Eukaryotic expression vectors comprising regulatory elements of the globin gene clusters

Abstract

The present invention pertains to the field of recombinant protein production. Novel expression cassettes comprising elements of the human globin gene clusters are provided which show enhanced expression rates of proteins or polypeptides of interest.

Claims

1. A method for recombinantly producing a polypeptide of interest, comprising the steps of (a) providing a host cell which comprises an expression cassette comprising, functionally linked to each other, (i) a locus control region comprising at least a functional part of the locus control region of the human -globin gene cluster or the human -globin gene cluster; (ii) a promoter region comprising at least a functional part of the promoter of the human .sup.A globin gene or a homologue thereof; and (iii) a coding region comprising a nucleic acid sequence encoding the polypeptide of interest and a nucleic acid sequence coding for a signal peptide for secretory expression; (b) culturing the host cell under conditions at which the host cell expresses and secretes the polypeptide of interest; and (c) isolating the polypeptide of interest.

2. The method according to claim 1, further comprising after step (c) the step of (d) formulating the polypeptide of interest as a pharmaceutical composition.

3. The method according to claim 1, wherein the locus control region of the expression cassette comprises the core element of the DNAse I hypersensitivity site 2 (HS2) of the human -globin gene cluster.

4. The method according to claim 3, wherein the locus control region of the expression cassette comprises the M1-core-M2 element of the DNAse I hypersensitivity site 2 (HS2) of the human -globin gene cluster.

5. The method according to claim 4, wherein the locus control region of the expression cassette comprises at least a part of the hypersensitivity site 2 (HS2) of the human -globin gene cluster which comprises the nucleic acid sequence of position 741 to 1109 of SEQ ID NO: 1.

6. The method according to claim 3, wherein the locus control region of the expression cassette further comprises the hypersensitivity site 3 (HS3) or a part thereof of the human -globin gene cluster, and/or the hypersensitivity site 4 (HS4) or a part thereof of the human -globin gene cluster.

7. The method according to claim 1, wherein the locus control region of the expression cassette comprises the hypersensitivity site 40 (HS40) or a part thereof of the human -globin gene cluster.

8. The method according to claim 1, wherein the promoter region of the expression cassette comprises nucleotides 384 to +36, with respect to the transcription initiation site, of the human .sup.A globin gene.

9. The method according to claim 1, wherein the expression cassette further comprises an enhancer region comprising at least a functional part of the 3 enhancer of the human .sup.A globin gene, functionally linked to the other elements of the expression cassette.

10. The method according to claim 1, wherein the polypeptide of interest is a glycoprotein or a part thereof.

11. The method according to claim 1, wherein the host cell is a white blood cell, blood precursor cell or leukemia cell, or a cell derived therefrom.

12. An expression cassette comprising, functionally linked to each other, (i) a locus control region comprising at least a functional part of the locus control region of the human -globin gene cluster or the human -globin gene cluster; (ii) a promoter region comprising at least a functional part of the promoter of the human .sup.A globin gene; (iii) a coding region comprising a nucleic acid sequence coding for a signal peptide for secretory expression; (iv) a transcription terminator region; and (v) an enhancer region comprising at least a functional part of the 3 enhancer of the human .sup.A globin gene; wherein the expression cassette does not comprise a nucleic acid sequence coding for the entire human .sup.A globin.

13. The expression cassette according to claim 12, wherein the locus control region comprises the core element of the DNAse I hypersensitivity site 2 (HS2) of the human -globin gene cluster.

14. The expression cassette according to claim 13, wherein the locus control region comprises the M1-core-M2 element of the DNAse I hypersensitivity site 2 (HS2) of the human -globin gene cluster.

15. The expression cassette according to claim 14, wherein the locus control region comprises at least a part of the hypersensitivity site 2 (HS2) of the human -globin gene cluster which comprises the nucleic acid sequence of position 741 to 1109 of SEQ ID NO: 1.

16. The expression cassette according to claim 13, wherein the locus control region further comprises the hypersensitivity site 3 (HS3) or a part thereof of the human -globin gene cluster, and/or the hypersensitivity site 4 (HS4) or a part thereof of the human -globin gene cluster.

17. The expression cassette according to claim 12, wherein the locus control region comprises the hypersensitivity site 40 (HS40) or a part thereof of the human -globin gene cluster.

18. The expression cassette according to claim 12, wherein the promoter region comprises nucleotides 384 to +36, with respect to the transcription initiation site, of the human .sup.A globin gene.

19. The expression cassette according to claim 12, wherein the enhancer region comprises the nucleic acid sequence of position 2136 to 2881 of SEQ ID NO: 1.

20. The expression cassette according to claim 12, further comprising a cloning site which comprises at least one recognition sequence of a restriction enzyme.

21. The expression cassette according to claim 12, wherein the coding region comprises a nucleic acid sequence coding for a polypeptide of interest.

22. A host cell comprising the expression cassette according to claim 12.

23. The host cell according to claim 22, being a white blood cell, blood precursor cell or leukemia cell, or a cell derived therefrom.

Description

FIGURES

(1) FIG. 1 shows the structure of the human globin gene clusters including the locus control region (LCR) with the different DNase hypersensitivity sites (HS) and the different globin genes. A: human -globin gene cluster on chromosome 11; B: human -globin gene cluster on chromosome 16.

(2) FIG. 2 shows the elements of exemplary expression cassettes as used in the vectors pHBG1A-E. HS: DNase hypersensitivity site; .sup.A-Prom: promoter of the .sup.A globin gene; CS: coding sequence/cloning site; pA: polyadenylation signal of the .sup.A globin gene; .sup.A-ENh: 3 enhancer of the .sup.A globin gene.

(3) FIG. 3 shows the factor VII protein yield obtained after transient transfection of different vectors comprising the coding sequence of factor VII. Vectors comprising the expression cassettes shown in FIG. 2 with the coding sequence of factor VII introduced into the cloning site and a gene encoding DHFR as amplifiable selectable marker were transiently transfected into NM-H9D8 cells. Total yield of factor VII was determined after cultivation. pEFdhfrmut(): control vector with factor VII coding sequence. The results of three independent experiments are shown.

(4) FIG. 4 shows a comparison of stable transfection of a vector according to the invention and a control vector encoding an antibody. NM-H9D8-E6Q12 cells were stably transfected with the control vector pEF or the vector pHB according to the invention. Both vectors comprise a coding sequence for an antibody and a gene encoding DHFR as amplifiable selectable marker. For amplification of the vector in the cells, the selection pressure, i.e. the concentration of the selection agent methotrexate in the culture medium, was stepwise increased. The graph shows the maximum selection pressure which was possible for the respective vector after a given cultivation time. A higher possible selection pressure (methotrexate concentration) indicates a stronger amplification of the vector in the transfected cells, which should result in a higher production of the protein of interest.

(5) FIG. 5 shows the pool productivity of the stably transfected cells of FIG. 4. The antibody production in picogram per cell per day is shown for the different selection pressures for the vector according to the invention and the control vector.

(6) FIG. 6 shows the increase in productivity of the stably transfected cells of FIG. 4 by amplification of the vector due to the selection pressure. The antibody production in picogram per cell per day is shown for the different selection pressures for the starting cell pool, for the cell pool after amplification and for single cell clones after amplification. A: control vector pEF; B: vector pHB according to the invention.

EXAMPLES

Example 1: Construction of Vectors Comprising the Human A Globin Promoter and Elements of the Locus Control Regions of the Human Globin Gene Clusters

(7) For construction of the globin vectors, the enhancer and promoter regions of a parent vector (e.g. pEF having a puromycin or neomycin resistance gene or a dhfr gene as selectable marker) were removed. The human .sup.A globin promoter, polyadenylation signal and 3 enhancer regions and different constructs of the locus control regions of the human globin gene clusters were synthesized and cloned into the vector at the appropriate sites. FIG. 2 shows exemplary constructs of the expression cassettes of the constructed vectors. Then a nucleic acid sequence coding for a polypeptide of interest was introduced into the cloning site.

Example 2: Transient Transfection of the Globin Vectors

(8) Transient transfection was performed with Lipofectamine LTX and Plus Reagent according to the manufacturer's instructions. Briefly, 210.sup.5 cells were seeded in 6-well plates during their logarithmic growth phase. Plasmid DNA was diluted in Opti-MEM I Reduced Serum Medium and PlusTm-Reagent. After an incubation time (15 min), Lipofectamine LTX was added to the solution. After further incubation (30 min), the mixture was dripped in the cell suspension. Expression was analyzed after 72 hours by ELISA. Higher protein titers were achieved by the vectors pHBG1Cdhfr, pHBG1Ddhfr and pHBG1Edhfr in comparison to the vector pEFdhfrmut() (FIG. 3).

Example 3: Stable Transfection of the Globin Vectors

(9) Transfection of the cell line NM-H9D8 was performed by nucleofection (Nucleofector Technology, Amaxa) using plasmid DNA of the two expression plasmids coding for the antibody heavy and light chain, respectively (both linearized) according to the manufacturer's instructions. For selection and amplification of antibody producing pools, methotrexate and puromycin were added at increasing concentrations and pools were screened for secretion of active antibody molecules.

(10) Pools transfected with pHB plasmids could be amplified in a shorter time period (FIG. 4) and led to higher protein levels (FIG. 5) which could be confirmed for resulting single cell clones, respectively (FIG. 6).

Identification of the Deposited Biological Material

(11) The cell lines DSM ACC 2606 and DSM ACC 2605 were deposited at the DSMZDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig (DE) by Nemod Biotherapeutics GmbH & Co. KG, Robert-Rssle-Str. 10, 13125 Berlin (DE) on Aug. 14, 2003. Glycotope is entitled to refer to these biological materials since they were in the meantime assigned from Nemod Biotherapeutics GmbH & Co. KG to Glycotope GmbH.

(12) The cell lines DSM ACC 2806, DSM ACC 2807, DSM ACC 2856, DSM ACC 2858 and DSM ACC 3078 were deposited at the DSMZDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrae 7B, 38124 Braunschweig (DE) by Glycotope GmbH, Robert-Rssle-Str. 10, 13125 Berlin (DE) on the dates indicated in the following table.

(13) TABLE-US-00001 Name of the Accession Date of Cell Line Number Depositor Deposition NM-F9 DSM ACC 2606 Nemod Aug. 14, 2003 Biotherapeutics NM-D4 DSM ACC 2605 Nemod Aug. 14, 2003 Biotherapeutics NM-H9D8 DSM ACC 2806 Glycotope GmbH Sep. 15, 2006 NM-H9D8-E6 DSM ACC 2807 Glycotope GmbH Oct. 5, 2006 NM-H9D8- DSM ACC 2856 Glycotope GmbH Aug. 8, 2007 E6Q12 GT-2x DSM ACC 2858 Glycotope GmbH Sep. 7, 2007 GT-5s DSM ACC 3078 Glycotope GmbH Jul. 28, 2010