrophobic amino acid in the central region is selected from the group consisting of alanine (Ala or A), isoleucine (He or I), leucine (Leu or L), phenylalanine (Phe or F), methionine (Met or M) and valine (VaI or V). Ala has a hydropathy index of 1.8. He has a hydropathy index of 4.5. Leu has a hydropathy index of 3.8. Phe has a hydropathy index of 2.8. Met has a hydropathy index of 1.9. VaI has a hydropathy index of 4.2. Preferably the central stretch of hydrophobic amino acids consists especially of Leu (L) with some occurrence of VaI, Ala, Phe and He. Preferably the central hydrophobic stretch has a length of 4 to 9 amino acid residues. Preferably the central hydrophobic region comprises 4 - 16 amino acid residues, particularly preferred 4-9 residues.

The present invention also relates to an expression cassette for the secretion of a heterologous protein from a mammalian host cell comprising a promoter, functionally linked to a DNA sequence encoding a signal peptide which is linked in frame to a DNA sequence encoding a heterologous protein, wherein the DNA sequence encoding the signal peptide is selected from a DNA sequence encoding the amino acid sequence MMRP [hydrophobic amino acid]nTSALA or a DNA sequence encoding the amino acid sequence MKT [hydrophobic amino acid]nCATVHC whereby n is an integer of 4 to 16 and the hydrophobic amino acid is selected from the group consisting of alanine, isoleucine, leucine, phenylalanine, methionine and valine.

In a particularly preferred embodiment the DNA sequence encoding the signal sequence is selected from SEQ ID No. 8 or SEQ ID No.10 or a DNA sequence encoding the amino acid sequence depicted in SEQ ID No. 7 or SEQ ID No. 9.

The present invention also relates to an expression cassette for the secretion of a heterologous protein from a mammalian host cell comprising a promoter, functionally linked to a DNA sequence encoding a signal peptide which is linked in frame to a DNA sequence encoding a heterologous protein, wherein the DNA sequence encoding the signal peptide is selected from the group consisting from SEQ ID No. 2, SEQ No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No.10 or a DNA sequence encoding an amino acid sequence depicted in SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7 or SEQ ID No. 9

In the context of the present invention an "expression cassette" is made up of one or more genes to be expressed and sequences controlling their expression such as a promoter/enhancer sequence, including any combination of cis-acting transcriptional control elements. The sequences controlling the expression of the gene, i.e. its transcription and the translation of the transcription product, are commonly referred to as regulatory unit. Most parts of the regulatory unit are located upstream of coding sequence of the heterologous gene and are operably linked thereto. The expression cassette may also contain a downstream 3' untranslated region comprising a polyadenylation site. The regulatory unit of the invention is either directly linked to the gene to be expressed, i.e. transcription unit, or is separated therefrom by intervening DNA such as for example by the 5 '-untranslated region of the heterologous gene. Preferably the expression cassette is flanked by one or more suitable restriction sites in order to enable the insertion of the expression cassette into a vector and/or its excision from a vector. Thus, the expression cassette according to the present invention can be used for the construction of an expression vector, in particular a mammalian expression vector.

In the context of the invention the terms "heterologous coding sequence", "heterologous gene sequence", "heterologous gene", "recombinant gene" or "gene of interest" are used interchangeably. These terms refer to a DNA sequence that codes for a recombinant or heterologous protein product that is sought to be expressed in the mammalian cell and harvested in high amount. The product of the gene can be a protein or polypeptide, but also a peptide. The heterologous gene sequence is naturally not present in the host cell and is derived from an organism of a different species.