MXPA06005307A - Plasmid system for multigene expression - Google Patents

Abstract

The present invention provides a plasmid system which facilitates the construction of a single amplifiable plasmid that, having the potential to accommodate may independent expression cassettes, has the ability to express multi-subunit complex proteinssuch as antibodies and receptors.

Description

PLASMING SYSTEM FOR THE EXPRESSION OF MULTIPLE GENES This application claims the benefit of the provisional patent application of E.U.A. No. 60 / 519,230, filed on November 12, 2003, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION The present invention relates to a plasmid system that facilitates the construction of an individual amplifiable expression plasmid, for multiple subunit proteins.

BACKGROUND OF THE INVENTION The development of any therapeutics with proteins based on mammalian cells requires an efficient expression system. Ideally, if a multiple subunit protein (e.g., an antibody) is to be produced, each polypeptide should be expressed from a single plasmid. The construction of expression vectors containing multiple genes, using commercially available expression plasmids, is problematic. Typically, multiple cloning sites (MCS), of the commonly available expression plasmids, are unsuitable for the insertion of multiple expression cassettes. The multiple cloning sites of the commonly available expression plasmids contain relatively few restriction sites. Ideally, an expression plasmid for the expression of multiple polypeptides would contain a large multiple cloning site containing many common and rare restriction sites. The present invention provides, among other things, an ideal generic plasmid expression system that can help to maintain uniformity in the construction of vectors, decrease the variability in downstream processing, simultaneously facilitate the realization of therapeutic projects with multiple proteins, and Significantly reduce the cycle time. The present invention includes said generic plasmid platform for expression in mammals, and its use for the production of various polypeptides. The platform is quite flexible to be used for the expression of simple proteins, such as interferon, as well as large complex proteins of multiple subunits, such as antibodies.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a system of plasmids comprising in separate containers: (a) a first universal transfer vector comprising the following first multiple cloning site: Bss Hll, Pme I, Sna B1, Hin dlll, Asp 718, Kpn I, Pae R71, Xho I, Sal I, Acc I, Hinc II, Cia I, Eco RV, Eco Rl, Pst i, Eco O109I, Eco O109I, Apa I, Xma I, Bsp El, Bam H1, Dsa I, Eag I , Ecl XI, Not I, Sac li, Xma III, Xba I, Sac I, Mlu I, Bel I, Bsr Gl and Bss Hil; (b) a second universal transfer vector comprising the following second multiple cloning site: Bss Hll, Sgr Al, Xma I, Rsr II, Spe I, Sna B1, Hin dlll, Asp 718, Kpn I, Pae R71; Xho I, Sal I, Acc I, Hinc II, Cia I, Eco RV, Eco Rl, Pst I, Eco O109I, Eco O109I, Apa I, Xma I, Bsp El, Bam H1, Dsa I, Eag I, Ecl XI , Not I, Sac II, Xma III, Xba I, Sac I, Nde I, Msc I, Nru I, Pac I and Bss Hll; and (c) an amplifiable vector comprising the following third multiple cloning site: Sgr Al, Srf I, Xma I, Spe I, Sac II, Rsr II, Pac I, Nru I, Not I, Nde I, Msc I, Mlu I, Kpn I, Fse 1, Bss Hll, Bsr Gl, Bsp El, Bel I, Bbv C1, Pme I, Bss Hll, Ase I and Xba I. In one embodiment of the invention, the plasmid system comprises: first universal transfer vector comprising the plasmid map of Figure 2, a second universal transfer vector comprising the plasmid map of Figure 1, and an amplifiable vector comprising the plasmid map of Figure 3. In another embodiment, the multiple cloning site of the first universal transfer vector comprises the nucleotide sequence set forth in SEQ ID NO: 11; the multiple cloning site of the second universal transfer vector comprises the nucleotide sequence set forth in SEQ ID NO: 12; and the multiple cloning site of the amplifiable vector comprises the nucleotide sequence set forth in SEQ ID NO: 10. In one embodiment of the invention, any of the universal transfer vectors or the amplifiable vector comprises a matrix binding region (MAR; for example, MAR of chicken lysozyme).

In another embodiment of the invention, the plasmid system comprises only the first and second universal transfer vectors (see above). In one embodiment of the invention, at least one of the plasmids comprises a promoter (eg, SR promoter, MMTV LTR, human cytomegalovirus immediate early promoter (hCMV) and localized murine cytomegalovirus (mCMV) immediate early promoter) towards the 5 'end of the multiple cloning site, or within it. Preferably, in this embodiment, the first universal transfer vector comprises the plasmid map of Figure 10; the second universal transfer vector comprises the plasmid map of Figure 11; and the amplifiable vector comprises e! plasmid map of Figure 9. In this embodiment, the first universal transfer vector can comprise the nucleotide sequence set forth in SEQ ID NO: 5; the second universal transfer vector comprises the nucleotide sequence set forth in SEQ ID NO: 4; and the amplifiable vector comprises the nucleotide sequence set forth in SEQ ID NO: 13. Another embodiment of the present invention includes the plasmid system, wherein at least one of the universal transfer vectors comprises a polyA / terminator addition site. located at the multiple cloning site, where the location of the polyA / terminator addition site is such, that a gene located at the multiple cloning site would be operably linked to the polyA / terminator addition site.

The vector amplifiable in the plasmid system of the invention may comprise a selectable marker for amplification, such as the DHFR gene. In one embodiment of the invention, the plasmid system of the present invention comprises in separate containers: (a) a first universal transfer vector comprising the nucleotide sequence set forth in SEQ ID NO: 2; (b) a second transfer vector. universal comprising the nucleotide sequence set forth in SEQ ID NO: 1; and (c) an amplifiable vector comprising the nucleotide sequence set forth in SEQ ID NO: 3. One embodiment of the invention includes a system of plasmids wherein the first or second universal transfer vector comprises a first series of one or more cassettes of expression, the other universal transfer vector comprises a second series of one or more expression cassettes, and the amplifiable vector comprises said first series and second series of expression cassettes; wherein the expression cassettes encode an immunoglobulin heavy chain and an immunoglobulin light chain (eg, anti-IGFR1, anti-IL10 or anti-IL5 immunoglobulin chains); for example, wherein (a) the first series of one or more expression cassettes comprises an anti-IL5 immunoglobulin heavy chain gene expression cassette, and the second series of one or more expression cassettes comprises an expression cassette of anti-IL5 immunoglobulin light chain gene; (b) the first series of one or more expression cassettes comprises an anti-IGFR1 immunoglobulin heavy chain gene expression cassette, and the second series of one or more expression cassettes comprises a light chain gene expression cassette of anti-IGFR1 immunoglobulin; (c) the first series of one or more expression cassettes comprises an expression cassette comprising a bicistronic gene expression cassette, whose bicistronic gene comprises an anti-IGFR1 immunoglobulin light chain gene and an alpha subunit gene of the receptor of IL2, wherein said genes are linked by an internal ribosome entry sequence (IRES), and the second series of one or more expression cassettes is an anti-IGFR1 immunoglobulin heavy chain gene expression cassette and a cassette of expression of hygromycin resistance gene (Hyg-b); or (d) the first series of one or more expression cassettes comprises an anti-IL10 immunoglobulin heavy chain gene expression cassette., and the second series of one or more expression cassettes comprises an anti-IL10 immunoglobulin light chain gene expression cassette and an hygromycin resistance gene expression cassette. In one embodiment of the invention, the amplifiable vector comprises a plasmid map as set forth in a selected figure of Figures 4 to 7. For example, the amplifiable vector may comprise a nucleotide sequence selected from SEQ ID NOs: 6-9 . In one embodiment of the present invention, the plasmid system includes the amplifiable vectors pinAlL10 / MAR (-); pAIL10V1 / pure / MAR (-); pAIGFRLCb2 / MAR (-) or pAIGFRLCb2V1 / pure / MAR (-). In one embodiment of the invention, the plasmids pinAIL10 / MAR (-); pAIL10V1 / pure / MAR (-); pAIGFRLCb2 / MAR (-) and pAIGFRLCb2V1 / pure / MAR (-), are characterized by figures 13 to 16, respectively. In another embodiment of the invention, the plasmids pinAIL10 / MAR (-); pAIL10V1 / pure / MAR (-); pAIGFRLCb2 / MAR (-) and pAIGFRI_Cb2V1 / pure / MAR (-), comprise a nucleotide sequence selected from SEQ ID NOs: 24-27. The present invention also provides a method for the expression of a protein comprising two or more types of polypeptide, comprising the steps of (a) introducing a series of one or more expression cassettes into a first universal transfer vector; (b) introducing one or more different expression cassettes into a second universal transfer vector; (c) moving the cassettes of the transfer vectors into an amplifiable vector; (d) cause the expression of said cassettes; and (e) optionally, isolating / purifying the polypeptide; wherein said vectors are provided in an equipment of the present invention. In one embodiment of the invention, the first universal transfer vector comprises the plasmid map of Figure 2 or Figure 10, or the nucleotide sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 5. In another embodiment, the second universal transfer vector comprises the plasmid map of Figure 1 or Figure 11, or the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 4. In another embodiment of the invention, the amplifiable vector comprises the plasmid map of Figure 3 or Figure 9, or the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 13. In one embodiment of the method, an anti-IGFR heavy chain or anti-IL10 heavy chain it is expressed in an amplifiable vector, comprising a MAR and the hygromycin resistance gene or the puromycin resistance gene, which is selected from pinAIL10 / MAR (-); pAIL10V1 / pure / MAR (-); pAIGFRLCb2 / MAR (-) and pAIGFRLCb2V1 / pure / MAR (-). In one embodiment of the invention, the plasmids pinAIL10 / MAR (-); pAIL10V1 / pure / MAR (-); pAIGFRLCb2 / MAR (-) and pAIGFRLCb2V1 / pure / MAR (-), are characterized by figures 13 to 16, respectively. In another embodiment of the invention, the plasmids pinAlLI 0 / MAR (-); pAIL10V1 / pure / MAR (-); pAIGFRLCb2 / MAR (-) and pAJGFRLCb2V1 / pure / MAR (-), comprise a nucleotide sequence selected from SEQ ID NOs: 24-27. In one embodiment of the method for the expression of a protein comprising two or more types of polypeptide, the expression cassettes encode an immunoglobulin light or heavy chain (eg, anti-IGFR1, anti-IL5 or anti-immunoglobulin chain). IL10); for example: (i) an expression cassette encodes an anti-IL5 immunoglobulin heavy chain, and the other expression cassette encodes an anti-IL5 immunoglobulin light chain; (ii) an expression cassette encoding an anti-IGFR1 immunoglobulin heavy chain (eg, SEQ ID NO: 17 or 21, or any polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18 or 22), and the other expression cassette encodes an anti-IGFR1 immunoglobulin light chain (eg, SEQ ID NO: 15 or 19, or any polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or 20); (iii) an expression cassette comprises a bicistronic gene encoding an anti-IGFR1 immunoglobulin light chain and an alpha subunit of the IL2 receptor, which are linked by an internal ribosome entry sequence (IRES), and the other expression cassette encodes an anti-IGFR1 and HYG-B immunoglobulin heavy chain; or (iv) an expression cassette encodes an anti-IL10 immunoglobulin heavy chain. and the other expression cassette encodes a light chain of anti-IL10 immunoglobulin and HYG-B. In one embodiment of the invention, the amplifiable vector comprises a plasmid map in a selected figure of Figures 4 to 7. The amplifiable vector can comprise a nucleotide sequence selected from SEQ ID NOs: 6-9. The scope of the present invention also encompasses any product obtained by any of the methods of the invention for the production of a polypeptide (e.g., any immunoglobulin chain, such as that of an anti-IGFR1, anti-IL5 or anti-IL10 antibody). In one embodiment of the method for the expression of a protein comprising two or more types of polypeptide, the expression is caused in a cell (e.g., a eukaryotic cell such as a CHO cell). The present invention also comprises a method for the production of an anti-lGFR1 antibody, comprising the steps of (a) introducing an expression cassette comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from SEQ ID NOs : 18 and 22, or an expression cassette comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 16 and 20, in a first universal transfer vector comprising the following first cloning site multiple: Bss Hll, Pme I, Sna B1, Hin dlll, Asp 718, Kpn I, Pae, R71, Xho I, Sal I, Acc I, Hinc II, Cia I, Eco RV, Eco Rl, Ps! I, Eco O109I, Eco O109I, Apa I, Xma I, Bsp El, Bam H1, Dsa I, Eag I, Ecl XI, Not I, Sac ll, Xma III, Xba I, Sac I, Mlu I, Bel I, Bsr Gl and Bss Hll (for example, pUHLS or PUHSRstopLS); (b) introducing the other expression cassette, not introduced into said first vector, into a second universal transfer vector comprising the following second multiple cloning site: Bss Hll, Sgr Al, Xma I, Rsr II, Spe I, Sna B1, Hin dlll, Asp 718, Kpn I, Pae R71; Xho I, Sal I, Acc I, Hinc II, Cia I, Eco RV, Eco Rl, Pst I, Eco O109I, Eco O109I, Apa I, Xma I, Bsp El, Bam H1, Dsa I, Eag I, Ecl XI , Not I, Sac II, Xma III, Xba I, Sac I, Nde I, Msc I, Nru I, Pac I and Bss Hll (for example, pULLS or PULSRstopLS); (c) optionally, moving the cassettes of the transfer vectors into an amplifiable vector comprising the following third multiple cloning site: Sgr Al, Srf l, Xma I, Spe I, Sac II, Rsr ll, Pac I, Nru I , Not I, Nde I, Msc I, Mlu I, Kpn I, Fse 1, Bss Hll, Bsr Gl, Bsp El, Bel I, Bbv C1, Pme I, Bss Hll, Ase I and Xba I (for example, pXBLS or pSRXBLS); (d) cause the expression of said cassettes; and (e) optionally isolating / purifying the antibody. The polynucleotide encoding a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 18 and 22, can comprise a nucleotide sequence selected from SEQ ID NOs: 17 and 21. The polynucleotide encoding a polypeptide comprising a sequence of amino acids selected from SEQ ID NOs: 16 and 20, can comprise a nucleotide sequence selected from SEQ ID NOs: 15 and 19. In one embodiment of the invention, the expression cassettes are operably linked to a human cytomegalovirus promoter (hCMV ). The scope of the present invention includes embodiments wherein the expression cassettes mentioned above, are linked to an immunoglobulin constant region such as that of either K or γ1 or γ2 or Δ3 or Δ4. The present invention also provides a kit comprising the plasmid system of the invention, and one or more components selected from: (i) sterile distilled water; (I) transforming reagents with calcium phosphate CaCI2 and 2X saline regulated in its pH with HEPES; (iii) transformation reagents with DEAE-dextran chloroquine e? f saline regulated in its pH with phosphate and saline regulated at its pH with phosphate; (iv) liposomes extruded with DOTAP / cholesterol; (v) competent transformation E. coli; (vi) Dulbecco / Vogt Modified Eagle minimum essential medium (DMEM); (vii) fetal calf serum; (viii) Luria broth medium; and (ix) paper instructions for the use of the plasmid system. One embodiment of the present invention includes a single-stranded or double-stranded polynucleotide (e.g., an initiating oligonucleotide), comprising a nucleotide sequence of SEQ ID NO: 10, 11 or 12. The present invention also includes a plasmid that comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-9.

- BRIEF DESCRIPTION OF THE FIGURES The scope of the present invention includes any plasmid or plasmid system containing a plasmid comprising a plasmid map substantially identical to any of the following plasmid maps: Figure 1. Plasmid map of the pULLS universal transfer vector. Amp: Start: 1955 End: 2812 Origin Col E1: Start: 1012 End: 1952 Multiple cloning site (MCS): Start: 620 End: 772 Origin f1 (+): Start: 3 End: 459.

Figure 2. Plasmid map of the universal transfer vector pUHLS. Amp: Start: 1949 End: 2806 MCS: Start: 620 End: 766 Origin f1 (+): Start: 3 End: 459 Origin Col E1: Start: 1006 End: 1946. Figure 3. Plasmid map of the amplifiable vector pXBLS. T antigen antigen (t Ag) of SV40: Start: 5431 End: 600 SV40 polyA signal: Start: 5184 End: 5432 MCS: Start: 5037 End: 5183 Ampicillin resistance (Amp): Start: 3965 End: 4828 pBR ORÍ: Start: 3207 End: 3207 Sequences of pBR322: Start: 3020 End: 5033 Sequences of pBR322: Start: 2811 End: 3019 Promoter of MMTV-LTR: Start: 1348 End: 2810 DHCP cDNA: Start: 601 End: 1347. Figure 4. Plasmid map of pAIL5V1. The heavy chain (VDJ-IgG4) and light chain (VDJ-IgK) anti-IL-5 antibody cassettes, driven by the CMV promoter, are inserted into the multiple cloning site of pXBLS, together with the cassette of Hygromycin B expression, directed by the TK promoter (TK / Hyg). Intrón de t Ag de SV40: Start: 12177 Finish: 600 Signal of poliA of SV40: Start: 11930 End: 12178 Promoter of CMV: Start: 11238 End: 11892 Starter / promoter site T7: Start: 11219 End: 11238 VDJ ( light chain anti-lL-5): Start: 10718 End: 11148 IG? (light chain anti-IL-5): Start: 10382 End: 10717 Beta globin polyA signal: Start: 10126 End: 10374 TK / Hyg: Start: 8161 End: 10033 Beta globin polyA signal: Start: 7877 End : 8115 IGG4-CH3 (heavy chain of anti-lL-5 antibody): Start: 7517 End: 7834 IGG4-CH2 (heavy chain of anti-IL-5 antibody): Start: 7087 End: 7419 IGG4-HINGE (heavy chain of anti-IL-5 antibody): Start: 6933 End: 6968 IGG4-CH1 (heavy chain of anti-IL-5 antibody): Start: 6247 End: 6540 VDJ (heavy chain of anti-IL-5 antibody): Start: 5813 End: 6247 T7 initiation site / promoter: Start: 5723 End: 5742 CMV Promoter: Start: 5069 End: 5723 APr (ampicillin resistance): Start: 3965 End: 4828 - PBR ORÍ: Start: 3207 End: 3207 Sequences of pBR322 : Start: 3020 End: 5033 Sequences of pBR322: Start: 2811 End: 3019 Promoter of MMTV-LTR: Start: 1348 End: 2810 DHFR cDNA: Start: 601 End: 1347. Figure 5. Plasmid map of pAIGFRV3. The heavy chain (VDJ-IgG4) and light chain (VDJ-IgK) anti-IGFR1 antibody cassettes, driven by the CMV promoter, are inserted into the multiple cloning site of pXBLS, together with the expression cassette of hygromycin B, directed by the TK promoter (TK / Hyg). The DHFR cDNA, together with its promoter (MMTV-LTR) for plasmid amplification, and the sequence encoding hygromycin B, together with its TK promoter, for selection in mammalian cells are shown. AP (R): Start: 3965 End: 4828 Non-genomic region of IgG1: Start: 7234 End: 8214 VDJ of IGFR1 of hybridoma 11 D8: Start: 8214 End: 8641 DHFR cDNA: Start: 601 End: 1347 Intron of t Ag of SV40: Start: 11603 End: 600 kappa chain of hu-antilGFR gene: Start: 9761 End: 10096 VDJ domain of hu-antilGFR gene for light chain: Start: 10097 End: 10477 Sequence of pBR322: Start: 2811 End: 3019 Sequence of pBR322: Start: 3020 End: 5033 TK-hygromycin: Start: 5053 End: 6925 Beta globin polyA signal: Start: 6971 End: 7209 Beta globin pA signal: Start: 9505 End: 9753 Signal of SV40 polyA: Start: 11356 End: 11604 MMTV-LTR: Start: 1348 End: 2810 CMV Promoter: Start: 10664 End: 11318 T7 Starter / Developer Site: Start: 8723 End: 8742 CMV Promoter: Start: 8742 End: 9396 T7 initiation / promoter site: Start: 10645 End: 10664 pBR ORIGIN: Start: 3207 End: 3207. Figure 6. Plasmid map of pAIG1 FR (-) IL2LS. This plasmid directs the expression of the anti-IGFR1 antibody and the membrane domain of the IL2 receptor. Three independent expression cassettes containing four genes including heavy and light chain anti-IGFR1, truncated IL2 receptor and hygromycin B, are incorporated into the multiple cloning site of pXBLS. Intron of t Ag of SV40: Start: 13066 End: 600 Signal of poliA of SV40: Start: 12819 End: 13067 CMV: Start: 12115 Finish: 12769 Site of initiation / promoter T7: Start: 12096 End: 12115 VDJ (light chain anti-IGFR1): Start: 11548 End: 11928 Kappa (Kap; light chain anti-IGFR1): Start: 11212 End: 11547 IRES: Start: 10621 End: 11195 IL-2R alpha: Start: 9787 End: 10615 Signal of beta globin poiiA (beta signal of pA globin): Start: 9505 End: 9753 CMV: Start: 8742 Finish: 9396 T7 initiation site / promoter: Start: 8723 End: 8742 VDJ (heavy chain anti-IGFR1 of hybridoma 11 D8): Start: 8214 End: 8641 lgG1 (heavy chain anti-IGFR1 hybridoma 11D8): Start: 7234 End: 8214 Beta globin polyA signal (pA beta globin signal): Start: 6971 End: 7209 TK-hygromycin: Start: 5053 End: 6925 Start: 3965 End: 4828 PBR ORÍ: Start: 3207 End: 3207 Sequences of pBR322: Start: 3020 End: 5033 Sequences of pBR322: Start: 2811 End: 3019 MMTV-LTR: Start: 1348 End: 2810 DHFR cDNA: Start: 601 End : 1347. Figure 7. Plasmid map of pAIL10V3. The plasmid directs the expression of anti-IL10. The heavy chain (VDJ-IgG4) and light chain (VDJ-IgK) anti-IL10 antibody cassettes, driven by the CMV promoter, are inserted into the multiple cloning site of pXBLS, together with the expression cassette of hygromycin B, directed by the TK promoter (TK / Hyg). The DHFR cDNA, together with its promoter (MMTV-LTR) for plasmid amplification, and the sequence encoding hygromycin B, together with its TK promoter, for selection in mammalian cells are shown. Intron of t Ag of SV40: Start: 11507 End: 600 Signal of poliA of SV40: Start: 11260 End: 11508 CMV: Start: 10568 End: 11222 Starter / developer site T7: Start: 10549 End: 10568 VDJ-lgK ( 12G8 light chain of rat anti-IL10 antibody): Start: 9739 End: 10468 Beta-globin polyA signal: Start: 9478 End: 9726 CMV Promoter: Start: 8715 End: 9369 T7 Starter / Promoter Site: Start: 8696 End: 8715 VDJ (heavy chain 12G8 of rat anti-IL-10 antibody): Start: 8214 End: 8644 Non-genomic region of lgG1 (12G8 heavy chain of rat anti-IL10 antibody): Start: 7234 End: 8214 Beta globin polyA signal: Start: 6971 End: 7209 TK promoter that directs the hygromycin gene (TK-hygromycin): Start 5053 End: 6925 APr: Start: 3965 End: 4828 PBR ORIGIN: Start: 3207 End: 3207 Sequences of pBR322: Start: 3020 End: 5033 Sequences of pBR322: Start: 2811 End: 3019 Promoter of MMTV-LTR: Start: 1348 End: 2810 DHCP cDNA: Start: 601 End : 1347. Figure 8. Plasmid map of pDSRG. This plasmid is deposited at the American Type Culture Collection (10801 University Boulevard; Manassas, Virginia 20110-2209), under catalog number 68233. The plasmid includes the SRa promoter, a strong SV40-based promoter, and the dihydrofolate reductase cDNA. (DHFR) for plasmid amplification in the presence of methotrexate in Chinese hamster ovary (CHO) dhfr (-) cells. Intron of t Ag of SV40: Start: 6371 End: 600 Signal of poliA of SV40: Start: 6124 End: 6372 Promoter SRa: Start: 5486 End: 6123 Sign of poliA of betá "globine: Start: 5038 End: 5298 APr: Start: 3965 End: 4828 Sequences of pBR322: Start: 3020 End: 5033 Sequences of pBR322: Start: 2811 End: 3019 Promoter of MMTV-LTR: Start: 1348 End: 2810 DHFR cDNA: Start: 601 End: 1347. Figure 9. Plasmid map of pSRXBLS pSRXBLS is the direct descendant of pDSRG that replaces its own multiple cloning site with a large multiple cloning site pSRXBLS is the progenitor plasmid of pXBLS SV Agtron of SV40: Start: 6065 End: 600 SV40 polyA signal: Start: 5818 End: 6066 SRa Promoter: Start: 5180 End: 5817 MCS: Start: 5038 End: 5179 Amp (R): Start: 3965 End: 4828 pBR ORIGIN: Start: 3207 End : 3207 Sequences of pBR322: Start: 3020 End: 5033 Sequences of pBR322: Start: 2811 End: 3019 Promoter of MMTV-LTR: Start: 1348 End: 2810 cDNA of DHFR: Start: 601 End: 1347. Figure 10. Plasmid map of pUHSRstopLS. pUHSRstopLS is the pUHLS-descending plasmid that has the SRa promoter and 249 base pairs of the chicken beta-globin terminator. This plasmid can only be used to express any gene of interest. Likewise, it can be used as a transfer vector to transfer a complete expression cassette from part of a protein complex with pXBLS, wherein all expression cassettes can be assembled into an individual plasmid. Amp: Start: 2975 Finish: 3832 Origin Col E1: Start: 2032 End: 2972 Promoter SRa with intron: Start: 955 End: 1764 Beta globin polyA signal: Start: 673 End: 911 Origin f1 (+): Start: 3 End: 459. Figure 11. Plasmid map of pULSRstopLS. pULSRstopLS is the pULLS-descending plasmid that has the SRa promoter and 249 base pairs of the chicken beta-globin terminator. This plasmid can only be used to express any gene of interest. Likewise, it can be used as a transfer vector to transfer a complete expression cassette from part of a protein complex with pXBLS, where all the expression cassettes can be assembled into a single plasmid. Amp: Start: 2981 End: 3838 Origin Col E1: Start: 2038 End: 2978 Beta globin polyA signal: Start: 1512 End: 1760 Promoter SRa: Start: 655: End: 1474 Origin f1 (+): Start: 3 End: 459. Figure 12? Plasmid map of pPAGOL This plasmid contains the SEA of chicken lysozyme of approximately 3kb Selexis (Geneva, Switzerland), flanked by Xbal and BamHL AP (R) sites (b / a-determinant Ap (r) gene): Start: 4165 End: 5022, Selexis Inc., MAR lys 5 ': Start: 1 End: 2960 P (LAC): Start: 3043 End: 3043 P (BLA) (b / a gene promoter): Start: 5057 End: 5057 Origin of ORI replication (ribonuclease digestion site H): Start: 3403 End: 3403.

Figure 13. Plasmid map of pinAIL10 / MAR (-). The figure describes the plasmid map, pinAIL10 / MAR (-), which has the MAR lysozyme MAR element juxtaposed to the heavy chain expression cassette of the anti-IL10 gene which contains the hygromycin resistance marker. AP (R): Start: 3695 End: 4828 MAR-lys (MAR-lys is the matrix binding region): Start: 5087 End: 8045 VDJ (anti-IL10 VDJ region (12G8)): Start: 8928 End: 9369 IgG1 (non-genomic region of IgG1): Start: 9374 End: 10354 DHFR cDNA: Start: 601 End: 1347 Intron of t Ag of SV40: Start: 14897 End: 600 VDJ-lgK (VDJ-lgK for light chain 12G8 (anti-IL10)): Start: 13026 End: 13755 pBR322: Start: 2811 End: 3019 pBR322: Start: 3020 End: 5033 TK-hygromycin: Start: 10663 Finish: 12672 Beta globin polyA signal: Start: 10379 End: 10617 beta globin pA signal: Start: 12765 End: 13013 SV40 polyA signal: Start: 14650 End: 14898 MMTV -LTR: Start: 1348 End: 2810 hCMV / intron (promoter of human CMV with hybrid intron): Start: 8077 End: 8918 hCMV / intron (promoter of human CMV and hybrid intron): Start: 13771 End: 14612 pBR ORÍ: Start: 3207 End: 3207. Figure 14. Plasmid map of pAIL10V1 / pure / MAR (-) . The figure describes the plasmid map, pAIL10 / pure / MAR (-), which has the chicken lysozyme MAR element juxtaposed to the heavy chain expression cassette of the anti-IL10 gene which contains the puromycin resistance marker instead of the hygromycin resistance marker. AP: Start: 3965 End: 4828 MAR-Lys (MAR-lys is the matrix binding region): Start: 5087 End: 8045 VDJ: Start (VDJ region of anti-IL10 (12G8)): Start: 8928 End: 9369 IgG1 (non-genomic region of lgG1): Start: 9374 End: 10354 PURE: Start: 11674 End: 12905 DHFR cDNA: Start: 601 End: 1347 Intron of t Ag of SV40: Start: 15070 End: 600 VDJ-lgK (VDJ-lgK for the light chain 12G8 (anti-IL-10)): Start: 13199 End: 13928 (complementary) pBR322: Start: 2811 End: 3019 pBR322: Start: 3020 End: 5033 Beta globin polyA signal: Start: 10379 End: 10617 Í SV40 polyA signal: Start: 10784 End: 10789 beta globin pA signal: Start: 12938 End: 13186 SV40 polyA signal: Start: 14823 End: 15071 MMTV-LTR: Start: 1348 End: 2810 hCMV / intron (human CMV promoter with hybrid intron): Start: 8077 End: 8918 HCMV-MIE: Start: 10902 End: 11660 HCMV / intron (human CMV promoter and hybrid intron): Start: 13944 End : 14785 (complementary) pBR ORÍ: Start: 3207 End: 3207. Figure 15. Plasmid map of pAIGFRLCb2 / MAR (-). The figure describes the plasmid map, which has the MAR lysozyme MAR element juxtaposed to the heavy chain expression cassette of the anti-IGFR1 gene which contains the hygromycin resistance marker. AP (R): Start: 3965 End: 4828 MAR-lys (MAR-lys is the matrix binding region): Start: 5087 End: 8045 VDJ (VDJ of IGFR1 of hybridoma 11 D8): Start: 8974 End: 9401 IgG (non-genomic region of IgG1): Start: 9401 End: 10381 DHFR cDNA: Start: 601 End: 1347 Intron of t Ag of SV40: Start: 14924 End: 600 Kappa (kappa chain): Start: 13063 End: 13386 VDJ (VDJ of IGFR1 (LCb, human germline sequence)): Start: 13387 End: 13764 pBR322: Start: 2811 End: 3019 pBR322: Start: 3020 End: 5033 TK-hygromycin: Start: 10690 End: 12699 Beta-globin polyA signal: Start: 10406 End: 10644 Beta-globin pA signal: Start: 12792 End: 13040 SV40 polyA signal: Start: 14677 End: 14925 MMTV-LTR: Start: 1348 End: 2810 hCMV / intron (human CMV promoter with hybrid intron): Start: 8077 End: 8918 hCMV / intron (human CMV promoter and hybrid intron): Start: 13786 End: 14627 pBR ORÍ: Start: 3207 End: 3207. Figure 16. Plasmid map of pAIGFRLCb2V1 / pure / MAR (-). The figure describes the plasmid map, which has the MAR element of chicken lysozyme juxtaposed to the expression cassette of heavy chain of the anti-lGFR1 gene containing the puromycin rtance marker in place of the hygromycin rtance marker. AP (R): Start: 3965 End: 4828 MAR-lys (MAR-lys is the matrix binding region): Start: 5087 End: 8045 VDJ (VDJ de-IGFR1 of hybridoma 11 D8): Start: 8974 End: 9401 lgG1 (non-genomic region of lgG1): Start: 9401 End: 10381 PURE (R): Start: 11701 End: 12932 DHFR cDNA: Start: 601 End: 1347 Intron of t Ag of SV40: Start: 15097 End: 600 Kappa (kappa chain): Start: 13236 End: 13559 VDJ (VDJ of IGFR1 (LCb, human germline sequence)): Start: 13560 End: 13937 pBR322: Start: 2811 End: 3019 pBR322: Start: 3020 End: 5033 Beta globin polyA signal: Start: 10406 End: 10644 SV40 polyA signal: Start: 10811 End: 10816 beta globin pA signal: Start: 12965 End: 13213 SV40 polyA signal: Start: 14850 End: 15098 MMTV-LTR: Start: 1348 End: 2810 hCMV / intron (human CMV promoter with hybrid intron): Start: 8077 End: 8918 HCMV-MIE: Start: 10929 End: 11687 hCMV / intron (promoter of human CMV and hybrid intron): Start: 13959 End: 14800 pBR ORIGIN: Start: 3207 End: 3207.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a system of plasmids useful for the expression of recombinant proteins in any cell, for example, in a mammalian cell, a bacterial cell, a yeast cell or an insect cell. The plasmid system is subject to any cell-based expression of a wide range of recombinant proteins, ranging from simple proteins such as interferon, to complex proteins such as antibodies. The system offers many common and rare restriction sites to accommodate a variety of expression cassettes. It also provides flexibility in the choice of various elements of an expression cassette, such as a promoter, enhancer and terminator, as well as a marker of antibiotic resistance. The plasmids can also be used with simple transfer vectors. The system offers potential for transient expression as well as stable expression. The pXBLS vector possesses the region encoding dihydrofolate reductase (DHFR) for selection and amplification of the plasmid in mammalian cells deficient in DHFR, eg, CHO DXB-11 and CHO DG44. In this way, the system can be used to isolate stable clones, using amplification and gene selection. The plasmid system includes two universal transfer plasmids, pUHLS and pULLS, which are useful for carrying out the expression of the parts of a complex protein such as an antibody. In this way, the system offers options for cotransfection with universal vectors, and individual transfection with pXBLS. The ability of the plasmid system to cause such segregated expression of several parts is advantageous, since it is sometimes necessary to make a deeper discernment in the expression of individual units of a multiple subunit protein to analyze the general expression of the complex protein. . The system can also be used to record the effect of directional variability, which results from the orientation of the multiple genes in the plasmid for the expression of multiple subunit proteins. In this way, a strategy can be reached in the placement of multiple expression cassettes for optimal expression of a complex protein. It has been shown that the plasmid system of the invention targets high levels of expression of multiple polypeptides including anti-IL5 antibody. anti-lGFR1 antibody, membrane domain of the IL2 receptor and anti-l10 antibody. Other proteins can also be expressed in the plasmid system of the invention, including interferon, fibrinogen, ion channels, bacterial porins (e.g., ompF), and the nicotinic acetylcholine receptor (nAChR). In one embodiment of the invention, the plasmid system comprises the light and heavy chain of the fully human monoclonal anti-IGFR1 antibody, 15H12 / 19D12, which may also be referred to as 15H12 or 19D12. Parts for the plasmid system can be provided separately or, conveniently, together as part of a team. The present invention includes any of the polynucleotides comprising or consisting of a nucleotide sequence set forth below in Table 1, individually or as part of a plasmid or kit system. The polynucleotides of the invention can be in any form, including circular, linear, double-stranded or single-stranded.

TABLE 1 Polynucleotides of the invention TABLE 1 (CONTINUED) Molecular Biology In accordance with the present invention, conventional techniques of recombinant DNA, microbiology and molecular biology can be used within the skill of the art. These techniques are explained in the literature. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, second edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (hereinafter, "Sambrook, et al., 1989"); DNA Cloning: A Practical Approach. volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Svnthesis (M. J. Gait ed., 1984); Nucleic Acid Hvbridization (B. D. Hames &S. J. Higgins eds. (1985)); Transcription And Traslation (B. D. Hames &S.J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide to Molecular Cloning (1984); F. M. Ausubel, ei al. (eds.), Current Protocols in Molecular Biology John Wiley & Sons, Inc. (1994). A "polynucleotide", "nucleic acid" or "nucleic acid molecule" includes the polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine, "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine; DNA molecules "), or any phosphoester analogue thereof, such as phosphorothioates and thioesters, in the form of a single chain, double-chain form, or otherwise. A "polynucleotide sequence", "nucleic acid sequence" or "nucleotide sequence", is a series of nucleotide bases (also referred to as "nucleotides") in a nucleic acid, such as DNA or RNA, and means any chain of two or more nucleotides. A "coding sequence" or a sequence "coding for" an expression product, such as RNA or peptide, is a sequence of nucleotides that, when expressed, results in the production of the product. The term "gene" means a DNA sequence that codes for, or corresponds to, a particular sequence of ribonucleotides or amino acids comprising one or more RNA molecules, proteins or complete enzymes, or part thereof, and may include or no regulatory DNA sequences, such as promoter sequences that determine, for example, the conditions under which the gene is expressed. Genes can be transcribed from DNA or RNA, which may or may not be translated into an amino acid sequence. As used herein, the term "oligonucleotide" refers to a nucleic acid, generally not more than about 300 nucleotides (eg, 30, 40, 50, 60, 70, 80, 90, 150, 175, 200, 250, 300), which can be hybridizable with a genomic DNA molecule, a cDNA molecule or a messenger RNA molecule encoding a gene, messenger RNA, cDNA, or other nucleic acid of interest. The oligonucleotides are usually single stranded, but can be double stranded. Oligonucleotides can be labeled, for example, by incorporation of 32 P-nucleotides, 3 H-nucleotides, 14 C-nucleotides, 35 S-nucleotides or nucleotides to which a tag, such as biotin, has been covalently conjugated. In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. In another embodiment, oligonucleotides (of which both can be labeled, or only one of them) can be used as PCR primers, for the cloning of the full length of the gene or a fragment thereof, or to detect the presence of nucleic acids . In general, oligonucleotides are prepared synthetically, preferably in a nucleic acid synthesizer. A "protein sequence", "peptide sequence" or "polypeptide sequence" or "amino acid sequence" refers to a series of two or more amino acids in a protein, peptide or polypeptide. The term "protein", "peptide" or "polypeptide", includes a continuous row of two or more amino acids. The term "isolated polynucleotide" or "isolated polypeptide", includes a polynucleotide (e.g., RNA or DNA molecule, or a mixed polymer), or a polypeptide, respectively, that is partially or completely separated from other components that are normally found in cells or in recombinant DNA expression systems or any other contaminant. These components include, but are not limited to, cell membranes, cell walls, ribosomes, polymerases, serum components and foreign genomic sequences. An isolated polynucleotide or polypeptide will preferably be an essentially homogeneous molecule composition, but may contain some heterogeneity. The term "DNA amplification by PCR", as used herein, includes the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixture of DNA sequences. For a description of the PCR, see Saiki, et al., Science (1988) 239: 487. Genes can be amplified, for example, in a plasmid in a cell. Cells harboring a plasmid containing an amplifiable selectable marker, but lacking an endogenous marker gene, such as DHFR, can be selected with increasing amounts of a selection agent, such as methotrexate (e.g., if the DHFR gene is in the plasmid). Typically, this procedure will increase the number of copies of the plasmid containing the selectable marker amplifiable in the cell. The term "host cell" includes any cell of any organism that has been selected, modified, transfected, transformed, developed or used or manipulated in any way, for the production of a substance by the cell, for example, expression or replication , by the cell, of a gene, a DNA or RNA or a protein. For example, a host cell can be a bacterium such as E. coli, or a eukaryotic cell such as a CHO cell. A "cassette" or an "expression cassette" refers to a DNA coding sequence or segment of DNA that codes for an expression product (e.g., peptide or RNA) that can be inserted into a vector at defined restriction sites . The DNA coding sequence can be operably linked to a promoter and / or a terminator and / or a polyA signal. The sequence of a nucleic acid can be determined by any method known in the art (eg, chemical sequencing or enzymatic sequencing). The "chemical sequencing" of DNA includes methods such as that of Maxam and Gilbert (Proc. Nati, Acad. Sci. USA (1977) 74: 560), in which the DNA is randomly digested using specific base reactions. The term "enzymatic sequencing" of DNA includes methods such as that of Sanger (Sanger, et al., Proc. Nati, Acad. Sci. USA (1977) 74: 5463). The present invention includes nucleic acids of the invention flanked by natural regulatory sequences (expression control), which can be associated with heterologous sequences, including promoters, internal ribosome entry sites (I RES) and other sequences of binding sites to ribosomes, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5 'and 3' non-coding regions, and the like. "Internal ribosome entry sites", "IRES" are commonly known in the art. Internal ribosome entry sites have been identified in several genes including elF4G (Johannes et al., RNA 4: 1500-1513 (1998)), DAP5 (Henis-Korenblit et al., Molecular and Cellular Biology 20: 496-506). (2000)); c-Myc (Stoneley et al., Molecular and Cellular Biology 20: 1162-1 169 (2000)), repression factor of NF-γ-b (Oumard et al., Molecular and Cellular Biology 20: 2755-2759 (2000 )), VEGF (Huez et al., Molecular and Cellular Biology 18: 6178-6190 (1998)), FGF-2 (Creancier et al., Journal of Cell Biology 150: 275-281 (2000)), PDGF-B (Bemstein et al., Journal of Biological Chemistry 272: 9356-9362 (1997)), inhibitor of X-linked apcptosis (XIAP) (Holcik et al., Oncogene 19: 4174-4177 (2000)), Apaf-1 ( Coldwell et al., Oncogene 19: 899-905 (2000)) and BiP (Macejak et al., Nature 353: 90-94 (1991)). In general, a "promoter" or "promoter sequence" is a DNA regulatory region capable of displaying binding to an RNA polymerase in a cell (e.g., directly or through other proteins or substances bound to the promoter), and start the transcription of a coding sequence. A promoter sequence is, in general, limited at its 3 'end by the transcription initiation site, and extends to the 5' end (5 'address), to include the minimum number of bases or elements necessary to initiate the transcription at any level. Within the promoter sequence can be found a transcription initiation site (conveniently defined, for example, by mapping with S1 nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. The promoter may be operably associated with other expression control sequences, including enhancer and repressor sequences, or with a nucleic acid of the invention. A coding sequence is "under the control of", "functionally associated with", "operably linked to" or "operably associated with" control sequences of transcription and translation in a cell, when the sequences direct or regulate the expression of the sequence. For example, a promoter operably linked to a gene will direct transcription mediated by RNA polymerase, from the coding sequence to RNA, preferably messenger RNA, which can then be spliced RNA (if it contains introns) and, optionally, translated into a protein encoded by the coding sequence. A polyA / terminator signal operably linked to a gene, terminates transcription of the gene in RNA, and directs the addition of a polyA signal in the RNA. The terms "expresses" and "expression" mean that they allow or that they cause the information in a gene sequence of RNA or DNA to become manifest, for example, by producing a protein activating the cellular functions involved in the transcription and translation of a corresponding gene. The terms "expresses" and "expression" include transcription of DNA to RNA, and RNA to protein. A DNA sequence is expressed in or by a cell, to form an "expression product", such as RNA (for example, messenger RNA) or a protein. It can also be said that the expression product itself is "expressed" by the cell. The term "transformation" means the introduction of a nucleic acid into a cell. The introduced sequence or gene can be referred to as a "clone". A host cell that receives the introduced DNA or RNA has been "transformed", and is a "transformant" or a "clone". The DNA or RNA introduced into a host cell can come from any source, which includes cells of the same genus or species as the host cell, or from cells of a different genus or species. Examples of transformation methods that are very well known in the art include liposome delivery, electroporation, CaP0 transformation, DEAE-dextran transformation, microinjection and viral infection. The present invention includes vectors comprising polynucleotides of the invention. The term "vector" can refer to a vehicle (eg, a plasmid) by which a DNA or RNA sequence can be introduced into a host cell to transform the host and, optionally, promoting the expression and / or replication of the introduced sequence. The polynucleotides of the invention can be expressed in an expression system * The term "expression system" means a host cell and compatible vector which, under suitable conditions, can express a protein or nucleic acid that is carried by the vector and introduced into the cell. the host cell. Common expression systems include host E. coli cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors such as plasmids, cosmids, BACs, YACs, and viruses, such as adenoviruses and viruses. adenoassociated (AAV).

Plasmids In one embodiment, the present invention comprises a kit comprising a first universal transfer vector comprising a multiple cloning site, an origin of replication and a selectable marker; a second universal transfer vector comprising a multiple cloning site, an origin of replication and a selectable marker and an amplifiable vector comprising a multiple cloning site, a promoter, an origin of replication or a chromosomal integration site, a site of polyadenylation and an amplifiable selectable marker. In general, multiple cloning sites comprise approximately 20, 25 or 30 restriction sites. The plasmids of the present invention can include any of several amplifiable labels known in the art. The use of amplifiable markers is discussed in Maniatis, Molecular Biology: A Laboratorv Manual. Cold Spring Harbor Laboratory, NY (1989)). Selectable markers useful for gene amplification in drug-resistant mammalian cells include DHFR (MTX (methotrexate) resistance) (Alt et al., J. Biol. Chem. 253: 1357 (1978); Wigler et al., Proc. Nati, Acad. Sci. USA 77: 3567 (1980)); metallothionein (cadmium resistance) (Beach et al., Proc. Nati, Acad. Sci. USA 78: 210 (1981)); CAD (resistance to N- (phosphonoacetyl) -l-aspartate (PALA)) (Wahl et al., J. Biol. Chem. 254: 8679 (1979)); adenylate deaminase (resistance to coformicin) (Debatisse et al., Mol Cell. Biol. 6: 1776 (1986)); IMP 5'-dehydrogenase (resistance to mycophenolic acid) (Huberman et al., Proc. Nati, Acad. Sel. USA 78: 3151 (1981)), and other markers known in the art (as reviewed, for example, in Kaufman et al., Meth. Enzymology 185: 537-566 (1988)). In one embodiment, the metallothionein IIA gene under the control of a metallothionein promoter is an amplifiable marker in cell lines such as CH0 C1. Amplification can be induced by the addition of Cd2 + or Zn2 + to the cell culture. The plasmids of the invention may include other non-amplifiable eukaryotic selectable markers known in the art. In one embodiment of the invention, the drug resistance marker is the hygromycin B gene, which confers hygromycin resistance. Other markers include the G418 resistance gene. The plasmids of the invention may also include a prokaryotic marker of antibiotic resistance, such as the ampicillin resistance gene or the kanamycin resistance gene. The plasmids of the invention can also include a matrix binding region (MAR). In general, MARs are DNA sequences capable of showing specific binding to nuclear proteins that are part of a nuclear matrix fibriiar analogous to the cytoskeleton. In one modality, the MAR is the MAR of chicken lysozyme. Promoters that can be used for the control of gene expression include, but are not limited to, SRa promoter (Takebe et al., Molec. And Cell Bio 8: 466-472 (1988)), the early human CMV immediate promoter. (Boshart et al., Cell 41: 521-530 (1985); Foecking et al., Gene 45: 101-105 (1986)), the immediate early promoter of mouse CMV, the early promoter region of SV40 (Benoist , et al., Nature 290: 304-310 (1981)), the immediate early promoter of Orgyia pseudotsugata, the herpes thymidine kinase promoter (Wagner et al., Proc. Nati. Acad. Sci. USA 78: 1441- 1445 (1981)), the regulatory sequences of the mefalothionein gene (Brinster, et al., Nature 296: 39-42 (1982)); prokaryotic expression vectors, such as the β-lactamase promoter (Villa-Komaroff, et al., Proc. Nati, Acad. Sci. USA 75: 3727-3731 (1978)), or the tac promoter (DeBoer, et al. ., Proc. Nati, Acad. Sci. USA 80: 21-25 (1983)); and promoter elements from yeasts or other fungi, such as the GAL1, GAL4 or GAL10 promoter, the ADH (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, or the alkaline phosphatase promoter. Viral long terminal repeat promoters, such as the long terminal repeat of the mouse mammary tumor virus (MMTV-LTR) (Fasel et al., EMBO J. 1 (1): 3-7 (1982)), the long terminal repeat of the sarcoma virus. moloney murines (Reddy et al., Proc. Nati, Acad. Sci. USA 77 (9): 5234-5238 (1980)), the long terminal repeat of the moloney murine leukemia virus (Van Beveren et al., Proc. Nati, Acad. Sci. USA 77 (6): 3307-3311 (1980)), the LTR of HIV (accession number of Genbank AB100245), the LTR of bovine foamy virus (accession number of Genbank NC_001831), the LTR 5 'of the RSV (Genbank access number K00087), the LTR of the VlH-2 (accession number of the Genbank NC_001722), a retroviral LTR of birds (Ju eí al., Cell 22: 379-386 (1980) ), and the LTR of the human herpes virus (accession number of Genb.ank NC_001806), can be included in the plasmids of the invention. Other acceptable promoters include the human CMV promoter, the human CMV5 promoter, the murine CMV promoter, the EF1a promoter, the SV40 promoter, a hybrid CMV promoter for specific expression in the liver (eg, obtained by conjugation of the immediate early promoter of CMV with the promoter elements of the transcription promoter of a1 -antitrypsin (HAT) or albumin (HAL) of human), or promoters for hepatoma-specific expression (e.g., wherein the promoter elements of the transcription of human albumin (HAL; about 1000 bp) or human a1-antitrypsin (HAT, approximately 2000 bp), combine with an 145-base-to-long-length enhancer of the precursor gene of a1-microglobulin and human bikunin (AMBP); HAL-AMBP and HAT-AMBP). In addition, bacterial promoters such as the R7 RNA polymerase promoter or the tac promoter can be used for the control of expression.

A promoter (e.g., the SRa promoter) can be bound to the cassette, and then moved in a transfer vector (e.g., pULLS or pUHLS). In another embodiment, the transfer vector may contain a promoter toward the 5 'end of the multiple cloning site (e.g., pULSRstopLS or pUHSRstopLS). When a gene, not linked to a promoter, is inserted into the multiple cloning site, it will be operably linked to the promoter towards the 5 'end. In another embodiment of the invention, a gene in a transfer vector, not linked to a promoter, can be moved into the amplifiable vector comprising a promoter (eg, SRa promoter) towards the 5 'end of the multiple cloning site ( for example, pSRXBLS). When the unbound gene is placed in the multiple cloning site, it will become operably linked to the promoter. The plasmids of the invention may also include a polyadenylation / terminator signal for the termination of the transcription of a gene in the plasmid, and for the addition of a polyA tail to the transcript. For example, the polyA signal / chicken beta-globin terminator can be included in a plasmid of the invention. Other acceptable polyA signals include the SV40 polyA signal, and the bovine growth hormone polyA signal. In one embodiment of the invention, the amplifiable vector comprises a multiple cloning site that includes the following restriction sites: Sgr Al, Srf I, Xma I, Spe I, Sac II, Rsr II, Pac I, Nru I, Not l , Nde I, Msc I, Mlu I, Kpn I, Fse 1, Bss Hll, Bsr Gl, Bsp El, Bel I, Bbv C1, Pme I, Bss Hll, Ase I and Xba I; for example, where the multiple cloning site of the amplifiable vector is that of pXBLS: ABCI Bell BsrGI Pme? Xbal EeeHII BbvCl BspEJ AAATC &GA.G GCGCGCCGTT TAAACCCTCA GCT &ATCA.TC CGGATGTACA TTTAGATCTC CGCGCGGCAA ATTTGGGAGT CGACTAGTAG GCCTACA.TGT Fsel Mlul Eael NruI BssHII Kpnx MSCJ. Nati 51 GCGCGCGGCC GGCCGGTACC ACGCGTTGGC CACATATGGC GGCCGCTCGC CGCGCGCCGG CCGGCCATGG GCGCA CCG GTGTATACCG CCGGCGAGCG Paci SacIX Srf I Xhol krui EsxII Spel Xrna.1 SgrAI 101 GATTAATTAA CGGACCGCCG CGGACTAGTG CCCGGGGG C CGGTGCTCGA CTUA TAATT GCCTGGCGGC GCCTGATCAC GGGCCCGGTG GCCACGAGCT Xhol 151 * GAAA &amp?; • '•' CTTOT: (SEQ ID NO: 10). . . . .

In one embodiment of the invention, a universal transfer vector comprises a multiple cloning site that includes the following restriction sites: Bss Hll, Pme I, Sna B1, Hin dlll, Asp 718, Kpn I, Pae R71, Xho I, Sal I, Acc I, Hinc II, Co. I, Eco RV, Eco Rl, Pst I, Eco O109I, Eco O109I, Apa I, Xma I, Bsp El, Bam H1, Dsa I, Eag I, Ecl XI, Not I , Sac II, Xma III, Xba I, Sac I, Mlu I, Bel I, Bsr Gl and Bss Hll; for example, where the multiple cloning site of the transfer vector is that of pUHLS: Ase I HirtdlII PaeE7I Pmel Bp BT Kpal Xma .l1 Xhol B = s? II BfrvCl Asp71S GGGGGCGCGC CGTTTAAACC CTCAGCTACG TAAAGCXTGG TACCCTCGAG CCCCCGCGCG GCAAA? TTGG GAGTCGATGC ATTTCGAACC ATGGGAGCTC Cla? BspEl HincII ScaRV PstI Exit X to Accl EcoRI Apal BarpHI 51 GTCGACATCG ATG &TATCGA AT CCTGCAG CAGCTGTAGC TACTATAGCT? AAGGACGTC CCCGGGGGGC CCAGGCCTCC Hot I M1-U? 3srGI Sac? BantHI Xbal BclJ Ssel 101 ATCCGCGGCC GCTCTAGAGA GCTCACGCGT TGATCATG A CAGGCCGGCC TAGGCGCCGG CGAGATCTCT CGAGTGCGCA ACTAGTA AT G CCGGCCGG Xmal BBSRII 151 AGCGCGCCCC TCGCGCGGGG (SEQ fD NO: 11).

A universal transfer vector may comprise a multiple cloning site that includes the following restriction sites: Bss Hll, Sgr Al, Xma I, Rsr ll, Spe I, Sna B1, Hin dlll, Asp 718, Kpn I, Pae R71; Xho I, Sal I, Acc I, Hinc II, Co. I, Eco RV, Eco Rl, Pst I, Eco O109I, Eco 0109I, Apa l, Xma I, Bsp El, Bam H1, Dsa I, Eag I, Ecl XI , Not I, Sac II, Xma III, Xba I, Sac I, Nde I, Msc I, Nru I, Pac I and Bss Hll; for example, where the multiple cloning site of the transfer vector is that of pULLS: B grAI Sn Bi Xínal Srfl Spel BssIlII Xmal RsrII Hiiid l? GGGGGCGCGC CACCGGTGGC CCGGGCCGGT CCGACTAGGGG ACGTAAAGCT CCCGCOCGCG GTGGCCÑ-GCG GGCCCGGCCA GGCTGATCAA TGCATTTCGA PaeR7I Clal BspEl Kpnl Hiucll EcoRV > s I left Xbol Hiixdlll Acc ~ BsoEI ApaX 51 TGG ACGCTC GAGGTCGACA TCGATGATAT CGAA TCCTG CAGGGGGCCCT ACCATGGGAG CTCCAGC? GT AGCTACT &TA GCGGAC GX'CCCCGGGA BspEI afcX Ndel KruI Sac! BamHI Xbs? Kscl Pací 101 CCGGAGGATC CGCGGCCGCT CTAGAGAGCT CCAT? TGTGG CC TCGCGAT GGCCTCCTAG GCGCCGGCGA GATCTCTCGA GGTATACACC GGTAGCGCTA PacI Xroal B? SHXJ 151 TAAGCG CGCCCC TTCGC GCGGGG (SEQ ID NO: 12).

The present invention contemplates amplifiable vectors or universal transfer vectors, comprising the multiple cloning sites referred to above in the orientation shown or in the opposite orientation. The plasmids of the present invention can be introduced into any cell line for the expression of the target polypeptides. In one embodiment of the invention, the plasmids are introduced into a mammalian cell line, preferably a Chinese hamster ovary (CHO) cell line. A commonly used cell line is the DHFR-CHO cell line, which can be transformed to the DHFR + phenotype using DHFR cDNA as an amplifiable dominant marker. One of said known DHFR-CHO cell lines is DX-B11 or DG-44. In another embodiment, the plasmids of the invention can be introduced into a lower eukaryotic cell line, such as S. cerevisiae, K. lactis, P. pastoris, C. albicans or A. fumlgatus. In addition, the plasmids of the invention can also be introduced into higher non-mammalian eukaryotic cell lines, such as insect cells (e.g., Drosophila megalogaster, sf9 cells, sf21 cells), amphibian cells (e.g., X. laevis) , plant cells (eg, A. thaliana) and bird cells. The plasmids of the invention can also be introduced into a bacterial cell. In one embodiment, competent E. coli cells are transformed. Examples of suitable E. coli include DH1, DH5, DH5a, XL1-Blue, SURE, SCS110, OneShot Top 10 and HB101. Plasmids can be introduced into a cell by any of the many methods that are commonly known in the art. For example, a plasmid of the invention can be used to transform a cell by the calcium phosphate precipitation method, electroporation, the DEAE-dextran method, or the liposome delivery method.

The plasmids of the invention can include any gene or combination of genes. In one embodiment of the invention, the plasmids include heavy and light chain immunoglobulin genes. The immunoglobulin chains can be part of antibodies that specifically recognize any antigen such as IL-5, IGFR1 or IL-10. Receptors or subunits of receivers, can also be expressed. For example, a gene encoding the IL-2 receptor or a portion thereof (eg, membrane domain) can be included in a plasmid of the invention. The patent application of E.U.A. No. 10 / 443,466, filed on May 22, 2003, which is hereby incorporated by reference in its entirety, discloses the nucleotide and amino acid sequences of the immunoglobulin heavy chain and light chain variable regions of anti-IGFR1 antibodies. Any of the heavy and light chain variable regions described herein can be incorporated into the plasmid system of the invention, and can be expressed. In one embodiment, the light chain variable region of anti-IGFR1 antibody is encoded by the nucleotide sequence set forth in SEQ ID NO: 15 or 19, or is any polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 15 or 19. NO: 16 or 20, and / or the heavy chain variable region of anti-IGFR1 antibody is encoded by the nucleotide sequence set forth in SEQ ID NO: 17 or 21, or is any polynucleotide that codes for a polypeptide comprising the sequence of amino acids of SEQ ID NO: 18 or 22. Each of the expression cassettes encoding the light and heavy immunoglobulin chain of the anti-IGFR1 antibody, can be entered in the multiple cloning site of pULLS or pUHLS. Preferably, the immunoglobulin light and heavy chains, in the expression cassettes, are linked to an immunoglobulin constant region such as? 1,? 4 or K. Preferably, the expression cassettes are then inserted into the amplifiable vector pXBLS , which is then introduced into a suitable cell to cause the expression of the light and heavy chains. For example, plasmid pA! GFRV3, which contains the immunoglobulin heavy and light chains of an anti-IGFR1 antibody, can be introduced into a mammalian dhfr'-cell line (eg, CHO-DXB11), wherein the chains they express themselves Equipment The plasmid system of the invention can be provided in equipment. The kits of the invention may include, in addition to the plasmid system, any reagent that can be used in the use of the plasmid system. In one embodiment, the kit includes reagents necessary for the transformation of the plasmids into mammalian cells. For example, the kit may include reagents for a transformation process with calcium phosphate: calcium chloride, pH regulator (eg, 2x saline regulated in its pH with HEPES) and sterile distilled water. In another embodiment, the kit includes reagents for a transformation with DEAE-dextran: chloroquine in PBS, DEAE-dextran in PBS and saline regulated in its pH with phosphate. In another embodiment, reagents for a transformation with liposomes are included in the kit: extruded liposomes of extruded liposomes with DOTAP / cholesterol. For example, the kit may include the transfection reagent based on cationic lipids Lipofectamine ™ (Invitrogen Life Technologies, Carlsbad, CA). The kit may include reagents required for the bacterial transformation of the plasmids of the invention. For example, the kit can include bacteria of competent transformation (for example, DH1, DH5, DH5a, XL1-Blue, SURE, SCS110, OneShot Top 10 or HB101). The kit may include reagents or growth media required to produce the growth medium. For example, in one embodiment, the kit may include fetal calf serum or DMEM (Dulbecco / Vogt modified Eagle's minimal essential eagle (Harry Eagle) medium) for the growth of mammalian cells. In another embodiment, the kit may contain powdered Luria broth medium or Luria broth plates containing a suitable antibiotic (eg, ampicillin or kanamycin) to inhibit the growth of bacteria. Components supplied in the equipment can be supplied in suitable jars or containers (for example, plastic or glass jars). The equipment may include instructions on proper labeling for storage, and instructions suitable for use.

Expression and purification of proteins The polypeptides produced in the plasmid system of the invention can be purified by standard methods including, but not limited to, precipitation with salts or alcohol, affinity chromatography (for example, used in conjunction with a label of purification), preparative disk gel electrophoresis, isoelectric focusing, high pressure liquid chromatography (HPLC), inverted phase high pressure liquid chromatography, gel filtration, partition chromatography and cation and anion exchange, and distribution by countercurrent Such purification methods are well known in the art and are described, for example, in "Guide to Protein Purification," Methods in Enzvmology: Vol. 182, M. Deutscher, ed., 1990, Academic Press, New York, NY. In particular where a polypeptide is being isolated from a cellular or tissue source, it is preferred to include one or more inhibitors of proteolytic enzymes in the test system, such as phenylmetanesulfonyl fluoride (PMSF), Pefabloc SC, pepstatin, leupeptin, chemostatin and EDTA The polypeptides of the invention can be fused with a second portion of polypeptide or polynucleotide, which can be referred to as a "tag". A label can be used, for example, to facilitate purification or detection of the polypeptide after expression. A fused polypeptide can be constructed, for example, by inserting into the reading frame a polynucleotide encoding the label at the 5 'or 3' end of the polynucleotide encoding the polypeptide to be expressed. The fused polynucleotide can then be expressed in the plasmid system of the invention. Such markers include glutathione-S-transferase (GST) markers, hexahistidine (His6), maltose-binding protein (MBP) markers, hemagglutinin (HA) markers, cellulose binding protein (CBP) markers, and myc markers. . Detectable labels such as 32P, 35S, 3H, 99mTc, 23l, 11 ln, 68Ga, 1dF, 125l, 31l, 1 3mln, 76Br, 67Ga, S9mTc, 123l, 111ln and 68Ga, can also be used to label polypeptides and polynucleotides of the invention. Methods for the construction and use of such fusions are very conventional and well known in the art.

EXAMPLES The following examples are provided to better describe the present invention, and should not be considered as a limitation thereof. The scope of the present invention includes all the plasmids set out below, and any of them, in the following examples, either individually or as part of a kit. Also included within the scope of the invention are all methods, and any of them, which are discussed below in the following examples.

EXAMPLE 1 Construction of the amplifiable cloning vectors, pSRXBLS and pXBLS This example describes the construction of mammalian expression vectors, pSRXBLS and pXBLS. A large multiple cloning site was inserted into the pDSRG plasmid towards the 3 'end of the SRa promoter, to generate pSRXBLS. pXBLS, a derivative of pSRXBLS, lacks any promoter. Both plasmids can serve as amplifiable vectors, in which more than one expression cassette can be easily inserted, for example, for light and heavy chain cDNA molecules of an antibody gene. A 155 bp multiple cloning site, pDSRG-xba-xho, was designed, synthesized by PCR, and was initially cloned into the TA cloning vector (Invitrogen, Carlsbad, CA). It was then cloned into the Xhol and Xbal sites of pDSRG, resulting in pSRXBLS. The SRa promoter was retained in the vector pSRXBLS. PSRXBLS was also digested with Xhol and Hindlll to remove the SRa promoter. The ends were then filled with Klenow enzyme and ligated again, regenerating the Hindlll site, to construct pXBLS.

EXAMPLE 2 Construction of the universal transfer vectors pUHLS, pULLS. and his descendants This example describes the construction of universal transfer vectors, each having a large multiple cloning site, and its descendants, each having a promoter and a polyA / terminator addition site. pUHLS and pULLS are the universal transfer vectors, and pUHSRstopLS and pULSRstopLS, are their corresponding descendants that possess the SRa promoter and the chicken beta-globin terminator. The plasmid system is constructed in this way, that different subunits of a large complex protein can be expressed in these vectors separately. Then, the expression cassettes for each subunit can be transferred to an individual vector, such as pXBLS or pSRXBLS, to facilitate the transfection, integration and equimolar production of a multiple subunit protein. Two sites of multiple cloning, universal plasmid primer 160 of base pairs, and universal plasmid primer 166 of base pairs, were designed to construct pUHLS and pULLS. Both cloning sites were synthesized by PCR, cloned into the TA cloning vector (Invitrogen), and then cloned into the BssHIl sites of the pCRScript vector (Stratagene). In this way, the original multiple cloning site of pCRScript was replaced with newly synthesized multiple cloning sites. The new vectors, pUHLS and pULLS, were derived from universal plasmid primer 1 and universal plasmid primer 2, respectively. A 249 base pair region of the chicken beta-globin terminator, derived from pDSRG by digestion with BamHI and Xbal in pUHLS and pULLS at the BamHI and Xbal sites, was inserted to generate pUHstopLS and pULstopLS, respectively. The SRa promoter and its accompanying intron, derived from pDSRG by digestion with Hindlll and Sali in pUHstopLS and pULstopLS in the Hindlll and Xhol sites, were inserted to generate pUHSRstopLS and pULSRstopLS, respectively.

EXAMPLE 3 Construction of pAIL5V1 The construction of pAIL5V1 for the expression of light and heavy antibody chains in a single vector is described herein. In addition to variations in their orientations, two types of plasmids have been constructed. The first has only the dhfr marker for selection with amplification. The second type of expression plasmid has the dhfr marker, together with the gene for hygromycin resistance (Hyg). This adds versatility, allowing selection with or without amplification. The heavy chain gene of the human anti-hulL5 monoclonal antibody (MAb) was isolated, and inserted into the vector pUHSRstopLS (see above) at the EcoRI and Xmal sites, to generate pUSRHLS. The light chain gene of the anti-hulL5 MAb was isolated, and inserted into pULSRstopLS at the EcoRI and Apal sites, to generate pUSRLLS. The minimal hCMV promoter, derived from pcDNA3.1 (Invitrogen; Carlsbad, CA) by digestion with Nrul and EcoRI, was replaced with the SRa promoter, which was removed by digestion with SnaBI and EcoRI, in pUSRHLS and pUSRLLS, to generate pUhCMVHLS and pUhCMVLLS. The TK-hygromycin gene (TK / Hyg) was inserted into pUhCMVHLS at the Fsel sites, to construct pUHhyg (+) hCMVLS and pUHhyg (-) hCMVLS. The light chain antibody cassette was transferred from pUhCMVLLS, by digestion with Pací and Srfl, to pXBLS, at the Pací and Srfl sites, to construct pAIL5L (-) hCMVLS and pAIL5L (+) hCMVLS. The heavy chain antibody cassette from pUHhyg (-) hCMVLS was transferred to pAIL5L (+) hCMVLS at the BssHIl sites, to generate PAIL5V1.

EXAMPLE 4 Construction of pAIGFRV3 CDNA molecules encoding the variable regions of a hybridoma expressing an anti-IGFR1 19D12 / 15H12 monoclonal antibody were isolated and cloned into TA cloning vectors (Invitrogen, Carlsbad, CA). The nucleotide and amino acid sequences of the heavy and light chain of the 19D12 / 15H12 antibody are set forth in the patent application of E.U.A. No. 10 / 443,466, filed May 22, 2003, which is hereby incorporated by reference in its entirety. the heavy chain of the EcoRI and Apal sites from the TA vector containing cDNA for the variable region of heavy chain of anti-IGFR1, the same sites pUhCMVHLS (see above) was transferred, to build pUhCMVIGFRHLS containing cDNAs for the light chain of anti-IGFR1. For selection, a TK-hygromycin resistance cassette was inserted in the Fsel site of pUhCMVIGFRHLS, to construct pUhCMVHyg (-) IGFRHLS. The light chain from the EcoRI and Bbsl sites of the TA plasmid was transferred to the same pUhCMVLLS sites (see above), to construct pUhCMVIGFRLLS. The whole light chain expression cassette was then transferred from pUhCMVIGFRLLS to pXBLS at the PacI and Srfl sites, to construct pAlGFRLLS. the expression cassette heavy chain together with the expression cassette hygromycin, to pAlGFRLLS transferred in BssHU sites to build pAIGFRVI and pAIGFRV3 (pIAGFRVI is essentially identical to pAIGFRV3, unless the orientation of the heavy chain and the genes of TK-Hyg are opposite).

EXAMPLE 5 Construction of pAIL10V3 CDNA molecules were isolated that code for the variable regions of 12G8, a rat antibody that recognizes IL-10. The heavy chain variable region of 12G8 was transferred to Kpnl and Apal sites of the pUhyg (-) IG1FRhuH plasmid, to construct pUIL10H. Plasmid pUhyg (-) IG1FrhuH possesses the modified cDNA for the variable region of IGFR1 together with lgG1 cDNA and the TK-hygromycin cassette. The light chain variable region of 12G8 was transferred to the EcoRI and Apal sites of pAIL5 (-) hCMVLS, to construct pAIL10 (-) L. The heavy chain expression cassette from pUIL10H was transferred to pA! L10 (-) L at the BssHI1 restriction sites, to construct PAIL10V3.

EXAMPLE 6 Construction of pAIG1FR (-) IL2LS PAIG1FR (-) IL2LS was constructed in a three-step procedure. The construction procedure was started with the transfer of an IRES-IL2Ra element to pULstopLS. The plasmid containing the IRES-IL2Ra, pme18IRES, was digested with Spel and NotI restriction enzymes, and the NotI site was completely filled using Klenow enzyme, to derive the ÍRES-IL2Ra element. Simultaneously, pULstopLS was digested with EcoRV and Spel enzymes, and the Spel site was filled using Klenow enzyme, and ligated with the IRES-IL2Ra element, to construct pULstoplRESIL2R. pULstoplRESIL2R was also digested with enzymes Spel and Xbal, and the Spel site was completely filled with Klénow enzyme. Also pUhCMVIGFRLLS was digested with XbaI and BspEI enzymes BspEI site and was completely filled using Klenow enzyme, and ligated with XbaI-SpeI fragment that was generated from pULstoplRESIL2R to build pUIGFRL-IRESIL2R. The IGFR1 heavy chain expression cassette was transferred from pUhyg (-) IG1FRhuH to pUIGFRL-lRESlL2R at BssHI1 restriction sites, to construct pAIG1FR (-) IL2LS.

EXAMPLE 7 Development of cell lines for the expression of the anti-IGFR1 19D12 monoclonal antibody In this example, the development and growth of cell lines for the expression of antibody 19D12 (LCF / HCA) are presented. DXB1-1--3e culture cells developed cells in MEM alpha medium with ribonucleosides and deoxyribonucleosides (GIBCO, 12571-063 catalog #; Gibco-Invitrogen Corp, Carlsbad, CA) plus 10% FBS (HyClone, Catalog # SH30071.03; Hyclone; Logan, UT). Selection medium with hygromycin. Cells were divided at 48 hours post-transfection. cells in alpha MEM medium ribonucleosides and deoxyribonucleosides (GIBCO, 12561-056 catalog #) were developed more than 10% dialyzed FBS (HyClone, Catalog # SH30079.03) plus hygromycin B (CLONTECH, catalog # 8057- 1; BD Biosciences-Clontech; Palo Alto, CA) at 300 μ / mL.

Subcloning medium. Subcloning was performed in MEM medium of MEM without ribonucleosides and deoxyribonucleosides (GIBCO, catalog # 12561-056) plus dialyzed FBS at 10% (HyClone, catalog # SH30079.03). Amplification medium with methotrexate. Amplification with methotrexate was carried out in MEM medium without ribonucleosides and deoxyribonucleosides (GIBCO, catalog # 12561-056) plus dialyzed FBS at 10% (HyClone, catalog # SH30079.03) plus MTX (Sigma, catalog # M8407; Sigma-Aldrich Corp; St. Louis, MO) at 20, 80 and 320 nM, respectively. Medium for adaptation to serum free suspension. Adaptation to serum-free suspension was carried out in protein-free CHO medium (Sigma, catalog # C5467) supplemented with 20 ml / L of 200 mM L-glutamine (GIBCO, catalog # 25030-081) and 10 ml / L of lipids (high in cholesterol) (Sigma, catalog # L4646). Provision medium for 3 liters production batch. L-glutamine-200 mM (GIBCO, catalog # 25030-081) and glucose solution (Sigma, catalog # G8769) were provided as the provision during production tests. Transfection and subcloning method. The DXB11 cells were trypsinized, counting them, and plated at 2 x 106 cells / T25 flask on the day before transfection, so that they become 50 to 90% confluent on the day of transfection. Transfections were performed using 2.5 μg of DNA (pAIGFRV3) / T25 flask and LipofectAMINE PLUS ™ reagent (GIBCO, catalog # 10964-013). According to the supplier's instructions, the DNA was first combined with PLUS reagent, the DNA-PLUS complex was mixed with LipofectAMINE reagent, and the DNA-PLUS-LipofectAMINE complex was then used to transfect the cells. The cells were incubated at 37 ° C, C02 at 5%, for 3 hours. After incubation, the culture medium of DXB11 cells was added to the desired volume, the cells and medium were transferred to a T75 flask, and the cells were grown for 2 days. The medium was exchanged with selection medium with hygromycin, and the cells were grown for 10 days to 2 weeks. Some cells were stacked at this stage, and the remaining cells were subcloned into 96-well plates. Subcloning was started in 96-well plates with subcloning medium. Individual clones were successively grown in 24-well plates, 6-well plates, T-25 flasks and T-75 flasks, after detection of satisfactory expression by ELISA at each step. Methotrexate medium was added in 20 to 30% confluent cultures for amplification. Methotrexate amplification was carried out at 20, 40, 80 and 320 nM for 10 days at 2 weeks. After amplification, the medium was exchanged with the subcloning medium, and the cells were allowed to develop to approximately 10% confluency. The cells underwent another round of subcloning at this stage. After the second round of subcloning, the cells were subjected to adaptation in serum free suspension culture with the medium designated in the T-25 flask stage. Serum was removed sequentially from the medium by dilution with serum free adaptation medium, and the cells were finally transferred to shake flasks with 2.5% serum. The remaining serum was removed by subsequent dilution (separation) of the cultures. The serum free culture was graduated to three liters.

EXAMPLE 8 Propagation of cells expressing anti-IL5 antibody Cells possessing pAIL5V1 are thawed from a frozen flask and propagated in suspension using protein-free Sigma CHO medium (C-5467 supplemented with 0.57 g / L L-glutamine). All cultures are maintained in an incubator at 37 ° C, C02 at 7.5%, or in an oscillating bag platform set at 37 ° C, and supplying C02 at 7.5%. The inoculation train begins in a shake flask, and is passed continuously and increased in proportion, until there is enough culture to start a 20 liter bag with a useful volume of 2 liters. When the bag reaches the predetermined separation criteria, it is extended in proportion to a useful volume of 10 liters. When the bag reaches the predetermined separation criteria it is separated, and the remaining culture is used to start another 20 liter oscillating bag (useful volume of 10 liters) in parallel. When the two oscillating bags reach the proper separation density, they are used to seed the production bioreactor. The shake flasks and the oscillating bags are typically separated at 1: 4 dilutions when the density of viable cells reaches 1-1.5 x 10 6 viable cells / mL. The inoculum deposit is diluted 1: 4 by entering the bioreactor. Flow chart illustrating the propagation procedure: Jar Defrosting SF-250V (medium C-5467 CHO supplemented with L-glutamine) mL, then at 60 mL (two steps) SF-1000V (medium C-5467 CHO supplemented with L-glutamine) 250 mL (one step), then create another SF-1000V with a useful volume of 250 mL ! L oscillating bag (medium C-5467 CHO supplemented with L-glutamine) 2 L (one step), then at 10 L (one step), then create another 20 L bag with a useful volume of 10 L i One-volume volume production bioreactor (medium C-5467 CHO supplemented with L-glutamine) 60 L (one step), then up to full volume (200L).

EXAMPLE 9 Procedure for purification of anti-IL10 antibody This example describes the procedure for the purification of the anti-IL10 antibody encoded by pAIL10V3, from a fermentation of CHO cells of 200 liters. The steps include: - Harvesting the cell culture supernatant by filtration with a CUNO filter positively charged in series with a 0.2 μm filter. - Affinity chromatography on rProtein-A Sepharose ™ Fast Flow from Amersham (4L) eluted by a pH 3.0 step. - Viral inactivation by incubation at pH 3.5 for 1 hour at 20-22 ° C, followed by pH adjustment at 5.5. - Cation exchange chromatography in EMD Fractogel® SE HiCap (4L) at pH 5.5 eluted with a gradient of 20 BV to NaCl at 250 mM. - Concentration (2x) / diafiltration (10x) in Tris at 200 mM, pH 8.0. - Anionic exchange chromatography in Q Sepharose ™ Fast Flow (4L) from Amersham in through flow mode. The maximum value not retained is grouped and adjusted to pH 5.5.

- Viral filtration with Planova filters: a Planova 35 filter of 0.1 m2 in series with 2 to 4 Planova 20 filters of 0.1 m2. - Final concentration (6-1 Ox) and diafiltration (10x) in 20 mM sodium acetate, followed by filtration (0.2 μm). This procedure gives material that is more than 99% pure by inverted phase HPLC. The overall performance is 70%.

EXAMPLE 10 Expression of anti-IGFR1 and anti-IL-10 antibodies In this example, expression plasmids were constructed which included the chains of anti-IGFR and anti-IL-10 antibodies, where the genes of the antibody chains were located in the plasmids, adjacent to an element of the MAR (Selexis Geneva, Switzerland; Kim et al., J. Biotechnol. 107 (2): 95-105 (2004); Stief et al., Nature 341: 343-345 (1989); Phi-Van et al., Mol. Cell Biol. 10: 2302-2307 (1990); Kalos et al., Mol. Ceil. Biol. 15: 198-207 (1995)). The MAR element is a DNA element of approximately 3 kb that facilitates the expression of a recombinant gene that is stably integrated into the host chromosome after its incorporation into the cell. The MAR element was inserted into the mammalian expression plasmids, pAILIOVi, having anti-IL10 together with the hygromycin expression cassette, pAIL10V1 / pure, having anti-IL10 together with the puromycin expression cassette in place of hygromycin, pAiGFRLCb2V1, which has anti-IGFR1 together with the hygromycin expression cassette, and pAIGFRLCb2V1 / pure, which has anti-IGFR1 together with the puromycin expression cassette in place of hygromycin. Each plasmid already contained four independent mammalian expression cassettes. The vector, pPAGOl, contained the DNA element of the matrix binding region (MAR) of chicken lysozyme of approximately 3 kb. One of the universal vectors, pULLS, was digested by restriction enzymes Age1 and BamH1, and ligated again, after final filling by Klenow enzyme, to construct the pULLSmod vector. Plasmid pPAGOl was digested by BamH1 and Xbal, to transfer the MAR element to pULLSmod at the same sites, to construct the pULMAR plasmid. The MAR element was finally transferred to the plasmids expressing anti-IL10 and anti-IGFR1. pULMAR was digested with BssHIl, and the fragment containing the MAR element was transferred to the Asc1 sites of the pAILIOVi, pAIL10V1 / pure plasmids, pAIGFRLCb2V1 and pAlGFRLCb2V1 / pure, to construct pinAIL10 / MAR (-), pAIL10V1 / pure / MAR (-), pAIGFRLCb2 / MAR (-) and pAIGFRLCb2 / pure / MAR (-), respectively. Plasmids containing MAR were introduced into the CHO cell line, DXB11 cells, and the chains of the antibodies were expressed. The expression of the antibody chains was confirmed by ELISA, as well as by HPLC analysis. In the HPLC analysis, the proteins isolated from the CHO cells were fractionated using an inverted phase column or a protein A column. Protein eluted spectrophotometrically was detected at A28onnv The present invention will not be limited in scope by the specific embodiments described in I presented. Of course, various modifications of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description. It is intended that said modifications be within the scope of the appended claims. Patents, patent applications, publications, product descriptions and protocols are cited throughout this application, and their description is incorporated herein in its entirety as a reference for all purposes. -

Claims (39)

NOVELTY OF THE INVENTION CLAIMS

1. - A system of plasmids comprising in separate containers: (a) a first universal transfer vector comprising the following first multiple cloning site: Bss Hll, Pme I, Sna B1, Hin dlll, Asp 718, Kpn I, Pae R71 , Xho I, Sal I, Acc I, Hinc II, Cia I, Eco RV, Eco Rl, Pst I, Eco O109I, Eco O109I, Apa I, Xma I, Bsp El, Bam H1, Dsa I, Eag I, Ecl XI, Not I, Sac II, Xma III, Xba I, Sac I, Mlu I, Bel I, Bsr Gl and Bss Hll; (b) a second universal transfer vector comprising the following second multiple cloning site: Bss Hll, Sgr Al, Xma I, Rsr II, Spe I, Sna B1, Hin dlll, Asp 718, Kpn I, Pae R71; Xho I, Sal I, Acc I, Hinc II, Cia I, Eco RV, Eco Rl, Pst I, Eco O1091, Eco O109I, Apa I, Xma I, Bsp El, Bam H1, Dsa I, Eag I, Ecl XI , Not I, Sac II, Xma III, Xba I, Sac I, Nde I, Msc I, Nru I, Pac I and Bss Hll; and (c) an amplifiable vector comprising the following third multiple cloning site: Sgr Al, Srf I, Xma I, Spe I, Sac II, Rsr II, Pac I, Nru I, Not I, Nde I, Msc I, Mlu I, Kpn I, Fse 1, Bss Hll, Bsr Gl, Bsp El, Bel I, Bbv C1, Pme I, Bss Hll, Ase I and Xba I.

2.- The plasmid system according to the claim 1, further characterized in that the first universal transfer vector comprises the plasmid map of Figure 2, the second universal transfer vector comprises the plasmid map of Figure 1, and the amplifiable vector comprises the plasmid map of Figure 3

3. The plasmid system according to claim 1, further characterized in that the multiple cloning site of the first universal transfer vector comprises the nucleotide sequence set forth in SEQ ID NO: 11.

4.- The plasmid system of according to claim 1, further characterized in that the multiple cloning site of the second universal transfer vector comprises the nucleotide sequence set forth in SEQ ID NO: 12.

5. The plasmid system according to claim 1, further characterized in that the multiple cloning site of the amplifiable vector comprises the nucleotide sequence set forth in SEQ ID NO: 1 0.

6.- The system of piásmidos in accordance with the claim 1, further characterized in that at least one of the plasmids comprises a promoter located towards or at the 5 'end of the multiple cloning site.

7. The plasmid system according to claim 6, further characterized in that the promoter is selected from the SRa promoter, long terminal repeat of mouse mammary tumor virus (MMTV-LTR), human cytomegalovirus immediate early promoter (hCMV) and immediate early promoter of murine cytomegalovirus (mCMV)).

8. - The plasmid system according to claim 7, further characterized in that the first universal transfer vector comprises the plasmid map of figure 10.

9. The plasmid system according to claim 7, further characterized in that the second The universal transfer vector comprises the plasmid map of Figure 11.

10. The plasmid system according to claim 7, further characterized in that the amplifiable vector comprises the plasmid map of figure 9.

11.- The system of plasmids according to claim 7, further characterized in that the first universal transfer vector comprises the nucleotide sequence set forth in SEQ ID NO: 5.

12. The plasmid system according to claim 7, further characterized in that the second vector Universal transfer comprises the nucleotide sequence set forth in SEQ ID NO: 4.

13.- The plasmid system in accordance with claim 7, further characterized in that the amplifiable vector comprises the nucleotide sequence set forth in SEQ ID NO: 13.

14. The plasmid system according to claim 1, further characterized in that at least one of the Universal transfer vectors comprise a polyA / terminator addition site.

15. The plasmid system according to claim 1, further characterized in that the amplifiable vector comprises a DHFR gene.

16. A system of plasmids comprising in separate containers: (a) a first universal transfer vector comprising the nucleotide sequence set forth in SEQ ID NO: 2; (b) a second universal transfer vector comprising the nucleotide sequence set forth in SEQ ID NO: 1; and (c) an amplifiable vector comprising the nucleotide sequence set forth in SEQ ID NO: 3.

17. The plasmid system according to claim 1, further characterized in that the cassettes encode an immunoglobulin light chain or a chain. heavy immunoglobulin.

18. The plasmid system according to claim 1, further characterized in that the first or second universal transfer vector comprises a first series of one or more expression cassettes, the other universal transfer vector comprises a second series of one or more. more expression cassettes, and the amplifiable vector comprises said first series and second series of expression cassettes; wherein: (a) the first set of one or more expression cassettes comprises an anti-IL5 immunoglobulin heavy chain gene expression cassette, and the second set of one or more expression cassettes comprises a gene expression cassette of light chain of anti-IL5 immunoglobulin; (b) the first series of one or more expression cassettes comprises an anti-IGFR1 immunoglobulin heavy chain gene expression cassette, and the second series of one or more expression cassettes comprises a light chain gene expression cassette of anti-IGFR1 immunoglobulin; (c) the first series of one or more expression cassettes comprises an expression cassette comprising a bicistronic gene expression cassette, whose bicistronic gene comprises an anti-IGFR1 immunoglobulin light chain gene and an alpha subunit gene of the receptor of IL2, wherein said genes are linked by an internal ribosome entry sequence (IRES), and the second series of one or more expression cassettes is an anti-IGFR1 immunoglobulin heavy chain gene expression cassette and a cassette of expression of hygromycin resistance gene; or (d) the first series of one or more expression cassettes comprises an anti-IL10 immunoglobulin heavy chain gene expression cassette, and the second series of one or more expression cassettes comprises a chain gene expression cassette light of anti-IL10 immunoglobulin and a hygromycin resistance gene expression cassette.

19. The plasmid system according to claim 18, further characterized in that the amplifiable vector comprises a plasmid map as set out in a selected figure of figures 4 to 7.

20. - The plasmid system according to claim 19, further characterized in that the amplifiable vector comprises a nucleotide sequence selected from SEQ ID NOs: 6-9.

21. The plasmid system according to claim 1, further characterized in that the first universal transfer vector, second universal transfer vector or the amplifiable vector, comprises a matrix binding region.

22. The plasmid system according to claim 21, further characterized in that the amplifiable vector comprises (a) a heavy chain of anti-IGFR1 antibody or an anti-IL10 antibody heavy chain, and (b) a resistance marker. to puromycin or a hygromycin resistance marker.

23. The plasmid system according to claim 21, further characterized in that the amplifiable vector is characterized by a plasmid map selected from the group consisting of figures 13 to 16.

24.- The plasmid system according to claim 21, further characterized in that the amplifiable vector comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24-27.

25. A method for the production of a protein comprising two or more types of polypeptide, comprising the steps of: (a) introducing a series of one or more expression cassettes into a first universal transfer vector comprising the following first multiple cloning site: Bss Hll, Pme I, Sna B1, Hin dlll, Asp 718, Kpn l, Pae R71, Xho I, Sal I, Acc I, Hinc II, Cia I, Eco RV, Eco Rl, Pst I , Eco O109I, Eco O109I, Apa I, Xma !, Bsp El, Bam H1, Dsa I, Eag I, Ecl XI, Not I, Sac ll, Xma III, Xba I, Sac I, Mlu I, Bel I, Bsr Gl and Bss Hll; (b) introducing a series of one or more different expression cassettes into a second universal transfer vector comprising the following second multiple cloning site: Bss Hll, Sgr Al, Xma I, Rsr II, Spe I, Sna B1, Hin dlll, Asp 718, Kpn I, Pae R71; Xho I, Sal I, Acc I, Hinc II, Cia I, Eco RV, Eco Rl, Pst I, Eco O1091, Eco O109I, Apa l, Xma I, Bsp El, Bam H1, Dsa I, Eag I, Ecl XI , Not I, Sac ll, Xma III, Xba I, Sac I, Nde I, Msc l, Nru I, Pac I and Bss Hll; (c) moving the cassettes of the transfer vectors into an amplifiable vector comprising the following third multiple cloning site: Sgr Al, Srf I, Xma I, Spe I, Sac II, Rsr II, Pac I, Nru I, Not I, Nde I, Msc I, Mlu I, Kpn I, Fse 1, Bss Hll, Bsr Gl, Bsp El, Bel I, Bbv C1, Pme l, Bss Hll, Ase I and Xba I; and (d) cause the expression of said cassettes.

26. The method according to claim 25, further characterized in that the first universal transfer vector, the second universal transfer vector or the amplifiable vector, comprises a matrix binding region.

27. The method according to claim 26, further characterized in that the amplifiable vector comprises (a) an anti-IGFR1 antibody heavy chain or an anti-IL10 antibody heavy chain, and (b) a puromycin resistance marker. or a hygromycin resistance marker.

28. The method according to claim 26, further characterized in that the amplifiable vector is characterized by a plasmid map selected from the group consisting of figures 13 to 16.

29.- The method according to claim 26, characterized in addition because the amplifiable vector comprises a sequence of nucleotides selected from the group consisting of SEQ ID NOs: 24-27.

30. The method according to claim 25, further characterized in that it comprises the purification of the protein.

31. The method according to claim 25, further characterized in that (a) an expression cassette encodes an anti-IL5 immunoglobulin heavy chain, and the other expression cassette encodes an anti-IL5 immunoglobulin light chain; (b) one expression cassette encodes an anti-IGFR1 immunoglobulin heavy chain, and the other expression cassette encodes an anti-IGFR1 immunoglobulin light chain; (C) an expression cassette comprising a bicistronic gene encoding an immunoglobulin light chain anti-IGFR1 and an alpha receptor subunit 1L2, which are linked by an internal sequence ribosome entry (I RES), and another expression cassette encodes an immunoglobulin heavy chain anti-IGFR1 and HYG-B; or (d) one expression cassette encodes an anti-IL10 immunoglobulin heavy chain, and the other expression cassette encodes an anti-IL10 and HYG-B immunoglobulin light chain.

32. A method for the production of an anti-IGFR1 antibody, comprising the steps of (a) introducing an expression cassette comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 18 and 22, or an expression cassette comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 16 and 20, in a first vector of universal transfer comprising the following first multiple cloning site: Bss Hll, Pme I, Sna B1, Hin dlll, Asp 718, Kpn I, Pae R71, Xho I, Sal I, Acc I, Hinc II, Cia I, Eco RV, Eco Rl, Pst I, Eco O109I, Eco O109I , Apa I, Xma I, Bsp El, Bam H1, Dsa I, Eag I, Ecl XI, Not I, Sac II, Xma III, Xba.l, Sac I, Mlu I, Bel I, Bsr Gl and Bss Hll; - (b) introducing the other expression cassette, not introduced into said first vector, into a second universal transfer vector comprising the following second multiple cloning site: Bss Hll, Sgr Al, Xma l, Rsr II, Spe I, Sna B1, Hin dlll, Asp 718, Kpn I, Pae R71; Xho I, Sal I, Acc I, Hinc II, Cia I, Eco RV, Eco Rl, Pst I, Eco O109I, Eco O109I, Apa l, Xma I, Bsp El, Bam H1, Dsa I, Eag I, Ecl XI , Not I, Sac ll, Xma III, Xba I, Sac I, Nde I, Msc I, Nru I, Pac and Bss Hll; (c) moving the cassettes of the transfer vectors into an amplifiable vector comprising the following third multiple cloning site: Sgr Al, Srf I, Xma I, Spe I, Sac II, Rsr II, Pac I, Nru I, Not I, Nde I, Msc I, Mlu I, Kpn I, Fse 1, Bss Hll, Bsr Gl, Bsp El, Bel I, Bbv C1, Pme l, Bss Hll, Ase I and Xba I; and (d) cause the expression of said cassettes.

33. - The method according to claim 32, further characterized in that it comprises the purification of the antibody.

34. The method according to claim 32, further characterized in that the polynucleotide encoding a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 18 and 22, comprises a nucleotide sequence selected from SEQ ID NOs: 17 and 21. 35.- The method according to claim 32, further characterized in that the polynucleotide encoding a polypeptide comprising a sequence of amino acids selected from SEQ ID NOs: 16 and 20, comprises a nucleotide sequence selected from SEQ ID NOs: 15 and 19. 36.- The method according to claim 32, further characterized in that the expression cassettes are operably linked to a cytomegalovirus promoter. human (hCMV). 37.- A device comprising the plasmid system according to claim 1, and one or more components selected from (i) sterile distilled water; (ii) transformation reagents with calcium phosphate CaCl2 and 2X saline regulated in its pH with HEPES; (iii) transformation reagents with DEAE-dextran chloroquine in saline regulated in its pH with phosphate and saline regulated in its pH with phosphate; (iv) liposomes extruded with DOTAP / cholesterol; (v) competent transformation E. coli; (vi) Dulbecco / Vogt Modified Eagle minimum essential medium (DMEM); (vii) fetal calf serum; (viii) Luria broth medium; and (ix) paper instructions for the use of the plasmid system. 38.- An initiating oligonucleotide comprising a nucleotide sequence selected from SEQ ID NOs: 10, 11 and 12. 39.- A plasmid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-9.